INTRODUCTION:

Nonalcoholic Fatty liver infection (NAFLD) is as a significant reason for liver disorder in India. Predominance of NAFLD in around 9% to 32% of overall population in India with higher commonness in those with overweight or obese and those with diabetes or prediabetes.¹

Term NAFLD includes patients with simple steatosis as also those with nonalcoholic steatohepatitis (NASH). NAFLD results in loss of productivity and reduced health-related quality of life (HRQOL), and also responsible for a major financial burden on a family.²

Presently, single therapy for NAFLD has not been clearly proven to be effective. Various treatment modalities used for NAFLD have included lifestyle modifications such as weight loss and exercise, treatment of risk factors like diabetes mellitus and hyperlipidemia, and use of insulin sensitizing agents such as biguanides (metformin), thiazolidinediones (rosiglitazone, pioglitazone) and antioxidants and various hepatoprotective agents.

There is an increase in the acceptability in the society for the use of herbal therapy by the patients including those with liver disease due to in appropriate therapy. Complementary therapies, such as herbal drugs, have also been tested for treatment of NAFLD.³ Several plants have been evaluated for effectiveness in a NAFLD like Vaccinium myrtillus, Hibiscus Sabdariffa, Hawthron leaf, etc.⁴Phyllanthus niruri, also known as gale of the wind and major stonebreaker, has been found to effective against liver damage. Lipid profile modulation of the plant extract were well demonstrated in experimental animals.⁵The present study aimed to evaluate the role of phyllanthin extracted from P. niruri in animal model (C57BL/6 mice) of NAFLD followed by the assessment of biochemical parameters. (Figure 1)

Figure 1: Effect of P. niruri on liver enzymes and lipid profile

METHODS AND METHODS:

Animal model:

Four-five week-old C57lb/6 mice (n=8 per group) were procured and housed in small animal research laboratory, PGIMER, Chandigarh. The mice were housed (three mice impound) in an mice test cage (temperature 24°C, humidity 50%, and a 12-hour on/off light cycle) with free access to water and a routine diet. After allowing one week of adjustment to the new environment, the 42 days old mice were randomized into different groups that will receive either a Methionine Choline deficient diet (MCD) diet or a routine diet. The “MCD diet contains neither methionine nor choline, while the standard diet contained 5.1g methionine per kg and 2.5g choline per kg”. The total duration of the experiment was 4 weeks. This experiment was performed at small animal research laboratory, PGIMER, Chandigarh, which is also approved by the Institutional Animal Ethics Committee (IAEC)

Formulation dosing was given through the mouth (Orally) and delivered into the stomach with the help of gastric gavage. The experimental protocol was approved by the IAEC at PGIMER. The study design has been showed in (figure 2).

Figure 2: Effect of P. niruri and Commercial Phyllanthin with and without combination of MCDD diet.

Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) has approved the C57BL/6 mice for current study from institutional animal ethics committee, PGIMER, Chandigarh (10575/2017-18).

Classification of different groups has been shown below:

Group I: Control (Saline)

Group II: Control + (200mg/kg) Plant Phyllanthin

Group III: Control + (400mg/kg) Plant Phyllanthin

Group IV: Methionine Choline Deficient Diet (MCDD)

Group V: MCDD + (200mg/kg) plant Phyllanthin

Group VI: MCDD + (400mg/kg) plant Phyllanthin

Group VII: Control + (2mg/kg) commercial phyllanthin

Group VIII: Control + (4mg/kg) commercial phyllanthin

Group XI: MCDD + (2mg/kg) commercial phyllanthin

Group X: MCDD + (4mg/kg) commercial Phyllanthin

Collection of Plant material and Authentication:

The plant P. niruri was collected in 2016-17 from the Bansathali Vidyapith Tonk, Rajasthan. The plant material was identified and authenticated taxonomically at Department of Biosciences and Biotechnology at Banasthali Vidyapith.

