Advanced Pharmacognosy Notes

T.B.EKNATH BABU

STUDENT AT ARULMIGU

KALASALINGAM COLLEGE OF

PHARMACY

THIS ADVANCED

PHARMACOGNOSY

NOTES BELONGINGS

TO

Dr. TAMILNADU M.G.R

MEDICAL UNIVERSITY

TAMILNADU

TRACER

TECHNIQUE

INTRODUCTION

Living plants considered as biosynthetic laboratory

p

r

i

m

a

r

y

as well as secondary metabolite. i) Different biosynthetic pathway: -

Shikmic acid pathway

Mevalonic acid pathway

Acetate pathway ii) Various intermediate and steps are involved in

biosynthetic pathway in plants can be investigated

by means of following techniques: - Tracer technique

Use of isolated organ

Grafting methods

Use of mutant strain

• Definition: - It can be defined as

technique which utilizes a

labelled compound to find out or

to trace the different intermediates

and various steps in biosynthetic

pathways in plants, at a given rate

& time.

OR

• In this technique different isotope,

mainly the radioactive isotopes

which are incorporated into

presumed precursor of plant

metabolites and are used as

marker in biogenic experiments.

The labelled compound can be

prepared by use of two types of

isotopes. » Radioactive isotopes. » Stable isotopes.

Radioactive isotopes: - [e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I decay with emission of radiation]

– For biological investigation – carbon &

hydrogen.

– For metabolic studies – S, P, and alkali

and alkaline earth metals are used.

– For studies on protein, alkaloids, and

amino acid – labelled nitrogen atom

give more specific information.

– 3H compound is commercially

available. vii) Stable isotopes: - [e.g. 2H, 13C, 15N, 18O]

– Used for labelling compounds as

possible intermediates in biosynthetic

pathways. – Usual method of detection are: – MASS spectroscopy [

15N,

18O]

– NMR spectroscopy [2H,

13C

SIGNIFICANCE OF TRACER TECHNIQUE

• Tracing of Biosynthetic Pathway: - e.g. By

incorporation of radioactive isotope of 14

C into

phenylalanine, the biosynthetic cyanogenetic glycoside prunasin, can be detected.

• Location & Quantity of compound containing

tracer: - 14

C labelled glucose is used for

determination of glucose in biological system • Different tracers for different studies: - For studies

on nitrogen and amino acid. (Labelled nitrogen give

specific information than carbon) • Convenient and suitable technique

CRITERIA FOR TRACER TECHIQUE

• The starting concentration of tracer

must be sufficient withstand resistance

with dilution in course of metabolism. • Proper Labelling: - for proper

labelling physical & chemical

nature of compound must be

known. • Labelled compound should involve in the

synthesis reaction. • Labelled should not damage the system to

which it is used.

ADVANTAGES

High sensitivity.

Applicable o all living organism.

Wide ranges of isotopes are available.

More reliable, easily administration & isolation

procedure.

Gives accurate result, if proper metabolic time

& technique applied.

LIMITATION

Kinetic effect

Chemical effect

Radiation effect

Radiochemical purity

High concentration distorting the result.

REQUIREMENT FOR TRACER TECHNIQUE

– Preparation of labelled compound. – Introduction of labelled compound into a biological

system. – Separation & determination of labelled compound in

various biochemical fractions at later time. I. Preparation of Labelled Compound: -

The labelled compound produce by growing chlorella in

atmosphere of14CO2.

All carbon compounds 14C labelled. The3H (tritium) labelled compound are commercially available. Tritium labelling is

effected by catalytic exchange in aqueous media by hydrogenation of unsaturated compound with tritium gas. Tritium is pure β – emitter of low intensity & its radiation energy is lower than 14C.

By the use of organic synthesis: -

CH3MgBr + 14

CO2

CH314

COOHMgBr+H2O

CH314

COOH

+

Mg(OH)Br

II. Introduction of labelled

compound: -

PRECAUTION: -

•The precursor should react at necessary site of synthesis

in plant.

•Plant at the experiment time should synthesize the

compound under investigation

•The dose given is for short period.

1. Root feeding

2. Stem feeding

3. Direct injection

4. Infiltration

5. Floating method

6. Spray technique

III. Separation and detection of compound: -

a) Geiger – Muller counter.

b) Liquid Scintillation counter.

c) Gas ionization chamber.

d) Bernstein – Bellentine counter.

e) Mass spectroscopy.

f) NMR eletrodemeter.

g) Autoradiography.

METHODS IN TRACER TECHNIQUE 1. PRECURSOR PRODUCT SEQUENCE: - In this technique, the

presumed precursor of the constituent under investigation on a labelled form

is fed into the plant and after a suitable time the constituent is isolated,

purified and radioactivity is determined. Disadvantage: - The radioactivity of isolated compound alone is not usually

sufficient evidence that the particular compound fed is direct precursor,

because substance may enter the general metabolic pathway and from there

may become randomly distributed through a whole range of product. Application: - •Stopping of hordenine production in barley seedling after 15 – 20 days of

germination. •Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura

stramonium. •This method is applied to the biogenesis of morphine & ergot alkaloids

2. DOUBLE & MULTIPLE LABELLING: - This method give the evidence for nature of biochemical

incorporation of precursor arises double & triple labelling. In this method specifically labelled precursor and

their subsequent degradation of recover product are more employed. Application: -

This method is extensively applied to study the biogenesis of plant secondarymetabolite. Used for study of morphine alkaloid.

E.g. Leete, use Doubly labeled lysine used to determine which hydrogen of lysine molecule was involved in

formation of piperidine ring of anabasine in Nicotina glauca.

N. glauca N

H N N

H

H2N 2- Anabasine

COO

Lysine - 2 - 14

C, ε − 15

Ν

N. glauca N

H2N H2N

N H

COOH

Anabasine

Lysine - 2 - 14

C, α − 15

Ν

3. COMPETITIVE FEEDING: - If incorporation is obtained it is necessary to

consider whether this infact, the normal route of synthesis in plant not the subsidiary

pathway. Competitive feeding can distinguish whether B & B‟ is normal intermediate

in the formation of C from A. B

OR

A C

A C A B C

Application:B'- A B' C

This method is used for elucidation of biogenesis of propane alkaloids.

Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of

alkaloids of Conium maculactum (hemlock) using 14C labelled compounds.

4. ISOTOPE INCORPORATION: - This

method provides information about the position

of bond cleavage & their formation during

reaction.

E.g. Glucose – 1- phosphatase cleavage as

catalyzed by alkaline phosphatase this reaction

occur with cleavage of either C – O bond or P –

O bond.

CH2OH

CH2OH

O

O

18

OH

+ H

2 O

OH OH +

H2PO

4

OPO3H

OH

OH

OH

OH

5. SEQUENTIAL ANALYSIS: - The principle of this method of investigation is to

grow plant in atmosphere of 14

CO 2 & then

analyze the plant at given time interval to obtain the sequence in which various correlated

compound become labelled.

Application: -

14

CO2& sequential analysis has been very successfully usedin elucidation of carbon in photosynthesis.

Determination of sequential

formation of opium hemlock

andtobacco alkaloids.

Exposure as less as 5 min.14

CO2, is used in detectingbiosynthetic sequence as –

Piperitone --------- (-) Menthone ------

---- (-) Menthol in

Mentha piperita.

APPLICATION OF TRACER TECHNIQUE

1. Study of squalene cyclization by use of 14C, 3H labelled

mevalonic acid. 2. Interrelationship among 4 – methyl sterols & 4, 4 dimethyl

sterols, by use of 14C acetate.

3. Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2- 14C mevalonate.

4. Study the formation of cinnamic acid in pathway of

coumarin from labelled coumarin. 5. Origin of carbon & nitrogen atoms of purine ring system

by use of 14C or 15N labelled precursor. 6. Study of formation of scopoletin by use of labelled

phenylalanine. 7. By use of 45Ca as tracer, - found that the uptake of calcium

by plants from the soil. (CaO & CaCO2). 8. By adding ammonium phosphate labelled with 32P of

known specific activity the uptake of phosphorus is followed by measuring the radioactivity as label reaches first in lower part of plant, than the upper part i.e.

branches, leaves etc.

T.B.EKNATH BABU (T.B.E.K.B) STUDENT AT A.K.C.P

ADVANCED PHARMACOGNOSY

PLANT TISSUE CULTURE TECHNIC

1. Introduction

Plant tissue culture can be defined as the in vitro manipulation of plant cells and tissues and is a keystone in

the foundation of plant biotechnology. It is useful for plant propagation and in the study of plant growth

regulators. It is generally required to manipulate and regenerate transgenic plants. Whole plants can be

regenerated under in vitro conditions using plant organs, tissues or single cells, by inoculating them in an

appropriate nutrient medium under sterile environment. Plant tissue culture relies on the fact that many plant

cells have the capacity to regenerate into a whole plant–a phenomena known as totipotency. Plant cells, cells

without cell walls (protoplasts), leaves, or roots can be used to generate a new plant on culture media

containing the necessary nutrients and plant growth regulators. Plant tissue culture was first attempted by

Haberlandt (1902). He grew palisade cells from leaves of various plants but they did not divide. In 1934,

White generated continuously growing cultures of meristematic cells of tomato on medium containing salts,

yeast extract and sucrose and vitamin B (pyridoxine, thiamine and nicotinic acid) and established the

importance of additives. In 1953, Miller and Skoog, University of Wisconsin – Madison discovered Kinetin,

a cytokine that plays an active role in organogenesis. Plant cell cultures are an attractive alternative source

to whole plants for the production of high-value secondary metabolites.

2. Advantages of plant tissue culture over conventional agricultural production

The most important advantage of in vitro grown plants is that it is independent of geographical variations,

seasonal variations and also environmental factors. It offers a defined production system, continuous supply

of products with uniform quality and yield. Novel compounds which are not generally found in the parent

plants can be produced in the in vitro grown plants through plant tissue culture. In addition, stereo- and

region- specific biotransformation of the plant cells can be performed for the production of bioactive

compounds from economical precursors. It is also independent of any political interference. Efficient

downstream recovery of products and rapidity of production are its added advantages (Figure 31.1).



Figure 31.1: Steps involved in the production of secondary metabolites from plant cell

3. Plant secondary metabolites

Plant products can be classified into primary plant metabolites and secondary metabolites. Primary plant

metabolites are essential for the survival of the plant. It consists of sugars, amino acids and nucleotides

synthesized by plants and are used to produce essential polymers. Typically primary metabolites are found

in all species within broad phylogenetic groupings, and are produced using the same metabolic pathway.

Secondary metabolites are the chemicals, which are not directly involved in the normal growth and

development, or reproduction of an organism. Secondary metabolites are not indispensable for the plants but

play a significant role in plant defense mechanisms. Primary metabolites essentially provide the basis for

normal growth and reproduction, while secondary metabolites for adaptation and interaction with the

environment. The economic importance of secondary metabolites lies in the fact that they can be used as

sources of industrially important natural products like colours, insecticides, antimicrobials, fragrances and

therapeutics. Therefore, plant tissue culture is being potentially used as an alternative for plant secondary

metabolite production. Majority of the plant secondary metabolites of interest to humankind fit into

categories which categorize secondary metabolites based on their biosynthetic origin. Secondary

metabolism in plants is activated only in particular stages of growth and development or during periods of

stress, limitation of nutrients or attack by micro-organisms.

Plants produce several bioactive compounds that are of importance in the healthcare, food, flavor and

cosmetics industries. Many pharmaceuticals are produced from the plant secondary metabolites. Currently,

many natural products are produced solely from massive quantities of whole plant parts. The source plants

are cultured in tropical, subtropical, geographically remote areas, which are subject to drought, disease and

changing land use patterns and other environmental factors.

Secondary metabolites can be derived from primary metabolites through modifications, like methylation,

hydroxylation and glycosylation. Secondary metabolites are naturally more complex than primary

metabolites and are classified on the basis of chemical structure (e.g., aromatic rings, sugar), composition



(containing nitrogen or not), their solubility in various solvents or the pathway by which they are

synthesized (Table 31.1). They have been classified into terpenes (composed entirely of carbon and

hydrogen), phenolics (composed of simple sugars, benzene rings, hydrogen and oxygen) and nitrogen and or

sulphur containing compounds (Figure 31.2). It has been observed that each plant family, genus and species

produces a characteristic mix of these bioactive compounds.

All plants produce secondary metabolites, which are specific to an individual species, genus and are

produced during specific environmental conditions which makes their extraction and purification difficult.

As a result, commercially available secondary metabolites, for example, pharmaceuticals, flavours,

fragrances and pesticides etc. are generally considered high value products as compared to primary

metabolites and they are considered to be fine chemicals.

Table 31.1: Classification of secondary metabolites

Figure 31.2: The production of secondary metabolites is tightly associated with the pathways of

primary/central metabolism, such as glycolysis, shikimate and production of aliphatic amino acids.

4. Strategies for enhanced production of secondary metabolites in plant cell cultures



4.1. Proper selection of cell lines

The heterogeneity within the cell population can be screened by selecting cell lines capable of accumulating

higher level of metabolites.

4.2. Manipulation of medium

The constituents of culture medium, like nutrients, phytohormones and also the culture conditions, like

temperature, light etc. influence the production of secondary metabolites. For e.g., if sucrose concentration

is increased from 3% to 5%, production of rosamarinic acid is increased by five times. In case of shikonin

production, IAA enhances the yield whereas 2,4-D and NAA are inhibitory.

4.3. Addition of Elicitors

Elicitors are the compounds which induce the production and accumulation of secondary metabolites in

plant cells. Elicitors produced within the plant cells include cell wall derived polysaccharides, like pectin,

pectic acid, cellulose etc. Product accumulation also occurs under stress conditions caused by physical or

chemical agents like UV, low or high temperature, antibiotics, salts of heavy metals, high salt

concentrations which are grouped under abiotic elicitors. Addition of these elicitors to the medium in low

concentration enhances the production of secondary metabolites.

4.4. Addition of precursors

Precursors are the compounds, whether exogenous or endogenous, that can be converted by living system

into useful compounds or secondary metabolites. It has been possible to enhance the biosynthesis of specific

secondary metabolites by feeding precursors to cell cultures. For example, amino acids have been added to

suspension culture media for production of tropane alkaloids, indole alkaloids. The amount of precursors is

usually lower in callus and cell cultures than in differentiated tissues. Phenylalanine acts as a precursor of

rosmarinic acid; addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production

of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N-

benzoylphenylisoserine side chain of taxol; supplementation of Taxus cuspidata cultures with phenylalanine

resulted in increased yields of taxol. The timing of precursor addition is critical for an optimum effect. The

effects of feedback inhibition must surely be considered when adding products of a metabolic pathway to

cultured cells.

4.5. Permeabilisation

Secondary metabolites produced in cells are often blocked in the vacuole. By manipulating the permeability

of cell membrane, they can be secreted out to the media. Permeabilisation can be achieved by electric pulse,

UV, pressure, sonication, heat, etc. Even charcoal can be added to medium to absorb secondary metabolites.

4.6. Immobilisation

Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called

immobilised plant cell cultures. Immobilization of plant cells allows better cell to cell contact and the cells

are also protected from high shear stresses. These immobilized systems can effectively increase the

productivity of secondary metabolites in a number of species. Elicitors can also be added to these systems to

stimulate secondary metabolism.



4.7. Limitations

• Production cost is often very high.

• Lack of information of the biosynthetic pathways of many compounds is a major drawback in the

improvement of their production.

• Trained technical manpower is required to operate bioreactors.

5. Advantages of cell, tissue and organ cultures as sources of secondary metabolites

5.1. Plant cell cultures

Once interesting bioactive compounds have been were identified from plant extracts, the first part of the

work consisted in collecting the largest genetic pool of plant individuals that produce the corresponding

bioactive substances. However, a major characteristic of secondary compounds is that their synthesis is

highly inducible, therefore, it is not certain, if a given extract is a good indicator of the plant potential for

producing the compounds. The ability of plant cell cultures to produce secondary metabolites came quite

late in the history of in vitro techniques. For a long time, it was believed that undifferentiated cells, such as

callus or cell suspension cultures were not able to produce secondary compounds, unlike differentiated cells

or specialized organs.

5.2. Callus culture

Callus is a mass of undifferentiated cells derived from plant tissues for use in biological research and

biotechnology. In plant biology, callus cells are those cells that cover a plant wound. To induce callus

development, plant tissues are surface sterilized and then plated onto in vitro tissue culture medium.

Different plant growth regulators, such as auxins, cytokinins, and gibberellins, are supplemented into the

medium to initiate callus formation. It is well known that callus can undergo somaclonal variations, usually

during several subculture cycles. This is a critical period where, due to in vitro variations, production of

secondary metabolite often varies from one subculture cycle to another. When genetic stability is reached, it

is necessary to screen the different cell (callus) lines according to their aptitudes to provide an efficient

secondary metabolite production. Hence, each callus must be assessed separately for its growth rate as well

as intracellular and extracellular metabolite concentrations. This allows an evaluation of the productivity of

each cell line so that only the best ones will be taken for further studies, for example, for production of the

desired compound in suspensions cultures.

5.3. Cell suspension cultures

Cell suspension cultures represent a good biological material for studying biosynthetic pathways. They

allow the recovery of a large amount of cells from which enzymes can be easily separated. Compared to cell

growth kinetics, which is usually an exponential curve, most secondary metabolites are often produced

during the stationary phase. This lack of production of compounds during the early stages can be explained

by carbon allocation mainly distributed for primary metabolism when growth is very active. On the other

hand, when growth stops, carbon is no longer required in large quantities for primary metabolism and

secondary compounds are more actively synthesized. However, some of the secondary plant products are

known to be growth-associated with undifferentiated cells, such as betalains and carotenoids.

5.4. Organ cultures



Plant organs are alternative to cell cultures for the production of plant secondary metabolites. Two types of

organs are generally considered for this objective: hairy roots and shoot cultures. A schematic representation

of various organized cultures, induced under in vitro conditions, is given in Figure 31.3.

5.4.1. Shoot cultures

Shoots exhibit some comparable properties to hairy roots, genetic stability and good capacities for

secondary metabolite production. They also provide the possibility of gaining a link between growth and the

production of secondary compounds.

5.4.2. Hairy root cultures

Hairy roots are obtained after the successful transformation of a plant with Agrobacterium rhizogenes. They

have received considerable attention of plant biotechnologists, for the production of secondary compounds.

They can be subcultured and indefinitely propagated on a synthetic medium without phytohormones and

usually display interesting growth capacities owing to the profusion of lateral roots. This growth can be

assimilated to an exponential model, when the number of generations of lateral roots becomes large.

Cell Suspension culture



Tissues and cells cultured in a agitated liquid medium produce a suspension of single cells and cells

clumps of few to may cell, these are called suspension cultures.

PROTOPLAST CULTURES

Isolated protoplasts have been described as "naked" cells because the cell wall has been removed by

either a mechanical or an enzymatic process.

Protoplasts can be induced to reform a cell and divide if placed in a suitable nutrient medium than

form callus.





Ovary/ovule culture

Ovary or ovule culture involves development of haploid from unfertilized cells of embryosac present in

ovary.



CELLULAR TOTIPOTENCY

In the preceding units of this course you have read that innumerable cells which constitute the body of a

higher plant or animal and containing identical genetic material can be traced to a single cell-the zygote.

During development cells undergo diverse structural and functional specialisation depending upon their

position in the body. Leaf cells bear chloroplasts and act as the site of photosynthesis. The colourless root

hairs perform the function of absorbing nutrients and water from the soil and some other cells become part

of the colourful petals. Normally fully differentiated cells do not revert back to a meristematic: state, which

suggests that the cells have undergone a permanent change. In earlier sections of this unit you have read that

the regenerative capacity is retained by all living cells of a plant. Several horticultural plants regenerate

whole plant from root, leafiand stem cuttings. Highly differentiated and mature cells such as those of pith

and cortex and highly specialised cells as those of microspores and endosperm,retain full potential to give

rise to full plants under suitable culture conditions. G. Haberlandt was the first to test this idea

experimentally. This endowment called "cellular totipotency" is unique to plants. Animal cells possibly

because of their higher degree of specialisation do not exhibit totipotency. Whole plant regeneration from

cultured cells may occur in one of the two pathways: ;)shoot bud differentiation, (organogenesis) and ii)

embryo formation (Embryogenesis). The Embryos are bipolar structures with no organic connection with

the parent tissue and can germinate directly into a complete plant. On the other hand, shoots are monopolar.

They need to be removed from the parent tissue and rooted to establish a plantlet. Often the same tissue can

be induced to form shoots or embryos by manipulating the components of the culture conditions. In the

following sub sections we will discuss organogenesis and embryogenesis in detail.

Organogenesis

Organogenesis refers to the differentiation of organs such as roots, shoots or flowers. Shoot bud

differentiation may occur directly from the explant or from the callus. The stimulus for organogenesis may

come from the medium, from the endogenous compounds produced by the cultured tissue or substances

carried over from the original explant. Organogenesis is chemically controlled by growth regulators. Skoog

while working with tobacco pith callus, observed that the addition of an auxin Indole Acetic Acid (IAA)

enhanced formation of roots and suppressed shoot differentiation. He further observed that adenine sulphate,

(Cytokinin) reversed the inhibition of auxin and promoted the formation of shoots. You should know that:



1) Organogenesis is contolled by a balance between cytokinin and auxin concentration i.e. it is their relative

rather than the absolute concentration that determines the nature of differentiation.

2. A relatively high auxin: Cytokinin ration induces root formation, whereas a high cytokinin: auxin ratio

favours shoot bud differentiation.

3. Differential response to exogenously applied growth regulators may be due to differences in the

endogenous levels of the hormones within the tissue. Organogenesis is a complex process. Whereas in the

cultured tissues of many species organogeiiesis can be demonstrated in this pattern, some plants, notably the

monocots, are exceptions.

Somatic Embryogenesis

The process of embryo development is called embryogenesis. It is not the monopoly of the egg to form an

embryo. Any cell of the female gametophyte (Embryo sac) or even of the sporophytic tissues around the

embryo sac may give rise to an embryo. Thus we can say that 'The phenomenon of embryogenesis is not

necessarily confined to the reproductive cycle". In this subsection we will discuss -,- some examples of

"embeos formed in culture", also referred to as "somatic - embryos". The first observation of somatic

embryos were made m Dacus Carota. Other plants in which the phenomenon has been studied in some detail

are Ranunculus scleratus, citrus and coflea spp. In Rarrunculus scleratus somatic as well as various floral

tissues, including anthers proliferated to form callus which, after limited unorganised growth differentiated

several embryos. These embryos germinated in situ and a fresh crop of embryos appeared on the surface of

the seedling. The embryos were derived from individual epidern~al cells of the hypocotyl

Citrus is commonly cited as an example of natural polyembryony

Figure 31.3: Guidelines for the production of secondary metabolites from plant organ cultures.

1. Laboratory Design and Development



The size of tissue culture lab and the amount and type of equipment used depend upon the nature of the

work to be undertaken and the funds available. A standard tissue culture laboratory should provide facilities

for:

• washing and storage of glassware, plasticware

• preparation, sterilization and storage of nutrient media

• aseptic manipulation of plant material

• maintenance of cultures under controlled temperature, light and humidity

• observation of cultures, data collection and photographic facility

• acclimatization of in vitro developed plants. The overall design must focus on maintaining aseptic

conditions.

At least three separate rooms should be available one for washing up, storage and media preparation (the

media preparation room); a second room, containing laminar-air-flow or clean air cabinets for dissection of

plant tissues and subculturing (dissection room or sterilization room); and the third room to incubate

cultures (culture room). This culture room should contain a culture observation table provided with

binoculars or stereozoom microscope and an adequate light source. Additionally, a green house facility is

required for hardening-off in vitro plantlets. For a commercial set-up, a more elaborate set-up is required.

1.1. Media preparation room

The washing area in the media room should be provided with brushes of various sizes and shapes, a large

sink, preferably lead-lined to resist acids and alkalis, and running hot and cold water. It should also have

large plastic buckets to soak the labware to be washed in detergent, hot-air oven to dry washed labware and

a dust-proof cupboard to store them. If the preparation of the medium and washing of the labware are done

in the same room, a temporary partition can be constructed between the two areas to guard any interference

in the two activities. A continuous supply of water is essential for media preparation and washing of

labware. A water distillation unit of around 2 litre/h, a Milli-Q water purification systems needs to be

installed.



Figure 2.1: A floor plan for plant tissue culture laboratory

1.2. Culture room

The room for maintaining cultures should be maintained at temperature 25 ±2°C, controlled by air

conditioners and heaters attached to a temperature controller are used. For higher or lower temperature

treatments, special incubators with built-in fluorescent light can be used outside the culture room. Cultures

are generally grown in diffuse light from cool, white, fluorescent tubes. Lights can be controlled with

automatic time clocks. Generally, a 16-hour day and 8-hour nights are used. The culture room requires

specially designed shelving to store cultures. Some laboratories have shelves along the walls, others have

them fitted onto angle-iron frames placed in a convenient position. Shelves can be made of rigid wire mesh,

wood or any building material that can be kept clean and dust-free. Insulation between the shelf lights and

the shelf above will ensure an even temperature around the cultures. While flasks, jars and petridishes can

be placed directly on the shelf or trays of suitable sizes, culture tubes require some sort of support. Metallic

wire racks or polypropylene racks, each with a holding capacity of 18-24 tubes, are suitable for the purpose.

1.3. Dissection room or sterilization room

This area should have restricted entry, which is needed to ensure the sterile conditions required for the

transfer operations. For sterile transfer operations, the laminar-air-flow cabinets are used. Temperature

control is essential in this room as the heat is produced continuously from the flames of burners in the

hoods. The room should be constructed in a way to minimize the dust particles and for easy cleaning.

Several precautions can be taken including the removal of shoes before entering the area.

The laminar horizontal flow sterile transfer cabinets are available in various sizes from many commercial

sources. They should be designed with horizontal air flow from the back to the front, and equipped with gas

cocks if gas burners are to be used. Electrical outlets are needed for use of electric sterilizers and

microscopes, and if weighing is to be done in the hoods. A stainless steel working platform is most durable,

easy to keep clean and to prevent the unwanted damage due to accidental fire. Sometimes it is fitted with



Ultraviolet light to maintain sterility inside the cabinet. UV light is a source of ozone, which can be

mutagenic, therefore, utmost care is to be taken while using this. Although UV light is not necessary, a short

exposure time of 3-5 min to cabinet is fine sometimes. Work can be started after 10-15 min of switching on

the air flow, and one can work uninterrupted for long hours.

