1.1. Diabetes is a serious disorder that is rampant across the globe. In India one in eight persons and one in six persons in Europe and USA are suffering from this disorder. Diabetes can be characterized into two groups viz., Type I and Type II. Among these, Type I is insulin independent and Type II is insulin dependent. The diabetes caused by Type I
can be treated and cured but not Type II. People do suffer from Type I diabetes for malfuction of
pancreas due to insufficient release of insulin. If
the pancreas stops functioning owing to many
reasons and does not secrete insulin, then that disorder is called Type II diabetes. Hence Type I
diabetes is still possible to be treated by stimulating the pancreas to secrete enough quantities of insulin. But Type II diabetes cannot be treated and supplementing insulin through external source (injection) is the only remedy at present. Scientists worldwide have been working hard to contain this disorder. In India several

pubescent on both sides with rounded or subcordate base and acute apex.
Flowers are small, yellow, in axillary ambate cymes. Fruits are slender, follicles upto 7.5 cms
long, seeds brown, glabrous, obovate with a thin,
broad marginal wing. Globally, the species is distributed in Paleotropics, tropical region of Africa, Asia. It is widely distributed in peninsular India. It can be propagated through seeds by sexual means and through stem cutting by vegetative means.
1.3 Gymnema sylvestre: AS A REMEDY
Amongst several herbs Gymnema sylvestre is considered to be one of the most useful herbs in treating Diabetes mellitus. It is known to grow throughout India and also in neighboring countries. It is believed that India is the center of origin

for this herb. It is known as Madhunashini (Kannada); Gokhru (Unani); Gurmar (Hindi); Meshasringi (Bengali, Sanskrit); Podapatri (Telugu); Shirukurinja (Tamil); Kharak (Arabian) ; Khar e Khasak (Persian); Masabedda (Singhalese); Mashasringi (Nepalese);
The herb has been critically investigated and found that it is highly useful in curing several ailments. Several clinical studies have been
conducted in animal manimalian models and in human beings to examine the utility and validity of the herb in containing the diabetes. All have opined that the blood sugar levels reduced after administering the drug compared to the control animals.
The herb is used to treat not only diabetes but also several other ailments. In literature we find all plant parts are useful. The leaves are

used to treat; inflamination, dyspepsia, jaundice, hemorrhoids, renal and vesicle calliculi, helminthiasis, cardiopathy, asthama, bronchitis, intermittent fever, amorrhea, conjunctivitis and leucoderma. The roots are used as emetic and expectorant. In some cases, the leaves are heated with sesame oil, to form an emulsion and used to treat eye diseases. The leaf paste of Gymnema sylvestre is externally used to cure mycosis of the toes. It is also used as hepatoprotective agent in curing liver disorders. In view of its importance in curing a number of ailments, the pharmaceutical industry is making use of the herb in many formulations.
1.4 BACKGROUND INFORMATION
Based on the ancient literature, humans have been using the herb for treating the ailments for better health. The biggest practical problem is

administration of drug. To administer the drug,
some of these questions have to be answered well before it is given to a patient/ viz.,
1. How much of G. sylvestre leaf should be packed in to each tablet?
2. If a known quantity of drug is packed whether all the tablets contain same active Ingredient, at all given times?
3.Whether the plant releases same amount of gymnemic acid (active principle) at all given times throughout the year?
These questions are relevant since the crude drug is collected from various places (states) of the country, at various seasons, having wide variety of agro-climatic diversity.

Most of these questions can be answered if the active principle is estimated accurately. Presently/ the estimation of gymnemic acid is done based on gravimetric method. It gives a very rough estimate of gymnemic acid because the extraction procedures followed to isolate gymnemic acid are not sensitive. The scheme of steps adapted in gravimetric method to convert all fractions of reactive gymnemic acids is not complete or getting converted into fractions that are not included and estimated. Hence there is a need to revisit the problem and refine the methodology for higher accuracy. We have examined the gravimetric method and found that the estimation is not accurate.
1.5 OBJECTIVES OF THE STUDY
Owing to these reasons, the applicants have cleared some of the bottlenecks, that would help the industry and also practitioners to decide on

