Title of Invention

A PROCESS FOR THE EXTRACTION OF FLAVONOID FROM THE AERIAL OF BARLERIA LUPULINA LINDL.

Abstract

There is provided a process for the extraction of flavonoid of the formula 3.5.7.3'.4' -pentahydioxy flavone (quercetin) from the aerial parts like barks, leaves, stem of the plant Barleria lupulina Lindl. comprising the following steps: a) Selecting the aerial parts of the plant and removing extraneous deposits such as mud etc, b) Pulverizing the dried material to a coarse powder, c) Subjecting the pulverized material to extraction with 90% methanol in one or several cycles, d) Combining the several extracts and subjecting the same to concentration to reduce the volume to about 1/4 the original volume, e) Subjecting the concentrated extract to re-fraction with petroleum ether at temperatures of around 60° to 80° C, f) Subjecting the aqueous portion to hydrolysation using dilute sulphuric acid (10%) boiling under reflux, g) Cooling and extracting the refluxed solution with solvent ether, h) Drying the organic layer with anhydrous sodium sulphate. i) Concentrating the resultant solution to about 1/4 th of its original volume followed by j) Subjecting the concentrated solution to polyamide column, chromatographic separation using polyamide MNCS-6 grade with n-butanol, acetic acid and water (BAW) and used in the ratio 4: 1: 5, where after the required material is recovered.

