PHYTOCHEMICAL INVESTIGATION OF LEPTADENIA RETICULATA

CONTENTS Introduction

Objectives

Materials and Methods

Results

Conclusion

Bibliography

INTRODUCTION

Leptadenia reticulata belonging to family Asclepiadaceae is a twining

shrub with slender glabrous or pubescent branches.

It is a climber with deeply cracked corky bark[2].

The roots are 0.5-2.5 cm in diameter with thin buff bark.

L. reticulata is found from the foot of the Himalaya, Punjab through

Central, Western and South India extending to Burma, Singapore and Sri

Lanka, ascending up to an altitude of 900 m[1].

The plant is stimulant and restorative.

It is occasionally used in nose, eye and ear troubles, while the leaves and

roots are useful in skin affections and wound.

The leaves are useful in asthma and cough and against ringworms.

Alkaloid leptidin, flavonoids apigenin, luteolin, isoquercetin, hyperoside

and rutin and steroids like stigmasterol, leptaculatin, reticulin are reported

as chief constituents in L. reticulata.

Synonyms[1]

Leptadenia(En)

Dodi (Gj)

Bhadjivai(Bn)

Jivanti (Sn)

Palaikkodi(Tml)

Kalasa (Tel)

Various plants used under jivanti name are[1]-

Sarcostemma brevistigma

Desmotrichum fimbriatum

Cimicifuga foetida

Leptadenia reticulata

Leptadenia reticulata (jivanti)

Parts used:

Roots, leaves, stems

Actions and common uses:

Roots and leaves:

Skin infections, wounds and inflammation.

Antibacterial, aphrodisiac, lactogenic and antipyretic.

OBJECTIVE

To find out various phytoconstituents and to evaluate

physicochemical parametres present in leptadenia

reticulata.

MATERIALS AND METHODS

Fresh roots of L. reticulata were collected in the month of March

2009 from Gandhinagar, Gujarat after taxonomic verification

and authenticated by the taxonomist at NISCAIR, New Delhi.

A voucher specimen (BMCPER/VS/0901) was deposited in the

Department of Pharmacognosy, Shri B. M. Shah College of

Pharmaceutical Education and research, Modasa.

The roots, after removal of soil and adhering material, was dried

at room temperature for 15-20 days under shade, powdered with

grinder to 40# and kept in airtight container at room temperature

for further study.

SCREENING OF PHYTOCHEMICAL COMPOUNDSCarbohydratesGlycosidesSterols and triterpenoidsSaponinsProteins and aminoacidsCoumarinsFlavonoidsTanninsPhenolicsAlkaloids

TEST FOR CARBOHYDRATES

(i) Molisch’s test

1 g powder of L. reticulata root was extracted with 10 mL ethanol for 15 min on a

boiling water bath and filtered. On addition of α-napthol and concentrated H2SO4, to

the filtrate a violet ring appeared indicating the presence of carbohydrates.

(ii) Fehling test

To the mixture of 1 ml Fehling A and 1 ml Fehling B solutions, boiled for 1 min, equal

volume of test solution was added. After heating it on boiling water bath for 5-10

minutes, formation of green colouration to red precipitates appeared which depends on

amount of carbohydrates.

(iii) Keller Killiani test

To 2 mL alcohol extract was add one drop of glacial acetic acid, one drop 5% FeCl3

and conc. H2SO4. No appearance of reddish brown colour at the junction of the two

liquid layers or bluish green upper layer indicates absence of 2-deoxy sugars.

TEST FOR GLYCOSIDES

The aqueous extract of L. reticulata was prepared by cold maceration

with 3% methanol-water for 7 days with occasional shaking.

(i) Legal test

Take 1 mL filtrate, add 3 mL sodium nitroprusside in pyridine and

KOH in methanol in a test tube.The alkaline layer does not turn to

blue indicating absence of cardiac glycoside.

(ii) Keller-killiani test

Take 1 mL filtrate; shake with 1mL of glacial acetic acid containing

traces of ferric chloride.

Carefully add 1 mL of concentrated sulphuric acid by the side of test

tubes. No blue colouration in acetic acid layer or no red color at the

junction of the two liquids indicates absence of glycosides.

