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  • Author or Editor: Mamta Shah x
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Withania somnifera , commonly known as Ashwagandha, is a valued herb in Ayurvedic medicine. Root leaves, and preparations of the plant are traditionally used as tonic, hypnotic, sedative and diuretic. W. somnifera mainly contains withanolides which are specific to the Solanaceae family. The biological activity of withanolides, especially withaferin A, has been studied extensively. Methods for analysis of withaferin A and withnolide D require acetylation before analysis, or separation time is extremely long. The main problem in identification of withaferin Awith other separated withanolides is that all withanolides absorb UV light, so without comparison with a standard it is not possible to identify withaferin A. The objectives of this paper are to present a new method of identification of withaferin A, using a destructive reagent, and an HPTLC method for quantification of the compound. Chromatography on silica with toluene-ethyl acetate-acetone 2:3:3 as mobile phase enabled good resolution of withaferin A without interference from other compounds present in W. somnifera . After spraying with anisaldehyde-sulfuric acid reagent and heating for 15 min at 105°C, characteristic orange fluorescence was observed for withaferin Aonly among all the spots resolved. When scanned at 214 nm the R F of withaferin A was 0.62. Interestingly, old root did not contain withaferin A whereas young root contained a large amount. The method was validated for accuracy, precision, specificity, linearity, and limits of detection and quantification. The method is simple, sensitive, and precise, and can be used for routine quality-control testing of W. somnifera .

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Inula cappa (family Compositae) is used in the Ayurvedic medicinal system for the treatment of bronchitis, diabetes, fever, hypertension, and rheumatism. The proposed high-performance thin-layer chromatography (HPTLC) study offers coherent evaluation of isoalantolactone, germacranolide, β-sitosterol, and lupeol from I. cappa root. Methanolic solutions of isoalantolactone, germacranolide, β-sitosterol, and lupeol were applied on an HPTLC plate and they were scanned at 525 nm. The mobile phase toluene—methanol (9.4:0.6, v/v) was used for all the phytochemicals. After development, all the plates were air-dried at room temperature, derivatized with anisaldehyde–sulfuric acid reagent and heated at 105°C. This study aids the identification of these compounds and provides an easy and simple method for the simultaneous estimation of these markers in the I. cappa roots. The method would serve as an expedient tool in routine analyses to corroborate the drug through good constancy.

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A sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantitative determination of oleanolic acid in the dried roots of Helicteres isora Linn. Total sapogenins were isolated from the roots, and their alcoholic extract was applied on silica gel G 60 F254 plates with toluene-ethyl acetate-glacial acetic acid, 7:3:0.1 (ν/ν/ν) as mobile phase. Detection and quantification were performed by densitometric scanning at 529 nm. The accuracy of the method was confirmed by conducting recovery studies at three different levels using the standard addition method. The average recovery of oleanolic acid was found to be 98.98%. The proposed HPTLC method provides good resolution of oleanolic acid from other constituents present in sapogenin extract of dried roots of H. isora and can be used for quantification of oleanolic acid present in the extract. The method is simple, accurate, and precise.

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Oldenlandia corymbosa Linn. (Rubiaceae) is an important herb traditionally used as a febrifuge and liver tonic. In this study, a high-performance thin-layer chromatography (HPTLC) method has been established for the quantification of four bioactive markers, oleanolic acid (OA), ursolic acid (UA), lupeol (LU), and stigmasterol (ST), in the whole plant of O. corymbosa. Separation was achieved on silica gel 60 F254 HPTLC plates using hexane-ethyl acetate-methanol (8.2:1.8:0.5, v/v) for oleanolic acid and ursolic acid; and toluene-methanol (9.4:0.6, v/v) for lupeol and stigmasterol as the mobile phases. The quantitation of the four markers was carried out using the densitometric scanning at 540 nm after derivatization using sulfuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r 2 = 0.9831–0.9979) in the concentration range of 1200–4200 ng for oleanolic acid, 400–1400 ng for ursolic acid, 100–500 ng for lupeol, and 500–2500 ng per spot for stigmasterol with respect to area. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for oleanolic acid, ursolic acid, lupeol, and stigmasterol were 98.77 to 99.12%, indicating the good reproducibility. Stigmasterol 1.19 ± 0.04% w/w was present at high concentration, and oleanolic acid 0.012 ± 0.006% w/w was present at low concentration in the whole plant powder. The proposed HPTLC method was found to be simple, precise, sensitive, accurate, reproducible, and robust.

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Convolvulus pluricaulis is a highly valued rasayana drug of the Ayurvedic system of medicine. It is one of the four drugs ( Convolvulus pluricaulis, Evolvulus alsinoides, Clitoria ternatea , and Canscora decussata ) described in Ayurveda as Shankhpushpi. Because C. pluricaulis is described and used as Shankhpushpi by most practitioners, the objective of this study was to develop an HPTLC method for generation of a distinct chemical profile of Shankhpushpi and for quantification of scopoletin in C. pluricaulis and in commercial formulations containing the plant. Because scopoletin is one of the major coumarins in C. pluricaulis , it was used as a chemical marker. Chromatography on silica gel, with toluene-ether, 1 + 1, saturated with 10% glacial acetic acid, as mobile phase enabled good resolution of scopoletin without interference from other compounds present in Shankhpushpi. Scopoletin was resolved at R F 0.31 as a blue fluorescent spot when scanned at λ =366 nm. The study was extended to the analysis of commercial samples and formulations containing Shankhpushpi. Scopoletin was found to be present in almost all the commercial samples and formulations. Three other coumarins of R F 0.47, 0.55, and 0.63 were present in some of the samples. The HPTLC results were confirmed by results from UV spectroscopic scanning of the samples at λ = 366 nm. The method was validated for linearity (400–1200 ng/spot), correlation coefficient (0.9881), accuracy (recovery = 98.3%), precision (or CV, %; intraday 1.5–3.3%, interday 3.5–5.5%), limit of detection (50 ng/spot), and limit of quantification (100 ng/spot). The method is simple, sensitive, and precise and can be used for routine quality-control testing of C. pluricaulis and its commercial formulations.

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Abstract

A validated UHPLC-PDA with an ESI-MS/MS method has been developed for simultaneous estimation of six bioactive alkaloids (magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine) in the different extracts of the roots of Berberis aristata DC (Family:Berberdiaceae). It is an important medicinal herb native to Northern Himalaya and commonly known as ‘daruharidra’, ‘daruhaldi’, ‘Indian barberry’ or ‘tree turmeric’. An insight into the research literature uncovered reports on isoquinoline alkaloids like magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine, and berberine as major bioactives in B. aristata roots, possessing different pharmacological and therapeutic effects. In the present study, these aforementioned alkaloids were separated on Phenomenex Luna®, 5 µm-C8 analytical column. The HPLC-MS analysis was performed at a flow rate of 0.90 mL min−1. Each alkaloid that is resolved was characterized by precursor ions and fragment ions with electrospray ionization (ESI) source in both positive and negative ionization using scan mode. The limit of detections (LODs) were 0.087, 0.727, 0.035, 0.124, 0.782 and 0.794 μg mL−1 for magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine, respectively. The proposed UHPLC-PDA method was fully validated according to international (ICH) guidelines and was found to be selective, sensitive and highly accurate for the concomitant estimation of the aforementioned symbolic bio-markers of B. aristata roots.

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