Efficacy of Phyllanthus niruri on improving liver functions in patients with alcoholic hepatitis: A double-blind randomized controlled trial.

INTRODUCTION: Phyllanthus niruri, a traditional herbal medicine, was found to be hepatoprotective as evidenced by several preclinical and clinical studies. However, to the best of our knowledge, there are no clinical trials available to date to evaluate its efficacy in alcoholic hepatitis.
MATERIALS AND METHODS: The present study is a block randomized, double-blind, parallel-arm placebo-controlled trial that was designed to assess the efficacy of P. niruri on the liver and renal function parameters, total oxidant and antioxidant levels in alcoholic hepatitis patients in comparison to placebo over a 4-week period. Patients were screened by CAGE questionnaire, and those with a confirmed diagnosis of mild-moderate alcoholic hepatitis based on laboratory findings and Maddrey's discriminant function score were randomly allocated to treatment and placebo arms. Clinical assessments were done at baseline, 2 weeks, and 4 weeks. A total of 454 patients were screened and 100 eligible patients were recruited for the study, and 71 were analyzed using the modified intention-to-treat approach.
RESULTS: Serum levels of liver and renal function parameters failed to demonstrate significant improvement with P. niruri. However, there was a statistically significant increase in the level of total antioxidants with P. niruri (P = 0.034) with an additional appetite stimulant activity (P = 0.03) in 4 weeks.
CONCLUSION: A 4-week administration of P. niruri in mild-moderate alcoholic hepatitis patients showed an improvement in the total antioxidant levels with an appetite stimulant activity compared to a placebo.

Introduction

Alcoholic hepatitis is one of the most prevalent liver disorders that carry significant morbidity and mortality all over the world.[1] Its burden is quite high, and the World Health Organization report states that the associated deaths are around 3.3 million sharing about 6% of the global mortality. It mainly occurs due to excess alcohol consumption on a chronic basis, and the disease onset and progression are also quite variable between the individuals.[2] However, the disease progression pattern is well defined ranging from initial hepatic steatosis to alcoholic hepatitis and liver cirrhosis and finally leading to hepatocellular carcinoma and death.[3] The common clinical manifestations may range from hepatomegaly, jaundice, and pain abdomen to other life-threatening complications such as portal hypertension, hepatic encephalopathy, hepatorenal problems, and coagulopathies with an increase in disease severity.[4] The CAGE questionnaire (common screening tool) and laboratory findings with a significant elevation in aspartate transaminase (AST) and alanine transaminase (ALT) by two to sevenfold, AST/ALT ratio >1 with elevated serum bilirubin, and prothrombin time are often considered as a confirmative diagnosis of alcoholic hepatitis.[56] Treatment options are usually based on the severity of alcoholic hepatitis. In mild-to-moderate alcoholic hepatitis (Maddrey's discriminant function (MDF) score of <32 and Model for End-Stage Liver Disease score <20], patients are started on benzodiazepines (like diazepam and lorazepam) with routine thiamine administration.[6] In severe alcoholic hepatitis, glucocorticoids like prednisolone are the drug of choice and pentoxifylline are given at times. However, many controversies exist with the current treatment options as they failed to provide a remarkable benefit. Furthermore, some of these drugs are contraindicated in certain conditions while the others had developed resistance, and even the side effect profile is quite troublesome in a majority of the patients. This has necessitated the development of newer drugs for the treatment of alcoholic hepatitis.[6]

Phyllanthus, the largest genus of the family Phyllanthaceae, includes 750–1200 wide variety of species each having a remarkable diversity and action.[7] Phyllanthus niruri is one of the commonly used species of this family for the treatment of liver and kidney disorders, asthma, constipation, and many others.[8] It is an annual herbal plant widely distributed all over the world and especially in tropical regions. It is called by different names in various places. In India, it is commonly referred to as “punarnava,” in Malaysia – as “dukunganak,” Spain – as “chanca piedra,” Brazil – as “quebra pedra,” and in Paraguay – as “praparai mi.”[9] Its role as a hepatoprotective agent is quite beneficial in the field of traditional medicine and is in use for many years.[8] However, it has gained significant interest among the researchers only in the mid-1960s after a thorough research was initiated to identify the active phytochemical constituents and to explore their pharmacological actions. Some of these active biological constituents include lignans, flavonoids, glycosides, alkaloids, sterols, lipids, and phenylpropanoids that were isolated from different parts of the plant such as root, stem, and leaf and are beneficial.[10]