Full Text

This invention relates to a process for the extraction of flavonoidal compound (quercetin) from the aerial parts of the plant Barleria lupulina Lindl. Prior Art and Drawback It is well known that flavonoid is isolated from sources such as plants. It is well known in Tamil Nadu. South India to use the whole plant of Barleria lupulina Lindl. (Acanthaceae) in traditional medicine of several human ailments. However, there has not been any attempt or suggestion for isolating flavonoids from the above plant source. Barleria lupulina Lindl. (Acanthaceae) is a well-known plant traditional medicine of India. The whole plant is used for various human ailments. In the preliminary qualitative chemical analysis methanol extract of the aerial parts of the plant showed positive test for the presence of steroids, flavonoids and tannins. In earlier study, the isolation of iridoid glucosides and alkaloid has been reported. More over, flavonoids are known to be use for the treatment of diabetes mellitus. We report herein the isolation, structural elucidation off a novel flavonol (quercetin) for the first time from the plant along with its anti-diabetic potential. Objects of the Invention It is an object of the invention to propose a method for the isolation and purification of flavonoid from Barleria lupulina Lindl. It is another object of the invention to propose such a process, which will be highly effective and produce material suited for the preparation of medicines. It is further object of this invention to propose such a method, which is not expensive and can be easily practiced. It is also another object of this invention to study the medicinal and structural characteristics of the isolated material. Z Brief Statement of the Invention According to this invention there is provided a process for the extraction of flavonoid of the formula 3.5.7.3'.4' - pentahydioxy flavone (quercetin) from the aerial parts like barks, leaves, stem of the plant Barleria lupulina Lindl. comprising the following steps: a) Selecting the aerial parts of the plant and removing extraneous deposits such as mud etc, b) Pulverizing the dried material to a coarse powder, c) Subjecting the pulverized material to extraction with 90% methanol in one or several cycles, d) Combining the several extracts and subjecting the same to concentration to reduce the volume to about 1/4 the original volume, e) Subjecting the concentrated extract to re-fraction with petroleum ether at temperatures of 60°,to 80° C, f) Subjecting the aqueous portion to hydrolysation using dilute sulphuric acid (10%) boiling under reflux, g) Cooling and extracting the refluxed solution with solvent ether, h) Drying the organic layer with anhydrous sodium sulphate. i) Concentrating the resultant solution to about 1/4 th of its original volume followed by j) Subjecting the concentrated solution to polyamide column, chromatographic separation using polyamide MNCS-6 grade with n-butanol, acetic acid and water (BAW) and used in the ratio 4: 1: 5, where after the required material is recovered. 3 Material and Method General: H and C- NMR spectra were recorded on a JNM-FX 100 FNMR spectrometer in DSMO-d6. IR spectra were taken with Perkin Elmer FTIR spectrophotometer in KBr discs (region of 4000-400 cm"1). Mass spectra were recorded in Shimadzu-qp-1000 (Quadraphor) mass spectrometer, UV spectrum was taken in Hitachi 2000 double beam UV visible spectrophotometer (200- 500 nm) and elemental analysis was performed with Perkin Elmer- Model 240 elemental analyzer. Column chromatography was carried out with polyamide MNSC 6 grade (E. Mark & Co. Ltd), eluting solvent of (chromatography grade) n-butanol: acetic acid: water (BAW-4:1:5) being used. Plant material: Aerial part such as leaves and bark of the plant of Barleria lupulina Lindl. (Acanthaceae) were collected and the taxonomical identification was established by the Botanical Survey of India, Shibpur, Howrah, India. The voucher specimen (J.S. -001) has been deposited at the herbarium of the Department of Pharmaceutical Technology, Jadavpur University, Calcutta, India. Extraction and isolation: Dried aerial parts of the plant Barkria lupuima (1 kg) were ground and exhaustively extracted with 90 % methanol (MeOH) The MeOH extract was concentrated under reduced pressure to afford a syrup (yield 125 g), which was refractronated with petroleum ether (60-80°C). The aqueous portion was hydrolyzed by addition of 10 % sulphuric acid, boiled under reflux for 2 h, cooled and extracted with solvent ether. This extract was concentrated to a small volume (l/4th) and subjected to polyamide column chromatographic separation with BAW (4: 1: 5) as eluting solvent system to afford a yellow coloured compound (50 mg). Structural elucidation: Isolated Yellow coloured powder melted (m. p) at the range of 310-312 (decomposed); UV ? max McOH nm: 256 (Band 1), 372 (Band II) with 5 % A1Cl3: 431 of band II; with 2 M NaOH: 325 of band I; with NaOAc: 268 of band I and after addition of H3BO3: 389 of band II. 1H-NMR (DMSO-d6): 8.4, 2.0 (H-5'-H-6' dd J=6.9, 7.7 Hz), 2.0 (H-2' d j= 7.5 Hz), 1.9 (H-6 d, J-6.2), 1.9 (H-8 d J=6.4 Hz); 13C-NMR (DMSO-d6): 160.735 (C-5), 98.203 (C-6), 163.893 (C-7), 93.370 (C-8), 156.151 (C-2), 135.737 (C-3), 175.849 (C-4), 121.986 (C-l'), 115.093 (C-21), 145.067 (C-3'X 147.708 (C-4'), 155.623 (C-5'), 120.003 (C-61) (Table. 1) IR (KBr discs) vmax in cm-1: 3419 and 2936 (aromatic ring structure), 1656 (quinonoid structure), 1456 (-C = C- bond), 1276 (phenolic-OH). C, H, N for C15H10O7 are C=59.51 %, H=3.30 %, N-Nil %. m/e: 302, 286, 273, 229,153 and 150. M. W= 302 (C15H10O7)11-13. Animais: Male Wistar rats weighing between 180-200 g were used in this study. Prior to experiment, animals were housed for at least one week in the laboratory animal 5 room. Housing conditions were maintained at 23±1 °C with 60 % humidity, in 12: 12 h light dark cycle Food and water were given ad libitum. Anti-diabetic evaluation: A modified Sharma's method was used14 The test drugs were prepared with 2 % v/v aqueous Tween 80. Oral administration; quercetin (20,40 mg/kg), glybenclamide (reference drug, 10 mg/kg) and 2 % aqueous Tween 80 (control) were administered to the alloxan-hyperglycemic rats and the plasma glucose concentration was estimated at 1.5, 3, 4.5 and 24 hours of drug treatment by ortho-toluidine method. Statistical analysis: The data are expressed as mean ± SE (n=6). ANOVA followed by Dunnefs t-test was employed to evaluate the statistical significant, p-values less than 0.05 imply significance. RESULT AND DISCUSSION The aerial parts of the plant Barleria lupullna was extracted with 90 % methanol and the extract was refractionated with petroleum ether (60-80°C). The aqueous layer was