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1433-1448 Page 1433 Vejendla.Ravikumar * Et Al Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y ISSN: 0975-766X Available Online through Research Article www.ijptonline.com DEVELOPMENT OF RP-HPLC METHOD FOR THE QUANTIFICATION OF KARANJIN IN THE SEED EXTRACTS OF PONGAMIA GLABRA Vejendla.Ravikumar*, Md.SirajuddinKhan 1, M.Manoj kumar 2 1Departmant of Pharmacy, Sri Indu Institute of Pharmacy, Hyderabad. 2Departmant of Pharmacy, Sir C.R.Reddy College of Pharmaceutical Sciences, Eluru. Email: [email protected] Received on 19-12-2010 Accepted on 01-01-2011 ABSTRACT: The main aim of the work, done by the author, is to estimate the active ingredient Karanjin, a cyclic furano- flavonoid, present in the different parts of seed extracts of karanja ( Pongamia glabra ) mentioned the structure in Fig.1. and to carry out the Quantitative Analysis of the Karanjin. Reporting the % of Karanjin present in the different solvent extracts namely: ethyl acetate, methanol & hexane of dry seed, kernel, shell and fresh seed & kernal of Pongamia glabra. By taking 200g. of each part found that in dry kernel maximum amount of 36.92g. is extracted by using methanol solvent, which was quantified by using HPLC was reported as 18.46% which is maximum when compared with other parts or by using other solvents. O O OCH3 O Fig1. Structure of Karanjin INTRODUCTION 1: The author thought to discuss some of the important features of flavonoid compounds as the present phytochmical work carried out was mainly concern with the flavonoid compounds. IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1433 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Flavonoids are yellow pigments, which occur in plant kingdom either in the free state or as glycosides or associated with tannins. These are also known as the anthoxanthins. Chemically, the flavones are hydroxylated derivative of flavone (2-phenyl-4-chromone), which are partially alkylated. In most of the flavones, positions 5 and 7 are hydroxylated and also one or more of positions 3, 4, 5 are also hydroxylated. Further, positions 3 1 and 51 are often methylated whereas positions 5, 7 and 4 1 are usually unmethylated. Figure: 2. Basic structure of flavone, flavonol, and flavanone (flavonoids). Quercetin is a flavonol with OH (hydroxy) group at 3, 5, 7, 3 1, 4 1 positions. Kaempferol is a flavonol with OH group at 3, 5, 7, 4 1 positions. Quercetagetin is a flavonol with OH group at 3, 5, 6, 7, 3 1, 4 1 position. Flavones show two absorption bands, one at 330-350 nm and other at 250-270 nm. Thus, it becomes possible to distinguish flavones from the anthocyanins on the basis of absorption bands and also by colour reactions. PROPERTIES OF FLAVONOIDS: 1 • Many flavones are yellow solids • Mostly flavones are soluble in water, ethanol, dilute acids and alkalis. • Flavones are precipitated by lead salt. • With ferric chloride, flavones give either a dull green or a red brown colour. • Shinoda test: Alcoholic solution of flavones in the presence of magnesium ribbon and concentrated hydrochloride gives magenta colour. IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1434 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y PHARMACOLOGICAL ACTIVITIES OF FLAVONOIDS: Flavonoids are phenolic compounds with nuclei arranged in a C6 - C3 - C6 configuration. They have several beneficial properties for plants. The co-evolution of plants and insects has resulted in different plant defense mechanisms, one of which is the accumulation of phytochemicals, which may affect feeding, or growth of insects. 1 For example, the resistance of soya bean, Glycine max L., to the cabbage looper, Trichoplusia ni, appears to be due to the presence of the leaf flavonoids, daidzein, glyceolin, sojagol and coumestrol.2 The generalist feeder, Spodoptera frugiperda, avoids plants known to contain flavone. 3 Similarly, the C-glycosyl flavone (maysin) in maize silk has antibiotic activities against corn earworm larvae, Helicoverpa zea (Boddie). 4 Flavonoids also function to shield plants from UV radiation, act as signaling molecules in plant bacterium symbioses, and are the primary pigments that attract pollinators and seed dispersers 5 although flavonoids are widely distributed among the flowering plants, particular classes of flavonoids have distinct functions in different plant groups. For example, flavonols are essential for male fertility in maize and petunia 6, but they do not appear to have a similar function in Arabidopsis. 7 Isoflavonoids are the major phytoalexins in legumes 8, whereas 3- deoxyanthocyanidins fulfill similar functions in Sorghum bicolor and other grasses 9 multiple mechanisms have been proposed to explain the diversity of phytochemicals between different plants. 