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A simple, sensitive, and rapid high-performance thin layer chromatographic (HPTLC) method has been established for estimation of piperine in commercial Ayurvedic formulations and in the fruits of Piper nigrum Linn, and Piper longum Linn. Chromatography was performed on aluminum foil HPTLC plates coated with 0.2 mm layers of silica gel F 254 , with hexane-acetone 6.5: 3.5 ( v/v ) as mobile phase. The development distance was 76 mm, the temperature 25 ± 5°C, and the chamber was saturated for 5 min. Piperine was quantified at 340 nm, its wavelength of maximum absorbance. Under the conditions used the R F of piperine was 0.33 and the limit of detection (LOD) was 4 ng per zone. The calibration plot was linear in the range of 10 to 60 ng per zone with a correlation coefficient of 0.9996. Recovery was in the range 98.76 to 100.70%. This HPTLC method was found to be reproducible, accurate, and precise and could be used to detect piperine at nanogram levels. The method is a very simple and cost-effective means of quantitative estimation of piperine in Ayurvedic formulations.

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A simple, precise, rapid, selective, and cost-effective high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous estimation of curcumin, piperine, and thymol in an ayurvedic formulation. Chromatography was performed on silica gel 60 F 254 plates with toluene-ethyl acetate-methanol, 9 + 1 + 0.5 ( v/v ), as mobile phase. Plates were developed to a distance of 8 cm at room temperature, without chamber saturation. The plates were scanned and the compounds were quantified at their wavelengths of maximum absorption, 420, 333, and 277 nm for curcumin, piperine, and thymol, respectively. The respective R F values of curcumin, piperine, and thymol were 0.23, 0.30, and 0.64. Response was a linear function of the amount applied to the plate in the ranges 50–250 ng, 10–60 ng, and 100–700 ng for curcumin, piperine, and thymol, respectively. LOD for curcumin, piperine, and thymol were 25, 5, and 50 ng, respectively. The mean results from assay of curcumin, piperine, and thymol in the ayurvedic formulation were found to be 0.85, 12.93, and 3.29 mg g −1 , respectively. The respective covariance for curcumin, piperine, and thymol was 0.78, 0.51, and 0.69%, respectively. Recovery was 100.41, 99.52, and 101.21% for curcumin, piperine and thymol, respectively. Rapid identification of curcumin, piperine, and thymol is also possible by spraying the plate with anisaldehyde in sulfuric acid reagent.

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An efficient, sensitive, and precise high-performance thin-layer chromatographic (HPTLC) method has been established for analysis of 6-gingerol in marketed Ayurvedic formulations and in the rhizomes of different varieties of Zingiber officinale . HPTLC separation was performed on aluminum foil-backed HPTLC plates coated with 0.2-mm layers of silica gel 60 F 254 , with n -hexane-acetone, 7.2:2.8 ( v/v ) as mobile phase. Plates were developed to a distance of 78 mm at 20 ± 4°C in a chamber previously saturated for 4 min. Under these condition the retention factor ( RF ) of 6-gingerol was 0.23 and the compound was quantified at 286 nm, its wavelength of maximum absorbance. The limits of detection and quantification were 40 and 150 ng per band, respectively. Response to 6-gingerol was a linear function of amount over the range 150 to 900 ng per band; the correlation coefficient was 0.9997, indicating a good relationship between peak area and amount. Recovery from 98.46 to 101.11% showed the accuracy of the method was excellent. The method is very accurate, simple, and cost effective, and enables sensitive quantitative analysis of 6-gingerol.

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, Dept. of Health, Govt. of India, 1978, pp. 178. Pharmacopoeial Standards of Ayurvedic Formulations, revised edn., Central Council for Research in Ayurveda & Siddha, Govt. of India, New Delhi, 1978, pp. 420

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Abstract  

Trikatu, an Ayurvedic formulation of three dried powder spices, ginger, black pepper and pipali in equal proportion is widely used to promote digestion, assimilation and bioavailibility of food. It works synergistically, and hence, is more effective than an equal amount of any of its three ingredients taken separately. Five different brands and its three constituents were analyzed for 31 elements by instrumental neutron activation analysis (INAA) using 5-minute and 6-hour thermal neutron irradiation followed by high-resolution γ-ray spectrometry. Heavy toxic metals Cd, Ni and Pb determined by atomic absorption spectrometry (AAS) were found below permissible limits. Most elements in different brands vary in a narrow range. Ginger is particularly enriched in Ca, Fe, Mg and Mn whereas black pepper is enriched in Cr, Se, P and Zn. Cu/Zn shows linear relationship (r = 0.92) with Cu whereas Fe and Mn exhibit inverse correlation (r = −0.89) in different brands. Hydro distillation of pipali yielded an essential oil whereby 10 organic constituents were identified by GC-MS. Also barbituric and tannic acids were isolated from the aqueous methanolic extract of pipali.

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The separation and estimation of natural products by chromatographic techniques such as high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) are widely preferred. The present work reveals the quantification of morphine (a regulated narcotic agent) in the Ayurvedic formulation ̀Kamini Vidrawan Raś using reversed-phase HPLC (RP-HPLC). The results obtained herein were compared with the results of earlier reported work using HPTLC. The HPTLC separation of morphine was performed on an aluminum-backed layer of silica gel 60 F254 using ethyl acetate–methanol–ammonia solution as the mobile phase, while RP-HPLC was performed on Kromasil C8-column (150 mm × 4.6 mm, 5 µm) using a mobile phase comprising of N-heptane sulfonate sodium salt–acetonitrile (70:30, v/v) at a flow rate of 1.5 mL min-1. The International Conference on Harmonisation (ICH) guidelines were followed for validation of both the chromatographic methods. Both the developed chromatographic methods are simple, rapid, accurate, and sensitive.

