Standardization of Anti-diabetic Poly Herbal Formulation

Standardization of Anti-diabetic Poly Herbal Formulation1. IntroductionNow-a-days most of au becausetic as wellhead as developing countries use Ayurvedic medicines then they uses it in past. They avoid use of allopathic drugs because of high risk of adverse effects. So, for the sake of community it is important to govern the dosage form available in market. Standardization of verbalism is evidence that the formulation contains constituents which it says to be contained.In present work formulation containing Gymnemic acid and Resveratrol has been studied. This formulation is anti-diabetic.Gymnemic Acid ( gymnasium) is major constituent isolated from leaves of plant Gymnema Sylvester belonging to family Asclepiadacea. (1) Plant has a property of masking the sugar test so it is known as GURMAR. Gymnemic acid is a triterpenoid saponin found in the leaves of Gymnema Sylvester. (2) Many studies shake off shown that oral administproportionn of Gymnema extract reduces serum glucose leve l and improves glucose tolerance in mildly diabetic rats. (3) Administration of water extract of Gymnema sylvestre leaves was found to increase serum insulin level suggesting its insulin releasing effect. (4) Number of beta cells at heart pancreatic tissue were increased which suggests a restorative effect of the Gymnema extract on pancreatic tissue. (5) So, from above it is now known that Gymnemic acid has the ability to decrease blood glucose level in diabetic patient which ultimately relives Diabetes.Resveratrol (reticuloendothelial system) (3,4,5-trihydroxy-trans-stilbene) is polyphenolic constituent isolated from plant Polygonum Cuspidatum belonging to family Polygnaceae. (6) It has been reported that Resveratrol has a variety of biological and pharmacological effects including antioxidant, anti-inflammatory, antiplatelet, anticarcinogenic effects, modulation of lipide metabolism and cardioprotection. (7) In pancreatic -cells, insulin secretion is linked to the oscillations in membrane potential, intracellular Ca2+ and metabolism. The variations in the ATP/ADP ratio control the conductance of ATP-dependent K+ channel leading to depolarization and periodic influx of Ca2+. The resultant membrane depolarization activates voltage dependent L-type Ca2+ channels and triggers intracellular Ca2+ release, elevating intracellular Ca2+ both in the cytosolic compartment and within the mitochondria, and thereby initiating insulin secretion. (8)From the survey of respective(a) literatures it is found that Gymnemic acid has been estimated by various analytical techniques like HPTLC, HPLC Gravimetery. (9) While Resveratrol had been estimated by HPLC and spectrometric techniques. (10) Not a private mode is reported for the estimation of both constituents simultaneously.Present work has been focused on estimation of both constituents simultaneously. Here estimation of both constituents was done by UV-Visible spectrometry and HPTLC. For estimation of both constituents simultaneously UV-visible Spectrophotometric and HPTLC methods were certain and validated.Figure 1 Gymnemic acidTable 1 Gymnemic acid GroupFigure 2 Resveratrol2. experimentalChemical ReagentsGymnemic acid telling Standard was extracted from the formulation. This relative standard was comp bed with standard obtained from Clearsynth TM Private Ltd (Andheri (w), Mumbai, India) . Then the prepared relative standard was used for methods. alike way, Resveratrol was extracted from formulation and then compared with standard Sigma-Aldrich constituent. Marketed formulation here used was Resveratrol plus (with Gymnemic acid) Zenith Nutritions containing 100mg Resveratrol and 500 mg Gymnemic acid per 2 capsules.All reagents for UV-Visible Spectrophotometry and HPTLC are M ethyl alcohol, Chloroform, Ethanol, Glacial Acetic Acid, water and Benzoyl Chloride. Methanol, Chloroform and Ethanol used were analytical grade purchased from merk solutions. Triple distill water was made in laborator y by distillation assembly. Benzoyl Chloride was purchased from SD fines Chemicals.InstrumentsUV-Visible Spectrophotometric was developed on a Shimadzu UV-Vis double beam spectrophotometer, model 2400 PC series, with spectral width of 1 nm, wavelength accuracy of 0.5nm and a pair of 10 mm matched quartz cells (Shimadzu , Japan). The HPTLC instrumentation consisted of a Linomat V experiment applicator with 100 L Hamilton syringe and a TLC III scanner controlled by WinCATS software (Camag, Muttenz, Switzerland) Merck TLC plates coated with 60F254 silica gelatine on aluminum sheets were used as stationary phase. The plates were developed in a Camag 10 x 10 cm touch through chamber that was antecedently saturated for 20 minutes with mobile phase.Spectrometric ConditionsGYM didnt contain chromophore in structure so it has to be derivatized for UV-Visible detection. Benzoyl Chloride was used as derivatizing agent. The solutions of GYM reticuloendothelial system were scanned in the spe ctrum mode from 200 to 400 nm, and from that 303nm for Gymnemic acid determination, 318.4 nm for Resveratrol determination and 349 nm for isoabsorptive shoot down for Q ratio method were selected for simultaneous estimation.Chromatography