A Laminar-air-flow cabinet has small motor to blow air which first passes through a coarse filter, where it

loses large particles, and subsequently through a fine filter known as „high efficiency particulate air

(HEPA). The HEPA filters remove particles larger than 0.3 µm, and the ultraclean air flows through the

working area. The velocity of the ultra clean air is about 27 ± 3 m min-1

which is adequate for preventing the

contamination of the working area as long as the flow is on. The flow of the air does not in any way hamper

the use of a spirit lamp or a Bunsen burner.

1.4. Greenhouse

The greenhouse facility is required to grow parent pants and to acclimatize in vitro raised plantlets. The size

and facility inside the green house vary with the requirement and depends on the funds available with the

laboratory. However, minimum facilities for maintaining humidity by fogging, misting or a fan and pad

system, reduced light, cooling system for summers and heating system for winters must be provided. It

would be desirable to have a potting room adjacent to this facility.

1.5. Equipments and apparatus

1.4.1. Media preparation area

• benches at a height suitable to work while standing

• pH meter is used to determine the pH of various media used for tissue culture. pH indicator paper can also

be used for the purpose but it is less accurate. The standard media pH is maintained at 5.8.

• hot-plate-cum-magnetic stirrer for dissolving chemicals and during media preparation

• an autoclave or domestic pressure cooker is crucial instrument for a tissue culture laboratory. High

pressure heat is needed to sterilize media, water, labware, forceps, needles etc. Certain spores from fungi

and bacteria can only be killed at a temperature of 121°C and 15 pounds per square inch (psi) for 15-20 min.

A caution should be taken while opening the door of autoclave and it should be open when the pressure

drops to zero. Opening the door immediately can lead to a rapid change in the temperature, resulting in

breakage of glassware and steam burning of operator.

• plastic carboys for storing distilled water required for media preparation and final washing of labware.

• balances near dry corner of the media room. High quality microbalance are required to weigh smallest of

the quantities. Additionally a top pan balance is required for less sensitive quantities.

• hot-air oven to keep autoclaved medium warm before pouring into vessels. It is also used for the dry heat

sterilization of clean glassware like, Petridishes, culture tubes, pipettes etc. Typical sterilizing conditions are

160-170 °C/1hr.

• Dish washer for cleaning glass pipettes in running water



1.4.2. Storage area

• a deep freezer (-20°C to -80°C) / refrigerator for storage of enzyme solutions, stock solutions plant

materials and all temperature-sensitive chemicals.

• microwave oven to melt agar solidified media

• Upright and inverted light microscope with camera attachment for recording the morphogenic responses

from various explants, calli, cells and protoplasts. Inverted microscope gives the clear views of cultures

settled at the bottom of Petridishes.

1.4.3. Dissection room

• laminar-air-flow cabinet within which tissue culture work can be carried out under sterilized environment

• glass bead sterilizer where temperature of beads is raised to 250°C in 15-20 min with 15 s cut off. Here

the sterilization of instruments is effecting by pushing them into the beads for 5-7 s. This is much safer

compare to the Bunsen burner heating of instruments like, forceps, needles, scalpels etc.

• binocular microscope to observe surface details and morphogenic responses of cultures and their possible

contamination.

• low speed table-top centrifuge to sediment cells or protoplasts

1.4.4. Culture room

• air (or heating / cooling system) to maintain 25±2 °C temperature

• racks for holding test-tubes

• lights to provide diffuse light and to maintain photoperiod

• shakers with various sized clamps for different sized flasks to grow cells in liquid medium

• thermostat and time clock for lights

• wall cabinets for dark incubation of cultures

1.4.5. Other apparatus

• beakers (100 mL, 250 mL, 1 L, 5 L)

• measuring cylinders (5 mL, 10 mL, 25 mL, 50 mL, 100 mL, 500 mL, 1L, 2 L, 5 L)

• graduated pipettes and teats

• reagent bottles for storing liquid chemicals and stock solutions (glass or plastic)

• culture tubes and flasks (glass or polypropylene or disposable)



• plastic baskets

• filter membrane, preferably nylon, of sizes 0.22 µm and 0.45 µm, holders and hypodermic syringes (for

solutions requiring filter sterilization)

• large forceps (blunt and fine points) and scalpels for dissecting and subculturing plant material.

• Scalpel handles (no. 3) and blades (no. 11)

• Chemicals and reagents for preparing culture media

• Disposable gloves and masks.

• Micropipettes of maximum volume size 5000 µL, 1000 µL, 500 µL, 250 µL, 100 µL

(A) Syringe with filter assembly fitted on conical flask,

(B) Disassembled filter assembly

Forceps and scalpels for dissection, Micropipettes .

Tissue culture media

1. Preparation and handling

The simplest method of preparing media is to use commercially available, dry, powdered media containing

mineral elements and growth regulators. By following the procedure written on the packets, dissolve the

powder in distilled or demineralized water (10% less than the final volume of the medium). After adding

sugar and other desired supplements like, plant growth regulators, make up the final volume with distilled

water, adjust the pH, add agar and then autoclave the medium.

An alternative method of media preparation is to prepare a series of concentrated stock solutions which can

be combined later as required. For preparing stock solutions and media, use glass-distilled or demineralized

water and chemicals of high purity, analytical reagent (AR) grade.

1.1. Composition of widely used tissue culture media

Both the media listed in the below tables 2 & 3 can be prepared from stock solutions of:

i. Macronutrients: As its name suggests, in plant tissue culture media these components provide the

elements which are required in large amounts (concentrations greater than 0.5 mmole l-1

) by cultured plant

cells. Macronutrients are usually considered to be carbon, nitrogen, phosphorous, magnesium, potassium,

calcium and sulphur.

ii. Micronutrients: It provides the elements that are required in trace amounts (concentrations less than 0.5

mmole l-1

) for plant growth and development. These include, manganese, copper, cobalt, boron, iron,

molybdenum, zinc and iodine.



iii. Iron source: It is considered the most important constituent and required for the formation of several

chlorophyll precursors and is a component of ferredoxins (proteins containing iron) which are important

oxidation : reduction reagents.

iv. Organic supplements (vitamins): Like animals, in plants too vitamins provide nutrition for healthy

growth and development. Although plants synthesize many vitamins under natural conditions and, therefore,

under in vitro conditions they are supplied from outside to maintain biosynthetic capacity of plant cells in

vitro. There are no firm rules as to what vitamins are essential for plant tissues and cell cultures. The only

two vitamins that are considered to be essential are myo-inositol and thiamine. Myo-inositol is considered to

be vitamin B and has many diverse roles in cellular metabolism and physiology. It is also involved in the

biosynthesis of vitamin C.

v. Carbon source: This is supplied in the form of carbohydrate. Plant cells and tissues in the culture medium

are heterotrophic and are dependent on external source of carbon. Sucrose is the preferred carbon source as

it is economical, readily available, relatively stable to autoclaving and readily assimilated by plant cells.

During sterilization (by autoclaving) of medium, sucrose gets hydrolyzed to glucose and fructose. Plant cells

in culture first utilize glucose and then fructose. Besides sucrose, other carbohydrates such as, lactose,

maltose, galactose are also used in culture media but with a very limited success.

Table 3.1: The media elements and their functions

The steps involved in preparing a medium are summarized below:

Add appropriate quantities of various stock solutions, including growth regulators and other special

supplements. Make up the final volume of the medium with distilled water.

Add and dissolve sucrose.

After mixing well, adjust the pH of the medium in the range of 5.5-5.8, using 0.1 N NaOH or 0.1 N HCl

(above 6.0 pH gives a fairly hard medium and pH below 5.0 does not allow satisfactory gelling of the agar).

Add agar, stir and heat to dissolve. Alternatively, heat in the autoclave at low pressure, or in a microwave

oven.

Once the agar is dissolved, pour the medium into culture vessels, cap and autoclave at 121°C for 15 to 20

min at 15 pounds per square inch (psi). If using pre-sterilized, non-autoclavable plastic culture vessels, the

medium may be autoclaved in flasks or media bottles. After autoclaving, allow the medium to cool to

around 60°C before pouring under aseptic conditions.



Allow the medium to cool to room temperature. Store in dust-free areas or refrigerate at 7°C

(temperature lower than 7°C alter the gel structure of the agar).

1.2. Gelling agents

The media listed above are only for liquids, often in plant cell culture a „semi-solid' medium is used. To

make a semi-solid medium, a gelling agent is added to the liquid medium before autoclaving. Gelling agents

are usually polymers that set on cooling after autoclaving.

i. Agar: Agar is obtained from red algae- Gelidium amansii . It is a mixture of polysaccharides. It is used as

a gelling agent due to the reasons: (a) It does not react with the media constituents (b) It is not digested by

plant enzymes and is stable at culture temperature.

ii. Agarose: It is obtained by purifying agar to remove the agaropectins. This is required where high gel

strength is needed, such as in single cell or protoplast cultures.

iii. Gelrite: It is produced by bacterium Pseudomonas elodea . It can be readily prepared in cold solution at

room temperature. It sets as a clear gel which assists easy observation of cultures and their possible

contamination. Unlike agar, the gel strength of gelrite is unaffected over a wide range of pH. However, few

plants show hyperhydricity on gelrite due to freely available water.

iv. Gelatin: It is used at a high concentration (10%) with a limited success. This is mainly because gelatin

melts at low temperature (25°C) and as a result the gelling property is lost.



1.3. Plant growth regulators

In addition to nutrients, four broad classes of growth regulators, such as, auxins, cytokinins, gibberellins and

abscisic acid are important in tissue culture. In contrast with animal hormones, the synthesis of a plant

growth regulator is often not localized in a specific tissue but may occur in many different tissues. They

may be transported and act in distant tissues and often have their action at the site of synthesis. Another

property of plant growth regulators is their lack of specificity- each of them influences a wide range of

processes.

The growth, differentiation, organogenesis and embryogenesis of tissues become feasible only on the

addition of one or more of these classes of growth regulators to a medium. In tissue culture, two classes of

plant growth regulators, cytokinins and auxins, are of major importance. Others, in particular, gibberellins,

ethylene and abscisic acid have been used occasionally. Auxins are found to influence cell elongation, cell

division, induction of primary vascular tissue, adventitious root formation, callus formation and fruit

growth. The cytokinins promote cell division and axillary shoot proliferation while auxins inhibit the

outgrowth of axillary buds. The auxin favours DNA duplication and cytokinins enable the separation of

chromosome. Besides, cytokinin in tissue culture media, promote adventitious shoot formation in callus

cultures or directly from the explants and, occasionally, inhibition of excessive root formation and are,

therefore, left out from rooting media. The ratio of plant growth regulators required for root or shoot

induction varies considerably with the tissue and is directly related to the amount of growth regulators

present at endogenous levels within the explants. In general, shoots are formed at high cytokinin and low

auxin concentrations in the medium, roots at low cytokinin and high auxin concentrations and callus at

intermediate concentrations of both plant growth regulators. Commonly used plant growth regulators are

listed in Table 4.

Stock solutions of growth regulators



1 molar = the molecular weight in g/l

1 mM = the molecular weight in mg/l

ppm = parts per million = mg/l

2. Establishing aseptic cultures

Plant tissue culture media contain sugar and so support the growth of many microorganisms (bacteria and

fungi). When these microorganisms reach a medium, they generally grow much faster than the cultured

plant materials. Their growth and toxic metabolites will affect, and may even kill, the tissue cultures. It is,

therefore, essential to maintain a completely aseptic environment inside the culture vessels.

There are several possible sources of contamination of the medium:

• the culture vessel

• the medium itself

• the explant (plant tissue)

• the environment of the transfer area

• the instruments used to handle plant material during establishment and subculture

• the environment of the culture room.

Autoclaving media will eliminate contamination from the culture vessel or the medium. In some cases,

substances such as gibberellic acid, abscisic acid (ABA), urea and certain vitamins are thermolabile and

break down upon autoclaving. These chemicals can be sterilized by membrane filtration using microfilters

of pore size 0.22-0.45 µm which is suitable enough to exclude pathogens. Later the filter sterilized

compound can be added to autoclaved medium cooled to around 40°C.

To prevent the environment of the culture room from being the source of contamination, keep the culture

room as dust- free as possible and remove contaminated cultures from the area as soon as they are detected.

Ideally, the culture room should be clean, filtered air which has passed through high efficiency particulate

air (HEPA) filters.

The transfer area in most laboratories is within a laminar air-flow cabinet. A laminar air-flow cabinet has a

small fan which blows air through a coarse filter to remove large dust particles and then through a fine

HEPA filter to remove microbes, their spores and other particles larger than 0.3 µm. The velocity of the air

coming out of the fine filter is about 27 ± 3 m/min, which keeps airborne microorganisms out of the

working area. The working area is swabbed with 70% alcohol (or equivalent) and instruments dipped in

70% alcohol, flamed and cooled before use.

Caution : Prolonged contact with alcohol can cause skin irritation, and other health problems can result from

the inhalation of fumes. Use ethanol rather than methanol, and surgical gloves when handling. Take care



with ultraviolet light as it can permanently damage eyes and promote skin cancer. Laminar flow cabinets

equipped with ultraviolet light for surface sterilization should be fitted with safety doors which can be

closed when ultraviolet light is used.

Plant surfaces carry a wide range of microorganisms. The tissue must be thoroughly surface-sterilized

before being placed on the nutrient medium. Discard cultures with fungal or bacterial contamination.

Solutions of sodium or calcium hypochlorite are usually effective in disinfecting plant tissues. Placing

tissues in a 0.5 to 1% solution of sodium hypochlorite for 10 to 15 minutes will disinfect most tissues.

Surface sterilants are toxic to plant tissues. Choose the concentration of the sterilizing agent and the length

of time to minimize tissue damage, which shows up as white, bleached areas. Other techniques for surface

sterilisation include dipping plant material for a few seconds in 90% ethanol or placing in running water for

30 minutes and 2 hours before disinfection.

Caution : Take care with powdered calcium hypochlorite as it is a powerful reducing agent. If calcium

hypochlorite is stored moist and the container opened later, it can explode. Store calcium hypochlorite in a

sealed container in a dry place.

A summary of the six steps commonly involved in establishing and maintaining aseptic plant tissue culture

follows.

i. Collect pieces of plant material (ex-plants) in a screw-cap bottle. Immerse them in a dilute solution of the

disinfectant containing a wetting agent. Replace the lid and store the bottle in the laminar air flow cabinet.

Shake the bottle two or three times during the sterilization period.

ii. Remove the lid and drain carefully. Thoroughly rinse the plant material in sterilized distilled water and

replace the lid. After shaking a few minutes, discard the water. Rinse two or three times more.

iii. Transfer the material to a pre-sterilized Petri-dishes or test-tubes.

iv. Sterilize the required instruments by dipping them in 70% ethanol and flamed them. Allow to cool.

Sterilize the instruments after each time they are used to handle tissue.

v. Prepare suitable explants from the surface sterilized material using sterilized instruments (scalpels,

needles, forceps, etc.).

vi. Quickly remove the lid of the culture vessel, transfer the explants on to the medium, flame the neck of

the vessel (only if glass) and replace the lid.

If handling aseptic plant materials during routine subculture, omit the first two steps.

Plant tissue culture techniques

1. Introduction

Plant tissue culture has become popular among horticulturists, plant breeders and industrialists because of

its varied practical applications. It is also being applied to study basic aspects of plant growth and

development. The discovery of the first cytokinin (kinetin) is based on plant tissue culture research.



The earliest application of plant tissue culture was to rescue hybrid embryos (Laibach, 1925, 1929), and the

technique became a routine aid with plant breeders to raise rare hybrids, which normally failed due to post-

zygotic sexual incompatibility. Currently, the most popular commercial application of plant tissue culture is

in clonal propagation of disease-free plants. In vitro clonal propagation, popularly called micropropagation,

offers many advantages over the conventional methods of vegetative propagation: (1) many species (e.g.

palms, papaya) which are not amenable to in vivo vegetative propagation are being multiplied in tissue

cultures, (2) the rate of multiplication in vitro is extremely rapid and can continue round the year,

independent of the season. Thus, over a million plants can be produced in a year starting from a small piece

of tissue. The enhanced rate of multiplication can considerably reduce the period between the selection of

plus trees and raising enough planting material for field trials. In tissue culture, propagation occurs under

pathogen and pest-free conditions.

An important contribution made through tissue culture is the revelation of the unique property of plant cells,

called “cellular totipotency”. The totipotency of plant cells was predicted in 1902 by Haberlandt and the

first true plant tissue culture on agar was established. Since then plant tissue culture techniques have greatly

evolved. The technique has developed around the concept that a cell has the capacity and ability to develop

into a whole organism irrespective of their nature of differentiation and ploidy level. Therefore, it forms the

backbone of the modern approach to crop improvement by genetic engineering. The principles involved in

plant tissue culture are very simple and primarily an attempt, whereby an explant can be to some extent

freed from inter-organ, inter-tissue and inter-cellular interactions and subjected to direct experimental

control.

Regeneration of plants from cultured cells has many other applications. Plant regeneration from cultured

cells is proving to be a rich source of genetic variability, called “somaclonal variation”. Several somaclones

have been processed into new cultivars. Regeneration of plants from microspore/pollen provides the most

reliable and rapid method to produce haploids, which are extremely valuable in plant breeding and genetics.

With haploids, homozygosity can be achieved in a single step, cutting down the breeding period to almost

half. This is particularly important for highly heterozygous, long-generation tree species. Pollen raised

plants also provide a unique opportunity to screen gametic variation at sporophytic level. This approach has

enabled selection of several gametoclones, which could be developed into new cultivars. Even the triploid

cells of endosperm are totipotent, which provides a direct and easy approach to regenerate triploid plants

difficult to raise in vivo.

The entire plant tissue culture techniques can be largely divided into two categories based on to establish a

particular objective in the plant species:

I. Quantitative Improvement

(Micropropagation)

Adventitious shoot proliferation (leaves, roots, bulbs, corm, seedling- explants etc.)

Nodal segment culture

Meristem/Shoot-tip culture

Somatic embryogenesis

Callus culture



II. Qualitative Improvement

Anther/ Microspore culture

Ovary/ Ovule culture

Endosperm culture

Cell culture

Protoplast culture

The above techniques are discussed in detail in subsequent chapters.

2. Micropropagation

Growing any part of the plant (explants) like, cells, tissues and organs, in an artificial medium under

controlled conditions (aseptic conditions) for obtaining large scale plant propagation is called

micropropagation. The basic concept of micropropagation is the plasticity, totipotency, differentiation,

dedifferentiation and redifferentiation, which provide the better understanding of the plant cell culture and

regeneration. Plants, due to their long life span, have the ability to withhold the extremes of conditions

unlike animals. The plasticity allows plants to alter their metabolism, growth and development to best suit

their environment. When plant cells and tissues are cultured in vitro , they generally exhibit a very high

degree of plasticity, which allows one type of tissue or organ to be initiated from another type. Hence,

whole plants can be subsequently regenerated and this regenerated whole plant has the capability to express

the total genetic potential of the parent plant. This is unique feature of plant cells and is not seen in animals.

Unlike animals, where differentiation is generally irreversible, in plants even highly mature and

differentiated cells retain the ability to regress to a meristematic state as long as they have an intact

membrane system and a viable nucleus. However, sieve tube elements and xylem elements do not divide

any more where the nuclei have started to disintegrate, According to Gautheret (1966) the degree of

regression a cell can undergo would depend on the cytological and physiological state of the cell. The

meristematic tissues are differentiated into simple or complex tissues called differentiation. Reversion of

mature tissues into meristematic state leading to the formation of callus is called dedifferentiation. The

ability of callus to develop into shoots or roots or embryoid is called redifferentiation. The inherent

potentiality of a plant cell to give rise to entire plant and its capacity is often retained even after the cell has

undergone final differentiation in the plant system is described as cellular totipotency.

2.1. Micropropagation vs. conventional method of propagation

All living plant cells, irrespective of their nature of specialization and ploidy level, have been shown to

regenerate plants via organogenesis or embryogenesis. The latter involves a highly specialized mode of

development that normally occurs only inside the seed, under the cover of several layers of parental tissues.

Consequently, the observation of developing embryos and their isolation in intact and living conditions for

experimental studies have been extremely difficult. In vitro production of embryos from somatic and

gametic cells has opened up the possibility of obtaining large numbers of embryos of different stages,

enabling investigations on cellular, genetic and physiological control of embryogenesis (induction, pattern

formation, organ differentiation and maturation). In vitroexpression of cellular totipotency and other

techniques of plant tissue culture have also facilitated and/ or accelerated the traditional methods of plant

improvement, propagation and conservation.



2.2. Micropropagation vs. vegetative propagation

The vegetative propagation has been conventionally used to raise genetically uniform large scale plants for

thousands of years. However, this technique is applicable to only limited number of species. In contrast to

this, micropropagation has several advantages which are summarized here:

i. The rapid multiplication of species difficult to multiply by conventional vegetative means. The technique

permits the production of elite clones of selected plants.

ii. The technique is independent of seasonal and geographical constraints.

iii. It enable large numbers of plants to be brought to the market place in lesser time which results in faster

return on the investment that went into the breeding work.

iv. To generate disease-free (particularly virus-free) parental plant stock.

v. To raise pure breeding lines by in vitro haploid and triploid plant development in lesser time.

vi. It can be utilized to raise new varieties and preservation of germplasm

vii. It offers constant production of secondary medicinal metabolites.

2.3. Cell differentiation

During in-vitro and in vivo cytodifferentiation (cell differentiation), the main emphasis has been on vascular

differentiation, especially tracheary elements (TEs). These can be easily observed by staining and can be

scored in macerated preparations of the tissues. Tissue differentiation goes on in a fixed manner and is the

characteristic of the species and the organs

2.4. Factors affecting vascular tissue differentiation Vascular differentiation is majorly affected qualitatively and quantitatively by two factors, auxin and

sucrose. Cytokinins and gibberellins also play an important role in the process of xylogenesis. Depending

upon the characteristics of different species, concentration of phytohormones, sucrose and other salt level

varies and accordingly it leads to the vascular tissue differentiation.

3. Micropropagation techniques

3.1. Strategies for propagation in vitro

Typical micropropagation system can be broadly divided into five distinct stages (Figure 4.1):

The stage zero is the selection of mother plant and preparation of explant.

The first stage is the initiation of a sterile culture of the explant in a particular enriched medium for specific

species.

The second stage includes initiation of cell division from almost any part of the plant system to initiate

regeneration or multiplication of shoots or other propagules from the explant. Adventitious shoot

proliferation is the most frequently used multiplication technique in micropropagation systems. The culture

media and growth conditions used in second stage need to be optimized for maximum rate of multiplication.



The third stage is the development of roots on the shoots to produce plantlets. Specialized media may or

may not be required to induce roots, depending upon the species.

The final or the fourth stage is to produce self-sufficient plants. This stage usually involves a hardening-off

process and acclimatization of plants in soil under green-house conditions for later transplanting to the field.

Mode of differentiation

Regenerants may differentiate either directly from the explants or indirectly via callusing. Dedifferentiation

favours unorganized cell growth and the resultant developed callus has meristems randomly distributed in

the callus. Most of these meristems, if provided appropriate invitro conditions, would differentiate shoot-

buds, roots or embryos.

Figure 4.1: Micropropagation stages

4. Trouble shooting

• Few explants exude dark colored compounds, like phenols, pigments etc which leach into the medium

from the cut ends of the explant. It results in the browning of tissues and the medium as well. The browning

of medium is associated with poor culture establishment and low regeneration capacity of the explants. This

can be overcome by:

i. minimizing the wounding of explants during isolation and surface disinfection to reduce this browning

response.

ii. washing or incubation of explants for 3-5 hrs in sterile distilled water to remove phenolics responsible

for browning of medium or explants.

iii. frequent subculture of explants with excision to fresh medium at regular intervals.



iv. initial establishment of cultures in liquid medium and later transfer to the semi-solid medium.

vi. culture of explants on porous substrate or paper bridges.

vi. addition of activated charcoal (AC) or polyvinylpyrrolidone (PVP) for adsorbtion of phenolics.

vii. antioxidants like ascorbic acid, citric acid etc. can also be used to prevent browning of tissues in culture.

• Appearance of vitrified tissues (hyperhydricity), a physiological disorder occurring in the in vitro cultures

due to which the tissues look transparent and fluffy resulting from excessive intake of water. Hyperhydricity

can be caused by a high concentration of cytokinin or low concentration of gelling agent or high water

retention capacity of explants if the container is tightly closed.

• Loss of regeneration ability in long-term cultures due to epigenetic variations (temporary variations) and

culture aging, including transition from juvenile to mature stage. Epigenetic variation are phenotypic

temporary variations which disappear as soon as the culture conditions are removed.

• Genotypic variations are also seen in the cultures, therefore, cytological, biochemical and molecular

analyses are required to confirm clonal fidelity of in vitro regenerants. Besides, morphological and

physiological testing is also required to remove undesired genetic variability.

Plant tissue culture

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs

under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely

used to produce clones of a plant in a method known as micropropagation. Different techniques in plant

tissue culture may offer certain advantages over traditional methods of propagation, including:

The production of exact copies of plants that produce particularly good flowers, fruits, or have other

desirable traits.

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.

The regeneration of whole plants from plant cells that have been genetically modified.

The production of plants in sterile containers that allows them to be moved with greatly reduced chances

of transmitting diseases, pests, and pathogens.

The production of plants from seeds that otherwise have very low chances of germinating and growing,

i.e.: orchids and Nepenthes.

To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned

stock' for horticulture and agriculture.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant

(totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can

often be used to generate a new plant on culture media given the required nutrients and plant hormones.

