Kedar Kumar Rout

Estimation of piperine in commercial Ayurvedic formulations

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 F254, 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 RF 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.

Antidiabetic effect of Streblus asper in streptozotocin-induced diabetic rats.

Context: In the Indian traditional system of medicine, Streblus asper Lour (Moraceae) is prescribed for the treatment of diabetes mellitus. Objective: In the present study, α-amyrin acetate isolated from S. asper, and the petroleum ether extract of S. asper stem bark (PESA) was screened for their antidiabetic properties in streptozotocin (STZ)-induced diabetic rats. Materials and methods: Successive Soxhlet extraction of the dried stem bark with petroleum ether and then with ethanol (95%) yielded petroleum ether and ethanol extracts, respectively, which were concentrated under reduced pressure. Hyperglycemia was induced in rats by STZ (50 mg/kg, b.w.). Twenty-four hours after STZ induction, respective groups of diabetic rats received PESA (100, 250 and 500 mg/kg, b.w.) and α-amyrin acetate (25, 50 and 75 mg/kg, b.w.) respectively, orally daily for 15 days. Glibenclamide (0.5 mg/kg, orally) served as a reference. Blood glucose levels were measured on every 5th day during the 15 days of treatment. The serum lipid profiles and biochemical parameters, viz., serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), insulin and glycosylated hemoglobin level, were measured. Results: PESA significantly (p < 0.01) normalized blood-glucose levels and serum biochemical parameters as compared with those of STZ controls. α-Amyrin acetate (75 mg/kg, b.w.) exhibited maximum glucose lowering effect (71.10%) in diabetic rats compared to the other dose (25, 50 mg/kg) at the end of the study. The protective effect was further confirmed by histopathological examination of the liver. Conclusion: PESA and α-amyrin acetate demonstrated remarkable antidiabetic activity in STZ-induced diabetic rats.

Simultaneous quantification of two bioactive lupane triterpenoids from Diospyros melanoxylon stem bark

A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of two bioactive lupane triterpenoids, namely, lupeol and betulin from Diospyros melanoxyon stem bark. Chromatographic separation was achieved on aluminium foil-backed HPTLC plates using ethyl acetate-hexane (1.8:8.2, v/v) as mobile phase. The compounds were quantified at their wave length of maximum absorbance in the range of 100–500 ng per spot. The instrumental precision was 0.82% and 1.07% (CV) and the repeatability of the method was 1.33% and 1.17% (CV), respectively, for lupeol and betulin. The minimum detectable amount was found to be 40 and 50 ng per spot for lupeol and betulin, respectively. The linear regression analysis data for the calibration plots showed a good linear relationship with r2 = 0.9996 for lupeol and 0.9997 for betulin. The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise, and has been successfully applied for the assay of these bioactive molecules in D. melanoxylon stem bark.

Efficient and sensitive method for quantitative analysis of 6-gingerol in marketed Ayurvedic formulation

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 F254, 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.

Bioassay Guided Fractionation and Hepatoprotective Activity of Oleanolic Acid Acetate Isolated from Vitex negundo Linn.

Abstract Hepatoprotective activity of dichloromethane-methanol extracts of Vitex negundo (Linn.) roots was tested against CCl -induced hepatotoxicity in rats in vivo by bioassay guided fractionation. The extract was fractionated by column chromatography and the resulting fractions were analyzed for their hepatoprotective action in rat liver explant cultures in-vitro. The active fraction was further purified by chromatographic method. The 1H & 13C NMR, IR and mass spectral studies of the isolated compound suggested its pentacyclic triterpenic nature, characterized as oleanolic acid acetate, was evaluated for its in vitro hepatoprotective activity. Dichloromethane-methanol extract of Vitex negundo (Linn) root showed a dose dependent in vivo hepatoprotective activity. Treatment of CCl -intoxcicated liver explant culture sets (in vitro) with oleanolic acid acetate at a concentration of 3.3 mg/ml attenuated the marker enzymes (GOT: 27.8±0.73 IU/l; GPT: 17.5±0.09 IU/l and ALP: 16.1±0.66 IU/l) level in culture supernatants significantly (P<0.001) as compared to the toxic. The study revealed significant anti-hepatoxic potential of Vitex negundo (Linn) roots extract and isolated oleanolic acid acetate.