Plant Phyllanthin extraction and analysis:

Briefly, extract was prepared from 20gm of dried leaves of P. niruri dried leaves, and finally lyophilized and stored at -20°C until the tests were performed. The purity was analyzed by HPTLC.

Establishment of Blood and Serum Biochemical Parameters:

Completion of four weeks to MCDD feeding, all the animals were fasted overnight and the body weight of each mouse was determined. They were anesthetized with sodium pentobarbital (60mg/kg intraperitoneal (i.p.). Five milliliters of the blood were taken via cardiac puncture from each test animal. All the samples were subjected to the liver function test as total bilirubin, liver transaminases (SGOT and SGPT), and lipid profile as total cholesterol, triglyceride, and HDL were performed using commercially available Erba Diagnostic kit.^6-11. The observations were recorded and comparisons were done to detect the detection in hepatic profile in all the groups with their respective controls.

Statistical Analysis:

Data were calculated as mean±standard deviation. Data were assessed to utilize one way analysis of variance (ANOVA) followed by Bonferroni post-hoc method of multiple comparison. P value <0.05 “was considered significant and the statistical analysis was performed using SPSS v21”.

RESULTS:

India is advancing into the diabetic capital of the world and NAFLD is developing as a significant reason for liver illness. Indeed, even truly lean Indians might be metabolically hefty. Epidemiological investigations recommend the commonness of NAFLD to associate with 9-32% when all is said in done Indian population.¹²

Focuses of upcoming treatments for non-alcoholic greasy liver ailment (NAFLD) all over again lipogenesis, fibroblast development factor; fecal microbial transfer, farnesoid X receptor, development hormone-delivering hormone, glucagon-like peptide, peroxisome proliferator-activated receptor. In spite of these drugs nowadays people interest towards the herbal medicine. Phyllanthus niruri has been used in folk medicine as antipyretic, analgesic, as well as to anti- inflammations or similar symptoms that might suggest anti-histamine effects.¹³Phyllanthus niruri to treat genitourinary infections, venereal diseases, kidney or bladder stones, as well as acting as a urinary inhibitor of calcium oxalate crystallization and as effective treatments for urolithiasis used to treat diabetes through blood glucose bringing down properties such as restraint of glucose ingestion and upgrade of glucose storage.¹⁴ Present study, focuses on the effect of P. niruri by combination of systematic targeting mechanism and treat the disease in experimental model of NAFLD.

Effect of animal weight:

Data revealed that mean weight (gm) in MCDD mice was significantly lower in comparison to control mice (18.03±2.29 vs. 33.08±2.38; P<0.0001). Mean weight was comparable in MCDD+ extract (200mg/kg and 400mg) in comparison to MCDD mice (20.32±5.10 vs. 18.16±1.08; P=NS) (Figure 3)

Figure 3: Comparison of animal weight in various groups

Plant extract normalized liver function:

Data revealed that serum bilirubin levels were significantly higher in MCDD mice in comparison to control mice (1.48±0.36 vs. 0.62±0.14; P=0.0003). It was comparable in control and plant phyllanthin at both 200mg/kg and 400mg/kg with (0.62±0.14 vs. 0.66± 0.08; P=0.557) and (0.62±0.14 vs. 0.61±0.09; P=0.886) respectively. While during administration of Phyllanthin it was 0.62±0.14 in control, (0.67±0.09; P=0.0.479) in (2mg/kg) and (0.61±0.05; P=0.872) in (4mg/kg) diet. The levels of serum bilirubin reduced in MCDD with 200mg/kg plant phyllanthin (1.48±0.36 vs. 0.66±0.08; P=0.0005) and MCDD + (400mg/kg) mice (1.48± 0.36 vs. 0.66±0.08; P=0.003). On another hand MCDD + phyllanthin (2mg/kg) mice (1.48±0.36 vs. 0.69±0.09; P=0.0004) and MCDD + phyllanthin (4mg/kg) mice (1.48± 0.36 vs. 0.61±0.05; P=0.002). The results indicate that serum bilirubin levels better responding 200mg/kg in plant phyllanthin and commercial phyllanthin was in 4mg/kg) (Table 1).

