ISOLATION AND IDENTIFICATION OF FUNGI ASSOCIATED WITH SWEET POTATO SPOILAGE

ISOLATION AND IDENTIFICATION OF FUNGI ASSOCIATEDWITH SWEET POTATO SPOILAGE

PRESENTED BY

TANIMU SANARIMAMMATRIC.NO: 10/66840

SUBMITTED TO

THE DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY

SCHOOL OFF APPLIED ARTS AND SCIENCE

THE FEDERAL POLYTECHNIC

P.M.B.55, BIDA

NIGER STATE.

IN PARTIAL FULFILMENT REQUIRED FOR THE AWARD OFNATIONAL DIPLOMA IN SCIENCE LABORATORY TECHNOLOGY.

FEBRUARY, 2013.1

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND INFORMATION

Maytenus senegalensis (Lam) excel is an African shrubs or trees

that go under the common name of Red spike-thorn, which

belong to the celastraccae family. Maytenus is one of the

most frequently used specie of the Maytenus Molina genus in

the African traditional medicine (along with M. Obscura, M.

peduncularis, M. putterlichwides, M. serrata and M. endata) (Novinger

2011).

More specifically Maytenus senegalensis is traditionally used as

anti microbial and anti inflammatory agents i:e. it is used

to treat respiratory ailments and inflammation. The use of

this anti inflammatory herbal drug is also common in other

African countries like Benin, Kenya, Zambia, Tanzania,

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Senegal and Zimbabwe. Maytenus senegalensis leaves are also used

to treat toothaches in India (Gurib and Muller, 2006).

In Zulu territories and Mozambique the leaves of Maytenus

senegalensis are macerated in water to be consumed twice a day

for the treatment of tuberculosis.

Recently, the anti-inflammatory activities of Maytenus

senegalensis ethanol extract (70%) were determined in Westar

albino rats by the carrageenan-induced paw edema method.

These extracts exhibited significant anti-inflammatory

activity (120mg/kg, per os), reducing edema by 51% and 35%

respectively. (INatse.Bio/med 2011)

1.2 ORIGIN OF MAYTENUS SENEGALENSIS

The origin of Maytenus senegalensis can be traced back to South

Africa, from the family celastraccae or confetti tree.

1.2.1 CULTIVATION OF MAYTENUS SENEGALENSIS

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The plant does not show a specific preference for a

particular soil type. It is more easily found where it grows

in groups. It also prefers a well drained humus rich fertile

soil (Huxley, 1992). It tolerates floods, heavy wind or

stagnant water. It is reported to have tolerated an animal

precipitation. Maytenus senegalensis is mainly cultivated in

East coast of Africa, Afghanistan, Indian and Arabia it is

best suited to tropical climates with a well distributed

rainfall of 1500 – 2000mm yearly from sea level to about

600mm altitude (Duke 1983). Maytenus senegalensis tolerates

warmer and more insipid climate than Amona senegalensis.

1.2.2 CLIMATIC CONDITION OF MAYTENUS SENEGALENSIS

Celastraceae is a large family comprising trees, shrubs and

woody lianas with a Gondwanan distribution (Simmons et al

2001). Geographically Maytenus senegalensisis apparently

confined from Ethiopia; westward to Angola Maytenus senegalensis

occupies a wide variety of habitats, from deciduous

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woodland, Hucket, sarab and woody grassland and also on

river band and swamp margins.

1.2.3 BOTANICAL DESCRIPTION

The Celastraceae classification at the generic level has

undergone changes. As currently circumscribed, the genus

Maytenus Molina, even after the reinstatement of Gumnosporia

is still clearly a heterogeneous group of species, a view

also supported by other authors (Rogers et al 1999) Maytenus

senegalensis is an evergreen shrub tree or more rarely a

shrublet, often straggling which grows up to 1m high,

charmed or with green to brown spines up to 24cm long

glabrous or very rarely with young branches lined or

angular. The lamina of Maytenus senegalensis is a pale to

deep green with a pale mudrib petiole up to 10mm long.

Spathilated, oblanceolated to ovated or elliptic margins up

to 9.5cm and 5cm.5

The flowers are dioecious, unscented with peduncle 1-30mm

long, pectical up to 7mm, circular to triangular lanceolate

sepals (Hutchings et al. 1996).

It has an untidy sparse, multi-stemmed evergreen shrub;it

branches into many long, thin whitish-grey branchlets and

twigs that curve down, with grey-green leaves towards the

tips.

The flowers bloom in dense, short racemes between the leaves

(May/June). The capsule is reddish, glubsoid or pyriform, 2-

6mm long, ovary 2-3 locular, smooth. Reddish-brown 1-2

seeds, glossy with a fleshy smooth rose-pink in yellowish

arid obliquely covering the lower (Fabricant et al. 2001)

1.3 OBJECTIVES OF THE STUDY

The objectives of this study seek to establish the

quantitative determination of phytochemical constituent of

Maytenus senegalensis.

