WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Sindhu et al. SJIF Impact Factor 2.786 Volume 4, Issue 03, 595-604. Research Article ISSN 2278 4357 HPTLC METHOD FOR QUANTIFICATION OF GARCINOL FROM DRY FRUIT RINDS OF GARCINIA INDICA AND ITS MARKET FORMULATION. Ankit M. Patel, Sindhu B. Ezhava*, Ishwarsinh S. Rathod, Mahesh T. Chhabria, Arpit H.Patwari Department of Quality Assurance, L.M College of Pharmacy, Gujarat Technological University, Ahmedabad-380009, India. Article Received on 26 Dec 2014, Revised on 21 Jan 2015, Accepted on 14 Feb 2015 *Correspondence for Author Dr. Sindhu B. Ezhava Department of Quality Assurance, L.M College of Pharmacy, Gujarat Technological University, Ahmedabad-380009, India. ABSTRACT Garcinol, polyisoprenylated benzophenone, a natural histone acetyltransferase inhibitor, extracted from dried fruit rinds of Garcinia indica. Garcinia indica is well spread in tropical rain forest of Western Ghats of India. High performance Thin Layer Chromatographic (HPTLC) method was developed for the estimation of Garcinol from dried fruit rinds of Garcinia indica and its marketed formulations. Chromatography was performed on silica gel G 60F 254 as stationary phase and the mobile phase optimized was Toluene: Ethyl acetate: Formic acid (4: 1: 0.5 v/v/v) with pre-saturation time of 30 minutes. The linearity was found to be within range 200-700 ng/spot with mean % recovery of 101.12 ±1.338 %.The proposed method was successfully applied for estimation of Garcinol in fruit rinds of Garcinia indica and the market formulations of Garcinia indica (Trayodashang Guggul by Pathanjali Ayurvedic Ltd. and slimmarz capsules by Medisys Biotech Pvt. Ltd). The amount of Garcinol was found to be 2.5, 0.701 and 0.760 %w/w in Garcinia indica fruit rinds, Trayodashang Guggul and slimmarz capsules respectively. KEYWORDS: Garcinol, Garcinia indica, HPTLC METHOD. INTRODUCTION Garcinia indica is well spread in tropical rain forest of Western Ghats of India. Garcinol, a polyisoprenylated benzophenone derivative, isolated from fruit rind of Garciniaindica has www.wjpps.com Vol 4, Issue 03, 2015. 595
been traditionally used and appreciated for centuries. It has shown promising antioxidative, antiglycation and free radical scavenging activities (Yamaguchi et al., 2000 a,b). It has been reported that garcinol can play an important role in the treatment of gastric ulcers caused by the hydroxyl radicals or chronic infection with Helicobacter pylori. Presently, treatment with Clarithromycin antibiotic is the therapy of choice for treating H. pylori infection, which, however,suffers from emergence of rapid resistance (Chatterjee et al., 2003, 2005). Garcinol may be a viable alternative to Clarithromycin. Garcinol shows antibacterial activity comparable to that of the antibiotic Vancomycin against Methicillin-resistant Staphylococcusaureus (Rukachaisirikul et al., 2005). Garcinol is also effective at blocking the formation of azoxy methane-induced colonic aberrantcrypt foci and induces apoptosis in human HL 60 cancer cells (Panet al., 2001; Tanaka et al., 2000). Another study has further demonstrated that cigarette smoke extract-induced COX-2 expression is blocked by Garcinol pre-treatment (Yang et al., 2009). Isogarcinol has also shown biological activities similar to that of garcinol and has been claimed to be an anti-inflammatory, antitumor, a lipase inhibitor, an anti-obesity agent and an antiulcer agent (Sang et al., 2001). In the present paper, an accurate HPTLC method for quantitative determination of Garcinol in herbal formulations is described. The proposed method was validated in compliance with ICH guidelines Figure 1.Chemical structure of Garcinol www.wjpps.com Vol 4, Issue 03, 2015. 596
