The project work embodied in this dissertation entitled; “EXTRACTION AND IDENTIFICATION OF PHYTOCHEMICAL CONSTITUENT OF THE PLANT A. INDICA”

1. “EXTRACTION AND IDENTIFICATION
OF PHYTOCHEMICAL CONSTITUENT
OF Aristolochia INDICA”
Presented by-
Abhisek Misra
Under the supervision of
Dr. Kazi Asraf Ali,
Assistant Professor,
Dr. B. C. Roy College of Pharmacy
& A.H.S , Durgapur

2. INTRODUCTION
 Name Of The Plant (Biological Name):- Aristolochia indica L.
Family:- Aristolochiaceae
Region:- Southern India (Kerala) & Srilanka
Genus:- Aristolochia
Species:-indica
Colour :- Greenish Whitish
Active Constituent:- Aristolochic Acid
Uses:- used in Ayurvedic, Sidda and Homeopathy systems of
medicines. Roots are widely used in joint pains and seeds in
inflammation, dry cough and dyspepsia. Also used as an antidote for
snake poisoning & as a blood purifier.

3. MATERIALS
A. Aristolochia indica plant – Stem part is obtained
from the forest of Bankura district of West Bengal.
B. Solvent like petroleum ether, Chloroform,
Methanol, mixture of water and methanol (50:50).
C. Distillation apparatus for solvent recovery.
D. glass apparatus like conical flask, round bottom
flask, condencer, test tube.
E. chemicals for identification like sulphuric acid,
hydrochloric acid, amonia.

4. PREPARATION OF PLANT SPECIMEN
 The process of preparing herbarium specimens can be divided into three
stages:-
1) Collection of the plant material in the field
2) Drying and pressing herbarium specimens
3) Botanical identification of the species and mounting the specimens
A.Indica Dried
Mounted on
paper sheet
Herbarium
transported
Stored in special
drawers Identified

5. STAGES OF PREPARING
HARBERIUM SPECIMEN
The process of preparing herbarium specimens by 3 stages are described below
1) Collection of the plant material in the field
2) Drying and pressing herbarium specimens
3) Botanical identification of the species and
mounting the specimens

6. EXTRACTION METHOD
The main extraction method for this process are:-
a) Petroleum Ether Extraction
b) Choloroform Extraction
c) Methanol Extraction
d) Water & Methanol Extraction (50:50)

7. PETROLEUM ETHER EXTRACTION
At first take 500ml of petroleum ether. Then the stem part of the supplied
plant is dipped into the solvent system and kept overnight. On next day
the mixture solvent system was transferred into the distillation apparatus
to carry on the distillation process so that we can recover the solvent
system and also get the crude product. The recovered petroleum ether
transferred into the jar. This process is repeated for 3 times to get the
crude product.
Fig. 1 Distillation process

8. CHOLOROFORM EXTRACTION
At first take 500ml of chloroform. Then the stem part of the supplied plant is
dipped into the solvent system and kept overnight. On next day, the
mixture solvent system is transferred into the distillation apparatus to carry
on the distillation process so that we can recover the solvent system and
also get the crude product.
The recovered Chloroform transferred into the jar.
This process is repeated for 3 times to get the
Crude product.
Fig. 2 Chloroform extract

9. METHANOL EXTRACTION
At first take 500ml of methanol. Then the stem part of the supplied plant is
dipped into the solvent system and kept overnight. On next day, the mixture
solvent system is transferred into the distillation apparatus to carry on the
distillation process so that we can recover the solvent system and also get
the crude product.
The recovered methanol transferred into the jar. This
process is repeated for 3 times to get the crude product.
Fig. 3 Methanol Recovery

10. Water & methanol(50:50) extraction
 In this process of extraction at first a mixture of methanol and water is
prepared in the ratio of 50:50 then the stem part of the supplied plant are
dipped into that mixture and left for over night .on the next day The
water methanol extraction is carried out in rotary evaporator. In this
process the methanol solvent is recovered from the mixture so that we
can get the crude product.

11. Identification test
The tests for this process are these :-
1. Test for alkaloids
2. Test for glycosides
3. Test for tannins
4. Saponin Test
5. Chromatographic Method

12. Alkaloid test
1) Dragendroff test: - 1 ml of Dragendroffs reagent (potassium
bismuth iodide solution) + crude product= An orange-red
precipitate.
2) Mayer’s test :- 1 ml of Mayer’s reagent (potassium mercuric
iodide solution) + crude product= Whitish or cream colour
precipitate.
3) Hager’s test :- 3 ml of Hager’s reagent(saturated aqueous
solution of picric acid) + crude product = Yellow
colour precipitate
1) Wagner’s test :- 2 ml of Wagner’s reagent
(iodine in potassium iodide) + crude product =
Reddish brown colour precipitate.
Fig. 4 Alkaloid test

13. Glycoside test
Borntrager’s test: - The crude product + dilute sulphuric
acid (boiled ,filter and cooled) The filtrate is extracted
with chloroform or benzene + dilute ammonia The
ammonical layer becomes pink to red.
Modified Anthraquinones test: - 1ml of drug + 5ml of 5%
solution of ferric chloride + 5ml dilute hydrochloric acid
(heat on water-bath for 5 minutes, cooled and shake gently)
+ organic solvent like benzene (Separate the organic solvent
layer) + equal volume of dilute ammonia A pinkish red
colour is formed in ammonical layer.

