INTRODUCTION:

Hepatic problems are global concerns as it is not only associated with developing countries but also to the developed countries^1,2. The liver is a vital metabolic organ as it performs biotransformation of food and drugs. It also helps in the detoxification and secretion of bile^3-5. Hepatotoxicity can be caused by chronic alcoholism, carbon tetrachloride, chlorinated hydrocarbons, certain drugs like cyclosporine, cisplatin, azathioprine, and doxorubicin, isoniazid, gases (CO₂ and O₂), and biological substances (Bacillus-Calmette–Guerin vaccine)^6-9_.It is well established that the overload of iron accumulation in the liver is associated with hepatic injury, fibrosis, and finally cirrhosis^10,11.

Ferric nitrilotriacetic acid (Fe-NTA) is used extensively to induce oxidative stress and is assumed to initiate free radical-mediated lipid peroxidation leading to the accumulation of lipid-derived oxidation products that cause liver injury. Further, excessive collagen deposition in the liver resulting in liver necrosis^12-14. Previous reports also provide a piece of evidence that it causes hepatic damage to HepG2 cells induces tumor, decreases the level of GSH, and renal damage^15-18. Hence, free radicals cause amendment in cellular signaling pathways responsible for the proliferation of cells. Iron is an indispensable micronutrient, plays a significant role in oxidative phosphorylation, transports O₂, synthesizes DNA, RNA, proteins, and regulates gene expression^19-22. Hibiscus rosasinensis Linn (Family Malvaceae), a plant with flowers widely distributed throughout the world. It has been reported to possess several medicinal values which include antipyretic, antifertility, anticomplementry²³, anti-spermatogenic and androgenic, anti-tumour^24,25, and anticonvulsant²⁶ activities. The use of flowers to treat cardiac complications has also been reported in the Ayurvedic book²⁷. It is also known to possess anti-diarrheal activity²⁸ and claimed to be a useful alternative for many disorders²⁹. On the other hand, other selected plant i.e. Butea monosperma (family Fabaceae) has been reported to have anticonvulsive, antidiabetic,^26,30 anti-inflammatory, hepatoprotective, anthelmintic, antioxidant and antistress^31-33. It is a commonly used Ayurvedic plant with important many medicinal values and is widely used by tribal and rural parts of India³⁴.

No reports are available on the hepatoprotective activity of aqueous extract of Hibiscus rosasinensis (AQEHR) and Butea monosperma (AQEBM) against iron overload. Hence, the present communication elaborates the pharmacological impact of AQEHR and AQEBM on Fe-NTA-induced hepatic oxidative damage in rats.

MATERIALS AND METHODS:

Plants and chemicals:

The flowers of Hibiscus rosasinensis were collected from the herbal garden of Lovely Professional University, Jalandhar, India, and the flowers of Butea monosperma were procured from Swaraj Pansari Suppliers, Jalandhar, India. Both were authenticated by the Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar having Reference No. 1042. Nitrile acetic acid Disodium salt was purchased from Tokyo Chemical Industry Co. Ltd., Japan. All the other chemicals used were of analytical grade and purchased from standard manufacturers.

Animals:

Male Wistar rats weighing 220-250g were procured from the National Institute of Pharmaceutical Education and Research (NIPER), Mohali, Punjab, India were used for the study. The animals were allowed to acclimatize under standard laboratory conditions in the animal house of the School of Pharmaceutical Sciences, Lovely Professional University, Punjab, India. The protocol was approved by the Institutional Animals Ethics Committee (954/ac/06/CPCSEA/11/16). They were housed in polypropylene cages and maintained at 25± 2˚C, relative humidity 65±10% under 12hrs light/ dark cycle. The animals were fed regularly and water ad libitum.

Preparation and screening of phytoconstituents:

Shade dried and powdered flowers of H. rosasinensis and B. monosperma were separately extracted with sterile distilled water (500mL). The solvent was further evaporated in a vacuum at 50°C giving an aqueous extract. Alkaloids test was conducted by boiling 0.5g aqueous extract (5ml) with 1% HCL followed by the addition of Mayer’s reagent^35,36. The test for the identification of carbohydrates was performed with a resorcinol solution³⁷. A copper acetate test was performed to detect diterpenes³⁸^,³⁹ was followed to test flavonoids. The identification of saponins was performed by a frothing test according to Sofowara⁴⁰. Glycosides were analyzed by Modified Borntrager’s test⁴¹.

Total phenolics and flavonoids content:

The determination of total phenolic content (TPC) in both the extracts was determined by the method described by Naskar et al., 2010^42,43, and total flavonoids content (TFC) determined by aluminium chloride⁴².

