Evaluation of microbial activity, standardization, characterization of total extract and active phytoconstituents of Roylea elegans A Synopsis Submitted in Partial Fulfilment for the Degree Of Doctor of Philosophy (Pharmacy) Supervised by Submitted by Dr. Anurekha Jain Neeru 1
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Evaluation of microbial activity, standardization, characterization of total extract and active phytoconstituents
of Roylea elegans
A Synopsis
Submitted in
Partial Fulfilment for the Degree
Of
Doctor of Philosophy
(Pharmacy)
Supervised by Submitted by Dr. Anurekha Jain Neeru
Department of Pharmaceutical SciencesJayotiVidyapeeth Women’s University
Jaipur (Rajasthan), IndiaJanuary, 2017
1
Table of Content
Outline Page No.
Introduction………………………….......................3-7
Objective of the Study……………………………….8
Review of Literature…………………………………9-13
Research Methodology…………………………….14-16
Activity Plan……………………………………….17
Bibliography…………………………………………18
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INTRODUCTION
Herbal medicine is defined as a plant part like leaf, seed, stem, roots, rhizomes flower and whole
plant and has an active principle which have any pharmacological activity or capable to treat any
disease. Plants are the natural source of herbal medicine and also natural sources of drugs. Plants
have been used in the treatment of various diseases from time immemorial. The traditional Indian
system of medicine, Ayurveda, Siddha, Unani systems, is based on the use of plants and other
natural substances. There are 2000,000 to 250,000 species of flowering plants growing on earth
which belong to 10,500 genera and about 300 families.
ADVANTAGES OF HERBAL MEDICINE
Herbal medicine have long history of use and better patient tolerance as well as
acceptance.
Medicinal plants have a renewable source, which is our only hope for sustainable supplies
of cheaper medicine for the world growing populations.
Availability of medicinal plants is not a problem especially in developing countries like
India having rich agro-climatic, cultural, and ethnic biodiversity.
The cultivation and processing of medicinal herbs and herbal products is environmental
friendly.
EXTRACTION- Extraction is defined as separation of mixture of constituents from crude drug
with the help of suitable solvents. In this method solvents are used according to polarity.
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 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.
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The extract thus obtained may be ready for use as a medicinal agent in the form oftinctures 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.
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. 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 flask 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.
STANDARDIZATION- Standardization is defined as the process in which crude drug or its
part is standardizing by different parameters to check the quality, safety and efficacy of
particular plant. Accounting toWHO it is the process involving the physicochemical evaluation
crude drug covering the aspects,as selection and handling of crude material,safety,efficacy and
stability assessment of finished product,documentation of safety and risk based on
experience,provision of product information to consumer and product promotion.
IMPORTANCE OF STANDARDIZATION- In olden times,vaidyas used to treat patients on
individual basis,and prepare drug according to the requirement of the patient. In almost all the
traditional system of medicine, the quality control aspect has been considered from its inspection
of itself Rishis,Vaidyas and Hakims. Unlike in olden times where traditional practitioners
prepared and tested the qualities of herbal medicines, the problem faced today are these of
economics of industrial scale production ,shelf life and distribution to long distances. These have
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necessitated development of modern and objective standards for evaluating the safety, quality and
efficacy of these medicines.
It is the cardinal responsibility of the regulatory authorities to ensure that the consumers
get the medication, which guarantee Purity, safety, potency and efficacy. Herbal product has been
enjoying renaissance among the customers through out the world. However,one of the
impedimentsin the acceptance of the Ayurvedic formulations the lack of standard quality control
profile. The quality of herbal medicine i.e. the profile of the constituents in the final product has
implication in efficacy and safety.
The task of lying down standards for quality control of herbal crude and their formulation
involves biological evaluation for a particular disease area, chemical profiling of the material and
lying down specification for the finished product. Therefore, incase of herbal drugs and product,
the word"Standardization"should encompass entire field of study from cultivation of medicinal
plant to its clinical application.
PARAMETERS OF STANDARDIZATION- The bioactive extract should be standardized on the
basis of active principles or major compounds along with the chromatographic fingerprints
(TLC,HPTLC, HPLC and GC). The standardization of crude drug materials includes the following
steps:
1. Authentication(Stage of collection, parts of the plant collected, regional status, botanical
identity like phytomorphology, microscopical and histological analysis, taxonomical identity,
etc.)
2. Foreign matter(herbs collected should be free from soil, insect parts or animal excreta,
etc.)
3. Organoleptic evaluation(sensory characters–taste, appearance,odor, feel of the drug,etc.)
4. Tissues of diagnostic importance present in the drug powder.
5. Ash values and extractive values.
6. Volatile matter
7. Moisture content determination
8. Chromatographic and spectroscopic evaluation. TLC, HPTLC, HPLC methods will
provide qualitative and semiquantitative information about the main active constituents present
in the crude drug as chemical markers in the TLC fingerprint evaluation of herbals(FEH).
9. Determination of heavy metals–e.g.cadmium,lead,arsenic,etc.
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10. Pesticide residue–WHO and FAO (Food and Agricultural Organization) setlimits of
pesticides, which are usually present in the herbs.These pesticides are mixed with the herbs
during the time of cultivation. Mainly pesticides like DDT, BHC, toxaphene, aldrin causes
eriousside- effects in human beings if the crude drugs are mixed with these agents.
11. Microbial contamination–usually medicinal plants containing bacteria and molds are
coming from soil and atmosphere. Analysis of the limits of E.coli and molds clearly throws
light towards the harvesting and production practices.The substance known as afflatoxins will
produces eriousside-effects if consumed along with the crude drugs. Afflatoxins should be
completely removed or should not be present.
