Recovery of o-coumaric acid from Gliricidia sepium leaves using ultrasonic-assisted extraction for bioconversion of dicoumarol as potential rodenticide anticoagulant

Gliricidia sepium is briefly a leguminous tree known as ‘pokok bunga Jepun’ which widely grown in Malaysia for fencing and shade. They are often associated with various traditional uses and medicinal values such as anti-inflammatory, anti-bacterial and anti-oxidant but very little research has been done on extraction for rodenticides application using this local bark size-plant to naturally reduce numerous rodents’ related problems. This research aims to develop an alternative to the chemical rodenticides that mostly used in agricultural sector. The work focuses on the two main processes including recovery of o-coumaric acid from ultrasonic assisted extraction and bioconversion into dicoumarol as superior anticoagulant compound through aerobic fermentation. Different experiments were designed to investigate the effects of various operating parameters on quantitative and qualitative aspects of G. sepium leaf extract. The effect of drying temperature on the quality of G. sepium leaf and drying kinetics were evaluated for determination of the optimum drying temperatures. The optimum drying temperatures of G. sepium leaves was 50°C as o-coumaric acid was chemically degraded and decrease in concentration above this temperature. Midilli model was the best suit model among the selected thin layer models in predicting the drying process. Based on the optimization analysis using RSM, it was found that 36.14 ±0.015 mg L-1 o-coumaric acid was produced with the optimum extraction temperature of 47°C, extraction time of 45 min and ratio of solvent to solid as of 10:1 (mL g-1). The study of bioconversion of o-coumaric acid to dicoumarol was conducted using fungal strain of Aspergillus fumigatus Fresenius NRRL 163 through submerged culture system. The fermentation process was done using shake flasks under optimized conditions (defined media, initial carbon and nitrogen concentrations at 10 and 2 g L-1) with constant parameters (temperature of 55°C and agitation 150rpm) which resulted in maximum dicoumarol and cell concentration of 0.322 U L-1 and 9.33 g L-1 respectively. Dicoumarol was chromatographically identified using HPLC with retention time of 2.29 minutes, characterized using FTIR and its molecular ions were confirmed using LCMS/MS as 336.6 m/z. The rodenticide anticoagulant activities were proven through in vitro anticoagulant assays using Activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT) tests in which the dicoumarol fraction demonstrated its potential anticoagulant activity with its ability to lengthen the coagulation time in aPTT test 43.8% higher than crude extract. The bioconversion process converting o-coumaric acid from G. sepium leaf extract into dicoumarol bioactive compound was successfully achieved indicating the plant potential as a source of natural rodenticide anticoagulant.

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