EXTRACTION, ISOLATION AND STRUCTURAL ELUCIDATION OF FLAVONOID FROM CHROZOPHORA PLICATA LEAVES AND EVALUATION OF ITS ANTIOXIDATIVE POTENTIALS

Objective: This investigation involves the extraction, isolation, and characterization of flavonoid from a Euphorbiaceae family plant Chrozophoraplicata followed by evaluation of its antioxidant principles.Methods: The dried leaves were subjected to sequential soxhlation with polar and nonpolar solvents. Methanolic extract reveals the presence of largeamount of flavonoids. Methanolic extract was subjected to isolation using column chromatographic analysis with solvents such as petroleum ether,chloroform, hexane, ethyl acetate, methanol, and water. Further, the isolated compound was subjected to thin layer chromatography technique andspectral analysis such as infrared, 1HNMR, 13CNMR, mass spectroscopy, and high performance thin layer chromatography (HPTLC) finger printingtechniques. The compound was evaluated for in vitro antioxidant studies using 2,2-diphenyl-1-picrylhydrazyl (DPPH), NO assay, reducing power assay,H2O2 scavenging assay, superoxide anion scavenging assay and β-Carotene linoleate system and in vivo antioxidative studies using carbon tetrachloride(CCl4), and acetaminophen intoxicated rats.Results: The compound was isolated in methanol:water in the ratio of 80:20 using column chromatographic technique. On the basis of phytochemical,chromatographic, and spectral analysis, the isolated compound was identified as kaempferol and finally with HPTLC finger printing technique it wasfound that the Rf value of the isolated compound was found to be 0.58 which is nearly similar to the Rf value of standard kaempferol (0.55). Hence,the isolated compound was confirmed as kaempferol and is structurally elucidated as 3,5,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one. Thiscompound was isolated for the first time from the C. plicata leaves. The in vitro antioxidant assay of isolated flavonoid has shown a dose-dependentincrease in free radical scavenging activity using DPPH, no assay, reducing power assay, H2O2 scavenging assay, superoxide anion scavenging assay, andβ-carotene linoleate system. Further, the methanolic extract of C. plicata (MECP) leaves was subjected to single dose acute toxicity study for 14 days infemale rats on the basis of OECD guidelines 423 and the therapeutically selected doses were 200 mg/kg and 400 mg/kg. In vivo antioxidant studies inCCl4 and acetaminophen intoxicated rats indicated that the MECP leaves have significantly decreased lipid peroxidation in a dose-dependent mannerand increased the levels of catalase, superoxide dismutase, and glutathione.Conclusions: By the above results, it was concluded that the isolated compound from C. plicata leaves was confirmed as kaempferol and it possessessignificant antioxidative potentials.Keywords: Chrozophora plicata leaves, Flavonoids, Extraction, Isolation, Characterization, Methanolic extract, Antioxidant activity, Carbontetrachloride, Acetaminophen

STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ALOGLIPTIN BENZOATE AND METFORMIN HYDROCHLORIDE IN TABLET DOSAGE FORM

Objective: A new simple, accurate, precise, economic and robust stability indicating RP-HPLC method has been developed and subsequently validated for the estimation of alogliptin and metformin in bulk and pharmaceutical dosage form.Methods: The HPLC separation was carried out by using hypersil BDS, C18 (250 x 4.6 mm, 5m.) column with a mobile phase comprising phosphate buffer and acetonitrile (48:52 % v/v) pH adjusted to 4.8 with orthophosphoric acid. The flow rate of the mobile phase was 1.0 ml/min and effluent was monitored at 210 nm using PDA detector. The retention time of alogliptin and metformin was 3.78 min and 2.78 min respectively. Forced degradation studies were conducted to know the stability of the drug samples under various stress conditions like acid, base, peroxide, and photolytic degradation according to ICH guidelines.Results: The results of this study showed excellent separation of the drug samples using developed method. The percentage recoveries were found 99.91 to 101.01% for alogliptin and 99.78-100.87% for metformin which is in the limits of acceptance. The calibration curve was plotted and the method was found to be linear over a range of 3-18 µg/ml and 125–750µg/ml of alogliptin and metformin respectively and regression data for calibration curve showed good linear relationship with r2= 0.9990 for the both alogliptin and metformin. In the study stability section, it was observed that there is no interference of the degradation products with drug samples.Conclusion: A new stability-indicating RP-HPLC method has been developed for estimation of alogliptin and metformin in bulk and pharmaceutical dosage form. The developed method was validated, and it was found to be simple, sensitive, precise, robust and it can be used for the routine analysis of alogliptin and metformin in both bulk and pharmaceutical dosage forms.Â

