Performance Thin Layer Chromatographic Method Research Articles

High performance thin layer chromatography (HPTLC) method development and validation for the simultaneous determination of paracetamol, caffeine, chlorpheniramine and phenylepherine in tablet formulation

AbstractA rapid, selective, and precise high performance thin layer chromatographic method was developed and validated for the simultaneous analysis of paracetamol, caffeine, phenylephrine and chlorpheniramine in tablets. The chromatographic analysis was carried out on glass plates pre-coated with silica gel 60 F254 as a stationary phase. The optimized mobile phase was methanol : n-butanol : toluene : acetic acid (8:6:4:0.2 v/v). TLC chamber of 10 × 20 cm was used with saturation time of 15 min. The retardation factor (RF) for chlorpheniramine, phenylephrine, caffeine and paracetamol was found to be 0.15 ± 0.02, 0.29 ± 0.02, 0.50 ± 0.02, 0.68 ± 0.02 respectively. Detection was carried out at 212 nm. Validation study was done following ICH Q2 (R1) guideline. The regression data for the calibration plots showed good linear relationship with R2 = 0.997 over the concentration range of 300–1,500 ng band−1 for caffeine, R2 = 0.996 over the concentration range of 100–500 ng band−1 for phenylephrine, R2 = 0.996 over the concentration range of 200–600 ng band−1 for chlorpheniramine, R2 = 0.998 over the concentration range of 400–2,400 ng band−1 for paracetamol. The method was validated for precision, accuracy and recovery. Minimum detectable amounts were found to be 304.9 ng band−1, 87.88 ng band−1, 117.18 ng band−1 and 143.06 ng band−1 for caffeine, phenylephrine, chlorpheniramine, and paracetamol respectively while the limit of quantification was found to be 923.95 ng band−1, 266.32 ng band−1, 355.11 ng band−1, and 433.53 ng band−1 in the same order. The method was successfully applied to analyze two marketed tablets in a selective and reproducible manner.

DEVELOPMENT AND VALIDATION OF HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF POSACONAZOLE IN FORMULATION

Objective: The study aims to developed and validate novel high performance thin layer chromatographic method for estimation of posaconazole in tablet dosage form and validate as per the international conference on harmonization (ICH) guidelines. Methods: The method was developed on silica gel 60 F254 HPTLC plates and the mobile phase was used as toluene: chloroform: ethanol in ratio (5:4:1 V/V/V). Densitometric analysis of Posaconazole was performed at the wavelength 265 nm. Results: The RF of Posaconazole was found to be 0.331. The calibration curve was found to be linear in the concentration range of 100–500 ng/band. The correlation coefficient is 0.9991 for Posaconazole. The precision result was found to be satisfactory, which indicate that the method is precise. The recovery value lies in the range of 98–102% indicating the accuracy of the method. Conclusion: A new, simpleaccurate, fast, economic, precise, and robust high performance thin layer chromatographic method was developed for the estimation of Posaconazole. The method is selective, specific it was applied in pharmaceutical analysis of Posaconazole tablet dosage form.

Quality control of marketed herbal products of Asparagus racemosus Willd. through high performance thin layer chromatography (HPTLC) analysis

Asparagus racemosus Willd. is a valuable medicinal plant which is used all over the world. There are several marketed products of A. racemosus. The high demand for this herb has increased the risk of adulteration in its commercial products. The adulterated herbal products might pose serious ill effects on health. Therefore, it is necessary to check the quality of marketed products in terms of the presence of their major bioactive compounds. The present study aimed to carry out the qualitative and quantitative analysis of Shatavarin IV in marketed products of A. racemosus through a validated high performance thin layer chromatography (HPTLC) method. Ten marketed products were analysed and all of them had shown the presence of Shatavarin IV which was quantified. The identification and quantification were done by taking a standard Shatavarin IV as reference. The Shatavarin IV was detected at Rf 0.4±0.05 and showed maximum absorption at 425 nm. The Shatavarin IV was quantified using a 6-point calibration curve having a standard deviation of 3.89 % with an R2 value of 0.9968. The amount of Shatavarin IV varied between 1.47±0.25 to 2.69±0.51 mg/g on a dry weight basis which is a normal range in the raw plant materials. Thus, the present findings would be a simple, reliable and cost-effective method for the quality determination of herbal products of A. racemosus. The developed HPTLC chromatograms would serve as a reference for the quality assessment of commercial products of A. racemosus in future.

