phytochemical and physicochemical evaluation of leptadenia reticulata

1. PHYTOCHEMICAL INVESTIGATION
OF LEPTADENIA RETICULATA

2. CONTENTS
 Introduction
 Objectives
 Materials and
 Results
 Conclusion
 Bibliography
Methods

3. INTRODUCTION

4. 
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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.

5. Synonyms[1]
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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

6. Leptadenia reticulata

7. Leptadenia reticulata (jivanti)
Parts used:
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Roots, leaves, stems
Actions and common uses:
Roots and leaves:
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Skin infections, wounds and inflammation.
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Antibacterial, aphrodisiac, lactogenic and antipyretic.

8. OBJECTIVE
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To find out various phytoconstituents and to evaluate
physicochemical parametres present in leptadenia
reticulata.

9. MATERIALS AND METHODS

10. 
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.
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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.

11. SCREENING OF PHYTOCHEMICAL
COMPOUNDS
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Carbohydrates
Glycosides
Sterols and triterpenoids
Saponins
Proteins and aminoacids
Coumarins
Flavonoids
Tannins
Phenolics
Alkaloids

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. (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.

19. 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

20. 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.

21. 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.

22. 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.

23. 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.

24. 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

25. MOISTURE CONTENT
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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

26. 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

27. 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

28. 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

29. EXTRACTIVE VALUES[3]
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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.

30. 2. WATER SOLUBLE EXTRACTIVE
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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.
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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.
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The percentage of water-soluble extractive was calculated
with reference to the air-dried powered drug (40#).

31. 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.

32. 4. CHLOROFORM SOLUBLE EXTRACTIVE
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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.
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It was then allowed to stand for 18 h and filtered rapidly to
prevent any loss during evaporation.
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25 mL of the filtrate was evaporated to dryness in a porcelain
dish and dried at 105˚C to a constant weight.
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The percentage of chloroform soluble extractive was
calculated with reference to the air-dried drug.

33. 5. PETROLEUM ETHER SOLUBLE EXTRACTIVE
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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.
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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.
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The percentage of petroleum ether soluble extractive was calculated
with reference to the air-dried drug.

34. RESULTS AND DISCUSSION

35. 
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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 .

38. CONCLUSION

39. 
The results indicated that the roots of plant L. reticulata contain
variety of phytoconstituents.
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Phytochemical studies on the extracts of L. reticulata (roots)
showed presence of
carbohydrates, coumarins, alkaloids, phytosterols flavonoids and
phenolic compounds.
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This information may be further used for isolation of various
compounds from Jivanti for treatment of diseases for human beings.
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The physicochemical evaluation of parameters for the quality and
purity of herbal drug and also gives information regarding the
authenticity of crude drug.

40. BIBLIOGRAPHY

41. 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.