Phytochemical constituents and antioxidant activity of extract from Torilis leptophylla (L.)

Abstract

In recent years phytochemistry and pharmacognosy appear as a primary focus of research. Torilis leptophylla of family Apiaceae has been used in folk medicine for the treatment of gastrointestinal illnesses in many areas of Pakistan and also the plant is traditionally used for its antimicrobial and anticancer properties. The present work describes the biological and various pharmaco-clinical effects of extract/fractions of T. leptophylla, with special focus on its possibility to counteract oxidative damage and fibrosis. At first, shade dried whole plant of T. leptophylla was extracted with methanol and the residue (TLM) was suspended in water and partitioned successively with n-hexane (TLH), chloroform (TLC), ethyl acetate (TLE) and n-butanol (TLB) while the remaining portion after filtration was used as residual aqueous fraction (TLA). The extract and fractions were subjected to preliminary phytochemical screening. The results showed the existence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids in TLM. Presence of anthraquinones and phlobatannins were not recorded in all fractions. The total phenolic contents (TPC) (121.9 ± 3.1 mg GAE/g extract) of TLM while total flavonoid contents (TFC) of TLE (60.9 ± 2.2 mg RTE/g extract), TLM (59.6 ± 1.5 mg RTE/g extract) and TLB (55.0 ± 2.5 mg RTE/g extract) were found significantly higher as compared to other solvent fractions. The data of LC-MS profile showed the existence of 13 flavonoids of which luteolin 7-O-glucoside, luteolin-O-pentoside, myricetin, apigenin derivative (apigenin +180), luteolin and apigenin 7-O-rutinoside were found common to most of the plant fractions. GC-MS analysis of TLM yielded 30 compounds of which di-(2-ethylhexyl) phthalate (45.93%) and triethylene glycol (18.77%) were major constituents whereas minor components were oleic acid, olein, 2-mono- and stigmasterol. The in vitro free-radical–scavenging activity of TLM and fractions was demonstrated for DPPH, ABTS, superoxide, •HO, H2O2, phosphomolybdenum, β-carotene bleaching, reducing power and iron chelating efficiency. TLM and all of its fractions exhibited potent free radical scavenging activities. The antibacterial activity of extract/fractions was assessed by measuring the inhibition zone diameters and by determining minimal inhibitory concentrations (MIC). The extract/fractions of T. leptophylla showed equal effectiveness against both Gram-positive and Gram-negative bacteria. Based on the MIC values, the most active were the TLM, TLH and TLA. Antifungal activity of extract/fractions, using agar tube dilution technique, was expressed in terms of mycelial growth inhibition. Against different fungal strains TLE, TLB and TLH showed distinct effectiveness, followed by TLM and TLC. Brine shrimp lethality assay was used for cytotoxicity while potato disc assay for antitumor activity. TLM (LD50 75.141) and TLC (LD50 90.656) were more cytotoxic than other fractions while TLM (IC50 114), TLH (IC50 119) and TLE (IC50128) had significant antitumor activity. Single doses of TLM (100 and 200 mg/kg i.p.) were compared with glibenclamide (10 mg/kg, i.p) for its antihyperglycemic effects against the intraperitoneal injection of 50% dextrose in water (1g/kg bw). Blood glucose levels estimated at 0h, 1h, 2h, 4h and 6h after administration of TLM (100 mg/kg bw) induced significant reduction (P < 0.01-0.001) while 200 mg/kg bw dose of TLM was more comparable to that of glibenclamide. In vivo carbon clearance assay (phagocytosis) and cyclophosphamide induced myelosuppression of TLM for immunomodulatory activity exhibited beneficial actions on immunity of immunosuppressed mice, indicating that it possesses a strong immunomodulatory potential. Further, TLM exhibited significant (P < 0.01-0.001) analgesic and anti-inflammatory activities via hot plate, acetic acid induced writhing test and carrageenan induced paw edema in rat models. The antidepressant activity was examined using Forced Swim Test (FST) in rats and this study showed that TLM exerted considerable (P < 0.01-0.001) antidepressant-like activity in FST at the oral dose of 100 and 200 mg/kg body weight in a dose dependent manner. To assess the protective effects of TLM (200, 400 mg/kg bw) against RIPE [Combination of four antituberculosis drugs, rifampicin (R), isoniazid (I), pyrazinamide (P) and ethambutol (E)] induced multiple organ toxicity in mice, TLM was administered to act on liver, kidney, brain, heart and lungs antioxidant status. RIPE administration in mice caused multi organ toxicity predominantly hepto-renal and neurotoxicity. Administration of RIPE caused a marked alteration in all the serum markers of experimental sera. Also the levels of total protein and albumin were significantly decreased in the serum of the RIPE treated animals. The levels of H2O2 and lipid peroxidation were increased in all the tissues exposed to RIPE. Besides, application of RIPE reduced the levels of antioxidant enzymes and cellular reserves of glutathione in all the tissues. Concurrent treatment with TLM dose dependently prevented this heave in the levels and restored the antioxidant enzyme and serum marker levels to almost normal. Apart from these, significant histological changes also revealed the protective nature of TLM against RIPE induced morphological derangement in all the tissues studied. Also TLM at both two levels efficiently inhibited DNA fragmentation in mice liver, kidney, brain, cardiac and lung tissues induced during administration of RIPE. Finally, high-throughput assay of inflammation-independent anti-fibrotic activities based on TGF-β1-induced total collagen accumulation in normal rat kidney fibroblasts (NRK-49F) was applied to examine the anti-fibrotic activities of TLM. Lactate dehydrogenase release assay and cell detachment index were used to monitor cytotoxicity. The Picro-Sirius red (PSR) Staining was used for the histological visualization of collagen using light microscopy and collagen was quantified by spectrophotometric analysis of PSR staining. TLM demonstrated anti-fibrotic effect at lower concentrations 32.65 and 15.6 μg/ml. TLM at these concentrations successfully suppressed the TGF-β1 induced total collagen accumulation, thus significantly lowered the PSR staining. Also these low concentrations induced little cell detachment and had low cytotoxicity. TLM at higher concentrations 125 mg/ml and 62.5 mg/ml effectively lowered PSR staining but this lowering effect is probably due to the increased cell detachment and high toxicity, thus their anti-fibrotic effect is coincided with cytotoxicity. Overall, results obtained could contribute to a better understanding of the potential health benefit of T. leptophylla. The plant has shown a remarkable antioxidant activity in both in vitro and in vivo model systems. Also the findings on plant‘s potential anti-fibrotic and antimicrobial activity if complied in a usable form may provide a new source of beneficial treatment to overcome the fatal effects of fibrosis and diseases due to microbes.