14. Tannin test
Gelatin test:- solution of tannin + aqueous solution of gelatin + sodium
chloride A white buff coloured precipitate is formed.
 Phenazone test:- A mixture of aqueous extract of a drug and sodium acid
phosphate is heated and cooled and filtered. A solution of phenazone is added
to the filtrate. A bulky coloured precipitate is formed.
Match stick test (Catechin test):- A match stick is dipped in aqueous
plant extract, dried near burner and moistened with concentrated hydrochloric
acid. On warming near flame, the matchstick wood turns pink or red due to
formation of phloroglucinol.

15. Saponnin test
 1ml crude product +5ml aquous solvent (shake for 15
min) soap like foaming indicates the presence of
saponin.

16. Chromatographic method
 PRINCIPLE- ADSORPTION
 SOLVENT USED-
1. Benzene: chloroform: petroleum-ether (6:3:1)
2. Benzene: chloroform: ether acetate (6:3:1)
3. Benzene: chloroform: ether acetate: methanol: water (3:3:2:1.5:0.5)
Fig. 5 TLC Spot detection in UV chamber

17. Result and Discussion
Test petroleum
ether
Chloroform methanol water-
methanol
Alkaloid test
Mayer”s test (-)ve (-)ve (-)ve (+)ve
Hager’s test (-)ve (-)ve (-)ve (+)ve
Wagner’s test (-)ve (-)ve (-)ve (-)ve
Dragendroff’s test (-)ve (-)ve (-)ve (-)ve
Tannin test
Match stick test (+)ve (-)ve (-)ve (-)ve
Glycoside test
Borntrager’s test (-)ve (-)ve (-)ve (-)ve
Modified
Borntrager’s test
(-)ve (-)ve (-)ve (-)ve

18. Result and DiscussionTest petroleum
ether
Chloroform methanol water-
methanol
Saponin test (-)ve (-)ve (-)ve (-)ve
Fig. 6 Representation of TLC using different solvent

19. Discussion
• After performing the above experiment we found that the
water-methanol extract was give (+) ve result in Mayer’s test
and Hager’s test. So it confirms the presence of alkaloids.
In case of petroleum ether, it shows the (+) ve result in match
stick test. So it confirms the presence of tannin.

20. Conclusion
Different types of extract are obtained successfully and
phytochemical constuents are identified by different chemical tests
like test for alkaloids, test for glycosides, test for saponins and test
for tannins. Chromatographic evaluation test showed a
distinguished spot on the TLC plate which indicated that chemical
constuents are there.

21. References
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Aristolochia indica plant extract, International Journal of
Pharmacognosy and Phytochemical Research 5(3): 168-172.
 Faisal M. et al. (2015), Pharmacognostical, phyotochemical and
toxicity profile of flower of Ishwari - Aristolochia indica, The Journal
of Phytopharmacology, 4(3): 133-138.
 Hossen M. et al. (2014), Phytochemical and biological
investigations on Aristolochia indica, Der Pharma Chemica, 6(3):
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Mall Mridula et al. (2011), Pharmacognostical and phytochemical
investigation of aerial parts of Aristolochia indica, International
journal research and publication, 2(4): 1282-1285.
Remya M et al. (2016), Direct regeneration and bioactive
components in A. Tangala,

22.  Umamaheshwari S. (2012), an evaluation and comparative
studies of root extract of Aristolochia indica and antibiotics on
Escherichia Coli, 5(3); Journal of pharmaceutical and scientific
innovation, (6): 61-63.
 Pramod V. Pattar (2012), In vitro Regeneration of Plantlets from
Leaf and Nodal explants of Aristolochia indica, Asian Pacific
Journal of Tropical Biomedicine, 488-493.
 Ramachandran et al. (2008), Effect of Aristolochia indica on
diuretics induced gout, Pharmacologyonline 1: 304-308.
 Reddy Pooja et al. (2015), Preclinical Evaluation of Anxiolytic
Activity of Aristolochia Indica, Scholars Academic Journal of
Pharmacy, 5(1): 6-8.

23. THANK YOU