EXPERIMENTAL 2.1 HERBAL MATERIALS AND CHEMICALS Standard Garcinol was purchased from Sigma Aldrich Ltd., Mumbai, India. Dried kokum fruit rinds were purchased from L.V.G. herbal store, Ahmedabad. Gujarat and identified by Dr. A. M. Shukla, botanist at M. G. Science College, Ahmedabad, Gujarat. TLC Aluminium sheets pre-coated with silica gel 60 F 254 20 20 cm 2 layer thickness- 0.2 mm (Merck, Germany) Rectified Spirit (Baroda Chemical Ltd., India) Methanol LR (Loba Chemicals, Mumbai, India) Ethyl Acetate (Finar Chemicals, Ahmedabad, India) Toluene Formic acid Acetic acid Hexane Accurately weighed standard Garcinol (25 mg) was transferred into 25 ml volumetric flask and diluted with methanol up to the mark. (1000 µg/ml of Garcinol). And working standard solution prepared. (100 µg/ml) In both Garcinia indica Formulations 1(Tryodashang Guggul,Patanjali Ayurvedic ltd.) and Fromulation2 (Slimmerz capsule, Medisys biotech pvt ltd.) the content of extract of Garcinia indica were 350 mg and 250 mg respectively. 2.2 CHROMATOGRAPHY The samples were spotted in the form of bands of width 3 mm with a Camag 100 microlitre syringe (Hamilton, Bonaduz, Switzerland) on silica gel pre-coated aluminium 60 F 254 plates, [20 cm 20 cm with 0.2 mm thickness; E. Merck, Germany)] using a Camag Linomat IV (Switzerland) sample applicator. The plates were pre-washed with methanol and activated at 60 C for 5 min prior to chromatography. A constant application rate of 0.1 μl/s was used and the space between two bands was 5 mm. The slit dimension was kept at 3 mm 0.1mm and the scanning speed was 20 mm/s. The monochromator bandwidth was set at 20 nm, each track was scanned three times and baseline correction was used. The mobile phase consisted of toluene: ethyl acetate: formic acid (4:1:0.5v/v/v) for chromatography run. Linear ascending development was carried out in a 10 cm 10 cm twin trough glass chamber www.wjpps.com Vol 4, Issue 03, 2015. 597
(Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 30 min at room temperature (25 C ± 2) at relative humidity of 60 % ± 5. The length of each chromatogram run was 8 cm. Following the development the TLC plates were dried in a current of air with the help of an air dryer in a wooden chamber with adequate ventilation. The flow rate in laboratory was maintained unidirectional (laminarflow, towards the exhaust). Scanning was performed using a Camag TLC Scanner III in the reflectance-absorbance mode at 279 nm and operated by CATS IV software. The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. Concentrations of the compound chromatographed were determined from the intensity of the diffused light. Evaluation was by peak areas with linear regression. The amount of Garcinol was computed from peak areas. 2.3 CALIBRATION CURVE Standard solution of Garcinol concentration of 100 μg/ ml prepared and 2, 3, 4, 5, 6 and 7 μl from standard solution applied in triplicate on TLC plate to obtain final concentration 200-700 ng/spot. The plate was then developed using the optimized mobile phase and the peak areas were plotted against the corresponding concentrations to obtain the calibration curves. [Q2A, ICH, Q2 (R1), 2005]. 3. RESULTS AND DISCUSSION 3.1 MOBILE PHASE DEVELOPMENT The mixtures of several mobile phases were tried to separate spot of Garcinol from other spots and get stable peak. The Solvent system used was Toluene: Ethyl Acetate: Formic Acid (4:1:0.5) was selected for estimation of Garcinol, which gave good resolution. Good Chromatogram in Figure 2 was attained with Rf value 0.49 ± 0.02. The absorption spectrum of Garcinol is shown in Figure 3. The wavelength 279 nm was used for Quantification of sample. www.wjpps.com Vol 4, Issue 03, 2015. 598
