DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF BERBERINE HYDROCHLORIDE AND EMBELIN IN POLYHERBAL FORMULATION

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1 DEVELPMENT AND VALIDATIN F ANALYTICAL METHD FR SIMULTANEUS ESTIMATIN F BERBERINE HYDRCHLRIDE AND EMBELIN IN PLYHERBAL FRMULATIN *ANKITA V GNDALIYA 1, KARTIK VIKANI 2, PANKAJ P KAPUPARA 3 ABSTRACT A reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of Berberine hydrochloride and Embelin in polyherbal formulations. The separation was achieved by C 18 (10 X 4.6 mm, 5 µm) column and 0.1% trifluroacetic acid and methanol as mobile phase, at a flow rate of 1 ml/min. Detection was carried out at 288 nm. Retention time of Berberine hydrochloride and Embelin were found to be 7.89 and 9.80 min, respectively. The method has been validated for linearity, accuracy and precision. Linearity for Berberine hydrochloride and Embelin were in the range of 10-30μg/ml. The percentage recoveries for Berberine hydrochloride and Embelin were found to be in range of % and %, respectively. Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of Berberine hydrochloride and Embelin in polyherbal formulations. KEYWRDS Berberine hydrochloride, Embelin, RP-HPLC, Validation, Herbal formulation AFFILIATIN 1. *Research Scholar, School of Pharmacy, RK University, Rajkot , Gujarat; Address: ankitagondaliya29@gmail.com; Contact number: *Author for Correspondence 2. Director, Aum Research Laboratory, Gandhinagar 3. Associate Professor, School of Pharmacy, RK University, Rajkot , Gujarat. 35

2 INTRDUCTIN Natural plants are source of raw materials for both traditional and modern system of medicine. Now-a-day about 80% of the world s population, living in developing countries still relay on traditional medicine based largely on the different species of plants for their primary health care. [1,2] Traditionally, herbal drugs have played a significant role in the management of both minor and major medical illnesses. Herbal drugs means a dosage form consisting of one or more herbs or processed herb in a specified quantities to provide specific nutritional, benefits, and/or other benefit meant for use to diagnosis treat, mitigate diseases of human beings or animal and/or alter the structure or physiology of human beings or animals. Herbal drugs are obtained by whole plant, cut plant part, powdered plant part, extract, essential oil, expressed juices and plant parts to treatment such as distillation, extraction, expression, fractionation, purification and concentration. [3,4,5] Berberine hydrochloride contains the natural plant substance Berberine. Berberine is an isoquinoline alkaloid isolated from roots and stem-bark of Berberis aristata. (Family: Berberidacease). Berberine hydrochloride is used as an Antidiarrhoeal and Antispasmodic [6]. ther uses found are as an Antifungal and antiprotozoal, [7] Anti-arrhythmic, [7] Antiinflammatory, [7] Antibiotic [8] and Antidiabetic. [8] Embelin is a natural benzoquinone, isolated from dried fruit of Embeliaribes. (Family: Myrsinaceae). Embelin is used as an antiapoptotic [9] and as a cosmetic agent. [10] It is also used as an antibacterial, [11] antidiabetic [12] and antidepressant. [13] Analytical methods are reported in literature for the estimation of the single molecule but not any simultaneous estimation method for determination of Berberine hydrochloride and embelin is reported. This work is aimed to develop and validate HPLC method for the simultaneous determination of Berberine hydrochloride and Embelin in herbal formulations. The method developed is accurate, precise and simple for these compounds in mixtures. Cl - N + Figure 1: Chemical Structure of Berberine hydrochloride. H H Figure 2: Chemical Structure of Embelin. 36

