Biochemical and medicinal evaluation of Trifolium repens

Abstract

Trifolium repens is also known as white clover, white trefoil, Dutch clover, creeping Trifolium, honeysuckle clover or ladino clover. It contains a high amount of rich sources of proteins and minerals. Valuation of the innovative therapeutic bioactive potential Trifolium repens derived extracts seems to have a strong tendency in experimental phytopharmacological research in a variety of treatments. The low cost of cultivation, the variety of different bioactive potential compounds and prevalent occurrence are advantages of Trifolium species. In this dissertation, different assays were performed on Trifolium repens leaves extract and their biological effects were studied. Trifolium repens L. leaves (Tr.Cme) extract was investigated for the bioactive secondary metabolites in the methanolic extract particularly phenolic antioxidants using LCHRMS & LC-DAD and evaluate its anticholinesterase and antioxidant activities. Tr.Cme and subsequent fractions, chloroform (Tr.Chf), ethyl acetate, Tr.Et, n-hexane (Tr.Hex), ethanol (Tr.Et) and aqueous (Tr.Aq) were tested for cholinesterase, α-glucosidase inhibitory and antioxidant potentials. A total number of 29 phenolic compounds were quantified and identified. The highest amount present was of tyrosol, quercetin-3-glucuronide, formononetin-7-glucoside, quercetin-3-O-glucoside, 3,4-di-O-caffeoylquinic acid, formononetin-7-glucoside-acetate, quercetin-3-O-glucoside, and formononetin. Among the tested fractions, Tr.Chf and Tr.Et were most active against AChE, BChE and free radicals. The current results of anti-cholinesterase and antiradical studies on T. repens further signify its potential application in neurodegenerative disorders. The aqueous extract of Trifolium repens (TR) leaves was analyzed for the phenolic profile using reversed phased HPLC-DAD and administered to mice against acetaminopheninduced hepatoxicity. Twenty-four phenolic compounds were reported. The highest amounts present were of kaempferol-3-(caffeoyldiglucoside)-7-glucoside, followed by p-coumaroyl-4- glucoside and daidzein-O-sulfate. 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 b 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 Trifolium repens leaves was administered to mice in different doses along with paracetamol. Paracetamol was found to significantly affected kidney weight, serum biochemistry of renal profile, serum electrolytes, reduced glutathione, and thiobarbituric acid reactive substances. These parameters were normalized by the coadministration of TR aqueous extract. Kidney histopathological examination revealed that paracetamol significantly induced nephrotoxicity, while the phenolic compounds present in TR extract act as therapeutic agents. Reduced glutathione and lipid peroxidation in the kidney were normalized by the administration of TR extract. The TR extract possesses a source of beneficial active phenolic compounds with nephroprotective function. The leaves of Trifolium repens were analyzed for carotenoids and chlorophylls composition and variations under the influence of different irrigated water such as well water, river, rain, lake, and spring waters. Correlations between samples and carotenoid compounds were determined by principal components analyses. Results revealed that nineteen carotenoids compounds were identified and quantified in Trifolium repens leaves. Phytofluene, pheophytin b, divinyl chlorophyll a, all-E-neoxanthin, hydroxyl-pheophytin a, hydroxyl-pheophytin a', all-E-auroxanthin, pheophytin a, pheophytin a', all-E-lutein, 9-Zlutein, 9'-Z-lutein, 13-Z-violaxanthin, chlorophyll b, 9-Z-neoxanthin, chlorophyll a, pyropheophytin a, and all-E-β-carotene were identified compounds. Total carotenoid and pigment contents were higher in well water samples followed by spring water and then rainwater. Significant variations & correlation were noted in individual carotenoid & pigment using principal components analyses revealed the importance of irrigation water. Similarly, the phenolic profile of Trifolium repens grown on different water origins was studied. Results reveal that in well-water sample 24 phenolic compounds were detected in which quercetin-7-O-glucuronide followed by kaempferol-3-O-sophoroside, (−)- Epigallocatechin, delphinidin-3,5-O-diglucoside and genistein were present in high amounts followed by spring water irrigated samples. Springwater samples contain 22 phenolic compounds in which ellagic acid, (−)-epigallocatechin, kaempferol-7-O-glucoside, delphinidin-3,5-O-diglucoside, and petunidin-3-O-glucoside were major compounds. In rainwater samples, 18 phenolic compounds identified including tri-O-galloylquinic acid, (−)- c epigallocatechin, petunidin-3-O-glucoside, and formononetin-O-glucuronic acid as the major compounds. Nineteen phenolics compounds were identified in river water samples, where delphinidin-3,5-O-diglucoside, petunidin-3-O-glucoside, kaempferol-3-malonylglucoside and (−)-epigallocatechin were -identified. These results showed that irrigation water plays an important role in the presence of different bioactive compounds in Trifolium repens. This study revealed that Trifolium repens contain important bioactive compounds such as phenolics, flavonoids, flavones, and carotenoids. These compounds showed variations based on irrigation water. The identified compounds showed hepatoprotective, and nephron-protective functions in animals. The plant extracts also revealed good antioxidants and other medicinal properties.