Techniques

Modern plant tissue culture is performed under aseptic conditions under HEPA filtered air provided by

a laminar flow cabinet. Living plant materials from the environment are naturally contaminated on their

surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting material

(explants) in chemical solutions (usually alcohol and sodium or calcium hypochlorite or mercuric

chloride[1]

is required. Mercuric chloride is seldom used as a plant sterilant today, unless other sterilizing

http://en.wikipedia.org/wiki/Micropropagation

http://en.wikipedia.org/wiki/Plant_tissue



http://en.wikipedia.org/wiki/Seed

http://en.wikipedia.org/wiki/Pollination

http://en.wikipedia.org/wiki/Seed

http://en.wikipedia.org/wiki/Genetic_engineering

http://en.wikipedia.org/wiki/Germinating

http://en.wikipedia.org/wiki/Orchids

http://en.wikipedia.org/wiki/Nepenthes

http://en.wikipedia.org/wiki/Horticulture

http://en.wikipedia.org/wiki/Totipotency

http://en.wikipedia.org/wiki/Protoplast

http://en.wikipedia.org/wiki/Plant_hormones

http://en.wikipedia.org/wiki/Aseptic_technique

http://en.wikipedia.org/wiki/HEPA

http://en.wikipedia.org/wiki/Laminar_flow_cabinet

http://en.wikipedia.org/wiki/Sterile_technique

http://en.wikipedia.org/wiki/Explant

http://en.wikipedia.org/wiki/Sodium_hypochlorite

http://en.wikipedia.org/wiki/Calcium_hypochlorite

http://en.wikipedia.org/wiki/Mercuric_chloride






agents are found to be ineffective, as it is dangerous to use, and is difficult to dispose of. Explants are then

usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid

medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally

composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are

prepared from liquid media with the addition of a gelling agent, usually purified agar. The composition of

the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or

amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For

example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may

yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells,

or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium

composition. As cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to

allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are

important in judging which pieces to culture and which to discard.

As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which,

when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.

Choice of explant

The tissue obtained from a plant to be cultured is called an explant based on work with certain model

systems particularly tobacco it has often been claimed that a totipotent explant can be grown from any part

of the plant and may include portions of shoots, leaves, stems, flowers, roots and single, undifferentiated

cells.,[citation needed]

however this has not been true for all plants.[3]

In many species explants of various organs

vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material

also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial

contamination is increased with inappropriate explants.

The specific differences in the regeneration potential of different organs and explants have various

explanations. The significant factors include differences in the stage of the cells in the cell cycle, the

availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the

cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip,

auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce

required growth regulating substances including auxins and cytokinins.

The pathways through which whole plants are regenerated from cells and tissues or explants such as

meristems broadly fall into three types:

1. The method in which explants that include a meristem (viz. the shoot tips or nodes) are grown on

appropriate media supplemented with plant growth regulators to induce proliferation of multiple

shoots, followed by rooting of the excised shoots to regenerate whole plants,

2. The method in which totipotency of cells is realized in the form of de novo organogenesis, either

directly in the form of induction of shoot meristems on the explants or indirectly via a callus

(unorganised mass of cells resulting from proliferation of cells of the explant) and plants are

regenerated through induction of roots on the resultant shoots,

3. Somatic embryogenesis, in which asexual adventive embryos (comparable to zygotic embryos in

their structure and development) are induced directly on explants or indirectly through a callus

phase.

The first method involving the meristems and induction of multiple shoots is the preferred method for the

micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue

culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has

http://en.wikipedia.org/wiki/Growth_medium

http://en.wikipedia.org/wiki/Auxin

http://en.wikipedia.org/wiki/Cytokinin

http://en.wikipedia.org/wiki/Callus_(cell_biology)

http://en.wikipedia.org/wiki/Explant

http://en.wikipedia.org/wiki/Wikipedia:Citation_needed

http://en.wikipedia.org/wiki/Plant_tissue_culture#cite_note-VasilThorpe1994-3

http://en.wikipedia.org/wiki/Haploid

http://en.wikipedia.org/wiki/Diploid

http://en.wikipedia.org/wiki/Meristematic

http://en.wikipedia.org/wiki/Auxins

http://en.wikipedia.org/wiki/Cytokinins



the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture

systems like bioreactors.

Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become

problematic during the tissue culture process. Certain soil microflora can form tight associations with the

root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without

injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the

tissue culture medium before there is significant growth of plant tissue.

Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed

from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of

the surface microflora do not form tight associations with the plant tissue. Such associations can usually be

found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.

An alternative for obtaining uncontaminated explants is to take explants from seedlings which are

aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration

of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for

seeds can be much more stringent than for vegetative tissues.

Tissue cultured plants are clones. If the original mother plant used to produce the first explants is susceptible

to a pathogen or environmental condition, the entire crop would be susceptible to the same problem.

Conversely, any positive traits would remain within the line also.

Applications

Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture. Applications include:

The commercial production of plants used as potting, landscape, and florist subjects, which uses

meristem and shoot culture to produce large numbers of identical individuals.

To conserve rare or endangered plant species.[4]

A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters,

e.g. herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds,

like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.[5]

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.

To cross-pollinate distantly related species and then tissue culture the resulting embryo which would

otherwise normally die (Embryo Rescue).

For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous

lines more rapidly in breeding programmes, usually by treatment with colchicine which causes doubling

of the chromosome number.

As a tissue for transformation, followed by either short-term testing of genetic constructs or

regeneration of transgenic plants.

Certain techniques such as meristem tip culture can be used to produce clean plant material

from virused stock, such as potatoes and many species of soft fruit.

Production of identical sterile hybrid species can be obtained.

http://en.wikipedia.org/wiki/Microflora

http://en.wikipedia.org/wiki/Forestry

http://en.wikipedia.org/wiki/Florist

http://en.wikipedia.org/wiki/Conservation_biology

http://en.wikipedia.org/wiki/Endangered_species

http://en.wikipedia.org/wiki/Plant_tissue_culture#cite_note-4

http://en.wikipedia.org/wiki/Plant_improvement

http://en.wikipedia.org/wiki/Herbicide

http://en.wikipedia.org/wiki/Bioreactor

http://en.wikipedia.org/wiki/Recombinant_proteins

http://en.wikipedia.org/wiki/Biopharmaceutical

http://en.wikipedia.org/wiki/Plant_tissue_culture#cite_note-5

http://en.wikipedia.org/wiki/Species

http://en.wikipedia.org/wiki/Somatic_fusion

http://en.wikipedia.org/wiki/Hybrid_(biology)

http://en.wikipedia.org/wiki/Embryo

http://en.wikipedia.org/wiki/Ploidy#Dihaploidy_and_polyhaploidy

http://en.wikipedia.org/wiki/Haploid

http://en.wikipedia.org/wiki/Colchicine

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Genetic_engineering

http://en.wikipedia.org/wiki/Transgenesis

http://en.wikipedia.org/wiki/Meristem

http://en.wikipedia.org/wiki/Virus

http://en.wikipedia.org/wiki/Potato



Callus Culture:

When the cells divide into an undifferentiated mass it is called as callus. Any part of a plant can be used to

produce the calli. It may be a stem, leaf, meristem or any other part. It is used to produce variations among

the plantlets. Callus formation is induced from plant tissues after surface sterilization and plating onto in

vitro tissue culture medium. Plant growth regulators, such as auxins, cytokinins, andgibberellins, are

supplemented into the medium to initiate callus formation or somatic embryogenesis. Plant callus is usually

derived from somatic tissues. The tissues used to initiate callus formation depends on plant species and

which tissues are available for explant culture. The cells that give rise to callus and somatic embryos usually

undergo rapid division or are partially undifferentiated such as meristematic tissue. In alfalfa,Medicago

truncatula, however callus and somatic embryos are derived from mesophyll cells that

undergo dedifferentiation.[17]

Plant hormones are used to initiate callus growth.

Specific auxin to cytokinin ratios in plant tissue culture medium give rise to an unorganized growing and

dividing mass of callus cells. Callus cultures are often broadly classified as being either compact or friable.

Friable calluses fall apart easily, and can be used to generate cell suspension cultures. Callus can directly

undergo direct organogenesis and/or embryogenesis where the cells will form an entirely new plant.

Callus induction and tissue culture

A callus cell culture is usually sustained on gel medium. Callus induction medium consists of agar and a

mixture of macronutrients and micronutrients for the given cell type. There are several types of basal salt

mixtures used in plant tissue culture, but most notably modified Murashige and Skoog medium,[13]

White's

medium,[14]

and woody plant medium.[15]

Vitamins are also provided to enhance growth such as Gamborg

B5 vitamins.[16]

For plant cells, enrichment with nitrogen, phosphorus, andpotassium is especially important.

Callus cells deaths

Callus can brown and die during culture, but the causes for callus browning are not well understood.

In Jatropha curcas callus cells, small organized callus cells became disorganized and varied in size after



http://en.wikipedia.org/wiki/Gibberellin

http://en.wikipedia.org/wiki/Somatic_Embryogenesis

http://en.wikipedia.org/wiki/Somatic

http://en.wikipedia.org/wiki/Explant_culture

http://en.wikipedia.org/wiki/Meristem

http://en.wikipedia.org/wiki/Medicago_truncatula



http://en.wikipedia.org/wiki/Mesophyll_tissue

http://en.wikipedia.org/wiki/Dedifferentiation

http://en.wikipedia.org/wiki/Callus_(cell_biology)#cite_note-17

http://en.wikipedia.org/wiki/Plant_hormones



http://en.wikipedia.org/wiki/Organogenesis

http://en.wikipedia.org/wiki/Embryogenesis

http://en.wikipedia.org/wiki/Macronutrient

http://en.wikipedia.org/wiki/Micronutrient

http://en.wikipedia.org/wiki/Murashige_and_Skoog_medium





http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Phosphorus

http://en.wikipedia.org/wiki/Potassium

http://en.wikipedia.org/wiki/Jatropha_curcas



browning occurred.[18]

Browning has also been associated with oxidation and phenolic compounds in both

explant tissues and explant secretions.

Suspension culture:

The callus produced from the explants are grown on nutrient solutions (that are semi solid) for a period of

time and they are induced to produce plants with new traits. A callus crumbles into smaller clumps and

single cells in liqu~d~medium by gentle agitation (100-120rPM) on a shaker. Shaking the cultures also

helps to aerate the cells. Such suspension cultures however rarely comprise single cells alone because cells

tend to aggregate in clusters of 2-100. Suspension cultures can be maintained indefinitely by inoculations of

known aliquot5 of cells to a fresh medium. This process is termed as "batch cultures". Alternatively, the

medium is replenished at regu lar intervals. This process is termed as "continuous culture". In the

continuous culture process at the time of replenishing the medium, cells are also harvested (open continuous

system) or the biomass is allowed t~*increase (close continuous system). Suspension cultures are useful in

studying problems related to cell biology including cell cycle and production of secondary metabolites like

alkaloids, steroids, glycosides, napthaquinones, flavones etc. which find medicinal and industrial

application. Pharmaceutical industries use large bioreactors for suspension cultures to obtain valuable

bioorganic compounds. A bioreactor is a vessel of glass or steel in which cells are cultured aseptically and

culture conditions are closely monitored. This results in higher yield of metabolites. In a bioreactor there is

provision for adding fresh medium, for harvesting cells, for the aeration of products, for mixing and

sampling, for controlling pH, 02 content and temperature Plant cells are immobilised in alginate, agarose,

polyacrylamide beads. Immobilisation of cells enables i) re-use of biomass by rotation of cells ii) separation

of cells from the medium and iii) leaching of metabolites in it . Immobilised cells are cultured in column

reactors. Column reactors are of different types with different agitation and flow systems. Such reactors may

be i) stirred tank type ii) air lift type iii) bubble column type and iv) rotating drum type.

11.3.2 Single Cell Culture

This is an important invitro technique which enables the cloning of selected cells. Single cells can be

obtained directly from plant organs by treatment with enzymes that dissolve middle lamellae. The separate

cells can sieve into liquid medium to start a suspension culture. The most widely used technique for single

cell culture is the Bergmann's method of Cell Plating and. Microchamber technique.

Bergmann's Method of Cell Plating:

In this method free cells are suspended in a liquid medium at a density twice the Plant Tissue And Organ

finally desired plating density. Melted agarcontaining medium of otherwise the Culture same composition as

the liquid medium is maintained at 35Oc in water bath. Equal volumes of the two media are mixed and

rapidly spread out in petri dishes in such a manner that the cells are evenly distributed and fixed in a thin

layer (about 1 mm thick) of the medium after it has cooled and solidified. The dishes are sealed with

parafilm. The cells to be followed are marked on the outside of the plate and before the colonies derived

from individual cells grow large enough to merge with each other. They are transferred to.separate plates.

(Fig. 11.3). Another popular method for single cell culture is the microchamber technique, developed by

Jones et al. (1960). In this method mechanically isolated single cells are cultured in separate droplets of

liquid medium. While Jones et al. used sterile microslides and three coverglasses to make microchamber, it

is now possible to buy pre-sterilised plastic plates with several microwells (Cuprak dishes). Individual cells

are cultured in separate wells each containing 0.25 ml of the liquid medium. The culture requirement of

single cells increases with decrease in the plating cell density, and the cell cultured in complete isolation

require a very complex culture medium. A simple medium conditioned by growing cell suspension for some

time rlso fulfils the requirements of single cell culture at low density


http://en.wikipedia.org/wiki/Browning_(chemical_process)

http://en.wikipedia.org/wiki/Natural_phenol



Clonal Propagation

Most cultivars of ornamental and fruit species and forest trees are highly heterozygous. Consequently, their

seed progeny is not true-to-type. To preserve the unique characters of selected cultivars of horticultural

plants nurserymen practise vegetative propagation, using stem, leaf or root cuttings or propagules such as

tubers, corms, bulbs or bulbils. For plants which do not set seeds, such as edible bananas,grapes, citrus,

petunia, rose and chrysanthemum, vegetative propagation is the only means of multiplication. A population

of plants derived from a single individual by vegetative propagation is genetically uniform and is called a

clone. The conventional methods of clonal propagation are slow and often not applicable. For example, the

only in-vivo method for clonal multiplication of cultivated orchids, which are complex hybrids,is "back-

bulb" propagation. It involves separating the oldest pseudobulbil to force the development of dormant buds.

This process allows, at best, doubling the plant number every year. Moreover, Diagrammatic summary of

steps involved in aseptic multiplication of plants. Shoot multiplication is achieved through enhanced axillary

branching adventitious budding from explants directly or after callusing The shoots are rooted individually

in a medium containing an auxin. The plantlets so obtained are transferred to well drained potting mix.

After maintaining them under high humidity for3-4 weeks the plants are transferred to ordinary glasshouse

or field conditions Plant multiplication involving a callus phase may occur via shoot bud differentiation or

somatic embryogenesis. In the latter case the rooting step is eliminated as the embryos possess a pre-formed

root primordiurn. monopodial orchids do not form pseudobulbils and, therefore, cannot be clonally

multiplied. In 1960, a French scientist, G.More1, described an in-vitro method for rapid clonal

multiplication of orchids. This revolutionised the orchid industry, and today tissue culture is the only

economically feasible method for clonal multiplication of orchids and is being widely used. In-vitro clonal

propagation, popularly called Micropropagation has been extended to a large number of species other than

orchids and is being practised on commercial scale for numerous ornamental and fruit bearing plant and

some forest trees. After the initiation of aseptic cultures micropropagation generally involves three steps:

Shoot multiplication, rooting and transplantation.

Shoot Multiplication:

This is the most important step with respect to the rate of propagation and genetic uniformity of the product.

The most reliable and, therefore, themost popular method of shoot multiplication is forced proliferation of

axillary shoots. For this, cultures are initiated from apical or nodal cuttings carrying one or more vegetative

buds. In the presence of a cytokinin alone or in combination with a low concentration of an auxin, such as

IAA or NAA, the pre-existing buds grow and produce 4-6 shoots (sometimes up to 30-40 shoots) within 3-4

weeks. By periodic removal of individual shoots and planting them on fresh medium of the original

composition, the shoot multiplication cycle can be repeated almost indefinitely, and a stock of large number

of shoots built up in a short period of time. Treatments with PGRs as described above can also help in a

rapid build up of shoots by inducing adventitious buds by the explant directly or after callusing. Somatic

embryogenesis, which generally occurs after callusing of the explant, is another method of micro

propagation. Somatic embryogenesis is not only fast, but may also allow partial automation of

micropropagation and the propagules so produced (somatic embryos) bear both, shoot and root meristems.

However, adventitive differentiation of shoots or somatic embryos, especially from callus tissue, has the risk

of genetic variability in the progeny. Such variation, that develops in tissue culture called "somaclonal

variation" is not desirable for micropropagation but is being exploited as a novel source of useful variations

for crop improvement.

Rooting:



Shoots produced through axillary branching or adventitious differentiation are rooted in-vitro on a medium

containing a suitable auxin, such as IAA, NAA or IBA. Alternatively, where possible, the shoots are treated

with auxin and directly planted in potting mixture for in-vivo rooting.

Transplantation:

The shoots or plantlets multiplied on a medium containing organic nutrients, show poor photosynthetic

capability. Moreover, in these plants mechanisms to prevent * loss of water from leaves are poorly

developed. Therefore, they require gradual acclimatization to the field conditions. In practice, the plants are

maintained under high humidity (80-90%) for 10-15 days after they are removed from culture vessels.

During the next few weeks the hu~idity around the plants is gradually lowered, before they are transferred

to naturtil conditions. The special merits of micropropagation are: i) it considerably increases the rate of

multiplication 2) high rate of multiplication can be maintained throughout the year, 3) the multiplied plants

are maintained in disease-free conditions 4) being free from microbes and insects valuable genotypes of

exotic plants can be multiplied for export purpose, and 5) small size of the propagules and their ability to

proliferate in a soil-less environment facilitates their convenient storage, handling and rapid transfer by air

across international quarantine baniers.

Uses of plant tissue culture

Plant tissue culture now has direct commercial applications as well as value in basic research into cell

biology, genetics and biochemistry. The techniques include culture of cells, anthers, ovules and embryos on

experimental to industrial scales, protoplast isolation and fusion, cell selection and meristem and bud

culture. Applications include:

micropropagation using meristem and shoot culture to produce large numbers of identical

individuals

screening programmes of cells, rather than plants for advantageous characters

large-scale growth of plant cells in liquid culture as a source of secondary products

crossing distantly related species by protoplast fusion and regeneration of the novel hybrid

production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in

breeding programmes

as a tissue for transformation, followed by either short-term testing of genetic constructs or

regeneration of transgenic plants

removal of viruses by propagation from meristematic tissues

IMPORTANCE AND HISTORICAL VIEW OF PLANT TISSUE CULTURE

Objective To begin with, one should know the importance of plant tissue culture in theimprovement of useful crop plants

and also the ways in which it has helped mankind. Planttissue culture forms an integral part of any plant

biotechnology activity. It offers an alternativeto conventional vegetative propagation. But, tissue culture

requires attention-to-detail andunless practiced as art and science, the entire process is ratherunforgiving. The

various objectives achievable or achieved by plant tissue culture may besummarized as under:

a. Crop Improvement As you all understand that for any crop improvement, conventional breeding methodsare employed which

involve six to seven generations of selfing and crossing- over to obtain apure line. With plant tissue culture

techniques, production of haploids through distant crossesor using pollen, anther or ovary culture, followed by

chromosome doubling, reduces this timeto two generations.

b. Micropropagation



Plant tissue culture techniques have also helped in large- scale production of plantsthrough micropropagation or

clonal propagation of plant species. Small amounts of tissue canbe used to raise hundreds or thousands of plants

in a continuous process. This is beingutilized by industries in India for commercial production of mainly

ornamental plants likeorchids and fruit trees, e.g., banana. Using this method, millions of genetically identical

plantscan be obtained from a single bud. This method has, therefore, become an alternative tovegetative

propagation. Shoot tip propagation is exploited intensively in horticulture and thenurseries for rapid clonal

propagation of many dicots, monocots and gymnosperms.

c. Genetic Transformation Tissue culture, in combination with genetic engineering is very useful in gene transfers.For example, the transfer

of a useful bacterial gene say, cry (crystal protein) gene from

Bacillus thuringiensis

, into a plant cell and, ultimately, regeneration of whole plants containing andexpressing this gene (transgenic

plants) can be achieved.

d. Production of Pathogen-free Plants Eradication of virus has been an outstanding contribution of tissue culture technology.It was found that even in

infected plants the cells of shoot tips are either free of virus or carry anegligible amount of the pathogen. Such

shoot tips are culturedin a suitable culture medium to obtain virus- free plants. This technique is economical

andused very frequently in horticulture, production of virus- free ornamentals etc.

e. Production of Secondary Metabolites Cultured plant cells are also known to produce biochemicals [secondary metabolites]like, alkaloids, terpenoids,

phenyl propanoids etc. of interest. The technology is now availableto the

industry. The commercial production of „shikonin‟[a naphthoquinone] from cell cultures

of Lithospermum erythrorhizon, has been particularly encouraging

Applications of immobilized enzymes

The first industrial use of an immobilized enzyme is amino acid acylase by Tanabe Seiyaku Company,

Japan, for the resolution of recemic mixtures of chemically synthesized amino acids. Amino acid acylase

catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino

acid, that can be easily separated, racemized and recycled. Some of the immobilized preparations used for

this purpose include enzyme immobilized by ionic binding to DEAE-sephadex and the enzyme entrapped as

microdroplets of its aqueous solution into fibres of cellulose triacetate by means of fibre wet spinning

developed by Snam Progetti. Rohm GmbH have immobilized this enzyme on macroporous beads made of

flexiglass-like material

By far, the most important application of immobilized enzymes in industry is for the conversion of glucose

syrups to high fructose syrups by the enzyme glucose isomerase95

. Some of the commercial preparations

have been listed. It is evident that most of the commercial preparations use either the adsorption or the

cross-linking technique. Application of glucose isomerase technology has gained considerable importance,

especially in nontropical countries that have abundant starch raw material. Unlike these countries, in tropical

countries like India, where sugarcane cultivation is abundant, the high fructose syrups can be obtained by a

simpler process of hydrolysis of sucrose using invertase. Compared to sucrose, invert sugar has a higher

humectancy, higher solubility and osmotic pressure. Historically, invertase is perhaps the first reported

enzyme in an immobilized form96

. A large number of immobilized invertase systems have been patented97

.

The possible use of whole cells of yeast as a source of invertase was demonstrated by D‟Souza and

Nadkarni as early as 1978. A systematic study has been carried out in our laboratory for the preparation of

invert sugar using immobilized invertase or the whole cells of yeast. These comprehensive studies carried



out on various aspects in our laboratory of utilizing immobilized whole-yeast have resulted in an industrial

process for the production of invert sugar.

L-aspartic acid is widely used in medicines and as a food additive. The enzyme aspartase catalyses a one-

step stereospecific addition of ammonia to the double bond of fumaric acid. The enzymes have been

immobilized using the whole cells of Escherichia coli. This is considered as the first industrial application

of an immobilized microbial cell. The initial process made use of polyacrylamide entrapment which was

later substituted with the carragenan treated with glutaraldehyde and hexamethylenediamine. Kyowa Hakko

Kogyo Co. uses Duolite A7, a phenolformaldehyde resin, for adsorbing aspartase used in their continuous

process99

. Other firms include Mitsubishi Petrochemical Co.100

and Purification Engineering Inc101

. Some of

the firms, specially in Japan like Tanabe Seiyaku and Kyowa Hakko, have used the immobilized fumarase

for the production of malic acid (for pharmaceutical use)94

. These processes make use of immobilized

nonviable cells of Brevibacterium ammoniagenes or B. flavus as a source of fumarase. Malic acid is

becoming of greater market interest as food acidulant in competition with citric acid. Studies from our

laboratory have shown the possibility of using immobilized mitochondria as a source of fumarase6.

One of the major applications of immobilized biocatalysts in dairy industry is in the preparation of lactose-

hydrolysed milk and whey, using b -galactosidase. A large population of lactose intolerants can consume

lactose-hydrolysed milk. This is of great significance in a country like India where lactose intolerance is

quite prevalent102

. Lactose hydrolysis also enhances the sweetness and solubility of the sugars, and can find

future potentials in preparation of a variety of dairy products. Lactose-hydrolysed whey may be used as a

component of whey-based beverages, leavening agents, feed stuffs, or may be fermented to produce ethanol

and yeast, thus converting an inexpensive byproduct into a highly nutritious, good quality food ingredient99

.

The first company to commercially hydrolyse lactose in milk by immobilized lactase was Centrale del Latte

of Milan, Italy, utilizing the Snamprogetti technology. The process makes use of a neutral lactase from yeast

entrapped in synthetic fibres103

. Specialist Dairy Ingredients, a joint venture between the Milk Marketing

Board of England and Wales and Corning, had set up an immobilized b -galctosidase plant in North Wales

for the production of lactose-hydrolysed whey. Unlike the milk, the acidic b -galactosidase of fungal origin

has been used for this purpose31

. Some of the commercial b -galactosidase systems have been summarized

in Table 3. An immobilized preparation obtained by cross-linking b -galactosidase in hen egg white

(lyophilized dry powder) has been used in our laboratory for the hydrolysis of lactose47

. A major problem in

the large-scale continuous processing of milk using immobilized enzyme is the microbial contamination

which has necessitated the introduction of intermittent sanitation steps. A co-immobilizate obtained by

binding of glucose oxidase on the microbial cell wall using Con A has been used to minimize the bacterial

contamination during the continuous hydrolysis of lactose by the initiation of the natural lacto-peroxidase

system in milk88

. A novel technique for the removal of lactose by heterogeneous fermentation of the milk

using immobilized viable cells of K. fragilis has also been developed10

.

One of the major applications of immobilized enzymes in pharmaceutical industry is the production of 6-

aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V, using

penicillin acylase (penicillin amidase)104

. More than 50% of 6-APA produced today is enzymatically using

the immobilized route. One of the major reasons for its success is in obtaining a purer product, thereby

minimizing the purification costs. The first setting up of industrial process for the production of 6-APA was

in 1970s simultaneously by Squibb (USA), Astra (Sweden) and Riga Biochemical Plant (USSR). Currently,

most of the pharmaceutical giants make use of this technology. A number of immobilized systems have

been patented or commercially produced for penicillin acylase which make use of a variety of techniques

either using the isolated enzyme or the whole cells100,105,106

. This is also one of the major applications of the

immobilized enzyme technology in India. Similar approach has also been used for the production of 7-

aminodeacetoxy-cephalosporanic acid, an intermediate in the production of semisynthetic cephalosporins.