An eco-friendly separation-based framework for quantitative determination and purity testing of an antihypertensive ternary pharmaceutical formulation

Designing new, verified methodologies with a focus on sustainability, analytical efficiency, simplicity, and the environment has become a major priority for pharmaceutical quality control units. In this way, sustainable and selective separation-based methodologies were designed and validated for the concurrent estimation of amiloride hydrochloride (AML), hydrochlorothiazide (HCT) and timolol maleate (TIM) in their fixed dose formulation (Moducren® Tablets) along with hydrochlorothiazide potential impurities, salamide (DSA) and chlorothiazide (CT). The first method is a high performance thin layer chromatographic method (HPTLC-densitometry). The first developed method employed silica gel HPTLC F254 plates as stationary phase using a chromatographic developing system composed of ethyl acetate–ethanol–water–ammonia (8.5:1:0.5:0.3, by volume). The separated drug bands were densito-metrically measured at 220.0 nm for AML, HCT, DSA and CT and at 295.0 nm for TIM. The linearity was assessed over a wide concentration range, 0.5–10 µg/band, 1.0–16.0 µg/band and 1.0–14 µg/band for AML, HCT and TIM, in order and 0.05–1.0 µg/band for each of DSA and CT. The second method is capillary zone electrophoresis (CZE). The electrophoretic separation was achieved using background electrolyte (BGE), borate buffer 40.0 mM with pH 9.0 ± 0.2, at applied voltage of + 15 kV with on-column diode array detection at 200.0 nm. The method linearity was reached over the concentration range of 20.0–160.0 µg/mL, 10.0–200.0 µg/mL, 10.0–120.0 µg/mL for AML, HCT and TIM, respectively and 10.0–100.0 µg/mL for DSA. The suggested methods were optimized to achieve best performance and validated agreeing with the ICH guidelines. Assessment of methods’ sustainability and greenness was performed using different greenness assessment tools.Graphical

Analytical Quality by Design (AQBD) Assisted Development and Validation of HPTLC Method for Estimation of Rottlerin in Topical Patch Formulation

Pharmacognosy Research,2023,15,2,267-276.DOI:10.5530/pres.15.2.029Published:February 2023Type:Original Article Authors:Kaumudee Bodas, Vaibhav M Shinde, Deshmukh Vishal, and Deshmukh Sheetal Author(s) affiliations:Kaumudee Bodas1, Vaibhav M Shinde1,*, Deshmukh Vishal2, Deshmukh Sheetal2 1Centre for Advanced Research in Pharmaceutical Sciences, Poona College of Pharmacy, Pune, Maharashtra, INDIA. 2Bharati Vidyapeeth (Deemed to be University), Yashwantrao Mohite Institute of Management, Karad, Maharashtra, INDIA. Abstract:Background: Rottlerin, a natural polyphenolic ketone is a major constituent of Mallotus philippensis (Lam.) Mull. Arg. plant. Although, less explored it has diverse array of therapeutic applications. Objectives: The current work aimed to develop novel, simple, speedy, accurate, potent High Performance Thin Layer Chromatography Method (HPTLC) to separate, and quantify rottlerin from the herbal topical patch In-House formulation. Materials and Methods: This was the foremost attempt to develop and validate HPTLC method using Analytical Quality by design (AQbD) approach. Box Behnken design (BBD) coupled with response surface methodology approach was followed for the systematic optimization. The end results of toluene content, chamber conditioning time, and solvent front on Rf value of rottlerin was studied. The optimised method was validated as per ICH guidelines. Results: For HPTLC, stationary phase used was aluminium plates 60F254 coated with silica gel as. AQbD optimised conditions were mobile phase proportion namely toluene: ethyl acetate: methanol: ammonia (5:4:2:0.2), development distance 80 mm, band width 6mm, and chamber saturation time 10 min. The method generated well resolved and compact bands at Rf of 0.41±0.05. The developed method exhibited high accuracy, precision, robustness, and recovery. The quantity of Rottlerin in developed herbal patch was found to be 134.8 ng/band. Conclusion: In a nutshell, the present study undeniably vouches for a QbD based method development approach for Rottlerin and can be extrapolated to estimate it in other herbal extracts, or marketed formulations Keywords:Analytical Quality by design (AQbD), HPTLC, Mallotus philippensis (Lam.) Mull. Arg., Rottlerin, Topical patch.View:PDF (1.15 MB)

Simultaneous densitometric determination of β-sitosterol and lupeol through validated HPTLC method in different plant parts of Uraria picta (Jacq.) Desv. ex DC. – A dashmool species

Abstractβ-sitosterol (BS) and lupeol (LU) exhibit a number of biological activities and are the important bioactive marker compounds in pharmaceutical science. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous quantification of these two compounds in leaves, stem and roots of Uraria picta, a critically endangered medicinal plant and one of the important constituents of ten plants ayurvedic formulation called Dashmoola. Standards and test samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene: methanol: chloroform (8:1:1, v/v/v) as a mobile phase. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BS and LU standards and those of test samples were found to be 0.53 ± 0.01 and 0.63 ± 0.01 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BS and LU, the linear regression data for the calibration plots revealed a satisfactory linear association with r2 = 0.995 and 0.998, respectively. Linear range for both BS and LU was 200–600 ng/band. Accuracy of the method was evaluated by performing recovery study at three different levels by standard addition methods with an average recovery of 99.86% and 101.07%. The results revealed that the leaf samples of U. picta contained highest concentration of BS (0.150 ± 0.02%) while its root samples confined the highest concentration of LU (0.149 ± 0.01%). The developed method can be applied for routine and quality control analysis in different herbal formulations containing U. picta species.

An ecofriendly multi-analyte high performance thin layer chromatographic densitometric determination of amoxicillin, metronidazole, and famotidine in their ternary mixtures and simulated gastric juice: A promising protocol for eradicating Helicobacter pylori.

Gastrointestinal tract disorders constitute a heavy burden to healthcare providers.To eradicate Helicobacter pylori infection, different triple therapy protocols have been proposed. Among which are combinations of proton pump inhibitors (e.g. omeprazole), histamine-2 receptor antagonists (e.g. famotidine), along with antibiotics (e.g. amoxicillin). In this work, a sensitive and accurate high performance thin layer chromatographic method was developed for the simultaneous determination of amoxicillin, metronidazole, and famotidine in bulk powder and laboratory prepared combined-tablet mixtures. Complete separation of the cited compounds was achieved using pre-coated silica gel plates with a mixture of methanol: chloroform: toluene: water: glacial acetic acid (5:2:1.5:0.5:0.1 v/v/v/v/v) as the mobile phase. The method was fully validated as per the international conference of harmonization guidelines. Good linearity, correlation coefficient of 0.9991, was obtained in the concentration ranges 0.1-1.6 μg band-1 (amoxicillin), 0.1-0.9 μg band-1 (metronidazole) and 0.1-0.9 μg band-1 (famotidine). Since the method allowed the determination of the three compounds in combined tablets with high degree of selectivity, accuracy, precision, with cost-effectiveness, it could be used for regular quality control. Moreover, the applicability of the proposed method was extended to the determination of the ternary mixture in simulated gastric juice. Method greenness was assessed using different green metrics. This article is protected by copyright. All rights reserved.

HPTLC Method Development and Validation for Simultaneous Estimation of Berberine, Ellagic Acid and Ferulic Acid in <i>Amrtadi Churna</i>

The objective of the current study was to develop a new simple and precise High Performance Thin Layer Chromatography (HPTLC) method for standardization of three biomarkers i.e., berberine, ellagic acid and ferulic acid in Amrtadi churna. Amrtadi churna is mainly used for hyperacidity and contains dried plant parts of Gokshur, Amla and Guduchi. The method was developed and validated using precoated silica gel at 60 F254 as the stationary phase and toluene:ethyl acetate:formic acid:methanol (6:6:1.6:0.4, v/v/v/v) as the mobile phase. The detection and quantification were performed at 319 nm and the Rf value obtained was 0.35±2 for berberine, 0.5±2 for ellagic acid and 0.74±2 for ferulic acid. The method was validated as per ICH guidelines in terms of linearity, precision, specificity, accuracy and robustness.

COMPARISON OF ACID DEGRADANT PRODUCT WITH METABOLIC PATHWAY OF REMOGLIFLOZIN ETABONATE IN DEVELOPED AND VALIDATED RP-HPTLC METHOD

Forced degradation studies in tablet and bulk formulations of remogliflozin etabonate have been developed using a precise and sensitive high performance thin layer chromatographic method. The silica gel RP-18 F254S plates were used as the stationary phase and ACN: water: ammonia solution (8:2:0.5 V/V/V) was used as the mobile phase for estimation. The proposed method was successfully validated, showing the Rf of the drug as 0.72 at 229 nm. The method was observed to be linear in the range of 500-3000 ng band-1 and then degradation was estimated by forced degradation pathway. In the forced degradation studies, the drug was found to be highly susceptible to both acid and base, including oxidative conditions, providing an active metabolite when remaining in contact in stressed conditions for a short time (30 min), while in longer duration (36 h), the drug provided another metabolite which on characterization was found to be inactive and predicted to be reported in the metabolic pathway

Method development and validation for quantification of six bioactive compounds (andrographolide, columbin, piperine, gallic, paracoumaric and oleanolic acids) by HPTLC.

During the spread of pandemic diseases, immunity boosting herbal drugs are taken as a preventive medicine. Kapacurak Kuṭinīr Cūraṇam is a Siddha drug used for flu like viral infections, cold and fever. Developing an analytical method to estimate the content of active phytoconstituents in such antiviral immune boosting drug will be useful in the phyto pharmaceutical industry. A precise, reliable and sensitive ordinary phase high performance thin layer chromatography (HPTLC) method has been developed and validated for identification and simultaneous estimation of six bioactive components namely like andrographolide, columbin, gallic acid, ρ-coumaric acid, piperine and oleanolic acid from any Indian traditional medicine, medicinal plant, drugs and food materials etc. The separation was achieved on silica gel 60F254 TLC plates using toluene: ethyl acetate: formic acid (7:3:0.5, v/v) as mobile phase. The gallic acid, ρ-coumaric acid, piperine markers were estimated using the densitometric scanning in absorption mode at 254nm. The densitometric scanning was done after derivatization (vanillin-sulphuric acid reagent) at λ=520nm for andrographolide, columbin and oleanolic acid. The linear regression analysis data for the calibration plots showed a correlation coefficient in the concentration range 1-5μg per band for the bioactive markers with respect to area. The method was validated for accuracy, precision, limit of detection (LOD), and quantitation of limit (LOQ). Developed method was accurate, precise and fast to ensure the quality of Kapacurak Kuṭinīr Cūraṇam.

Method Development and Validation for the estimation of Amlodipine and Perindopril in Bulk and Pharmaceutical Dosage Form

Literature survey reveals that few spectrophotometric, high performance liquid chromatographic (HPLC)and High performance thin layer chromatographic (HPTLC)methods were reported for the estimation of Amlodipine and perindopril in bulk and combined pharmaceutical dosage forms. But there is no any stability indicating high performance liquid chromatography study is reported for amlodipine. So the present study was aimed to development of simple, accurate, rapid and economical HPLC stability indicating assay method were established for the determination of Amlodipine and Peridopril in bulk and tablet dosage form. A High-performance liquid chromatograph WATERS, software: Empower 2,2695 separation module, 996 PDA detector, using Phenomenex Luna C18 (4.6mm×250mm) 5µm or equivalent column, with mobile phase composition of Methanol: Phosphate Buffer pH3.0 (70:30v/v) was used. The flow rate of 1.0ml min-1 and effluent was detected at 230nm. The retention time of Amlodipine and Perindopril was found to be 1.870min and 2.499minutes respectively. Linearity was observed over concentration range of 10-50µg ml-1 for Amlodipine and 16-80µg ml-1 for Perindopril respectively. The accuracy of the proposed method was determined by recovery studies and the Amlodipine was found to be 99.1% and Perindopril was found to be 98.8% respectively. The proposed method is applicable to routine analysis of Amlodipine and Perindopril in bulk and pharmaceutical formulations. The proposed method was validated for various ICH parameters like linearity, limit of detection, limits of quantification, accuracy, precision, range and specificity.

Assessment Of Antiarthritic Potential Of Carica Papaya L. Leaves Extract And Fractions: Histopathological Analysis

Carica papaya L has been traditionally claimed for curing inflammation hence the present work exemplifies the anti-arthritic potential of crude leaves extract and its fractions in arthritic models. Screening on this plant has established presence of kaempferol which might possess antiarthritic properties. Kaempferol was quantitatively estimated by high performance thin layer chromatographic method in chloroform fraction and was found to be 0.08% w/w. Arthritis was induced by intraplantar administration considering formaldehyde (2%v/v), turpentine oil emulsion (10%v/v) and complete Freund’s adjuvant (1%v/v) in left hind paw of rats. Paw oedema, body weight, haematological parameters, level of serum enzyme markers, biochemical parameters, interleukin-6 and histopathological analysis were observed in all groups. Present experimental work justifies that in chronic arthritis, iinvolvement of interleukin-6 is associated with non-articular manifestations considering joint inflammation including anaemia. The plant extract and chloroform fraction possess dose dependent antiarthritic effect in acute and complete Freund’s adjuvant induced arthritic model by ameliorating the level of serum enzyme biomarkers, haematological and biochemical parameters, body weight abnormalities, interleukin-6. The experimental explanation established that extract and its fraction of Carica papaya L. revealed anti-arthritic potential.

Assessment Of Antiarthritic Potential Of Carica Papaya L. Leaves Extract And Fractions: Histopathological Analysis

Carica papaya L has been traditionally claimed for curing inflammation hence the present work exemplifies the anti-arthritic potential of crude leaves extract and its fractions in arthritic models. Screening on this plant has established presence of kaempferol which might possess antiarthritic properties. Kaempferol was quantitatively estimated by high performance thin layer chromatographic method in chloroform fraction and was found to be 0.08% w/w. Arthritis was induced by intraplantar administration considering formaldehyde (2%v/v), turpentine oil emulsion (10%v/v) and complete Freund’s adjuvant (1%v/v) in left hind paw of rats. Paw oedema, body weight, haematological parameters, level of serum enzyme markers, biochemical parameters, interleukin-6 and histopathological analysis were observed in all groups. Present experimental work justifies that in chronic arthritis, iinvolvement of interleukin-6 is associated with non-articular manifestations considering joint inflammation including anaemia. The plant extract and chloroform fraction possess dose dependent antiarthritic effect in acute and complete Freund’s adjuvant induced arthritic model by ameliorating the level of serum enzyme biomarkers, haematological and biochemical parameters, body weight abnormalities, interleukin-6. The experimental explanation established that extract and its fraction of Carica papaya L. revealed anti-arthritic potential.

Stability Indicating HPTLC Method for Bedaquiline Fumarate

Bedaquiline fumarate is an anti-TB treatment that was approved in 2012 and has a novel mechanism of action for fighting multidrug resistance (MDR). Bedaquiline fumarate, a diarylquinoline having significant bactericidal and sterilising activity, has been licenced by the US Food and Drug Administration,for the treatment of multidrug-resistant tuberculosis (MDR-TB).A simple, sensitive and stability indicating High Performance Thin Layer Chromatography method has been developed and validated for estimation of Bedaquiline fumarate as per ICH guidelines. Method was developed with silica gel 60 F254TLC plates using Acetonitrile: Ethyl acetate (7:3v/v) as mobile phase with densitometric detection at 229 nm. This system was found to give compact bands for Bedaquiline fumarate at (Rf 0.53 ±0.01). The linearity was observed in the range of200-1000 ng/band. Bedaquiline was exposed to different stress degradation conditions like hydrolysis at different pH, oxidation, photolytic and thermal degradation. The proposed method can used for routine quantitative determination of Bedaquiline.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND PIOGLITAZONE IN PHARMACEUTICAL DOSAGE FORM

A simple, specific, accurate and precise High Performance Thin Layer Chromatography (HPTLC) method was developed which can separate and quantitatively estimate formulation of Metformin Hydrochloride and Pioglitazone The chromatographic separation for Metformin Hydrochloride and Pioglitazone in HPTLC method separation was achieved with the mobile phase consisting of Toluene: Methanol: Triethylamine (6: 4: 0.1 % v/v/v/v). The detection of spot was carried out at 230 nm and the Rf values Metformin Hydrochloride and Pioglitazone were found to be 0.25±0.03 and 0.47±0.04.. Ths method was validated accordance with ICH guidelines. The recovery these combination were above 96%. The developed method are free from interference due to excipients present in tablets. These method are rapid and simple, do not require any sample preparation and is suitable for routine quality control analysis Keywords: HPTLC ,Method validation, Quantitative analysis

Simultaneous Estimation of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in Combined Dosage Form by HPTLC Method

A simple, step by step, cost-effective, accurate, sensitive, and selective validated High Performance Thin Layer Chromatographic method for simultaneous determination of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in syrup formulation has been developed and validated. HPTLC separation was achieved on Merck aluminum HPTLC plates precoated with silica gel 60 F254. The solvent system comprised of Chloroform: Methanol: Ethyl acetate: Acetic acid: Formic acid (7.0:1.4: 0.8:1.0:0.5 v/v). Densitometric detection wavelength at 200 nm was used in reflectance-absorbance mode. The retention factors were found to be 0.32±0.02, 0.55± 0.02, and 0.72±0.02, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin respectively. Results were found to be linear over a range of 200 - 700ng band-1, 1000 - 7000ng band-1, and 400-1200ng band-1 for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively. The percent assay was found to be 98.47%, 98.47%, and 99.23%, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively in marketed formulation. The developed densitometric method was validated by following the International Council on Harmonisation (ICH) guideline. The developed and validated chromatographic method can be applied for routine quality control of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in the combined syrup dosage form used in study.

Principal Component Analysis and DoE-Based AQbD Approach to Multipurpose HPTLC Method for Synchronous Estimation of Multiple FDCs of Metformin HCl, Repaglinide, Glibenclamide and Pioglitazone HCl.

Metformin HCl (MET) is a mostly used antidiabetic drug and several fixed-dose combinations are available in the market with other antidiabetic drugs for the treatment of insulin-independent diabetes. Numerous chromatographic methods have been reported for the estimation of multiple fixed dose combination (FDC) of MET but each FDC needs a separate and dedicated chromatographic method for analysis. No chromatographic method has been reported yet in the literature which promotes synchronous estimation of multiple FDCs of MET to minimize organic solvent consumption, time and cost of analysis. Hence, the multipurpose-high performance thin layer chromatography (HPTLC) method was developed for the synchronous estimation of FDCs of MET by the implementation of an enhanced analytical quality by design approach. Principal component analysis was applied for the identification of critical method risk parameters. Box-Behnken design was applied for response surface analysis. The method operable design ranges was navigated and the control strategy was framed. The developed method was applied for the analysis of multiple FDCs of metformin. The developed method can synchronously analyze multiple FDCs of MET which required several chromatographic methods. Hence, the present method is multipurpose-HPTLC for the synchronous estimation of multiple FDCs of MET which required minimum organic solvent, time and cost of analysis.

HPTLC Profiling and Antibacterial Efficacy of Melia Azedarach Linn. Leaf Extracts Against Secondary Bacterial Pathogens of Dermatophytosis

Medicinal plants are potential source of antimicrobial agents, used traditionally to treat various human microbial infections worldwide. The present study was aimed to determine the antibacterial efficacy of Melia azedarach Linn. leaf extracts against secondary bacterial pathogens of dermatophytosis such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus and to investigate the presence of phytocompounds through High Performance Thin Layer Chromatographic (HPTLC) method of the potential extract. The leaf extracts of the selected plants were tested for antibacterial potentiality using disc diffusion method and gentamicin (10µg/disc) was used as positive control. The results revealed that the positive control had more inhibitory effect than the solvent extracts used. Comparatively, acetone extract of M. azedarach leaves was more effective against two test pathogens, S. aureus (12.93±0.65 mm) at 150 µl/disc and P. aeruginosa (11.5±0.10 mm) at 100 µl/disc concentration with significant difference of p≤0.05 using one-way analysis of variance (ANOVA). The varying degree of extract concentrations has a greater influence in the inhibitory effect against test pathogens. The different Rf values, maximum percentage concentration, area percentage of polyvalent chemical constituents was recorded in HPTLC profiling of acetone leaf extract, where the maximum percentage concentration was found to be 14.07% at 0.09 Rf. The HPTLC studies has confirmed that the compounds present in the acetone extract might be responsible for the inhibitory effect against the bacterial pathogens and are more soluble in semi-polar solvent. Therefore, the present investigation forms the basis as preliminary study of antibacterial efficacy of M. azedarach leaf extracts and phytocompound HPTLC profiling of potential extract, which could be used for quality evaluation of compound and standardisation of drug in future work.

Marker Based Standardization of Herbal Sunscreen Formulation by Using Validated High-Performance Thin Layer Chromatography Method

Plant based products are worldwide used and trusted for the care and cure of the health. Herbal sunscreen formulations are gaining popularity for their effectiveness and are devoid of side effects. The present investigation is an effort made to develop herbal sunscreen cream containing methanolic extracts of different plants and its standardization for the presence of bioactive compounds. The leaves of Cymbopogon citratus (Stapf), fruit peel of Punica granatum (Linn), flowers of Butea monosperma (Lam.) and leaves of Neolamarckia cadamba (Roxb.) were selected for the preparation of sunscreen cream. The standardization of herbal formulations is very important to determine its quality based on the concentration of their active phytoconstituents. The herbal cream F-5 (2 % w/w) imparted its sun-protective and antioxidant property. It shows free radicals scavenging activity due the presence of flavonoids and phenolic compounds. High performance thin layer chromatography method is used to determine the quality and quantity of the sun protective phytoconstituents present in the product. The method was validated according to ICH guidelines for the estimation butrin (BT), isobutrin (IBT), quercetin (QC), apigenin (API), chlorogenic acid (CA) and gallic acid (GA) using the optimized solvent systems. The estimation of bioactive markers was carried out on silica gel precoated thin layer chromatography plates with 60F254 as the stationary phase and Camag TC scanner III for densitometric scanning. The average Rf values for the markers were found to be 0.46 for BT, 0.57 for IBT, 0.50 for QC, 0.57 for API, 0.66 for CA and 0.42 for GA. The developed HPTLC method was linear with correlation coefficient 0.999 for BT, 0.998 for IBT, QC, API and 0.9966 for CA and 0.9989 for GA. Limit of detection (LOD) and limit of quantification (LOQ) were recorded. The developed analytical method for quantitative determination of phytoconstituents was found efficient, simple, accurate, and validated.

Estimation of Gallic Acid in Triphala Using Enzymatic Hydrolysis

A well-known Ayurvedic formulation, Triphala has numerous health benefits like appetite stimulation, controls diabetes, reduces cholesterol, relieves stress, alleviates inflammation and treats a variety of bacterial and fungal infections along with digestive problems. The phytoconstituents flavonoids, tannins, polyphenols and many chemical compounds are responsible for its claimed health benefits. One of the very significant compounds is gallic acid, which is reported to possess hepatoprotective, anti-inflammatory, antineoplastic and antioxidant properties that are beneficial in treatment of several diseases. In the present work using gallic acid as a marker, an attempt has been made to extract gallic acid from tannins present in Triphala by enzymatic hydrolysis and subsequent estimation of total gallic acid content by high performance thin layer chromatography (HPTLC). Rhizopus oryzae fungus is cultured for the production of enzyme tannase, which converts tannins into gallic acid. The extracted gallic acid is estimated in the biocatalyzed sample at optimal process parameters by high performance thin layer chromatography (HPTLC) method. The gallic acid content after treatment with enzyme was found to be doubled 0.296 ± 0.018 mg/ml, Rf value 0.37 at 254nm which was 0.197 ± 0.008 mg/ml and Rf value was 0.37 prior to enzymatic treatment. Gallic acid in Triphala is present in free form as well as the constituent of tannins. There is a remarkable yield of gallic acid from tannins using the enzyme Tannase in the conversion process. Application of such advanced technology increases the gallic acid yield from response surfaces in herbal and ayurvedic products containing gallic acid. The extraction condition of phenolic compounds can be optimised using enzyme hydrolysis. The effective factors including the solid to solvent ratio, enzyme concentration, particle size and extraction time can be optimised. Keywords: Triphala, Gallic acid, Tannin, Rhizopus oryzae, Tannase, HPTLC