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
Linn. Chromatography was performed on aluminum foil HPTLC plates coated with 0.2 mm layers of silica gel F
, with hexane-acetone 6.5: 3.5 (
) 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
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.
A high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous analysis of eugenol and piperine in the Siddha formulation Amukkara choornam. TLC was performed on aluminum foil-backed silica gel 60F
plates with toluene-ethyl acetate 9:3 as mobile phase. Calibration plots for both eugenol and piperine were linear in the range 1–12 μg. The polynomial regression data for the calibration plots were indicative of good linear relationships with
= 0.9979 and 0.9980 for eugenol and piperine respectively. The
values of eugenol and piperine were 0.73 ± 0.03 and 0.28 ± 0.01, respectively. The method was validated for sensitivity, accuracy, precision, and robustness. Minimum detectable amounts were 0.121 ng per band for eugenol and 0.0102 μg per band for piperine. The eugenol and piperine content of the formulation were 0.207 μg g
and 1.992 mg g
, respectively. Recovery of eugenol and piperine was greater than 97%. The method enabled simple, sensitive, precise, accurate, and specific analysis of eugenol and piperine in the formulation.
Authors:Anagha Rajopadhye, Anuradha Upadhye, and Arvind Mujumdar
Piperine, an alkaloid with diverse biological activity commonly occurring in fruits of Piper sp., has high commercial, economical, and medicinal value. In this communication densitometric estimation of piperine from fruits of Piper nigrum L., processed Piper nigrum L. (white pepper), Piper longum L., Piper retrofractum Vahl, Piper cubeba Hunter, and Piper betle L. is reported. Extracts of these fruits and a standard solution of piperine were applied to silica gel F254 HPTLC plates and the plates were developed in a twin-trough chamber with toluene-ethyl acetate-diethyl ether 6:3:1 as mobile phase. The plates were scanned at 337 nm and quantification of piperine was based on a predetermined calibration plot obtained by chromatography of 15 to 75 ng standard. The quantity of piperine was highest in fruits of Piper nigrum L. and least in Piper betle L. The trend was Piper nigrum L. > Piper longum L. > processed Piper nigrum L. > Piper retrofractum Vahl > Piper cubeba Hunter > Piper betle L. The HPTLC method was found to enable simple, convenient, rapid screening and quantification of the active marker piperine in six fruits of Piper sp. commonly used in the Indian traditional system of medicine.
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
plates with toluene-ethyl acetate-methanol, 9 + 1 + 0.5 (
), 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
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
, 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.
Authors:Kilambi Pundarikakshudu, Anilkumar Sharma, Chaitanya Bhatt, and Niranjan Kanaki
The fruit of Piper longum Linn. (family: Piperaceae), known as pippali in India, is a reputed drug of Ayurveda (the Indian system of medicine). It contains many amides of piperic acid among which piperine and piperlongumine are important from biological point of view. In the present work, we quantified these two marker compounds from the fruits of P. longum by high-performance thin-layer chromatography (HPTLC) with densitometry. The method was found to be precise. Relative standard deviation (RSD) values for intra-day analyses were in the range of 1.05 to1.07% (piperine) and 1.12 to 1.16% (piperlongumine) and, for inter-day analyses, the values were in the range of 1.11 to 1.21% (piperine) and 1.02 to1.18% (piperlongumine). Instrumental RSD values were 0.64 and 0.78% for piperine and piperlongumine, respectively. Accuracy of the method was evaluated by carrying a recovery study at three different levels for the two compounds. Average recoveries were found to be 99.23% for piperine and 99.26% for piperlongumine. P. longum fruits obtained from Bombay market showed 0.213 ± 0.009% w/w piperine and 0.364 ± 0.014% w/w piperlongumine when analyzed by the proposed method. Similarly, sample fruits obtained from Ahmedabad market had 0.85 ± 0.026% piperine and 2.70 ± 0.065% of piperlongumine. This TLC-densitometric method was found to be simple, precise, specific, sensitive, and accurate. So it can be used in routine quality control for the simultaneous analysis of piperine and piperlongumine from P. longum fruits and piperine alone from Piper nigrum fruits.
In this article, a simple and sensitive stress-induced high-performance thin-layer chromatography (HPTLC) method is proposed for the quantitative analysis of piperine (a bioactive marker) in the alcoholic extracts of five different marketed powders (P1–P5) and dried seeds (P6) of black pepper (Piper nigrum). HPTLC was performed on glass-backed silica gel 60 F254 HPTLC plates with toluene, ethyl acetate, and glacial acetic acid (7:3:0.5; v/v) as the mobile phase. Scanning and quantification of the developed HPTLC plate (λ = 280 nm) furnished compact and intense spots of piperine at RF = 0.40 ± 0.01. For the proposed method, the estimated regression equation (Y) and r2 were 9.473X + 2262.23 and 0.997, respectively, in the range of 100–900 ng, the limit of detection (LOD) and limit of quantification (LOQ) were 19.63 and 59.47 ng, respectively. The quantified piperine values (% w/w) in P1, P2, P3, P4, P5, and P6 were 1.75, 2.71, 1.21, 1.67, 2.45, and 1.80, respectively. In the stress study, the treatment of piperine with acid, alkali, and hydrogen peroxide showed its complete degradation. On the other hand, there was no effect of dry or moist heat, and photochemical and ultraviolet light (λ = 254 nm) on the stability of piperine. The present maiden report on a validated HPTLC method for the stress induced analysis of piperine, therefore, would help in the selection of good-quality marketed products. Furthermore, the developed method may help in selecting or rejecting the in-process procedural conditions for making the products and formulations more efficacious and safe.
Authors:Subrata De, Pankaj Nariya, and Nikhil Jirankalgikar
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.
Authors:Anilkumar S. Sharma, Chaitanya J. Bhatt, and Kilambi Pundarikakshudu
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.