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1.4 JUSTIFICATION OF STUDY

Establishment of the quality control parameters necessary to

profit herbal materials as raw materials for the manufacture

of herbal medicines is one of the goals of this study.

The use of Maytenus senegalensis as an anti-inflammatory agent

has been documented but however no studies were found to be

related to the botanical identification of Maytenus senegalensis

stem as herbal drugs and in sequence, these kind of study

have been conducted and the methodology includes the

analysis of the whole fragmentized and powdered stem of

Maytenus senegalensis by phytochemical determination or

screening of the plant stem.

1.5 SCOPE OF THE STUDY

The scope of the study includes the following

• Identification of plant.

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• Collection of the plant for the study

• Phytochemical determination of the plant.

1.6 LIMITATION OF STUDY

The limitations were encountered during the study.

Difficulties involved in identification and collection of

the plant due to geographical distribution of the plant.

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 MEDICINAL PLANT

About half the worlds medicinal components are still derived

or obtained from plants and other biota are, in general more

important in developing countries than in industrialized

nation. But even in these, where the focus is very much on

chemical discovery and synthesis of pharmaceutical, drug

products from biota are major contributors to the human

health services sectors of the economy, and plant derived

drugs contributed to the economy each year.

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It is also noteworthy that some of the most important drugs

of the past 50 years or so, which have revolutionized modern

medical practices, have almost all first been isolated from

plants and often from plants which for one purpose or

another have been employed in primitive or ancient societies

(schulkes 1986). These wonder drugs include the curare

altraloids; penicillin and other antibiotics; cortisone,

reserpine; vincoleublastine, the vetratrum alkaloid

podophyllotoxin; strophantine and other new therapeutically

agent (Schulkes and Swain 1976).

2.1.1 GLOBAL IMPORTANCE OF MEDICINAL PLANT

Ancient man is known to have utilized plants as drugs for

millennia based on current knowledge, at least in the west;

it is know that extracts of some of these plants are useful

in a crude. Form i: eafropa-belladonnes Tincture as an

antispasmodic, Ranvolfia serpentine roots for hypertension

and as a tranquilizer, Papaversomniferum extract or tincture

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as an analgesic etc. further, we know that at least 121

chemical substance of known structure are still extracted

from plants that are useful as drugs throughout the world

(Anon, 1982).

A large number of plants are used in traditional medical

practices, and have been for more than 3000 years, such as

in Chinese traditional medicine, Ayurvedic medium, clnani

medicine, etc, most of which probably exert therapeutic

effects and would be proven as such if they were properly

evaluated by western standard. Still further, plants have

been employed for centuries by primitive cultures; most of

these are less likely to pass the test o modern experimental

verification of efficacy. Finally, there are a large number

of so-called herbal remedies, mainly sold in health food

stores in developed countries many of which remain to be

verified for their real therapeutic effect. (Farnsworth and

Morris, 1976)

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2.1.2 USES OF MAYTENUS SENEGALENSIS AS A MEDICINAL PLANT

The root and bark of Maytenus senegalensis are used

traditionally in folk medicine of some African region for

the treatment of number of ailments which includes chest

paws, rheumatism, snake bites, diarrhea, eye infection and

dyspepsia (indigestion). (http://www.metafro.bd/prelude.)

The extract of the root and barks is used to treat severe

headache, an analgesic for skin rashes, muscles spasms,

excessive sweating and fever. The root of Maytenus senegalensis

chipped into beer has been used in Zambia as an aphrodisiac.

The roots which are slightly bitter are also mildly laxative

and are used in various part of tropical Africa for gastro

intestinal troubles, especially dysentery and a poultice of

the green leaves has been put on sores in Tanzania (Sosa et

al. 2007).

The leaves are used for malaria, yellow fever and

Trypanosomiasis. It is also used in fertility problems and

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also reduces the over excitement of brain in epileptic

patient. (Sosa et al, 2007).

2.2 PHYTOCHEMICAL

Phytochemical, also referred to as plant constituents are

active ingredients which have an effect on the body. There

is a wide range of phytochemical including single to complex

carbohydrates, colour, odour, glycosides e.t.c. There are

thousands of plant chemicals and new ones are still being

discovered (Lesley 1993).

Phytochemical as free radical scavengers, defusing free

radical damage, thus reducing wear and tear of the body.

When some phytochemicals are added to the diet, the capacity

of human genes to protect and restore optimal health is much

greater than previously recognized (Daniel 1997). Medical

research into these powerful plants extracts reveals a

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wealth of health benefits including the reduction of the

risk of cancer (Dragstael 1993)

Many phytochemicals are showing great promise as diseases

fighters in the body, boosting production of activities of

enzymes, which act by blocking carcinogenus, suppressing

malignant cells, or interfering with the process that can

cause heart diseases and stroke (Michael, 2005). As an

example, homocystein in is an amino acid produced by the

body, usually after eating meat, which has been established

to cause atherosclerosis, a build-up of fat and other

materials on the inside of arteries. Research has proved

that chefs deficient in, folic acid, vitamin B6 and B12 are

associated with higher blood levels of homocysteine and a

higher incidence of heart diseases and stroke adding

nutritional supplement to foods such as beans, potatoes,

banana and broccoli can reduce elevated homocysteine level,

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lowering the risk of heart attack and stroke (Davidson,

2002).

