PLANT PROFILE

Tinospora Sinesis

Family : Menispermaceae

Synonymes :
English : Gulancha tinopora, Tinospora

Hindi : Gulancha. Jungly Giloy, Amrita

Sanskrit : Guduchi, Amrita

Marathi : Gulvel

Distribution : Throughout India in forests.

Plant:
A large extensively spreading glabours, perennial deciduous twiner with
succulent stems; leaves simple; flowers yellow in lax racemes, male flowers in
clusters, female flowers usually solitary; fruits drupes, red when ripe.

Part uses : Stems, leaves, roots and whole plant.

Properties and uses:

Plant is widely used in Ayurvedic systems of medicine for its general tonic,
anti-diabetic, anti-malarial, anti-allergic and aphrodisiac properties. It is also
claimed to be useful in skin diseases, vomiting, anaemia, piles, chronic fever and
emaciation.

1
 The stem is bitter, stomachic, antipyretic and astringent. It is powdered
and made into and infusion, used as alterant. Starch from stem and root (Jungly
Giloe Satwa) have nutrient property and is used in chronic diarrhea and
dysentery. Juice of tehfresh plant is used as a powerful diuretic and is also used
in gonorrhea. All part of decoction products are mentioned in Ayurvedic text
books for use in joint diseases. The root is known for its anti-stress, anti-leprotic
and anti-malarial activities. Various part of plants are shown in fig.

2
 TINOSPORA SINESIS
• The chemical examination of stem of Tinospora Sinesis was carried out.
The petroleum ether, chloroform, ethanol extracts of the stem were carried
out and their yields were measured.

• The alkaloids from the stems of Tinospora Sinesis have been precipitated
with Dragendorff reagent.

• The alcoholic extract of fresh stems of Tinospora Sinesis was found to

contain two bitters (tinosporide and cordifolide), one alkaloid (tinosporin)
and δ -sitostrol.

• Concentrated alcoholic extracts of fresh stems on fractional distillation

reported to have three bitter compounds i.e. tinosporon, tinosporic acid
and tinosporal and their structures were established by IR absorption
spectra.

• Antidiabetic effect of aqueous and alcoholic extracts of the stems of

Tinospora Sinesis was reported.

• Two compounds were isolated from petroleum ether extract of leaves of

Tinospora Sinesis which were identified by TLC on silica gel G plates as
octacosanol and β -sitosterol.

• From the alcoholic extract of fresh stems, β -sitosterol and a compound

named tinosporidine have been isolated. Petroleum ether extract of the
leaves gave heptacosanol and a carbonyl compound named cordifolone.

• Tinosporide, a diterpene isolated from stems of Tinospora Sinesis

reported with molecular formula, C23H26O8 and structure was assigned on
spectral grounds.

3
• A new clerodane furanolactone with molecular formula, C20H22O8 has been
isolated from stem and its structure was established by 1H NMR and C13
NMR.

• The stem wood of Tinospora Sinesis had yielded a novel 18 norclerodane

diterpene O- glucoside which has been named tinosporaside and its
structure can be assigned on the basis of NMR studies.

• A novel furanoid diterpene glucoside, C26H34O11 was isolated from the hot
chloroform extract and its structure was determined by spectroscopic and
chemical methods.

• Structure of columbin, a diterpenoid furanolactone was established by IR

and NMR spectra.

• The chemistry and pharmacology of Tinospora Sinesis was studied which

includes the constituents which are present in stems, leaves, roots and
aqueous extract alcoholic extract gives anti-stress and immunomodulatory
activity.

• Several glycosides were isolated, as polyacetates, from butanol fraction of

stems and structure of 3 new norditerpene furan glycosides, Cordifoliside
A, B and C were established by 1D and 2D NMR spectroscopy.

• Phenylpropanoid glycosides ad tetra hydro furofuran lignan glycosides

were isolated from the plant drugs Tinospora Sinesis and Drypetus
roxburghii.

• Tinosponone and tinocordioside have been isolated from stem and

structure were established by spectroscopic studies and chemical
correlation.

4
• Cordifoliside D and E were isolated as tetracetates from polar butanol
extract and structural elucidations and relative configurations were based
on high resolution 1 D and 2D NMR spectroscopy.

