Advanced Herbal drug technology,A Presentation on
Extraction, isolation and standardization of Phytochemicals in Crude extract of Tinospora Cordifolia (Giloy, gulvel,giloe, Amrita,garo).It Shows presence of flavonoids and Alkaloids which shows Anti-cancer,Anti-oxidants, Anti-viral, Anti-inflammatory and Anti-allergic activity by boosting host immune system. it also involves different test for identification of Alkaloids, flavonoids, saponins,tanins, glycoside.
5. Habitat : Tropical India and extending from Kumaon to Assam
and Myanmar, Bihar, Konkan to Sri Lanka.
Pharmacognosy of Tinospora Cordifolia
1. Stems - Fleshy
2. Roots - long thread like, aerial, arise from branches.
3. Bark - Thin, greyish or creamy white in colour, when peeled
fleshy stem is exposed.
4. Leaves - Cordate (heart shaped), membranous, juicy.
5. Flowers - Bloom during summer
a. Male flower - Small, yellow or green coloured occur in
clusters.
b. Female flower - Occur singly.
6. Fruits - Pea shaped, fleshy, shiny turn red when boiled. Occur
in winter
7. Seeds - curved, pea sized.
Fig 1:Tinospora Cordifolia {Leaf}
6. Biological Source
Fig 2:Leaves of Tinospora Cordifolia Fig 3 :Dried stem of Tinospora Cordifolia
Fig 4:
fresh stem of Tinospora Cordifolia
7. Different methods of Identification
of plants
• Expert Determination
• Recognition
• Comparison
• The Use of Keys and Similar Devices (Synopses, Outlines, etc.
9. • soxhlet extraction
• aqueous extraction
• alcoholic extraction
• Grinding
• simple decoction
• Supercritical fluid extraction
• microwave-assisted extraction
Different extraction methods
Fig 5 :Decoction of stem part of Tinospora Cordifolia
10. Isolation and purification technique
Analysis by UV-Spectroscopy.
TLC-Thin layer chromatography.
HPLC.
HPTLC.
Column Chromatography.
11. Qualitative analysis of phytochemicals
a) Molisch’s test OBSERVATION
Sample of plant extract was taken in a
test tube. Then 20% alcoholic solution
and concentrated sulphuric acid, which is
freshly prepared is added in to test tube
along the sides
developed reddish violet and purple Ring
at junction between two liquids
Test for carbohydrate:
b) Benedict’s test OBSERVATION
Taken a test tube, which contain small
amount of plant extracts sample. In a test
tube added small quantity of benedict’s
solution and mix properly. Then boiled
this sample mixture for two minutes and
cool it.
Solution appears GREEN,YELLOW Or RED
depending on the amount of reducing
sugar present in test solution
12. FEHLING’S TEST OBSERVATION
Mix 1ml of Fehling's A & B Solution and equal
volume of Test solution ;boil for 10 min in
boiling water bath.
First yellow then brick red precipitate is seen.
Test for Monosaccharides
BARFOED’S TEST OBSERVATION
Mix equal volume of barfoed’s solution and
test solution .heat for 1 to 2 min in boiling
water bath and cool .
red precipitate is observed
13. Test for Pentose sugar
BIAL’s ORCINOL TEST OBSERVATION
To boiling Bial’s reagent add few drop of
test solution.
GREEN PURPLE colouration appears
ANILINE ACETATE TEST OBSERVATION
A piece of paper, previously impregnated
with aniline acetate is exposed to the
vapour from the sample solution.
A bright PINK OR RED colour appears on
paper.
Test for Hexose sugar
SELIWANOFF’s TEST OBSERVATION
For ketose like fructose heat 30ml of
seliwanoff reagent and 1ml of test
solution in bearing water bath for 1 to 3
min.
Red colour is formed.
14. Test for Alkaloid’s
Dragendorff’s test OBSERVATION
Taken a few mg of extracts sample and dissolved in
5ml water. Then 2 M hydrochloric acid added until
an acid reaction developed. In this mixture, 1ml of
dragendorff’s reagent (potassium bismuth iodine
solutions) was added.
orange red/BROWN precipitate is observed.