Immobilized oxidoreductases are gaining considerable importance in biotechnology to carry out synthetic

transformations. Of particular significance in this regard are oxidoreductase-mediated asymmetric synthesis

of amino acids, steroids and other pharmaceuticals and a host of speciality chemicals. They play a major

role in clinical diagnosis and other analytical applications like the biosensors. Future applications for

oxidoreductases can be in areas as diverse as polymer synthesis, pollution control, and oxygenation of

hydrocarbons107

. Immobilized glucose oxidase can find application in the production of gluconic acid,

removal of oxygen from beverages, and in the removal of glucose from eggs prior to dehydration in order to

prevent Maillard reaction. Studies carried out in this direction in our laboratory have shown that glucose can

be removed from egg, using glucose oxidase and catalase which are co-immobilized either on polycationic

cotton cloth57

or in hen egg white foam matrix50

. Alternatively, glucose can also be removed by rapid

heterogeneous fermentation of egg melange, using immobilized yeast108

. Immobilized D-amino acid oxidase

has been investigated for the production of keto acid analogues of the amino acids, which find application in

the management of chronic uremia. Keto acids can be obtained using either L- or D-amino acid oxidases.

The use of D-amino acid oxidase has the advantage of simultaneous separation of natural L-isomer

from DL-recemates along with the conversion of D-isomer to the corresponding keto acid which can then be

transamina-ted in the body to give the L-amino acid. Of the several microorganisms screened, the triangular

yeast T.variabilis was found to be the most potent source of D-amino acid oxidase with the ability to

deaminate most of the D-amino acids109

. The permeabilized cells entrapped either in radiation polymerized

acrylamide24

Ca-alginate23

or gelatin25

have shown promise in the preparation of a -keto acids. Another

interesting enzyme that can be used profitably in immobilized form is catalase for the destruction of

hydrogen peroxide employed in the cold sterilization of milk. A few reports are available on its

immobilization using yeast cells11,22

.

Lipase catalyses a series of different reactions. Although they were designed by nature to cleave the ester

bonds of triacylglycerols (hydrolysis), lipase are also able to catalyse the reverse reaction under

microaqueous conditions, viz. formation of ester bonds between alcohol and carboxylic acid moieties. These

two basic processes can be combined in a sequential fashion to give rise to a set of reactions generally

termed as interesterification. Immobilized lipases have been investigated for both these processes. Lipases

possess a variety of industrial potentials starting from use in detergents; leather treatment controlled

hydrolysis of milk fat for acceleration of cheese ripening; hydrolysis, glycerolysis and alcoholysis of bulk

fats and oils; production of optically pure compounds, flavours, etc. Lipases are spontaneously soluble in

aqueous phase but their natural substrates (lipids) are not. Although use of proper organic solvents as an

emulsifier helps in overcoming the problem of intimate contact between the substrate and enzyme, the

practical use of lipases in such psuedohomogeneous reactions poses technological difficulties. Varieties of

approaches to solve these, using immobilized lipases, have recently been reviewed110

.

Significant research has also been carried out on the immobilization and use of glucoamylase. This is an

example of an immobilized enzyme that probably is not competitive with the free enzyme and hence has not

found large-scale industrial application111

. This is mainly because soluble enzyme is cheap and has been

used for over two decades in a very optimized process without technical problems. Immobilization has also

not found to significantly enhance the thermostability of amylase111

. Immobilized renin or other proteases

might allow for the continuous coagulation of milk for cheese manufacture112

. One of the major limitations

in the use of enzymes which act on macromolecular substrates or particulate or colloidal substrates like

starch or cellulose pectin or proteins has been the low retention of their realistic activities with natural

substrates due to the steric hindrance. Efforts have been made to minimize these problems by attaching

enzymes through spacer arms113

. In this direction, application of tris (hydroxymethyl) phosphine as a

coupling agent114

may have future potentials for the immobilization of enzymes which act on

macromolecular substrates. Other problem, when particulate materials are used as the substrates for an

enzyme, is difficulty in the separation of the immobilized enzyme from the final mixture. Efforts have been



made in this direction to magnetize the bicatalyst either by directly binding the enzyme on magnetic

materials (magnetite or stainless steel powder) or by co-entrapping magnetic material so that they can be

recovered using an external magnet98,115

. Magnetized biocatalysts also help in the fabrication of

magnetofluidized bed reactor116

.

A variety of biologically active peptides are gaining importance in various fields including in pharmaceuti-

cal industries and in food industries as sweeteners, flavourings, antioxidants and nutritional supplements.

Proteases have emerged over the last two decades as powerful catalysts for the synthesis and modification of

peptides. The field of immobilized proteases may have a future role in this area117,118

. One of the important

large scale applications will be in the synthesis of peptide sweetener using immobilized enzymes like the

thermolysin119

. Proteolytic enzymes, such as subtilisin, a-chymotrypsin, papain, ficin or bromelain, which

have been immobilized by covalent binding, adsorption or cross-linking to polymeric supports are used

(Bayer AG) to resolve A N-acyl-DL-phenylglycine ester racemate, yielding N-acyl-D-esters or N-acyl-D-

amides and N-acyl-L-acids100

. Immobilized aminopeptidases have been used to separate DL-

phenylgycinamide racemates100

. SNAM-Progetti SpA-UK have used the immobilized hydropyrimidine

hydrolase to prepare D-carmamyl amino acids and the corresponding D-amino acids from various

substituted hydantoins100

.

Application of Immobilized Cells

Immobilization of plant cells is considered to be of importance in research and development in plant cell

cultures, because of the potential benefits that could be provided (24, 92):

a) The extended viability of cells in the stationary (and producing) stage, enabling maintenance of biomass

over a prolonged time period;

b) Simplified downstream processing (if products are secreted);

c) The (putative) promotion of differentiation, linked with enhanced secondary metabolism;

d) Higher cell density enabling a reduced bioreactor size, thereby reducing costs and the risk of

contamination;

e) Reduced shear sensitivity (especially with entrapped cells);

f) Promotion of secondary metabolite secretion, in some cases;

g) Flow-through reactors can be used enabling greater flow rates;

h) Minimization of fluid viscosity increase, which in cell suspension causes mixing and aeration problems.

An immobilization system which could maintain viable cells over an extended period of time and release the

bulk of the product into the extracellular medium in a stable form, could dramatically reduce the costs of

phytochemicals production in plant cell culture (1). However, an immobilized system also has the problems

described below:

a) Immobilization is normally limited to cases where production is decoupled from cell growth;

b) The initial biomass must be grown in suspension;



c) Secretion of product into the extracellularly medium is imperative;

d) Where secretion occurs there may be problems of extracellular degradation of the products;

e) When gel entrapment is used, the gel matrix introduces an additional diffusion barrier.

Due to these problems, a system with commercial potential has not yet been developed in plant tissue

cultures. However, various immobilization methods have been developed, ie., entrapment, adsorption and

covalent coupling.

Some preliminary results have been obtained with immobilized cells. Early work with C. roseus, showed

that agar, agarose and carageenan were all suitable immobilization matrices suitable for maintenance of cell

viability; but alginate was superior in terms of ajmalicine production (93). The accumulation of serpentine

by C. roseus and anthraquinones by Morinda citrifolia were both enhanced in the immobilized state when

compared with freely suspended cells. It should be noted however, that the possibility that alginate acts as

an elicitor of secondary metabolism cannot be ruled out (94). Agar has been shown to stimulate shikonin

accumulation in L. erythrorhizon cultures (95). Lambe and Rosevear (96) have successfully

immobilized C. roseus cells in polyacrylamide with alginate and observed prolonged viability and increased

productivity.

Adsorption immobilization has been successfully used with a number of plant

species. Capsicum frutescens cells immobilized on polyurethane foam produced 50 times as much capsaicin

as suspension cells (97). Similarly, Solanum nigrum cells accumulated glycoalkaloids to levels exceeding

those found in suspensions. Datura innoxia cells accumulated tropane alkaloids with a profile similar to that

of the intact plant, whilst in free suspensions productivity was markedly suppressed (98). In general it

appears that mild immobilization either through gel entrapment or surface adsorption enhances productivity

and prolongs the viability of cultured cells.

As described in the section on Biotransformation, immobilized cells can also be used as biocatalysts for

biotransformations. Such a system compares favourably with the use of freely suspended cells since, in the

case of immobilization, the catalyst is theoretically reusable and the product is easily separated from the

biomass. The most appropriate example is that of the 12-hydroxylation of ß-methyldigitoxin to ß-

methyldigoxin with alginate-entrapped Digitalis lanata cells (99). The enzyme activity was maintained by

the immobilized cultures for a period of 61 days. Furthermore, the product was located in the extracellular

medium. Mild permeabilization of the cells may enable biotransformation rates to be increased.

Polyurethane-immobilized C. frutescens cells fed capsaicin precursors produced this metabolite at levels of

up to 10 times those of non-fed cultures (98). DiCosmo et al. (48) found that glass fibres can be used as a

carrier of plant cells to produce useful plant metabolites. Papaver somniforum cells were immobilized on

fabric of loosely woven polyester fibres arranged in a spiral configuration on stainless steel support frame

by Kurz et al. (100) to produce sanguinarine, an antibiotic in oral hygiene. The yield was 3.6 mg/g-fw. by

immobilized cells and was more than twice as much as by suspension cells.

INDUSTRIAL PRODUCTION OF PHYTOCONSTITUTIONS:

Process for the preparation of sennosides A and B:

A. Extraction, evaporation and washing



16 kg of senna pods are digested for 3 days in a mixture of 10 liters of methanol and 10 liters of tap water.

The solution is circulated with a pump in order to make the extraction more effective. The temperature is

maintained at 20° to 30° C. After the specified time the solution is drained, and the pods washed with 4 l of

a 50% methanol solution. The obtained methanol-water-solution is distilled under vacuum at 40° C. The

distillation is stopped when the density of the distillation residue is 1.25 kg/l. The distillation residue is

extracted with 8 l of n-butanol. The extraction is carrried out in a suitable reaction vessel by mixing the

solution for 1.5 hours. The stirring is discontinued and the layers are left to separate over the night. After

separation, the aqueous phase is poured into the reduction vessel.

B. Reduction

To the raw aqueous sennoside solution in the reduction vessel at a temperature of 25° C. a 50% sodium

hydroxide solution is added until the pH is 8.3. 120 to 150 g of lye is consumed. To the solution 500 g of

sodium dithionite is added and the mixture is stirred for two hours. 3 l of water is added, whereafter the pH

is adjusted with sulphuric acid to a value of 4.7. l g of rheinanthrone-8-glucoside crystals are added and

adjusted with sulphuric acid to a pH of 2.9. The mixture is cooled to 10° C., where it is kept for two hours.

The crystallized rheinanthrone-8-glucoside is filtered onto a filter and washed with 1500 ml of hot water.

The precipitate is dried by sucking nitrogen through the filter under vacuum, whereby appr. 560 g of

rheinanthrone-8-glucoside is obtained, which contains about 20% moisture.

C. Oxidation of rheinanthrone-8-glucoside

560 g of rheinanthrone-8-glucoside containing 15-20% moisture is slurried in 6000 ml of 80%

(vol./vol.)isopropanol at +5° to +10° C. The rheinanthrone-8glucoside is made to dissolve by adding

triethylamine to a pH of appr. 8. The pH may not rise above 8.5. Appr. 200 ml of triethylamine is consumed.

Thereafter 50 g of OH- active carbon is added and the introduction of pressurized air into the mixture is

started by bubbling through a sinter at a rate of appr. 3 liters in a minute. Air is bubbled for appr. 2 1/2hours,

the temperature of the reaction mixture being 5° to 10° C.

When the reaction is complete, the mixture is filtered through filter cardboard and adjusted with

concentrated hydrochloric acid (about 200 ml) to a pH-value of 1.5 to 2.0. The mixture is left to crystallize

over night at room temperature while stirring. The obtained precipitate is filtered through cardboard, washed

with 500 ml of isopropanol and dried in a vacuum chamber at a temperature of not more than appr. 40° C.

The yield is appr. 310 g (appr. 62.2% calculated from the rheinanthrone-8-glucoside).

D. Preparation of the calcium salt

300 g of sennoside A + B acid is slurried in 1800 ml of water and dissolved by adding a calcium hydroxide-

water slurry (30 g Ca(OH)2 +150 ml of water). The addition is continued to a pH value of 8±0.5 and appr.

110 ml of lime slurry is consumed to dissolve the acid. Thereafter the pH is adjusted with weak

hydrochloric acid (40 ml; 1:10 dilution) to a pH-value of 6.7 in the course of one hour, while making sure

that the pH stays in the range of 6.7 to 6.9. Within 1/2hour 1000 ml of a 90% methanol solution and

thereafter, during appr. 2 hours, 4400 ml pure (100%) methanol are added. The mixture is stirred for another

hour and filtered through cardboard. The precipitate is washed with a small amount of methanol.

The precipitate is dried at a temperature of at the most 40° C. over night and weighed. The yield is appr. 317

g (100% ) as air-dry and 285 g (appr. 91% ) as vacuum dry calculated from the sennoside A + B acid, of

which the sennoside content is appr. 82%.



Vinca Alkaloids

The dimeric indole alkaloids, vinblastine and vincristine have become highly valued drugs in cancer

chemotherapy due to their potent antitumor activity against various leukemias, Hodgkin's disease and solid

tumors. They are currently produced commercially by extraction from Catharanthus roseus (Apocyanaceae)

plants, but the process is not efficient because of very low concentrations of the alkaloids in the plant. It was

reported that the concentration of both vinblastine and vincristine was only 0.0005% as a dry weight basis.

In order to produce these useful anticancer drugs much more efficiently, many scientists have tried to apply

plant tissue culture technology. In fact, a large number of papers related to this approach have been

presented since the first research carried out by Carew et al. in 1966 (196). However, production of both

alkaloids by de novo synthesis using the callus or the suspension cultured cell of C. roseus is so far not

promising because the productivity of the cultured cells reported was so far very low.

Misawa and his colleagues of Allelix Inc. in Canada (197-199) studied on production of vinblastine by an

alternative way in collaboration with Kurz of the National Research Council of Canada and Kutney of

University of British Columbia, and established an economically feasible process consisting of production

of catharanthine by plant cell fermentation and a simple chemical or an enzymatic coupling.

The vinblastine molecule is derived from two monomeric alkaloids, catharanthine and vindoline as shown in

Fig. 8. The concentration of vindoline in the intact C. roseus plant is approximately 0.2% as a dry weight

basis, which is much a higher level than catharanthine, and the cost of vindoline is less expensive compared

to catharanthine and vinblastine. The Allelix group, therefore, investigated the production of catharanthine

by a cell suspension culture process with a selected C. roseus cell line induced from anthers on Gamborg's

B5 medium containing 2% sucrose, 1 mg/L 2,4-D and 0.1 mg/L kinetin. The cells were grown in 250 ml

flasks containing 60 ml of MS liquid medium supplemented with 3% sucrose, 1 mg/L NAA and 0.1 mg/L

kinetin under continuous diffuse light on a rotary shaker (250 r.p.m.) at 25° C. In experiments for

optimization of catharanthine production, they transferred 7 day old cells to a test medium and subcultured

for 3 passages. In the 4th passage, 60 ml cultures were harvested in triplicate after 2 or 3 weeks growth, and

the cell mass and alkaloid content were determined.

Figure 8: Chemical Structures of Catharanthine, Vindoline, Vinblastine and Vincristine

The results showed that the MS medium was the most favourable for catharanthine production but the

optimal levels of phytohormones for the growth and the production were varied in different cell lines. For



example, one line required no phytohormones but another line required 0.1 mg/L NAA and 0.1 mg/L

kinetin. Addition of various chemically defined compounds to the medium as "inducers" was found to

stimulate the production efficiently. Among them effects of vanadyl sulphate, abscisic acid and NaCl on the

production of catharanthine were significant (200). Based on the conditions optimized by using flasks,

Smart et al (201) scaled up the cultures to 10, 30 and 100 L-air lift fermentors. When abscisic acid was

added to the culture as an elicitor on the 7th day of cultivation, the final titer of catharanthine was raised to

85 mg/L in a 30 L fermentor.

The second stage in this project, the Allelix's group tried to couple enzymatically or chemically

catharanthine produced by the cell culture process with commercially available vindoline. As an enzyme

source for the coupling, a crude preparation obtained by 70% ammonium sulphate precipitation from the

cultured cells of C. roseus was used. The reaction mixture containing both monomeric alkaloids, Tris buffer

(pH 7.0) and the enzyme preparation was incubated at 30° C and for 3 hours. It was determined that the

enzyme reaction gave various dimeric alkaloids including vinamidine, 3-(R)-hydroxyvinamidine and 3'4'-

anhydrovinblastine. Leurosine and catharine, oxidized derivatives of anhydrovinblastine, were also detected

in the early stages of the incubation. They found that MnCl2 and either FAD or FMN stimulated the

coupling. Although neither vinblastine nor vincristine was detected in the mixture, it was recognized that a

substantial amount of anhydrovinblastine was formed as a major coupling product when an excess amount

of sodium borohydride was added to the mixture after incubation.

In order to investigate properties of the coupling enzyme(s) it was partially purified with gel filtration and

isoelectric focusing and five isozymes were obtained by Endo et al. (202). One of them had MW 15,000 and

the other four had the same MW (37,000). All of these isozymes were shown to have peroxidase activity.

Using the partially purified enzymes, anhydrovinblastine was formed with a conversion yield of about 50%.

Formation of vinblastine from vincristine as detected by Goodbody et al. (203) using a crude enzyme

preparation obtained from suspension cultured cells of C. roseus. The highest yield of conversion obtained

was 13% from 0.13 mg anhydrovinblastine in 1 ml of the reaction mixture after 3 hours incubation at 30° C,

Ph 7.0.

During the course of these studies on coupling mechanisms, they found that ferric ion catalyzed the

coupling reaction significantly in the absence of the enzyme. It is of interest that the products of the

chemical coupling were not only anhydrovinblastine but also vinblastine. The yields of both alkaloids were

52.8% and 12.3%, respectively after 3 hours incubation at 30° C, pH 7.0. These products including

catharanthine were analyzed by high resolution mass spectrometry as further confirmation of their

identification. Circular dichroism confirmed that a-coupling exists between the 2 monomeric units of both

vinblastine and vincristine produced either enzymatically or chemically.

This is a novel and an efficient process to produce an antitumor drug, vinblastine, and is likely to be applied

commercially. The technology was transferred from the Canadian company to a Japanese company, Mitsui

Petrochemicals Industry for further development.

Hara et al. (204) of Mitsui Petrochemical could increase the yield of catharanthine up to 150 mg/L in the

MS medium supplemented with 1 mg/L NAA and 0.1 mg/L kinetin using the best producing cell line

isolated from Allelix's cell line. The stimulating activity of NaCl and KCl on alkaloid production was also

confirmed. Furthermore, the scientists of Mitsui employed high-cell density cultures and reported yields of

catharanthine of 230 mg/L/week (205).



The yield of vinblastine by the chemical coupling reaction was also improved by the same group; addition

of ferric chloride, oxalate, maleate and sodium borohydrate stimulated the yield of vinblastine from

anhydrovinblastine up to 50% (206).

Bede et al. (206) also investigated the production of anhydrovinblastine. They employed a two-enzyme

system containing horseradish peroxidase and glucose oxidase to catalyze the formation of

anhydrovinblastine from catharanthine and vindoline. Although peroxidase requires hydrogen peroxide for

the coupling reaction, its presence in excess in the reaction mixture may inhibit the reaction. But addition of

glucose oxidase was used to allow the controlled, continuous production of hydrogen peroxide at low levels,

minimizing oxidative reactions. Both enzymes were immobilized on Euperight C beads, an oxirane matrix,

and the system was shown in catalyze the coupling reaction.

Podophyllotoxin

Podophyllum pelatatum, May apple, which is a common herb in eastern North America contains an

antitumor lignan, podophyllotoxin. It is active to KB cells and is used against certain virus diseases and skin

cancer (190). A semi-synthetic derivative of podophyllotoxin, etoposide (V-16), was found to be active

against brain tumor, lymphosarcoma and Hodgkins' disease and was approved by the FDA in the U.S.

Bristol-Myers Squibb is one of the largest manufacturers of the drug.

Production of podophyllotoxin by P. pelatum cell cultures was first attempted by Kadkade (191) and he

found that a combination of 2,4-D and kinetin in the medium supported the highest amount of its

production. Red light stimulated the production.

Sakata et al. of Nippon Oil (192) induced embryogenic roots from a callus of the plant in a liquid MS

medium supplemented 1 mg/L NAA, 0.2 mg/L kinetin and 500 mg/L casein hydrolysates. The roots were

then transferred to the medium without growth regulators. They detected 1.6% of podophyllotoxin in the

dried tissues, which was 6 times higher level than that in a mother plant.

To increase the yield of podophyllotoxin, Woerdenberg et al. in the Netherlands (193) added a complex of a

precursor, coniferyl alcohol, and ß-cyclodextrin to Podophyllum hexandrum cell suspension cultures.

Addition of 3 mM coniferyl alcohol complex gave 0.013% podophyllotoxin of the cells on a dry weight

basis but the cultures without the precursor produced only 0.003%. ß-D-glucoside of coniferyl alcohol,

coniferin, was a more potent precursor in terms of the yield of the anticancer compound (0.055%), but

unfortunately this compound is not commercially available. The same authors reported that cell suspension

cultures of Callitris drummondii (conifer) also accumulated podophyllotoxin-ß-D-glucose. In the dark, the

cells produced approximately 0.02% podophyllotoxin of the dry cell mass and 85-90% of the lignans were

the ß-D-glucoside form, while in the light the yield of podophyllotoxin-ß-D-glucose increased to 0.11%.

Smooly et al. (194) reported that callus tissues and suspension culture cells of Lilium album produced

podophyllotoxin. One of the cell lines produced 0.3% podophyllotoxin of dried cells together with small

amounts of 5-methylpodophyllotoxin, lariciresinol and pinoresinol after 3 weeks of cultivation. The callus

tissue induced from P. hexandrum was reported by Heyenga (195) to produce podophyllotoxin, 4'-

demethyl-podophyllotoxin and podophyllotoxin-4-0-glucoside when the callus was incubated in B5 medium

containing 2,4-Dichlorophenoxyacetic acid, gibberellic acid and 6-benzylaminopurine. The levels of

podophyllotoxin and its derivatives were similar to those in the mother plant.

QUINOLINES.

http://en.wikipedia.org/wiki/Quinoline



The Skraup synthesis is a chemical reaction used to synthesize quinolines. It is named after the Czech

chemist Zdenko Hans Skraup (1850-1910). In the archetypal Skraup reaction, aniline is heated with sulfuric

acid, glycerol, and an oxidizing agent such as nitrobenzene to yield quinoline.[1][2][3][4]

In this example, nitrobenzene serves as both the solvent and the oxidizing agent. The reaction, which

otherwise has a reputation for being violent ("the Chemical Inquisition"), is typically conducted in the

presence of ferrous sulfate.[5]

Arsenic acid may be used instead of nitrobenzene and the former is better

since the reaction is less violent.[6]

8-aminoquinolines• Drugs in this group have amino group at position 8 of quinoline ring• Important

members of this family include 1- Pamaquine 2- Primaquine, etc.

2. • Such drugs have OCH3 group at position 6• This molecule has antimalarial activity but when side chain

is introduced at amino group antimalarial activity is intensified e.g pamaquine• It causes hemolysis of RBCs

Diethyl amino pentyl side chain

3. • It contains tertiary amino group and when it is converted into primary amino group the compound is

called primaquine, which is – Less toxic – Well tolerated – It is the most commonly used agent in this group

in the treatment of malaria

4. • OCH3 is not necessary for antimalarial activity but when replaced by OC2H5 the compound became –

less active – Toxic in nature• OCH3 when replaced by CH3 the compound become inactive• Introduction of

halogens increases toxicity• Presence of quinoline ring is necessary for antimalarial activity. When pyridine

ring is converted to piperidine (saturated) the compound became inactive

5. • Pentyl side chain gives maximum activity, increase or decrease of chain result is reduction of activity.•

The branched side chain when converted into straight chain pentaquine is obtained• It has less antimalarial

activity as compared to both pamaquine and primaquine

6. Chemical synthesis (pamaquine)• Glycerol undergoes dehydration to produce propene aldehyde•

Dehydrating agent is sulphuric acid

7. • Addition reaction of propene aldehyde and 4 methoxy 2-nitro aniline to form 4 methoxy 2- nitro

propene aldehyde

8. • Tautomerization: 4 methoxy 2-nitro propene aldehyde (keto form) converted in enol form

9. • Enol form undergoes cyclization to form 8 nitro 6 methoxy dihydroquinoline which then oxidized to

form 8 nitro 6 methoxy quinoline

10. • 6 methoxy 8 nitro quinoline undergoes reduction to form 8 amino 6 methoxy quinoline

11. • 8 amino 6 methoxy quinoline reacts with 2 chloro diethyl amino pentane to form pamaquine

http://en.wikipedia.org/wiki/Chemical_reaction

http://en.wikipedia.org/wiki/Quinoline

http://en.wikipedia.org/wiki/Zdenko_Hans_Skraup

http://en.wikipedia.org/wiki/Aniline

http://en.wikipedia.org/wiki/Sulfuric_acid



http://en.wikipedia.org/wiki/Glycerol

http://en.wikipedia.org/wiki/Redox

http://en.wikipedia.org/wiki/Nitrobenzene

http://en.wikipedia.org/wiki/Skraup_reaction#cite_note-1





http://en.wikipedia.org/wiki/Nitrobenzene

http://en.wikipedia.org/wiki/Solvent

http://en.wikipedia.org/wiki/Iron(II)_sulfate


http://en.wikipedia.org/wiki/Arsenic_acid

http://en.wikipedia.org/wiki/Skraup_reaction#cite_note-finar-6

http://en.wikipedia.org/wiki/File:Skraup_Reaction_Scheme.png



12. Therapeutic uses• Active against hepatic stage of plasmodium• Provide radical cure hepatic stage of P.

vivax and P. ovale• It also acts at gametocytes, hence used as prophylactic drugs• Used in combination with

chloroquine for complete eradication of malaria• Side effect: hemolysis in G6 phosphate dehydrogenase

deficient people

Aplication of Quinoline: Quinoline is used in the manufacture of dyes.Quinoline and quinoline derivative

can be usedin manufacturing a wide range of Food Colors, Lake Colors, Salt Free Dyes, etc. which

areextensively used as Dyes in various industries, such as Food, Pharmaceutical and Cosmetic. Quinoline is

used in the preparation of hydroxyquinoline sulfate and niacin. It has also used asa solvent for resins and

terpenes. Quinoline is mainly used as a feedstock in the production of other specialty chemicals. Itsprincipal

use is as a precursor to 8-hydroxyquinoline, which is a versatile chelating agent andprecursor to pesticides.