The data suggest that SGOT and SGPT levels were significantly higher in MCDD mice in comparison to control mice (50.17±7.68 vs. 17.15±4.86; P <0.0001, 71.18±9.13 vs. 25.28±4.69; P <0.0001). SGOT and SGPT levels were comparable in control + extract (200mg/kg) mice (17.23±2.40 vs. 17.15±4.86; P=0.972, 31.8±5.8 vs. 25.28±4.69; P=0.057) and 400mg/kg fed mice showed the non significant changes in SGOT and SGPT levels (P=0.865; P=0.445) respectively. Control + phyllanthin (2mg/kg) mice were also checked SGOT and SGPT levels (14.64±2.02 vs. 17.15±4.86; P=0.270, 25.30±5.69 vs. 25.28±4.69; P=0.995) respectively and control + phyllanthin (4mg/kg) mice (17.73±4.53 vs. 17.15±0.835; P=0.872, 28.24±4.70 vs. 25.28±4.69; P=0.330). In the MCDD + extract (200mg/kg) mice is significantly lower in 400mg/kg diet group (P=0.0009). SGOT and SGPT levels were significantly lower in MCDD + phyllanthin (2mg/kg) mice (15.3±1.04 vs. 50.17±7.68; P=0.0004) as compared to the MCDD+ phyllanthin (4mg/kg) mice (17.65±6.38 vs. 50.17±7.68; P=0.002) when compared with SGOT levels in MCDD mice (Table 1).

Table 1: Liver Function test in different groups

GROUPS	BILIRUBIN	SGOT	SGPT
Control	0.62±0.14	17.15±4.86	25.28±4.69
Control + Extract (200 mg)	0.80±0.08	17.23±2.40	31.8±5.3
Control + Extract (400 mg)	0.41±0.13	17.63±4.70	27.67±5.67
MCDD	1.48± 0.36	50.17±7.68	71.18±9.13
MCDD +Extract (200 mg)	0.69±0.14	33.97±18.63	29.4±14.39
MCDD + Extract (400 mg)	0.85±0.14	23.3±11.87	21.80±6.12
Control + Phyllanthin (2 mg/Kg)	0.27±0.09	14.64±2.02	25.30±5.69
Control + Phyllanthin (4 mg/Kg)	0.61±0.05	17.73±4.53	28.24±4.70
MCDD + Phyllanthin (2 mg/Kg)	0.70±0.04	15.3±1.04	22.92±3.55
MCDD + Phyllanthin (4 mg/Kg)	0.78±0.06	17.65±6.38	24.10±1.04

Plant extracts improved lipid profile:

Data revealed that cholesterol levels were significantly higher in MCDD mice in comparison to control mice (237.5±39.36 vs. 136.0±16.98; P=0.0002). It was comparable in control + Plant phyllanthin at both 200mg/kg and 400mg/kg (139.37±13.17 vs. 136.0±16.98; 131.62±12.72 vs. 136.0±16.98; with P=0.713; P=0.624) respectively. While administration of Phyllanthin it was 136.0±16.98 in control, 140.13±19.27; P=0.702 and 136.73±5.62; P=0.817 in 4mg/kg. Indicating that 2mg/kg dosage is better that 4mg/kg. The levels of Cholesterol reduced in MCDD with 200mg/kg plant phyllanthin (237.5±39.36 vs. 154.8±11.32; P<0.00001) and MCDD + 400mg/kg mice (237.5±39.36 vs. 151.93±15.07; P<0.00001). We also compared the MCDD + phyllanthin (2mg/kg) mice (237.5±39.36 vs. 138.9±21.20; P<0.00001) and MCDD + phyllanthin (4mg/kg) mice (237.5±39.36 vs. 138.63±13.71; P=0.0005) (Table 2).

