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THERAPEUTIC EFFECT OF NYMPHAEA ALBA LINN. FLOWERS AGAINST ISONIAZID-INDUCED HEPATOTOXICITY: AN EXPERIMENTAL STUDY

Objective: This study was designed to evaluate the effect of ethanolic extract of Nymphaea alba (NAEE) Linn. flowers on liver marker enzymes, histology, and antioxidant tests against isoniazid (INH)-induced hepatotoxicity in rats. Methods: Wistar albino rats were treated with INH (50 mg/kg) for 28 days to induce hepatotoxicity. Silymarin (100 mg/kg) and NAEE Linn flowers in 200 mg/kg and 400 mg/kg doses, respectively, were used as standard and test drugs. Liver marker enzymes and histological examination of livers were performed to demonstrate the effect of NAEE against INH-induced hepatotoxicity. Catalase [CAT], glutathione, and malondialdehyde (MDA) levels were estimated to evaluate the antioxidant property of the NAEE extract. Results: The NAEE extract in 200 mg/kg and 400 mg/kg doses significantly decreased INH-induced elevation of liver marker enzymes as well as oxidative stress markers (CAT, glutathione, and MDA) in rats. Changes in biochemical parameters were supported by histological improvements of the liver. Conclusion: The NAEE Linn flowers in 200 mg/kg and 400 mg/kg doses showed a significant reversal of hepatic damage which was induced by INH in rats.


INTRODUCTION
Tuberculosis is one among the oldest diseases known to affect the human being and is one of the major causes of death worldwide [1]. Isoniazid (INH) is the preferred drug in both chemoprophylaxis and treatment of tuberculosis [2,3]. Daily intake can lead to moderate elevation in liver enzymes and severe hepatic damage (especially, hepatic necrosis) in 3-20% and 1-2% of patients, respectively [3,4]. Recent experimental studies have shown that one of the mechanisms responsible for INH-induced hepatic injury is oxidative stress. Free oxygen radicals may result in oxidative damage by lipid peroxidation. One of the aldehyde compounds generated from lipid peroxidation is malondialdehyde (MDA), which can be used to evaluate this damage [2]. The decrease of catalase (CAT) and glutathione (GSH) levels and increase of MDA level are closely related to oxidative stress, and the normalization of these levels by drugs or chemicals is extremely important for the attenuation of oxidative tissue damage [5][6][7][8].
Nymphaea alba Linn. (Nymphaeaceae) is mainly found in tanks and ponds throughout the warmer parts of India and Africa. The flowers are white, and they contain various phytoconstituents such as alkaloids, carbohydrates (polysaccharides), glycosides, steroids, flavonoids, tannin, and phenolic compound [9]. All parts of the plant have medicinal uses in the traditional system of medicine. It is employed as an antiinflammatory, anodyne, astringent, antiscrophulatic, cardiotonic, demulcent, sedative, and aphrodisiac. It also produces sedative and calming effects on the nervous system and is useful in the treatment of anxiety, insomnia, and similar disorders [10][11][12]. The leaves and rhizomes of N. alba possess antioxidant activity [13,14].
The aim of the present study was therefore to investigate the therapeutic effect of ethanolic extract of N. alba (NAEE) Linn. flowers against INHinduced hepatotoxicity in rats.

Institutional Animal Ethical Committee (IAEC) approval
The Institutional Animal Ethics Committee approved the experimental protocol (Registration No. 401/Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) dated 09.05.2012). All experiments were carried out in accordance with the rules and regulations of IAEC and CPCSEA.

Preparation of extract
The flowers of N. alba Linn. were procured from Dawakhana Tibbiya College, A.M.U., Aligarh, and identified by Prof. S. H. Afaq, Pharmacognosy Section, Department of Ilmul Advia, A.K.T.C., A.M.U., Aligarh, Uttar Pradesh, India. Shade-dried flowers were coarsely powdered and then subjected to extraction. Extraction of powder in ethanol was done 72 h using Soxhlet apparatus. The extract was filtered using Whatman No. 1 filter paper, evaporated on a water bath at 50°C until it dried completely, and stored in the refrigerator for further use. The yield of NAEE was found to be 10.6%.

Experimental animals
Thirty-six healthy Wistar albino rats weighing 150-200 g of either sex procured from Central Animal House of the Institute were housed under standard condition (temperature 27 ± 2°C, Humidity 30-70%, and 12 h light/dark cycles) and fed with standard pellet diet and water ad libitum. All the animals were acclimatized to laboratory conditions for 1 week before the experimental procedure.

Experimental design
The animals were divided in 6 groups comprising of 6 animals each. All drugs were administered orally. The groups were treated as follows: • Group-I: Normal saline (1 mL/kg) for 28 days. • Group-II: INH (50 mg/kg) [2] for 28 days. • Group-III: INH (50 mg/kg) for 28 days followed by normal saline (1 mL/kg) for 15 days. • Group-IV: INH (50 mg/kg) for 28 days followed by silymarin (50 mg/kg) [15] for 15 days. Animals were sacrificed under sodium pentobarbitone (50 mg/kg intraperitoneally). Rats of Groups-I and II were sacrificed on the 29 th day, while animals of Groups III-VI were sacrificed on the 44 th day. Blood sample was taken by cardiac puncture for the estimation of liver function tests, and liver was dissected out for antioxidant tests and histological examination in each animal.

Biochemical investigations
The serum separated from blood was used for biochemical analysis of liver function (AST, ALT, and ALP) [16,17].

Antioxidant tests
The homogenate of the liver (in 10% weight/volume of phosphate buffer [0.2 M, pH-6.6]) tissue was used to perform in vivo antioxidant tests such as CAT [18], reduced GSH [19], and MDA [20].