Several in vitro studies, preclinical studies, and clinical trials were conducted to assess the beneficial role of P. niruri, and many of these studies had shown promising results in hepatitis B virus (HBV) infection and kidney stones.[111213] However, they are not sufficient enough to gain conclusive evidence as the studies had poor methodological quality and a small sample size. Hence, to consider the use of P. niruri as a treatment option, well-designed studies with an adequate follow-up should be done. Furthermore, the selection of the herb is cumbersome due to other misleading species, resulting in a disparity in the authentication process in several regions of the world. With these associated lacunae, the available data on P. niruri is not confirmative with regard to efficacy and safety.[1415] With this background, the present study aimed to evaluate the efficacy of a 4-week administration of P. niruri on the improvement of liver functions in alcoholic hepatitis patients.

Materials and Methods

Trial design and ethics

The trial was a block-randomized, double-blind, parallel-arm, placebo-controlled phase 2 trial of P. niruri in patients with alcoholic hepatitis exclusively from a tertiary center, JIPMER, Puducherry. The trial protocol was approved by the Institute Ethics Committee (IEC, Human Studies), JIPMER (JIP/IEC/2016/1022, dated February 16, 2017), which follows the ethical guidelines for biomedical and health research set by the Indian Council of Medical Research, 2017. The protocol was registered with Clinical Trials Registry-India (CTRI/2017/05/008712). From the eligible patients willing to take part in the study, a written informed consent was taken before the trial initiation. A patient information sheet with all details of the study procedure was given to the participants. The intervention arm included P. niruri and the parallel-arm received placebo. The trial was conducted in Medicine and Psychiatry Outpatient Departments and Wards, and from the alcohol de-addiction counseling centers organized by the Department of Psychiatry, JIPMER, on every Tuesdays and Saturdays.

Study drugs

The dry extract of the entire herb of the P. niruri (Bhumi Amla, 10:1) was obtained from Konark Herbals and Healthcare, Mumbai, India (Batch No.: KH/PN/026/16-17 of July 2016) and is the Food and Drug Administration and Current Good Manufacturing Practices approved. The shelf life of the extract was 3 years, and it was obtained along with the certificate of analysis (Certificate No.: KHHC/16-17/08/556) after proper authentication and standardization. The pure extract was filled in empty capsules with the help of a capsule filling machine, and each unit dosage form was equivalent to 500 mg of P. niruri. The dose selection of P. niruri (dose: 500 mg twice daily which is equivalent to 1000 g/day) was done based on the previous literature and clinical expertise from an ayurvedic physician, considering the safety and efficacy parameters.[12131415]

The placebo used in our study was maize starch IP grade, especially used for pharmaceutical preparations and research purposes (Batch No.: STIP-1702314 of February 2017) from Sayaji Industries Ltd., Ahmedabad, India, with the certificate of analysis. Its shelf life was 5 years and was filled in similar-looking empty capsules of 500 mg each. Thirty capsules each of P. niruri and placebo were then packaged and sealed in identical bottles to be administered to the patients.

The plant extract and placebo were analyzed for microbial contamination (Escherichia coli, Salmonella, Shigella, yeast/molds) and heavy metals (lead, mercury, arsenic, and cadmium) and were complying with the standard values. The quantity of P. niruri used in our study was equivalent to the other commercial preparations commonly available in the market.

Trial population

Inclusion criteria

Alcoholic hepatitis patients of either gender aged 18–65 years with a present history of alcohol abuse as confirmed by CAGE questionnaire score ≥2

Patients with elevated liver enzymes levels, with AST/ALT ratio >2.