the quantity of drug based on gynmnemic acid (active principle) content of the drug.
l.A process for isolation, estimation, and quantification of gymnemic acid from the dried leaves of G. sylvestre.
2. A process for conversion of total gymnemic acid to deacyl gymnemic acid (DAGA) .
3. Detailed process for estimation of total gymnemic acid in the leaves of G. sylvestre using HPLC technique as DAGA.
2.0 PRIOR ART
Many scientists have attempted extraction of gymnemic acids from the leaves of Gymnema sylvestre* The work was first initiated by Hooper (1887), but the work did not prosper till 1959,
until Warren and Pf f afmann11 did try to isolate gymnemic acid and some sugars. From 1966 onwards, quite a few number of scientists isolated gymnemic acids, gymnemagenin, acyl moieties and sugars,
There has been quite a lot of information on fractionation of gymnemic acids and their structural elucidation. The main objectives of these studies were to isolate various fractions of gymnemic acid and not total gymnemic acid per se.
A patent has been registered at USPTO about
the use of Gymnema sylvestre for the treatment of
diabetes mellitus (Patent No: 5,900,240 filed on May 4th, 1999) .

3.0 PROBLEMS IN THE PRIOR ART
3.1 SCANTY INFORMATION
The leaves are used in the industry mainly for its active ingredient i.e., gymnemic acid. The applicants have reviewed the published literature for quant3ification of gymnemic acids, but they
could find scanty information on procedure to quantify the gymnemic acid. When fractionation of gymnemic acids is made the summation of fractionation will not lead to total gymnemic acids. This may be due unknown errors and procedural defects. Hence, to overcome this problem the applicants have aimed to develop a protocol for quantification of gymnemic acids.
compound to an extent of 98% while estimating gymnemic acid in HPLC, hence accurate estimation is
possible using our technique.
3.3 ABSENCE OF PROTOCOL FOR TOTAL GYMNEMIC ACID
One of the biggest problems that the industry is facing is conversion of all fractions of gymnemic acids to single framework of gymnemic acid, which is essential for quantification of gymnemic acid in gravimetric method. Estimation of various fractions of gymnemic acids would not necessarily sum up to total gymnemic acid due procedural sensitivities.
For these reasons the estimation of gymnemic acid is a limitation.

4.0 HOW THE INVENTOR SEEKS TO SOLVE THE PROBLEM?
The applicants overcome the problem by
defining the process to first isolate and
characterize the compound - gymnemic acid. This is tedious process since there is no published
information.
The second step is purification of the compound. Since purity is the major constraint we plan to purify using several methodologies. The purified compound will be characterized for its chemical integrity.
The third step is guantification by conversion of all fractions of gymnemic acid to a single fragment of total gymnemic acid. A new methodology based on the inputs of workers will be developed.
5.0 WHAT ARE THE DIFFERENT STEPS INVOLVED IN
DEVELOPMENT OF PROTOCOL
5.1 DIFFERENT STEPS INVOLVED IN ESTIMATION OF
TOTAL GYMNEMIC ACID
Raw material was subjected to successive extraction using Petroleum ether, chloroform, 1:1 ethanol: water, and butanol to get the butanolic extract which was dried and mixed in water, this is
subjected to pH adjustment with cone, HCL, the precipitate was subjected to successive extraction with the same above solvent and followed by extraction with ethyl acetate and subjected to various charcoal purification to get the total gymnemic acid whi ch was subjected to column chromatography to get the pure gymnemic acid. Different steps involved in estimation of gymnemic
acid are essentially described in Step No, 7.1
through schematic diagram.