3 MATERIALS AND METHDS Chemicals and reagents Drugs which are used were extracted in Aum research laboratories and are of acceptable characterization and purity. HPLC grade methanol and triflouroacetic acid were procured from Finar Chemicals Ltd. Commercial herbal formulation was purchased from the local market which contain Berberis aristata and Embeliaribes. The pharmaceutical dosage form used in the study was Sudarshan tablet manufactured in India by Emani Limited. Instrumentation and chromatographic condition HPLC of Agilent Technologies (Model: 1220 Infinity LC) with auto sampler was used. C 18 (10 X 4.6 mm, 5 µm) Column was used for chromatographic separation. Mobile phase consists of 0.1% triflouroacetic acid in water (A) and methanol (B) in gradient system (table 1). The flow rate was set 1 ml/min. wavelength selected for detection was 288 nm. Injection volume was 20 µl. Preparation of standard stock solution Table 1: Mobile phase gradient. Time (min.) A (ml) B (ml) Accurately 1 mg of Berberine hydrochloride and Embelin were weighed separately and transferred to two different 10 ml volumetric flask. Each of them was dissolved in few ml of methanol and ultrasonicated for few minutes. The volume was made up to the mark with methanol to obtain final solutions containing 100 μg/ml of Berberine hydrochloride and 100 μg/ml of Embelin. Preparation of standard solution of binary mixtures of Berberine hydrochloride and Embelin Accurately 1 mg of Berberine hydrochloride and 1 mg of Embelin were weighed and transferred in 10 ml volumetric flask. Both drug were dissolved in methanol and ultrasonicated for few minutes. The volume was made up to the mark with methanol to obtain a binary mixture containing 100 μg/ml of Berberine hydrochloride and 100 μg/ml of Embelin. From that solution appropriate dilution was made to prepare a solution containing 20 μg/ml of Berberine hydrochloride and 20 μg/ml of Embelin. Preparation of mobile phase Here HPLC grade 500 ml methanol was ultrsonicated for 20 minutes on ultrasonicator (A). Accurately measured out 0.5 ml of trifluoroacetic acid and transferred in to 500 ml 37

4 volumetric flask. Volume was made up with water up to mark to got 0.1% TFA in water and the prepared solution ultrsonicated for 20 minutes on ultrasonicator(b). Chromatographic separation 20 µl solutions of Berberine hydrochloride and Embelin were injected in column. The chromatogram was run for appropriate minutes with mobile phase gradiently which was previously degassed. The chromatogram was stopped after separation achieved completely. Data related to peak like area, height, retention time, resolution etc. were recorded using software. Method validation Method validation was performed as per the guidelines. During the method validation, parameters evaluated include accuracy, precision, linearity, limit of detection, limit of quantification and Robustness. RESULTS AND DISCUSSIN ptimization of chromatographic conditions ptimization of chromatographic conditions plays an important role in the successful development of analytical methods. During the selection of mobile phase, Different mobile phases like acetonitrile : methanol, water : methanol and methanol : 0.1% trifluoroacetic acid were tried in order to find the best conditions for the separation of Berberine hydrochloride and Embelin. It was found that methanol and 0.1% trifluoroacetic acid in gradient system gave satisfactory results as compared to other mobile phases. This mobile phase system was tried with different proportions in gradient system. Finally the gradient selected as given in table 1. Figure 3. Chromatograph of standard Berberine hydrochloride and Embelin. 38

5 Table 2: Data for Berberine hydrochloride and Embelin. Parameters Berberine hydrochloride Embelin Retention time (min.) Theoritical plates Resolution Asymmetry Validation parameters Linearity The linearity of the method was determined at five concentration levels ranging from μg/ml for both drug Berberine hydrochloride and Embelin. The equation for regression line was y = x (R 2 = 0.999) for Berberine hydrochloride and y = x (R 2 = 0.999) for Embelin. Figure 4. calibration curve of Embelin. Figure 5. calibration curve of Embelin. 39

6 LD and LQ Calibration curve was repeated six times and the standard of the intercepts was calculated. Then LD and LQ were calculated as follow: Where, SD = Deviation of intercepts of calibration curves. Specificity Table 3. LD and LQ data for Berberine hydrochloride and Embelin. Drug LD (μg/ml) LQ (μg/ml) Berberine hydrochloride Embelin The specificity of the HPLC method was determined by complete separation of Berberine hydrochloride and Embelin with parameters like retention time (t R ), resolution (R S ) and tailing factor (T f ). Here tailing factor for peaks of Berberine hydrochloride and Embelin was less than 2 % and resolution was also more than 1.5%. The average retention time ± standard for Berberine hydrochloride and Embelin were found to be ± and ± respectively for six determinations. The peaks obtained for Berberine hydrochloride and Embelin were sharp and have clear baseline separation. Table 4: Statistical validation of specificity. Drug Mean retention time (min.) Deviation Berberine hydrochloride Embelin Precision Results should be expressed as Relative Deviation (RSD) or coefficient variance. 40

7 Table 5: Statistical validation of precision data. Drugs Precision % Mean drug found %Relative standard Repeatability Berberine hydrochloride Intraday precision Interday precision Repeatability Embelin Intraday precision Interday precision *n = 6 The result has shown that the recovery of Berberine hydrochloride and Embelin were in the range of % to %. The relative standard was less than 1 %. Accuracy The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 50, 100 and 150% and the percentage recovery was calculated and presented in Table 6 and 7. Table 6: Statistical validation of recovery studies for Berberine hydrochloride Concentration level (%) % Mean recovery % Relative standard Table 7: Statistical validation of recovery studies for Embelin. *n = 3 Concentration Level (%) % Mean Recovery Deviation % Relative Deviation *n = 3 41

