III.

MATERIALS AND METHODS

Collection of Plant Materials

Medicinal plants chosen for the investigation were procured from

Visakhapatnam district situated in the southern part of Andhra Pradesh

respectively. These medicinal plants were collected from their

geo-ecological habitats based on their folkloric use as they were

obtained from traditional medical practitioners from the areas. The plant

materials were cleaned and powdered. The botanical names, family names,

English names and parts used are presented in Table-1 and Fig. 1.

Preparation of Plant Extracts

The chosen plant materials were carefully selected by using previous

work, folklore reports as well as records of flora gamble for the study and

the raw materials were collected from several locations of Andhra Pradesh,

during the flowering periods of these plants. The collected plants were

identified taxonomically, by ethno botanist, Department of Botany, Andhra

University, Visakhapatnam. The plant parts were collected, cleaned to free

from residual soil and air-dried at room temperature. The leaves and stems

were harvested; later the stem barks peeled off while still fresh, made

into small portions and dried at room temperature under shade for one

month. The dried plant materials were then ground to a fine powder using

a laboratory mill and passed through a 24 mesh sieve to generate a

homogeneous powder. The powder was then stored in a temperature-

controlled, dry and dark storage room until extraction. The coarsely

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powdered materials weighed and extracted with hexane, chloroform and

methanol in sequential order of polarity using a soxhlet extractor for five to

six hours at temperature not exceeding the boiling point of the solvent. For

each gram of dry material 2ml of solvent was used. The extracted solvents

were filtered through whatman no-1 filter paper and subsequently

concentrated under reduced pressure (in vacuum at 40°c) using a rotary

evaporator. The residue obtained were designated as crude extracts and

stored in a freezer at - 20°C until assayed.

Preliminary Qualitative Screening of Phytochemicals

Phyto-medicine represents one of the most important fields of

traditional medicine all over the world and are of prime importance to the

health of individuals and communities. The medicinal values of these

economically important plant species is due to presence of some chemical

substances which produce a definite physiological action on human body

like alkaloids, tannins, flavonoids and saponin etc. (Khan et al., 1990; Edeoga

et al., 2005). Phytochemical screening assay was used to identify the presence

of the various types of Phytochemicals in a mixture and an important tool in

bioactive compound analyses. The different qualitative chemical tests were

performed for establishing the profile of the leaf extracts for its chemical

composition. Preliminary phytochemical screening of alkaloids, glycosides/

reducing sugars, coumarins, flavonoids, phenols, quinones, tannins,

terpenoids and saponins present in plant extracts were performed using

standard methods. The following tests were performed to detect various

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phyto-constituents present in them. A brief summary of the experimental

procedures for the various phytochemical screening methods for the

secondary metabolites were given below.

Detection of Alkaloids

Plant Extracts were dissolved individually in diluted HCL

(Hydrochloric acid) and filtered.

Mayer’s Test

Filtrates were treated with Mayer‟s reagent (dissolve 1.36grms of

mercuric chloride and 5 gm of potassium iodide in 100ml of distilled water).

Formation of yellow color precipitate indicates presence of alkaloids.

Detection of Cardiac Glycosides

2ml of Glacial acetic acid and few drops of 5% ferric chloride were

added to 0.5% of the extract, this was under layered with 1ml of

concentrated sulphuric acid. Formation of brown ring at the interface

indicates the presence of cardiac glycoside.

Detection of Coumarins

10% NaoH was added to 1 ml of plant extract. Formation of yellow

colour indicates the presence of coumarins.

Detection of Flavonoids

5ml of dilute Ammonia solution was added to a portion of aqueous

filtrate of each plant extract followed by addition of concentrated sulphuric

acid. If a yellow colouration of solution is observed in each extract, indicates

the presence of flavonoids.

63
Detection of Glycosides

Legal’s Test: 3ml of chloroform and 10% ammonium solution was

added to 2ml of plant extract. Formation of pink colour indicates the

presence of glycosides.

Detection of Phenols

Ferric Chloride Test

Extract was treated with 3 to 4 drops of 5% ferric chloride solution.

Formation of bluish black colour indicates the presence of phenols.

Detection of Quinones

1 ml of concentrated sulphuric acid was added to 1 ml of each plant

extract. Formation of red colour indicates the presence of quinones.

