Several techniques have been used to determine the antioxidant activity in vitro in order to allow rapid screening of substances since substances that have low antioxidant activity in vitro, will probably show little activity in vivo. Free radicals are known to play a definite role in a wide variety of pathological manifestations. Antioxidants fight against free radicals and protect us from various diseases. They exert their action either by scavenging the reactive oxygen species or protecting the antioxidant defense mechanisms .
The electron donation ability of natural products can be measured by 2,2′-diphenyl-1- picrylhydrazyl radical (DPPH) purple-coloured solution bleaching . The method is based on scavenging of DPPH through the addition of a radical species or antioxidant that decolourizes the DPPH solution. The degree of colour change is proportional to the concentration and potency of the antioxidants. A large decrease in the absorbance of the reaction mixture indicates significant free radical scavenging activity of the compound under test . In the present study among all the fractions tested, n-butanol, chloroform and ethyl acetate showed significantly higher inhibition percentage and positively correlated with total phenolic content. Results of this study suggest that the plant extract contain phytochemical constituents that are capable of donating hydrogen to a free radical to scavenge the potential damage.
Superoxide radical is considered a major biological source of reactive oxygen species . Although superoxide anion is a weak oxidant, it gives rise to generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress . The results of our study revealed that TLM, TLC and TLB had effective capacity of scavenging for superoxide radical and correlated with total flavonoid content thus suggesting its antioxidant potential.
The antioxidant capacity of the fractions was measured spectrophotometrically through phosphomolybdenum method, based on the reduction of Mo (VI) to Mo (V) by the test sample and the subsequent formation of green phosphate/Mo (V) compounds with a maximum absorption at 765 nm. The present study demonstrated that TLB exhibited the highest antioxidant capacity for phosphomolybdate reduction. Recent studies have shown that many flavonoid and related polyphenols contribute significantly to the phosphomolybdate scavenging activity of medicinal plants [37, 38].
Hydroxyl radical is one of the potent reactive oxygen species in the biological system. It reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes damage to cell [5, 39]. The hydroxyl radical is regarded as a detrimental species in pathophysiological processes and capable of damaging almost every molecule of biological system and contributes to carcinogenesis, mutagenesis and cytotoxicity . Hydroxyl radicals were produced by the reaction of H2O2 and the ferrous that would react with 2-deoxyribose. The reaction was stopped by adding TBA reagent that would give a red colour if the malonaldehyde was formed as the result of the reaction between the radical and 2-deoxyribose. Hydroxyl radical scavenging capacity of an extract is directly proportional to its antioxidant activity which is depicted by the low intensity of red colour . All fractions of T. leptophylla when added to the reaction mixture actively scavenged the hydroxyl radicals and prevented the degradation of 2-deoxyribose.
Hydrogen peroxide occurs naturally at low concentration levels in the air, water, human body, plants, microorganisms and food . H2O2 is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (·OH) that can initiate lipid peroxidation and cause DNA damage . Ethyl acetate fraction of T. leptophylla efficiently scavenged hydrogen peroxide which may be attributed to the presence of phenolic groups that could donate electrons to hydrogen peroxide, thereby neutralizing it into water.
ABTS radical scavenging assay involves a method that generates a blue/green ABTS+ chromophore via the reaction of ABTS and potassium persulfate. The ABTS radical cation is generated by the oxidation of ABTS with potassium persulfate, its reduction in the presence of hydrogen-donating antioxidants is measured spectrophotometrically at 745 nm. All the fractions possessed strong ABTS scavenging activity an observation that is supported by other researchers .
In reducing power assay, the yellow colour of the test solution changes to green depending on the reducing power of the test specimen. The presence of the reductants in the solution causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by absorbance measurement at 700 nm. Previous reports suggested that the reducing properties have been shown to exert antioxidant action by donating of a hydrogen atom to break the free radical chain . Increasing absorbance at 700 nm indicates an increase in reducing ability. The antioxidants present in the fractions of T. leptophylla caused their reduction of Fe3+
/ ferricyanide complex to the ferrous form, and thus proved the reducing power.
Plant materials rich in phenolics are increasingly being used in the food industry because they retard oxidative degradation of lipids and improve the quality and nutritional value of food . Phenolic compounds are considered secondary metabolites and these phytochemical compounds derived from phenylalanine and tyrosine occur ubiquitously in plants and are diversified . The methanol extract exhibited the highest total phenolics content, whereas the contents obtained with residual aqueous fraction were much smaller that is in agreement with other reports . Phenolic compounds of plants are also very important because their hydroxyl groups confer scavenging ability.
Phenolic compounds of plants fall into several categories; chief among these are the flavonoids which have potent antioxidant activities . Flavonoids are naturally occurring in plants and are thought to have positive effects on human health. Studies on flavonoidic derivatives have shown a wide range of antibacterial, antiviral, anti inflammatory, anticancer, and anti-allergic activities [48, 49]. Flavonoids have been shown to be highly effective scavengers of most oxidizing molecules, including singlet oxygen, and various free radicals  implicated in several diseases. So comparable with the findings in the literature for other extracts of plant products  our results suggested that phenolic acids and flavonoids may be the major contributors for the antioxidant activity as the EC50 values of radical scavenging activity of various soluble fractions of T. leptophylla and the contents of phenolics or flavonoids exhibited significant correlation. However, non significant correlation was found in case of hydrogen peroxide radical scavenging. It is known that different phenolic compounds have different responses in the Folin-Ciocalteu method. Similarly the molecular antioxidant response of phenolic compounds varies remarkably, depending on their chemical structure . In addition, there may be some interference rising from other chemical components present in the extract, such as sugars or ascorbic acid .
Liver damage is very common since liver has to detoxicate a lot many toxic substances. There are several chemicals that have been known to induce hepatotoxicity by producing the reactive species which form covalent bonds with the lipids of the tissue [53, 54]. Liver injury due to CCl4 in rats was first reported in 1936  and has been widely and successfully used by many investigators [56, 57]. Carbon tetrachloride is metabolized by cytochrome P-450 in endoplasmic reticulum and mitochondria with the formation of CCl3O-, a reactive oxidative free radical, which initiates lipid peroxidation [58, 59].
A large reserve of reduced glutathione is present in hepatocytes for detoxification of free radicals. However, oxidative stress results in toxicity when the rate at which the ROS are generated exceeds the cell capacity for their removal [60, 61]. Most hepatotoxic chemicals damage liver by inducing, directly or indirectly, lipid peroxidation. MDA is one of the end products in the lipid peroxidation process . In our in vivo study elevation in levels of end products of lipid peroxidation in liver of rats treated with CCl4 were observed. The increase in MDA levels in liver suggests enhanced lipid peroxidation leading to tissue damage . Treatment with TLM significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection of extract is due to its antioxidant effect .
GSH is an intracellular reductant and protects cells against free radicals, peroxides and other toxic compounds. GSH is a naturally occurring substance that is abundant in many living creatures; GSH depletion increases the sensitivity of cells to various aggressions leading to tissue disorder and injury . In the present study we demonstrated the effectiveness of the extract by using CCl4 induced rats and found that exogenous TLM supplementation elevated GSH levels in rats with CCl4 treatment and thus might provide a mean of recovering reduced GSH levels and to prevent tissue disorders and injuries. Therefore, it is valid to consider that TLM, because of its antioxidant property as the extract has confirmed the presence of saponins, might be capable of protecting the hepatic tissue from CCl4-induced injury and inflammatory changes. Saponins are natural products, which have been shown to possess antioxidant properties [65, 66].