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Posted: June 11th, 2023
Standardization of Anti-diabetic Poly Herbal Formulation
1. Introduction
Now-a-days most of developed as well 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 standardize the dosage form available in market. Standardization of formulation is evidence that the formulation contains constituents which it says to be contained.
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In present work formulation containing Gymnemic acid and Resveratrol has been studied. This formulation is anti-diabetic.
Gymnemic Acid (GYM) 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 have shown that oral administration of Gymnema extract reduces serum glucose level 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 within 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 (RES) (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 lipid metabolism and cardioprotection. (7) In pancreatic β-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+ channels 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 various 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 single method is reported for the estimation of both constituents simultaneously.
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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 developed and validated.
Figure 1: Gymnemic acid
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Table 1: Gymnemic acid Group
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Gymnemic acid I Is My Privacy Protected?100%! We encrypt everything—your details stay secret. Papers are custom, original, and yours alone, so no one will ever know you used us. |
Tigloyl |
Aryl |
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Gymnemic acid II |
2-methoxybutyl oly Is AI Involved in Writing?Nope—all human, all the time. Our writers are pros with real degrees, crafting unique papers with expertise AI can’t replicate, checked for originality. |
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Gymnemic acid III |
2-methoxybutyl oly |
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Gymnemic acid IV |
Tigloyl |
H Figure 2: Resveratrol 2. Experimental Chemical & Reagents Gymnemic acid relative Standard was extracted from the formulation. This relative standard was compared with standard obtained from Clearsynth TM Private Ltd (Andheri (w), Mumbai, India) . Then the prepared relative standard was used for methods. Same 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 Nutrition’s} containing 100mg Resveratrol and 500 mg Gymnemic acid per 2 capsules. Will My Paper Be Unique?Guaranteed—100%! We write every piece from scratch—no AI, no copying—just fresh, well-researched work with proper citations, crafted by real experts. You can grab a plagiarism report to see it’s 95%+ original, giving you total peace of mind it’s one-of-a-kind and ready to impress. All reagents for UV-Visible Spectrophotometry and HPTLC are Methanol, 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 laboratory by distillation assembly. Benzoyl Chloride was purchased from SD fines Chemicals. Instruments UV-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 sample applicator with 100 µL Hamilton syringe and a TLC III scanner controlled by WinCATS software (Camag, Muttenz, Switzerland) Merck TLC plates coated with 60F254 silica gel on aluminum sheets were used as stationary phase. The plates were developed in a Camag 10 x 10 cm twin through chamber that was previously saturated for 20 minutes with mobile phase. Spectrometric Conditions Can You Use Any Citation Format?Yep—APA, Chicago, Harvard, MLA, Turabian, you name it! Our writers customize every detail to fit your assignment’s needs, ensuring it meets academic standards down to the last footnote or bibliography entry. They’re pros at making your paper look sharp and compliant, no matter the style guide. GYM didn’t 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 & RES were scanned in the spectrum 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 point for Q ratio method were selected for simultaneous estimation. Chromatography Condition The solutions were spotted in the form of hands of 5 mm width on pre-coated silica gel 60F254 aluminum plates using a Camag 100 µL sample applicator syringe. They were activated at 110 oC in an oven for 20 minutes before sample application. A constant application rate 0.1µL/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 heated in hot air oven at 105oC for 5 minutes. Then plates were scanned at 575nm in TLC III scanner. Preparation of solutions Can I Change My Order Details?For sure—you’re not locked in! Chat with your writer anytime through our handy system to update instructions, tweak the focus, or toss in new specifics, and they’ll adjust on the fly, even if they’re mid-draft. It’s all about keeping your paper exactly how you want it, hassle-free. Preparation of Standard solution for UV Resveratrol relative standard 10 mg was accurately weighed which is transferred to 10 ml clean volumetric flask. This much quantity was dissolved in 10 ml of ethanol to produce 1000µg/ 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 µg/ 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 triple distill water to produce 10000 µg/ ml standard stock solution. From standard stock solution 1 ml was transferred to 10 ml volumetric flask diluted with methanol to produce 1000 µg/ ml working standard solution. Benzoyl