• The structure of cordioside, isolated from the stem was characterized on

the basis of NMR spectroscopy.

• Jatrorrhizine, iscolumbin, palmatine, tetrahydro disaccharides were

isolated and screened for immunostimulant activity and structure were
elucidated on the basis of spectroscopic evidences.

• Palmatosides C and F have been isolated as tetracetates from butanol

fraction and structure were elucidated by NMR studies.

• By using NMR spectra, structure of steroid, isolated from the ethyl acetate
extract of aerial parts of Tinospora Sinesis has been established.

• A new daucane-type sesquiterpenes, tinocordifolioside and tinocordifolin

has been isolated from the stems and their structure were established by
detailed spectroscopic studies.

• Two photoecdysones viz. ecdysterone, makisterone A and N-trans

feruloyltyramine were isolated from butanol fraction of methanol extract of
stems and structure were elucidated by NMR studies.

• The active principle of Tinospora Sinesis were found to possess anti-

complementary and immuno-modulatery activities. Syringin and cordiol
inhibited invitro immunohemolysis of antibody coated sheep erythrocytes
by guinea pig serum.
• Prince, P.S.M. et. al. reported hypolipidaemic and antioxidant activities of
Tinospora Sinesis roots in experimental diabetes.

5
• Menon, V.P. et. al. reported hypoglycemic and other related actions
of aqueous extract of Tinospora Sinesis roots in allloxan-induced diabetic
rats.

• Grover. J.K. et. al. reported anti-hyperglycemic effect of Eugenia

jambolana and Tinospora Sinesis in experimental diabetes and their
effects on key metabolic enzymes involved in carbohydrate metabolism.

• Kar, A., et. al. reported comparative evaluation of hypoglycemic activity of

30 Indian medicinal plant in alloxan-induced diabetic rats which includes
Tinospora Sinesis.

• Grover, J.K., et. al. reported 45 medicinal plants and their products (active,
natural principles and crude extracts) for anti-diabetic potential which
includes Tinospora Sinesis.

• The structure of tinosporide has been revised from the previously (Ahmad
1978) suggested structure on the basis of its IR, NMR and mass spectra
and by chemical correlations.

• Guduchisatwa, an Ayurvedic drug derived from Tinospora Sinesis was

mainly composed of a 1 → 4 – linked glucan. Chemical and enzymatic
studies on the glucan revealed that they have both similarities and
differences from amylase.

• The stems of Tinospora Sinesis were found to contain magflorine and its
biogenetic precursor tembetarine and identified by their methyl derivatives.

• The occurrence of quaternary Alkaloids was studied. The components

were proterberine bases, berberine and palmatine, with jatrorrhizine, an

6
 occasional minor constituent, and the apomorphine base, magnoflorine.
The presence of choline was also reported.

• A new diterpenoid furanolactone has been isolated from the stem of

Tinospora Sinesis. Its structure was established by 1H NMR and C13 NMR
studies.

• Systematic chemical investigation of Tinospora Sinesis yielded a new

phenolic lignan was shown by a combination of spectroscopic and
chemical methods.

• A new clerodene diterpenoid, C20H21O7, has been isolated from stem and
its structure was established by spectroscopic means and by comparison
with closely related clerodene derivatives.

• Chloroform extract of stem yielded new clerodene derivative with

molecular formula, C20H22O8 and structure was established by IR spectra.

7
OBJECTIVE
Diabetes mellitus, a common metabolic disorder known as fast spreading
disease with varied etiology and is associated with variety of irreversible
complications. The modern management of diabetes, in spite of newer
developments, remains unsatisfactory. Presently used antidiabetic drugs have
been found to have limitations in therapeutic use, primarily, because or their
undesirable and untoward effects.

Chlorpropamide like sulphonylureas causes severe cholistatic jaundice

and occasionally pulmonary eosinophilia, whereas, hypoglycemia, allergic
reactions like leucopenia, thrombocytopenia, agranulocytosis, etc. and
cardiovascular complications i.e. stroke and myocardial infarction being common
side effects of other sulfonylureas.

Biguanides are associated with less severe reactions like anorexia,

nausea and abdnominal discomfort. Phenformin is contractindicated in patients
with liver, kidney and cardiac diseases because of its ability to produce lactic
acidosis.