Another discovery has focused attention on the importance of

phytochemicals; pregnant women with the deficient in folic

acid have a higher incidence of babies with spinal rifida

and other neutral tube defects. These devastating birth

defects results from incomplete development of the foetal

brain, spinal cord, skull and spinal column, yet the

majority of cases are completely preventable with a healthy

diet (Davidson, 2003).

2.3.0 PHYTOCHEMICAL IN INDUSTRIES

2.3.1 PHYTOCHEMICAL IN FOOD INDUSTRIES

Phytochemical such as anthocyanidins constitute a large

family of differently coloured compounds and occurs in

countless mixtures in all parts of higher plants. They are

15

of great economic importance as fruit pigments and thus are

used in fruit puas, with and some beverages (Bendich, 1989).

2.3.2 PHYTOCHEMICAL IN CHEMICAL AND PHARMACEUTICAL

INDUSTRIES.

A bio-chemically active ingredients obtained from a legume

(Lonchocarpus species) is rotenone. This phytochemical is

useful as an insecticide and has been used as fish poison.

Rotenone is unstable in light and heat. It is very toxic to

fish, one of its main uses by nature people over the

centuries being to paralyze fish for capture and

consumption. Crystalline rotenone has an acute oral LD 50 of

60, 132 and 300mg/kg for guinea pigs, rats and rabbits

(Matumura, 1985). Acute poisoning in animals is

characterized by an initial respiration stimulation followed

by respiratory arrest (Shimkim and Anderson, 1936).

The anaesthetic-like action of nerves appears to be related

to the ability of rotenone to block electron transport in16

mitochondria by inhibiting oxidation linked to NADH2, thus

resulting in nerves conduction blockage (Brien, 1996).

Rotenone has been used tropically for treatment of head

lice, scabies and other ecto-parasite (Komori, 1994).

2.4 DIFFERENT TYPES OF PHYTOCHEMICALS IN PLANTS AND THEIR

FUNCTIONS.

TANNIN: (Commonly referred to as tarmac acid) are water

soluble poly-phenols that are present in many plants foods.

They have been reported to be responsible for decrease in

feed intake, growth rate, and feed efficiency, net

metabolism of energy and protein digestibility in

experimental animals. Therefore, foods rich in tannins are

considered to be of low nutritional value.

However, recent findings indicate that the major effect of

tannins was not due to their inhibition of food consumption

or digestion but rather the decreased efficiency is

converting the absorbed nutrients to new body substances.17

Incidences of certain cancers such as esophageal cancer

have been reported to be related to the consumption of

tannins – rich food such as betel nuts and herbal teas,

suggesting that tannins might be carcinogenic.

However, other reports indicate that carcinogenic

activities of tannins might be related to component

associated with tannins rather than tannins themselves.

(Biblack, 2000).

ALKALOIDS: Naturally alkaloids are nitrogenous compounds

that constitute the pharmacologically active basic principle

of flowering plants (Nchube et al, 2002). Alkaloids have

been divided into three (3) major classes depending on the

precursor of the final structure. The true alkaloids are

derived from amino acids which are basic and contain

Nitrogen in heterocyclic ring e.g. nicotine common alkaloids

ring structure includes the pyrimidine, pyrroles, indoles,

pyrrolidenes, I-soquinolines and piperidines. These

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compounds interfere with neutron-transmission and this

attribute to their ability to intercolate with DNA (Bidlack,

2000) Indoquinoline alkaloids from cryptoplepsis,

sanquinolenta displayed actively against a number of grain

negative bacterial and yeast (Silva et al. 1996).

TERPENSE: These comprise of the largest class of

phytonutrients. The most intensely studied terpense are

carotenoids, as evidenced by the many recent studied on beta

carotene. Terpense functions as anti-oxidants, protecting

lipids, blood and other body fluid from assault by free

radical oxygen species including singlet oxygen, hydroxyl

peroxide and superoxide radicals (Walker, 1993).

PHENOLS: These are obtained in berries and grapes. The

lactstanding phytonutrients feature of phenols is their

ability to block specific enzymes that cause inflammation.

They also protect. Platelet from clumping (Walker, 1993).

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FLAVONOIDS:These constitute one of the most characteristics

disease of compounds in higher plants many flavonoids are

recognized as flower pigments in higher plants. The

biological activities of flavonoids includes action against

allergies, inflammation, free radical, hepatotoxins,

platelets aggregation, microbes, ulcers, burns and tumors.

Flavonoids also inhibit specific enzymes. For example,

Flavonoids block the amgisotesin converting enzyme (ACE)

that raises blood pressure. Additionally, flavonoids blocks

the enzymes that produces oestrogen and thus reducing the

risk of oestrogen induced cancers, flavonoids also appear to

refined development of cataracts in individuals with inborn

errors in sugar metabolism such as diabetes (Davidson,

2002).