Detection of Tannins

Ferric Chloride Test

5mg of extract was dissolved in 5ml of distilled water and then few

drops of 5% ferric chloride solution were added. The formation of blue green

color indicates tannins.

Detection of Terpenoids

Salkowski Test

5ml of each extract was mixed in 2ml of chloroform, and concentrated

sulphuric acid was added carefully until to form a layer. If a reddish brown

coloration of the interface is formed it indicates positive result for the

presence of terpenoids.

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Detection of Saponins

2ml of distilled water was added to each plant extracts and shaken in

a graduated cylinder for 15 min for proper mixing. Formation of 1 cm foam

indicates the presence of saponins.

Quantitative Determination of Phytochemicals in Studied Medicinal

Plants

The quantity of various phyto-chemicals presented in studied

medicinal plants were determined as follows

Total Phenolic Content

Total phenolic content was determined using Folin-Ciocalteau

reagent (FC reagent) and gallic acid used as the standard according to the

method by Rasineni et al. (2008). About 500 mg of milled plant material

powder was weighed and homogenized in 10 ml of n-hexane. The

homogenate was centrifuged at 10,000 × g for 20 minutes and the

supernatant was used for the determination of total phenolics as follows.

About 0.5 ml of Folin-Ciocalteau 2 N reagent was added to 2.5 ml of the

supernatant and then followed the addition of 2 ml of 10% sodium carbonate

in ethanol. The mixture was incubated for 5 minutes at 20ºC and then the

absorbance read in triplicates at wavelength of 650 nm.

The gallic acid standard was prepared by dissolving 100 mg of Gallic

acid (SDS Lab- Chem Industry Bombay-India), in 100 ml of distilled water to

make the standard stock. This was serially diluted into the working range of;

0.5, 1, 2, 4, 8 and 16 mg/100 ml. To each 2.5 ml of the serially diluted

standard, 2 ml of the 10% (w/v) sodium carbonate solution and 0.5 ml of

65
Folin-Ciocalteau 2N were added and incubated for 5 minutes. The

absorbance was read at 750 nm before 15 minutes. The mixture of distilled

water, 10% sodium carbonate and Folin-Ciocalteau 2N was used as the

blank. The absorbencies were read using UV-VIS spectrophotometer (UV-

1700 Pharmaspec, UV-VIS Spectrophotomer, Shimadzu Japan). The total

phenol content was expressed as mg/g dry weight Gallic acid equivalent.

Flavonoids

5 ml of 2% aluminium trichloride in methanol with the same volume

of aqueous plant extract. The absorbance was then read at 420 nm after one

hour of incubation against a blank sample of 5 ml extract solution with 5 ml

methanol without aluminium trichloride. The total flavonoids content was

determined using a standard curve of pyrocatechol ranging from 0.5 to 16

mg/100 ml, and the results expressed as mg/g of pyrocatechol equivalents.

Alkaloids

Alkaloids were determined according to the method by Edeoga et al.,

2005) with slight modifications as follows: 2.5 g of the plant material powder

was extracted using 100 ml of 20% acetic acid in ethanol. The solution was

covered for almost 4 hours. Filtrate was concentrated to 25 ml. Concentrated

ammonium hydroxide was added stepwise to attain precipitation. The

whole solution was kept as such so that precipitate was settled properly.

Collected precipitate was washed with dilute ammonium hydroxide and

finally filtered. Filtrate was discarded and pellet obtained was dried and

weighed (Edeoga et al., 2005).

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Saponins

Similarly, saponin was determined as per the method by Edeoga et al.

(2005) with slight modifications: 10 g of a sample was mixed with 100 ml of

20% aqueous ethanol. The mixture was kept for 4 hours on water bath

shaker at 55°C. Filtrate was again extracted in same manner. The combined

extract was concentrated to 40 ml over water bath at 90°C. The obtained

concentrate was transferred into a separating funnel and 10 ml of diethyl

ether was added to it. After shaking vigorously aqueous layer was recovered

and ether layer was discarded. The process was repeated and to the aqueous

layer n-butanol was added. The whole mixture was washed in separating

funnel twice with 10 ml 5% of aqueous NaCl. Upper part was retained and

heated in water bath till to evaporation. Latter it was dried in oven to a

constant weight (Edeoga et al., 2005).