ConditionThe solutions were placementted in the form of hands of 5 mm width on pre-coated silica gel 60F254 aluminum plates using a Camag 100 L try applicator syringe. They were activated at 110 oC in an oven for 20 minutes before sample application. A constant application rate 0.1L/s was applied and bandwidth was 9 mm between two bands. Spotted plates were developed in twin through chamber which is previously saturated for 20 minutes with mobile phase containing Chloroform Methanol Glacial acetic acid (13 4 0.5 %). The plates were developed for 8 cm run length. The plates were dried by hair dryer and then post derivatization done by dipping plates in Vanillin-Sulphuric acid solution. Then it is heat in hot air oven at 105oC for 5 minutes. Then plates were scanned at 575nm in TLC III scanner.Preparation of solutionsPreparation of Standard solution for UVResveratrol relative standard 10 mg was accurately weighed which is transferred to 10 ml clean volumetric flask. This a good deal quantity was dissolved in 10 ml of ethanol to take a leak 1000g/ ml standard stock solution. From standard stock solution 1 ml is transferred to another 10 ml of volumetric flask and volume was made up with methanol to produce 100 g/ ml working standard stock solution.Gymnemic acid relative standard 100 mg was accurately weighed which is transferred to 10 ml clean volumetric flask. This much quantity was dissolved in 10 ml of terzetto distill water to produce 10000 g/ ml standard stock solution. From standard stock solution 1 ml was transferred to 10 ml volumetric flask diluted with methanol to produce 1000 g/ ml working standard solution.Benzoyl chloride was diluted in ethanol first and then in methanol lastly to produce 125 g/ ml solution which was used as derivatizing solution.Preparation of sample solution for UV10 capsules shells were removed and powders from those capsules were mixed and from that weight equivalent to 10 mg Resveratrol and 50 mg Gymnemic acid was weighed accurately and transferred to 10 ml of volumetric flask volume made up with mixture of ethanol water (11). From this solution 1 ml solution was taken diluted with mixture of ethanol water (11).this solution is working sample solution further dilution done by the same mixture.Preparation of standard solution for HPTLCResveratrol relative standard 20 mg and Gymnemic acid relative standard 100 mg was accurately weighed and transferred to two polar 10 ml volumetric flask respectively in which weighed quantity was dissolved in 10 ml ethanol water (11) mixture to produce RES 2000 g/ ml and GYM 10000 g/ ml standard stock solution. From these solution 5 ml solution was transferred to 10 ml volumetric flask diluted with ethanol water (11) up to 10 ml t o produce RES 1000 g/ ml and GYM 5000 g/ ml working standard stock solution.Preparation of sample solution for HPTLC10 capsules shells were removed and powders from those capsules were mixed from that weight equivalent to 20 mg RES and 100 mg Gym was weighed. That much amount of powder was accurately transferred to 10 ml volumetric flask and dissolved in ethanol water (11) mixture. From this solution 5 ml was taken and filtered with 0.45 m filter sized syringe filter. This solution was then diluted with mixture of ethanol water (11) up to 10 ml solution to produce RES 1000 g/ ml equivalent and GYM 5000 g/ ml equivalent. bank check method verificationPreparation of calibration sheerFor UV-visible Spectrophotometric method individual solutions were prepared in methanol from working standard stock solution to produce 5-25 g/ ml RES and 25- 125 g/ ml GYM solution. Benzoyl chloride 10 L was added to each solution. Then these solutions were analyzed in methanol at tercet different wave lengths at 303 nm, 318.4 nm and 349 nm. Calibration curve here made up was absorbance v/s closeness.For HPTLC method different aliquots of were taken from standard stock solution to make final concentration of RES 1000 g/ ml and GYM 5000 g/ ml in the same solution. Then different aliquots were spotted on activated TLC plate. The concentration of RES was varied between (5-25) g/ spot while for GYM it was (25-125) g/ spot. Then plate was developed in mobile phase and was developed to scan as mention above at 575 nm. Calibration curve here made was peak area v/s concentration of constituents. truth (recovery)For UV-visible spectrophotometer solution of standard RES was added to previously analyzed sample solution at three different levels (80%, 100 % and 120%). Same procedure been followed to accommodate a GYM accuracy by adding standard stock solution at three different levels (80%, 100 % and 120%). Amount of standard RES added was (8, 10 and 12 g/ ml) to 10 g/ ml sample solution. A mount of standard GYM added was (40, 50 and 60 g/ ml) to 50 g/ ml sample solution.For HPTLC known amount standard solution of RES (8, 10 12 g/ ml) and GYM (40, 50 60 g/ ml) added to previously analyzed sample solution having concentration of RES 10 g/ spot and GYM 50 g/ spot.PrecisionThe intra-day and inter-day clearcutness of proposed methods were determined by estimating corresponding responses three times on the same day and on three different days for three different concentrations. For UV-Visible spectroscopy RES