Toxicological studies of methiocarb in (in vivo and in vitro) models and protective effect of grape seed extracts

Mohammed Alsaif

Methiocarb is a pesticide that is widely used in the world in agriculture and biocontrol programs for many purposes. However, it is classified by WHO as a highly hazardous pesticide. Despite a variety of agricultural benefits, it has been pronounced as toxic to human and animals and harmful to environment. Grape seeds and pomace are waste materials generated from juice production and winemaking. They have a high concentration of phenolic compounds that have beneficial health properties such as antioxidant, antimicrobial and anti-inflammatory activities. This research investigated the role of polyphenolic compounds extracted from grape seeds in prevention and/or alleviation of the toxic effects of MC in cultured cells, earthworm and rat models. Grape seed extract (GSE) was prepared from defatted grape seed powder using ethanol/water (50:50 v/v) and compared with two commercial grape seed products (GC obtained by a proprietary process for water extracted grape seed extraction process and T which is a tannin-rich fraction prepared from grape seeds by a proprietary process). Four methods were used to test the antioxidant capacity of the three grape seed extracts, DPPH, ABTS, ORAC and FRAP assays. Also, the individual phenolic compounds were determined using high pressure liquid chromatography mass spectroscopy (HPLC-MS). A total of 17 phenolic compounds were identified in the three grape seed extract (GSE, GC and T) types. Significant differences (p < 0.05) in the total and individual phenolic compounds were detected among the three grape seed extracts. Accordingly, the antioxidant activities were also significantly variable among the three extracts. The GC extract exhibited the highest total phenolic concentration followed by T and GSE. GC and T extracts showed higher (p < 0.05) antioxidant activities than the GSE extract demonstrating a strong correlation between the phenolic compound concentration and antioxidant activity values. The MC was found to be toxic to L929 and HepG2 cells at concentrations of ≥15 and ≥31 µg/ml, respectively. The grape seed extract at concentration of 40 and 80 µg/ml were able to protect L929 and HepG2 cells against cytotoxicity induced by MC at different efficacy levels. Exposure of A. caliginosa earthworms to MC (0.125 mg/ml for 24 h) induced a significant decrease in antioxidant enzymes (SOD, CAT, and GPx) activities. The observed reduction in the antioxidant enzymes activities in the earthworms was prevented by the GC extract. Other extracts (GSE and T) had no significant effect. Treatment of rats with MC at a concentration of 10 mg/Kg bw for 10 days significantly (p < 0.05) decrease the rat body weight, activity of antioxidant enzymes (SOD, CAT, and GPx) and led to histopathological changes in the liver and kidney tissues. The three grape seed extracts partially prevented the damage induced by MC. Pre-treatment with the three grape seed extracts (100 mg/kg b.w. for 20 days) did not improve body weight loss. However, pre-treatment with GC extract restored all the antioxidant enzyme activities to the normal levels. Pre-treatment with T extract improved the activities of CAT and GPx enzymes significantly (p < 0.05) but not SOD enzyme. The treatment with GSE extract improved the activity of SOD and GPx enzymes significantly but not the CAT enzyme. The GC extract alleviated protein oxidation induced by the MC in all three models (cells, earthworm, rat). The treatment with T and GSE extracts alleviated protein oxidation only in the rat model. The GC extract had the best protective effect against oxidative stress compared with the two other extracts (GSE and T). The results obtained from this study indicated that grape seed extracts from winery waste, exhibit strong antioxidant activities and can be used as a good source of natural antioxidants and could potentially be useful to reduce the toxic effects of MC. These biological activities appear to be dependent on the type of extraction system used to obtain the extracts.

Toxicological studies of methiocarb in (in vivo and in vitro) models and protective effect of grape seed extracts

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