Tannins

The tannins were determined as follows: 2 g of plant powder was

extracted thrice in 70% acetone. After centrifugation the sample supernatant

was removed. Different aliquots were taken and final volume to 3 ml was

adjusted by distilled water. The solutions after vortexing were mixed with 1

ml of 0.016M K₃Fe (CN)₆, followed by 1 ml of 0.02 M FeCl₃ in 0.10 M HCl.

The Vortex process was repeated and the tubes were kept as such for 15 min.

The stabilizer was prepared in the ration of 3:1:1 of water, H₃PO₄ and 1%

gum Arabic, and then 5 ml was added to the mixture followed by re-

vortexing. Absorbance was measured at 700 nm against blank. Standard

67
curve was plotted using various concentrations of 0.001M Gallic acid

(Gurib-Fakim, 2006).

Physico-Chemical Properties

Studied plant materials were evaluated by following physicochemical

parameters physical constants:

Extractive Values

It is employed for material to which as yet no suitable chemical or

biological assays exist. Extractive values determine amount of active

constituents present in a given plant material in a given solvent. Percentage

of dry extract was calculated in terms of air-dried stem powder.

Water Soluble Extractive Value

Accurately weighed 5 gm of powdered drug was taken in the glass

stopper conical flask. The powder was macerated with 25 ml of distilled

water for 6 hours with frequent shaking, then allowed to stand for 18 hours.

After completion of 18 hours, filtered the content in a flask and transferred

the filtrate in tarred flat bottom porcelain dish. Then filtrate was evaporated

to dryness on boiling water bath and finally dried at 105oC for 6 hours. Later

it was cooled in desiccators for 30 min and weighed. Finally content of

extractable matter was calculated in milligrams per gram of air dried

material. The results of extractive value are given in table 7.

Alcohol Soluble Extractive Value

Accurately weighed 5gm of powdered drug placed in the glass

stopper conical flask and macerated with 25 ml of methanol (95%) for 6

hours with frequent shaking, the mixture allowed standing for 18 hours.
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After completion of 18 hours, the sample was filtered rapidly taking care not

to lose any solvent. The filtrate was transferred in tarred flat bottom

porcelain dish. Filtrate was evaporated to dryness on water bath, dried at

1050C for 6 hours cooled in desiccators for 30 min and weighed. Finally

content of extractable matter was calculated in milligrams per gram of air

dried material.

Loss on Drying (LOD)

Loss on drying is the loss in weight in percent w/w resulting from

loss of water and volatile matter of any kind that can be driven off under

specific conditions. 2 gm of air-dried drug reduced to powder was placed in

a crucible of silica. Originally the crucible was cleaned and dried and weight

of empty dried crucible was taken. The powder was spread in a thin uniform

layer. The crucible was then placed in the oven at 105oC. The powder was

dried for 4 hours and cooled in a desecrator to room temperature and weight

of the cooled crucible plus powder was noted.

Ash Values

Ash values are indicative to some extent of care taken in collection

and preparation of drug for market and of foreign matter content of natural

drug. The object of ash preparation is to remove all traces of organic material

interfering in an analysis of inorganic elements.

Total Ash Value

Total Ash value is quite reliable parameter to judge types of

adulteration. This method is designed to measure total amount of material

69
remaining after ignition. It includes both physiological ash and non-

physiological ash. The physiological ash is derived from plant tissue itself

and non-physiological ash is residue of extraneous matter (e.g. sand and

soil) adhering to plant surface. 2 gm of air-dried powder was taken in tarred

platinum crucible. Drug material was spread in fine even layer at bottom of

the platinum crucible. This platinum crucible with drug material was kept in

muffle furnace for ignition at high temperature. Temperature of furnace

increased gradually up to 450ºC. The material was kept at this temperature

for 6 hours till complete ignition of drug occurred, that is till complete white

colored ash was obtained, intermittent weighing was also done and heating

continued till constant weight of crucible. Crucible was then taken out from

furnace, cooled and weighed. The total ash was calculated by subtracting the

weight of crucible with ash of drug after ignition from weight of crucible

with drug powder before ignition. Percentage of total ash was calculated

with reference to air-dried drug.

Acid Insoluble Ash Value

The acid insoluble ash value has been undertaken to remove

variations caused by calcium oxalate. The ash obtained in the total ash

method was taken and boiled with 25 ml of 2N hydrochloric acid for 5 min.

Insoluble matter was collected on ash less filter paper and washed with hot

water. The material was further ignited and weighed. Percentage of acid

insoluble ash was calculated with reference to air dried material.