Our study observed that triglycerides levels were significantly higher in MCDD mice in comparison to control mice (135.53±9.19 vs. 75.68±27.65; P=0.0005). Triglycerides levels were comparable in control + phyllanthin (200mg/kg-74.67±25.00 vs. 75.68±27.65; P=0.948) and 400mg/kg 83.56±32.31 vs. 75.68±27.65; P=0.660). Phyllanthin (2mg/kg) mice (75.68±27.65; P=0.561) and (4mg/kg - 59.56±40.49 vs. 75.68±27.65; P=0.439). Triglycerides levels were significantly lower in plant phyllanthin 200mg/kg (P<0.0001) and 400mg/kg (P=0.0009) when compared with triglycerides levels in MCDD mice. In another group MCDD + phyllanthin (2mg/kg) mice (135.53±9.19 vs. 63.76±29.86; P=0.0002) and MCDD + phyllanthin (4 mg/kg) mice (135.53±9.19 vs. 78.5±20.97; P=0.0001) when compared with triglycerides levels in MCDD mice (Table 2).

MCDD mice had significantly lower levels of HDL in comparison to control mice (23.00±7.16 vs. 36.42±3.39; P=0.006). Administration of 200mg/kg and 400mg/kg plant phyllanthin significantly raised HDL in MCDD mice (56.59±4.79 and 55.4±3.51 respectively) (P<0.0001). Between 2mg/kg and 4mg/kg HDL Phyllanthin significantly increased in 2mg/kg group (P=0.0009, P=0.013) respectively. HDL levels were significantly higher in MCDD + extract (200mg/kg) mice (56.59±4.79 vs. 23.00±7.16; P<0.0001) and 400 mg/kg (55.4±3.51 vs. 23.00±7.16; P=0.0009) HDL levels were significantly higher in MCDD + phyllanthin (2mg/kg) mice (51.49±6.67 vs. 23.00±7.16; P<0.0001) as compared to 2mg/kg (Table -2).

Table 2: Lipid profile in different groups

Groups	Cholesterol	Triglycerides	HDL
Control	136.0±16.98	75.68±27.65	36.42±3.39
Control + Extract (200 mg)	139.37± 13.17	74.67±25.00	40.83±2.89
Control + Extract (400 mg)	131.62± 12.72	83.56±32.31	51.46±2.74
MCDD	237.5±39.36	135.53±9.19	23.00±7.16
MCDD +Extract (200 mg)	154.8±11.32	66.4±23.79	56.59±4.79
MCDD + Extract (400 mg)	151.93± 15.07	65.03±35.96	55.4±3.51
Control + Phyllanthin (2 mg/Kg)	140.13± 19.27	63.3±42.14	49.45±6.00
Control + Phyllanthin (4 mg/Kg)	136.73±5.62	59.56±40.49	45.13±6.18
MCDD + Phyllanthin (2 mg/Kg)	138.9±21.20	63.76±29.86	51.49±6.67
MCDD + Phyllanthin (4 mg/Kg)	138.63± 13.71	78.5±20.97	49.0±4.36

DISCUSSION:

This study demonstrated the lipid lowering and hepatoprotective effect of a phyllanthin extracted from P. niruri on experimental model of NAFLD. This study showed that MCD-fed mice lost significant weight in comparison to control mice. It has been recently shown that C57BL/6J mice when fed MCD diet, a significant reduction in weight was observed at 4 weeks.¹⁵Pervious reports suggested that MCD diet brings about a fast beginning of the NASH phenotype with lobular aggravation and expanding (30-60days).¹⁶These MCD-fed animals have not been shown any other metabolic features of human NAFLD, such as obesity, peripheral insulin resistance and dyslipidemia.