10,11 From a pharmaceutical perspective, flavonoids possess a remarkable spectrum of biochemical and pharmacological activities. Different flavonoids exhibit antioxidative, antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory properties. 12,13 Catechin and catechin derivatives, oligomeric proanthocyanidins, quercetin and quercetin chalcone, Ginkgo flavone glycosides, silymarin, and others are utilized in the prevention and treatment protocols for cardiovascular disease, cancer, asthma, periodontal disease, liver disease, cataracts and macular degeneration. 14-18 Kuntz et al 19 reported that dietary polyphenols could have a significant role in the prevention of colon cancer by blocking hyper- proliferation of the epithelium and by promoting apoptosis. IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1435 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y BOTANICAL CHARACTERISTICS AND SIGNIFICANCE OF P. GLABRA: TAXONOMICAL CLASSIFICATION: [20] Kingdom : plantae (plants) Sub kingdom : tracheobionta (vascular plants) Super division : spermatophyte (seed plants) Division : magnoliophyta (flowering plants) Class : liliopsida (monocotyledons) Sub class : arecidae Order : arecales Family : leguminacea Genus : Pongamia Species : glabra / pinnata VERNACULAR NAMES: [21] Telugu : Gaanuga, Pungu Tamil : Ponga, Pongam. Oriya : Koranja. Kanada : Honge. Assam : Karchaw. USES OF PLANT PONGAMIA: [22] Traditional Uses: The wood of the plant is used in building material, ploughs, combs, yokes, oil-mills and solid wheel of a cart and as fuel. The oil extract from the seeds is used in lamps by the weaker section of the society and also in soap making. IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1436 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Seeds are used as fish poison. Leaves are used as fodder, as manure for rice and sugarcane fields. Pharmacological Uses: The fresh bark of P.pinnata is used internally to cure bleeding piles. The root and the bark are bitter, anthelmintic and used in vaginal and skin diseases. A poultice of the leaves is applied on ulcers infected with worms. Seeds are anthelmintic, bitter, acrid and carminative they are useful in inflammations, pectoral diseases, chronic fever, hemorrhoids and anemia. The oil is styptic, anthelmintic, good in leprosy, piles, ulcers, chronic fever and pain in liver. A decoction of dried flowers is given for diabetes. The juice of the roots is used for cleaning teeth, strengthening gums, against gonorrhea and cleaning foul ulcers. The juice of the plant is used for treating diarrhea, cough, leprosy and gonorrhea. Aqueous extract of stem bark exhibits significant CNS sedative and antipyretic activities. MATERIALS: All chemicals were analytical grade: Methanol from Qualigens fine chemicals (Mumbai, India), Millipore- Water used for the preparation of mobile phase solutions, was obtained from All Quartz Double Distilled, Bhanu Scientific Instruments Company Pvt. Ltd. (Bangalore, India). All the above solutions were de-gassed in an ultrasonic bath (Sonicator), for 30min. Stock solution of karanjin for generating standard curves were prepared by dissolving 1.2mg of compound in 20ml s.v.f with methanol to yield concentration of 6000mg/ml. Working standard solution of karanjin obtained by diluting 1, 0.8, 0.6, 0.4, 0.2, 0.1ml in 10ml standard volumetric flask so as to get 60, 48, 37, 24, 12, 6µg/ml concentration of karanjin respectively to get a calibration curve IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1437 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Chromatography was performed on HPLC equipment consisting of LC-10AS pumps, SPD – 10A UV- Visible detector, and an injector equipped with a 20ul sample loop (Rheodyne, USA). Analytical separation was on column packed with 3µm Waters LC - 18 stationary phase (Waters, USA). The dimension of the separation column was 150 x 4.6mm i.d. with 3um particle size. Data and chromatograms were collected using C-7RA chromatopac software system (Shimadzu, Japan). Dissolution of the compound was enhanced by sonication on Bandlin-Sonerex (Bandelin, Berlin). UV spectra of karanjin for selecting the working wavelength of detection were recorded on Cintra 5 UV-Visible spectrophotometer (GBC Scientific equipments, Australia). METHADOLOGY: A collected dried seed were crushed into powder and was taken into column weighed amount of powder as 200g. Kept socking for about 12hours with the solvent Hexane so as to remove fat solubles, then after run with Methanol and collect the methanol extract and kept for concentration through rotovapour so as to get the methanol extract
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