8 The result has shown that the recovery of Berberine hydrochloride and Embelin were in the range of % to %.The relative standard was less than 1 %. Robustness The evaluation of robustness should be considered during the development phase and depends upon the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters. The parameters included flow rate, and ph of mobile phase. Berberine hydrochloride (20 µg/ml), and Embelin (20 µg/ml) were injected into sample injector of HPLC three times under different parameters like deliberate variations in flow rate and ph of mobile phase. The results are shown in table 8. Table 8: Statistical validation of robustness studies. Condition Change in flow rate Change in ph of 0.1 % of TFA in mobile phase Drug Berberine hydrochloride Mean of retention time (min.) % Relative standard Embelin Berberine hydrochloride Embelin Analysis of marketed formulation Sample A quantity of tablet powder equivalent to 0.2 mg of Berberine hydrochloride and Embelin was weighed and extracted in 10 ml of methanol by keeping it on ultrasonic bath for 25 to 30 minutes. Filtered it through filter paper. The final solution was obtained has concentration of 20 μg/ml of berberine hydrochloride and 20 μg/ml of Embelin. This solution was analysed using the developed method. The results for analysis of marketed formulation are given in table 9. Figure 6: Chromatogram for marketed formulation. 42

9 Table 9: Statistical validation of marketed formulation. Brand Drug % Mean recovery % Relative standard Sudarshan Tablet Berberine hydrochloride Embelin CNCLUSIN From the results obtained, it is obvious that the proposed HPLC method is applicable for the determination of Berberine hydrochloride and Embelin in polyherbal formulation. The results obtained indicates that the proposed method for the estimation of Berberine hydrochloride and Embelin is simple, specific, rapid, linear, accurate, precise and robust for intended use. It can be used in the routine quality control of polyherbal dosage forms in industries for determination of Berberine hydrochloride and Embelin. From the chromatograph of marketed formulation, it can be concluded that if rest of the peaks are identified for particular compound then this developed HPLC method may be used for estimation for that drugs. Thus this method may be used for simultaneous estimation of more than five drugs. REFERENCES 1. Agarwal SS, Paridhavi M. Herbal drug technology. University Press. Hyderabad, 2007;1: Ali M. Pharmacognosy and Plant Cultivation, 1 st ed. New Delhi, CBS Publishers and Distributers, 2008: Kumar T. ization of herbal drugs a review. Int journal of universal pharmacy and bio sciences. 2013; 2(4): Handa SS, Rakesh DD. Compendium of medicinal and aromatic plants asia. ICS UNID. 2006;1: Kennedy JH. Analytical Chemistry-Principals. Saunders college publishing, New York, 1990;2: Shamkuwar PB, Pawar DP. Antidiarrhoeal and antispasmodic effect of Berberis aristata. International journal of pharmacognosy and phytochemical research 2013;5(1): Singh A, Duggal S, Kaur N, et al. Berberine: Alkaloid with wide spectrum of pharmacological activities. Journal of Natural Products 2010;3: Hao M, Li SY, Sun CK, Jingyu-Xu, et al. Amelioration effects of berberine on diabetic microendothelial injury model by the combination of high glucose and advanced glycation end products in vitro. Eur J Pharmac 2011;654(3):

10 9. Ahn K, Sethi G, Aggarwal B. Embelin, an inhibitor of X chromosome linked inhibitor-of-apoptosis protein, blocks nuclear factor-kb (NF-kB) signalling pathway leading to suppression of NF-kB-regulated antiapoptotic and metastatic gene products. Molecular pharmacology 2007;71: Radhakrishnan N, Kavitha V, Raja S, et al.embelin-a natural potential cosmetic agent. J. Appl 2011;29: Radhakrishnan N, Mandal AB. A potential antibacterial agent Embelin, a natural benzoquinone extracted from Embeliaribes. Biology and Medicine 2011;3 (2): Sahu AK, Gautam MK, Deshmukh PT, et al. Effect of embelin on lithium-induced nephrogenic diabetes insipidus in albino rats 2013; S729-S Gupta G, Kazmi I, Afzal M, et al. Antidepressant-like activity of Embelin isolated from Embeliaribes. Phytopharmacology 2013;4(1):