12. Radioactive contamination–Microbial growth in herbals are usually avoided by
irradiation. This process may sterilize the plant material but the radio activity hazard should be
taken into account. The radioactivity of the plant samples should be checked accordingly to the
guideline of International Atomic Energy(IAE) in Vienna and that of WHO.
ISOLATION- Isolation of single phytoconstituents from mixture of phytoconstituents with the
help of suitable solvent. Solvents are used according to polarity.
IDENTIFICATION AND CHARACTERIZATION- Due to the fact that plant extracts usually
occur as a combination of various type of bioactive compounds or phytochemicals with different
polarities, their separation still remains a big challenge for the process of identification
andcharacterization of bioactive compounds. It is a common practice in isolation of these
bioactive compounds that a number ofdifferent separation techniques such as TLC, column
chromatography, flash chromatography, Sephadex chromatography and HPLC, should be used to
obtain pure compounds. The pure compounds are then used for the determination of structure and
biological activity. Besides that, non-chromatographic techniques such as immunoassay, which
use monoclonal antibodies (MAbs), phytochemical screening assay, Fourier-transform infrared
spectroscopy (FTIR), can also be used to obtain and facilitate the identification of the bioactive
compounds.
FLAVONOID- The term “flavonoid” is generally used to describe a broad collection of natural
products that include a C6-C3-C6 carbon framework, or more specifically a
phenylbenzopyranfunctionality. Depending on the position of the linkage of the aromatic ring
to the benzopyrano (chromano) moiety, this group of natural products may be divided into
three classes: the flavonoids (2-phenylbenzopyrans) 1, isoflavonoids (3-benzopyrans) 2, and
6
the neoflavonoids (4-benzopyrans) 3. These groups usually share a common chalcone
precursor, and therefore are biogenetically and structurally related.
2-PHENYLBENZOPYRANS (C6-C3-C6 BACKBONE)-Based on the degree of oxidation and
saturation present in the heterocyclic C-ring, the flavonoids may be divided into the different
groups.
ISOFLAVONOIDS- The isoflavonoids are a distinctive subclass of the flavonoids. These
compounds possess a 3-phenylchroman skeleton that is biogenetically derived by 1,2-aryl
migration in a 2-phenylchroman precursor. Despite their limited distribution in the plant
kingdom, isoflavonoids are remarkably diverse as far as structural variations are concerned.
This arises not only from the number and complexity of substituents on the basic 3-
phenylchroman system, but also from the different oxidation levels and presence of additional
heterocyclic rings. Isoflavonoids are subdivided into the different groups.
NEOFLAVONOIDS- The neoflavonoids are structurally and biogenetically closely related to
the flavonoids and the isoflavonoids and comprise the 4-arylcoumarins (4-aryl-2H-1-
benzopyran-2-ones), 3,4-dihydro-4-arylcoumarins, and neoflavenes.
MINOR FLAVONOIDS- Natural products such as chalcones and aurones also contain a C6-
C3-C6 backbone and are considered to be minor flavonoids. These groups of compounds
include the 2′-hydroxychalcones, 2′-OH-dihydrochalcones, 2′-OH-retro-chalcone, aurones (2-
benzylidenecoumaranone), and auronols.
ANTIMICROBIAL AGENTS- Antimicrobial agents have the abilitythat a drugkills or suppresses
the growth of microorganisms.
MECHANISMS OF ANTIMICROBIAL AGENTS
A. Inhibition of cell wall synthesis
B. Inhibition of functions of cellular membrane
C. Inhibition of protein synthesis
D. Inhibition of nucleic acid synthesis
E. Inhibition of folic acid synthesis
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F. Antiviral agents
PLANTS USED AS ANTIMICROBIAL AGENTS- Natural antimicrobials are secondary
metabolites that can be found in plants, animals, and microorganisms.Plants,especially herbs and
spices, are been given more attention in natural antimicrobial research, however; this chapter
focuseson the newly discoveries in fruits and vegetables.Plants are the largest biochemical and
pharmaceuticalstores ever known on our planet. These living stores are able togenerate endless
biochemical compounds. In their living, humanand animals are using only a small portion (1
to10%) of plantsavailable on Earth (250,000 to 500,000 species). Although, plants have many
other defense mechanismsagainst micro and macro organisms, but in this review we focusonly
on their chemical products of important pharmaceuticalcharacteristics (secondary metabolites).
Medicinal plants are rich ina numerous variety of secondary metabolites of
antimicrobialproperties such as saponines, tannins, alkaloids, alkenyl phenols,glycoalkaloids,
flavonoids, sesquiterpenes lactones, terpenoids andphorbol esters. Plants are able to develop
new, faster and naturalantimicrobials and then man-made remedies, and that is explaining why
plants succeed in its fightingagainst microbes since millions of years while human failed.
1. Garciniamangostana
2. Caesalpiniacoriaria
3. Psidiumguajava
4. Commiphoramolmol
5. Boswelliapapyrifera
6. Centratherumpunctatum
7. Pelargoniumsidoides
8. Thonningiasanguinea
9. Acacia nilotica,
10. Cinnamomumzeylanicum
11. Syzygiumaromaticum
12. Cinnamomumcassia
13. Prosopisjuliflora
ANTIBACTERIAL AGENTS- Anything that destroys bacteria or suppresses their growth or
their ability to reproduce. Heat, chemicals such as chlorine, and antibiotic drugs all have
antibacterial properties.
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MECHANISM OF ACTION- Antibacterial agents act against bacterial infection either by killing
the bacterium or by arresting its growth. They do this by targeting bacterial DNA and its
associated processes, attacking bacterial metabolic processes including protein synthesis, or
interfering with bacterial cell wall synthesis and function.
7. Mounyr, B., Moulay, S.,2016. Methods for in vitro evaluating antimicrobial activity, pp. 71-
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