concentrations were 8, 10 and 12 g/ ml measured at wavelengths 318.4 nm and 349 nm.GYM concentrations were 45, 50 and 60 g/ ml measured at wavelengths 303 nm and 349 nm. For HPTLC three different dilutions were made having both RES and GYM in those solutions ranging (RES 9, 10 and 12.5 g/spot) and GYM (45, 50 and 62.5 g/ spot).For repeatability in HPTLC, sample solution containing 10 g/spot RES and 50 g/ spot GYM was measured in terms of response.LOD LOQThe sensiti vity of analytical method was evaluated by determining LOD and LOQ. They are measured by following equationsLOD 3.3 / SLOQ 10 SHere, is standard deviation of intercept and S is slope in linearity equation.SpecificityFor HPTLC spots were scanned for its purity with standard sample and checked whether they give a same response or not. This was done by spectral scanning in WinCats HPTLC.RobustnessThe hardiness of methods was studied by analyzing the same samples of RES and GYM with deliberate change in parameters. The changes in responses were noted. For HPTLC, spots scanned at ( 2 nm) and mobile phase ratio change was performed. For UV-Visible method solutions were scanned at ( 2 nm).3. Results and DiscussionsSimultaneous estimation of RES and GYM was difficult task because they are isolated from herbal source and they have RES GYM ratio 15 in marketed formulations.System suitability parametersSystem suitability run for HPTLC was performed before each validation run. fiver replica te spots were made. Parameters monitored were Rf value and Peak areas of them. (Table 2)Table 2 System suitability Parameters HPTLCOptimization of MethodFor HPTLCVarious experimental conditions such as the mobile phase and the wavelength of detection were optimized to achieve accurate, precise and reproducible results for the estimation of RES and GYM. Good resolution and sharp peaks with minimum tailing of these drugs (Rf RES 0.78, Rf GYM 0.236) was obtained by using mobile phase Chloroform Methanol Glacial acetic acid (13 4 0.5%) at wavelength of detection 575 nm.Figure 3 optimized Chromatogram of HPTLC, RES (10 g/spot) GYM ( 50 g/spot)Figure 4 Wavelength selection for HPTLC of RES and GYMFor UV-Visible SpectrophotometricForm overlain spectra (Figure 5) of methanolic solution of RES and GYM three wavelengths were finalized for analysis 303 nm, 318.4 nm and 349 nm. The method here used for simultaneous estimation is Q ratio method. Here both of constituents are measured at 349 nm isoabsorptive point and 303 nm and 318.4 nm GYM and RES respectively.Figure 5 wavelength selection after derivatization of GYM, BCL (Benzoyl Chloride) and RESMethod validation of proposed methodsOptimized methods were validated in compliance with ICH guidelines. The results of various parameters are discussed followingLinearityFor UV Spectrophotometric method, linear correlation was obtained between absorbance and concentration for RES 5-25 g/ ml at 318.4 nm 349 nm and GYM 25-125 g/ ml 303 nm 349nm.( Table 3)For HPTLC method, linear correlations were obtained between peak area and concentration of RES was 5-25 g/spot and GYM was 25- 125g/spot. (Table 4)AccuracyThe percentage recovery values of RES and Gym were obtained in the range of 98% to 103 %, and relative standard deviation values for both constituents in two methods were less then 2%, it shows that methods are accurate for both constituents. Values are shown in table 3 and 4.PrecisionInter-day and intra-day variation in quantification of RES and GYM showed that RSD values were always less than 2% during the analysis by both methods. These low RSD values show that methods are precise. Values of precision studies for UV spectrometry and HPTLC are in table 3 and 4 respectively.LOD and LOQFor UV-Visible spectrometry method LOD and LOQ for RES was found to be 0.09g/ml and 0.28g/ml respectively. LOD and LOQ for GYM were 0.63g/ml and 1.92g/ml respectively.For HPTLC method LOD and LOQ for RES were 0.065 g/spot and 0.20 g/ spot respectively. LOD and LOQ for GYM were 1.2 g/spot and 3.87 g/spot respectively.SpecificityFor HPTLC method, a good correlation was obtained between standard and sample spectra of RES and GYM correlation suggests that there in no affray in quantification of RES and GYM.RobustnessRobustness of the methods was studied by performing assays of RES and GYM in capsule formulation. The parameters are deliberately altered and changes were recorded. Assay values were calculated in the changed parameters. Methods proved to be robust, because assay values in changed parameters were within limits.Assay of marketed formulationAssay of Resveratrol plus from ZENITH nutrition was performed in both the methods. Results of assay were compared with corresponding amounts claimed on capsule. The assay results are shown in table 3 4.Table 3 UV-Visible method Validation parametersTable 4 HPTLC Validation ParametersConclusionDeveloped HPTLC and UV-Visible Spectrophotometric method was found to be simple, accurate, precise, rapid, sensitive and robust for the estimation of RES and GYM in combined dosage form. The validation data and recovery shows that methods are free from inferences of excipients used in formulations. Thus method is useful for both constituents to be estimated by both methods.Referencesx