Thiazolidinedione derivatives like rosiglitazone, pioglitazone and

troglitazone are always at a high risk of liver toxicity. They are always prescribed
with a constant check on liver toxicity.

Thus there is continuous need to develop and antidiabetic drug in view of

alarming rise in diabetic patients worldwide.

The search for natural sources still leads to drug discovery and drug
design, and has established with unexpectedly fast developing biotechnology
and biomedical fields. The humanity is turning back towards increasing reliance
on herbal medicine.

8
 Literature survey revealed that the stem of plant Tinospora Sinesis
(Menispermaceae) is traditional in Indian system of medicine for treatment of
diabetes.

The constituents of Tinospora Sinesis stem are Diterpenoids (Clerodane

derivative), Alkaloids, Steroids, (β -Sitosterol), Lignan (Phenolic lignan),
Octacosanol, Heptacosanol and Nonacosa-15-one.

Therefore the objective of present study was to screen different extracts

for antidiabetic activity and to isolate the constituent responsible fro antidiabetic
activity.

9
PLAN OF WORK
1. Procurement of plant material.
2. Drying and size reduction of stems.
3. Extraction with –
(a) Petroleum ether (60-80º C)
(b) Benzene
(c) Chloroform
(d) Ethyl acetate
4. Determination of hypoglycemic activity of each extract by using Alloxan-
induced diabetic rats.
5. Preliminary phytochemical screening of active extract to isolate the
presence of active constituent.
6. Thin layer chromatography of active extract to determine the presence of
number of chemical constituents.
7. High performance thin layer chromatography (HPTLC) of active extract to
confirm the number of constituents present.
8. Separation of chemical constituents of active extract by using column
chromatography.
9. Phytochemical screening of the separated constituents.
10. Screening of hypoglycemic activity of separated constituents by
using Alloxan-induced diabetic rats.
11. Physio-chemical studies of separated constituents:
(a) Melting point
(b) UV spectra
(c) IR spectra
(d) CHN analysis
(e) Mass spectra
(f) NMR spectra

10
Experimental

LIST OF CHEMICALS AND EQUIPMENTS USED

List of Chemicals
1. Petroleum ether (60-80) (LR)
2. Benzene (LR)
3. Chloroform (LR)
4. Ethyl acetate (LR)
5. Methanol (AR)
6. Chloroform (AR)
7. Acetic acid
8. Hydrochloric acid
9. Silica gel G (For TLC)
10. Silica gel (for column chlromatography) (60-120 mesh)
11. GOD / POD Kit

List of Equipments
1. Single pan balance
2. pH meter
3. Centrifuge
4. UV spectrophotometer
5. Soxhlet apparatus
6. Chromatographic column
7. TLC chamber and plates
8. H.P.T.L.C.
9. IR.
10. Melting point apparatus

11
PROCUREMENT OF PLANT MATERIAL

The plant of Tinospora Sinesis (Wild) was procured form indigenous Drug
Market of Nagpur. Identification of the plant material was through an examination
of flowering top specimen. Identity of the plant material was confirmed by the
outer surface which was grayish brown to almost black in colour, longitudinally
wrinkled which showed a soft pale yellow or dirty white surface with marked
wedge-shaped structure brought about by radiating medullary rays. The plant
material was identified and authenticated from the Department of Botany, Nagpur
University Campus, Nagpur. The herbarium sheet and authentication certificate
should below :

The authentication number is Acc. No. 7/1.

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13
 DRYING AND SIZE REDUCTION
Stems were subjected to drying in normal environmental conditions under
shade and then subjected for size reduction to coarse power by pulverization.

EXTRACTION
Powdered stems (250 g) were charged into soxhlet apparatus and
successive hot continuous extraction was carried out using following solvents
1. Petroleum ether (60-80º C)
2. Benzene
3. Chloroform
4. Ethyl acetate

Powdered stem (5 kg) were extracted with each of the solvent.

Each time before extraction with the next solvent, the powdered material
was air dried. Each time extract was filtered using suction and each extract was
concentrated by distilling the excess of solvent to obtain the crude extractive. The
drug was extracted with each solvent until complete extraction was affected
(about 40 cycles).