Its 2- and 4-methyl derivatives are precursors to cyanine dyes. Oxidationof quinoline affords quinolinic acid

(pyridine-2,3-dicarboxylic acid), a precursor to the herbicidesold under the name "Assert". Non-Cancer:

Quinoline is an irritant of the eye and respiratory tract. Acute inhalationoverexposure to quinoline vapors in

humans may cause signs and symptoms such as headaches,dizziness and nausea, and coma. Quinoline

overexposure has also been reported to cause injuryto the cornea, retina, and optic nerve.

MENTHOL MANUFACTURING PROCESS & TECHNOLOGY:

The leaves of the "Mentha Arvenisis" are subjected to steam distillation, the distillation products are

condensed and separated into peppermint oil and water. The crude mint oil then obtained is refined by

vacuum filteration and then chilled to about 5-10 degree C to obtain Menthol Crystals. The crystals, thus

formed are centrifuged and obtain about 45% yield of menthol. The spent oil is treated with sodium

hydroxide and Boric Acid while crystalline borate esters which are formed are separated and decomposed

by steam. The Menthol thus released is recovered by crystallisation under reduced temperatures and

centrifuging. The mother liquor is distilled to obtain dementholised peppermint oil. The overall yield of

menthol is about 50% and an equal amount of dementhonised oil is obtained as co-product.

INDUSTRIAL PRODUCTION OF CITRIC ACID

Microorganism: Aspergillus niger (mainly), Candida yeast (from carbohydrates or n-alkanes)

Citric acid production is mixed growth associated, mainly take place under nitrogen and phosphate

limitation after growth has ceased.

Medium requirements for high production:

- Carbon source: molasses or sugar solution.

- Na-ferrocyanide is added to reduce Iron (1.3 ppm) and

manganese (<0.1ppm).

- High dissolved oxygen concentration

- High sugar concentration

- pH<2

- Temperature: 30oC

Bioreactor: batch or fed-batch (100m3)

- 5-25×106 A. niger spores/L may be introduced to the fermentor.



- Aeration is provided to the fermenter by air sparging (0.1-0.4

vvm)

- Temperature is controlled by cooling coil.

- Agitation: 50-100rpm to avoid shear damage on molds.

- Fed-batch is used to reduce substrate inhibition and prolong the

production phase one or two days after growth cessation.

- Volumetric yield: 130 kg/m3

Separation:

- The biomass is separated by filtration

- The liquid is transferred to recovery process:

- Separation of citric acid from the liquid: precipitation calcium hydroxide is added to obtain calcium

citrate tetrahydrate → wash the precipitate→ dissolve it with dilute sulfuric acid, yield citric acid and

calcium sulfate precipitate → bleach and crystallization → anhydrous or monohydrate citric acid.

- Microorganism: S. cerevisae for hexose

Candida sp. for lactose or pentose

Genetically modified E. coli

- Ethanol production is growth-associated with S. cerevisae.

- Medium requirements for high production

- Carbon source: sugar cane, starch materials (e.g. corn, wheat), cellulosic materials (?!). yield: 0.51

g ethanol/g glucose.

- N, P, minerals.

- Anaerobic

- 100g/L glucose are inhibitory for yeast.

- 5% (v/v) of ethanol are inhibitory for yeast.

- pH:4-6 for 30-35 oC.

Bioreactor: batch, continuous or with cell recycle

95% conversion of sugars with a residence time of

40 h in batch reactor

21 h in continuous reactor without cell recycle

1.6 h in continuous reactor with cell cycle



By-products: glycerol, acetic acid, succinic acid.

Separation:

- Distillation to obtaining 95% (w/w) of ethanol-water mixture, followed by

- Molecular sieves to removing water from the mixture to get anhydrous ethanol.

Purification of Citric acid

• A typical method used for purification of citric acid from a

fermentation broth involves two major purification techniques:

precipitation and filtration.

• The following schematic displays a generic citric acid purification scheme:



To crack the calcium citrate precipitate, sulfuric acid is needed. The temperature of this reaction should stay

below 60ºC. The reaction will produce free citric acid and a new precipitate, calcium sulfate, which will

need to be removed later. The stoichiometric coefficients for this reaction are all one.

In this filter, the calcium sulfate is washed away from the citric acid and the leftover biomass is removed.

Again, the contaminants that were present in the fermentation broth can be removed by additional filtration

means, such as microfiltration or ultra filtration.

Applications :

Food

• Used as flavoring and preservative in food and beverages.

• Can be added to e.g. ice cream as an emulsifying agent to keep fats from separating, to caramel to

prevent sucrose crystallization, or to recipes in place of fresh lemon juice.

• Citric acid is used with sodium bicarbonate in a wide range of effervescentformulae, both for

ingestion (e.g., powders and tablets) and for personal care (e.g., bath salts, bath bombs, and cleaning

of grease).

• Citric acid is also often used in cleaning products and sodas or fizzy drinks.

Cleaning and Chelating agent

• Used to remove scale from boilers and evaporators.

• Can be used to soften water, which makes it useful in soaps and laundry detergents.

• In industry, it is used to dissolve rust from steel.

• Can be used in shampoo to wash out wax and coloring from the hair.

Cosmetics and pharmaceuticals



• Citric acid is widely used as a pH adjusting agent in creams and gels of all kinds.

• Citric acid is commonly used as a buffer to increase the solubility of brown heroin.

• Citric acid is used as one of the active ingredients in the production of antiviral tissues.

Dyeing

• Citric acid can be used in food coloring to balance the pH level of a normally basic dye.

• It is used as an odorless alternative to white vinegar for home dyeing with acid dyes.

Photography

• Citric acid can be used as a lower-odor stop bath as part of the process for developing photographic

film.

INDUSTRIAL PRODUCTION OF DIOSGENIN:

DIOSGENIN TUBERS COLLECTED---------- WASHED ------- DRIED----------- EXTRACTED WITH

HOT WATER OR 90% ETHANOL FOR 6 HRS……………………… ALCOHOLIC EXTRACT

CONCENTRATED UNDER VACUUM………….. FILTER IT ……….. FILTERATE + SOLVENT

ETHER OR LEAD ACETATE SOLUTION…………HYDROLYSIS BY ACID ……………………..

EXTRACTED WITH PET. ETHER……….EVAPORATE SOLVENT …………………… DIOSGENIN

COLLECTED, DRIED AND PACKED .

INDUSTRIAL PRODUCTION OF SOLSODINE

SOLSODINE BY TWO METHODS METHOD 1 B. METHOD 2

METHOD. 1 Dried berries is powdered-------- Oil is removed------------- Defatted is extracted with ethanol--

------------------- Resultant is filtered , Concentrated & Treat with HCl & Reflux ---------------- Extract is

made alkaline by ammonia…………. Reflux for 1 hr……………. Filter it…………………Dry and wash

Residue ……………. Mix in chloroform …………. Evaporate solvent……….. Solasodine , solid residue is

obtained.

METHOD. 2 Powdered drug + ethanol-------- Soxhlation 6 hrs.------------- Solvent distilled off……………

Concentrated to syrupy mass ---------Add 5 ml HCl , Boil …….. Reflux for 2 hr……………. Cool it &

Filter………… Residue + Boil water………. Adjust pH-9 by NH 3 (10%) …………. Boil under reflux for

2 hrs ……… Cool & Filter…..Dry Ppt ……….. Solasodine , solid residue is obtained.

Atropine

The final problem in the synthesis, the combination of tropine and tropic acid, was overcome by a

Fischer-Speier esterification [13]. The acid and alcohol were heated together in the presence of HCl to

yield atropine



The biosynthesis of atropine starting from L-Phenylalanine first undergoes

a transamination forming phenylpyruvic acid which is then reduced to phenyl-lactic acid.[14]

Coenzyme A

then couples phenyl-lactic acid with tropine forming littorine, which then undergoes a radical rearrangement

initiated with aP450 enzyme forming hyoscyamine aldehyde.[14]

A dehydrogenase then reduces the aldehyde

to a primary alcohol making (-)-hyoscamine, which upon racemization forms atropine.

Atropine is a naturally occurring tropane alkaloid extracted from deadly nightshade (Atropa belladonna),

Jimson weed (Datura stramonium),mandrake (Mandragora officinarum) and other plants of the

family Solanaceae. It is a secondary metabolite of these plants and serves as a drugwith a wide variety of

effects.

http://en.wikipedia.org/wiki/Phenylalanine

http://en.wikipedia.org/wiki/Transamination

http://en.wikipedia.org/wiki/Phenylpyruvic_acid

http://en.wikipedia.org/wiki/Atropine#cite_note-dewick-14

http://en.wikipedia.org/wiki/Tropine

http://en.wikipedia.org/wiki/Littorine

http://en.wikipedia.org/wiki/P450

http://en.wikipedia.org/wiki/Atropine#cite_note-dewick-14

http://en.wikipedia.org/wiki/Dehydrogenase

http://en.wikipedia.org/wiki/Tropane_alkaloid

http://en.wikipedia.org/wiki/Atropa_belladonna

http://en.wikipedia.org/wiki/Datura_stramonium

http://en.wikipedia.org/wiki/Mandrake_(plant)

http://en.wikipedia.org/wiki/Mandragora_officinarum

http://en.wikipedia.org/wiki/Solanaceae

http://en.wikipedia.org/wiki/Secondary_metabolite

http://en.wikipedia.org/wiki/Drug



In general, atropine counters the "rest and digest" activity of glands regulated by the parasympathetic

nervous system. This occurs because atropine is a competitive antagonist of the muscarinic acetylcholine

receptors (acetylcholine being the main neurotransmitter used by the parasympathetic nervous system).

Atropine dilates the pupils, increases heart rate, and reduces salivation and other secretions.

Chemistry[edit]

Ergometrine, 1-hydroxymethylethylamide lysergic acid, is synthesized by esterification of D-lysergic

acid using 2-aminopropanol indimethylformamide and direct treatment of the reaction mixture

with phosgene.[5]

http://en.wikipedia.org/wiki/Gland

http://en.wikipedia.org/wiki/Parasympathetic_nervous_system



http://en.wikipedia.org/wiki/Competitive_antagonist

http://en.wikipedia.org/wiki/Muscarinic_acetylcholine_receptor



http://en.wikipedia.org/wiki/Acetylcholine

http://en.wikipedia.org/wiki/Neurotransmitter

http://en.wikipedia.org/w/index.php?title=Ergometrine&action=edit&section=6

http://en.wikipedia.org/wiki/Lysergic_acid



http://en.wikipedia.org/wiki/Dimethylformamide

http://en.wikipedia.org/wiki/Phosgene

http://en.wikipedia.org/wiki/Ergometrine#cite_note-5

http://en.wikipedia.org/wiki/File:Ergonovine_synthesis.png





Diosgenin is steroidal sapogenin obtained from the tubers of Dioscorea species of the family Dioscoreacece.

Chemically is a steroidal sapogenin. Sole source for steroidal contraceptives, topical hormones, estrogens,

progestogen, androgen and sex hormone .

Chemical nature Diosgenin is hydrolytic product of saponin Dioscin . Saponins – plant constituent which

bring about frothing in an aqueous solution. Historically used for their detergent properties. Properties:

Frothing property : hydrophobic large molecules (C 27 - 30 ) glycone (hydrophilic) makes the molecule

capable of lowering surface tension in water. Hemolytic property : destroys RBC by hemolysis (toxic to

cold-blooded animals - fish poison)

Methods of isolation:

Methods of isolation (1) Alcoholic extraction method :

Dioscorea tubers are cut into small pieces & dried under sun

↓

Dried tubers are powdered , extracted with ethanol / methanol , twice for 6-8 hrs

↓

filter & filterate is concentrated to a syrupy liquid

↓

the concentrated liq. Is then hydrolysed using an acid , HCl or H2SO4 for 2 -12 hrs

↓

85% of diosgenin is ppted ↓ Ppts are filtered , washed with water

↓

purification with alcohol

(2) Acid hydrolysis method

Dried rhizomes are powdered (20#) and first subjected to hydrolysis by refluxing with 5% HCl for 2 hours.

↓

The hydrolyzed mass is filtered, washed twice with water and then twice with 5% sodium bicarbonate

solution.

↓

It is finaly washed with water till the washing are neutral. The residue thus obtained is dried and futher

extracted with toluene for 8 hours.

↓

The toluene extract concentrated during which diosgenin gets precipitated.

↓

Diosgenin filtered, washed with little hexane and dried(40-60 o c) to yield about 95% pure product.

Identification tests Thin Layer Chromatography:

Identification tests Thin Layer Chromatography Stationary phase : silica gel G Solvent system : Toluene

:Ethyl acetate (7:3) Spraying reagent : Anisaldehyde in sulfuric acid Standard solution : Dissolve Std

.diosgenin 1mg in 1 ml chloroform. Test solution : Dissolve residue obtained through isolation in

chloroform Rf ( for diosgenin ) : 0.62 14

Chemical Tests:

Chemical Tests 1.Libermann-Burchard test : Treat the extract with few drops of acetic anhydride , boil and

cool. Then add conc .sulphuric acid from the sides of test tube , brown ring is at the junction of two layers

&upper layer turns green (steroids) and formation of deep red colour (triterpenoids)



2. Libermann „s reaction : mix 3 ml extract + 3 ml acetic anhydride heat & cool add few drops of conc

H2SO4 Blue color obtained 15 Nikita modi L.M.college of pharmacy

3.Salkowski test : 2ml extract + 2ml CHCl3 +2ml conc H2SO4 CHCl3 layer appears red and acid layer

shows greenish yellow florescence 4.Sulfur powder test : Add small amount of sulfur powder to the test

solution , it sinks at the bottom. Steroid present.

PRODUCTION AND UTILIZATION OF podophyllotoxin

It is obtained from the dried rhizomes and root of Podophyllum hexandrum Family: Berberidaceae

ISOLATION OF PODOPHYLLOTOXIN:

ISOLATION OF PODOPHYLLOTOXIN DRIED RHIZOME POWDER------------- EXTRACT WITH

ETHANOL----------SOXHLATION----------------- DISTILLATION------------------ CONCENTRATE TO

SYRUPY MASS ---------------------- ADD (HCL + H 2 O)--------------- COOL AT 5 0 C---------------

ALLOWED TO STAND FOR 2 HRS--------------- FILTER UNDER VACUUM--------------------WASH

RESIDUE WITH ACIDIFIED WATER------------ COOL BELOW 5 0 C------------- RESIDUE + HOT

ALCOHOL (90%)-------------- FILTER & EVAPORATE------------------ DRY RESIDUE TO CONSTANT

WEIGHT AT 80 0 C.

STRUCTURE OF SOLASODINE

Solasidine is obtained from the whole plant . Solanum xanthocarpum And dried full growth berries of

Solanum khasianum Family:Solanaceae



ISOLATION OF SOLSODINE BY TWO METHODS METHOD 1 B. METHOD 2

METHOD. 1 Dried berries is powdered-------- Oil is removed------------- Defatted is extracted with ethanol--

------------------- Resultant is filtered , Concentrated & Treat with HCl & Reflux ---------------- Extract is

made alkaline by ammonia…………. Reflux for 1 hr……………. Filter it…………………Dry and wash

Residue ……………. Mix in chloroform …………. Evaporate solvent……….. Solasodine , solid residue is

obtained.

METHOD. 2 Powdered drug + ethanol-------- Soxhlation 6 hrs.------------- Solvent distilled off……………

Concentrated to syrupy mass ---------Add 5 ml HCl , Boil …….. Reflux for 2 hr……………. Cool it &

Filter………… Residue + Boil water………. Adjust pH-9 by NH 3 (10%) …………. Boil under reflux for

2 hrs ……… Cool & Filter…..Dry Ppt ……….. Solasodine , solid residue is obtained.

UTILIZATION OF SOLASODINE Used as a precursor for steroidal synthesis. It is first converted to 16-

dehydropregnalone acetate which acts as a precursor for steroidal synthesis like Corticosteroids, Pregnane

Used in synthesis of Sex hormones and Oral contraceptives. Shows Antispermatogenic Activity Used as

Hypocholestremic Agent Used as Antiatherosclerotic Agent .

Quinine

Biological Sources It is obtained from the bark of Cinchona calisaya Wedd; Cinchona officinalis Linn.

belonging to family Rubiaceae.

Thalleioquin Test: Add to 2-3 ml of a weakly acidic solution of a quinine salt a few drops of bromine-

water followed by 0.5 ml of strong ammonia solution, a distinct and characteristic emerald green colour is

produced. The coloured product is termed as thalleioquin, the chemical composition of which is yet to be

established. This test is so sensitive that quinine may be detected to a concentration as low as 0.005%.

Basic Structures of Cinchona Alkaloids The various quinoline alkaloids, which possess potent medicinal

activities are, namely: quinine, quinidine, cinchonine, and cinchonidine.







Identification test1.(Vitali – Morin reaction): - Alkaloid/ atropine (1µg) + Drop of H SO 2 4 Evaporate to

dryness Indicates Which produce Add 0.3ml of 3% solution of presence of atropine bright purple

color KOH in methyl alcohol2. On addition of AgNO3 solution to solution Yellowish of hyoscine



hydrobromide white ppt Insoluble – HNO3 Soluble – Dil. NH3

T.B.E.K.B

TLC• 1% solution of atropine dissolved in 2N acetic acid isspotted over silica gel G plate and eluted in the

solventsystem of strong NH3 solution – methanol (! : 5 : 100).• TLC plate is spread with an acidified

iodoplatinate solution.•Rf – 0.18.•Solvent system – Acetone – 0.5 sosium chloride solution.•Spraying

reagents – Dragondroff‟s reagent.

Chemistry & Properties•Melting point – 115oC to 116oC.•Molecular formula – C17H23NO3.

Identification test for Citric Acid:

Add a few milligrams of your substance to a solution containing 15mL of pyridine and 5mL of acetic

anhydride. If citric acid is present, a bright red color is produced.

Manufacturing Process & Technology:

The leaves of the "Mentha Arvenisis" are subjected to steam distillation, the distillation products are

condensed and separated into peppermint oil and water. The crude mint oil then obtained is refined by

vacuum filteration and then chilled to about 5-10 degree C to obtain Menthol Crystals. The crystals, thus

formed are centrifuged and obtain about 45% yield of menthol. The spent oil is treated with sodium

hydroxide and Boric Acid while crystalline borate esters which are formed are separated and decomposed

by steam. The Menthol thus released is recovered by crystallisation under reduced temperatures and

centrifuging. The mother liquor is distilled to obtain dementholised peppermint oil. The overall yield of

menthol is about 50% and an equal amount of dementhonised oil is obtained as co-product.

Another commercial process is the Haarmann-Reimer process. This process starts from m-cresol which is

alkylated with propene to thymol. This compound is hydrogenatedin the next step. Racemic menthol is

isolated by fractional distillation. The enantiomers are separated by chiral resolution in reaction with methyl

benzoate, selective crystallisation followed by hydrolysis. Racemic menthol can also be formed by

hydrogenation of pulegone.

Chemical Tests

1. When 10 mg crystals menthol are first dissolved in 4 drops of concentrated sulphuric acid and then a

few drops of vanillin sulphuric acid reagent are added it shows an orange yellow colouration that ultimately

changes to violet on the addition of a few drops of water.

http://en.wikipedia.org/wiki/M-cresol

http://en.wikipedia.org/wiki/Propene

http://en.wikipedia.org/wiki/Thymol

http://en.wikipedia.org/wiki/Hydrogenation

http://en.wikipedia.org/wiki/Fractional_distillation

http://en.wikipedia.org/wiki/Chiral_resolution

http://en.wikipedia.org/wiki/Methyl_benzoate



http://en.wikipedia.org/wiki/Racemic

http://en.wikipedia.org/wiki/Pulegone



Definition of Electrophoresis

Electrophoresis is a separations technique that is based on the the mobility of ions in an electric field.

Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a

positive electrode.For safety reasons one electrode is usually at ground and the other is biased positively or

negatively. Ions have different migrationrates depending on their total charge, size, and shape, and can

therefore be separated. Instrumentation An electrode apparatus consists of a high-voltage supply, electrodes,

buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or

a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually

used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the

support is stained to visualize the separated components.

Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains

a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At

this pH the protein is netural and no longer migrates, i.e, it is focused into a sharp band on the gel.

Schematic of zone electrophoresis apparatus

Specific electrophoretic techniques

disc electrophoresis

capillary electrophoresis

gel electrophoresis (SDS-PAGE)

http://www.chemicool.com/definition/electrophoresis.html


http://www.chemicool.com/definition/field.html

http://www.chemicool.com/definition/migration.html

http://www.chemicool.com/definition/buffer.html

http://www.chemicool.com/definition/cellulose.html

http://www.chemicool.com/definition/capillary.html

http://www.chemicool.com/definition/tube.html

http://www.chemicool.com/definition/capillary.html

http://www.chemicool.com/definition/dna.html

http://www.chemicool.com/definition/resolution.html

http://www.chemicool.com/definition/isoelectric.html

http://www.chemicool.com/definition/gel.html

http://www.chemicool.com/definition/ph.html


http://www.chemicool.com/definition/isoelectric.html


http://www.chemicool.com/definition/band.html

http://www.chemicool.com/definition/gel.html





BIOSYNTHESIS OF ANTI BIOTICS:





EXPORT POTENTIAL



PREPARATION OF ALLERGENIC EXTRACT: · Grinding

· Defatting

· Extraction

· Clarification

· Dialysis

· Concentration

· Sterilization

· Lypholization

· Testing

· Standardization

· Storage

GRINDING: The material to be extracted must be ground or subdivided in order to effect efficient extraction of the

allergens. Household blenders or small plant mills can be used for dried materials, while juicers or food

grinders can be used for those containing much moisture. Materials such as hairs, feathers and textiles

should be divided finely with shears.

DEFATTING:



Many allergenic substances, including all pollens, should be defatted before final extraction. Ether and

petroleum ether are used most commonly for this purpose but alcohols occasionally may be included in the

menstruum. Defatting provides a clearer final extract and also removes irritants found in large amounts in

some substances, e.g. Coffee, tea, cottonseed, pepper, mustard, ginger. The extract obtained in the defatting

process may be used in the preparation of some patch-testing substances.

EXTRACTION: The extraction procedures in current use are based upon the assumption that allergens are water-soluble

proteins or glycoproteins although the identity of only a few is known. Extraction is carried out normally for

24 to 72 hours in a cold room using sterile, pyrogen-free buffered saline, coca's solution or similar aqueous

menstruum of pH 8.

Buffered Saline

Sodium chloride 5 gm

Monobasic potassium phosphate 0.36 gm

Dibasic sodium phosphate, anhydrous 7 gm

Phenol crystals 4 gm

Water for injection USP, to make 1000 mL

Coca‟s Solution

Sodium chloride 5 gm

Phenol crystals 5 gm

Sodium bicarbonate 2.5 gm

Water for injection USP, to make 1000 mL

CLARIFICATION: After extraction the mixture is clarified by coarse filtration.

DIALYSIS: Some extracts are dialyzed against saline or running tap water to remove irritants or coloring agents. Most

pollens require no dialysis but some substances (e.g. house dust, mustard, potato, spinach, beets) give nearly

universally positive reactions unless dialyzed.

CONCENTRATION: Concentration of the extract, where required, may be achieved by a number of methods but care should be

taken not to alter the allergens.

STERILIZATION: The processed extract is sterilized by filtration, usually through a cellulose membrane filter. Prefilters

usually are required but asbestos should not be used since it may adsorb some immunogens and may be

carcinogenic.

LYPHOLIZATION: Freeze-dried pollen extracts are prepared essentially as described above except that water rather than

electrolyte solution is used as the extracting medium. The lyophilized products are reconstituted with

buffered saline at time of use.

TESTING:



The extracts are thermolabile and must be sterilized by filtration, and sterility tests for both aerobic and

anaerobic microorganisms must be performed on the finished products. Toxicity testing usually is

performed in guinea pigs and recommended particularly for autogenous extracts where unknown toxic

constituents may be present. Recent concerns for possible mycotoxin contaminants in mold extracts or from

mold contamination of other substances have resulted in more intensive efforts to detect and eliminate these

toxins.

STANDARDIZATION: Most allergenic extracts carry the statement “No US standard of potency”. Although the first standardized

allergenic extract was licensed in 1982 and there has been much progress realized in this area, there are still

no completely satisfactory means of assaying allergenic extracts and expressing their potency.

The two most common measures of allergenic potency are by weight/volume (w/v) and the protein nitrogen

unit (PNU). Weight/volume is the weight of allergenic substances extracted per volume of extracting fluid.

For example, a 1:50 extract is prepared by extracting 1 g of substance with 50 mL of solvent and decimal

dilutions of this extract provide 1:500, 1:5000, etc concentrations. The protein nitrogen units also are listed

often along with the w/v concentration on commercial products and 1 mg of protein nitrogen equals 100,000

PNU. The allergenic protein is virtually always a small and variable part of the total protein, and neither the

PNU nor weight/volume standards correlate consistently with each other or clinical potency.

Units of Potency for Allergenic Extracts

Unit Description Used

Weight/volume (w/v) Allergen (g) per volume

(mL) of extracting fluid Worldwide

Protein Nitrogen Unit (PNU) 1 mg protein N = 100,000

PNU Worldwide

Allergy Unit (AU) Skin testing to endpoint US

Biological Unit (BU) Skin testing relative to

histamine Europe

Three general methods are used to estimate potency better in the preparation of standardized allergenic

extracts.

Specific allergens in an extract are compared to those in the reference standard by immunoelectrophoresis.