Radical scavenging activity of extracts:

DPPH Radical Scavenging activity:

The free radical scavenging activity of AQEHR and AQEBM was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) using a standard method described by Pithayanukul et al., 2009, Mon et al., 2011), the reduction of DPPH was measured by recording by the decrease in absorbance at 518 nm^44,45.

Scavenging of H₂O₂:

Superoxide anion scavenging activity was measured by the spectroscopic method⁴⁶. The absorbance was recorded at 230nm was determined after 10 min against a blank solution containing phosphate buffer without H₂O₂.

Reducing power assay:

The reducing power of the extract was determined according to Ebrahimzadeh et al., 2009⁴⁷. The absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicated an increase in reducing power capacity⁴⁸.

Phosphomolybdenum antioxidant assay:

A 0.3mL extract (2mg/mL) mixed with 3mL of prepared reagent (0.6 M sulphuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate), and the reaction mixture was incubated at 95°C for 90min. The absorbance of the solution was measured at 695nm using a UV-visible spectrophotometer against blank after cooling to room temperature⁴⁹.

Preparation of Ferric nitrilotriacetate (Fe-NTA) solution:

Ferric nitrilotriacetate (Fe-NTA) solution was prepared fresh immediately before its use. The aqueous solution of ferric nitrate (9mg/kg body weight) was mixed disodium salt of NTA (36mg/kg body weight) and the pH was adjusted to 7.4 at room temperature with sodium bicarbonate solution with magnetic stirring^19,50.

AQEHR = Aqueous extract of Hibiscus rosasinensis, AQEBM = Aqueous extract of Butea monosperma

Fig.1. FeNTA induced hepatotoxicity model

Induction of hepatotoxicity and experimental protocol:

Animals were allocated into 7 groups (6 rats each). The group I will receive daily i.p. injection of appropriate volumes of saline for 8 days and served as a normal control group. The animals in the other 6 groups received an i.p. injection of Fe-NTA for 8 successive days. 6, 9, 12, and 15mg Fe/Kg body weight was administrated for 1^st 2 days, 2^nd 2 days, 3^rd 2 days, and last 2 days respectively. Rats of group II received an overload of iron without further treatment considered as the experimental control. Silymarin (50mg/Kg) was administrated orally to the rats of group III. Rats of group IV and V were treated with 200 and 400mg/kg of AQEBM respectively while rats of group VI and VII received 200 and 400mg/kg of AQEBM (Fig.1.). Treatment started 3 days before and concurrently with iron administration for 8 days. At the end of the experimental periods, animals were sacrificed by the decapitation method. Blood will be collected into plain centrifuge tubes and EDTA-containing centrifuge tubes and the liver of each animal was isolated immediately, washed with ice-cold saline, and blotted dry⁵¹.

Assessment of hepatoprotective activity:

Effect on biochemical parameters:

Just before the scarification of animals blood samples was collected in test tubes from retro-orbital sinus for determining Serum glutamic pyruvic transaminase (SGPT)⁵², serum glutamic-oxaloacetic transaminase (SGOT)⁵³, Alkaline phosphatase (ALP)⁵⁴, bilirubin⁵⁵, cholesterol⁵⁶, HDL[14] land triglycerides level using standard kits.

Estimation of oxidative biomarkers in liver tissue:

Isolated liver perfused with saline solution and homogenized in the cold environment of phosphate-buffered saline (PBS) with pH 7.4. The homogenates were centrifuged at 800g for 5 min at 4°C to remove nuclear debris. The collected supernatant undergoes centrifugation at 10,500g for 20 min at 4°C to obtain the post mitochondrial supernatant (PMS). Now this PMS was used to assay catalase^57,58, reduced glutathione (GSH)⁵⁹, and thiobarbituric acid reactive substances (TBARS) in terms of malondialdehyde (MDA)^60,61.

Histopathological studies:

After draining the blood, liver samples were excised and washed with normal saline and processed separately for histological observations. A portion of each liver was fixed in 10% buffered formalin for histopathological studies Liver tissues were dehydrated with alcohol and their paraffin section were cut in 5mm thickness, and were stained with alum hematoxylin and eosin.

Statistical analysis:

Statistical analysis was carried out by a one-way analysis of variance (ANOVA) followed by Dunnett’s test and the results were expressed as mean±S.E.M for 6 rats in each group.