ISOFLAVONES: These phytonutrients are formed in beans and

other legumes, the enzymes that promote tumor growth. People

who consume traditional diets rich soy foods rarely

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experience breast, uterine and prostate cancer. Isoflavines

have gained popularity as an aid for those who consume

alcohol (Davidson, 2002).

STEROID: These occurs in most plant species green and

yellow vegetables contain significant amount their seeds

concentrate the steroid. These valuable phytonutrients are

present in the seed of pumpkins, yams, soy rice and herbs,

steroid function in competing with dietary cholesterols for

uptake in the intestine. It blocks the uptake of cholesterol

and facilitate its excretion from the body steroids also

block the development of tumors in colon, breast and

prostate glands. (Walker, 1993.)

ANTHRAQUINONE:- Athraquinone compounds have the formula

STRI where R1 and R2 are methyl R3 propyl or R1 and R2 are

ethyl and R3 is methyl and A is a cosmetically acceptable

anion, when employed in an isatinlamine dye system for the

dyeing of hair enable the hair colorist to develop a full

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range of shades for the isatinlamine dye system. The

compounds of formular where in R1 and R2 are methyl and R3

is propyl is preferred. Compositions and a method of use

employing same are disclosed (Evans, 1989).

VOLATILE OIL: Posses biological and anti-inflammatory

effects. oils from annamonium, osmophloeum have been shown

to possess anti-bacterial activity against Escherichiacoli,

Enterococusfaeculis, Staphylococcusaureus andVibrioporahaemolyricus with

cinnamaldehydes being the anti bacterial components isolated

(Nchube et al; 2007, Bidlack, 2000 and Wallace, 2004)

GLYCOIDES: - Glycoside is antimicrobial compounds in

plants that contain carbohydrates molecules; steroid and

triterpense can accumulate as glycosides conjugates in

substantial quantity in plants. These glycosides which

include steroids glycol alkaloids are commonly referred to

as saponms. (Nchube et al; 2007)

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CHAPTER THREE

3.0 MATERIAL AND METHODOLOGY

3.1 MATERIALS/REAGENTS

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Maytenus senegalensis (confiffetti tee),Automated pipette,

Beaker, Buchner funnel, Volumetric flask ,Reflux condenser,

Whatman paper, Spectrophotometer, Conical flask, Separating

funnel, Water bath, watch.

REAGENTS

Methanol, Parafilm, Alcohol, 10% of Hcl, Mgo, Sucrose,

Conc.H2SO4, Selenium, Isobutyl Alcohol, Benzene.

3.2 COLLECTION OF PLANT SPECIMENS

The stem of Maytenus Senegalensis was collected from a tree

along Lemu road near Bida town Niger state. The stem was

identified and authenticated by a botanist in biology unit

of The Department of Science Laboratory Technology, The

Federal Polytechnic Bida Niger, State.

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3.3 METHODS

3.3.1 PREPARATION OF SAMPLE

The fresh stems were air dried at room temperature until

they are dried. The dried stem was them blended using a

blending machine and stored in a clean glassware container

until need for the analysis.

3.3.2 AQUEOUS EXTRACTION

20g of powdered stem was extracted with 200ml of different

solvent like methanol, benzene, chloroform, alcohol,

parafilm etc. by using water extraction method. The

extraction was carried out for 1hr, 11/2hr differently and

the solvent used was recovered by refluxing back so as to

get a concentrated extract of the tree stem.

3.4.0 PHYTOCHEMICAL DETERMINATION

25

3.4.1 QUANTITATIVE ANALYSIS

3.5.0 TANNIN

0.20ml of sample was measured into a 50ml beaker, 20ml of

50% methanol was added and covered with parafilm and placed

in a water bath at 77-80oC for 1 hour. It was shaking

thoroughly to ensure a uniform mixing. The extract was

quantitatively filtered using a double layered Whatman No 41

filter paper into a 100ml volumetric flask, 20ml water

added, 2.5ml folin-Denis reagent and 10ml of 17% na2Co3 were

added and mixed properly. The mixture was made up to mark

with water mixed well and allowed to stand for 20min. The

bluish –green color which developed at the end of range 0-

10ppm were treated similarly as 1ml sample above.

The absorbance of the Tannic acid standard solutions as well

as samples were read after color development on a spectronic

21D spectrophotometer at a wavelength of 760nm. % Tannin was

calculated using the formula:26

%TANNIN =absorbance of sample X average gradient factor X

Dilution factor

Vol. of

extract taken X 10,000

3.5.2 ALKALOIDS

This is a distillation and titrimetric procedure 2ml of

sample extract was weighed into a 100ml beaker and 20mls of

80% absolute alcohol added to give a smooth paste. The

mixture was transferred to a 250ml flask and more alcohol

added to make up to 100ml and 1g magnesium oxide added. The

mixture was digested in a boiling water bath for 1.5hrs

under a reflux air condenser with occasional shaking. The

mixture was filtered while hot through a small Buchner

funnel. The residue was returned to the flask and re-

digested for 30min with 50ml alcohol after which the alcohol

will be evaporated, adding hot water to replace the alcohol

lost. When all the alcohol has been removed, 3 drops of 10%27

HCL was added. The whole solution was later transferred into

a 250ml volumetric flask 5ml of zinc acetate solution and

5ml of potassium ferrocyanide solution was added, thoroughly

mixed to give a homogenous solution.

The flask was allowed to stand for a few minutes, filtered

through a dry filter paper and 10ml of the filtrate was

transferred into a seperatory funnel and the alkaloids

present were extracted vigorously by shaking with five

successive portions of chloroform. The residue obtained was

dissolved in 10ml hot distilled water and transferred into a

kjeldahl tube with the addition of 0.20g sucrose and 10ml

Conc.H2SO4 and 0.02g selenium for digestion to a colorless

solution to determine %N by Kjeldahl distillation method.

%Nitrogen got is converted to % total alkaloid by

multiplying by a factor of 3.26 i.e. % Total alkaloid = %N X

3.26

3.5.3 SAPONIN

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The Spectrophotometric method of Brunner (1984) was used for

Saponin Analysis.

1ml of sample extract was weighed into a 250ml beaker and

100ml of isobutyl alcohol was added. The mixture was shaken

on a UDY shaker for 5 hours to ensure uniform mixing.

Thereafter the mixture was filtered through a whatman No1

filter paper into a 100ml beaker and 20ml of 40% saturated

solution of magnesium carbonate was added. The mixture

obtained with saturated MgCO3 was again filtered through a

Whatman No1 filter paper to obtain a clear colorless

solution. 1ml of the colorless solution, was pipetted into

50ml volumetric flask and 2ml of 5% FeCL3 solution was

added and made up to mark with distilled water. It was

allowed to stand for 30min for blood red color to develop.

0-10ppm standard Saponin solutions were prepared from

saponin stock solution. The standard solutions were treated

similarly with 2ml of 5% FeCL3 solution as done for 1ml

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sample above. The absorbance of the sample as well as

standard saponin solutions were read after color development

in a Jenway V6300 Spectrophotometer at a wavelength of

380min

% Saponin = Absorbance of sample X gradient factor X

dilution factor

Vol. of sample

X10000

3.5.4 PHLOBATANNIN

0.50ml of sample extract was weighed into 50ml beaker .20ml

of 50% Methanol was added and covered with parafilm and

placed in a water bath set at 77-800C for 1 hour.

The mixture was properly shaken to ensure uniform mixing and

later filtered through a Whatman No 1 Filter paper into a

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50ml volumetric flask using aqueous methanol to rinse, and

make up to mark with distilled water.

1ml of the sample extract was pipetted into a 50ml

volumetric flask, 20ml water 2.5ml Folin-Dennis reagent and

10ml of 17% Sodium carbonate were added to the solution in

the 50ml Flask. This mixture was homogenized thoroughly for

20mins.0-5mg/ml of Phlobatannin standard concentration were

prepared from100mg/ml phlobatannin stock solution and

treated like sample above.

The absorbances of standard solutions as well as sample were

read on a Spectronic 21D spectrophotometer at a wavelength

of 550nm.%Phlobatannin was calculated using the formula:

Absorbance of sample X gradient factor X dilution

factor

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Wt. of sample

X 10,000

3.5.5 PHENOL

0.20ml of sample extract was weighed into a 50ml beaker,

20ml of acetone was added and homogenize properly for 1hr to

prevent lumping. The mixture was filtered through a Whatman

No.1 filter paper into a 100ml Volumetric Flask using

acetone to rinse and made up to mark with distilled water

with thorough mixing.

1ml of sample extract was pipetted into 50ml Volumetric

flask,20ml water added,3ml of phosphomolybdic acid added

followed by the addition of 5ml of 23% NaCO3 and mixed

thoroughly, made up to mark with distilled water and allowed

to stand for 10min to develop bluish-green colour.

Standard Phenol of concentration range 0-10mg/ml were

prepared from 100mg/l stock Phenol solution from Sigma-

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Aldrich chemicals, U.S.A. The absorbances of sample as well

as that of standard concentrations of Phenol were read on a

Digital Spectrophotometer at a wavelength of 510nm.The

percentage Phenol is calculated using the formula:

The percentage Phenol = Absorbance of sample X gradient

factor X dilution factor

Vol. of

sample extracts X 10,000

3.5.6 FLAVONOIDS

0.50ml of sample extract was weighed into a 100ml beaker and

80ml of 95% Ethanol added and stirred with a glass rod to

prevent lumping. The mixture was filtered through a Whatman

No.1. Filter into a 100ml volumetric flask and made up to

mark with Ethanol. 1ml of the extract was pipetted into 50ml

volumetric flask, four drops of Conc.HCL added via a

dropping pipette after which 0.5g of magnesium turnings

33

added to develop a magenta red coloration. Standard

flavonoid solution of range 0-5ppm were prepared from 100ppm

stock solution and treated in a similar way with HCL and

magnesium turnings like sample. The absorbance of magenta

red coloration of sample and standard solutions were read on

a digital Jenway V6300 Spectrophotometer at a wavelength of

520nm. The percentage flavonoid is calculated using the

formula:

% flavonoids = Absorbance of sample X average gradient

factor X dilution factor

Vol. of sample X 10,000

3.5.7 GLYCOSIDE

1.0ml of extract was pipette into a 250ml Conical Flask.50ml

Chloroform was added and shaken on a Vortex Mixer for 1hr.