5.2 DIFFERENT STEPS INVOLVED IN CONVERSION OF TOTAL GYMNEMIC ACID TO DECAYL GYMNEMIC ACID -SCHEMATIC REPRESENTATION
Total gymnemic acid

Converted to deacyl gymnemic acid (DAGA) by
saponification using 3 % potassium hydroxide in
alcohol and refluxed for 20 minutes

T
To give crude DAGA which was loaded to Sephadex LH - 20 containing column and eluted with water to get
purified DAGA.
This was characterized by Thin Layer chromatography (TLC), High Performance Liquid chromatography (HPLC), Infra Red Spectroscopy

(IRS), Nuclear Magnetic Resonance (NMR) and Liquid Chromatography Mass Spectroscopy (LCMS). Using this DAGA molecule, possessing the basic framework of all the gymnemic acid present, a novel method was identified to analysis total gymnemic acid as DAGA using HPLC instrument, the mobile phase being orthophosphoric acid buffer and acetonitrile.
6.0 APPARATUS REQUIRED Plant material
Plant material - leaves of Gymnema sylvestre (dried)
Instruments
Balance - lKg balance Mettler balance ± 10 gms ,
Electronic balance ± 1 mg. Spatula.

pH meter - Elico table top
Roto evapourator, BUCHI, Water bath, Vacuums o ven with heater
Glass apparatus
Volumetric Flasks - Various capacity - 10, 25, 50, - 100, 250, 500, 1000 ml
Round Bottom Flasks - 5000 ml
Funnel,
Separating Funnels, Columns for chromatography, .Pipettes - 2.0, 5.0, 10.0, 25.0 ml, Burette - 25, 50 ml,
Chemicals
Buffer solutions (4.0, 7.0 and 9.2) Silica gel for column chromatography. TLC Precoated plate silica gel G 60. Sephadex LH-20

Petroleum ether
Chloroform
Ethanol
Butanol
Hydrochloric acid Silicic acid Ethyl acetate Activated charcoal Potassium Hydroxide
Filter paper
Whatman No.1, Whatman No.4 0, Blotting papers,
Glass wares
Small glass vials - 5 ml, 10ml, 20 ml.

7.0 FLOW CHART
7.1 FLOW CHART FOR TOTAL GYMNEMIC ACID

Transferred this dried Crude gymnemic acid and silicic acid Mass to 5 litres three neck bottle


Concentrated this ethyl acetate to give brownish black precipitate, which was dried completely of ethyl acetate.


Loaded into column using ethyl acetate and methanol in increasing polarity

Crude DAGA was loaded to column containing Sephadex LH - 20 and eluted with water to get purified DAGA.

This was characterized by TLC, HPLC, IR, NMR and LCMS. Using this DAGA molecule, which is nothing but the basic framework of all the gymnemic acid present. A novel method was identified to analysis total gymnemic acid as DAGA using HPLC instrument, the mobile phase being orthophosphoric acid buffer and acetonitrile. STATEMENT OF INVENTION:-This invention provides for,
A process for making 98% pure total gymnemic acid stabilized as deacyl gymnemic acid from Gymnema sylvestre leaves comprising of the following steps:
(i) The dried leaves of Gymnema sylvestre are treated with organic solvents such as petroleum ether and chloroform separately and in succession, optionally in a hot water bath to facilitate maximum extraction of total Gymnemic acid, wherein the petroleum

ether removes the unwanted substances such as chlorophyll, and the chloroform dissolves the gymnemic acid
(ii) The chloroform slurry devoid of any ether residue is treated with 50% ethanol (diluted with distilled water) mixed well and subsequently the ethanol is removed by evaporation from the slurry, comprising gymnemic acid,
(iii)
The resultant slurry, after removal of ethanol is dried at 70 to 90 degrees Celsius,
(iv)
The dried residue is further dissolved in water followed by acidification to pH 1.5 to 2.5., resulting in crude gymnemic acid precipitated as black, shiny, resinous mass,
(v) The said resinous mass is further subjected to purification by boiling the same with