chloride was diluted in ethanol first and then in methanol lastly to produce 125 µg/ ml solution which was used as derivatizing solution. How Do I Order a Paper?It’s a breeze—submit your order online with a few clicks, then track progress with drafts as your writer brings it to life. Once it’s ready, download it from your account, review it, and release payment only when you’re totally satisfied—easy, affordable help whenever you need it. Plus, you can reach out to support 24/7 if you’ve got questions along the way! Preparation of sample solution for UV 10 capsule’s 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 (1:1). From this solution 1 ml solution was taken diluted with mixture of ethanol: water (1:1).this solution is working sample solution further dilution done by the same mixture. Preparation of standard solution for HPTLC Resveratrol relative standard 20 mg and Gymnemic acid relative standard 100 mg was accurately weighed and transferred to two different 10 ml volumetric flask respectively in which weighed quantity was dissolved in 10 ml ethanol : water (1:1) mixture to produce RES 2000 µg/ ml and GYM 10000 µg/ ml standard stock solution. From these solution 5 ml solution was transferred to 10 ml volumetric flask diluted with ethanol: water (1:1) up to 10 ml to produce RES 1000 µg/ ml and GYM 5000 µg/ ml working standard stock solution. How Quick Can You Write?Need it fast? We can whip up a top-quality paper in 24 hours—fully researched and polished, no corners cut. Just pick your deadline when you order, and we’ll hustle to make it happen, even for those nail-biting, last-minute turnarounds you didn’t see coming. Preparation of sample solution for HPTLC 10 capsule’s 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 (1:1) mixture. From this solution 5 ml was taken and filtered with 0.45 µm filter sized syringe filter. This solution was then diluted with mixture of ethanol: water (1:1) up to 10 ml solution to produce RES 1000 µg/ ml equivalent and GYM 5000 µg/ ml equivalent. Assay method validation Preparation of calibration curve For UV-visible Spectrophotometric method individual solutions were prepared in methanol from working standard stock solution to produce 5-25 µg/ ml RES and 25- 125 µg/ ml GYM solution. Benzoyl chloride 10 µL was added to each solution. Then these solutions were analyzed in methanol at three different wavelengths at 303 nm, 318.4 nm and 349 nm. Calibration curve here made up was absorbance v/s concentration. Can You Handle Tough Topics?Absolutely—bring it on! Our writers, many with advanced degrees like Master’s or PhDs, thrive on challenges and dive deep into any subject, from obscure history to cutting-edge science. They’ll craft a standout paper with thorough research and clear writing, tailored to wow your professor. For HPTLC method different aliquots of were taken from standard stock solution to make final concentration of RES 1000 µg/ ml and GYM 5000 µg/ ml in the same solution. Then different aliquots were spotted on activated TLC plate. The concentration of RES was varied between (5-25) µg/ spot while for GYM it was (25-125) µg/ 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. Accuracy (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 have 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 µg/ ml) to 10 µg/ ml sample solution. Amount of standard GYM added was (40, 50 and 60 µg/ ml) to 50 µg/ ml sample solution. For HPTLC known amount standard solution of RES (8, 10 & 12 µg/ ml) and GYM (40, 50 & 60 µg/ ml) added to previously analyzed sample solution having concentration of RES 10 µg/ spot and GYM 50 µg/ spot. Precision How Do You Match Professor Expectations?We follow your rubric to a T—structure, evidence, tone. Editors refine it, ensuring it’s polished and ready to impress your prof. The intra-day and inter-day precision 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 µg/ ml measured at wavelengths 318.4 nm and 349 nm.GYM concentrations were 45, 50 and 60 µg/ 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 µg/spot) and GYM (45, 50 and 62.5 µg/ spot). For repeatability in HPTLC, sample solution containing 10 µg/spot RES and 50 µg/ spot GYM was measured in terms of response. LOD & LOQ The sensitivity of analytical method was evaluated by determining LOD and LOQ. They are measured by following equations: How Do You Edit My Work?Send us your draft and goals—our editors enhance clarity, fix errors, and keep your style. You’ll get a pro-level paper fast. LOD: 3.3 ï³/ S LOQ: 10 ï³ï€¯ï€ S Here, ï³ is standard deviation of intercept and S is slope in linearity equation. Specificity For 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. Can You Brainstorm Topics?Yep! We’ll suggest ideas tailored to your field—engaging and manageable. Pick one, and we’ll build it into a killer paper. Robustness The robustness 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 Discussions Simultaneous estimation of RES and GYM was difficult task because they are isolated from herbal source and they have RES: GYM ratio 1:5 in marketed formulations. System suitability parameters System suitability run for HPTLC was performed before each validation run. Five replicate spots were made. Parameters monitored were Rf value and Peak areas of them. (Table 2) Table 2: System suitability Parameters HPTLC Do You Offer Fast Edits?Yes! Need a quick fix? Our editors can polish your paper in hours—perfect for tight deadlines and top grades.
