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The aqueous extract of Trifolium repens (TR) leaves was analyzed for the phenolic profile using reversed phase HPLC‐DAD and administered to mice against acetaminophen‐induced hepatoxicity. Twenty‐four phenolic compounds were identified and quantified. The highest amounts present were of kaempferol‐3‐(caffeoyldiglucoside)‐7‐glucoside (983.7 µg/ml), followed by p‐coumaroyl‐4‐glucoside (905.6 µg/ml) and daidzein‐ O ‐sulfate (808.3 µg/ml). The aqueous extract was administered to mice along with acetaminophen at different doses. Acetaminophen was found to significantly alter body weight, serum biochemistry, and hematological indices of mice, which were ameliorated by the co‐administration of aqueous extract. Liver histopathological studies revealed that acetaminophen significantly induced toxicity, while TR aqueous extract provides curative functions. Lipid peroxidation and total reduced glutathione in the liver were also normalized by the aqueous extract of TR. The aqueous extract of TR was rich in important phenolic compounds, which can be used as a source of beneficial bioactive compounds with hepato‐protective function. Practical applications Acetaminophen has been widely used as antipyretic and analgesic. However, the major complication reported is hepatotoxicity. Synthetic or conventional drugs used for hepatic diseases or against hepatotoxicity are insufficient and causes severe side effects. For this purpose, traditional medicinal plants or nutraceuticals are used to decrease in the side effects of different hepatotoxic medicine are demanding. Food and neutraceuticals are rich in important polyphenolic compounds which are the best antioxidants. This study was aimed to evaluate the phenolic composition of aqueous extract of Trifolium repens and its potential protective action against the acetaminophen‐induced toxicity in mice. This study showed for the first time that the aqueous extract of TR was protective against the hepatotoxicity induced by acetaminophen.
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