Diuretic Activity of Ethanol Extract of Piper attenuatum Leaves Might Be Due to the Inhibition of Carbonic Anhydrase Enzyme:An in vivo and in silico Investigation

Background:Ayurvedic system of medicine is well established for prevention and treatment of renal problems.There are vast number of medicinal plants mentioned in Ayurvedic system of medicine,including Piper attenuatum Buch.-Ham.ex Miq.,which has been known to exhibit diuretic properties.Objective:To investigate possible mechanism and diuretic activity of ethanol extract of P.attenuatum leaves.Methods:Present study reports an in vivo diuretic activity of ethanol extracts of P.attenuatum leaves.Twenty-four Sprague-Dawley male rats were deprived from food and water for 20 h,followed by being divided into four groups to receive normal saline,Furosemide,and two doses of the ethanol extract,respectively.Further,the ethanol extract were subjected for prelimnary phytochemical screening test,and analytical investigation was performed via high-performance thin-layer chromatography(HPTLC),followed by effectiveness exploration of putative bioactive compounds from P.attenuatum against human carbonic anhydrase(hCA)enzyme using molecular docking tool.Results:During phytochemicals screening,several groups of compounds such as amides,terpenoids,proteins,flavonoids,and glycosides have been identified.Further,HPTLC reveals presence of carbohydrates,proteins,amino acids,steroids,alkaloids,flavonoids,tannins,glycosides,and terpenoids.Significant diuretic action was revealed for both 200 mg/kg bw and 400 mg/kg bw of ethanol extract of P.attenuatum leaves.Interestingly,molecular docking analysis demonstrated greater binding affinity of compounds such as Cepharadione A,Nor-cepharadione B,Galbelgin,Crotepoxide,and Pipoxide chlorohydrin out of total 23 studied compounds against hCA isoforms(a key biomarker for diuretic)and score is comparable with standard drug Acetazolamide.Conclusion:It was concluded from the present studies that ethanol extract of P.attenuatum leaves exhibits signifi-cant diuretic activity.The activity may be correlated due to the presence of Cepharadione A,Norcepharadione B,Galbelgin,Crotepoxide,and Pipoxide chlorohydrin as key component responsible for inhibition of hCA isoforms.