TEST FOR ANTHRAQUINONE GLYCOSIDES(i) Borntrager’s test

Take powder drug, extracted with ether; add ethereal extract of

ammonia to the filtrate. After shaking aqueous layer does not

show pink red or violet color indicating absence of anthraquinone

glycoside.

(ii) Modified Borntrager’s test

Take aqueous extract of drug, add ferric chloride and dilute HCl,

heat, cool and filter. Shake filtrate with ether, separate ethereal

layer and shake with dilute ammonia. The aqueous layer does not

show rose pink to cherry red color indicating absence of

anthraquinone glycoside.

TEST FOR STEROLS AND TRITERPENOIDS[4]

(i) Liberman Buchardt test

1 g powdered drug was moistened with 1.0 mL of acetic

anhydride and 2 drops of sulphuric acid on a clean tile and

observe the color gained by the powder. Purple to violet color

confirm the presence of sterols and triterpenoids.

(ii) Salkowski reaction

To 2 mL of methanol extract of plant, add 2 mL chloroform

and 2 mL concentrated H2SO4 and shake well. Chloroform

layer appeared red and acid layer showed greenish yellow

fluorescence confirm the presence of sterols and triterpenoids

in drug.

TEST FOR SAPONINS

(i) Froth test

Shake vigorously 0.1 g of powder of drug with 5mL of

distilled water in a test tube for 30 second and keep aside for

20 min. No persistent frothing indicates absence of saponins.

(ii) Haemolytic Zone

Mix 0.5 mL blood with gelatin solution (3 g gelatin powder

dissolved in 100 mL of 0.85% NaCl solution) at 60˚C and

taken on a glass slide. A thick section of plant was placed on

it. No observation of haemolytic zone confirms the absence

of saponins.

TEST FOR PROTEINS AND AMINOACIDS

100 mg of methanol extract of L. reticulata root was dissolved in 10

mL of water and filtered. Filtrates were used to test the presence of

proteins and amino acids.

(i) Millon’s test

Add 2 mL of filtrate in 2 mL of Millon’s reagent in a test tube and

heated in a water bath for 5 minutes,cooled and add few drops of

NaNO2 solution. Formation of white precipitates turn to red upon

heating indicates presence of proteins and amino acids.

(ii) Ninhydrin test

Take 2 mL of filtrate, add 2-3 drops of Ninhydrin reagent in a test

tube and boiled for 2 minutes. Formation of distinct blue colour

indicates presence of amino acids.

(iii) Biuret test

Take 2 mL of filtrate, add 2 mL of 10% NaOH in a test tube and

heat for 10 min, add a drop of 7% of CuSO4 distinct violet

colouration indicates presence of proteins.

TEST FOR COUMARINS

(i) Test with Ammonia

To a drop of ammonia on a filter paper, a drop ofaqueous extract

of drug observation of green fluorescence verify the presence of

coumarins.

(ii) Test with Hydroxylamine hydrochloride

Ethereal extract of drug was treated with one drop of saturated

alcoholic hydroxylamine HCl and a drop of alcoholic KOH. It

was heated, cooled and acidified with 0.5 N HCl and add a drop

of 1 %w/v FeCl3. Distinct violet coloration indicates the

presence of coumarin

TEST FOR FLAVONOIDS[5]

(i) Shinoda test

1g of powdered drug was extracted with 10mL of ethanol (95 %v/v) for 15 min

on a boiling water bath and filtered. To the filtrate add small piece of

magnesium ribbon and 3 to 4 drops of concentrated H2SO4. Formation of light

pink colour confirms the presence of flavanoids.

(ii) Fluroscence test

1g powder drug was extracted with 15 mL methanol for 2 min. on a boiling

water bath,

filtered while hot and evaporated to dryness. To the residue add 0.3 mL boric

acid solution (3 %w/v) and 1 mL oxalic acid solution (10 %w/v) and

evaporated to dryness. The residue was dissolved in 10 mL ether; ethereal layer

shows greenish fluorescence under UV light indicates presence of flavanoids.

TEST FOR TANNINS

For following tests, aqueous extract of L. reticulata root

powder (10 g) was prepared by refluxing with 50 mL water

for about 1h on water bath.