NEW METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF FEBUXOSTAT IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY

Objective: To develop a new method and validate the same for the determination of Febuxostat (FBS) in human plasma by liquid chromatography–mass spectrometry (LCMS).Methods: The present method utilized reversed-phase high-performance liquid chromatography with tandem mass spectroscopy. Febuxostat D9 (FBS D9) was used as internal standard (IS). The analyte and internal standard were separated from human plasma by using solid phase extraction method. Zorbax Eclipse XDB, C8, 100 mm x 4.6 mm, 3.5 µm column was used and HPLC grade acetonitrile, 5 millimolar (mM) ammonium format (80: 20, v/v) as mobile phase, detected by mass spectrometry operating in positive ion and multiple reaction monitoring modes.Results: The parent and production transitions for FBS and internal standard were at m/z 317.1→261.0 and 326.1→262.0 respectively. The method was validated for system suitability, specificity, carryover effect, linearity, precision, accuracy, matrix effect, sensitivity and stability. The linearity range was from 20.131 ng/ml to10015. 534 ng/ml with a correlation coefficient of 0.999. Precision results (%CV) across six quality control samples were within the limit. The percentage recovery of FBS and internal standard from matrix samples was found to be 76.57% and 75.03% respectively.Conclusion: Present study describes new LC-MS method for the quantification of FBS in a pharmaceutical formulation. According to validation results, it was found to be a simple, sensitive, accurate and precise method and also free from any kind of interference. Therefore the proposed analytical method can be used for routine analysis for the estimation of FBS in its formulation

Preventive effect of Anogeissus latifolia in High Fructose Diet Induced Insulin Resistance and Metabolic Dyslipidemia

Anogeissus latifolia (Combretaceae) has been used in traditional system of medicine in India for over many years, and widely used to enhance the immune system and against various ailments. During the last decade many in-vitro, in-vivo and analytical studies witnessed and justified the traditional claim of this plant. The study was aimed to evaluate the preventive effect of Anogeissus latifolia bark in insulin resistance and metabolic dyslipidemia induced by high fructose diet feeding in rats. At the end of the treatment period, the plasma from normal control, diabetic, reference standard and Anogeissus latifolia extract (300mg/kg) treated animals were subjected to estimation of insulin, lipid profile, liver marker enzymes and kidney function test. Anogeissus latifolia was found to attenuate insulin resistance and metabolic dyslipidemia induced by adverse effects of fructose. This study validates the traditional use and shows a possible beneficial effect of the plant in the treatment of diabetes mellitus. Key Words: Anogeissus latifolia, High Fructose Diet, Insulin Resistance, Metabolic Disorder.

A Validated Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Aliskiren Hemifumarate and Amlodipine Besylate in Pharmaceutical Dosage Form

The present study describes the stability indicating RP-HPLC method for simultaneous estimation of aliskiren hemifumarate and amlodipine besylate in pharmaceutical dosage forms. The proposed RP-HPLC method was developed by using waters 2695 separation module equipped with PDA detector and chromatographic separation was carried on C-8 Inertsil ODS (150 × 4.6 mm, 5 µ) column at a flow rate of 1 mL/min and the run time is 10 min. The mobile phase consisted of phosphate buffer and acetonitrile in the ratio of 40 : 60% v/v and pH was adjusted to 3 with orthophosphoric acid and eluents were scanned using PDA detector at 237 nm. The retention time of aliskiren and amlodipine was found to be 3.98 and 5.14 min, respectively. A linearity response was observed in the concentration range of 30–225 µg/mL for aliskiren and 2–15 µg/mL for amlodipine, respectively. Limit of detection and limit of quantification for aliskiren are 0.161 µg/mL and 0.489 µg/mL and for amlodipine are 0.133 µg/mL and 0.404 µg/mL, respectively. The stability indicating method was developed by subjecting the drugs to stress conditions such as acid and base hydrolysis, oxidation, and photo- and thermal degradation and the degraded products formed were resolved successfully from the samples

Application of Digital (Motic) and Scanning electron microscope in histological study of leaf of Tecoma gaudichaudi DC (Bignoniaceae)

Tecoma gaudichaudi DC (Bignoniaceae) is a shrub or a small tree found in various regions of Maharashtra. The objective of the study was to develop various standardization parameters for the evaluation of leaves of this plant. Microscopy, powder characteristics and scanning electron microscopy (SEM) of leaves were observed and results were recorded. Histological study of leaves shows underlined palisade cells on the upper epidermal surface, anomocytic stomata on the lower epidermis with covering (2–3 cells) and glandular trichomes etc. The specific observed characteristics such as types of stomata and trichomes were predominantly observed on the lower epidermal surface of the leaf. Physicochemical analysis such as extractive value includes petroleum ether, ethanol, ethyl acetate and aqueous soluble extractive values of 3.08, 10.50, 8.5, and 13.4% w/w respectively; extracts were analysed by chemical test and showed presence of flavonoids, tannins, steroids and triterpenoids etc. Fluorescence analysis is one of the parameters that help in the analysis of chemical constituents. So these parameters are useful in the authentication of T. gaudichaudi DC and can play a vital role in authentication and standardization of botanicals