Fig.2. Chromatogram of Garcinol (600 ng spot) (Rf = 0.49±0.02), Mobile phase: toluene: ethyl acetate: formic acid (4:1:0.5, v/v/v). 3.2 Method validation 3.2.1 Specificity Fig.3. Absorption spectra of Garcinol and peak purity of Garcinol The specificity of method was ascertained by standard Garcinol and samples (Formulation 1 and Formulation 2). The spots of standard Garcinol and samples (Formulation 1 and Formulation 2) were spotted on TLC plate in triplicate and run. The spot for Garcinol in the samples were confirmed by comparing the Rf values of the spot with that of the standard. The validation parameters for the proposed method are shown in Table 1. www.wjpps.com Vol 4, Issue 03, 2015. 599
Table.1. Validation Parameters for Garcinol by HPTLC method Sr. No Parameters Garcinol 1 Linearity range 200-700 ng/spot 2 Correlation coefficient Slope Intercept 0.9981 6.827 385.84 3 Precision a. Repeatability(CV) Repeatability of measurement of peak area (<1%) Repeatability of sample application (<2%) 0.60 0.72 b. Reproducibility(CV) Intraday precision Interday precision 0.40-1.88 1.32-2.12 4 Accuracy(% recovery) 101.12±1.338 5 Limit of detection 50 ng/spot (practically) 28 ng/spot (By equation) 6 Limit of quantification 84 ng/spot (by equation) 7 Specificity Specific 3.2.2 LINEARITY, LIMITS OF QUANTIFICATION AND DETECTION Calibration plots of peak area against concentration were linear in the range 200-700 ng/spot Garcinol. The calibration lines were represented by linear equation y=6.8271x + 385.84 For this equation the correlation coefficient, r 2 was 0.9981. The LOQ and LOD were calculated as 28 ng/spot and 84ng/spot, respectively. The linearity range was found to be 200-700 ng/spot. 3.2.3 PRECISION The repeatability of sample application and measurement of the peak area was expressed in terms of % RSD. The % RSD was found to be less than 2.0 in all cases indicate no significant variations in the analysis of Garcinol at the concentration of 200, 300, 400, 500,600,700ng/spot. www.wjpps.com Vol 4, Issue 03, 2015. 600
3.2.4 ACCURACY The accuracy was studied by the standard addition technique. Three different levels of standard wereadded to the previously analyzed samples, each level being repeated thrice. The average percentage recovery of garcinol was 101.12 %. Herbal Formulations Table.2.Results from recovery analysis (n=3). Amount of sample taken [g] A Amount of Garcinol present in (A) [ng/spot] B Amount of Garcinol added to (A)[ng] C Total Garcinol taken (B+C) [ng] D Total garcinol found [ng] E % Recovery E/Dx100 (mean) 1 301 100 401 402.65 100.41 1 1 301 200 501 506.32 101.06 1 301 300 601 612.35 101.88 Average percentage recovery 101.12 3.2.5 ESTIMATION OF GARCINOL FROM DRY FRUIT RINDS OF Garcinia indica The optimized solvent system was used for the estimation of the Garcinol from fruit rinds of Garcinia indica. The resolution was good and components were observed at different Rf value, As shown in figure.4. The method was used to determine the Garcinol content in fruit rinds of Garcinia indica.the result shown in Table 3.The low RSD values are indicate the high accuracy and precision of the method. 