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Water Soluble Ash Value

The ash obtained from total Ash was taken, boiled with 25 ml water

for 5 min. All insoluble matter was collected on ash less filter paper, washed

with hot water and ignited for 15 min at the temperature not exceeding

450ºC. The percentage of water-soluble ash was calculated by subtracting

weight of insoluble matter from weight of total ash. The difference between

weights represents water-soluble ash. Percentage of water-soluble ash was

calculated with reference to air-dried drug.

Moisture Content

Moisture was determined by oven drying method (AOAC, 2000).

Medicinal plant powder was well-mixed and 2g was accurately weighed

in clean, dried crucible. The crucible was allowed in an oven at 100-105ºC

for 6-12 hour until a constant weight was obtained. Then the crucible was

placed in the desiccator for 30 minutes to cool. After cooling it was weighed

again, the percent moisture content was calculated by the following formula:

In Vitro Antioxidant Activities of the Plant Extracts

Ferric Reducing Antioxidant Power (FRAP) Assay

Total antioxidant potential of hexane, chloroform and methanol

extracts of twenty medicinal plants were determined using FRAP assay. All

solutions were used on the day of preparation. An amount of 200 μl

extracted samples were mixed with 3 ml FRAP reagent in test tubes and

vortexed. Blank samples were prepared for both methanol and deionized

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water extracted samples. Both samples and blank were incubated in water

bath for 30 minutes at 37ºC and the absorbance of the samples was

determined against blank at 593 nm. Series of stock solution at 200, 400, 800,

1200 and 1600 μM were prepared (r2 = 0.9944) using aqueous solution of

FeSO4.7H2O as standard curve. The values obtained were expressed as µM

of ferrous equivalent Fe (II) per gram of freeze dried sample.

DPPH Radical Scavenging Assay

Free radical scavenging is one of the mechanisms involved in

antioxidant action, a good antioxidant (AH) able to scavenge the DPPH (1,1

Di phenyl 2-picryl hydrazyl) radical and retain its own stability due to its

reduction ability as shown in the equation below.

DPPH* + AH DPPH H + A*

Procedure

Plant extracts were tested for the scavenging effect on DPPH radical

method, 2ml of extract solution of different solvents (Hexane, Chloroform

and Methanol) were taken in different concentration (5, 50, 100 and 400μg)

to which 2 ml of 0.4 m mol/L DPPH methanolic solution was added.

Solution containing 2 ml of methanol and 2 ml of the DPPH solution was

used as negative control and synthetic antioxidant ascorbic acid was used as

positive control. Different concentrations were kept in the dark at room

temperature for 30 min. The scavenging activity of the DPPH was

determined by measuring the absorbance at 517 nm until the reaction

reached the steady state, using a spectrophotometer. All the determination

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was performed five times.

The DPPH radical scavenging activity was calculated using the following

equation.

% inhibition = (1- A1/A0) x100

A1 and A0 are the absorbance of the tested sample and control respectively.

Anti-Diabetic Assays

In vitro Anti-Diabetic Studies

α-Amylase Inhibition Assay

Inhibition of α-amylase by the crude methanol extracts. Briefly,

porcine pancreatic α-amylase (Sigma) was dissolved in ice-cold distilled

water (5 units/ml solution). Potato starch (1% w/v) in 20 mM phosphate

buffer (pH 6.9) containing 6.7 mM sodium chloride was used as the

substrate solution. Plant extract (40 μl) was mixed with 40 μl porcine

pancreatic α- amylase and 80 μl of 20 mM phosphate buffered saline (pH

6.9). Tubes were pre incubated for 15 min at 37OC and then 1% potato starch

(40 μl) was added to all the tubes. Final concentration of plant extract used

during screening was 1mg/ml. Series of final concentrations used for the

extracts with higher inhibitory effects included 20, 40, 60, 80, 100µg/ml. µg/

ml for all the medicinal plant extracts. Control was carried out in the absence

of plant extract or standard inhibitor. Test blanks were conducted in the

presence of plant extracts without α-amylase. A blank reaction was carried

out with 40μl methanol replacing the plant extract. Acarbose (Sigma) (200

µg/ ml) was used as the standard inhibitor. Reaction mixtures were

73
incubated for 15 min at 37°C. Dinitrosalicylic acid color reagent (96 mM 3,5-

dinitrosalicylic acid, 5.31 M sodium potassium tartarate in 2 M NaOH) was

added (100 μl) to all the tubes and was kept immediately in a water bath at

85°C for 15 min. Distilled water (900 μl) was added to each tube and the

absorbance was measured at 540 nm.