Dataset on leaf surface and elemental study of four species of Bignoniaceae family by SEM-EDAX

The data presented in this article are related to the scanning electron microscope and elemental studies in the four species of Bignoniaceae namely Tecoma gaudichaudi DC (Sample 1), Tecoma capensis (Thunb.) Lindl. (Sample 2), Tecoma stans (L.) Juss.Ex Kunth (Sample 3), Tabebuia rosea (Bertol.) (Sample 4). The SEM images were obtained for permanent record. The abaxial and adaxial surfaces of each species were carefully studied. In addition to this, the consistent occurrence of anomocytic stomata in all four species of this family shows that morphological and taxonomically all the species are very close and intimate.The elemental data on leaf samples of all four species were performed and total eight important components were present such as C, O, Mg, Al, Si, Cl, K, Ca. These elements are useful, so identification of inorganic components of these species defiantly helps to promote as dietary elements. Keywords: Bignoniaceae, Tecoma, Tabebuia, Tecoma gaudichaudi DC, Tecoma capensis (Thunb.) Lindl, Tecoma stans (L.) Juss.ex Kunth, Tabebuia rosea (Bertol), Scanning electron microscopy, Elemental analysi

A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations

In present study HPTLC method was developed and validated for the determination of friedelin in Putranjiva roxburghii Wall (family: Euphorbiaceae) leaf, bark extract and in polyherbal formulations. Analysis of samples were performed on TLC aluminium precoated plate (60 F254) by using mobile phase toluene: chloroform (9:1 v/v). Plate was derivatized with vanillin sulphuric acid and scanned at 580 nm. Developed method found to give compact spot for friedelin at Rf value 0.43 ± 0.01. The method was validated using International Council for Harmonization (ICH) guidelines including linearity, precision, accuracy, and robustness. Friedelin was found to be present in leaf extract of Putranjiva roxburghii Wall (0.003% w/w), in bark (0.04% w/w), formulation 1 (0.002% w/w) and formulation 2 (0.035% w/w). A good linearity relationship was found to be (100–500 ng spot−1) with correlation coefficient (r2) value of 0.9892 for friedelin. Limit of detection and limit of quantitation was found to be 32.15, 97.44 ng/band respectively for friedelin. The developed method was found to be accurate and precise with 0.78%, 0.9% (%RSD) for interday and intraday precision. Accuracy of the method was performed by recovery studies at three different concentration levels and the average percentage recovery was found to be 98.55% for friedelin. The proposed method for the quantitation of friedelin was found to be simple, specific, accurate and robust in Putranjiva roxburghii Wall and polyherbal formulations

Data on investigation of hypoglycemic, anti-cholesteremic, in vivo antioxidant and pancreatic beta cell protective effect of Putranjiva roxburghii Wall bark in streptozotocin-induced diabetic rats

The data existing in this article are associated to the antidiabetic activity of ethyl acetate extract of Putranjiva roxburghii Wall barks (EAPR) in streptozotocin (STZ) induced diabetic rats at a dose of 250 & 500 mg/kg by oral route for 21 days. The phytochemical screening of the extract was carried out by gas chromatography and mass spectrometry. Diabetes was induced by streptozotocin (50 mg/kg; i.p), EAPR (250 & 500 mg/kg; b.wt) and standard Insulin (6 IU/animal; subcutaneous; o.i.d) were administered to the diabetic rats. Body weight and blood glucose were estimated weekly. Cholesterol, SOD and CAT were estimated in the blood serum on 21 days of the investigation period. Oral administration of EAPR (500 mg/kg) significant rises in the body weight, decrease in the blood glucose and total cholesterol and restore function of SOD and CAT enzymes (P < 0.05). Current data were also supported by histological study, necrosis was observed in the diabetic rat pancreas; however, necrosis was less observable in treated groups. These findings reveal that an ethyl acetate extract of Putranjiva roxburghii Wall barks shows antihyperglycemic, anti-cholesterolemic, antioxidant and improved the cell density of β-cells of islets of Langerhans in diabetic rats. Keywords: Putranjiva roxburghii Wall barks, Streptozotocin, Antihyperglycemic, Anti-cholesterolemic, Antioxidan