Optimization of Method For HPTLC Various 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 µg/spot) & GYM ( 50 µg/spot) Can You Add Charts or Stats?Definitely! Our writers can include data analysis or visuals—charts, graphs—making your paper sharp and evidence-rich. Figure 4: Wavelength selection for HPTLC of RES and GYM For UV-Visible Spectrophotometric Form 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 RES Method validation of proposed methods Optimized methods were validated in compliance with ICH guidelines. The results of various parameters are discussed following: Linearity For UV Spectrophotometric method, linear correlation was obtained between absorbance and concentration for RES 5-25 µg/ ml at 318.4 nm & 349 nm and GYM 25-125 µg/ ml 303 nm & 349nm.( Table 3) What About Multi-Part Projects?We’ve got it—each section delivered on time, cohesive and high-quality. We’ll manage the whole journey for you. For HPTLC method, linear correlations were obtained between peak area and concentration of RES was 5-25 µg/spot and GYM was 25- 125µg/spot. (Table 4) Accuracy The 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. Precision Inter-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. Do You Adapt to International Rules?Yes! UK, US, or Aussie standards—we’ll tailor your paper to fit your school’s norms perfectly. LOD and LOQ For UV-Visible spectrometry method LOD and LOQ for RES was found to be 0.09µg/ml and 0.28µg/ml respectively. LOD and LOQ for GYM were 0.63µg/ml and 1.92µg/ml respectively. For HPTLC method LOD and LOQ for RES were 0.065 µg/spot and 0.20 µg/ spot respectively. LOD and LOQ for GYM were 1.2 µg/spot and 3.87 µg/spot respectively. Specificity For HPTLC method, a good correlation was obtained between standard and sample spectra of RES and GYM correlation suggests that there in no interference in quantification of RES and GYM. What does a complex assignment mean?If your assignment needs a writer with some niche know-how, we call it complex. For these, we tap into our pool of narrow-field specialists, who charge a bit more than our standard writers. That means we might add up to 20% to your original order price. Subjects like finance, architecture, engineering, IT, chemistry, physics, and a few others fall into this bucket—you’ll see a little note about it under the discipline field when you’re filling out the form. If you pick “Other” as your discipline, our support team will take a look too. If they think it’s tricky, that same 20% bump might apply. We’ll keep you in the loop either way! Robustness Robustness 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 formulation Assay 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. Who is my writer? How can I communicate with him/her?Our writers come from all corners of the globe, and we’re picky about who we bring on board. They’ve passed tough tests in English and their subject areas, and we’ve checked their IDs to confirm they’ve got a master’s or PhD. Plus, we run training sessions on formatting and academic writing to keep their skills sharp. You’ll get to chat with your writer through a handy messenger on your personal order page. We’ll shoot you an email when new messages pop up, but it’s a good idea to swing by your page now and then so you don’t miss anything important from them. Table 3: UV-Visible method Validation parameters
Table 4: HPTLC Validation Parameters
Conclusion Developed 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
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