Calculation of Percentage Inhibition of Enzyme Activity

Percentage inhibition was calculated using the following formula.

(Absorbance of Test-Absorbance of Test Blank) X

100 Percentage inhibition
------------------------------------------------------------- = 100
(Absorbance of Control-Absorbance of Control Blank)

α-Glucosidase Inhibition Assay

The α-glucosidase inhibition was determined using the method as

described by Elya et al., [14]. Briefly, 200 μl of 67 mM sodium phosphate

buffer (pH 6.8) and 120 μl of 10 mM p-Nitrophenyl α-D- Glucopyranoside

(Sigma) was added to tests, control and the blanks. Plant extract (40μl) was

added to the test and test blank. The mixtures were pre incubated for 15 min

at 37°C. After incubation, 40 μl of 0.1U α-glucosidase from Saccharomyces

cerevisiae (Sigma) was added to the tests and control. Final concentration of

plant extract used during screening was 0.5 mg/ml. Series of final

concentrations used for the extracts with higher inhibitory effects included

20, 40, 60, 80, 100µg/ ml for all the medicinal plant extracts. Control was

carried out in the absence of plant extract or standard inhibitor. The reaction

mixture was incubated for 15 min at 37°C. Reaction was terminated by

74
adding 200 mM sodium carbonate (800 μl). The hydrolysis of α-D-

glucopyranoside to p-nitrophenol was measured at 405 nm. Acarbose (200

µg/ ml) was used as the standard inhibitor.

Calculation of IC50

The concentration of the extract that inhibits 50% of the enzyme

activity (IC50) was calculated. Extracts with high inhibitory activity were

analyzed using a series of suitable extract concentrations. IC50 values

were determined by plotting percent o f inhibition (Y axis) versus log10

extract concentration (X axis) and calculated by logarithmic regression

analysis from the mean inhibitory values.

Statistical Analysis

All experiments were performed three times. Each experiment was

carried out in triplicates. Data are expressed as mean ± standard deviation.

Statistical analysis was performed using ANOVA. Values of p which

were <0.05 were considered as significant.

Phytochemical Investigation of Mucuna pruriens (L.) DC

Isolation and Separation Methods

The preliminary in-vitro screening for antimicrobial activity of

Mucuna pruriens (L.) DC was showed excellent biological activities of anti-

oxidant and anti-diabetic assays even at low concentrations. Although some

phytochemical aspects have been recorded on the plant, the author has

considered it to isolate the bioactive molecules in view of their anti-diabetic

importance. So, the extracts were subjected to column chromatography for

75
separation of pure compounds by gradient elution method.

Phytochemical Investigation

All the plant materials of Mucuna pruriens were collected in and

around laboratory zone. The collected plant materials were chopped into

small pieces and shade dried 7 - 8 days, adhering dust particles must be

removed. The dried plant materials pulverized into coarse powder in a

grinding machine. The coarse powder material of Mucuna pruriens was

weighed and packed in soxhlet apparatus. Material extracted with hexane,

chloroform and methanol in sequential order of polarity using a soxhlet

extractor. Porcelin beads were added in the round bottom flask to prevent

bumping. For each gram of dry material 3ml of solvent (Hexane, Chloroform

and Methanol) was added in the round bottom flask and was heated to

extract the plant extract in a water bath using the adjustable rheostat. Hot

vapor of solvent (Hexane, Chloroform and Methanol) passes through the

percolator tube and then vapor drops in the soxhlet tube and it degrade the

cell wall of the plant materials and constituents of plants will be extracted

out and the extraction was done up to 72hr by 3 batches. The extract was

collected and concentrated by evaporating solvents using rotary evaporator

under reduced pressure. Extract became viscous which were dried on

vaccum oven. The residue obtained were designated as crude extracts and

stored in a room temperature until assayed.

Obtained Crude Extracts gms

Hexane crude extract 117

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Chloroform crude extract 138.7

Methanol crude extract 286.4

Column Chromatography

This method is based upon the differential absorption of the various

components of a mixture on a suitable adsorbent such as silica gel or

alumina. Since some compounds are more strongly adsorbed than the other,

they travelled through column at different rates through a stationary phase

under the influence of a mobile phase and thus get separated. Fractions of

250 ml each were collected distilled and the corresponding residues was

collected in the fractionation tube and kept for crystallization.