Exclusion criteria

Patients who are terminally ill (encephalopathy, spontaneous bacterial peritonitis, and hepatorenal syndrome) with severe alcoholic hepatitis (MDF ≥32)

Patients with viral and drug-induced hepatitis

Patients with comorbid conditions such as coronary artery disease and peripheral vascular disease

Pregnant women or lactating mothers and women in reproductive age group not practicing contraceptive measures

Patients who had participated in other trials in the past 3 months

Patients on herbal medications.

Trial procedures

The patients and the primary investigators were blinded during the entire study period. Following brief history taking and physical examination, participants were randomized to receive oral capsules of either P. niruri or placebo twice daily preferably after food for 4 weeks. Block randomization (block sizes 2, 4, and 6) with an allocation ratio of 1:1 was done using Statistical Analysis System software. Serially numbered sealed containers were used to obscure the allocation sequence from the primary investigator associated with the recruitment and assessment of the patients. Randomization and serial numbering of the containers were done by a third person, who was not a part of the study. Capsules of P. niruri and placebo were identical in appearance, color, size, and smell, and were prepacked in separate identical bottles and are numbered consecutively based on the randomization sequence. The blinding was decoded only after the study completion, and the analysis was performed.

Clinical assessments were done with serum at baseline, 2 weeks, and 4 weeks after study completion. At each visit, blood samples were collected for biochemical analysis and to measure the total oxidant and antioxidant capacity. Patients were encouraged to come to the hospital for their subsequent follow-up visits and were strictly advised to abstain from alcohol during the entire study period. During their follow-up visits, they are personally interviewed for symptomatic relief, other associated symptoms, and adverse drug reactions, if any, and were recorded. To check for adherence, patients were asked to bring the empty bottles during their follow-up visits and the capsules not taken were counted and the reasons for nontaking were noted. They were also reminded by telephonic conversation every week.

Outcomes

The primary endpoints were to assess the efficacy of a 4-week administration of P. niruri on normalization of hepatic transaminase enzymes (AST and ALT), improvement of alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), bilirubin, albumin, urea, creatinine, prothrombin time, and serum lipid profile (total cholesterol and triglycerides) in patients with alcoholic hepatitis in comparison to placebo. The secondary endpoints were to assess the effect of P. niruri on the total oxidant and antioxidant capacity in patients with alcoholic hepatitis.

Laboratory analysis

The serological investigations were carried out in Central Laboratory, JIPMER. AST, ALT, and GGT were measured by calorimetric assay kits, ALP by para-nitrophenyl phosphate method, bilirubin by Jendrassik and Grof method, albumin by bromocresol green method, urea by glutamate dehydrogenase method, creatinine by Jaffe (initial rate) method using alkaline picrate, total cholesterol by cholesterol oxidase peroxidase method, and triglycerides by GPO-POD (glycerol phosphate oxidase peroxidase) method. Apart from this, another 3 ml of blood was collected at each visit for measuring the total oxidant and total antioxidant capacity. The blood was centrifuged at 4000 RPM for 5 min. The serum thus separated was and stored in a deep freezer (−80°C) for further analysis by the enzyme-linked immunosorbent assay (ELISA) kit method (obtained from Shanghai Korean Biotech Co., Ltd; Brand: Smart Prime ELISA). The ELISA kit assays are based on the biotin double-antibody sandwich technology.[1617]

Statistical analysis

The values were expressed as mean ± standard deviation for parametric data and in terms of the median (interquartile range) for nonparametric data. Normality testing was done using Kolmogorov–Smirnov test. The measured parameters such as AST, ALT, ALP, GGT, urea, creatinine, albumin, bilirubin, prothrombin time, lipid profile (total cholesterol and triglycerides), and total oxidant and antioxidant levels at baseline, 2 weeks, and 4 weeks were compared between the two study groups by two-way repeated measures analysis of variance. The frequency of adverse reactions between the two groups was compared by Chi-square test. P < 0.05 was interpreted to be statistically significant. Post hoc analysis was done by Bonferroni test. Statistical analysis was performed using SPSS version 19.0 (IBM Corp., Armonk, N.Y., USA).

