Tinospora Sinesis
Family : Menispermaceae
Synonymes :
English : Gulancha tinopora, Tinospora
Marathi : Gulvel
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.
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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.
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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.
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• A new clerodane furanolactone with molecular formula, C20H22O8 has been
isolated from stem and its structure was established by 1H NMR and C13
NMR.
• A novel furanoid diterpene glucoside, C26H34O11 was isolated from the hot
chloroform extract and its structure was determined by spectroscopic and
chemical methods.
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• 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.
• By using NMR spectra, structure of steroid, isolated from the ethyl acetate
extract of aerial parts of Tinospora Sinesis has been established.
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• 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 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.
• The stems of Tinospora Sinesis were found to contain magflorine and its
biogenetic precursor tembetarine and identified by their methyl derivatives.
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occasional minor constituent, and the apomorphine base, magnoflorine.
The presence of choline was also reported.
• 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.
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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.
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.
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Literature survey revealed that the stem of plant Tinospora Sinesis
(Menispermaceae) is traditional in Indian system of medicine for treatment of
diabetes.
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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
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Experimental
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
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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 :
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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
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).
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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.
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)
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The ethyl acetate extract was subjected to preliminary phytochemical
screening for the detection of various plant constituents.
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.
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
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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
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
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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.
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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.
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The Rf value was calculated as follows:
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.
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.
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
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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.
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.
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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 +
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The ethyl acetate extract showed presence of diterpenoids, as active
constituents.
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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.
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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.
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
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2. U.V. Spectrum of constituents
Table 12
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BIBILIOGRAPHY
Arbid M.S.(2002) .
723 .
• Kiritikar K.R. Basu b.d.;Indian medicinal plants Vol II ;second edition third
• Kiritikar K.R. Basu b.d.;Indian medicinal plants Vol II ;sudhindra nath ebasu
Distributers.
• www.Google.com
• www.pubmed.com
• www.siencedirect.vom
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