Thin layer chromatography (TLC)

For separation of the mixtures and testing the homogeneity of the

isolated compounds Thin Layer Chromatography was used. Silica gel-G for

TLC (with 2% CaSo4) was used as adsorbent and for visualization of the

compounds, 5 % alcoholic sulphuric acid was used. For sterol separation

silver nitrate impregnated silica gel TLC plates were prepared and used. The

ratio of distance travelled by the solute to the distance travelled by the

solvent front has been taken as Rf value. The purification of each fraction

was affected by extensive re-chromatography over silica gel columns and re-

crystallization from suitable solvents and the purity of the compounds was

as obtained by homogeneity over silica gel (G60-120mesh) TLC.

Melting Point

All the melting points were taken on Boeitus micro melting point

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apparatus. The degree of temperature is expressed in terms of Centigrade.

Chemical Tests

For confirmation of compounds the following chemical tests were

carried out.

Liebermann-Burchard Test

This test indicates the presence of sterols and terpenoids. Either the

compound or the plant extract was dissolved in chloroform and added two

to three drops of acetic anhydride and a drop of concentrated sulphuric acid

through the test tube wall. A brown ring or pink color indicates the presence

of terpenoids and play of colors (Pink-blue-green) shows the presence of

sterols.

Ferric Chloride Test

When alcoholic ferric chloride solution is added to the compound, an

olive green or blue color indicates the presence of phenolic compounds and

flavonoids. The production of colors with ferric chloride is characteristic of

3, 5 and 8- hydroxy flavones but not 6, 7 or 4- hydroxyl flavones. 3-hydroxy

flavones give usually brown color and 5- hydroxy flavones give green or

olive green or purple color.

Shinoda’s Test

The presence of flavonols, flavones, and their glycosides can be

confirmed by testing the methanolic or alcoholic plant extract or compound

treated with magnesium and hydrochloric acid. The reaction mixture

develops pink, scarlet or crimson red color, indicates the presence of

flavonoids.
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Lead Acetate Test

Basic lead acetate will give colored precipitates with most of the

flavonoids, polyphenolics. While the neutral lead acetate form precipitate

with compounds containing O-dihydroxyl grouping. The 5-hydroxy

flavones give yellow color and 3, 4-hydroxyl groupings give orange red

color with lead acetate solution.

Wilson’s Boric and Citric Acid Test

This test confirms the presence of 5-hydroxyl group in flavonoid

nucleus. And yellow color appears with Wilson‟s boric and citric acid

reagent indicates the presence of hydroxyl group in flavonoids.

Wieffering Test

Wieffering test reagent consists of acetic acid (10ml), copper sulphate

solution (1ml in 0.2%) and hydrochloric acid (0.5ml concentration). The

above reagent is added to the test solution and heated on a small flame,

which develops the following colors blue, red-violet, brown-violet indicated

the presence of irridoids.

Bargellini Test

For identifying the 5, 6, 7, or 3‟4‟5‟- trihydroxy system in flavones,

Bargellini test was performed. In this the methanolic or alcoholic solution of

the substance or plant extract was treated with sodium amalgam and adds

one or two drops concentrated sulphuric acid. A green color develops or a

green flakes separate that indicates the presence of a trihydroxy system.

Labet Test
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 Several flavones and isoflavones contain methylene dioxy group, are

directed by the production of green color when treated with gallic acid and

sulphuric acid.

Durham’s Test

When concentrated nitric acid is added to the compound, green color

appears which slowly changed to the red and reverting to green by the

addition of ammonia.

Identification of Alkaloids by Precipitation Methods

Mayor’s Reagent (potassium mercuric iodide solution)

1.35gms of HgCl2 was dissolved in 60ml of distilled water. 5 gm of

potassium iodide was dissolved in 10ml of distilled water and made it up to

100ml with distilled water. 5ml of extract was added to mayor‟s reagent.

Cream or pale yellow colored precipitate was obtained.

Wagner’s reagent (iodide and potassium iodide solution)

2mgs of KI and 1.25gms of iodide were dissolved in 100ml of distilled

water. The plant extract was treated with Wagner‟s reagent. Brown or

reddish brown color precipitate was obtained.