EVALUATION OF HYPOGLYCEMIC ACTIVITY OF EACH EXTRACT

The determination of blood glucose was most frequently carried out test in
the laboratories for screening of hypoglycemic activity and it was done using
glucose oxidase / peroxides method. Other methods used were done in three
stages.
1. Precipitation of the blood proteins.
Reduction, ether of an alkaline cupric copper solution to cuprous oxide, or of
alkaline potassium ferricyanide to ferrocyanide.

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Working Reagent Preparation

Reagent 2 was mixed with the volume of Regent 1 indicated on the label.
The reconstituted reagent was stable up to three months at 2-8º C or one month
at 20-25º C.

Laboratory Procedure for End point Test

Blank Standard Sample
Working Reagent 5.0 ml 5.0 ml 5.0 ml
Standard - 5.0 ml -
Sample - - 0.05

Blank, standard and sample were mixed properly and optical density was
read after 20 min. incubation at room temperature.

Calculations :
Glucose (mg/d1) = O.D. Sample
---------------------- X 100
O.D. (Standard)

Where : O.D. = Optical Density

PRELIMINARY PHYTOCHEMICAL SCREENING OF ETHYL ACETATE EXTRACT

The plant may be considered as a biosynthetic laboratory for a multitude

of compounds like alkaloids, diterpenoids, sterols, phenolic lignans, glycosides,
etc. that exerts physiological effect. The compounds that are responsible for
therapeutic effect are usually the secondary metabolites. A systematic study of a
crude drug embraces through consideration of both primary and secondary
metabolites derived as a result of plant constituents.

15
 The ethyl acetate extract was subjected to preliminary phytochemical
screening for the detection of various plant constituents.

1) Test for Sterols and Terpenoids

i) Salkowski Reaction
To a few mg of the residue in 2 ml chloroform, conc. Sulphuric acid (2 ml)
was added. The reagents were stirred for few minutes. The red colour was
developed in chloroform layer and acid layer gave greenish yellow fluorescence
indicating the presence of sterols.

ii) Liebermann-Burchard Reaction

To a few mg of the residue, chloroform was dissolved, few ml of acetic
anhydride and 2 drops of concentrated sulphuric acid from the side of the test
tube were added. The greenish transient colour indicates the presence of sterols.
The pinkish red colour indicates presence of terpenoids.

2) Test for Alkanoids

To a few ml of each extract, 5 ml of 1.5 v/v HCI was added and filtered.
Those filtrates were used for testing alkaloids.

i) Dragendroff’s Reagent
To solution A (17 g of bismuth subnitrate + 200 g tartaric acid + 800 ml
distilled water), solution B (160 g potassium iodide + 400 ml distilled water) was
added and mixed in 1:1 v/v, from this solution, working standard was prepared by
taking 50 ml of this solution and adding 100 g of tartaric acid and making up to
500 ml with distilled water. The above reagent was sprayed on a filter paper and
the paper was dried. The sample solution in dilute hydrochloric acid was applied
on the paper using a capillary tube.

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Formation of orange red colour indicated the presence of alkaloids in them.

ii) Mayers Reagent (Potassium Mercuric Oxide)

To 1.38 g of mercuric chloride, 60 ml of distilled water and 5 g of
potassium iodide in 10 ml of distilled water were added. The solution mixed and
diluted to 100 ml with distilled water.
To a little of the extract dilute HCI was added in a watch glass, few drops
(excess dissolves the precipitate) of the reagent were added. Formation of cream
coloured precipitate showed the presence of alkaloid.

iii) Wagner’s Reagent (Iodine – Potassium Iodide)

To 1.27 g of iodine & 2 g of potassium iodide, 5 ml of water was added
and solution was diluted to 100 ml, to this reagent when a little or the filtrates (i.e.
Acid solutions of the extracts) was added, a brown flocculent precipitated which
indicate the presence of alkaloids.

3) Test for Saponins

Foam Test
To a few mg of residue of each extract in a test tube, small amount of
sodium bicarbonate and water was added separately and shaken vigorously,
stable froth was obtained which indicate the presence of saponins

4) Cardiac Glycosides
Keller Killiani Test
To a few mg of the residue of the extract, 5 ml alcohol (70%) was added
and boiled for two to three minutes and filtered. To the filtrate, 2 ml of water and
strong lead acetate solution were added. The solution was filtered and the filtrate
was extracted with chloroform. The chloroform layer separated

17
and evaporated slowly in a porcelain dish. To the residue, 1 ml of glacial acetic
acid containing one drop of ferric chloride was added and this was carefully
transferred to the test tube containing 2 ml of sulphuric acid. A reddish brown ring
at the junction of two layers indicated positive results for the deoxy sugars and
therefore cardiac glycosides.

