Effects of Picrorhiza kurroa (Katuki rhizome) on Antioxidant Activity:

Effects of Picrorhiza kurroa (Katuki rhizome) on Antioxidant Activity: A clinical study

The present study was carried out to investigate the efficacy and safety of the Effects of Picrorhiza kurroa (Katuki rhizome) on Antioxidant Activity

Treatment Offered

The fresh leaves of P. kurroa were dried and used in this investigation were collected in the months of August and September 2011, from Holi-Nala village, district Chamba situated at an elevation of 2800-3200 m above sea level located in the mid-hills of the Western Himalayas (Dhauladhar range). The plant material was authenticated in-house by a taxonomist of the institute and the voucher specimen deposited in the herbarium of the CSIR-IHBT, Palampur, India (voucher # PLP 11694). 2,2€²-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2€²-diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid were purchased from Sigma-Aldrich Chemie, Steinheim, Germany. HPLC grade solvents (acetonitrile and water) were purchased from J. T. Baker, USA. All other solvents and chemicals were of analytical grade and obtained from S. D. Fine-Chem. Ltd., Mumbai, India.

Extraction, fractionation, and isolation:

The fresh plant material was dried at 40±5° and crushed properly. One kilogram of powdered material was extracted with ethanol: water (95:5, v/v) (1Ã—7 l, 1Ã—3.5 l, 6Ã—1 l). The ethanol solutions were combined and dried in a rota evaporator at 40±5° (130 g). The crude ethanol extract (100 g) was suspended in water and successively extracted with hexane (3Ã—250 ml), chloroform (3Ã—250 ml), ethyl acetate (3Ã—250 ml), and n-butanol (3Ã—250 ml) and evaporation of the solvents at reduced pressure gave 6.6 g of n-hexane, 7.0 g of chloroform, 1.5 g of ethyl acetate and 23.2 g of n-butanol extract. These extracts were lyophilized and kept in the dark at +4° until tested. Isolation of compounds through column chromatography was started by using 20.0 g of extract from the mixed obtained fractions of n-butanol. A slurry of the extract was prepared by dissolving it in a minimum volume of methanol followed by adsorbing the extract over silica gel (mesh size 230-400, 20.0 g). The slurry of the extract was uniformly packed over a dry silica gel column (mesh size 230-400, 455.0 g, 35Ã—7.0 cm) for column chromatography. Elution of components was started through column chromatography using an isocratic solvent system (ethyl acetate:chloroform:methanol: water; 15:8:2:0.5). Fractions of 100 ml each were collected in a conical flask. TLC (silica gel F₂₅₄) of all individual fractions were developed using a solvent system (ethyl acetate:chloroform: methanol: water; 15:8:4:1; 12:8:8:2) and then viewed under UV chamber at both the wavelengths (254 and 366 nm) followed by spraying with iodine in iodine chamber and finally sprayed with vanillin sulfuric acid as visualizing reagent. Based on the TLC profile of the fractions, similar fractions were pooled and then dried in rotavapor under reduced pressure at a temperature of about 40±5°. After drying all the fractions, pooled fractions were obtained. Fraction no. 70-80 were precipitated to obtain luteolin-5-O-glucoside (compound 1, fig. 1a).

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Results

The structures of the isolated compounds were determined as luteolin-5-O-glucoside (compound 1, fig 1) and picein (compound 2, fig 1) with the help of NMR data, LC-MS/MS analysis, and also get compared with spectroscopic data of literature[21,22]. The content of picein in the different extracts of P. kurroa was quantified by the HPLC method. The optimum HPLC separation of picein was achieved using acetonitrile: water (10:90%, v/v). The HPLC chromatogram obtained is shown in fig 2. The retention time was 6.5 min for picein. Linearity was confirmed by the construction of a calibration curve. For this curve, standard solutions were prepared at eight concentrations, and chromatograms were recorded. The correlation coefficient obtained for picein was 0.9999 (Table 1). The linear range was 1.56-200 Î¼g/ml for picein. The limits of detection and limits of quantification were 2.34 and 7.81 Î¼g/ml, respectively. The picein content was determined to be 20.09, 10.68, and 10.63% in butanol, ethyl acetate, and ethanol extract, respectively. The total antioxidant activity of P. kurroa leaves extract was evaluated by two tests, DPPH and ABTS^·+ free radical. Their ability to scavenge those free radicals at different concentrations was analyzed. The percentage inhibition of absorbance was calculated and plotted as a function of the concentration of the extract and of standard. The results of IC₅₀ of DPPH and ABTS radical scavenging activity assays are shown in Table 2. The antioxidant capacity was expressed as IC₅₀, which is the concentration of an antioxidant needed to trap 50% of DPPH and ABTS absorbance. Consequently, a low IC₅₀ value indicates a high antioxidant capacity. The antioxidant activity of parent extract, different fractions, and isolated compounds of P. kurroa was determined by comparing the IC₅₀ values evaluated by DPPH and ABTS^·+assays. The IC₅₀ values of ascorbic acid, compound 1, butanol, ethyl acetate fractions, and ethanol extract determined by DPPH assay were 0.81, 1.04, 37.12, 39.58, and 67.48 Î¼g for 2.0 ml of 0.100 mM DPPH solution, respectively. The results demonstrated that fractions showed less antioxidant activity as compare to the standard (ascorbic acid) and isolated compound 1. Butanol fraction of P. kurroa showed maximum activity against DPPH and ABTS^·+free radical followed by ethyl acetate and ethanol fractions. The IC₅₀ values of ascorbic acid, compound 1, butanol, ethyl acetate fractions, and ethanol extract determined by ABTS assay were 2.59, 4.02, 29.48, 33.24, and 48.36 Î¼g for diluted ABTS (A=0.700±0.020) solution, respectively. However, isolated compound 2 (picein) was lacking with significant antioxidant activity in both assays. Both DPPH and ABTS assays showed the nearly identical antioxidant potential of fractions

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Conclusion

The antioxidant and radical-scavenging activity of parent extract, fractions, and isolated compound of P. kurroa leaves indicate its role toward various oxidative stress-related diseases, as a food supplement and source of natural antioxidants. This study discloses that the isolated compound 1, butanol, and ethyl acetate fractions found to be promising with antioxidant potential as compared to parent ethanol extract. Thus, isolated molecules and fractions can be considered for further detailed pharmacological studies to develop a new natural product for the treatment of oxidative stress-related diseases.

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