Antioxidant and hepatoprotective potential of Lawsonia inermis L. leaves against 2-acetylaminofluorene induced hepatic damage in male Wistar rats

Plant kingdom is regarded as gold repository of natural antioxidant constituents that can delay or prevent oxidation of other substances when ingested in daily diet [61]. During the last decade, bioprospection of phytoconstituents with nutritional and pharmaceutical importance is gaining popularity [62]. Numerous literature reports have demonstrated that medicinal plant extracts possess antioxidant and genoprotective activity [63–67]. Crude extracts or isolated molecules from medicinal plants not only possess antioxidant potential but they are even more potent than BHT, Vitamin E etc. in various in vitro experiments [68–70]. Mitochondrion is chief source of reactive oxygen species like superoxide anions (O₂^.-). Dismutation of superoxide anions radicals by superoxide dismutase enzyme leads to the formation of hydrogen peroxide (H₂O₂). Hydrogen peroxide undergoes interaction with transition metal ions viz. Fe²⁺ or Cu⁺ to generate hydroxyl radicals (OH^.). Hydroxyl radicals (OH^.) cause number of harmful activities such as initiating lipid peroxidation and causing alterations in DNA [71]. But-LI showed statistically significant dose-dependent hydroxyl radical scavenging potential of 79.86% at highest tested concentration. Analysis of one way ANOVA showed F-ratio of 50.29 which was found to be statistically significant at p???0.05. Regression analysis showed regression equation of y?=?16.71ln(x)-33.63; r?=?0.9969). The value of correlation coefficient was found to be significant at p???0.001. Since the fraction contained numerous polyphenolic constituents in appreciable amount as reported in our earlier publication [51], the hydroxyl radical scavenging activity of the fraction might be attributed to these compounds. Similar results were reported by Thind et al. [72] who investigated root extracts of Schleichera oleosa for antioxidant activity using deoxyribose degradation assay and reported extracts as good hydroxyl radical scavengers. Kaur and Arora [73] evaluated antiradical potential of methanol extract of Chukrasia tabularis leaves using deoxyribose degradation assay and reported that extract possessed promising hydroxyl radical scavenging activity in deoxyribose degradation assay.

Reactive oxygen species are mainly generated in the mitochondria as byproduct of cellular metabolic processes and can affect biomolecules by causing damage [74]. Reactive intermediates resulting from oxidative stress can target membrane bilayers by causing lipid peroxidation. Polyunsaturated fatty acids in the membranes undergo lipid peroxidation resulting in lipoperoxyl radical (LOO^.) generation, which attack lipids to form lipid hydroperoxides (LOOH)^. and lipid radicals. Lipid hydroperoxides are known to be unstable and can give rise to peroxyl and alkoxyl radicals and decompose to produce various secondary products. The breakdown products of lipid peroxides include malondialdehyde, hexanal, 4-hydroxynonenal etc. which are highly reactive [75–78]. 4-hydroxynonenal is of electrophilic nature and reacts with glutathione, proteins and also with DNA at higher concentration [79, 80]. In the present investigation, it was found that But-LI moderately inhibited the lipid peroxidation dose dependently (y?=?8.548ln(x)-0.680; r?=?0.9904). The value of correlation coefficient was found to be significant at p???0.01. Earlier, we have reported that But-LI fraction harbours high amount of catechin, chlorogenic acid, ellagic acid and kaempferol while phytoconstituents such as gallic acid, epicatechin and quercetin were found to be present in moderate amount [51]. The lipid peroxidation inhibitory activity of the fraction might be due to various polyphenolic constituents present in it. Nakchat et al. [81] studied antioxidant activity including anti-lipid peroxidation activity of boiling water Tamarind seed coat extract and reported that extract effectively inhibited the lipid peroxidation. HPLC analysis of Tamarind seed coat extract showed presence of phenolics constitiuents such as (+)-catechin, (?)-epicatechin and procyanidin B2 which may be resposnsible for its antioxidant activities. Mulla and Swamy [82] studied antioxidant activity of polyphenolic extract of Portulaca quadrifida and reported that extract showed antilipid peroxidation activity of 71% with IC₅₀ value of 370.33?±?2.91 ?g/ml. Results of FRAP assay revealed that But-LI fraction also possessed dose-dependent reducing potential. Analysis of results using one way ANOVA showed F-ratio of 49.94 which was found to be statistically significant at p???0.05. Regression analysis showed regression equation of y?=?0.003×-0.007; r?=?0.9979. The value of correlation coefficient was found to be significant at p???0.001. Reducing ability of the fraction may be due to polyphenols present in it. Singh et al. [83] studied leaf, fruit and seed extract of Moringa oleifera for antioxidant activity and reported that leaf extract possessed good reducing potential in reducing power assay. HPLC analysis of the extract demonstrated the presence of phenolic constituents such as gallic acid, chlorogenic acid, kaempferol, quercetin, ellagic acid, ferulic acid, and vanillin. Soobrattee et al. [84] studied various polphenolic phytochemicals for reducing potential in FRAP assay. Gallic acid, ellagic acid, chlorogenic acid, quercetin, kaempferol, (?)-epicatechin and (+)-catechin exhibited FRAP value of 5.25, 4.39, 3.22, 7.39, 1.95, 2.90 and 2.47 mmol Fe (II)/L respectively.