Legal Test
To a residue, pyridine and sodium nitroprusside solution was added and
make it alkaline. The pink colour indicates the presence of cardiac glycoside.

5) Cyanogenetic Glycosides

Guignard Test

To 2 g of moist shredded powder stem in a test tube, few drops of

chloroform was added to enhance the enzymatic activity. Sodium picrate solution
(5 g sodium carbonate, 0.5 g of picric acid, water q.s. 100 ml) was prepared &
strip of filter paper were then moistened with this solution. The strips were
removed, dried and then inserted between the split cork stopper which was
exercised to ensure that the paper strip did not touch the inner sides of the test
tube. The test tube was then warmed at 30-35º C for half an hour. Absence of a
red color on paper indicated the absence of HCN evolution and hence the
absence of cyanogenetic glycosides,

6) Test for Anthraquinone Glycisides

Bontrager Test

To a little of the extract, 5 ml of 10% sulfuric acid was added and boiled for
a few minutes and filtered while hot. The filtrate was cooled and shaken with
benzene. The benzene layer was separated and shaken with half of it’s

18
Volume of 10% ammonia. Ammonical layer acquired pink colour indicating the
presence of anthraquinone.

7) Test of Sugars

Mollisch’s Test

To a few mg of extracts in test tube, 0.5 ml. of water was added and mixed
with two drops of 10% solution of naptol in alcohol, then 1 ml of conc. H 2SO4
through the sides of inclined test tube was added, so that the acid from a layer
beneath the aqueous solution at the common surface of the liquids, shake and
was added allowed the mixture to stand for two minutes, then 5 ml of water. A
dull violet precipitate appeared. It showed the presence of sugar.

8) Test for Lignans

To a few mg of extract, 500 ml of conc. HCI and 2% furfuralhehyde were

added. The development of red colour indicated the presence of lignans.

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THIN LAYER CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT

1) Preparation of Plate
Silica gel G TLC plates were prepared by coating glass plates with slurry
composed of 18 g of adsorbent and 45 ml of water. After air drying for 30 min. at
room temperature (27º C) Plates were activated in an oven at 105º C for 1 hr.
The plates were cleaned from back side and from edges. Before using
these plates, they were marked with scale, fixing the distance to be travelled by
solvent from using sharp pin.

2) Application of Sample
For applying samples on plates, glass capillaries were used. The distance
between two spots was kept at minimum of 1 cm.

3) Development of Chromatogram
The chromatogram was developed by the ascending technique in which
plate was immersed in the developing solvent to a depth of 0.5 cm. The chamber
used was lined with sheets of filter paper which dip into the solvent, this ensures
that the chamber was saturated with solvent vapour. Development was allowed
to proceed until the solvent front has been traveled the required distance (usually
10-15 cm), then the plate was removed from the chamber and solvent front was
immediately marked with a pointed object. The plate was dried using heat or a
current of air as appropriate.
Spray reagents used for visualizing components on chromatogram are
benzoyl chloride-sulfuric acid reagent and modified Liberman-Burchard reagent.

20
The Rf value was calculated as follows:

Rf = Distance travelled by the sample

-------------------------------------------
Distance travelld by the solvent

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY OF ETHYL

ACETATE EXTRACT

1) Selection of Solvent System

The solvent system which was developed for TLC was used for HPTLC
and it showed same result was obtained.

2) Application of Sample
A small quantity of extract was dissolved in ethyl acetate and 5 ul sample
was applied on precoated plate with the help of Linomet IV applicator.

3) Development of Chromatogram
A rectangular twin trough glass chamber was used in the experiment. To
avoid insufficient chamber saturation and the undesirable edge effect, a smooth
filter paper was placed in the glass chamber and was allowed to be soaked in the
developing solvent. The moistened paper was pressed against the walls of the
chamber so that it adheres to the walls. The chamber was allowed to saturate for
15 min. before use. The experiment was carried out at room temperature in
diffused day light.