Dragendroff’s reagent (potassium bismuth iodine solution)

Solution A: 1.7gms of basic bismuth nitrate and 20gms of tartaric acid

are dissolved in 80ml of distilled water.

Solution B: 16gms of KI is dissolved in 100ml of distilled water. Stock

solution: 1:1 (v/v- mixture of A and B solution as prepared). This solution

may be stored in the refrigerator. This reagent (5ml) can be diluted by

adding 50ml of 20% tartaric acid.

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 The plant extracts were treated with Wagner‟s reagent brown or

reddish brown colored precipitate was obtained.

Hager’s reagent: (Saturated solution of picric acid)

The plant extracts were treated with Hager‟s reagent yellow colored

precipitate was obtained.

Identification of Alkaloids by Color Reagent

Purine, Xanthin group of alkaloids (e.g. Caffeine) do not respond to

the precipitation method tests. Color reagent like potassium chloride with

caffeine, mineral acids for colchicines, or p-distinct amino-benzaldicide with

indole alkaloids produce distinct colors.

Identification and Characterization of Compounds

The basic techniques for the structural elucidation of natural products

have not changed much in the past few years. For identification,

characterization and structural elucidation of the compounds spectroscopic

methods like UV, IR, 1H NMR and 13C NMR spectroscopy, Mass Spectra,

Optical rotation and Circular dichroism were used. As a result of computer

software revolution, the level of automation that has been brought to

instrumentation has changed.

A wide range of NMR techniques that are available to the natural

product chemist interested in the structure elucidation of new metabolites is

almost bewildering. The most useful NMR technique in structure

elucidation and unambiguous assignment are studied by COSY

(mononuclear 2D technique that is used to correlate the chemical shifts of 1H

81
nuclei which are J-coupled to one another) DQF-COSY, HOHAHA, NOESY,

ROESY, HETCOR, HMQC, HMBC, FLOCK, selective INEPT, and COLOC.

Substantial progress has also been made in the field of mass

spectroscopy; particularly in terms of quadrate desk-top instruments which

linked to gas chromatography systems serves as a powerful analytical tool.

The fast atom Bombardment mass spectroscopy, GC-MS analysis and

plasma-Desorption mass spectroscopy (PD-MS) are newer techniques that

are developed in the field of mass spectroscopy.

For flavonoids UV spectral data with shift reagents like NaOMe,

AlCl3/HCl, NaOAc and AlCl3/H3Bo3 were used. IR spectral data has been

recorded on KBr disc. For ¹H NMR (90 and 200 MHz) spectral data CCl4,

CDCl3, and DMSO-d6 solvents were used with TMS as internal standard.

The IR and UV spectra are recorded on Shimadzu IR and UV

spectrophotometers and ¹H NMR spectra were recorded on JOEL Ex 90 FT

NMR spectrometer.

Spectral properties

UV spectrum

The UV absorption spectrum of sterols are characteristic of the

functional groups present and are in good agreement with those calculated

from Woodward‟s and its main use is to detect conjugation.

 Simple acyclic dienes : 217-222nm.

 Diene heteroannular : 230-240nm

 Diene homoannular : 256-260nm

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These values changes with the number of substitutes.

Infra-Red spectrum (IR)

The infra-red spectroscopy is extremely valuable in elucidating the

structures of the unknown sterols. The more important groups that are

detected by the IR spectra are O-H, C=O, C=C, C=C-C=C, etc.

Nuclear Magnetic Resonance spectrum (NMR)

NMR spectroscopy has been used in structural studies of sterols, but

complete analysis of complex molecules is extremely difficult. These appear

to have a fingerprint region which is characteristic of C-H, CH2 protons in

the nucleus. The NMR spectra of the sterols helps in the identification of

double bonds as olefinic protons appear in the downfield region of NMR

spectrum.

Mass Spectroscopy (MS)

Mass spectrum is very useful for structural analysis of sterols. Sterols

usually give an abundant molecule ion and so it is easy to determine the

molecular weight and the molecular formulae. Four common peaks usually

observed are

 [M-R]+, where R is a side chain.

 [M-(R+C2)]+, where mass C2 is C3H6.

 [M-15]+, due to loss of an angular methyl group.

 [M-(R+C2+13)]+, because of this general fragmentation pattern, it is

possible to detect the presence of steroid nucleus.

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