The mixture was filtered into 100ml conical flask and 10ml

34

pyridine; 2ml of 2% sodium nitroprusside were added, shaken

thoroughly for 10 minutes. 3ml of 20% NaOH was later added

to develop a brownish yellow colour.

Glycoside standard of concentrations which range from 0-

5mg/ml were prepared from 100mg/ml stock Glycoside standard.

The series of standards 0-5mg/ml were treated similarly like

sample above.

The absorbances of sample as well as standards were read on

a Spectronic 21D Digital Spectrophotometer at a wavelength

of 510nm. % Glycoside was calculated using the formula:

% Glycoside = Absorbance of sample X gradient factor X

dilution factor

Vol. of sample

extracts X 10000

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3.5.8 STEROIDS

0.50ml of sample extract was weighed into a 100ml

beaker .20ml of Chloroform-Methanol (2:1) mixture was added

to dissolve the extract upon shaking for 30minutes on a

shaker. The whole mixture was later filtered through a

Whatman No.1filter paper into another dry clean 100ml

Conical Flask/Beaker.

The resultant residue was repeatedly treated with

Chloroform-Methanol mixture until free of Steroids.1ml of

the filtrate was pipetted into a 30ml test tube and 5ml of

alcoholic KOH was added and shaken thoroughly to obtain a

homogenous mixture. The mixture was later placed in a water

bath set at 370C-400C for 90minutes.It was cooled to room

temperature and 10 ml of petroleum ether added followed by

the addition of 5ml distilled water. This was evaporated to

dryness on the water bath. 6ml of Liebermann Burchard36

reagent was added to the residue in dry bottle and

absorbance taken at a wavelength of 620nm on a Spectronic

21D digital Spectrophotometer.

Standard Steroids of concentration of 0-4mg/ml were prepared

from 100mg/ml stock steroid solution and treated similarly

like sample as above. % Steroid was calculated using the

formula:

% Steroid = Absorbance of Sample X Gradient X Dilution

Factor

Vol. of sample extracts X 10000

3.5.9 CARDENOLIDES

0.50ml of extract was accurately weighed into 100ml beaker

followed by the addition of 50ml of Chloroform to dissolve

the extract.0.20 of Sodium bicarbonate powder(NaHCO3) was

added after complete dissolution of extract in Chloroform to

37

remove any free acid. The mixture was later transferred into

a 250ml Separatory funnel and thoroughly shaken to allow the

two layers to separate.5 drops of acetic anhydride was

added to allow the mixture to be cleared and free of cloudy

suspensions. This was filtered through a Whatman No 1 Filter

paper into 100ml Volumetric Flask and made up to mark with

chloroform

Standard cardenolides solutions of concentration 0-10mg/ml

were prepared from 100mg/ml stock cardenolide solution and

treated similarly as sample above to obtain the gradient

factor. % Cardenolide was calculated using the formula:

% Cardenolide =Absorbance of sample X gradient factor X

dilution factor

Vol. of

sample X 10000

3.5.10 ANTHRAQUINONES

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0.50ml of sample extract was weighed into 250ml beaker and

60ml benzene added and stirred with a glass rod to prevent

lumping. This was filtered into 100ml volumetric flask using

Whatman No.1 filter paper.10ml of filtrate was pipetted into

another 100ml volumetric flask and 0.2% Zinc dust was added

followed by the addition of 50ml hot 5% NaOH solution. The

mixture was heated just below boiling point for five minutes

and then rapidly filtered and wash once in water. The

filtrate was again heated with another 50ml of 5% NaOH to

develop a red colour.

Standard Anthraquinone solution of range 0-5mg/l were

prepared from 100mg/l stock Anthraquinone and treated in a

similar way with 0.2% Zinc dust and NaOH like sample. The

absorbances of sample as well as that of standard

concentrations were read on a Digital Spectrophotometer at a

wavelength of 640nm.The percentage anthraquinone is

calculated using the formula:

39

Percentage anthraquinone =Absorbance of sample X gradient

factor X dilution factor

Vol. of

sample X 10,000

3.5.11 TERPENE

0.50ml of sample extract was weighed into a 50ml Conical

Flask, 20ml of 2:1 Chloroform-Methanol mixture was added,

shaked thoroughly and allowed to stand for 15minutes.The

mixture was later centrifuged for another

15minutes.Supernatant obtained was discarded, and the

precipitate was re-washed with another 20ml chloroform-

methanol mixture for re-centrifugation.