RESULTS AND DISCUSSION:

The yield of AQEHR and AQEBM was 16 and 19% w/w respectively. The preliminary phytochemical analysis of the AQEHR and AQEBM extract showed the presence of alkaloids, carbohydrates, flavonoids, saponins, diterpenes, and glycosides. The AQEHR was found to contain 24.73µg/mg total phenolic content expressed as gallic acid equivalent (GAE, µg/mg of extract) and 21.87µg/mg total flavonoid content expressed as quercetin equivalent (QUE, µg/mg of extract). The AQEBM was found to contain 34.27µg/mg total phenolic content expressed as gallic acid equivalent (GAE, µg/mg of extract) and 27.39µg/mg total flavonoid content expressed as quercetin equivalent (QUE, µg/mg of extract).

Free radical scavenging potential of AQEHR and AQEBM:

The scavenging potential of both the extracts at their different concentration is depicted in figure.2. DPPH radical is scavenged by extracts indicates the ability of the antioxidant of both the extracts and the color changes from purple to yellow after reduction was accessed by its decrease in absorbance at wavelength 518nm. Radical scavenging activity increased with an increasing percentage of free radical inhibition (Fig. 2a). Scavenging of H₂O₂ by AQEHR and AQEBM was 49.92% and 65.07% respectively (Fig. 2b). Ascorbic acid (ASA) was taken as a reference and showed a 95.31% activity. IC₅₀ value obtained in DPPH assay was 10.4±0.37, 20.55±0.51, and 14.8±0.49µg/mL for ASA, AQEH, and AQEBM respectively while in H₂O₂scavenging assay it was recorded as9.06±0.24, 18.74±0.38, and 14.73±0.23 µg/mL for ASA, AQEH and AQEBM respectively. The reductive capabilities of AQEH and AQEBM were investigated and this capacity increases with the increase in the concentration of the extract (Fig. 2c).

Phosphomolybdenum antioxidant assay is based on the reduction of Mo (VI)-Mo (V) by the extracts and subsequent formation of a green phosphate/Mo (V) complex at acidic pH⁶². The extracts demonstrated electron-donating capacity and thus they may act as radical chain terminators (Fig. 2d)., the transformation of reactive free radical species into stable non-reactive products⁶³. Hence the dose-dependent antioxidant capacity of both the extracts was recorded. It is very important to observe that AQEBM exhibits a better response concerning AQEHR.

Effect of AQEHR and AQEBM on biochemical parameters:

Fe-NTA inflicted damage to the hepatic system and it was investigated by several biochemical parameters. Level of SGOT, SGPT, ALP, and bilirubin was accessed in terms of the recoding of liver function tests (LVT). Due to iron overload, the level of SGOT, SGPT, and ALP increased to 188.35 ± 1.71, 132.39 ± 3.07, and 113.06 ± 3.23 IU/L respectively (Table.1.). Treatment with AQEBM at 200 and 400 mg/kg significantly reduced the level of SGOT to 106.25 ± 1.99 and 93.91 ± 1.40, SGPT to 92.99 ± 2.24 and 80.42 ± 2.79 IU/L, ALP to 58.11 ± 1.25 and 50.35 ± 1.52 IU/L. Similarly, AQEHR at 200 and 400 mg/kg significantly reduced the level of SGOT to 141.64 ± 0.98 and 126.62 ± 2.69 IU/L, SGPT to 122.44 ± 2.63 and 114.87±2.95 IU/L, ALP to 85.02 ± 1.87 and 76.52 ± 1.23 IU/L. AQEBM was able to reduce the level of SGOT, SGPT, and ALP to 50.23, 39.68 and 55.70 % at the dose of 400mg/kg and AQEHR was able to reduce the level of SGOT, SGPT, and ALP to 32.84, 13.9, and 32.31% at the dose of 400mg/kg. Hence, it is very interesting that AQEBM showed a prominent effect in comparison to AQEHR on Fe-NTA induced SGOT, SGPT, and ALP.

Table.1. Effect of the AQEHR and AQEBM on biochemical parameters in FeNTA induced hepatotoxicity