3.2.6 ESTIMATION OF GARCINOL IN HERBAL FORMULATIONS The optimized solvent system was used for the estimation of the Garcinol from Formulation 1 and Formulation 2. The resolution was good and components were observed at different Rf value, as shown in (Figure. 5, Figure. 6). The method was used to determine the Garcinol content in Formulation 1 and Formulation 2. The results are shown in Table 3. The low RSD values are indicate the high accuracy and precision of the method. Table.3. Results from HPTLC analysis of Garcinol Herbal Formulations Component Amount found by proposed method [% w/w] RSD [%] (n = 3) Fruit rinds Garcinol 2.55 1.00 1 Garcinol 0.701 0.42 2 Garcinol 0.760 1.04 www.wjpps.com Vol 4, Issue 03, 2015. 601
Fig.4. HPTLC chromatogram of Fruit rind extract (3 µl spot); (Rf: 0.49±0.02); mobile phase: toluene: ethyl acetate:formic acid (4:1:0.5, v/v/v) Fig.5. HPTLC chromatogram of Formulation 1 (20 µl spot); (Rf: 0.49±0.02); mobile phase: toluene: ethyl acetate:formic acid (4:1:0.5, v/v/v) Fig.6. HPTLC chromatogram of Formulation 2 (20 µl spot); (Rf: 0.49±0.02); mobile phase: toluene: ethyl acetate:formic acid (4:1:0.5, v/v/v) www.wjpps.com Vol 4, Issue 03, 2015. 602
CONCLUSION The developed HPTLC technique is a simple, precise specific and accurate for the determination of Garcinol. Statistical analysis proves that the method is reproducibleand selective for the analysis of Garcinol. Since the proposed mobile phase effectively resolves Garcinol, the method can be used for qualitative as well as quantitative analysis of Garcinol in herbal formulations. REFERENCES 1. Balsubramanyam.K, Altaf.M, Varier.R.A, Swaminathan.V, Ravindran.A,Sadhale. P.P and Kandu. T.K. PolyisoprenylatedBenzophenone, Garcinol, a Natural Histone Acetyltransferase Inhibitor, Represses Chromatin Transcription and Alters Global Gene Expression. Journal of Biological Chemistry, 2004; 279: 33716-33726. 2. Sutar.R.L, Mane.S.P and Ghosh.J.S. Antimicrobial activity of extracts of dried kokum (GarciniaindicaC).International Food Research Journal, 2012; 19:1207-1210. 3. Deore.A.B, Sapakal.V.D, Dashputre.N.L and Naikwade.N.S. Antiulcer activity of Garciniaindicalinn fruit rinds. Journal of Applied Pharmaceutical Science, 2011; 05: 151-154. 4. Khatib.N.A, Pawase.K, and Patil.P.A. Evaluation of anti-inflammatory activity of Garciniaindica fruit rinds extract in Wister rats. International Journal of Research in Ayurveda & Pharmacy, 2010; 02: 449-454. 5. Darji.K.K, Shetgiri.P and D mello.p.m. Evaluation of antioxidant and antihyperlipidemic activity of extract of Garciniaindica. International Journal of Pharmaceutical Science and Research, 2010; 01: 175-181. 6. Pike, B, Biomol. Material safety data sheet for Garcinol September 2012 www.enzolifesciences.com/bml-gr343/garcinol.html 7. Kaur.R, Chattopadhyay.S.K, Tandon.S and Sharma.S. Large scale extraction of the fruits of Garciniaindica for the isolation of new and known polyisoprenylatedbenzophenone derivatives. Industrial Crops and Products, 2012; 37: 420-426. 8. Padhye.S, Ahmad.A, Oswal.N and Fazlul. (2009). Emerging role of Garcinol, the antioxidant chalcone from GarciniaindicaChoisy and its synthetic analogs. Journal of Hematology& Oncology, 2009; 38: 1-13. 9. Krishnamurthy.N, Lewis.Y.S, Ravindranath.B. On the structures of Garcinol, isogarcinol and camboginol.tetrahedron Letters, 1981; 22: 793-796. www.wjpps.com Vol 4, Issue 03, 2015. 603
10. ICH draft guideline on validation of analytical procedures: Definitions and terminology ICHQ2 (R1); federal register, IFPMA, Switzerland, 1995. www.wjpps.com Vol 4, Issue 03, 2015. 604