Mice fed the MCD diet for a month indicated to the advancement in the plasma AST and ALT levels. Circulating ALT and AST levels are effective to hepatocyte damage in NAFLD. ALT level were reduced in the mice that received both plant phyllanthin extract as well as commercial phyllanthin compared to that in the MCD-diet mice. Similar observation demonstrated that have shown that liver ALT levels were also rapidly doubled in high fat diet animals and P. niruri significantly decreased ALT levels in comparison to HFD animals.¹⁷ The increased serum liver enzymes (ALT and AST) in MCDD mice compared to control suggest that necrosis of hepatocytes that outcome in the effusion of transaminase, and this also can be assigned to the destruction in the histostructural morality of the hepatocytes.

The data also suggests that significantly increase in levels of cholesterol and triglyceride while decrease in HDL indicated in MCD-fed mice in NAFLD model. Present study indicated that plant phyllanthin and Commercial phyllanthin were responsible for improving the overall biochemical parameters due to damage caused by MCDD in NAFLD C57BL/6 mice model (Figure 3). Out of the two applications plant phyllanthin indicates statistically significant results. Addition to P. niruri significantly improved lipid profile with Deranged lipid profile in MCD-fed animals has been shown earlier.¹⁸ It has also shown that serum lipids were decreased by P. niruri extract orally fed (250 mg/kg b.w.) to the triton WR-1339 induced hyperlipidemic mice. Long term” feeding of this drug (100mg/kg b.w.) in animals simultaneously fed with cholesterol (25 mg/kg b.w.) for 30 days caused lowering in the lipids and apoprotein levels of VLDL and LDL in experimental” animals.⁵

The species P. niruri (Linn.) have also been shown to have inhibitory activity against Hepatitis B virus.¹⁹Recent Study have also shown reduced transaminase and hepatic damage in CCl₄-induced mice after P. niruri treatment.²⁰ The study suggests the hepatoprotective action of the plant phyllanthin and it can be suggest for the pre clinical studies after selection of experimental validation.

CONCLUSION:

The rate of NAFLD is increasing globally and its therapies are associated with certain complications. Therefore, there is an urgent need to find an alternative therapy for the cure with less or no side effects. Phyllanthin, a lignan from plant is an excellent supplement for the cure suggestive by folk medicine. The present study indicated that Methoinine choline deficient diet (MCDD) cause to development of fatty liver disease as NAFLD. It causes a drastic alteration in the liver profile. Alternatively, phyllanthin from plant and commercial source were used as therapeutic agents. Study indicated better efficacy of plant phyllanthin (200 mg/kg) in comparison to commercial phyllanthin (4 mg/kg) plant phyllanthin significantly improve the biochemical parameters including (lipid profile, liver function test) in NAFLD C57BL/6 mice model. Therefore the formulation can be further recommended for pre clinical trials for the treatment of NAFLD.

ACKNOWLEDGEMENTS:

The authors are grateful to Professor Aditya Shastri, Vice-Chancellor, Banasthali Vidyapith, Rajasthan and Bioscience and Biotechnology Department, Banasthali Vidyapith for providing research facilities and constant motivation. We also provide thanks to DST-CURIE for providing financial assistance for conducting our research work.

CONFLICT OF INTEREST:

Disclosure of potential conflicts of interest indicates that there is no conflict of interest during the study conduct in any of the authors. The study is conducted in compliance with Ethical Standards.

CONFLICT OF INTEREST:

All authors declare they have no conflict of interest.

REFERENCES:

1. Duseja A. Nonalcoholic fatty liver disease in India – a lot done, yet more required! Indian J Gastroenterol. 2010; 29: 217–225

2. Iqbal U, Perumpail BJ, Akhtar D, Kim D, Ahmed A. The Epidemiology, Risk Profiling and Diagnostic Challenges of Nonalcoholic Fatty Liver Disease. Medicines. 2019; 6: 41.