Procedure
The plate was dipped in a saturated chromatographic chamber containing
the solvent system and was allowed to elute upto 8 cm and was air dried. The
Spots were scanned in CAMAG TLC scanner-3.

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Chromatographic Condition
Following are the chromatographic conditions required to get and effective
resolutions by selected mobile phase.

Stationary phase : HPTLC precoated, silica gel 60 F254 (Merck, Germany

Size : 5 X 10 cm
Developing chamber : Twin trough glass chamber
Mode of application : Band
Band size : 5 mm
Separation technique : Ascending
Temperature : 20 ± 5º C
Saturation time : 15 min.
Scanning wavelength : 350 nm
Scanning mode : Absorbance / Reflectance

Column Chromatography of Ethyl Acetate Extract

1) Selection of Mobile Phase

The solvent system development for TLC and HPTLC was used as mobile
phase for column chromatography. Alternation in the composition of the eluting
solvent was achieved by adding the second solvent gradually to a reservoir of the
first with efficient mixing. This is known as gradient elution which, with proper
choice of adsorbent and gradient reduce tailing of the compound on the column.

2) Preparation of the Column

The slurry of Silica gel G (60-120 mesh size) was prepared by mixing the
adsorbent with the mobile solvent. Solvent poured into the column. A cotton wool
was placed at the base of the column and an air bubble inside the

22
cotton wool were removed. Small amount of sand poured into the column in
order to provide flat base. Slurry of adsorbent poured gradually and allowed to
settle. The air entrapped was removed by stirring with glass rod. A filter paper
disc was placed above the adsorbent. Sand was added. The excess solvent then
run off of until the level falls of 1 cm above the top layer of sand.

3) Preparation and Application of Sample

A saturated solution was prepared by dissolving the extract in methanol. 5

ml of sample was applied to the column.

4) Collection of Fractions
5 ml fraction was collected each time.

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SUCCESSIVE SOLVENT EXTRACTION OF STEMS OF TINOSPORA SINESIS
On successive solvent extraction of the dried coarse powdered stems of
Tinospora Sinesis with different solvents, differing in their polarity, resulted in
separation of constituents of different polarity range present in different extracts.
The colour, consistency and percentage extractive values were included in
table 1.

Table No.1: The Colour Consistency and Percentage Extractive Values of

Extracts
Sr. Solvent Color and Consistency % Extractive
No. Value w/w
1 Petroleum ether (60-80º C) Greenish yellow, Oily 4.2
2 Benzene Brown, Semisolid 0.49
3 Chloroform Greenish Brown, Semisolid 0.20
4 Ethyl Acetate Dark Brown, Solid 1.9

SCREENING OF HYPOGLYCEMIC ACTIVITY OF DIFFERENT EXTRACTS

All extracts obtained were subjected fro evaluation of hypoglycemic

activity in Alloxan-induced diabetic rats.

Metformin was taken as standards and administration of Metformin (500

mg/kg) showed significant hypoglycemic activity in Alloxan-induced diabetic rats
as compared with control.

Among the various extracts, only ethyl acetate extract significantly

decreased blood glucose level as compared to the control group. Other extracts
did not show significant decrease in blood glucose level.

24
Phytochemical examination of ethyl acetate extract:
Different test were performed to identify the classes of compounds present
in the extract which shows the presence of saponins and sugars. It is shown in
table 5.
Table 5
Plant constituents Test Inference
1. Sterols a. Salkowski reaction -
b. Liberman Burchard reaction +
2. Alkaloid a. Dragondorf’s reagent -
Mayer’s reagent -
c. Wagner’s reagent -
3. Saponin a. Foam test +
4. Cardiac glycoside a. Keller Killiani test -
b. Legal test -
c. Kedde’s test -
5. Cyanogenic glycoside a. Guignard test -
6. Anthraquinone glycoside a. Bontrager;s test -
7. Flavonoids Magnesium turnings test -
8. Sugars Mollisch’s test +

Table 6. Thin Layer chromatography of ethyl acetate extract

Constituent Rf value in solvent system
I II III
1 0.0875 0.1043 0.0163
2 0.1586 0.2935 0.7096
3 0.1708 0.3114 0.8112
4 0.1992 0.3495 0.9154
Listed in the order of Rf values
Solvent systems: I - Chloroform–formicacid-methanol (4:1:1)
II - Chloroform-acetic-acid-methanol (5:1:1)
III - Chloroform-methanol-acetic acid (10:1:1)
PHYTOCHEMICAL SCREENING OF ETHYL ACETATE EXTRACT
As the ethyl acetate extract showed hypoglycemic activity, further
screening of ethyl acetate extract was performed to isolate active constituents in
the extract. The results showed in table 7.