Histological examination of liver
The histological assessment of liver damage was done by scoring of structural changes described by the National Health Services, Maryland, USA [21]. The parameters were as follows: Degeneration (0 = no degeneration, 1 = few vacuolated cells per lesion, 2 = more than 10 vacuolated cells per lesion, 3 = one to two rows of vacuolated cells around necrotic zone per lesion, and 4 = more than two rows of vacuolated cells around necrotic zone per lesion), necrosis (0 = no necrosis, 1 = focal necrosis of one or two cells per lesion, 2 = focal necrosis of more than two cells per lesion, 3 = massive centrilobular necrosis, and 4 = massive centrilobular necrosis with necrotic tissue bridging the central vein), fibrosis (0 = normal appearance of liver, 1 = central necrosis, hydropic degeneration, no fibrosis, 2 = fibrous tissue in periportal area only, 3 = fibrous tissues insinuating surrounding hepatic parenchyma, and 4 = formation of pseudo lobules), and regeneration (0 = no regeneration, 1 = mild, 2 = moderate, and 3 = excellent).

Statistical analysis
The data were analyzed using one-way analysis of variance with post hoc Tukey test for biochemical parameters and Mann-Whitney U-test for comparing histopathology score. The values are represented as mean ± standard error of mean. p<0.05 was considered statistically significant.

Effect of NAEE on biochemical tests
The therapeutic effect of N. alba Linn. flowers was measured against INH-induced hepatotoxicity as shown in Table 1 Table 1).

Effect of NAEE on antioxidant tests
In the experiment, the antioxidant activity was evaluated as the amount of CAT consumed per minute and total GSH present in liver tissue.
There was a significant decrease in the levels and activity of GSH and CAT in INH alone treated group. NAEE 200 and 400 mg/kg showed an increase in the levels of CAT (p<0.001), thereby suggesting a correction in oxidative stress. There was a significant increase in the levels of GSH (p<0.001) and a decrease in MDA (p<0.001) levels in both NAEE 200 and NAEE 400 extracts treated groups as compared to negative control ( Table 1).

Effect of NAEE on histological findings
INH administration in Group-II gave rise to necrosis, fibrosis, and degeneration of hepatic microstructure. In Group-III, there was degeneration along with bridging fibrosis of hepatic microstructure but no regenerating foci. Silymarin-treated group showed near normal hepatic microstructure. In NAEE-treated groups, there was increased regenerating foci (Fig. 1a-e). NAEE 200 and 400 mg/kg administrations resulted in a significant increase in regenerating foci and decrease in fibrosis and degeneration as compared to INH only group (p<0.001) ( Table 2).

DISCUSSION
Drug-induced hepatotoxicity is the serious adverse effect of the currently used antitubercular chemotherapeutic agents containing INH (H), rifampicin, and pyrazinamide [2]. The serum levels of hepatic enzymes primarily reflect the degree of liver damage and have been  frequently used as a diagnostic marker for hepatotoxicity [22,23]. INH (50 mg/kg) administration produced hepatotoxicity which was evident from the deranged biochemical and histological parameters.
INH is used extensively for prophylaxis as well as treatment of tuberculosis, but significant hepatotoxicity has often been reported. Hepatotoxicity after INH treatment is encountered in approximately 2% of cases, which if not recognized timely can have fatal outcome [24]. INH gets converted to acetylisoniazid by the enzyme N-acetyl transferase-2 which is eliminated by the kidney; acetylisoniazid is further transformed into acetylhydrazine and then to potential hepatotoxic metabolite acetyl diazine by the CYP enzymes which generates reactive acetyl onium ion, acetyl radical, and ketene, which causes irreversible damage to the liver tissue [25].
The NAEE Linn. flowers in 200 mg/kg and 400 mg/kg dose were therapeutically effective against INH-induced hepatotoxicity in rats. Both biochemical and histological parameters showed marked improvement with the restoration of liver functions. Both the doses decreased the derangement of liver function parameters, but this effect was more dominant in the high dose, i.e., 400 mg/kg dose group. Rats treated with NAEE showed significant reduction in the serum bilirubin, AST ALT, and ALP levels at both the doses. Since in Group-III there was no improvement in above parameters, it can be taken into account that the improvement in NAEE-treated groups was not due to self-healing process. There was further significant improvement in the antioxidants levels in NAEE-treated groups. This may be due to the prevention of lipid peroxidation as manifested by the decrease in MDA levels in NAEEtreated groups. The therapeutic benefit provided by NAEE was similar to that offered by the standard drug silymarin.
The findings of the biochemical analysis were further supported by histological examination of the liver tissues from the test group. NAEE in both the doses protected or maintained the liver tissue morphology evidenced by a reduction in the degeneration, necrosis, and fibrosis score in the histopathological examination. Regenerative nodules were likewise seen in the liver samples from both the test groups. Hence, the findings of the histological analysis also indicate that protection of the hepatocytes against damage induced by INH was significant in NAEEtreated groups. These findings were comparable to the prevailing drug silymarin.
One of the proposed mechanisms for INH-induced hepatic injury is oxidative stress [2]. Generation of free radicals damages the hepatocytes. The curative effect of extract can be explained by its antioxidant activity as demonstrated in the study. Previous studies demonstrate the presence of flavonoids and phenolic compounds in NAEE Linn [7]. The presence of these compounds might be responsible for the antioxidant activity.

CONCLUSION
The NAEE Linn. flowers in 200 mg/kg and 400 mg/kg doses showed therapeutically beneficial effect against INH-induced hepatotoxicity in rats.