The sample size was calculated by using Power and Sample Size software version 3.1.2 (Nashville, TN, USA) for all the primary objectives, and the highest value was taken which was the proportion of patients with normalization of hepatic transaminase enzymes at the end of 4 weeks. For sample size calculation, there was no previous clinical data to compare between P. niruri and placebo in alcoholic hepatitis patients. Therefore, based on clinical expertise, a 30% better response with P. niruri when compared to placebo was considered clinically significant. With an alpha error (α) of 5% and power 80%, it was found that 42 subjects are required in each group. Upon inclusion of a dropout rate of 10% and nonadherence of 10%, the final estimated sample size was 50 subjects in each group. Modified intention-to-treat (mITT) approach was followed for analyzing the data. The last observation carried forward imputation method was used for the missing values of the parameters in the last (second) visit when the patient is either lost to follow-up or discontinued medication due to some other reasons before the study completion.

Results

Four hundred and fifty-four patients with a confirmed diagnosis of alcoholic hepatitis were screened and 100 patients meeting the inclusion criteria were recruited for the study. The study duration was 1 year and 5 months from July 2017 to November 2018.

The CONSORT flowchart is depicted in Figure 1.

Figure 1

The CONSORT flowchart of study participants

Fifty patients each were allotted to the study groups. Of those, 53 patients completed all the study-related visits, and based on mITT analysis, 34 patients in the P. niruri group (32% dropout rate) and 37 patients in the placebo group (26% dropout rate) were analyzed. Baseline demographic features of the participants were similar across both the groups [Table 1].

Table 1

Baseline demographic characteristics of the study participants across the trial groups

Baseline characteristics	Phyllanthus niruri (n=50)	Placebo (n=50)	P
Age (years)*	40.94±8.67	40.96±9.26	0.70
Male, n (%)	50 (100)	50 (100)	-
Height (m)*	1.65±0.08	1.66±0.08	0.63
Weight (kg)*	61.22±10.78	61.16±9.01	0.97
BMI (kg/m2)*	22.22±3.46	22.01±3.05	0.75
Smokers, n (%)	28 (56)	23 (46)	1.00
Family history of alcohol problems, n (%)	28 (56)	32 (64)	1.00
Alcohol consumption (units/day), n (%)
≤15	27 (54)	28 (56)	0.296
>15	23 (46)	22 (44)	0.829

*Data are expressed as mean±SD, other parameters are expressed as n (%). SD=Standard deviation

The patients during the entire trial period had received treatment with benzodiazepines (either lorazepam/diazepam) along with routine thiamine supplementation and Vitamin B complex. The clinical characteristics of the patients at baseline are given in Table 2. Almost all the baseline clinical characteristics were similar across the trial groups except for AST that showed a difference between the groups (P = 0.02).

Table 2

Baseline clinical characteristics of the study participants across the groups

Baseline characteristics	Phyllanthus niruri (n=50)	Placebo (n=50)	P
AST (U/mL)	137.5 (101-225.25)	200.5 (131-273.75)	0.02
ALT (U/mL)	81.5 (52.75-130.75)	88.5 (65-159.5)	0.11
ALP (U/mL)	177 (111.5-257.75)	149 (105.25-213)	0.46
GGT (U/mL)	255 (109-504)	332 (172-741)	0.10
Bilirubin (mg/dL)	1.13 (0.71-1.46)	1.52 (0.75-2.3)	0.09
Albumin (mg/dL)*	3.71±0.70	3.65±0.70	0.67
INR	1.12 (0.96-1.27)	1.09 (0.95-1.21)	0.89
Creatinine (mg/dL)	0.88 (0.77-1.03)	0.83 (0.74-0.97)	0.24
Urea (mg/dL)	17 (13-23.5)	16 (12-21)	0.34
Total cholesterol (mg/dL)	207 (152.75-245.25)	204 (161-254)	0.74
Triglycerides (mg/dL)	118.5 (89.75-160.25)	124 (93-191)	0.56
Total oxidant levels	3.18 (2.33-4.29)	3.2 (1.71-4.67)	0.87
Total antioxidant levels	10.26 (4.78-20.03)	13.25 (4.7-20.86)	0.48