Groups	Treat-ment	Dose (mg/ Kg)	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)	Cholesterol (mg/dL)	Triglycerides (mg/dL)	Direct Bilirubin (mg/dL)	Total bilirubin (mg/dL)	Protein (TP) (g/dL)
I	Control	-	73.05±1.09	62.79±2.10	27.05±1.47	123.75±4.47	132.12±1.31	0.115±0.007	0.65±0.01	5.56±0.12
II	FeNTA	Iron over load	188.35±1.71	132.39±3.07	113.06±3.23	647.17±4.81	106.03±1.95	0.620±0.015	2.39±0.01	4.24±0.15
III	Silymarin	50	81.15±1.48^**	70.60±2.06^**	34.68±1.20^**	153.16±4.82^**	129.03±1.11^**	0.116±0.008^**	0.65±0.01^**	5.34±0.03^**
IV	AQEBM	200	106.25±1.99^**	92.99±2.24^**	58.11±1.25^**	343.66±5.47^**	119.20±1.56^**	0.270±0.023^**	1.41±0.08^**	5.03±0.06^**
V	AQEBM	400	93.91±1.40^**	80.42±2.79^**	50.35±1.52^**	253.10±4.92^**	122.10±1.37^**	0.181±0.019^**	1.16±0.04^**	5.25±0.07^**
VI	AQEHR	200	141.64±0.98^**	122.44±2.63^*	85.02±1.87^**	531.42±4.54^**	113.91±1.77^**	0.508±0.034^**	1.86±0.03^**	4.49±0.06
VII	AQEHR	400	126.62±2.69^**	114.87±2.95^**	76.52±1.23^**	409.03±5.29^**	117.61±1.28^**	0.341±0.033^**	1.61±0.09^**	4.62±0.12^*

Results expressed as Mean ± SEM (n=6). Statistical analysis was performed using One-way ANOVA followed by Dunnett test. * p< 0.05; ** p< 0.01. All groups are compared with FeNTA. Where, ASA = ascorbic acid, AQEHR = aqueous extract of H. rosasinensis, AQEBM = aqueous extract of B. monosperma

AQEBM and AQEHR treated groups have shown dose-dependent hepatoprotective activity in the determination of total protein (g/dL) level (TP). Treatment with silymarin ameliorated the level of TP to 5.34±0.03g/dL. AQEBM at 200 and 400mg/kg significantly increased the level of TP to 5.03±0.06 and 5.25±0.07g/dL respectively. Similarly, AQEHR at 200 and 400mg/kg increased the level of TP to 4.49±0.06 and 4.62±0.12 g/dL respectively. An increase in the level of iron also resulted in the reduction of serum total protein levels in the present study. The reduction in the total protein is attributed to the initial damage in the endoplasmic reticulum which results in the loss of cytochrome P₄₅₀ enzymes. All treated groups enhanced the synthesis of TP which accelerates the regeneration process and the protection of liver cells that as demonstrated in table.1.

Effect of AQEHR and AQEBM on oxidative biomarkers:

The level of catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) level decrease due to overload of iron-induced by Fe-NTA in rats. Treatment with silymarin significantly ameliorated the level of CAT, GSH, and MDA to 46.56±0.67, 252.60±8.54, and 80.49±0.22 respectively. AQEBM at 200 and 400mg/kg significantly improve the level of CAT to 34.40±0.53 and 40.35±0.67 IU/100mg, GSH to 142.63±4.78 and 190.24±3.24µM/100mg, MDA to 116.86±0.22 and 92.92±0.41nM/mg respectively. Similarly, AQEHR at 200 and 400 mg/kg significantly reduced the level of catalase to 23.61±0.66 and 31.40±0.53IU/100mg, GSH to 73.46±4.30 and 102.94±5.73µmol/100mg, MDA to 153.22±0.22 and 135.04±0.22nM/mg respectively (Fig.3). It is worth exploring that the changes in the levels of these oxidative tissue markers were protected by the AQEBM and AQEHR.

Histopathological evaluation of liver:

Treatment of AQEBM and AQEHR at both doses preserved hepatic architecture. Hepatocytes showed improvement in cords with no fatty changes, edema, or appearance of hemorrhage. High doses of both the extracts exhibited better response but 400 mg/kg of AQEBM exhibited architecture closer to the normal (Fig.4.).

Epidemiological studies, as well as experimental results, provide evidence that oxidative stress plays a central role in the etiopathogenesis of diabetes, cancer, arthritis, and neurological problems^66,67. Administration of antioxidant defense can delay the onset and progression of these disorders. Therefore, there is a scope to exploring safe and effective antioxidants compounds. Plant sources or their derived products potentiate the defense system of the human body. It can be safer over synthetic drugs even at higher doses.

Fig: 3.a. Effect of AQEBM and AQEHR on a. CAT (µM H₂O₂/min/100 mg protein), b. GSH (µM/100 mg) c. MDA (nM/mg) in iron overloaded induced hepatotoxicity.

Hence, the huge scientific body of research is trying to discover plants with potential antioxidant profiles. Fe-NTA causes hepatic as well as renal injury due to its well-known oxidant nature. Long-term administration of Fe-NTA causes accumulation of iron in hepatic parenchyma and development of renal tumors⁶⁸. Idiopathic mochromotoris is a similar kind of condition of iron overload^12,19.