Mayer’s test OBSERVATION
2ml of plant extracts sample was taken and 2 - 3
drops of Mayer’s reagent was added (potassium
mercuric iodine solution) in the test tube.
it formed dull white precipitate
Wagner’s test OBSERVATION
Acidify the plant extract sample with hydrochloric
acid (1.5% v/v) and added a few drop of Wagner’s
reagent (iodine potassium iodide solution) in the
test tube.
reddish brown precipitates formed.
15. Hager’s test OBSERVATION
Add 2-3ml of test solution with few drop of hager’s
Reagent.
Yellow precipitate is observed
Murexide test OBSERVATION
For purine alkaloids-3-4 ml test solution and 3-4
drops of concentrated HNO3 .evoprate to dry and
then cool and add 2 drop of NH40H
Purple colour is observed
Ionic acid test OBSERVATION
Test solution treated with tannic acid solution. Gives buff colour precipitate
16. Test for glycosides
Legal’s test OBSERVATION
Taken a extracts sample and dissolved in pyridine then
added sodium nitroprusside solution. Make this
solution completely alkaline
PINK to RED colour appear.
Baljet’s test OBSERVATION
Taken a plant extracts sample in the test tube and
added sodium picrate solution.
yellow to orange colour produce.
Borntrager’s test OBSERVATION
The test solution of plant extract was added in few ml
of dilute sulphuric acid solution. This solution was
filtered. Then Chloroform and ether was added in to
filtrate and shaken well. In this solution ammonia was
added and separated the organic layer.
layer showed pink, red or violet colour.
17. Test for saponins
TEST OBSERVATION
1ml of alcoholic sample extract was taken and diluted
with 20ml of distilled water. This solution was shaked
for 15 min in graduated cylinder.
foam layer of 1cm is observed
Test for flavonoids
Shinoda test OBSERVATION
Taken the alcoholic sample extract in the test tube and
5-10 drops of hydrochloric acid added in the sample.
Then small pieces of magnesium added in tubes.
Reddish pink or brown colour observed.
Alkaline reagent test OBSERVATION
Plant extracts sample was mixed with 2ml of 2% NaOH
solution. It produced yellow colour. In this solution, 2
drops of diluted acids was added.
yellow colour changed into colourless
18. Test for tannins
TEST OBSERVATION
Taken the sample of plant extracts in the test tube and
added ferric chloride solution.
dark blue or greenish black colour appears.
TEST OBSERVATION
Taken the sample extracts and added potassium
cyanide.
deep red colour produced.
TEST OBSERVATION
Potassium dichromate was added in to sample extracts. Yellow precipitate was formed.
TEST OBSERVATION
Lead acetate solution White precipitate was formed
19. Test for protein and amino acid
Biuret’s test OBSERVATION
Taken 2-3 ml of sample extract and added 1 ml sodium
hydroxide solutions (40%) and 2 drops of copper
sulphate solution (1%) and mixed properly.
show a pinkish - violet and purple - violet colour.
Ninhydrin’s test OBSERVATION
Plant extracts sample mixed with freshly prepared 2
drops of 0.2% ninhydrin solution and heated to boiling
for 1-2 min and allowed cooling.
Blue colour appear.
Xanthoprotein test OBSERVATION
Extracts sample was taken in test tube and added conc.
nitric acid. A white precipitate was obtained and upon
heating turns to yellow and cool the solution carefully.
20% sodium hydroxide solution added in excess.
produce orange colour .
20. Test of fats or fixed oils
Using sodium hydroxide OBSERVATION
The extract was mixed in one ml 1 % of copper
sulphate solution then 10% sodium hydroxide solution
was added.
Blue colour seen.
Saponification OBSERVATION
plant extracts was taken and mixed with 2% sodium
carbonate solution. Shaked vigorously and boiled. A
clean soapy solution was formed cooled and few drops
of conc. HCl was added.
observed that fatty separate out and float up.
21. Estimation of total flavonoids content (TFC) OBSERVATION
Estimation of total flavonoids component was based
on aluminum chloride (AlCl3) method18 . Taken 50 mg
quercetin component and dissolved in 50 ml methanol.
Then different aliquots of 5-25µg/ml were prepared in
methanol. Quercetin was used as a standard. 10gm of
dried extracts of plant were dissolved with 10ml
methanol and filter. Three ml (1mg/ml) of this extract
was used for the estimation of flavonoids. Take 3 ml of
extract or standard and added 1 ml of 2% AlCl3
methanolic solution, then allowed this mixture to stand
at room temperature for 60 min. Then absorbance was
measured at 420 nm by spectrophotometer.