The resultant precipitate was dissolved in 40ml of 10%

Sodium Deodocyl Sulphate solution.1ml of 0.01M Ferric

Chloride solution was added to the above at 30s interval

shaked well, and allowed to stand 30minutes.Standard

40

Terpenes of concentration range 0-5mg/ml were prepared from

100mg/l stock Terpenes solution from Sigma-Aldrich

chemicals, U.S.A. The absorbances of sample as well as that

of standard concentrations of Terpenes were read on a

Digital Spectrophotometer at a wavelength of 510nm.The

percentage Terpene is calculated using the formula:

%terpene = Absorbance of sample X gradient factor X

dilution factor

Vol. of

sample extracts X 10,000

41

CHAPTER FOUR

4.0 RESULT

Table 1: Result showing the amount of some phytochemicals inaqueous extract of Maystenus senegalensis Stem

Phytochemical Amount (%)

Alkaloids 0.29±0.00

Tannins 0.07±0.00

Phlobatannins 0.12±0.00

Saponins 0.30±0.00

Anthraquinones 0.06±0.00

Cardenolides 0.03±0.00

Phenol 0.05±0.00

42

Cardicglycosides

0.09±0.00

Amounts are mean values of duplicate determinations

And expressed in Mean±SEM

4.1 DISCUSSION OF RESULT

The quantitative phytochemical screenings were carried out

on aqueous extract of Maytenus senegalensis where alkaloid,

Tannins, Phlobatonnous, Saponins, anthraquinone, Flavonoids,

Steroids and Cardenolides were totally absent in the

extract.

Medicinal properties of plants are normally dependent on the

presence of certain phytochemical principles such as

alkaloids, anthraquinones, glycosides, Saponins, tannins and

Flavonoids which are the bioactive bases responsible for the

pharmacological property (Ebana et al, 1993). Medicinal

plants contain these pharmacologically active principles

which over the years have been exploited in traditional

43

medicinal practice for the treatment of various ailments

(Adebanjo et al,1983).

Tannins have been reported to prevent the development of

micro-organisms by precipitation microbial protein and

making nutritional proteins unavailable to them (Egwim et

al, 2002). Tannins have been found to form irreversible

complexes with prolinerich protein (Shimada,2006) resulting

in the inhibition of cell protein synthesis. Parekh and

Chanda (2007) reported that tannins are known to react with

proteins to provide the typical tannin. Effect which is

important for the treatment of inflamed ulcerated tissues

Herbs that have tannins as their main components are

astringent in nature and are used for treating intestinal

disorders such as diarrhea and dysentery (Dharmananda,2003).

These observations therefore suggest the use of aqueous

extract of Maytenus senegalensis leaf in herbal cure remedies

for diarrhea and dysentery. the biological activities of

44

tannins showed that tannins have anticancer activity and can

be used in cancer prevention, thus suggesting that aqueous

extract of Maytenus senegalensis stem has potential as a

source of important bioactive molecules for the treatment

and prevention of cancer.

Alkaloids is another secondary metabolite compound that is

of medicinal importance. Classes of alkaloids are among the

highest poisons known, some classes of alkaloids have also

been proved to be useful in correcting some renal disorders

(Egwin et al, 2002). One of the most common biological

properties of alkaloids is their toxicity against cells of

foreign organisms. These activities have been widely studied

for their potential use in the elimination and reduction of

human cancer cell lives (Nobori et al, 1994). Alkaloids

which are one of the largest groups of phytochemicals in

plants have amazing effects on humans and this has led to

the development of powerful pain killer medications.

45

According to (Just et al, 1998) Saponins was found to be

present in aqueous extract of Maytenus senegalensis stem and

has supported the usefulness of this plant in managing

inflammation. Steroidal compounds present in aqueous extract

of Maytenus senegalensis stem are of importance and interest due

to possible relationship they have with various anabolic

hormones including sex hormones (Okwu, 2001). Earlier study

of Quinlan et al, (2002) on steroidal extracts from some

medicinal plants revealed antibacterial activities on some

bacterial isolation. Antiviral property of steroids was also

confirmed by (Neumann et al, 2004).

The appreciable amount of phytochemicals of pharmacological

importance observed in this study in the aqueous extract of

Maytenus senegalensis stem had established the scientific bases

of future tradition and belief behind the use of Maytenus

senegalensis plant in the treatment of various diseases. To

46

buttress this point, Maytenus senegalensis has multiple uses

such as pharmacological and economic uses.

CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

The result of the work done supports the facts that the

medicinal plants possess various active compounds or

substances which are useful for therapeutic purpose and has

effect against some diseases.

The quantitative screening of aqueous extract of Maytenus

senegalensis could help, this might account for the reasons

why they (i:e medicinal plant) are used for treatment of

diseases.

5.2 RECOMMENDATION

47

Research institution should focus their attention on

rural areas for medicinal plants, since they are more

abundant there and possible load for a very way of

conserving this medicinal plant in a garden.