Two systems of bioassay based upon skin testing in patients sensitive to the particular extract are used

presently o establish the potency of a reference standard. The Nordic system is used in Europe and potency

is expressed in biological unit (BU). The American system adopted by FDA expresses potency in terms of

allergy unit (AU).

Radioallergosorbent-inhibition (RAST-inhibiton) tests are used widely to evaluate allergenic extracts.

RAST-inhibition (not skin testing) is the main methods of comparing different batches of standardized

allergenic extracts with reference standards.

Standard extracts represent a major improvement in allergenic extracts and, in general, probably are more

potent than the conventional extracts. However, a standardized extract can be either more or less potent than

the corresponding w/v extract and the two should never be used interchangeably. The same general

principles of administration apply to both standardized and conventional extracts.

Because allergenic extracts are not standardized completely the appropriate dosage for immunotherapy must

be determined clinically. The initial dilution of extract, starting dose and progression of dosage must be

determined clinically. The initial dilution of extract, starting dose and progression of dosage must be

determined carefully on the basis of the patient‟s history and sensitivity tests. Because dilute extracts tend to

lose activity more rapidly, the first dose from a more concentrated vial generally should be the same or less

than the previous dose. Also, it is common to reduce the dose whenever a new lot of extract is started and

then build the dose back to the maintenance level over a period of several weeks.



STORAGE: Allergenic extracts tend to show reduced potency within a matter of weeks or months after their preparation,

but there have been few detailed studies on the stability of these products. Both high temperatures and

freezing usually have deleterious effects, and the latter may cause agglomeration of adjuvant extracts. Some

extracts also contain proteolytic enzymes and these may contribute to decomposition of the allergens. Both

glycerinated and lyophilized products are more stable than aqueous extracts. Very dilute extracts tend to

lose potency by adsorption to the surfaces of containers and syringes and thus usually are prepared close to

the time of use. Several studies have shown that the inclusion of Tween 80, Teen 20 or human serum

albumin reduces or adsorption but a more-complete investigation of this problem is required. The adjuvant

extracts should not be diluted with either phosphate buffered saline or Coca‟s solution since these may cause

partial release of allergen; normal saline containing 0.4% phenol is a satisfactory diluents. The adjuvant

extracts may be mixed with one another but should not be mixed with other types of extracts.

All allergenic extracts should be refrigerated at 2 to 8 ̊ and freezing should be avoided. The expiration date

for aqueous extracts is usually 18 months, while for glycerinated scratch test and bulk extracts is usually 3

years. Lyophilized products have an expiration date of 4 years or 18 months after reconstitution, so long as

the time falls within the original 4 years. Care must be exercised in changing to new lots or different

dilutions of extracts because of possible variations in potency. It generally is recommended that quantities of

extract sufficient to last the patient for 1 year be prepared to avoid frequent changes in extracts.

Classification of allergens in plants

1.1 Inhalent allergens Inhalent allergens from grass or tree pollens, house dust mite and animal dander are the major

substances that are capable of provoking type I hyperresponsiveness. Among those allergens, one of the

most common ones is pollen of plants[3]. An individual who has hypersensitivity to pollen often suffers

from seasonal allergic rhinitis or extrinsic asthma. Weeds, grasses and trees are common sources of pollen,

and high concentrations of these pollen allergens in the air surrounding us correspond well to pollen-related

hypersensitivity disease. The major and most widespread allergenic components of pollen is the group I

allergens. Thus, the allergy caused by these allergens is often termed "seasonal". These allergenic proteins

in pollens with molecular weight about 30 kD are quickly and profusely released by grass pollen upon

hydration[4]. In recent years, research in this area has focused on the characterization of relevant grass

pollen allergens because as many as approximately 40% of allergic individuals start their symptoms

immediately after contacting with grass pollens[5].

1.2 Ingestent allergens Ingestent allergens often refer to substances inducing allergy after the sensitized individuals eating a

certain food. Typical symptoms of this type allergy include mouth or throat itching and lip swelling. In

recent years, an increase in tree nut and peanut allergy has been reported in Europe and in US. For example,

peanut and/or tree nut allergy affect approximately 1.1% of US population, corresponding to 3 million

individuals at risk of adverse reaction to these foods[6]. Of these individuals, 50% considered in the survey

performed by Sicherer et al.[7] were reactive to peanut, 30% to walnut and 10% to almond, while only 4%

were reactive to both peanut and tree nut. In previous reports, the percentage of allergic individuals

symptomatically reactive to two or more nuts has been found to be nearly 10%, which corresponds to at

least half a million individuals in the world reactive to two related nuts. On the other hand, a recent study

reported that approximately 35% patients with pollen allergy were also sensitive to fresh fruits and

vegetables[8].

1.3 Contactent allergens Latex is the most important contactent allergens in plants. Since the late 1980's, this immediate-type

allergy provoked by natural rubber products has been reported around the world[9]. It is now known as latex

allergy. It also can be induced by wide-ranging latex products. In addition, allergy to exotic fruit is



frequently reported in studies on latex-allergic subjects. Subjects suffering from the latex-fruit syndrome

become primarily sensitized to latex and then develop food allergy as a result of cross-reacting IgE against

protein, such as in banana and avocado[10]. Nowadays, plant defense-related proteins induced by stress

were reported as a main kind of latex allergen.

2 The biological functions of allergens in plants

In last decade, with the implementation of molecular biological techniques in the field of allergen

characterization, the sequence, nature, and three-dimensional structure of several important allergens have

been revealed. Application of molecular cloning techniques also enable us to understand the natural

functions of the IgE-binding proteins in plants. There are at least three major biological functions for the

allergens in plants.

2.1 Calcium-binding protein In plant molecular biology, calcium in pollen is recognized as an essential constituent of in vitro pollen-

germination media and a potential chemoattractant guiding pollen growth. In 1999, Rozwadowski et al.[11]

characterized calcium-binding protein from Brassica and Arabidopsis pollen. By sequence comparison, the

protein was revealed as a part of a family of pollen allergens identified recently in several evolutionarily

distant dicot and monocot plants. The protein also has strong immunoreactivity to IgE from a human subject

allergic to Brassica pollen[12]. In addition, the members in the two EF-hand allergen families share an

average sequence identity of 77%, which is of comparable magnitude within and outside the calcium-

binding domain. In fact, several kinds of plant allergens with EF-hand calcium-binding domains have been

identified in birch[13], Bermuda grass[14] and rapeseed[12]. Calcium binding plant proteins have now been

discovered as relevant cross-reactive allergens, and the EF-hand domain is the major epitope for antibody

reorganization in those allergens[15].

2.2 Pathogenesis-related protein (PR protein) PR proteins which represent an important group of human allergens can be up-regulated in plants in

response to stressors such as freezing, drought, temperature, fungi, viruses or bacteria infection. So far,

several allergenic PR proteins have been biochemically characterized. They belong to different PR protein

groups (there are 10 groups of PR proteins in nature). For example, Jun a 3, the allergen in mountain cedar,

was found to be homologous to the PR-5 protein group. Plant allergens Bet v 1 (in birch), Mal d1 (in apple)

and Dau c 1 (in carrot) are members of PR protein 10 group[16]. Similarly, the major allergen in rubber Hev

b6[17] and its metabolic products, Bar r 2 (in turnip)[18] and Pers a 1 (in avocado)[19] have the properties

of chitinase, and belong to the PR protein 4 group. Investigation of potential common functions and

structures of PR-proteins will uncover some "law" of allergens in plants and will explain the reason for

cross-reaction phenomena in plant allergens.

2.3 Expansins A cell wall-loosening agent, is extracellular protein that promotes plant cell wall enlargement by

disrupting noncovalent bonding between cellulose microfibrils and matrix polymers[20]. When the first

expansin complementary DNA was sequenced, BLAST searches in GenBank revealed a distant sequence

similarity to a group of grass allergens called group-1 allergen. It was characterized further that group-1

allergens in plants were indeed structurally and functionally related to expansin, and that their vegetative

homologs comprise a second family of expansins, such as LolｐＩ(in ryegrass), Ory s I (in rice) and Zea m

I (in maize)[4]. But different from the original group of expansin, this group of expansin in pollens could

only induce extension in the cell walls of grass and was not effective on the walls from dicotyledons[21].

Recently, the cell wall-loosening agents in pollens have been named as β-expansin family, in order to

distinguish it from the original group of expansins, which are now called α-expansins.

In type I hyperresponsiveness, there are varieties of cross-reaction between allergens in different plants[22].

The common conservative domain and/or isotope among different allergens in plants are the radical cause of

these phenomena. Thus, research on identification and characterization of allergens and their structures and

biological functions will be benefit for the diagnosis and treatment of pollen related allergic diseases.

3 Progress in gene cloning and recombinant protein production of plant allergens



Allergen-specific immunotherapy (SIT) represents one of the few curative approaches toward type I

hyperresponsiveness[23]. But, there are three major problems associated with SIT: first, presently SIT is

performed with natural allergen extracts, containing mixtures of allergens, nonallergenic and/or toxic

proteins, and other macromolecules, which are hard to standardize. Second, systemic administration of

allergen can cause severe IgE-mediated side effects during the treatment on patients, and third,

therapeutically effective dose often cannot be achieved because of non-standardized extracts or side effects.

With the clarification of the nature, sequence and three-dimensional structure of several important

allergens, molecular level recognization of allergens and IgE antibodies will become available. To date,

cDNA sequence of 60 pollen allergens from 27 plant species have been deposited in the allergen databank

(www.allergen.com). Since pure and standardized recombinant allergens can be formulated to replace

natural extracts, using genetic engineered allergens for SIT become a possible and promising method for

immunotherapy. In last decade, a variety of recombinant allergens from plants, mites, molds, mammals and

insects have been expressed using various systems, such as E.coli[24], Pichia[25] and plants[26]. Moreover,

the recombinant allergens can be engineered to reduce the risk of the IgE-mediated side effects. The

molecules with reduced allergenicity (hypoallergen) would not lead to anaphylactic reaction upon injection

and would allow higher-dose administration of allergen, which has showed to be more effective in symptom

reduction than low dose. In this way, high dose of allergen can be administered to allergic patients, which

increases the efficacy of the treatment. Based on this consideration, site-directed mutant and comformation

has been applied in the recombination of hypoallergens[27, 28]. The clinical use of these products may lead

to not only improve diagnostic specificity and sensitivity but also safer and more effective immunotherapy.

4 Summary

As the most widespread species on the earth, plant is a part of the human normal life. It is hard to avoid

plant allergens from trees, grasses and weeds. Although specific immunotherapy represents a curative

approach toward allergy, the mechanism operating in SIT still remains not completely understood. In recent

statistics, there has been a significant increase in the prevalence of allergic disease over the past 2 to 3

decades. Currently, more than 130 million people suffer from the asthma and the numbers are

increasing[29]. There is a research considering that air pollutants from industry and automobiles are

cofactors contributing to recent increase in allergic disease and asthma[30]. On the other hand, man cannot

ignorance transgenic plants are widespread in the modern world, it could be the source for new kind of

allergens.

Hallucinogen

Hallucinogens are a general group of pharmacological agents that can be divided into three broad

categories: psychedelics, dissociatives, and deliriants. These classes of psychoactive drugs have in

common that they can cause subjective changes in perception, thought, emotion and consciousness. Unlike

other psychoactive drugs, such as stimulants and opioids, these drugs do not merely amplify familiar states

of mind, but rather induce experiences that are qualitatively different from those of ordinary consciousness.

These experiences are often compared to non-ordinary forms of consciousness such

as trance,meditation, dreams, or insanity.

L. E. Hollister's criteria for establishing that a drug is hallucinogenic is:

in proportion to other effects, changes in thought, perception, and mood should predominate;

intellectual or memory impairment should be minimal;

stupor, narcosis, or excessive stimulation should not be an integral effect;

autonomic nervous system side effects should be minimal; and

addictive craving should be absent.

Not all drugs produce the same effect and even the same drug can produce different effects in the same

individual on different occasions.

http://en.wikipedia.org/wiki/Pharmacology

http://en.wikipedia.org/wiki/Psychedelic_drug

http://en.wikipedia.org/wiki/Dissociative

http://en.wikipedia.org/wiki/Deliriant

http://en.wikipedia.org/wiki/Psychoactive_drugs

http://en.wikipedia.org/wiki/Perception

http://en.wikipedia.org/wiki/Thought

http://en.wikipedia.org/wiki/Emotion

http://en.wikipedia.org/wiki/Consciousness

http://en.wikipedia.org/wiki/Psychoactive_drug

http://en.wikipedia.org/wiki/Stimulants

http://en.wikipedia.org/wiki/Opioids

http://en.wikipedia.org/wiki/Trance

http://en.wikipedia.org/wiki/Meditation

http://en.wikipedia.org/wiki/Dreams

http://en.wikipedia.org/wiki/Insanity



Dissociatives

Dissociatives produce analgesia, amnesia and catalepsy at anesthetic doses.[10]

They also produce a sense of

detachment from the surrounding environment, hence "the state has been designated as dissociative

anesthesia since the patient truly seems disassociated from his environment."[11]

Dissociative symptoms

include the disruption or compartmentalization of "...the usually integrated functions of consciousness,

memory, identity or perception."[12]p. 523

Dissociation of sensory input can cause derealization, the

perception of the outside world as being dream-like or unreal. Other dissociative experiences

include depersonalization, which includes feeling detached from one's body; feeling unreal; feeling able to

observe one's actions but not actively take control; being unable to recognize one's self in the mirror while

maintaining rational awareness that the image in the mirror is the same person.[13][14][15]

Simeon (2004)

offered "...common descriptions of depersonalisation experiences: watching oneself from a distance (similar

to watching a movie); candid out-of-body experiences; a sense of just going through the motions; one part

of the self acting/participating while the other part is observing;...."[16]

The primary dissociatives achieve their effect through blocking the signals received by the NMDA

receptor set (NMDA receptor antagonism) and

include ketamine, phencyclidine(PCP), dextromethorphan (DXM), and nitrous oxide.[17][18][19]

However,

dissociation is also remarkably administered by salvinorin A's (the active constituent in Salvia

divinorumshown to the left) potent κ-opioid receptor agonism[20]

and is notably the most potent

psychoactive chemical harnessed directly from the plant kingdom.[citation needed]

Some dissociatives can have CNS depressant effects, thereby carrying similar risks as opioids, which can

slow breathing or heart rate to levels resulting in death (when using very high doses). DXM in higher doses

can increase heart rate and blood pressure and still depress respiration. Inversely, PCP can have more

unpredictable effects and has often been classified as a stimulant and a depressant in some texts along with

being as a dissociative. While many have reported that they "feel no pain" while under the effects of PCP,

DXM and Ketamine, this does not fall under the usual classification of anesthetics in recreational doses

(anesthetic doses of DXM may be dangerous). Rather, true to their name, they process pain as a kind of "far

away" sensation; pain, although present, becomes a disembodied experience and there is much less emotion

associated with it. As for probably the most common dissociative, nitrous oxide, the principal risk seems to

be due to oxygen deprivation. Injury from falling is also a danger, as nitrous oxide may cause sudden loss of

consciousness, an effect of oxygen deprivation. Because of the high level of physical activity and relative

imperviousness to pain induced by PCP, some deaths have been reported due to the release of myoglobin

from ruptured muscle cells. High amounts of myoglobin can induce renal shutdown.[21]

Along with most, if

not all of the chemicals in this article, none of the dissociatives have any physically addictive properties,

though psychological addiction has been observed.

Many users of dissociatives have been concerned about the possibility of NMDA antagonist neurotoxicity

(NAN). This concern is partly due to William E. White, the author of the DXM FAQ, who claimed that

dissociatives definitely cause brain damage.[22]

The argument was criticized on the basis of lack of

evidence[23]

and White retracted his claim.[24]

White's claims and the ensuing criticism surrounded original

research by John Olney.

In 1989, John Olney discovered that neuronal vacuolation and other cytotoxic changes ("lesions") occurred

in brains of rats administered NMDA antagonists, including PCP and ketamine.[25]

Repeated doses of

NMDA antagonists led to cellular tolerance and hence continuous exposure to NMDA antagonists did not

lead to cumulative neurotoxic effects. Antihistamines such as diphenhydramine, barbiturates and even

diazepam have been found to prevent NAN.[26]

LSD and DOB have also been found to prevent NAN.[27]

http://en.wikipedia.org/wiki/Hallucinogen#cite_note-10



http://en.wikipedia.org/wiki/Derealization

http://en.wikipedia.org/wiki/Depersonalization






http://en.wikipedia.org/wiki/Out-of-body_experience


http://en.wikipedia.org/wiki/NMDA_receptor



http://en.wikipedia.org/wiki/NMDA_receptor_antagonist

http://en.wikipedia.org/wiki/Ketamine

http://en.wikipedia.org/wiki/Phencyclidine

http://en.wikipedia.org/wiki/Dextromethorphan

http://en.wikipedia.org/wiki/Nitrous_oxide






http://en.wikipedia.org/wiki/Salvinorin_A

http://en.wikipedia.org/wiki/Salvia_divinorum



http://en.wikipedia.org/wiki/Kappa_Opioid_receptor


http://en.wikipedia.org/wiki/Wikipedia:Citation_needed

http://en.wikipedia.org/wiki/Central_nervous_system

http://en.wikipedia.org/wiki/Depressant

http://en.wikipedia.org/wiki/Opioids

http://en.wikipedia.org/wiki/Nitrous_oxide

http://en.wikipedia.org/wiki/Hypoxia_(medical)

http://en.wikipedia.org/wiki/Kidney


http://en.wikipedia.org/wiki/DXM_FAQ




http://en.wikipedia.org/wiki/John_Olney

http://en.wikipedia.org/wiki/John_Olney

http://en.wikipedia.org/wiki/Phencyclidine

http://en.wikipedia.org/wiki/Ketamine



http://en.wikipedia.org/wiki/Lysergic_acid_diethylamide

http://en.wikipedia.org/wiki/2,5-Dimethoxy-4-bromoamphetamine




Deliriants

Deliriants, as their name implies, induce a state of delirium in the user, characterized by extreme confusion

and an inability to control one's actions. They are called deliriants because their subjective effects are

similar to the experiences of people with delirious fevers.

Included in this group are such plants as Atropa belladonna (deadly nightshade), Brugmansia species

(Angel's Trumpet), Datura stramonium (Jimson weed), Hyoscyamus niger(henbane), Mandragora

officinarum (mandrake), and Myristica fragrans (nutmeg), as well as a number of pharmaceutical drugs,

when taken in very high doses, such asdiphenhydramine (Benadryl) and its close

relative dimenhydrinate (Dramamine). Uncured tobacco is also a deliriant due to its intoxicatingly high

levels of nicotine.[28]

In addition to the dangers of being far more disconnected from reality than with other drugs and retaining a

truly fragmented dissociation from regular consciousness without being immobilized, the anticholinergics

are toxic, carry the risk of death by overdose, and also include a number of uncomfortable side effects.

These side effects usually includedehydration and mydriasis (dilation of the pupils).

Most modern-day psychonauts who use deliriants report similar or identical hallucinations and challenges.

For example, diphenhydramine, as well as dimenhydrinate, when taken in a high enough dosage, often are

reported to evoke vivid, dark, and entity-like hallucinations, peripheral disturbances, feelings of being alone

but simultaneously of being watched, and hallucinations of real things ceasing to exist. Deliriants also may

cause confusion or even rage, and thus have been used by ancient peoples as a stimulant before going into

battle.

Traditional use

Psychedelics have a long history of traditional use in medicine and religion, where they are prized for their

perceived ability to promote physical and mental healing. In this context, they are often known

asentheogens. Native American practitioners using mescaline-containing cacti (most notably peyote, San

Pedro, and Peruvian torch) have reported success against alcoholism, and Mazatec practitioners routinely

use psilocybin mushrooms for divination and healing. Ayahuasca, which contains the powerful

psychedelic DMT, is used in Peru and other parts of South America for spiritual and physical healing as

well as in religious festivals.

Taxonomy

Hallucinogens can be classified by their subjective effects, mechanisms of action, and chemical structure.

These classifications often correlate to some extent. In this article, they are classified

as psychedelics,dissociatives, and deliriants, preferably entirely to the exclusion of the inaccurate word

hallucinogen, but the reader is well advised to consider that this particular classification is not universally

accepted. The taxonomy used here attempts to blend these three approaches in order to provide as clear and

accessible an overview as possible.

Almost all hallucinogens contain nitrogen and are therefore classified as alkaloids. THC and salvinorin

A are exceptions. Many hallucinogens have chemical structures similar to those of human neurotransmitters,

such as serotonin, and temporarily modify the action of neurotransmitters and/or receptor sites.

http://en.wikipedia.org/wiki/Delirium

http://en.wikipedia.org/wiki/Atropa_belladonna

http://en.wikipedia.org/wiki/Brugmansia

http://en.wikipedia.org/wiki/Datura_stramonium

http://en.wikipedia.org/wiki/Hyoscyamus_niger




http://en.wikipedia.org/wiki/Nutmeg

http://en.wikipedia.org/wiki/Diphenhydramine

http://en.wikipedia.org/wiki/Dimenhydrinate

http://en.wikipedia.org/wiki/Curing_of_tobacco

http://en.wikipedia.org/wiki/Nicotine


http://en.wikipedia.org/wiki/Dehydration

http://en.wikipedia.org/wiki/Mydriasis

http://en.wikipedia.org/wiki/Psychonaut

http://en.wikipedia.org/wiki/Entheogens

http://en.wikipedia.org/wiki/Indigenous_peoples_of_the_Americas

http://en.wikipedia.org/wiki/List_of_psychedelic_plants#Mescaline

http://en.wikipedia.org/wiki/Peyote

http://en.wikipedia.org/wiki/Echinopsis_pachanoi



http://en.wikipedia.org/wiki/Echinopsis_peruviana

http://en.wikipedia.org/wiki/Alcoholism

http://en.wikipedia.org/wiki/Mazatec_people

http://en.wikipedia.org/wiki/Psilocybin_mushroom

http://en.wikipedia.org/wiki/Ayahuasca

http://en.wikipedia.org/wiki/Dimethyltryptamine

http://en.wikipedia.org/wiki/Psychedelic

http://en.wikipedia.org/wiki/Dissociative

http://en.wikipedia.org/wiki/Deliriant

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Alkaloids

http://en.wikipedia.org/wiki/Tetrahydrocannabinol




http://en.wikipedia.org/wiki/Serotonin







Causes:

Causes of teratogenesis can broadly be classified as:

Toxic substances, such as, for humans, drugs in pregnancy and environmental toxins in pregnancy.

Vertically transmitted infection

Lack of nutrients. For example, lack of folic acid in the nutrition in pregnancy for humans can result

in spina bifida.

Physical restraint. An example is Potter syndrome due to oligohydramnios in humans.

Genetic disorders

PLANT TOXIC PART SYMPTOMS

HOUSE PLANTS

Hyacinth, Narcissus,

Daffodil Bulbs Nausea, vomiting, diarrhea. May be fatal.

Oleander Leaves, branches Extremely poisonous. Affects the heart, produces

severe digestive upset and has caused death.

Dieffenbachia (Dumb

Cane), Elephant Ear All parts

Intense burning and irritation of the mouth and

tongue. Death can occur if base of the tongue swells

enough to block the air passage of the throat.

Rosary Pea, Castor

Bean Seeds

Fatal. A single Rosary Pea seed has caused death.

One or two Castor Bean seeds are near the lethal dose

for adults.

FLOWER GARDEN PLANTS

Larkspur Young plant, seeds Digestive upset, nervous excitement, depression. May

be fatal.

Monkshood Fleshy roots Digestive upset and nervous excitement.

Autumn Crocus, Star of

Bethlehem Bulbs Vomiting and nervous excitement.

Lily-of-the-Valley Leaves, flowers Irregular heart beat and pulse, usually accompanied

by digestive upset and mental confusion.

Iris Underground stems Severe-but not usually serious-digestive upset.

Foxglove Leaves

Large amounts cause dangerously irregular heartbeat

and pulse, usually digestive upset and mental

confusion. May be fatal.

Bleeding Heart Foliage, roots May be poisonous in large amounts. Has proved fatal

to cattle.

VEGETABLE GARDEN PLANTS

Rhubarb Leaf blade Fatal. Large amounts of raw or cooked leaves can

cause convulsions, coma, followed rapidly by death.

ORNAMENTAL PLANTS

Daphne Berries Fatal. A few berries can kill a child.

http://en.wikipedia.org/wiki/Drugs_in_pregnancy

http://en.wikipedia.org/wiki/Environmental_toxins_and_fetal_development

http://en.wikipedia.org/wiki/Vertically_transmitted_infection

http://en.wikipedia.org/wiki/Nutrition_in_pregnancy

http://en.wikipedia.org/wiki/Spina_bifida

http://en.wikipedia.org/wiki/Potter_syndrome

http://en.wikipedia.org/wiki/Oligohydramnios

http://en.wikipedia.org/wiki/Genetic_disorder



Wisteria Seeds, pods Mild to severe digestive upset. Many children are

poisoned by this plant.

Golden Chain

Bean-like capsules in

which the seeds are

suspended

Severe poisoning. Excitement, staggering,

convulsions and coma. May be fatal.

Laurels,

Rhododendrons,

Azaleas

All parts Fatal. Produces nausea and vomiting, depression,

difficult breathing, prostration and coma.

Jasmine Berries Fatal. Digestive disturbance and nervous symptoms.

Lantana Camara (Red

Sage) Green berries

Fatal. Affects lungs, kidneys, heart and nervous

system. Grows in the southern U.S. And in moderate

climates.

Yew Berries, foliage Fatal. Foliage more toxic than berries. Death is

usually sudden without warning symptoms.

TREES AND SHRUBS

Wild and cultivated

cherries Twigs, foliage

Fatal. Contains a compound that releases cyanide

when eaten. Gasping, excitement and prostration are

common symptoms.

Oaks Foliage, acorns

Affects kidneys gradually. Symptoms appear only

after several days or weeks. Takes a large amount for

poisoning.

Elderberry All parts, especially

roots

Children have been poisoned by using pieces of the

pithy stems for blowguns. Nausea and digestive

upset.

Black Locust Bark, sprouts, foliage Children have suffered nausea, weakness and

depression after chewing the bark and seeds.