Concentration and absorbance was noted.
Estimation of total flavonoids content (TFC)
22. • leaf and stem extract of Tinospora Cordifolia shows presence of :
a) Flavonoids [Luteolin,kaempferol,quercetin]
b) proteins
c) saponins,
d) amino acids
e) sugars.
• Three different solvents (chloroform, methanol, and ethanol) were used to obtain
leaf and stem extracts used for qualitative phytochemicals screening of plants
using standard phytochemical tests.
• Methanol extract of stem showed that most of phytochemicals present in it
Phytochemical screening
23. Sr. No. Constituents Chloroform Methanolic Ethanolic
Leaves Stem Leaves Stem Leaves Stem
1. Alkaloids
2. Glycosides
3. Flavonoids
4. Phenolics
5. Amino Acids
6. Carbohydrate
7. Proteins
8. Saponins
9. Diterpines
Leaves Stem Leaves Stem Leaves Stem
A A P P A P
A A P P P P
P P P P P P
A A A P A A
A A A P P P
P A A A A A
A A A P P P
A A A P P P
P P A P P P
P = presence ; A = absenc
Table: Result of phytochemical analysis of T. cordifolia
{at http://jddtonline.info Journal of Drug Delivery and Therapeutic}
24. The active principles from T. cordifolia enhance host immune system by
increasing immunoglobulin and blood leukocyte levels and by the
stimulation of stem cell proliferation.
It has the ability to reduce solid tumour volume by 58.8%, which is
comparable to cyclophosphamide, a known chemotherapeutic agent.
These immunostimulating properties can be used in the prevention of
tumour mediated immunosuppression and hence could be a drug choice for
various cancers.
• Anticancer Activity
USES
25. • Tinospora cordifolia has been studied for its anti allergic
effect.
• It was found that T cordifolia provided significant relief
from sneezing, nasal discharge, nasal obstruction.
Anti Allergic Activity
26. • It has been observed that Tinospora cordifolia
exhibited excellent antioxidant activity in methanol,
ethanol and water extracts.
• The observed high due to presence of flavonoids.
Antioxidant Activity
28. WHO GUIDELINES FOR QUALITY STANDARDIZED HERBAL FORMULATIONS
1) Quality control of crude drugs material, plant preparations and finished
products.
2) Stability assessment and shelf life
3) Safety assessment; documentation of safety based on experience or
toxicological studies.
4) Assessment of efficacy and biological activity evaluations. The bioactive extract
should be standardized on the basis of active principles or major compounds
along with the chromatographic fingerprints (TLC, HPLC, and GC).
Generally, all medicines , whether they are synthetic or of plant origin,
should fulfil the basic requirement of being safe and effective.
29. Future scopes
Due to Presence of flavonoids it possess a number of medicinal
benefits, including anticancer, antioxidant, anti-inflammatory,
and antiviral properties.
30. • In this study we conclude that the presence of phytochemical study of
Flavonoids, Saponins, cardiac glycoside, Terpenoids.
• Hence these results clearly indicate that this plant can be used for the
further development of phytomedicine for medicinal purpose.
Conclusion
31. References
1. Indian Medicinal Plants , An Illustrated Dictionary By C.P. Khare ,Page no
662 -663, 776. Springer Science+Business Media, LLC Publication.
2. Textbook of Pharmacognosy and Phytochemistry by Shah and Seth ,Page
no 540-541 ,ELSEVIER A division of Reed Elsevier India Private Limited.
3. Pharmacognosy and Phytochemistry by Kokate ,Page no 9.101 -102-103.
4. https://www.researchgate.net/publication/275346992
5. Garg P, Garg R, Qualitative and quantitative analysis of leaves and stem of
Tinospora cordifolia in different solvent extract, Journal of Drug Delivery
and Therapeutics. 2018; 8(5-s):259-264 .
DOI: http://dx.doi.org/10.22270/jddt.v8i5-s.1967
32. 6. An overview of advancesin the standardization of herbal drugs Neeraj
Choudhary andBhupinder Singh Sekhon* PCTE Institute of Pharmacy, Near
Baddowal Cantt. (Ludhiana) -142021, India. *Email:sekhon224@yahoo.co
7. https://www.researchgate.net/publication/298426911 A review on the
Standardization of herbal medicine
