Title of Invention


Abstract Now, it is evident that insulin therapy is not a panacea for diabetes mellitus. This realization has promoted a great deal of research toward the development of more effective ways of treating the disease and has led to the discovery of several orally active agents. Metformin hydrochloride is an orally active hypoglycemic agent-Root covering of Flemingia macrophylla was cleaned, dried and powdered. The experiment was undertaken with aqueous decoction of root covering of F. macrophylla using metformin hydrochloride as the standard. Micro HPLC was used as a tool for detection of metformin hydrochloride. The chromatograms obtained during HPLC Studies clearly represented the presence of this compound in the sample.
(39 of 1970)
[See sectionlO]
(a) Dr. Shipra Roy,
A-92, Minal Residency, J.K.Road, Bhopal - 462023 (M.P.) India,
Nationality: Indian;
(b) Mrs. Venu Agarwal,
B-134,Alkapuri, Bhopal - 462024 (M.P.) India, Nationality: Indian
"An improved Micra HPLC method for identification of Metformin hydrochloride in root of Flemingia macrophylla."
The following specification (particularly) describes the nature of the invention and the manner in which it is to be performed.

"An improved Micro HPLC method for identification of Metformin hydrochloride in root of Flemingia macrophylla."
Field of invention
It is a branch of medicinal chemistry, deals with phytochemistry, or plant
chemistry, and is concerned with the enormous variety of organic substances that are elaborated and accumulated by plants and deals with the chemical structures of these substances, their biosynthesis, turnover and metabolism, their natural distribution and their biological function.
Background of invention
It is evident that insulin therapy, as currently practiced, is not a panacea
for diabetes mellitus. This realization has promoted a great deal of research toward the development of more effective ways of treating the disease and has led to the discovery of several orally active agents, including the sulphonylureas and the biguanides.
The applications of microcolumn HPLC have grown significantly in recent years. The trends in HPLC are to employ continuously smaller diameter columns to reduce solvent consumption, enhance sample detection. The role of microcolumn HPLC is also increasingly important in limited sample. With a very limited sample quantity and sample concentration, micro HPLC offers the best solution for the analysis
Object of invention
Search for plant drugs, without any side toxic effects. With a very limited
sample quantity and sample concentration, capillary HPLC offers the best solution for the analysis.

The findings observed in high pressure liquid chromatography indicates the presence of metformin hydrochlonde in the test sample . Metformin is an antihyperglycemic agent which improves glucose tolerance in patients with type 2 diabetes, lowering both basal and postprandial plasma glucose Its pharmacologic mechanisms of action aro different from other classes of oral antihyperglycemic agents. Metformin decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Unlike sulfonylureas, metformin does not produce hypoglycemia in either patients with type 2 diabetes or normal subjects and does not cause hyperinsulinemia. With metformin therapy, insulin secretion remains unchanged while fasting insulin levels and day-long plasma insulin response may actually decrease. Metformin hydrochloride is a biguanide, which does not create any adverse effects on the metabolic reactions.The presence of this metformin hydrochloride was confirmed in the root cover of Flemingia macrophylla was done by using Micro HPLC.
Brief Discription Of Accompanying Drawing
Drawing 1 shows the peak and the retention time of standard solution of metformin hydrochloride.Drawing 2 shows the similar peak of aqueous solution of Flemingia macrophylla.
Detailed Description
Micro High Pressure Liquid Chromatography was the method employed. Large reduction in solvent consumption and therefore solvent operation and toxic waste disposal cost, very low flow rates used with microcolumns make it the method of choice for HPLC-MS, particularly the bench-top MS with electrospray and nanospray interfaces. Low flow rates used in microcolumns facilitate the unified approach to chromatography. These major advantages are the result of

the fact, that small particles such as 3 and 5 urn can be efficiently packed in microcolumn and therefore same separation chemistry and elution pattern as that obtained from a 4 6 mm id, column can be expected. As a result of smaller column diameters for microcolumn HPLC, the flow rate requirement is also reduced and the elution peak volume (flow rate times peak width at half-height) is therefore reduced by a factor equal to the ratio of the cross-sectional areas for the 4.6 mm to the microcolumns.
The stationary phase bonded to a porous polymer is held in a narrow bore stainless steel column and the liquid mobile phase is forced through under considerable pressure with the help of a suitable pumping system. The mobile phase is a miscible solvent mixture. The compounds are monitored as they elute off the column by means of a detector, usually measuring in the ultra violet or visible regions of the spectrum.
HPLC was carried out under following conditions
Column Octadecyl silane (ODS) C-18/15cm.
Particle size 3 micro meter
Inner diameter of column 5 mm
Mobile phase 50:50 , Water: Methanol
Wave Length 220 nm
Flow rate 0.2 ml/min.
Instrument used TSP ( Thermo Seperation Product ) make micro
HPLC system with UV-VIS detector.
Reagents: HPLC Grade water and methanol from Emerck India, standard solution, sample.
Standard Solution: Aqueous solution of metformin hydrochloride (0.1666 mg/ml) was used as standard.

Test Sample of Flamingia macrophylla: 500mg of sample powder was submerged in 3ml-distilled water. After keeping for 6 hours, it was stirred for 30 min Then it was filtered in 10 ml volumetric flask and diluted the solution 20 times.
The ehromatograms obtained during HPLC Studies are represented in drawing 1 and 2. The retention time and characteristic peak was recorded. The standard sample was run in micro HPLC under the similar conditions already mentioned. The test sample was also run under the same experimental conditions. For the test sample at the same retention time the characteristic peak was observed. This confirms the presence of same compound i.e metformin hydrochloride in the test sample.
Thus with the help of micro HPLC , a new method has been evolved with which the presence of a compound in herbal produt having the same pharmacological effect as that of a synthetic compound , can be proved
Metformin hydrochloride is a synthetic compound used in synthetic drugs for the treatment of diabetes . This compound was found to be present in the root cover of Flemingia macrophylla . The above mentioned method was used for this purpose.

We claim
(i) An improved Micro HPLC method for identifying Metformin hydrochloride in root cover of Flemingia macrophylla having the same pharmacological effect for the treatment of Diabetes comprising of
a) preparing the standard solution,
b) preparing the aqueous solution of root cover of Flemingia macrophylla,

c) allowed the standard solution of stage (a) and aqueous solution at stage (b) into Micro HPLC.
d) the Micro High Pressure Liquid Chromatography characterized in that column used octadecyl silane, particle sized 3 micrometer, inner diameter of column 5mm, mobile phase of water and methanol (in 50:50 ratio), wavelength 220 nm and flow rate adjusted to 0.2 ml / min.
e) the powdered root cover of Flemingia macrophylla submerged in distilled water for 6 hours, stirred for 30 minutes followed by filtration and diluted the filtrate twenty times.
(li) A method as claimed in claim (i) wherein the standard solution prepared using water.
Dated 26 day of May 2003
( Shrpra Roy )




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Patent Number 210394
Indian Patent Application Number 541/MUM/2003
PG Journal Number 43/2008
Publication Date 24-Oct-2008
Grant Date 03-Oct-2007
Date of Filing 28-May-2003
Name of Patentee DR. SHIPRA ROY
Applicant Address A-92, MINAL RESIDENCY, J K ROAD, BHOPAL.
# Inventor's Name Inventor's Address
PCT International Classification Number A61B1/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA