Amit Srivastava

HPTLC METHOD FOR QUANTIFICATION OF VALERENIC ACID IN AYURVEDIC DRUG JATAMANSI AND ITS SUBSTITUTES

Objective of the present study was quantification of valerenic acid in rhizome of three plant species which is generally traded under the name of Jatamansi. A simple, rapid, cost-effective and accurate high performance thin layer chromatographic method has been developed for quantification of valerenic acid in Valeriana jatamansi, Nardostachys jatamansi, and Selinum vaginatum, which is one of the stable compounds and designated as a key marker compound. Separation and quantification of valerenic acid was achieved by HPTLC using ternary mobile phase of toluene: ethyl acetate: formic acid (80:20:5 v/v/v) on precoated silica gel 60F254 aluminum plate and densitometric determination was carried out in λ280 absorption-reflectance UV mode by deuterium lamp.

ISOLATION AND QUANTIFICATION OF VANILLIN THROUGH FLASH & HPTLC CHROMATOGRAPHIC TECHNIQUES FROM DECALEPIS HAMILTONII WIGHT AND ARN ROOT AND THEIR ANTIOXIDANT STUDIES

Decalepis hamiltonii Wight and Arn (family: Asclepiadaceae) roots are widely used in folk and traditional medicine in India. In the present study, we isolated vanillin through flash chromatography, and performed quantification and validation through HPTLC in the root of D. hamiltonii. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to ICH guidelines. Separation and quantification was achieved using toluene:ethyl acetate (9.0:1.0 v/v) as a binary mobile phase on precoated silica gel 60 F254 aluminum plates on HPTLC. The HPTLC densitometry was carried out at a wavelength of 254 nm in the absorption-reflectance mode. The antioxidant potential of D. hamiltonii root sample was also studied by the 2,2-Diphenyl-2-picryllhydrazyl (DPPH) method. Aqueous plant extract showed 54.67% radical scavenging ability at 0.1 mg/mL concentration.

HPTLC-densitometric determination and kinetic studies on antioxidant potential of monomeric phenolic acids (MPAs) from Bergenia species

The aim of the present communication is the development of validated HPTLC method for simultaneous separation, detection, comparative quantification of monomeric phenolic acids (MPAs), such as vanillic acid (VA), syringic acid (SYA), gallic acid (GA), protocatechuic acid (PCA) in Bergenia species viz. Bergenia ciliata (BC) and Bergenia stracheyi (BS) (Paashanbheda; family Saxifragraceae) and Kinetics studies on antioxidant activity of focused metabolites. The analyses were performed on HPTLC pre-coated silica gel 60F254 plates with optimized solvent system toluene : ethyl acetate : formic acid (5 : 4 : 1 v/v/v) as mobile phase. Densitometric detection of MPAs was performed at 280 nm (λ max) wavelength. The contents of MPAs in both species were found (% in 10 mg ml−1) 0.007 ± 0.1–0.003 ± 0.4 (VA) (y = 3.326x − 1103, regression coefficient r = 0.998), 0.017 ± 0.4 − 0.002 ± 0.5 (SYA) (y = 3.410x − 1009, r = 0.998), 0.024 ± 0.2 − 0.012 ± 0.2 (GA) (y = 5.349x − 240.2, r = 0.999) and 0.027 ± 0.6 − 0.018 ± 0.2 (y = 3.6x − 461.5, r = 0.995). Quantitative variation was assumed as a result of samples collected from different altitudinal range. Two antioxidant assays DPPH and β-carotene were used kinetically in antioxidant potential assessment. Among both the species BC had higher DPPH antioxidant activity and antiradical kinetics than BS, MPAs and positive controls (TOCO), (BHT). Whilst in β-carotene assay highest antiradical activity was reported in PCA kinetically despite BHT than others. However, the deviation in CAA values of BC and BS extracts were very close to the PCA value. EC50 values, rate constant (k), rate of reaction (dx/dt), half-life and average life were also measured in both assays. On the basis of finding it can be concluded that the investigated MPAs were actively involved in antioxidant properties. The kinetic studies of MPAs revealed that H atom transfer from phenolic moieties to the ROS predicts the reactivity of antioxidants.

Simultaneous Quantification of Bergenin, Epicatechin, (+)-Catechin, and Gallicin in Bergenia ciliata Using High Performance Liquid Chromatography

γBergenia ciliata (Paashanbheda, Saxifragraceae) has been used for centuries in Ayurvedic formulations for a number of ailments related to antiurolithiasis, as an anti-inflamatory, for antitussive activity, and as an antioxidant. In the present communication, four samples of Bergenia ciliata were collected from varying altitudes of Uttrakhand, India, and an analytical HPLC method was developed and validated for the separation and quantification of Bergenin, Epicatechin, (+)-Catechin, and Gallicin (A-D) in Bergenia ciliata. HPLC analysis was carried out onto a reverse phase column (Reodein; RP-C18; size 4.5 × 250 mm, 5.0 µm) with mobile phase started with gradient composition prepared from water:phosphoric acid 99.6:0.3 (component A) and acetonitrile:water:phosphoric acid 79.6:20:0.3 (component B). Scanning wavelength λmax 280 nm was used in the determination of compounds using HPLC. The contents of targeted compounds A–D were found positively correlated with the altitudes of collecting site. However, good correlation was only found in compound A and B contents (r = 0.6892, 0.6398) compared to compound C and D contents (r = 0.1297, 0.3245).

Phytochemical and Nutritional Evaluation of Amorphophallus campanulatus (Roxb.) Blume Corm

Amorphophallus campanulatus (Roxb.) Blume (Araceae) is commonly known as Elephant foot yam. Corms are used in India in curries and pickles and are ascribed in vitiated conditions of vata and kapha, arthralgia, elephantiasis, tumours, inflammations, haemorrhages, vomiting, cough, bronchitis, asthma, anorexia, dyspepsia, flatulence, colic constipation, helminthiasis hepatopathy, splenopathy, amenorrhoea, dysmenorrhoea, seminal weakness, fatigue, anaemia and general debility. The present communication deals with the detailed pharmacognostic evaluation of the corm sample using light microscopy, WHO recommended physico-chemical determinations and phytochemical procedures. The physico-chemical, morphological and histological parameters presented in this paper may be proposed as parameters to establish the authenticity of A. campanulatus corms. A detailed nutritional analysis has also been carried out for quantitative evaluation of active nutrient components to determine calorific value for edible usage. HPTLC analysis showed the presence of β-sitosterol as marker compound in different extracts and fractions.

Comparative Botanical and Phytochemical Evaluation of Medicinally Important Stem Bark of Ficus species

Abstract Objective To evaluate the pharmacognostical comparison (Botanical study, physicochemical parameters, HPTLC analysis) in stem barks of four Ficus species (family-Moraceae) viz. F. religiosa L, F. glomerata Roxb, F. retusa auct. & F. carica . Methods Estimation of Phytochemical markers viz: β-sitosterol and lupeol was quantified by HPTLC method and antioxidant studies by carried by DPPH method. Results HPTLC method showed considerable amount of variation with two reference standard viz: β-sitosterol and lupeol content in stem bark of F. religiosa , F. glomerata, F. retusa & F. carica and it were found 0.084, 0.041, 0.059 & 0.131 and 0.020, 0.043, 0.069 & 0.049 respectively. The antioxidant potential of ethanolic extract of stem bark of F. religiosa , F. retusa , F. glomerata & F. carica were found 46.86%, 42.56%, 31.25% & 25.63% at 0.1mg/mL concentration. Conclusion The present work was taken up with a view to lay down standards which will contribute significantly to quality control of these medicinally useful Ficus species. It also provides suitable criteria to differentiate the stem barks of four Ficus species.

Evaluation of Phenolic Content Recoveries in Hydrolyzed Extracts of Bergenia ciliata Using RP-HPLC, GC–MS after Silylation, and Validation through Antioxidant Potential

Methanol extracts of Bergenia ciliata treated with chemical and nonchemical medium were comparatively analyzed for optimum recovery of phenolic (PH) and flavonoid (FV) compounds such as Gallic acid, Bergenin, Gallicin, and (+)-Catechin using RP-HPLC coupled to photo diode array detector. The gas chromatography–mass spectrometry method (GC–MS) was also used for identification of targeted compounds after silylation. Methanol extract was treated separately in acid, base, hydrogen peroxide, neutral, and dry heat medium, analyzed and compared using HPLC. After treatment, drastic variation in phenolic content was observed. Out of five treatments, acid treatment was found to enhance the recovery of all studied phenolic compounds, namely Gallic acid (0.415 ± 0.12), Bergenin (1.91 ± 0.14), Gallicin (0.409 ± 0.17), and (+)-Catechin (18.66 ± 0.15). Dry heat and neutral heat specifically enhances the recovery of Bergenin (2.95 ± 0.12), (3.29 ± 0.18), whereas base and hydrogen peroxide did not show significant content variation. Acid treated extract has shown, as expected, maximum antioxidant potential compared to nontreated extracts.

Simultaneous Quantification of Syringic Acid and Kaempferol in Extracts of Bergenia Species Using Validated High-Performance Thin-Layer Chromatographic-Densitometric Method

A rapid, sensitive, selective and robust quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for separation and quantification of syringic acid (SYA) and kaempferol (KML) in the hydrolyzed extracts of Bergenia ciliata and Bergenia stracheyi. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using toluene : ethyl acetate : formic acid (5 : 4: 1, v/v/v) as the mobile phase. The quantification of SYA and KML was carried out using a densitometric reflection/absorption mode at 290 nm. A dense spot of SYA and KML appeared on the developed plate at a retention factor value of 0.61 ± 0.02 and 0.70 ± 0.01. A precise and accurate quantification was performed using linear regression analysis by plotting the peak area vs concentration 100-600 ng/band (correlation coefficient: r = 0.997, regression coefficient: R(2) = 0.996) for SYA and 100-600 ng/band (correlation coefficient: r = 0.995, regression coefficient: R(2) = 0.991) for KML. The developed method was validated in terms of accuracy, recovery and inter- and intraday study as per International Conference on Harmonisation guidelines. The limit of detection and limit of quantification of SYA and KML were determined, respectively, as 91.63, 142.26 and 277.67, 431.09 ng. The statistical data analysis showed that the method is reproducible and selective for the estimation of SYA and KML in extracts of B. ciliata and B. stracheyi.

Simultaneous reverse-phase HPLC determination of major antioxidant phenolics in Commelina benghalensis L. tubers

Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correl...

Reversed-phase high-performance Liquid Chromatography-ultraviolet Photodiode Array Detector Validated Simultaneous Quantification of six Bioactive Phenolic Acids in Roscoea purpurea Tubers and their In vitro Cytotoxic Potential against Various Cell Lines

Background: Roscoea purpurea or Roscoea procera Wall. (Zingiberaceae) is traditionally used for nutrition and in the treatment of various ailments. Objective: Simultaneous reversed-phase high-performance liquid chromatography-ultraviolet (RP-HPLC) photodiode array detector identification of phenolic acids (PAs) was carried out in whole extract of tuber and their cytotoxic potential was estimated along with radical scavenging action. Bioactivity guided fractionation was also done to check the response potential against the same assay. Materials and Methods: Identification and method validation was performed on RP-HPLC column and in vitro assays were used for bioactivity. Results: Protocatechuic acid, syringic acid, ferulic acid, rutin, apigenin, and kaempferol were quantified as 0.774%, 0.064%, 0.265%, 1.125%, 0.128%, and 0.528%, respectively. Validated method for simultaneous determination of PAs was found to be accurate, reproducible, and linearity was observed between peak area response and concentration. Recovery of identified PAs was within the acceptable limit of 97.40–104.05%. Significant pharmacological response was observed in whole extract against in vitro cytotoxic assay, that is, Sulforhodamine B assay, however, fractionation results in decreased action potential. Similar pattern of results were observed in the antioxidant assay, as total phenolic content and total flavonoid content were highest in whole extract and decreases with fractionation. Radical scavenging activity was prominent in chloroform fraction, exhibiting IC50 at 0.25 mg/mL. Conclusion: Study, thus, reveals that R. purpurea exhibit significant efficacy in cytotoxic activity with the potentiality of scavenging free radicals due the presence of PAs as reported through RP-HPLC. SUMMARY Proto-catechuic acid, syringic acid, ferulic acid, rutin, apigenin and kaempferol were quantified as 0.774, 0.064, 0.265, 1.125, 0.128 and 0.528 % Preliminary cytotoxic activity revealed that whole extract of R. purpurea exhibit promising effect and after fractionation the potentiation of action reduces The radical scavenging potential of whole extract and fractions are well reflected by TPC, TFC and DPPH assay.