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Summary

In this chapter, an isocratic reverse phase HPLC determination of mimosine was developed and validated in an anti-psoriatic topically applied formulation “Lajjalu”. The chromatography was performed on a C18 column with water-orthophosphoric acid (98.8:0.2, υ/υ) as a mobile phase with a pH of 3.0 at a flow rate of 1.0 mL min−1. Detection was performed at 284 nm, and a sharp peak was obtained for mimosine at a retention time of 2.62 ± 0.01 min. Linear regression analysis data for the calibration plot showed a good linear relationship between response curve and concentration in the range of 0.050–5000 ng mL−1 and the regression coefficient was 0.9998 with the linear regression equation y = 4766.8x−17726. The detection (LOD) and quantification (LOQ) limits were 10.3 and 35.6 ng mL−1, respectively. The wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase imply the method is suitable for routine quantification of mimosine with high precision and accuracy.

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Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and Cosmetics Act of India. Its usefulness has been stated in erectile dysfunction, impotence, and premature ejaculation. Opium is the major ingredient of formulation which is highly addictive as it contains narcotic alkaloids (morphine, codeine, and thebaine). Opium is a natural product; hence, the morphine content varies from 4–21%. The aim of this study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography— densitometry method for the quantitative estimation of morphine in the tablets of the said Ayurvedic medicines. Aluminum-backed silica gel F254 (20 cm × 10 cm) was used as the stationary phase and ethyl acetate, methanol, and ammonia (85:10:5, v/v) as the mobile phase. The R F value for morphine was 0.36, and the quantitative evaluation of the bands over plates was performed in the reflectance—absorbance mode at 280 nm. The regression analysis of the calibration plot showed good linear relationship between peak area vs. morphine concentration. Linearity was found in the range of 400 to 1200 ng per band, and the amount of morphine was estimated by comparing the peak area of the standard morphine.

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Trikatu Churna is an important formulation in Ayurveda — the Traditional System of Indian Medicine. It consists of fine powders of fruits of Piper nigrum L., Piper longum L., and rhizomes of Zingiber officinale Roscoe in equal proportions. Piperine, present in both P. nigrum and P. longum, is considered to be responsible for the improvement of digestion and bioavailability enhancement of many medicaments. Gingerols and 6-shogaol are key chemical molecules in Z. officinale. Piperlongumine is present in P. longum fruits but absent in the fruits of P. nigrum. We report a validated high-performance thin-layer chromatography (HPTLC) method for the determination of piperine, piperlongumine, and 6-shogaol in these herbs and in Trikatu Churna. Piperine, piperlongumine, and 6-shogaol resolved well in n-hexane—ethyl acetate (8:2) on precoated silica gel 60 F254 plates. The absorption maxima for piperine, piperlongumine, and 6-shogaol were found to be 327, 272 and 235 nm, respectively. Linearity for the corresponding markers was observed between 0.1–0.5, 0.2–1.0, and 0.1–1.6 μg spot−1, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were 28 and 100, 56 and 200, and 32 and 100 ng for piperine, piperlongumine, and 6-shogaol, respectively. Recovery experiments showed 99.6%, 99.5%, and 99.7% recoveries for piperine, piperlongumine, and 6-shogaol, respectively. P. nigrum fruits from Delhi and Ahmedabad had around 2.0% w/w piperine, while fruits of P. longum from these markets were analyzed for 0.8% and 0.6% w/w piperine. Piperlongumine was not found in both samples of P. nigrum, while the fruits of P. longum had 0.36% and 0.26% w/w piperlongumine. Z. officinale from Delhi had 0.19% w/w of 6-shogaol as against 0.16% w/w found in the sample from Ahmedabad. Plant materials procured from Delhi were employed for the preparation of Trikatu Churna which showed 96.5%, 95%, and 103% w/w of the expected values of piperine, piperlongumine, and 6-shogaol, respectively. The present method is simple, reproducible, and reliable which can be applied for the routine analysis of Trikatu Churna and its ingredients in polyherbal formulations.

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Piperine and gallic acid are of different chemical natures — piperine is an alkaloid, while gallic acid a phenolic compound. They are used as marker compounds in many plant-based formulations. A highperformance thin-layer chromatographic (HPTLC)-densitometric method has been developed and validated for the simultaneous quantification of piperine and gallic acid as such and in pharmaceutical dosage forms. Toluene-ethyl acetate (3:7) was used as mobile phase and scanning was done at 254 and 340 nm. The method was validated with respect to linearity, reproducibility, specificity, accuracy, precision, robustness, and ruggedness. Both compounds showed good linearities in the range of 250–1750 ng. LOD and LOQ for piperine were 9.98 and 33.29 ng, while for gallic acid 25 and 83.33 ng. Average % RSD values of precision for piperine and gallic acid were 0.46% and 0.72%, respectively. % Recovery was 96–103%. The method is accurate, reproducible, cost-effective, and can be used in routine analysis.

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