More antimicrobial testing should be conducted using

different test organisms especially clinical isolated

organisms since the plant show significant activity

against the organisms used.

Further more investigation on the plant Maytenus

senegalensis is also imperative. This investigation

includes antimicrobial properties of Maytenus

senegalensis.

It is recommended that effects should be geared towards

extraction of aqueous extract of antimicrobial

substances from plant origin, so that toxicity

associated with synthetic antimicrobial substances

could be reduced or if possible eliminated

48

REFERNCES49

Foraz, M; Mohammed K, Naysannch, G and Hammed, R.V. (2003):Phytochemical

screening of some species of Iranian plants.Iranian journal of Pharmaceutical Research3:77-82.

MC. Graw-Hill-encyclopedia (1787): Flavonoids improvescirculation on the conary

Arteries.Okerulu and A (2001): Method for Phytochemical analysis.

Parekh and Chanda (2007)

Chlorine, Nitrogen and Potassium, in plantand man. Modern Biology Pp 120-150.

Osei A. (2001): Calcium as source of limestone uses ofMagnesium, Sulphur and

Sodium, Calcium, Chlorine and Phosphorus.Osei Ababio Text Book edition Pp. 120 – 125.

Okerulu and A (2001): Method for Phytochemical analysis.

Caveli, A. j, JSI Food Agric, 1955, 6,479 procedures fordetermination of Nitrogen and phosphorus in plant.

Davies W.L analyst (London), 1932, 57, 79 procedures fordetermination of Chloride in plants.

50

Susan E. Eichorn, (1999): Historical development ofinorganic element Plant Biology

Pp.518 – 521.

Perkin Elmer Crop, (1968): Analytical methods for AtomicAbsorption Spectrometry,

Perkin-Elmer corp.

Piper, C.S. (1944): Soil and plant analysis, inter sciencepublisher, Inc. New York

Association of official analysis Chemist(A.O.A.C) (1970) Official Method of AnalysisEd. 11, Washington.

Spencer R. (1961): Department Memorandum No. 30 Herald,W.R.1966. Calcium and

Magnesium P.999 in C.A Black (e.d). Methods ofsoil analysis Part 2 Amer. Soc. Agron.Madison, Wis.

Perkin-Elmer corp (1996): Analytical Method for AtomicAbsorption Spectrometry,

Perkin – Elmer Corp. Nwwalk Connectible

Susan E. Eichhorn, (1999): History of Inorganic analysis.

51

APPENDIX 1

Alkaloids

Column1

Mean 0.29

StandardError 0.002

Median 0.29

Mode #N/A

52

StandardDeviation

0.002828

SampleVariance 8E-06

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.004

Minimum 0.288

Maximum 0.292

Sum 0.58

Count 2

APPENDIX 2

Tannins

Column1

53

Mean 0.07

StandardError 0.01

Median 0.07

Mode #N/A

StandardDeviation

0.014142

SampleVariance 0.0002

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.02

Minimum 0.06

Maximum 0.08

Sum 0.14

Count 2

54

APPENDIX 3

Phlobatannins

Column1

Mean 0.12

StandardError 0.01

Median 0.12

Mode #N/A

StandardDeviation

0.014142

SampleVariance 0.0002

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.02

Minimum 0.11

55

Maximum 0.13

Sum 0.24

Count 2

APPENDIX 4

Saponins

56

Column1

Mean 0.3025

StandardError 0.0015

Median 0.3025

Mode #N/A

StandardDeviation

0.002121

SampleVariance

4.5E-06

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.003

Minimum 0.301

Maximum 0.304

Sum 0.605

Count 2

APPENDIX 5

Anthraquinone

57

Column1

Mean 0.06

StandardError 0.01

Median 0.06

Mode #N/A

StandardDeviation

0.014142

SampleVariance 0.0002

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.02

Minimum 0.05

Maximum 0.07

Sum 0.12

Count 2

APPENDIX 6

Cardenolides

Column1

Mean 0.025

StandardError 0.005

Median 0.025

58

Mode #N/A

StandardDeviation

0.007071

SampleVariance 5E-05

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.01

Minimum 0.02

Maximum 0.03

Sum 0.05

Count 2

APPENDIX 6

59

Phenol

Column1

Mean 0.045

StandardError 0.002

Median 0.045

Mode #N/A

StandardDeviation

0.002828

SampleVariance 8E-06

Kurtosis#DIV/0!

Skewness#DIV/0!

Range 0.004

Minimum 0.043

Maximum 0.047

Sum 0.09

Count 2

60

APPENDIX 7

Cardio glycosides

Column1

Mean 0.0925

StandardError 0.0015

Median 0.0925

Mode #N/A

StandardDeviation

0.002121

SampleVariance

4.5E-06

Kurtosis#DIV/0!

Skewness#DIV/0!

61

Range 0.003

Minimum 0.091

Maximum 0.094

Sum 0.185

Count 2

62