*Values expressed as mean±SD, other data expressed as median (IQR). AST=Aspartate transaminase, ALT=Alanine transaminase, ALP=Alkaline phosphatase, GGT=Gamma-glutamyl transferase, INR=International normalized ratio, SD=Standard deviation, IQR=Interquartile range

Efficacy of Phyllanthus niruri on hepatic transaminases

Among the 100 patients recruited for the study, the data of 71 patients were analyzed by following the mITT approach. The hepatic transaminases, namely AST and ALT, were calculated and compared between the two groups considering the three different time points (baseline, 2 weeks, and 4 weeks) to assess the pharmacological effect of P. niruri if any. There was an improvement in hepatic transaminases between the groups over time with P. niruri although it was not statistically significant [Table 3].

Table 3

Outcome and comparison of liver and renal parameters between the study groups

Parameters	Phyllanthus niruri (n=34)		Placebo (n=37)		P
	2 weeks	4 weeks	2 weeks	4 weeks
AST	48.79±27.83	42.70±27.77	75.43±62.9	70.29±63.39	0.276
ALT	45.50±41.65	33.73±21.45	51.05±51	49.27±50.35	0.791
ALP	140.29±97.88	138.79±100	151.54±78.97	157.43±104.47	0.194
GGTP	152±130	117.10±115.55	184.08±148.19	153.80±181.52	0.286
Bilirubin	0.98±0.79	0.95±0.77	1.23±1.29	1.14±1	0.352
Albumin	3.83±0.57	3.76±0.67	3.74±0.65	3.75±0.60	0.361
INR	1.10±0.21	1.17±0.54	1.17±0.34	1.15±0.29	0.469
Creatinine	0.95±0.29	0.95±0.30	0.86±0.13	0.86±0.14	0.976
Urea	18.20±10.43	19.05±8.53	15.08±4.60	16.94±7.4	0.687
Total cholesterol	170.64±60.49	165±45.33	179.83±45.76	177.77±34.98	0.942
Triglycerides	130.44±66.59	134.23±87.28	156.43±162.43	159.54±198.55	0.723

Data expressed as mean±SD. Test statistics: Two-way repeated measures ANOVA (done for three time points: Baseline, 2 weeks, and 4 weeks). AST=Aspartate transaminase, ALT=Alanine transaminase, ALP=Alkaline phosphatase, GGTP=Gamma-glutamyl transpeptidase, INR=International normalized ratio, SD=Standard deviation

Efficacy of Phyllanthus niruri on other liver and renal parameters

The other liver and renal parameters that are implicated in liver diseases such as ALP, GGTP, serum bilirubin, serum albumin, international normalized ratio (INR), serum creatinine, urea, total cholesterol, and triglycerides were also evaluated to assess the actual effect of P. niruri on these parameters. There was no significant difference between the study groups [Table 3].

Efficacy of Phyllanthus niruri on total oxidant and antioxidant status

The effect of P. niruri on the levels of total oxidants and antioxidants was assessed between the trial groups. It was found that there was an increase in the level of total antioxidants, which was statistically significant [Table 4]. Furthermore, during the study period, some of the participants reported increased appetite. The comparison of appetite-stimulating activity across the trial groups is shown in Table 5.