PLANTS IN WOODED AREAS

Jack-in-the-Pulpit All parts, especially

roots

Like Dumb Cane, contains small needle-like crystals

of calcium oxalate that cause intense irritation and

burning of the mouth and tongue.

Moonseed Berries Blue, purple color, resembling wild grapes. May be

fatal.

Mayapple Apple, foliage, roots

Contains at least 16 active toxic principles, primarily

in the roots. Children often eat the apple with no ill

effects, but several apples may cause diarrhea.

Mistletoe Berries Fatal. Both children and adults have died from eating

the berries.

PLANTS IN SWAMP OR MOIST AREAS

Water Hemlock All parts Fatal. Violent and painful convulsions. A number of

people have died from hemlock.



PLANTS IN FIELDS

Buttercups All parts Irritant juices may severely injure the digestive

system.

Nightshade All parts, especially the

unripened berry

Fatal. Intense digestive disturbance and nervous

symptoms.

Poison Hemlock All parts Fatal. Resembles a large wild carrot.

Jimson Weed (Thorn

Apple) All parts

Abnormal thirst, distorted sight, delirium,

incoherence and coma. Common cause of poisoning.

Has proved fatal.

First Aid

Workers who have come in contact with poisonous plants should:

Immediately rinse skin with rubbing alcohol, specialized poison plant washes, degreasing soap (such

as dishwashing soap) or detergent, and lots of water.

o Rinse frequently so that wash solutions do not dry on the skin and further spread the urushiol.

Scrub under nails with a brush.

Apply wet compresses, calamine lotion, or hydrocortisone cream to the skin to reduce itching and

blistering.

o Follow the directions on any creams and lotions. Do not apply to broken skin, such as open

blisters.

o Oatmeal baths may relieve itching.

An antihistamine such as diphenhydramine (Benadryl) can be taken to help relieve itching.

o Follow directions on the package.

o Drowsiness may occur.

o If children come in contact with work clothing contaminated with urushiol, a pediatrician

should be contacted to determine appropriate dosage.

In severe cases or if the rash is on the face or genitals, seek professional medical attention.

Treatment

Severe allergic reactions (anaphylaxis) need to be treated with a medicine called epinephrine, which can be

life saving when given right away. If you use epinephrine, call 911 and go straight to the hospital.

The best way to reduce symptoms is to avoid what causes your allergies. This is especially important for

food and drug allergies.

There are several types of medications to prevent and treat allergies. Which medicine your doctor

recommends depends on the type and severity of your symptoms, your age, and overall health.

Illnesses that are caused by allergies (such as asthma, hay fever, and eczema) may need other treatments.

Medications that can be used to treat allergies include:

ANTIHISTAMINES



Antihistamines are available over-the-counter and by prescription. They are available in many forms,

including:

Capsules and pills

Eye drops

Injection

Liquid

Nasal spray

CORTICOSTEROIDS

Anti-inflammatory medications (corticosteroids) are available in many forms, including:

Creams and ointment for the skin

Eye drops

Nasal spray

Lung inhaler

Patients with severe allergic symptoms may be prescribed corticosteroid pills or injections for short periods

of time.

DECONGESTANTS

Decongestants can help relieve a stuffy nose. Do not use decongestant nasal spray for more than several

days, because they can cause a "rebound" effect and make the congestion worse. Decongestants in pill form

do not cause this problem. People with high blood pressure, heart problems, or prostate enlargement should

use decongestants with caution.

OTHER MEDICINES

Leukotriene inhibitors are medicines that block the substances that trigger allergies. Zafirlukast (Accolate)

and montelukast (Singulair) are approved for people with asthma and indoor and outdoor allergies.

ALLERGY SHOTS

Allergy shots (immunotherapy) are sometimes recommended if you cannot avoid the allergen and your

symptoms are hard to control. Allergy shots keep your body from over-reacting to the allergen. You will get

regular injections of the allergen. Each dose is slightly larger than the last dose until a maximum dose is

reached. These shots do not work for everybody and you will have to visit the doctor often.

Allergy Tests

Allergy testing involves having a skin or blood test to find out what substance, orallergen, may trigger

an allergic response in a person. Skin tests are usually done because they are rapid, reliable, and generally

less expensive than blood tests, but either type of test may be used.

Skin tests

A small amount of a suspected allergen is placed on or below the skin to see if a reaction develops. There

are three types of skin tests:

Skin prick test. This test is done by placing a drop of a solution containing a possible allergen on the skin,

and a series of scratches or needle pricks allows the solution to enter the skin. If the skin develops a red,

raised itchy area (called a wheal), it usually means that the person is allergic to that allergen. This is called a

positive reaction.

Intradermal test. During this test, a small amount of the allergen solution is injected into the skin. An

intradermal allergy test may be done when a substance does not cause a reaction in the skin prick test but is

still suspected as an allergen for that person. The intradermal test is more sensitive than the skin prick test

but is more often positive in people who do not have symptoms to that allergen (false-positive test results).

http://www.webmd.com/hw-popup/allergen

http://www.webmd.com/hw-popup/allergic-reaction

http://www.webmd.com/skin-problems-and-treatments/picture-of-the-skin

http://www.webmd.com/heart/anatomy-picture-of-blood

http://www.webmd.com/allergies/default.htm

http://www.webmd.com/hw-popup/false-positive-test-result



Skin patch test. For a skin patch test, the allergen solution is placed on a pad that is taped to the skin for 24

to 72 hours. This test is used to detect a skin allergy called contact dermatitis.

Blood test

Allergy blood tests look for substances in the blood called antibodies. Blood tests are not as sensitive as skin

tests but are often used for people who are not able to have skin tests.

The most common type of blood test used is the enzyme-linked immunosorbent assay (ELISA, EIA). It

measures the blood level of a type of antibody (called immunoglobulin E, or IgE) that the body may make in

response to certain allergens. IgE levels are often higher in people who have allergies or asthma.

Other lab testing methods, such as radioallergosorbent testing (RAST) or an immunoassay capture test

(ImmunoCAP, UniCAP, or Pharmacia CAP), may be used to provide more information.

Challenge testing: Challenge testing is when small amounts of a suspected allergen are introduced to the

body orally, through inhalation, or other routes. Except for testing food and medication allergies, challenges

are rarely performed. When this type of testing is chosen, it must be closely supervised by an allergist.

Elimination/Challenge tests: This testing method is used most often with foods or medicines. A patient

with a suspected allergen is instructed to modify his/her diet to totally avoid that allergen for determined

time. If the patient experiences significant improvement, he/she may then be “challenged” by reintroducing

the allergen to see if symptoms can be reproduced.

Patch testing: Patch testing is used to help ascertain the cause of skin contact allergy, or contact dermatitis.

Adhesive patches, usually treated with a number of common allergic chemicals or skin sensitizers, are

applied to the back. The skin is then examined for possible local reactions at least twice, usually at 48 hours

after application of the patch and again two or three days later.

Unreliable tests: There are other types of allergy testing methods that the that are unreliable including

applied kinesiology (allergy testing through muscle relaxation), cytotoxicity testing, urine autoinjection,

skin titration (Rinkel method), and provocative and neutralization (subcutaneous) testing or sublingual

provocation.

How the Test Is Performed

There are three common methods of allergy skin testing.

The skin prick test involves:

Placing a small amount of substances that may be causing your symptoms on the skin, most often on the

forearm, upper arm, or back.

Then, the skin is pricked so the allergen goes under the skin's surface.

The health care provider closely watches the skin for swelling and redness or other signs of a reaction.

Results are usually seen within 15-20 minutes.

Several allergens can be tested at the same time.

The intradermal skin test involves:

Injecting a small amount of allergen into the skin.

Then the health care provider watches for a reaction at the site.

This test is more likely to be used to find out if you are allergic to something specific, such as bee venom or

penicillin.

Patch testing is a method to diagnose the cause of skin reactions that occur after the substance touches the

skin.

Possible allergens are taped to the skin for 48 hours.

The health care provider will look at the area in 72 - 96 hours.

http://www.webmd.com/hw-popup/contact-dermatitis-8212

http://www.webmd.com/hw-popup/antibodies

http://www.webmd.com/asthma/default.htm

http://en.wikipedia.org/wiki/Contact_dermatitis

http://health.nytimes.com/health/guides/symptoms/swelling/overview.html



Differential diagnosis

Before a diagnosis of allergic disease can be confirmed, other possible causes of the presenting symptoms

should be considered. Vasomotor rhinitis, for example, is one of many maladies that shares symptoms with

allergic rhinitis, underscoring the need for professional differential diagnosis. Once a diagnosis of asthma,

rhinitis, anaphylaxis, or other allergic disease has been made, there are several methods for discovering the

causative agent of that allergy.

ALTERNATIVE SYSTEM OF MEDICINES

Siddha Principles

Like all other medicine systems, Siddha system of medicine also has some underlying principles and concepts. These fundamental principles bear resemblance to that of ancient Ayurveda. According to Siddha system, the human body, food and the drugs are the replica of the universe, irrespective of their origin. Moreover, they believe that the universe holds two main entities namely, matter and energy. Siddhars call them Siva (male) and Shakti (female). The two are inseparable and co-exist as matter cannot subsist without the energy in it and vice versa. They are also the primordial elements, Bhutas, known as Munn (solid), Neer (fluid), Thee (radiance), Vayu (gas) and Veli (ether). These are present in every substance in varied proportions. Also, Earth, Water, Fire, Air and Ether are the manifestations of these elements. Even the human body is made up of these five elements in different permutations. It also considers that it is an assortment of three humours, seven basic tissues and the waste products produced by the body such as faeces, urine and sweat. The food ingested by the humans is regarded as the elementary building material of the body, which in turn is converted into humours, body tissues and waste products. Besides, the food and drugs also contains mixture of five elements. However, when the equilibrium of humors, considered as health, is disturbed, it leads to disease or sickness. Drugs constituting varying proportion of the elements are responsible for therapeutic actions and results. Apart from this, Siddha system also lays down the concept of salvation in life. The exponents of this system emphasize on achievement of this state via medicines and meditation.

Principles of Ayurveda

Ayurveda is a holistic healing science which comprises of two words, Ayu and Veda. Ayu means life

and Vedameans knowledge or science. So the literal meaning of the word Ayurveda is the science of

life. Ayurveda is a science dealing not only with treatment of some diseases but is a complete way of

life.

Ayurveda aims at making a happy, healthy and peaceful society. The two most important aims

of Ayurveda are:

+ To maintain the health of healthy people

+ To cure the diseases of sick people

A Person is seen in Ayurveda as a unique individual made up of five primary elements.

http://en.wikipedia.org/wiki/Vasomotor_rhinitis

http://en.wikipedia.org/wiki/Illness

http://en.wikipedia.org/wiki/Asthma

http://en.wikipedia.org/wiki/Anaphylaxis



These elements are ether (space), air, fire,water and earth.

Just as in nature, we too have these five elements in us. When any of these elements are

imbalanced in the environment , they will in turn have an influence on us. The foods we eat and

the weather are just two examples of the influence of these elements . While we are a composite of

these five primary elements, certain elements are seen to have an ability to combine to create

various physiological functions.

The elements combine with Ether and Air in dominence to form what is known in Ayurveda as Vata

Dosha. Vatagoverns the principle of movement and therefore can be seen as the force which directs

nerve impulses, circulation, respiration and elemination etc.,

The elements with Fire and Water in dominence combine to form the Pitta Dosha . The Pitta

Dosha is responsible for the process of transformation or metabolism. The transformation of foods

into nutrients that our bodies can assimilate is an example of a Pitta function. Pitta is also

responsible for metabolism in the organ and tissue systems as well as cellular metabolism.

Finally, it is predominantly the water and earth elements which combine to form the Kapha

Dosha. Kapha is responsible for growth, adding structure unit by unit. It also offers protection , for

example, in form of the cerebral-spinal fluid,which protects the brain and spinal column. The

mucousal lining of the stomach is another example of the function of Kapha Dosha protecting the

tissues.

We are all made up of unique proportions

of Vata,Pitta and Kapha. These ratios of the Doshas

vary in each individual and because of

this Ayurveda sees each person as a special mixture

that accounts for our diversity.

Ayurveda gives us a model to look at each individual

as a unique makeup of the three doshas and to

thereby design treatment protocols that specifically

address a persons health challenges. When any of the

doshas become accumulated, Ayurveda will suggest

specific lifestyle and nutritional guidelines to assist

the individual in reducing the dosha that has become

excessive. Also herbal medicines will be suggested , to

cure the imbalance and the disease.

Understanding this main principle of Ayurveda , it

offers us an explanation as to why one person

responds differently to a treatment or diet than

another and why persons with the same disease might

yet require different treatments and medications.

Other important basic principles of Ayurveda which are briefly mentioned here are:

1. Dhatus- These are the basic tissues which maintain and nourish the body. They are seven in

number namely- rasa(chyle), raktha(blood), mamsa(muscles),meda(fatty tissue),

asthi(bone), majja(marrow) and sukla(reprodutive tissue). Proper amount of each dhatu

and their balanced function is very important for good health.

2. Mala- These are the waste materials produced as a result of various metabolic activities in

the body. They are mainly urine, feaces, sweat etc. Proper elimination of the malas is

equally important for good health. Accumulation of malas causes many diseases in the body.

3. Srotas- These are different types of channels which are responsible for transportation of



food, dhatus,malas and doshas. Proper functioning of srotas is necessary for transporting

different materials to the site of their requirement. Blockage of srotas causes many diseases.

4. Agni- These are different types of enzymes responsible for digestion and transforming one

material to another.

All these factors should function in a proper balance for good health. They are inter-related and

are directly or indirectly responsible for maintaining equilibrium of the tridoshas.

Balance and Harmony of the Three Doshas

When the three Doshas are well harmonised and function in a balanced manner, it results in good

nourishment and well-being of the individual . But when there is imbalance or disharmony within

or between them, it will result in elemental imbalance , leading to various kinds of ailments.

The Ayurvedic concept of physical health revolves round these three Doshas and its primary

purpose is to help maintain them in a balanced state and thus to prevent disease.This humoral

theory is not unique to the ancient Indian Medicine : The Yin and Yang theory in chinese medicine

and the Hippocratic theory of four humours in Greek medicine are also very similar.

The Qualities of the Three Doshas

The three Doshas possess qualities and their increase or decrease in the system depends upon the

similar or antagonistic qualities of everything ingested.

Vata is : dry, cold, light, mobile, clear, rough, subtle

Pitta is : slightly oily, hot, intense, light, fluid,free flowing, foul smelling.

Kapha is: oily, cold, heavy, stable, viscid, smooth, soft

Both Vata and Pitta are light and only Kapha is heavy.

Both Vata and Kapha are cold and only Pitta is hot.

Both Pitta and Kapha are moist and oily and only Vata is dry.

Anything dry almost always increases Vata , anything hot

increases Pittaand anything heavy , Kapha.

Puffed rice is dry, cold light and rough - overindulgence in puffed

rice therefore is likely to increase Vata in the overindulger.

Mustard oil is oily , hot , intense , fluid , strong-smelling and liquid

and increases Pitta in the consumer.

Yoghurt , which , being creamy, cold, heavy, viscid, smooth and

soft , is the very image of Kapha , adds to the body's Kapha when

eaten.

All Five elemets , as expressed through Vata, Pitta and Kapha , are

essential to life, working together to create health or produce

disease. No one dosha can produce or sustain life - all three must

work together , each in its own way.



Principles of Unani

The Unani system recognises that disease is an unnatural process and symptoms of a disease are body's reaction to noxious factors from its surroundings. The chief function of the physician is to aid the natural force of the body, which is termed as Tabi‟at (Physis). Unani medicine is based on four Humor theories, which are Dam (Blood), Balgham (Phlegm), Safra (Yellow bile) and Sauda (black bile). The body has the power of self-control to maintain an optimum balance of these humors, which is called as Quwwat-e-Mudabbirah-e-Badan (Medicatrix Naturae).

The essential constituents and the working principles of the body, according to Unani system of medicine, can be classified into seven main groups:

1. Arkaan (Elements)

2. Mizaj (Temperament)

3. Akhlaat (Humors)

4. A‟za (Organs)

5. Arwaah. (Pneuma)

6. Quwa (Faculties of Power)

7. Afa‟al (Actions)

Unani system of medicine believes that Arkaan (elements), which are broadly divided in four categories i.e. Earth (Arz), Water (Maa‟), Air (Hawa), and Fire (Naar), are bricks of human structure and have their own temperament. After mixing and interaction of these principle elements in a particular ratio, a new structure comes into existence having its own temperament (Mizaj).

Mizaj (Temperament) of each and every individual varies widely as per composition as well as other surrounding factors and circumstances. Normal temperament is defined as a condition in which a person survives comfortably with all symptoms of healthy life.

Akhlaat (Humors) are liquid components of body which run through different channels inside the body and provide nutrition to the whole tissues of the body and maintain normal health. These humors have been named, according to their colour as Dam (Blood), Balgham (Phlegm), Safra (Yellow bile) and Sauda (black bile), which are red, white, yellow and black in colour respectively. Equilibrium of these humors is mainly responsible for health. Any alteration/deviation in quality or quantity from optimum position may lead to disease.

A‟zaa (Organs) are solid components of the body which are composed of different types of tissues. Organs collectively form a system. Through these organized systems, body performs its routine activity. Certain organs are specified as vital organs (A,zaa-e- Raeesah) of the body which include Heart, Brain, Liver, Testes/ Ovaries and these are responsible for vital functioning of the body and play major role in continuity and propagation of life.

Arwah (Pneuma) are the gaseous components of the body mainly consisting of Naseem (Oxygen), which runs in the body through blood in dissolved form. It is the basic source of life, which provides energy for all body activities.



Quwa (Faculties of Power) are nothing but ability to perform. All organs have been assigned a particular type of action on the basis of their nature and compositions. The main four faculties of power are Quwwat-e-Haiwaniyah, Quwwat-e-Nafsaaniyah, Quwwat-e-Tab‟iyah and Quwwat-e-Tanaasuliyah and the four vitals organs i.e. Heart, Brain, Liver, Testes/ Ovaries are responsible for these powers respectively.

Afa‟al (Functions) are bodily activities essential for fulfilling the objectives of the body. [The organs and also testimony perform these to the presence of power in them.] Principles of Homeopathy

Similia Similibus Curanter

This is the law of similars. It states that 'that which can cause can cure'. The onion, which produces tears in the eye and irritation (similar to a cold), can be used as a homeopathic medicine to cure colds which have irritating tears. The early Indians recognised this principle and states that Vishasya Vishamevam Aushadam and Samaha Samena Shantihi, but it was Dr.Samuel Hahnemann, who through his studies and experiments on the various medicines available in nature, practically proved the law.

Simplex Similimum Minimum

This principle consists of three words. The first is Simplex i.e : simple medicines not compound should be prescribed. This is the doctrine of single remedy. Mixture of medicines or polypharmacy is not allowed. Only one medicine must be given at a time. Similimum - As discussed previously the totality of symptoms of the patient must be taken. This will yield a picture which corresponds to one medicine, the similimum, which must be given. That medicine which has been tested on various provers and has produced similar symptoms as that of the patient is the similar remedy. Minimum - A low dosage of medicine is recommended. In homeopathy less is more, so medicines of low potency and given at long intervals have a better impact. Hahnemann, in fact used to give just one dose of the medicine and wait to see the reaction over a period of time.

Principle of Individualisation

Treat the patient, not the disease. This is the most important doctrine of homeopathy. Not two human beings are alike and so the medicines used for their treatment need not be alike. Homeopathic medicines are prescribed based on the totality of symptoms of that individual. So, the name of the disease is not important to the doctor who tries to get a complete picture of the patient - his symptoms,the modalities of symptoms, his likes and disliked, his environment, etc to arrive at the individualised remedy - which is the similimum.

Principle of Potentisation

Homeopathic medicines are diluted in alcohol or milk-sugar(lactose) to make them more palatable and also to reduce the harmful effects. It has been found that the more the medicine is diluted, the more effective and powerful it becomes. So, the process of the dilution is called as potentisation and the medicines are referred to as potencies.The crude homeopathic medicine(eg : Cinchona/Lachesis) is triturated in alcohol to yield the mother tincture. The mother tincture is denoted by the symbol ø. Potency : 1x potency of the medicine signifies 1 part of mother tincture diluted with 9 parts of alcohol / milk sugar. 2x potency is 1x of medicine diluted with 9 parts of sugar milk / alcohol. 1C potency is mother tincture diluted with 99 parts. 1M potency is mother tincture diluted with 999 parts. Low potency : 1x, 3x , 6x (3c), 12x (6c)

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Medium potency : 12x, 30x, 30c High potency : 200c, 1M, 20 M , CM, LM, etc. Law of Direction The law of direction of cure proposed by Dr.Constantine Hering states that - "As a patient recovers from a disease, the symptoms move from within outwards, from above downwards,from centre to circumference and disappear in the reverse order of their appearance" A patient suffering from a skin disease may use various medicines which suppress this disorder and send it into the body and it may manifest as athma. So, when this patient takes homeopathic medicine, the asthma is replaced with the skin infection and then finally the skin infection leaves to yield a cure. Three-legged stool This principle attributed to the elder Lippe(Dr.Adolph Lippe) states that while prescribing a medicine, three leading symptoms of that medicine should match the symptoms of the patient. Just as a stool with three legs is more stable than a stool with one leg, medicine given on the basis of atleast three key symptoms is more reliable than that treated with one symptom. Thus, a careful study is required to apply this law. Use of Materia Medica The Materia Medica is a dictionary of homeopathic medicines and their symptoms. It is a book which is the final authority on homeopathy. The materia medica contains the list of symptoms experienced by provers of the medicine. The symptoms are arranged in a systematic order - Mind (symptoms related to mind/mental), Head, Eyes, etc.. It is not required for a doctor to memorise or remember all the contents of the Materia Medica. What is required is to understand the nature / keynotes of each remedy. A number of materia medicas have been authored. Prominent among them are Kent'sLectures, Hering's Guiding Symptoms, Allen's Keynotes, etc. Repertorisation The repertory is an index to the Materia Medica. It is a book containing all possible symptoms arranged in alphabetical order for each of the organs of the body. The physican has to regularly refer this book to find out the medicines which have produced in a prover, the symptoms of the patient. Only, through correct usage of the repertory, can the job of prescription be made easier.



Types of formulation used in alternative system of medicine.

Definition :

Ayurvedic medicines are all the medicines intended for internal or external use, for or in the diagnosis

treatment, mitigation or prevention of disease or disorder in human beings or animal and

manufactured exclusively in accordance with the formulae described in the authorative books of

Ayurvedic Systems of medicine specified in the first schedule of the Drug and Cosmetic act 1940.

Ayurvedic Dosage Forms

LiquidAsava

Arishta

ArkaKwahaTaila

Dravaka

Netrabindu

Semisolides

Avaleha

Lepa

Matras

Kalka

Swarasa

Kajjali

Praash

Solid

Vatika

Gutika

Churna

Bhasma

Ksharas

Nasyas

Sattva









HERBALS AND THEIR FORMULATIONS

Tinctures are concentrated herbal extracts that are made using alcohol and chopped herbs. The tincture is

especially effective in drawing out the essential compounds of plants, especially those that are fibrous or

woody, and from roots and resins.[1]

Since this method ensures that the herbs and their nutrients can be

preserved for a long time, it is often mentioned in herbal books and remedies as a preferred way of using

herbs.

In addition, many herbalists love tinctures for other beneficial reasons, such as their being easy to carry,

their utility for long-term treatments, and their ability to be absorbed rapidly, as well as allowing for

immediate dosage changes.[2]

As well, should the tincture prove bitter, it's easily added to juice to disguise

the flavor. Another benefit of tinctures is that they keep nutrients from the plants in a stable, soluble form

and they retain the volatile and semi-volatile ingredients that are otherwise lost in heat-treatment and

processing of dry herbal extracts.

Fresh Herb • Finely chop or grind clean herb to release juice and expose surface area.

• Fill jar 2/3 to 3/4 with herb. ~ OR ~ Fill jar 1/4 to ½ with roots.

• Pour alcohol over the herbs. Cover completely!

• Jar should appear full of herb, but herb should move freely when shaken.

Dried Herb • Use finely cut herbal material.

• Fill jar 1/2 to 3/4 with herb ~ OR ~ Fill jar 1/4 to 1/3 with roots.

• Pour alcohol over the herbs. Cover completely!

• Roots will expand by ½ their size when reconstituted!

Purchase quality alcohol. The preferred type of alcohol for producing a tincture isvodka.[3]

This is owing

to its being colorless, odorless, and fairly flavorless. If you cannot obtain vodka, brandy, rum, or whiskey

can be substituted. Whatever alcohol is chosen, it must be 80 proof (namely, 40% alcohol) to prevent

mildewing of the plant material in the bottle.It is also possible to make a tincture from quality apple cider

vinegar or glycerin.[4]

The alternatives may work better where the patient refuses alcohol.

Use a suitable container. The container for the tincture should be glass or ceramic. Avoid using metallic or

plastic containers because these can react with the tincture or leach dangerous chemicals over time. Items

such as a Mason jar, a glass bottle with an attached stopper, etc., are ideal for steeping a tincture. In

addition, you will need to get some small dark glass tincture bottles for storing the tincture in once it has

been made; these bottles should have a tight screw-on or tight clip-on lid to prevent air intrusion during

storage but to allow for ease of use. Ensure that all containers are both washed clean and sterilized prior to

use.

Prepare the tincture. You can prepare a tincture by measurement or by sight; it really depends on your

level of comfort with simply adding herbs and judging by eye, or whether you feel more comfortable adding

them by measured weight. Also, you should know whether you want to add fresh, powdered, or dried

herbs to the tincture. Some suggestions for adding the herbs in the order of fresh, powdered, or dried are as

follows:

Add enough fresh chopped herbs to fill the glass container. Cover with alcohol.[5]

Add 4 ounces (113g) of powdered herb with 1 pint (473ml) of alcohol (orvinegar/glycerin).[6]

Add 7 ounces (198g) of dried herb material to 35 fluid ounces (1 liter) of alcohol (or vinegar/glycerin).

Using a butter knife, stir around the edge of the glass container to ensure that air bubbles are broken.