Several plant extracts and phytochemicals are reported to modulate the mammalian antioxidant enzymes system and provide protective effects against cellular damage [85–88]. Liver is the main organ responsible for detoxification processes occurring in the body. In the liver cells, endoplasmic reticulum is the primary site of metabolism. Hence, this metabolism is termed as hepatic metabolism. Besides liver, there are extrahepatic sites of metabolism which include organs such as lungs, kidney, skin, epithelial cells of gastrointestinal tract, adrenals and placenta [89–92]. Liver injury in response to various chemicals results in the leakage of serum enzymes into the blood circulation, thus causing increase in their level in the serum [93]. Sehrawat et al. [94] reported that 2-AAF administration to rats increased the level of SGOT and SGPT enzymes in serum. In another study, Hasan and Sultana [34] reported that 2-AAF treated rats demonstrated high level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In the present investigation results obtained from serum toxicity markers such as SGOT, SGPT, ALP demonstrated significant increase on treatment with 2-AAF as compared to normal control. 2-AAF induced 2.22, 1.72 and 5.68 fold enhancements in SGOT, SGPT and ALP levels respectively. On co-administration of rats with 2-AAF and varying doses of But-LI, there was significant decrease in these serum parameters and the serum enzymes levels were restored towards normal control levels. The But-LI fraction alone did not induce any increase in the values of these markers and results were statistically not different to the normal control and vehicle control group at p???0.05, reflecting non-toxic nature of But-LI fraction.

A study carried out by Selvanayaki and Ananthi [48] reported aqueous extract of Lawsonia inermis seeds with potent hepatoprotective effects against paracetamol induced hepatic damage in male rats. Extract significantly reduced the levels of various serum enzymes viz. aspartate aminotransferase (AST), acid phosphatase (ACP), alkaline aminotransferase (ALT), alkaline phosphatase (ALP) etc. altered by paracetamol treatment. Recently, Mohamed et al. [95] reported hepatoprotective potential of methanol extract of L. inermis leaves against carbon tetrachloride (CCl₄)-induced hepatic damage. It was found that extract treatment significantly protected rats from hepatic damage induced by CCl₄.

Lipid peroxidation is critical marker of oxidative stress and is coupled with various diseases including cancer [96, 97]. Malondialdehyde (MDA) and lipid hydroperoxides are produced as the result of lipid peroxidation of polyunsaturated fatty acids [86, 92]. Results of the present investigation demonstrated that 2-AAF treatment resulted in 2.94 fold increase in the MDA level in rats. Further, treatment of rats with But-LI along with 2-AAF reversed the effect of 2-AAF as reflected from lower level of MDA. Our results are in agreement with previous studies [34, 88, 94, 95], in which natural plant products effectively reduced lipid peroxidation induced in response to various toxicants. Further, results of histopathological examination were also in concordance with results of other parameters and provided supportive evidence regarding protective potential of Lawsonia inermis (But-LI) fraction. It was found that 2-AAF administration to the male Wistar rats caused severe damage to the liver tissue, since it showed various histopathological alterations such as moderate piecemeal necrosis, mild confluent and spotty necrosis, moderate portal inflammation etc. with necroinflammatory score of 6 out of 18. The untreated, vehicle and negative control group rats did not demonstrate such pathologies in their liver tissue and necroinflammatory score was found to be zero. All the 3 doses (100, 200 and 400 mg/kg bw) provided protection against damage induced by 2-AAF with necroinflammatory score of 1 out of 18 at highest tested dose (400 mg/kg bw) and histoarchitecture of the animals in these groups was comparable to untreated control group. Hepatoprotection can be achieved either by reinstating the normal hepatic physiology or by diminishing the toxic damaging effect induced by toxicant [98]. The in vivo protective activity of But-LI of Lawsonia inermis against 2-AAF could be attributed to the various polyphenolic phytochemicals present in the fraction.