methanol and charcoal and passing the same over resin column to get a clear solution of total gymnemic acid which is nearly clear,
) The resultant product of step 5, being total gymnemic acid, the same is stabilized as deacyl gymnemic acid by saponification process using 2.5 to 4% potassium hydroxide solution or any other alkali of same concentration in alcohol and refluxed for 20 minutes to get a crude deacyl gymnemic acid which is now passed through a sephadex column and eluted out using distilled water to obtain purified deacyl gymnemic acid.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:-
FIGURE 1. The above photo of the isolation of total gymnemic acid depicts the flow of isolation of the gymnemic acid using various solvents followed by further purification with column chromatography and the TLC carried out using the pure total gymnemic acid using the mobile phase containing I PA: NH3: CHCL3 at the ratio of 8:3:1. The TLC plate after
eluting on the mobile phase was dried at 105° C and followed by spraying with Acetic anhydride and chloroform (1:1) and followed by drying and than spraying with 10 % sulphuric acid and drying.
PE : Petroleum ether
CHCL3 : Chloroform
ETH : Ethanol
BUT : Butanol
ETH.ACT : Ethyl acetate

STD 1 : GA from external source
STD 2 : GA isolated from the authors
FIGURE 2. The above photo depicts the total gymnemic acid having been converted DAGA after
saponification using alcoholic potassium hydroxide. The above TLC is done using a mobile phase containing n-butanol: ethyl methyl ketone: formic acid: water (5:3:1:1) . The TLC plate after eluting on the mobile phase was dried at 105° C and followed by spraying with Acetic anhydride and chloroform (1:1) and followed by drying and than spraying with 10 % sulphuric acid and drying.

CLAIMS:
WE CLAIM: -
1. A process for making 98% pure total gymnemic acid stabilized as deacyl gymnemic acid from Gymnema sylvestre leaves comprising of the following steps:
(i) The dried leaves of Gymnema sylvestre are treated with organic solvents such as petroleum ether and chloroform separately and in succession, optionally in a hot water bath to facilitate maximum extraction of total Gymnemic acid, wherein the petroleum ether removes the unwanted substances such as chlorophyll, and the chloroform dissolves the gymnemic acid
(ii) The chloroform slurry devoid of any ether residue is treated with 50% ethanol (diluted

removed by
comprising
with ditilled watet) mixed well and
subsequently the ethanol is evaporation from the slurry, gymnemic acid,
i) The resultant slurry, after removal of ethanol is dried at 70 to 90 degrees Celsius,
) The dried residue is further dissolved in water followed by acidification to pH 1.5 to 2.5., resulting in crude gymnemic acid precipitated as black, shiny, resinous mass,
The said resinous mass is further subjected to purification by boiling the same with methanol and charcoal and passing the same over resin column to get a clear solution of total gymnemic acid which is nearly clear,
The resultant product of step 5, being total gymnemic acid, the same is stabilized as

deacyl gymmemic acid by saponification process using 2.5 to 4% potassium hydroxide solution or any other alkali of same concentration in alcohol and refluxed for 20 minutes to get a crude deacyl gymnemic acid which is now passed through a sephadex column and eluted out using distilled water to obtain purified deacyl gymnemic acid.
2. A process as claimed in claim No. 1 wherein the
dried leaves are first treated with petroleum ether and chloroform in succession and separately, optionally in hot water bath atleast thrice in succession, to facilitate maximum dissolution of gymnemic acid in the chloroform solvent.
3. A process as claimed in claim No.l wherein the
chloroform resi due, i s treated with 50 % ethanol (diluted with distilled water) and the same is further subjected to hot water bath.
4. A process as claimed in claim No. 1 wherein the resulting slurry, after ethanol/water evaporation and ethanol treatment, is dried at 70 to 90 degree Celsius and the dried extract is dissolved in water followed by acidification to pH. 1.5 to 2.5, when the gymnemic acid precipitates into black resinous mass.
5. A process as claimed in claim No. 1 wherein the
gymnemic acid precipitate is boiled with charcoal
and methanol and then filtered by sephadex filled
- in column.
6. A process as claimed in claim No. 1 wherein the
total gymnemic acid is stabilized as deacyl
gymnemic acid by saponification process by using 3% potassium hydroxide solutions or any suitable
alkali in alcohol and refluxed for 20 minutes to
get a crude deacyl gymnemic acid which is now
passed through a sephadex column and eluted out using distilled water to obtain purified deacyl gymnemic acid,
7. A process for making 98% pure total gymnemic acid as substantially herein described and as illustrated in the examples furnished herein.