hydrolyzed with 10 % sulphuric acid and boiled under reflux for 2 hours. Then it was extracted with solvent ether and concentrated. This extract has been subjected to polyamide (MNSC-6 grade) column chromatography, yielded a yellow coloured compound. was identified as(3, 5, 7, 3', 4'-pentahydroxy flavone (quercstion)by the aid of UV, FTIR spectral data; by comparison of its H-NMR, C- 6 NMR and Mass spectral data with those of can authentic sample as well as available features. The UV spectrum of the compound m methanol (MeOH) was indicative of a fiavonoidal aglycone and UV spectral shift of the isolated compound with 5 % AlCl3 (59 nm bathochromic shift), 2M NaOH (69 nm bathochromic shift), NaOAc (12 nm bathochromic shift) and NaOAc + H3BO3 (17 nm bathochromic shift) indicated the free 5-OH, 4'-OH, 7-OH and 3', 4'-OH respectively confirmed the structure of a 3, 5, 7, 3', 4'-pentahydroxy flavone (quercetin). The isolated compound showed intense peak with a highest mass number at m/e 302 and it provides the molecular weight of the isolated compound. The molecular formula was established as C15H10O7. The structure of the compound was further confirmed by 'H-NMR in DMSOd6, which revealedH-2-H-6' meta coupling (2 Hz) at 7,5 ppm, H-6-H-5' ortho coupling (8.4 Hz) at 7.5 ppm and 6.9 ppm respectively, and another meta coupling attributed between H-6-H-8 (1.9 Hz) at 6.2 ppm and 6.4 ppm respectively (Table 1). These results have identical characteristics in every respect with authentic sample of quercetin. The C-NMR also conformed with the authentic sample. Furthermore, the structure of the isolated compound was confirmed by FTIR spectra. The compound showed the presence of quinonoid and - C = C - bond as well as presence of phenolic - OH group. The (vmax) observed at 3419, 2936, J656, 1456 and 1276 cm'1 which are exactly tallied with the values of authentic sample (quercetin). 7 The chemical structure of the isolated compound assigned as quercetin with additional support of elemental analysis (C= 59.51 %, H= 3.30 %) and mixed melting point (310-3I2°C). Anti-diabetic activity of quercetin was evaluated on alloxan-induced diabetic rats. Groups of animals fasted for 18 hours and were made diabetic by injecting them intra venously in the tail vein with 70 mg/kg body weight of alloxan monohydrate every third day for three doses. After 7 days of alloxan injection, blood glucose levels of the surviving rats were determined by ortho-toluidihe method. Rats with blood glucose levels of 150-350 mg/100 ml were considered diabetic and divided into groups of 6 animals each and administered with test drugs. Glybenclamide was used as a positive control treatment The glucose level of the experimental groups were estimated at 1.5, 3,4.5 and 24 hours of drug treatment by ortho-toluidine method. The maximum activity (p 9 found to have an inhibitory effect on the production of lipid peroxide in-viva. On the basis of the literature citation, the anti-diabetic potential of quercetin might be due to free radical scavenging property and antioxidant nature. However, further comprehensive pharmacological investigations require to elucidate the exact mechanism underlying this effect 9 Table 1. 1H- and13CNMR-spectroscopic data of the isolated compound from Jussiaea suffruticosa. Linn.in DMSO- d6(J9n HZ) Table 2. Effect of isolated quercetin on alloxan-induced diabetic rats Values are expressed as mean ± SE. Significance was evaluated by Student's-t test (n=6). p values were calculated versus control. a-p Data in parenthesis indicate percentage reduction of blood glucose level. 11 We Claim: 1 A process for the extraction of flavonoid (of the formula 3.5.7.3' 4- pentahydioxy flavone (quercetin) from the aerial parts (ike barks, leaves, stem of the plant Barieria lupulina Linda comprising the following steps:. a) selecting the aerial parts of the plant and removing extraneous deposits such as mud etc, b) pulverizing the dried material to a coarse powder, c) subjecting the pulverized material to extraction with 90% methanol in one or several cycles, d) combining the several extracts and subjecting the same to concentration to reduce the volume to about 1/4 the original volume, e) subjecting the concentrated extract to re-fractionation with petroleum ether at temperatures of 600 to 80° C, f) subjecting the aqueous portion to hydrolysation using dilute sulphuric acid (10%) boiling under refluxm g) cooling and extracting the refluxed solution with solvent ether,. h) drying the organic layer with anhydrous sodium sulphate. i) concentrating the resultant solution to about 1/4th of its original volume followed by j) Subjecting the concentrated solution to polyamide column, chromatographic separation using polyamide MNSC-6 grade with n-butanol acetic acid and water (BAW) and used in the ratio of 4 1: 5, whereafter the required material is recovered. 2. A process for the extraction of flavonoid of the formula 3.5.7.3',4'- pentahydioxy flavone (quercetin) from the aerial parts like barks, leaves, stem of the plant Barieria lupulina Linda substantially as herein described with reference to the examples. 12 There is provided a process for the extraction of flavonoid of the formula 3.5.7.3'.4' -pentahydioxy flavone (quercetin) from the aerial parts like barks, leaves, stem of the plant Barleria lupulina Lindl. comprising the following steps: a) Selecting the aerial parts of the plant and removing extraneous deposits such as mud etc, b) Pulverizing the dried material to a coarse powder, c) Subjecting the pulverized material to extraction with 90% methanol in one or several cycles, d) Combining the several extracts and subjecting the same to concentration to reduce the volume to about 1/4 the original volume, e) Subjecting the concentrated extract to re-fraction with petroleum ether at temperatures of around 60° to 80° C, f) Subjecting the aqueous portion to hydrolysation using dilute sulphuric acid (10%) boiling under reflux, g) Cooling and extracting the refluxed solution with solvent ether, h) Drying the organic layer with anhydrous sodium sulphate. i) Concentrating the resultant solution to about 1/4 th of its original volume followed by j) Subjecting the concentrated solution to polyamide column, chromatographic separation using polyamide MNCS-6 grade with n-butanol, acetic acid and water (BAW) and used in the ratio 4: 1: 5, where after the required material is recovered.

Documents:

00230-kol-2003 abstract.pdf

00230-kol-2003 claims.pdf

00230-kol-2003 correspondence.pdf

00230-kol-2003 description(complete).pdf

00230-kol-2003 form-1.pdf

00230-kol-2003 form-18.pdf

00230-kol-2003 form-2.pdf

00230-kol-2003 form-3.pdf

00230-kol-2003 form-5.pdf

00230-kol-2003 letters patent.pdf

00230-kol-2003 p.a.pdf