(i) Test with gelatin

Add 2-3 mL of aqueous extract to 1 %w/w gelatin solution

containing NaCl. No formation of heavy white precipitates

indicates absence of tannins.

(ii) Reaction with lead acetate

Add the aqueous extract in 2 mL of 10 %w/w solution of lead

acetate no formation of precipitates indicates absence of

tannins.

TEST FOR PHENOLICS

(i) Test with FeCl3

A drop of freshly prepared FeCl3 solution was added in

methanol extracts of drug. Observation of brownish green color

indicates presence of phenolic compounds.

(ii) Test with Folin ciocalteu reagent

A drop of methanol extract was added in a drop of Folin

ciocalteu reagent. Observation of bluish green color confirms the

presence of phenolic compound.

TEST FOR ALKALOIDS[6]

(i) Dragendroff’s test

1 g powdered drug was extracted with 20ml alcohol by refluxing for 15

min and filtered; filtrate was evaporated to dryness. The residue was

dissolved in 15 mL 2N H2SO4 and filtered. After making alkaline, the

filtrate was extracted with chloroform. The residue was treated with

Dragondroff’s reagent. Development of orange precipitates indicates

presence of alkaloids.

(ii) Hager’s test

Dissolve 100 mg methanol extract of drug in 10mL of 0.1 N dilute HCl

and filtered. Two ml of the filtrate was added in Hager’s reagent;

formation of yellow precipitates indicates the presence of alkaloids.

DETERMINATION OF PHYSICOCHEMICAL PARAMETRES

Physicochemical parametres has been evaluated on the basis of WHO standards Moisture content Total Ash value Acid insoluble ash Water soluble ash Water soluble extractive Alcohol soluble extractive Acetone soluble extractive Chloroform extractive Petroleum ether extractive

MOISTURE CONTENT

An accurately weighed (3 g) shade-dried root powdered of L. reticulata was and taken in a tarred glass bottle.

The crude drug was heated at 105ºC in an oven till a constant weight.

Percentage moisture content of the sample was calculated with reference to the shade-dried material using formula.

% Moisture content= loss in wt of sample x 100 wt of sample

ASH VALUES[3]

1. TOTAL ASH Weighed accurately 2 g root powder of L.reticulata,

incinerated in a crucible at a temperature 500-600˚C in a muffle furnace till carbon free ash was obtained.

It was then cooled, weighed and percentage of total ash was calculated with reference to the air-dried drug using following equation.

% Total ash= wt of total ash x 100 wt of crude drug

2. ACID INSOLUBLE ASH Ash, above obtained, was boiled for 5min with 25mL of 70 g/L

hydrochloric acid and filtered using an ashless filter paper. Insoluble matter retain on filter paper was washed with hot water

and filter paper was burnt to a constant weight in a muffle furnace. The percentage of acid-insoluble ash was calculated with reference

to the air-dried powered drug (40#) using following equation.

% Acid insoluble ash value = wt of acid insoluble ash x 100 wt of crude drug

3. WATER SOLUBLE ASH Total ash was boiled for 5min with 25 mL water and insoluble

matter collected on an ash-less filter paper was washed with hot water and ignited for 15min at a temperature not exceed ing 450˚C in a muffle furnace.

Difference in weight of ash and weight of water insoluble matter gave the weight of water-soluble ash.

The percentage of water-soluble ash was calculated with reference to the air-dried powered drug (40#) using formula

% Water soluble ash value = wt of total ash-wt of water insoluble ash x 100 wt of crude drug

EXTRACTIVE VALUES[3]

Extractive values of root powder of L. reticulata were

determined using following methods.

1. ALCOHOL SOLUBLE EXTRACTIVE

Air-dried powdered (4g) of L. reticulata root (40#) was

macerated with 100 mL of alcohol in a closed flask for 24 h,

shaking frequently at an interval of 6h.

It was then allowed to stand for 18 h and filtered rapidly to

prevent any loss during evaporation.

Evaporate 25 ml of the filtrate to dryness in a porcelain dish and

dried at 105˚C to a constant weight.

The percentage of alcohol soluble extractive was calculated with

reference to the airdried drug.

2. WATER SOLUBLE EXTRACTIVE

Air-dried powder (4 g) of L. reticulata root was soaked in 100

mL of water in a closed flask for 1h with frequent shakings.

It was then boiled gently for 1 h on water bath; cooled and

weighed and re adjusted the weight. 25 mL of the filtrate was

evaporated to dryness in a porcelain dish and dried at 105˚C

to a constant weight.

The percentage of water-soluble extractive was calculated

with reference to the air-dried powered drug (40#).

3. ACETONE SOLUBLE EXTRACTIVE 4 g of the air-dried powder of L. reticulata (40#) root was

macerated with 100 mL of acetone in a closed flask for 24 h, shaking frequently at an interval of 6 h.

It was allowed to stand for 18 h and filtered rapidly to prevent any loss during evaporation.

25 mL of the filtrate was evaporated to dryness in a porcelain dish and dried at 105˚C to a constant weight.

The percentage of acetone soluble extractive was calculated with reference to the air-dried drug.

4. CHLOROFORM SOLUBLE EXTRACTIVE Take 4 g of the air-dried powder of L. reticulata (40#) and

macerated with 100 mL of chloroform in a closed flask for 24 h, shaking frequently at an interval of 6 h.

It was then allowed to stand for 18 h and filtered rapidly to prevent any loss during evaporation.

25 mL of the filtrate was evaporated to dryness in a porcelain dish and dried at 105˚C to a constant weight.

The percentage of chloroform soluble extractive was calculated with reference to the air-dried drug.

5. PETROLEUM ETHER SOLUBLE EXTRACTIVE

4 g of the air-dried powder of L. reticulata (40#) root was

macerated with 100 mL of petroleum ether in a closed flask for

24 h, shaking frequently at an interval of 6 h.

It was allowed to stand for 18 h and filtered rapidly to prevent

any loss during evaporation. 25 mL of the filtrate was

evaporated to dryness in a porcelain dish and dried at 105˚C to a

constant weight.

The percentage of petroleum ether soluble extractive was

calculated with reference to the air-dried drug.

RESULTS AND DISCUSSION

L. reticulata was subjected to systematic physiochemical and phytochemical screening by extracting with various organic solvents to determine the amount of soluble constituents in a given amount of medicinal plant material.

The data generated is helpful in determining the quality and purity of a crude drug, especially in powdered form.

The objective of reducing the vegetable drug to its ash is to remove all traces of organic matter, which may otherwise interfere in an analytical determination.

On incineration, crude drugs leave an ash usually consisting of carbonates, phosphates and silicates of sodium, potassium, calcium and magnesium.

The determination of ash is useful for detecting low-grade products, exhausted drugs and excess of sandy or earthy matter; it is more especially applicable to powdered drugs.

Phytochemical analysis was performed on the root powder and it was found to contain carbohydrates, steroids, coumarins, flavonoids, phenolics and alkaloids .

CONCLUSION

The results indicated that the roots of plant L. reticulata contain

variety of phytoconstituents.

Phytochemical studies on the extracts of L. reticulata (roots)

showed presence of carbohydrates, coumarins, alkaloids,

phytosterols flavonoids and phenolic compounds.

This information may be further used for isolation of various

compounds from Jivanti for treatment of diseases for human beings.

The physicochemical evaluation of parameters for the quality and

purity of herbal drug and also gives information regarding the

authenticity of crude drug.

BIBLIOGRAPHY

1. Gupta AK, Quality Standards of Indian Medicinal Plants Vol. 3, ICMR,

New Delhi. 2003; 236-245.

2. Patel RI, Forest Flora of Gujarat State,Forest Department, Gujarat State,

Baroda.1971; 198.

3. Anonymous, WHO guidelines,1st Edition, AITBS Publishers and

Distributors: New Delhi. 2002; pp28, 30, 41, 46.

4. Kokate CK, Purohit AP, Gokhale SB, Pharmacognosy, 12th ed. Nirali

prakashan, Pune.1999; pp.145-155.

5. List PH, Horhammer L, Hager Hand buchder pharmazeutischem praxis,

Springer Verlag Band 1, Berlin. 1967; pp. 256.

6. Sim SK, Medicinal plant glycosides,University of Toronto Press,

Toronto.1968; 2:25.