Fig: 4. Histological structure of the liver (HE staining, original magnification 40X),

Where, ASA = ascorbic acid, AQEHR = aqueous extract of H. rosasinensis, AQEBM = aqueous extract of B. monosperma

Fe-NTA mediates its action by inducing oxidative stress in the liver⁶⁹ and production of 4-ydroxynonenal significantly causing toxicity of Fe-NTA because of lipid peroxidation^16,70-72. It also causes depletion of GSH, polyunsaturated fatty acids contents, tocopherol^13,73, antioxidant enzymes due to protein carbonyl formation, and iron-catalyzed hydroxyl radicals^74,75. The present investigation was designed to investigate more effective antioxidant agents and their possible ameliorating effect on certain biochemical alterations associated with chronic Fe-NTA-induced liver injury in rats.

AQEHR and AQEBM were found to contain phenolic and flavonoids contents but AQEBM was found rich in these components. Since free radicals are known to generate oxidative stress and destroy cellular components. We evaluated the antioxidant potential of both the extracts at their different concentration. AQEHR and AQEBM scavenge free radicals in concentrations dependent manner in DPPH, H₂O₂, Phosphomolybdenum, and reducing power assay. In all assays of antioxidant potential AQEBM exhibited better antioxidant properties and expected to protect biochemical and biological components from oxidative damage. Products of reactive oxygen species are known to harm biological systems as they are highly reactive and can modify the structural integrity as well as the normal function of various cellular components. Consequently, ameliorations in Fe-NTA mediated toxicity and hepatic damage in the present investigation are possibly recorded due to a reduction in oxidative damage. In our present report, Fe-NTA altered the normal level of hepatic enzymes like SGOT, SGPT, and ALP. It also results in abnormal levels of bilirubin, protein, cholesterol, and triglycerides. Treatment with AQEHR and AQEBM were able to control the elevated levels of SGPT, SGOT, ALP, bilirubin, protein, and cholesterol significantly in a dose-dependent manner.

These findings suggest that extracts can protect the biochemical alterations of the liver. We further scrutinized the ability of AQEHR and AQEBM the protection of endogenous hepatic antioxidants such as GSH, CAT, and MDA levels. Such a study gave a clear correlation with Fe-NTA-induced hepatoxicity also altered these oxidative biomarkers. AQEHR and AQEBM significantly defended oxidative stress induced by Fe-NTA and inhibited hepatic injury. Apart from this, these findings were also supported by histological studies, wherein the extracts were also found to protect pathological changes and maintain hepatic architecture. Finally, in all results, AQEBM was found superior over AQEHR.

CONCLUSION:

The present study deciphers the free radical scavenging, antioxidant, and hepatoprotective potential of AQEHR and AQEBM. The preliminary study indicates the availability of phenolic and flavonoids components in both the extracts and AQEBM possesses its higher amount. It may be the reason that the extract is capable of protecting against oxidative damage to lipids and proteins and also of increasing/maintaining the levels of antioxidant molecules and enzymes in vivo.

CONFLICT OF INTEREST:

The author declares no conflict of interest.

REFERENCES:

1. Ingawale DK, Mandlik SK, Naik SR 2014. Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism (s): a critical discussion. Environmental Toxicology and Pharmacology 37(1):118-133.

2. Zachariah SM, Aleykutty N, Viswanad V, Halima O 2012. An Overview on Hepatoprotective Activity of Natural Products. Research Journal of Pharmacy and Technology 5(3):317.

3. Rowland I, Gibson G, Heinken A, Scott K, Swann J, Thiele I, Tuohy K 2018. Gut microbiota functions: metabolism of nutrients and other food components. European Journal of Nutrition 57(1):1-24.

4. Patel J, Reddy V, Kumar G 2015. Evaluation of hepatoprotective activity of ethanolic extract of Diospyros melanoxylon (Roxb) leaves against CCl4 induced hepatotoxicity in albino rats. Research Journal of Pharmacy and Technology 8(5):571-574.

5. Patel A, Kushwah P, Pillai S, Raghuvanshi A, Deshmukh N 2017. Formulation and evaluation of Herbal Hand wash containing Ethanolic extract of Glycyrrhiza glabra root extract. Res J Pharm Technol 10(1):55-57.

6. Abboud G, Kaplowitz N 2007. Drug-induced liver injury. Drug Safety 30(4):277-294.

7. Kaplowitz N 2004. Drug-induced liver injury. Clinical Infectious Diseases 38(Supplement_2):S44-S48.

8. Acocella G 1978. Clinical pharmacokinetics of rifampicin. Clinical Pharmacokinetics 3(2):108-127.

9. Aithal GP, Day CP 2007. Nonsteroidal anti-inflammatory drug–induced hepatotoxicity. Clinics in Liver Disease 11(3):563-575.