Development of a Sensitive HPTLC Method for Quantification of Nimbolide in Azadirachta indica and Its Dosage Form

An improved and sensitive High Performance Thin-Layer Chromatography (HPTLC) method has been developed for determination of anticancer compound, nimbolide in different parts of Azadirachta indica and its dosage form. A quick and simple ultrasonication technique was used for the preparation of sample solutions. Separation of the components was achieved on precoated TLC plates by using optimized tertiary mobile phase consisting of n-hexane:ethyl acetate:acetic acid (6:4:0.2, v/v/v) with a solvent migration distance of 68 mm. Densitometric quantification was performed after derivatization of the plate with methanol-sulfuric acid reagent in reflection/absorption mode at 515 nm. A linear response of nimbolide was obtained over the range of 200-1400 ng/spot with a correlation coefficient of r(2) = 0.99968, indicating good relationship between concentration and peak area. The method sensitivity was found to be increased by performing the analysis after derivatization with methanol-sulfuric acid reagent. The limit of detection and limit of quantification were found to be 70 and 200 ng/spot, respectively. The obtained recovery range from 96.70 to 98.01% with an average of 97.46% proves excellent accuracy of the method. ICH protocol was followed for validation of the HPTLC method in terms of precision, repeatability and accuracy. The developed method was found to be highly sensitive and the mobile phase efficiently separated nimbolide from other components. The maximum content of nimbolide was found in leaves. Further, the developed HPTLC method can be applied successfully for the marker evaluation of the formulations containing A. indica.

Standardization of Ayurvedic formulations containing Aloe vera by quantification of a marker compound

HPTLC has been used for quantitative analysis of aloin, as marker compound, in commercial Ayurvedic preparations, in the solid dosage form, containing Aloe vera Linn as major herbal ingredient. Chromatography was performed on aluminum plates coated with 0.2-mm layers of silica gel 60 F254, with ethyl acetate—methanol—water 10:1.4:1 (v/v) as ternary mobile phase after saturation at 30 ± 4°C for 5–7 min. The development distance was 70 mm. Aloin was quantified at 360 nm, its wave length of maximum absorbance. The limits of detection and quantification were 10 and 20 ng per band, respectively. Regression analysis of the calibration data revealed a good linear relationship (r = 0.9998) between peak area response and concentration in the range 20 to 100 ng per band. Instrumental precision and the repeatability of the method were 0.44% and 0.89% (CV), respectively. The accuracy of the method, determined by measurement of recovery at three different levels, was in the range 98.13 to 99.75%. These results are indicative of the excellent reliability, reproducibility, accuracy, and precision of the method. The method has been successfully used for analysis of aloin in commercial formulations containing Aloe vera.

Development and validation of HPTLC method for quantification of the antidiabetic compound α-amyrin acetate in Streblus asper Lour

The bioactive molecule, α-amyrin acetate, was isolated from the n-hexane extract Streblus asper stem bark by column chromatography. The structure of the compound was characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for its quantification in S. asper leaf, stem bark, and root. Chromatographic separation of the compound was achieved on high-performance precoated TLC plates by using optimized binary mobile phase consisting of n-hexane-ethyl acetate (9.6:0.4, v/v). HPTLC scanning was performed at 472 nm after derivatization of the plate with methanol-sulphuric acid reagent in reflection/absorption mode. The method was validated according to International Conference on Harmonization (ICH) protocol for specificity, sensitivity, linearity, accuracy, precision, repeatability, and robustness. The developed method is found to be very simple, rapid, precise, sensitive, and accurate for the quantification of α-amyrin acetate in S. asper.

HPTLC Quantification and Antimicrobial Activity of Ursolic Acid from Diospyros melanoxylon

An important bioactive molecule, ursolic acid was isolated from the leaves of Diospyros melanoxylon and characterized with help of physical and spectroscopic data viz. m. p, IR, 1H, and 13C NMR. A high-performance thin-layer chromatography method has also been developed and validated for its quantification in D. melanoxylon leaves. The high-performance thin-layer chromatography analysis was performed on high performance thin-layer chromatography plates using chloroform-methanol (9.5:0.5, v/v) as mobile phase. The compound was quantified at 540 nm after derivatzation with sulphuric acid reagent. The sensitivity of the method with respect to limit of detection and limit of quantification were found to be 20 and 40 ng per spot. The response was obtained as a linear function of peak area and concentration in the range of 50 to 450 ng per spot with correlation coefficient of r2 = 0.9998. The method showed excellent accuracy greater than 97.54% with acceptable precession and was successfully validated according to International Conference of Harmonization protocols. Antimicrobial screenings of ursolic acid revealed potent activity against two Gram-positive bacteria viz. Staphylococcus aureus and Enterococcus faecalis and three fungal starins viz. Aspergillus niger, Candida tropicalis, and Candida albicans.