25
 The ethyl acetate extract showed presence of diterpenoids, as active
constituents.

Table No. 7: Phytochemical Screening of Ethyl Acetate Extract

Sr. Plant Constituents Tests/Reactions Inference
No.
1 Sterol i) Salkowaski - Ve
ii)ibermannBurchard + Ve
2. Alkaloids i) Dragendroff’s - Ve
ii) Wagner’s - Ve
iii) Mayer’s - Ve
3 Terpenoids i) Libermann Burchurd + Ve
4 Cardiac Glycosides i) Kellar Killani - Ve
ii) Legal - Ve
5 Cynogenetic Glycosides i) Bontrager’s - Ve
6 Anthraquinone Glycosides i) Bontrager’s - Ve
7 Saponins i) Foam + Ve
8 Sugars i) Mollish - Ve
9 Lignan i) Methoxylene Group - Ve

+ Ve → Presence of Chemical Constituents.

- Ve → Absence of Chemical Constituents

THIN LAYER CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT

The ethyl acetate extract was subjected to TLC. Various solvent systems
were tried, of which the most suitable was Chloroform: Methanol: acetic acid
(9:1:0.1). The spots were located by iodine vapour chamber and UV lamp. The
ethyl acetate extract showed 5 spots, which indicate the

26
COLUMN CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT
The constituents were separated by column chromatography using gradient
elution technique. The constituents separated were collected in fractions
indicated in Table 8.

Table No.8 Column Chromatography of Ethyl Acetate Extract

Solvent Fraction Constituents Detection
(5 ml)
Chloroform : Methanol 1-8 V
( 9 : 1) 9-15 V, IV Detection on
16-19 IV Preparative TLC
Chloroform : Methanol 20-24 IV, III using ( 9 :1 )
(7 : 3) 25-28 III Chloroform :
29-.31 III, II Methanol solvent
Chloroform : Methanol 32-35 II system
(5 : 5) 36-40 II, I
40-45 I

Purification : Purification of the constituents obtained was performed by

preparative TLC.

PHYTOCHEMICAL SCREENING OF SEPARATED CONSTITUENTS

The separated constituents were subjected to Liberman-Burchard test for
the presence of terpenoids. Of the 5 constituents, constituents 2, 3, 4 and 5
confirmed terpenoids.

Table No. 9: Phytochemical Screening of Separated Constituents

Sr. No. Constituents Test for Terpenoids
1. 1 - ve
2. 2 + ve
3. 3 + ve
4. 4 + ve
5. 5 + ve
+ ve → Presence of terpenoide.
- ve → Absence of terpenoids.

27
Silica gel chromatography of ethylacetate extract.

Table 10
Solvent Fractions Constituents Detection
(5 ml each)
Chloroform-methanol 1-17 None -
(10:1) 18-21 IV II
Chloroform-methanol 2225 IV, III II
(7:3) 26-29 III II
3035 III, II II
Chloroform-methanol 41-45 - -
(5:5) 46-51 I I
Detection by TLC in solvent system

Purification
Purification of the compounds obtained was done by preparative TLC.

Physicochemical data of constituents:

1. Melting Point
Melting points of the separated constituents were shown in the
table 11.

Table 11
Sr. No. Constituent Melting point (ºC)
1 1 212.4-216.8
2 2 198.4-201.8
3 3 226.6-228.2
4 4 280.1-282.3

28
2. U.V. Spectrum of constituents

U.V. spectrum was taken by dissolving the constituents in methanol. The

absorbance maximas are shown in table 12.

Table 12

Sr. No. Constituent Absorbance

Maximas (nm)
1 1 220
2 2 225, 269
3 3 222, 257, 370
4 4 223, 270, 229

29
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