10. Masini A, Ceccarelli D, Giovannini F, Montosi G, Garuti C, Pietrangelo A 2000. Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin. Journal of Bioenergetics and Biomembranes 32(2):175-182.

11. Nimbalkar V, Pansare P, Nishane B 2015. Screening Methods for Hepatoprotective Agents in Experimental Animal's. Research Journal of Pharmacy and Technology 8(12):1725.

12. Kaur G, Jabbar Z, Athar M, Alam MS 2006. Punica granatum (pomegranate) flower extract possesses potent antioxidant activity and abrogates Fe-NTA induced hepatotoxicity in mice. Food and Chemical Toxicology 44(7):984-993.

13. Deiana M, Aruoma OI, Rosa A, Crobu V, Casu V, Piga R, Dessi MA 2001. The effect of ferric-nitrilotriacetic acid on the profile of polyunsaturated fatty acids in the kidney and liver of rats. Toxicology Letters 123(2-3):125-133.

14. Poornima K, Chella Perumal P, Gopalakrishnan VK 2014. Protective effect of ethanolic extract of Tabernaemontana divaricata (L.) R. Br. against DEN and Fe NTA induced liver necrosis in Wistar Albino rats. BioMed Research International 2014.

15. Sakurai K, Cederbaum AI 1998. Oxidative stress and cytotoxicity induced by ferric-nitrilotriacetate in HepG2 cells that express cytochrome P450 2E1. Molecular Pharmacology 54(6):1024-1035.

16. Iqbal M, Sharma S, Rezazadeh H, Hasan N, Abdulla M, Athar M 1996. Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury. Redox Report 2(6):385-391.

17. Agarwal MK, Iqbal M, Athar M 2005. Vitamin E inhibits hepatic oxidative stress, toxicity and hyperproliferation in rats treated with the renal carcinogen ferric nitrilotriacetate. Redox Report 10(2):62-70.

18. Prabha S, Rao M, Kumar MR 2017. Evaluation of in vitro Antioxidant, Antibacterial and Anticancer activities of leaf extracts of Cleome rutidosperma. Research Journal of Pharmacy and Technology 10(8):2492-2496.

19. Lone IA, Kaur G, Athar M, Alam MS 2007. Protective effect of Rumex patientia (English Spinach) roots on ferric nitrilotriacetate (Fe-NTA) induced hepatic oxidative stress and tumor promotion response. Food and Chemical Toxicology 45(10):1821-1829.

20. Thomson AM, Rogers JT, Leedman PJ 1999. Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation. The International Journal of Biochemistry and Cell Biology 31(10):1139-1152.

21. Verma P, Srivastava S, Rao CV 2018. Hepatoprotective effect of Ethanolic Extract of Holarrhena antidysenterica against Paracetamol Induced Toxicity in Wistar Rats. Research Journal of Pharmacy and Technology 11(4):1633-1639.

22. Venkatalakshmi P, Ragadevi M 2012. Protective effect of tinospora cordifolia linn., on paracetamol and alcohol induced hepatotoxicity in albino rats. Research Journal of Pharmacy and Technology 5(2):281.

23. Shimizu N, Tomoda M, Suzuki I, Takada K 1993. Plant mucilages. XLIII. A representative mucilage with biological activity from the leaves of Hibiscus rosa-sinensis. Biological and Pharmaceutical Bulletin 16(8):735-739.

24. Reddy C, Murthy D, Patil S 1997. Antispermatogenic and androgenic activities of various extracts of Hibiscus rosa sinesis in albino mice. Indian Journal of Experimental Biology 35(11):1170-1174.

25. Serrame E, Lim-Sylianco C 1995. Anti-tumor promoting activity of decoctions and expressed juices from Philippine Medicinal plants. Philippine Journal of Science 124(3):275-281.

26. Kasture V, Chopde C, Deshmukh V 2000. Anticonvulsive activity of Albizzia lebbeck, Hibiscus rosa sinesis and Butea monosperma in experimental animals. Journal of Ethnopharmacology 71(1-2):65-75.

27. Sharma U 1994. Cure of heart diseases with ayurvedic drugs. Sachitra Ayurved 47:95-96.

28. Jadhav V, Thorat R, Kadam V, Sathe N 2009. Traditional medicinal uses of Hibiscus rosa-sinensis. J Pharm Res 2(8):1220-1222.

29. Obi FO, Usenu IA, Osayande JO 1998. Prevention of carbon tetrachloride-induced hepatotoxicity in the rat by H. rosasinensis anthocyanin extract administered in ethanol. Toxicology 131(2-3):93-98.