3. Yang JD, Abdelmalek MF, Pang H, Guy CD, Smith AD, Diehl AM et al. Gender and Menopause Impact Severity of Fibrosis Among Patients with Nonalcoholic Steatohepatitis. Hepatology 2014, 59, 1406-1414.

4. Yao, Yu-Jie Qiao, Ya-Li Zhao, Xu-Feng Tao et.al. Herbal medicines and nonalcoholic fatty liver disease. Hong World J Gastroenterol 2016; 30: 6890-6905

5. Khanna AK, Rizvi F, Chander R. Lipid lowering activity of Phyllanthus niruri in hyperlipemic mice. J Ethnopharmacol. 2002; 82: 19-22

6. Pearlman FC, Lee RT. Detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents. Clin Chem. 1974; 20: 447-53

7. Thomas L. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). In: Thomas L, editor. Clinical Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books Verlagsgesellschaft; 1998. p. 55-65

8. Bradley DW, Maynard JE, Emery G, Webster H. Transaminase activities in serum of long-term hemodialysis patients. Clin Chem. 1972; 18: 1442

9. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974; 20: 470-5

10. Rifai N, Bachorik PS, Alberts JJ. Lipids, lipoproteins and apolipoproteins. In: Burtis CA, Ashwood ER, editors Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W. Saunders Company; 1999. p. 809-61

11. Burstein M, Scholnick HR, Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J Lipid Res. 1970; 11: 583-95.

12. Townsend SA, Newsome PN. Non-alcoholic fatty liver disease in 2016. Br Med Bull. 2016; 119: 143–156

13. Ong, H., and Norzalina, J. Malay herbal medicine in Gemencheh, Negri Sembilan, Malaysia. Fitoterapia. 1999; 70: 10-14.

14. C.O. Okoli, I.C. Obidike, A.C. Ezike , P.A. Akah, and O.A. Salawu. Studies on the possible mechanisms of antidiabetic activity of extract of aerial parts of Phyllanthus niruri. Pharmaceutical Biology, 2011; 49: 248-255.

15. Kim S-B, Kang O-H, Lee Y-S, Han S-H, Ahn Y-S, Cha S-W, et al. Hepatoprotective Effect and Synergism of Bisdemethoycurcumin against MCD Diet-Induced Nonalcoholic Fatty Liver Disease in Mice. PLoS ONE 2016; 11: e0147745

16. Van Herck MA, Vonghia L, Francque SM. Animal Models of Nonalcoholic Fatty Liver Disease-A Starter's Guide. Nutrients. 2017; 9: 1072.

17. Al Zarzour RH, Ahmad M, Asmawi MZ, et al. Phyllanthus Niruri Standardized Extract Alleviates the Progression of Non-Alcoholic Fatty Liver Disease and Decreases Atherosclerotic Risk in Sprague-Dawley Mice. Nutrients. 2017; 9: 766.

18. Zahra AA, Mehmet B, Alshawsh MA, Ali HM, Hadi HA, Abdulla MA. Protective Role of Phyllanthus niruri Extract against Thioacetamide-Induced Liver Cirrhosis in Rat Model. Evidence-Based Complementary and Alternative Medicine. 2012: Article ID 241583.

19. Blumberg BS, Millman I, Venkateswaran PS, Thyagarajan SP. Hepatitis B Virus and hepatocellular carcinoma-treatment of HBV carriers with Phyllanthus amarus. Cancer Detect Prevent. 1989; 1.

20. Manjrekar AP, Jisha V, Bag PP, Adhikary B, Pai MM , Hegde A et al. Effect of Phyllanthus niruri Linn. Treatment on liver, kidney and testes in CCl4 induced hepatotoxic mice. Indian Journal of Experimental Biology. 2008; 46: 514-520

Received on 03.06.2020 Modified on 29.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4685-4690.

DOI: 10.52711/0974-360X.2021.00814