In vitro and ex vivo approach for anti-urolithiatic potential of bioactive fractions of gokhru with simultaneous HPLC analysis of six major metabolites and their exploration in rat plasma
Authors
Ikshit Sharma
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard (Hamdard University), New Delhi, India
Washim Khan
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard (Hamdard University), New Delhi, India
Sayeed Ahmad
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard (Hamdard University), New Delhi, India
Context: Tribulus terrestris L. (Zygophyllaceae) fruits have long been used in traditional systems of medicine for the treatment of various urinary diseases including urolithiasis.
Objective: To explore the anti-urolithiatic potential of gokhru and to develop an analytical method for quantitative estimation of metabolites for its quality control.
Materials and methods: Aqueous extract of gokhru fruit was prepared through maceration followed by decoction to produce a mother extract, which was further used for polarity-based fractionations. In vitro and ex vivo anti-urolithiatic activity of mother extract and fractions at different concentration (100–1000 μg/mL) were carried out using aggregation assay in synthetic urine and in rat plasma, however, nucleation assay for 30 min was done using confocal microscopy. A simultaneous HPLC method has been developed for quantification of diosgenin, catechin, rutin, gallic acid, tannic acid and quercetin in mother extract and in fractions.
Results: The extraction resulted in 14.5% of w/w mother extract, however, polarity-based fractionation yielded 2.1, 2.6, 1.5, 1.3 and 6.1% w/w of hexane, toluene, dichloromethane (DCM), n-butanol and water fractions, respectively. In vitro and ex vivo studies showed a significant anti-urolithiatic potential of n-butanol fraction. Further, HPLC analysis revealed significantly (p < 0.01) higher content of quercetin (1.95 ± 0.41% w/w), diosgenin (12.75 ± 0.18% w/w) and tannic acid (9.81 ± 0.47% w/w) in n-butanol fraction as compared to others fractions.
Discussion and conclusion: In vitro and ex vivo studies demonstrated potent anti-urolithiatic activity of n-butanol fraction which can be developed as new phytopharmaceuticals for urolithiasis. HPLC method can be used for quality control and pharmacokinetic studies of gokhru.
Keywords: Tribulus terrestris, fractionations, aggregation, turbidity, nucleation, HPLC quantification
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