30. Somani R, Kasture S, Singhai AK 2006. Antidiabetic potential of Butea monosperma in rats. Fitoterapia 77(2):86-90.

31. Shahavi V, Desai S 2008. Anti-inflammatory activity of Butea monosperma flowers. Fitoterapia 79(2):82-85.

32. Bhatwadekar A, Chintawar S, Logade N, Somani R, Kasture VS, Kasture S 1999. Antistress activity of Butea monosperma flowers. Indian Journal of Pharmacology 31(2):153-155.

33. Wagner H, Geyer B, Fiebig M, Kiso Y, Hikino H 1986. Isoputrin and butrin, the antihepatotoxic principles of Butea monosperma flowers1. Planta Medica 52(02):77-79.

34. Burlia D, Khadeb A 2007. Phcog Rev.: Plant Review A Comprehensive review on Butea monosperma (Lam.) Kuntze. Pharmacognosy Reviews 1(2).

35. Harborne J 1998, Phytochemical Methods: A guide to modern techniques of plant analysis. Champman and Hall, London.

36. Herborne J 1973. Phytochemical methods. A guide to modern Techniques of Plant Analysis: 5-11.

37. Wall ME, Eddy CR, McClennan ML, Klumpp ME 1952. Detection and estimation of steroidal sapogenins in plant tissue. Analytical Chemistry 24(8):1337-1341.

38. Roopashree T, Dang R, Rani S, Narendra C 2008. Antibacterial activity of antipsoriatic herbs: Cassia tora, Momordica charantia and Calendula officinalis. International Journal of Applied Research in Natural Products 1(3):20-28.

39. Kapoor L, Singh A, Kapoor S 1969. Survey of Indian plants for saponins, alkaloids and flavonoids. I.

40. Sofowara A 1993. Medicinal plants and Traditional medicines in africa. chichester john, willeyandsons. New York:97-145.

41. De S, Dey Y, Ghosh A 2010. Phytochemical investigation and chromatographic evaluation of the different extracts of tuber of Amorphaphallus paeoniifolius (Araceae). Int J Pharm Biol Res 1(5):150-157.

42. Naskar S, Islam A, Mazumder U, Saha P, Haldar P, Gupta M 2010. In vitro and in vivo antioxidant potential of hydromethanolic extract of Phoenix dactylifera fruits. Journal of Scientific Research 2(1):144-157.

43. Prior RL, Cao G, Martin A, Sofic E, McEwen J, O'Brien C, Lischner N, Ehlenfeldt M, Kalt W, Krewer G 1998. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. Journal of Agricultural and Food Chemistry 46(7):2686-2693.

44. Pithayanukul P, Nithitanakool S, Bavovada R 2009. Hepatoprotective potential of extracts from seeds of Areca catechu and nutgalls of Quercus Infectoria. Molecules 14(12):4987-5000.

45. Mon M, Maw S, Oo Z 2011. Quantitative determination of free radical scavenging activity and anti-tumor activity of some Myanmar herbal plants. World Acad Sci Eng Technol 75:524-530.

46. Czochra M, Widensk A 2002. Spectrophotometric determination of H2O2 activity. Anal Chem Acta 452:177-184.

47. Ebrahimzadeh M, Nabavi S, Nabavi S 2009. Essential oil Composition and Antioxidant Activity of Pterocaryaﬁvaxinifolia. Pak J Biol Sci 12(13):957-963.

48. Nabavi SF, Ebrahimzadeh MA, Nabavi SM, Eslami B 2010. Antioxidant activity of flower, stem and leaf extracts of Ferula gummosa Boiss. Grasasy Aceites 61(3):244-250.

49. Sudha G, Priya MS, Shree RI, Vadivukkarasi S 2011. In vitro free radical scavenging activity of raw pepino fruit (Solanum muricatum aiton). Int J Curr Pharm Res 3(2):137-140.

50. Awai M, Narasaki M, Yamanoi Y, Seno S 1979. Induction of diabetes in animals by parenteral administration of ferric nitrilotriacetate. A model of experimental hemochromatosis. The American Journal of Pathology 95(3):663.

51. El-Maraghy SA, Rizk SM, El-Sawalhi MM 2009. Hepatoprotective potential of crocin and curcumin against iron overload-induced biochemical alterations in rat. Afr J Biochem Res 3(5):215-221.

52. Bradley D, Maynard J, Emery G, Webster H 1972. Transaminase activities in serum of long-term hemodialysis patients. Clinical Chemistry 18(11):1442-1442.