Seal the container. Place it into a cool, dark area; a cupboard shelf works best. The container should be

stored there for 8 days to a month.[7]

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Shake the container regularly. Humbart Santillo recommends shaking it twice a day for 14 days,[8]

while

James Wong recommends shaking it occasionally.[9]

Be sure to label the steeping tincture so that you know what it is and the date on which it was made. Keep it

out of the reach of children and pets.

Strain the tincture. Once the steeping time is finished (either the tincture instructions you're following will

inform you of this or you'll know already from experience but if not, about two weeks is a good steeping

time), strain the tincture as follows:

Place a muslin cloth across a sieve. Place a large bowl underneath to catch the strained liquid.

Gently pour the steeped liquid through the muslin-lined sieve. The muslin will capture the plant material

and the liquid will pass through into the bowl underneath.

Press the herb material with a wooden or bamboo spoon to squeeze out some more liquid, and lastly, twist

the muslin to extract any leftover liquid from the herbs.

Burdock Root Extract Natural healers use this

herb as an effective

blood purifier, believing

that it rids the body of

toxins. Excellent for

arthritis and applied

externally for skin

problems. Burdock is

still used today as a

diuretic, and to support

the healing of chronic

acne and psoriasis.

Butchers Broom

Extract For centuries European

herbalists have used this

herb to relieve water

retention and to treat the

discomfort and pain

caused by poor

circulation in the legs.

This plant contains

steroid-like compounds

that may constrict veins

and reduce inflammation

caused by arthritis and

rheumatism.

Capsicum Tincture Capsicum tincture

produces a local

stimulant and analgesic

effect. Use in cases of

pain along the spinal

nerves and other nerve

endings, nerve root

syndrome, inflammation

of the voluntary muscles,

lower back pain, and

pain in the hips. Do not

use in case of

hypersensitivity.

How to Make Herbal Syrups

Herbal syrups are not hard to make and are a good alternative way to prepare some mixtures especially

some of the herbs that are really bitter. Some of the herbs are bitter which serves a natural purpose – it keeps

us from overusing and also stimulates digestive juices. The bitterness can also make us not want to take the

medicine, especially children. Syrups help in this area and also it can extend the storage life of the herb.

Syrups are good to use for colds and flu and to soothe a sore throat.

Make sure you never use honey for children younger than one to two years old.

First decide which herb you want to use

For a basic syrup you can use a infusion or decoction that you have made.

Put 1 part infusion or decoction to 1 part honey or sugar in a saucepan

Gently heat this until the sugar or honey is completely dissolved.

Cool slightly and pour into clean glass or ceramic jars or bottles

Keep in the refrigerator for three to six months

You can use 1 to 2 teaspoons of the syrup up to three times a day.

A really popular herbal syrup is elderberry syrup, you can make it with fresh or dried berries. There are

red and blue elderberries, the red ones have seeds that are toxic so use the blue ones. Or just buy some dried

elderberries. It is good for cold and flu among other things.



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Put a cup of fresh elderberries or a half cup of dried berries in a saucepan

Pour in 3 cups of water

Bring it to a boil then simmer for 30 minutes

Smash the berries

Strain the berry mixture with a strainer and cheesecloth

Stir in the honey

Pour into a bottle and label

Store in the refrigerator for a few month

Take 1 teaspoon every 2-3 hours while sick.

Some use it regularly with food as you would syrup. Elder is VERY high in bioflavonoids and is a great

antioxidant. (They say for children under 2 years you can add the syrup to hot water and it will kill any

microbes in the honey that might make them sick, or just use sugar.)

What is a cream?

Creams generally consist of two basic components, an oil phase and an aqueous phase. A cream is formed

when the oil phase is successfully emulsified into the aqueous phase, producing an oil in water emulsion of

stable and solid consistency at room temperatures.

Functions of a cream.

A cream can be successfully used to deliver and hold nutrients and medications on the skin's surface. Both

the oil and aqueous components can be used as a carrier. The skin has a limited capacity to absorb many oils

and some chemical compounds and is responsive to surface medications such as herbal extracts, and to

vibrational energies such as Flower essences.

Procedure

1. Make the CREAM BASE by measuring the oil components into the larger jug and the aqueous

components into the smaller jug. The oil phase must contain the emulsifier. Heat both in the water

bath saucepans so they do not come in direct contact with the heating element and are thus protected

from being over-heated.

2. Stir the components regularly with the spatula to distribute the heat and use the stick thermometer to

measure the temperature. The usual temperature before mixing for the making of a stable emulsion is

80C for the aqueous component and 70C for the oils.

3. When the aqueous and oil components are at the required temperature and any waxes have melted,

mix the two together by removing both jugs out of the baths and away from the heating elements and

pouring the water component into the oil. Use vigorous stirring or preferably, a hand-held Bamix

type stick blender to make an emulsion.

4. Do this for 1-2 minutes to allow the emulsion to form. Avoid blending air into the liquid, pulse the

blender and keep the blender head well under the liquid.

5. Quickly cool the mix to around 55C by sitting the jug in the cold-water bath as you stir the emulsion.

6. Add the remaining ingredients including any tinctures and specialized oils, and omega 3 and

fragrance oils at this time with constant stirring. Remove any set cream from the sides and bottom of

the jug. Use a little gentle water bath heat if required. Blend again and avoid blending air into the

emulsion..

7. Allow the liquid emulsion to sit for a minute or two and tap the base of the jug to remove air

bubbles.

8. When at 44C or showing signs of thickening (i.e. starting to set, usually around 42C) pour into

ready, uncapped jars. Attention is needed as the cream can set quickly and a little hot water bath heat

may be required to finish the pouring.



Capping and labeling

1. Allow the cream to cool until "cold to touch" before capping as condensation can occur on the inside

of the lid and drop onto the surface of the cream and lead to mould growth.

2. Before capping, check both the cream in the jar and the cap for any surface contaminants.

3. When the cream has set, apply a label including the name for the cream, date of expiry, storage

advice, batch number and it is best to include details of the ingredients and how the cream is to be

used.

Assessment of quality

Pharmaceutical assessment

This should cover all important aspects of the quality assessment of herbal medicines. It should be sufficient

to make reference to a pharmacopoeial monograph if one exists. If no such monograph is available, a

monograph must be supplied and should be set out as in an official pharmacopoeia.

All procedures should be in accordance with good manufacturing practices.

Crude plant material

The botanical definition, including genus, species and authority, should be given to ensure correct

identification of a plant. A definition and description of the part of the plant from which the medicine is

made (e.g. leaf flower, root) should be provided, together with an indication of whether fresh, dried or

traditionally processed material is used. The active and characteristic constituents should be specified and, if

possible content limits should be defined. Foreign matter, impurities and microbial content should be

defined or limited. Voucher specimens, representing each lot of plant material processed, should be

authenticated by a qualified botanist and should be stored for at least a 10-year period. A lot number should

be assigned and this should appear on the product label.

Plant preparations

Plant preparations include comminuted or powdered plant materials, extracts, tinctures, fatty or essential

oils, expressed juices and preparations whose production involves fractionation, purification or

concentration. The manufacturing procedure should be described in detail. If other substances are added

during manufacture in order to adjust the plant preparation to a certain level of active or characteristic

constituents or for any other purpose, the added substances should be mentioned in the manufacturing

procedures. A method for identification and, where possible, assay of the plant preparation should be added.

If identification of an active principle is not possible, it should be sufficient to identify a characteristic

substance or mixture of substances (e.g. “chromatographic fingerprint”) to ensure consistent quality of the

preparation.

Finished product

The manufacturing procedure and formula, including the amount of excipients, should be described in

detail. A finished product specification should be defined. A method of identification and, where possible,

quantification of the plant material in the finished product should be defined. If the identification of an

active principle is not possible, it should be sufficient to identify a characteristic substance or mixture of

substances (e.g. “chromatographic fingerprint”) to ensure consistent quality of the product. The finished

product should comply with general requirements for particular dosage forms.



For imported finished products, confirmation of the regulatory status in the country of origin should be

required. The WHO Certification Scheme on the Quality of Pharmaceutical Products Moving in

International Commerce should be applied.

Stability

The physical and chemical stability of the product in the container in which it is to be marketed should be

tested under defined storage conditions and the shelf-life should be established.

Assessment of safety

This should cover all relevant aspects of the safety assessment of a medicinal product. A guiding principle

should be that, if the product has been traditionally used without demonstrated harm, no specific restrictive

regulatory action should be undertaken unless new evidence demands a revised risk - benefit assessment.

A review of the relevant literature should be provided with original articles or references to the original

articles. If official monograph/review results exist, reference can be made to them. However, although long-

term use without any evidence of risk may indicate that a medicine is harmless, it is not always certain how

far one can rely solely on long-term usage to provide assurance of innocuity in the light of concern

expressed in recent years over the long-term hazards of some herbal medicines.

Reported side-effects should be documented according to normal pharmaco-vigilance practices.

Toxicological studies

Toxicological studies, if available, should be part of the assessment. Literature should be indicated as above.

Documentation of safety based on experience

As a basic rule, documentation of a long period of use should be taken into consideration when assessing

safety. This means that, when there are no detailed toxicological studies, documented experience of long-

term use without evidence of safety problems should form the basis of the risk assessment. However, even

in cases of drugs used over a long period, chronic toxicological risks may have occurred but may not have

been recognized. The period of use, the health disorders treated, the number of users and the countries with

experience should be specified. If a toxicological risk is known, toxicity data must be submitted. The

assessment of risk, whether independent of dose or related to dose, should be documented. In the latter case,

the dosage specification must be an important part of the risk assessment. An explanation of the risks should

be given, if possible. Potential for misuse, abuse or dependence must be documented. If long-term

traditional use cannot be documented or there are doubts on safety, toxicity data should be submitted.

Assessment of efficacy

This should cover all important aspects of efficacy assessment. A review of the relevant literature should be

carried out and copies provided of the original articles or proper references made to them. Research studies,

if they exist, should be taken into account.

Activity

The pharmacological and clinical effects of the active ingredients and, if known, their constituents with

therapeutic activity should be specified or described.



Evidence required to support indications

The indication(s) for the use of the medicine should be specified. In the case of traditional medicines, the

requirements for proof of efficacy should depend on the kind of indication. For treatment of minor disorders

and for non-specific indications, some relaxation in requirements for proof of efficacy may be justified,

taking into account the extent of traditional use. The same considerations may apply to prophylactic use.

Individual experiences recorded in reports from physicians, traditional health practitioners or treated

patients should be taken into account.

Where traditional use has not been established, appropriate clinical evidence should be required.

Combination products

As many herbal remedies consist of a combination of several active ingredients, and as experience of the use

of traditional remedies is often based on combination products, assessment should differentiate between old

and new combination products. Identical requirements for the assessment of old and new combinations

would result in inappropriate assessment of certain traditional medicines.

In the case of traditionally used combination products, the documentation of traditional use (such as

classical texts of Ayurveda, traditional Chinese medicine, Unani, Siddha) and experience may serve as

evidence.

An explanation of a new combination of well known substances, including effective dose ranges and

compatibility, should be required in addition to the documentation of traditional knowledge of each single

ingredient. Each active ingredient must contribute to the efficacy of the medicine.

Clinical studies may be required to justify the efficacy of a new ingredient and its positive effect on the total

combination.

Intended use

Product information for the consumer

Product labels and package inserts should be understandable to the consumer or patient. The package

information should include all necessary information on the proper use of the product.

The following elements of information will usually suffice:

• name of the product

• quantitative list of active ingredient(s)

• dosage form

• indications

- dosage (if appropriate, specified for children and the elderly)

- mode of administration

- duration of use

- major adverse effects, if any

- overdosage information



- contraindications, warnings, precautions and major drug interactions

- use during pregnancy and lactation

• expiry date

• lot number

• holder of the marketing authorization.

Identification of the active ingredient(s) by the Latin botanical name, in addition to the common name in the

language of preference of the national regulatory authority, is recommended.

Sometimes not all information that is ideally required may be available, so drug regulatory authorities

should determine their minimal requirements.

Promotion

Advertisements and other promotional material directed to health personnel and the general public should be

fully consistent with the approved package information.

Utilization of these guidelines

These guidelines for the assessment of herbal medicines are intended to facilitate the work of regulatory

authorities, scientific bodies and industry in the development, assessment and registration of such products.

The assessment should reflect the scientific knowledge gathered in that field. Such assessment could be the

basis for future classification of herbal medicines in different parts of the world. Other types of traditional

medicines in addition to herbal products may be assessed in a similar way.

The effective regulation and control of herbal medicines moving in international commerce also requires

close liaison between national institutions that are able to keep under regular review all aspects of

production and use of herbal medicines, as well as to conduct or sponsor evaluative studies of their efficacy,

toxicity, safety, acceptability, cost and relative value compared with other drugs used in modern medicine.

Extraction techniques of Medicinal plants

Extraction, as the term is used pharmaceutically, involves the separation of medicinally active

portions of plant or animal tissues from the inactive or inert components by using selective solvents in

standard extraction procedures. The products so obtained from plants are relatively impure liquids,

semisolids or powders intended only for oral or external use.

These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular

(semisolid) extracts and powdered extracts. Such preparations popularly have been called galenicals, named

after Galen, the second century Greek physician. The purposes of standardized extraction procedures for

crude drugs are to attain the therapeutically desired portion and to eliminate the inert material by treatment

with a selective solvent known as menstruum.



The extract thus obtained may be ready for use as a medicinal agent in the form of tinctures and fluid

extracts, it may be further processed to be incorporated in any dosage form such as tablets or capsules, or it

may be fractionated to isolate individual chemical entities such as ajmalicine, hyoscine and vincristine,

which are modern drugs. Thus, standardization of extraction procedures contributes significantly to the final

quality of the herbal drug.

Circularly extraction

Methods of Extraction of Medicinal Plants

Maceration

In this process, the whole or coarsely powdered crude drug is placed in a stoppered container with

the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation

until the soluble matter has dissolved. The mixture then is strained, the marc (the damp solid material) is

pressed, and the combined liquids are clarified by filtration or decantation after standing.

Infusion

Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or

boiling water. These are dilute solutions of the readily soluble constituents of crude drugs.

Digestion

This is a form of maceration in which gentle heat is used during the process of extraction. It is used

when moderately elevated temperature is not objectionable. The solvent efficiency of the menstruum is

thereby increased.

Decoction In this process, the crude drug is boiled in a specified volume of water for a defined time; it is then cooled



and strained or filtered. This procedure is suitable for extracting water-soluble, heat-stable constituents. This

process is typically used in preparation of Ayurvedic extracts called “quath” or “kawath”. The starting ratio

of crude drug to water is fixed, e.g. 1:4 or 1:16; the volume is then brought down to one-fourth its original

volume by boiling during the extraction procedure. Then, the concentrated extract is filtered and used as

such or processed further.

Percolation This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and

fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is generally used. The solid

ingredients are moistened with an appropriate amount of the specified menstruum and allowed to stand for

approximately 4 h in a well closed container, after which the mass is packed and the top of the percolator is

closed. Additional menstruum is added to form a shallow layer above the mass, and the mixture is allowed

to macerate in the closed percolator for 24 h. The outlet of the percolator then is opened and the liquid

contained therein is allowed to drip slowly. Additional menstruum is added as required, until the percolate

measures about three-quarters of the required volume of the finished product. The marc is then pressed and

the expressed liquid is added to the percolate. Sufficient menstruum is added to produce the required

volume, and the mixed liquid is clarified by filtration or by standing followed by decanting.

Hot Continuous Extraction (Soxhlet) In this method, the finely ground crude drug is placed in a porous bag or “thimble” made of strong

filter paper, which is placed in chamber E of the Soxhlet apparatus (Figure 2). The extracting solvent in

flask A is heated, and its vapors condense in condenser D. The condensed extractant drips into the thimble

containing the crude drug, and extracts it by contact. When the level of liquid in chamber E rises to the top

of siphon tube C, the liquid contents of chamber E siphon into fl ask A. This process is continuous and is



carried out until a drop of solvent from the siphon tube does not leave residue when evaporated. The

advantage of this method, compared to previously described methods, is that large amounts of drug can be

extracted with a much smaller quantity of solvent. This effects tremendous economy in terms of time,

energy and consequently financial inputs. At small scale, it is employed as a batch process only, but it

becomes much more economical and viable when converted into a continuous extraction procedure on

medium or large scale.

Aqueous Alcoholic Extraction by Fermentation

Some medicinal preparations of Ayurveda (like asava and arista) adopt the technique of

fermentation for extracting the active principles. The extraction procedure involves soaking the crude drug,

in the form of either a powder or a decoction (kasaya), for a specified period of time, during which it

undergoes fermentation and generates alcohol in situ; this facilitates the extraction of the active constituents

contained in the plant material. The alcohol thus generated also serves as a preservative. If the fermentation

is to be carried out in an earthen vessel, it should not be new: water should first be boiled in the vessel. In

large-scale manufacture, wooden vats, porcelain jars or metal vessels are used in place of earthen vessels.

Some examples of such preparations are karpurasava, kanakasava, dasmularista. In Ayurveda, this method

is not yet standardized but, with the extraordinarily high degree of advancement in fermentation technology,

it should not be difficult to standardize this technique of extraction for the production of herbal drug

extracts.

Counter-current Extraction

In counter-current extraction (CCE), wet raw material is pulverized using toothed disc disintegrators



to produce a fine slurry. In this process, the material to be extracted is moved in one direction (generally in

the form of a fine slurry) within a cylindrical extractor where it comes in contact with extraction solvent.

The further the starting material moves, the more concentrated the extract becomes. Complete extraction is

thus possible when the quantities of solvent and material and their flow rates are optimized. The process is

highly efficient, requiring little time and posing no risk from high temperature. Finally, sufficiently

concentrated extract comes out at one end of the extractor while the marc (practically free of visible solvent)

falls out from the other end. This extraction process has significant advantages: iii) A unit quantity of the plant material can be extracted with much smaller volume of solvent as

compared to other methods like maceration, decoction, percolation.

iv) CCE is commonly done at room temperature, which spares the thermolabile constituents from exposure

to heat which is employed in most other techniques.

v) As the pulverization of the drug is done under wet conditions, the heat generated during comminution is

neutralized by water. This again spares the thermolabile constituents from exposure to heat.

vi) The extraction procedure has been rated to be more efficient and effective than continuous hot

extraction.

Ultrasound Extraction (Sonication)

The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz;

this increases the permeability of cell walls and produces cavitation. Although the process is useful in some

cases, like extraction of rauwolfia root, its large-scale application is limited due to the higher costs. One

disadvantage of the procedure is the occasional but known deleterious effect of ultrasound energy (more

than 20 kHz) on the active constituents of medicinal plants through formation of free radicals and

consequently undesirable changes in the drug molecules.



Supercritical Fluid Extraction Supercritical fluid extraction (SFE) is an alternative sample preparation method with general goals of

reduced use of organic solvents and increased sample throughput. The factors to consider include

temperature, pressure, sample volume, analyte collection, modifier (cosolvent) addition, flow and pressure

control, and restrictors. Generally, cylindrical extraction vessels are used for SFE and their performance is

good beyond any doubt.

The collection of the extracted analyte following SFE is another important step: significant analyte

loss can occur during this step, leading the analyst to believe that the actual efficiency was poor.

There are many advantages to the use of CO2 as the extracting fluid. In addition to its favorable

physical properties, carbon dioxide is inexpensive, safe and abundant. But while carbon dioxide is the

preferred fluid for SFE, it possesses several polarity limitations. Solvent polarity is important when

extracting polar solutes and when strong analyte-matrix interactions are present. Organic solvents are

frequently added to the carbon dioxide extracting fluid to alleviate the polarity limitations. Of late, instead

of carbon dioxide, argon is being used because it is inexpensive and more inert. The component recovery

rates generally increase with increasing pressure or temperature: the highest recovery rates in case of argon

are obtained at 500 atm and 150° C.

The extraction procedure possesses distinct advantages: i) The extraction of constituents at low temperature, which strictly avoids damage from heat and some

organic solvents.

ii) No solvent residues. iii) Environmentally friendly extraction procedure.

The largest area of growth in the development of SFE has been the rapid expansion of its

applications. SFE finds extensive application in the extraction of pesticides, environmental samples, foods

and fragrances, essential oils, polymers and natural products. The major deterrent in the commercial

application of the extraction process is its prohibitive capital investment.



Phytonics Process

A new solvent based on hydrofluorocarbon-134a and a new technology to optimize its remarkable

properties in the extraction of plant materials offer significant environmental advantages and health and

safety benefits over traditional processes for the production of high quality natural fragrant oils, flavors and

biological extracts. Advanced Phytonics Limited (Manchester, UK) has developed this patented technology

termed “phytonics process”. The products mostly extracted by this process are fragrant components of

essential oils and biological or phytopharmacological extracts which can be used directly without further

physical or chemical treatment.

The properties of the new generation of fluorocarbon solvents have been applied to the extraction of

plant materials. The core of the solvent is 1,1,2,2-tetrafluoroethane, better known as hydrofluorocarbon-

134a (HFC-134a). This product was developed as a replacement for chlorofluorocarbons. The boiling point

of this solvent is -25° C. It is not flammable or toxic. Unlike chlorofluorocarbons, it does not deplete the

ozone layer. It has a vapor pressure of 5.6 bar at ambient temperature. By most standards this is a poor

solvent. For example, it does not mix with mineral oils or triglycerides and it does not dissolve plant wastes.

The process is advantageous in that the solvents can be customized: by using modified solvents with

HFC-134a, the process can be made highly selective in extracting a specific class of phytoconstituents.

Similarly, other modified solvents can be used to extract a broader spectrum of components. The biological

products made by this process have extremely low residual solvent. The residuals are invariably less than 20

parts per billion and are frequently below levels of detection. These solvents are neither acidic nor alkaline

and, therefore, have only minimal potential reaction effects on the botanical materials. The processing plant

is totally sealed so that the solvents are continually recycled and fully recovered at the end of each

production cycle. The only utility needed to operate these systems is electricity and, even then, they do no

consume much energy. There is no scope for the escape of the solvents. Even if some solvents do escape,

they contain no chlorine and therefore pose no threat to the ozone layer. The waste biomass from these

plants is dry and “ecofriendly” to handle

Advantages of the Process

• Unlike other processes that employ high temperatures, the phytonics process is cool and gentle and its

products are never damaged by exposure to temperatures in excess of ambient.

• No vacuum stripping is needed which, in other processes, leads to the loss of precious volatiles. • The process is carried out entirely at neutral pH and, in the absence of oxygen, the products never suffer

acid hydrolysis damage or oxidation.

• The technique is highly selective, offering a choice of operating conditions and hence a choice of end



products.

• It is less threatening to the environment. • It requires a minimum amount of electrical energy. • It releases no harmful emissions into the atmosphere and the resultant waste products (spent biomass) are

innocuous and pose no effluent disposal problems.

• The solvents used in the technique are not flammable, toxic or ozone depleting. • The solvents are completely recycled within the system. Applications The phytonics process can be used for extraction in biotechnology (e.g for the production of antibiotics), in

the herbal drug industry, in the food, essential oil and flavor industries, and in the production of other

pharmacologically active products. In particular, it is used in the production of top quality pharmaceutical-

grade extracts, pharmacologically active intermediates, antibiotic extracts and phytopharmaceuticals.

However, the fact that it is used in all these areas in no way prevents its use in other areas. The technique is

being used in the extraction of high-quality essential oils, oleoresins, natural food colors, flavors and

aromatic oils from all manner of plant materials. The technique is also used in refining crude products

obtained from other extraction processes. It provides extraction without waxes or other contaminants. It

helps remove many biocides from contaminated biomass.

Parameters for Selecting an Appropriate Extraction Method i) Authentication of plant material should be done before performing extraction. Any foreign matter should

be completely eliminated.

ii) Use the right plant part and, for quality control purposes, record the age of plant and the time, season and

place of collection.

iii) Conditions used for drying the plant material largely depend on the nature of its chemical constituents.

Hot or cold blowing air flow for drying is generally preferred. If a crude drug with high moisture content is

to be used for extraction, suitable weight corrections should be incorporated. iv) Grinding methods should be specified and techniques that generate heat should be avoided as much as

possible.

v) Powdered plant material should be passed through suitable sieves to get the required particles of uniform

size.

vi) Nature of constituents:

a) If the therapeutic value lies in non-polar constituents, a non-polar solvent may be used. For

example, lupeol is the active constituent of Crataeva nurvala and, for its extraction, hexane is



generally used. Likewise, for plants like Bacopa monnieri and Centella asiatica, the active

constituents are glycosides and hence a polar solvent like aqueous methanol may be used.

b) If the constituents are thermolabile, extraction methods like cold maceration, percolation and

CCE are preferred.

For thermostable constituents, Soxhlet extraction (if nonaqueous solvents are used) and decoction (if

water is the menstruum) are useful.

c) Suitable precautions should be taken when dealing with constituents that degrade while being kept

in organic solvents, e.g. flavonoids and phenyl propanoids.

d) In case of hot extraction, higher than required temperature should be avoided. Some glycosides

are likely to break upon continuous exposure to higher temperature.

e) Standardization of time of extraction is important, as:

• Insufficient time means incomplete extraction.

• If the extraction time is longer, unwanted constituents may also be extracted. For example,

if tea is boiled for too long, tannins are extracted which impart astringency to the final

preparation.

f) The number of extractions required for complete extraction is as important as the duration of each

extraction.

viii) The quality of water or menstruum used should be specified and controlled. ix) Concentration and drying procedures should ensure the safety and stability of the active constituents.

Drying under reduced pressure (e.g. using a Rotavapor) is widely used. Lyophilization, although expensive,

is increasingly employed. x) The design and material of fabrication of the extractor are also to be taken into consideration. xi) Analytical parameters of the final extract, such as TLC and HPLC fingerprints, should be documented to

monitor the quality of different batches of the extracts.

Source: Sukhdev Swami Handa, Suman Preet Singh Khanuja, Gennaro Longo, Dev Dutt Rakesh. 2008. Extraction

technologies for medicinal and aromatic plants, International centre for science and high technology.



T.B.E.K.B STUDENT

AT

ARUL MIGU

KALASALINGAM

COLLEGE OF

PHARMACY

Advanced Pharmacognosy Notes

Science