53. Rej R, Fasce CF, Vanderlinde RE 1973. Increased aspartate aminotransferase activity of serum after in vitro supplementation with pyridoxal phosphate. Clinical Chemistry 19(1):92-98.

54. MacComb R, Bowers G 1972. Alkaline phosphatase activity in serum. Clin Chem 18(97):12.

55. Pearlman FC, Lee RT 1974. Detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents. Clinical Chemistry 20(4):447-453.

56. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC 1974. Enzymatic determination of total serum cholesterol. Clinical Chemistry 20(4):470-475.

57. Basu S, Das M, Datta G 2013. Protective activity of ethanolic extract of Amorphophallus campanulatus against ethanol induced hepatotoxicity in rats. Int J Pharm Pharm Sci 5:412-417.

58. Luck H 1963. Spectrophotometric method for the estimation of catalase. Methods of Enzymatic Analysis.

59. Draper H, Hadley M. 1990. [43] Malondialdehyde determination as index of lipid Peroxidation. Methods in Enzymology, ed.: Elsevier. p 421-431.

60. Ohkawa H, Ohishi N, Yagi K 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95(2):351-358.

61. Ellman GL 1959. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82(1):70-77.

62. Saha MR, Alam MA, Akter R, Jahangir R 2008. In vitro free radical scavenging activity of Ixora coccinea L. Bangladesh Journal of Pharmacology 3(2):90-96.

63. Lobo VC, Phatak A, Chandra N 2010. Antioxidant and free radical scavenging activity of Hygrophila schulli (Buch.-Ham.) Almeida and Almeida. seeds. Adv Biores 1(2):72-78.

64. Uzunhisarcikli M, Kalender Y 2011. Protective effects of vitamins C and E against hepatotoxicity induced by methyl parathion in rats. Ecotoxicology and Environmental Safety 74(7):2112-2118.

65. Anusha M, Venkateswarlu M, Prabhakaran V, Taj SS, Kumari BP, Ranganayakulu D 2011. Hepatoprotective activity of aqueous extract of Portulaca oleracea in combination with lycopene in rats. Indian Journal of Pharmacology 43(5):563.

66. Hogg N. Seminars in reproductive endocrinology, 1998, pp 241-248.

67. Farber JL 1994. Mechanisms of cell injury by activated oxygen species. Environmental Health Perspectives 102(suppl 10):17-24.

68. Toyokuni S 1996. Iron-induced carcinogenesis: the role of redox regulation. Free Radical Biology and Medicine 20(4):553-566.

69. Matos HR, Capelozzi VL, Gomes OF, Di Mascio P, Medeiros MH 2001. Lycopene inhibits DNA damage and liver necrosis in rats treated with ferric nitrilotriacetate. Archives of Biochemistry and Biophysics 396(2):171-177.

70. Iqbal M, Athar M 1998. Attenuation of iron-nitrilotriacetate (Fe-NTA)-mediated renal oxidative stress, toxicity and hyperproliferative response by the prophylactic treatment of rats with garlic oil. Food and Chemical Toxicology 36(6):485-495.

71. Iqbal M, Giri U, Athar M 1995. Ferric nitrilotriacetate (Fe-NTA) is a potent hepatic tumor promoter and acts through the generation of oxidative stress. Biochemical and Biophysical Research Communications 212(2):557-563.

72. Iqbal M, Giri U, Giri DK, Alam M, Athar M 1999. Age-dependent renal accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins following parenteral administration of ferric nitrilotriacetate commensurate with its differential toxicity: implications for the involvement of HNE–protein adducts in oxidative stress and carcinogenesis. Archives of Biochemistry and Biophysics 365(1):101-112.

73. Deiana M, Rosa A, Casu V, Piga R, Dessı̀ MA, Aruoma OI 2004. L-ergothioneine modulates oxidative damage in the kidney and liver of rats in vivo: studies upon the profile of polyunsaturated fatty acids. Clinical Nutrition 23(2):183-193.

74. Athar M, Iqbal M 1998. Ferric nitrilotriacetate promotes N-diethylnitrosamine-induced renal tumorigenesis in the rat: implications for the involvement of oxidative stress. Carcinogenesis 19(6):1133-1139.

75. Ye Sf, Hou Zq, Zhang Qq 2007. Protective effects of Phellinus linteus extract against Iron overload‐mediated oxidative stress in cultured rat hepatocytes. Phytotherapy Research 21(10):948-953.

Received on 07.04.2021 Modified on 20.11.2021

Research J. Pharm. and Tech. 2022; 15(7):3213-3220.

DOI: 10.52711/0974-360X.2022.00539