Table 4

Outcome and comparison of total oxidants and antioxidants between the study groups

Parameters	Phyllanthus niruri (n=34)		Placebo (n=37)		P
	2 weeks	4 weeks	2 weeks	4 weeks
Total oxidants	3.72±3.07	4.32±3.02	3.55±1.77	3.61±1.91	0.298
Total antioxidants	13.67±10.94	14.45±10.43	12.44±7.39	11.83±7.01	0.034*

*Indicates P<0.05. Data expressed as mean±SD. Test statistics: Two-way repeated measures ANOVA (done for three time points: Baseline, 2 weeks, and 4 weeks). SD=Standard deviation

Table 5

The appetite-stimulating activity between the study groups

Parameter	Phyllanthus niruri (n=34)*	Placebo (n=37)*	P
Appetite stimulation	9 (21.42)	3 (9.37)	0.03$

*Data expressed as n (%), $P<0.05. Test statistics: Chi-square test

Safety

The prevalence of adverse events in the P. niruri and placebo groups was 57% and 43%, respectively [Table 6]. Nausea (32.35%) and vomiting (26.47%) were the most common adverse reactions reported in the P. niruri group. One serious adverse event (SAE) was reported in a patient of the P. niruri group 1 month after study completion. The patient is a case of type II diabetes mellitus with chronic alcoholism-related liver disease and was admitted to the hospital. He later developed bilateral subdural and subarachnoid hemorrhage resulting in the death of the individual. The SAE was reported to the IEC, and the causality assessment was done categorizing it as “unlikely.”

Table 6

Distribution of adverse events in the study groups

AE	Phyllanthus niruri (n=34)*	Placebo (n=37)*	P
Nausea	11 (32.35)	4 (10.81)	0.026$
Vomiting	9 (26.47)	3 (8.11)	0.039$
Gastritis	4 (11.76)	5 (13.51)	0.824
Fatigue	4 (11.76)	3 (8.11)	0.605
Abdominal pain	2 (5.88)	5 (13.51)	0.28
Visual impairment	2 (5.88)	0	-
Decreased appetite	0	5 (13.51)	-
Hiccups	0	1 (2.70)	-
Facial puffiness	0	1 (2.70)	-
Hypoglycaemia	0	1 (2.70)	-

*Data expressed as number of AE (%), $P<0.05. Test statistics: Chi-square test. AE=Adverse events

Discussion

P. niruri, one of the traditional herbal medicines, is commonly used for the treatment of several liver and kidney disorders, particularly HBV infection and urolithiasis with no reported SAEs to date.[12] It was also found to have a potent antioxidant effect characterized by a modest increase in the levels of ascorbic acid and gallic acid when administered to healthy individuals.[18] However, a majority of these studies were not reliable to give conclusive evidence and to establish P. niruri as a treatment option due to their low methodological quality, small sample size, and inadequate duration for the conduct of study and follow-up, and lacking to provide proper details on randomization and allocation concealment techniques followed in these studies.[12]

In our study, the hepatoprotective, renoprotective, and antioxidant role of P. niruri was evaluated. It was found that a 4-week administration of P. niruri extract to patients with mild-to-moderate alcoholic hepatitis did not result in a significant improvement in the levels of AST, ALT, ALP, GGTP, albumin, bilirubin, INR, renal parameters (creatinine and urea), total cholesterol, triglycerides, and total oxidants. There was an increase in total antioxidant levels over 4 weeks with P. niruri compared to placebo. Furthermore, the appetite stimulant activity of P. niruri in comparison to placebo was observed.

In the current study, the baseline AST levels were not similar across the study groups and showed a statistically significant difference. This is because a large number of patients with AST levels >400 were allocated to the placebo group. Over a period of 4 weeks, we did not observe any difference in hepatic transaminases between the groups. However, contrary to our results, a few studies suggested the hepatoprotective role of P. niruri. Phytoconstituents in P. niruri such as phyllanthin, triacontanol, and hypophyllanthin demonstrated a significant hepatoprotective effect by hepatic regeneration as evidenced by few preclinical studies.[19] This too suggests the healing properties of the liver with P. niruri that can play a role in improving the levels of hepatic transaminases. Another preclinical study conducted using a standardized extract of P. niruri evaluated its effects on liver and renal function parameters in rats with nonalcoholic fatty liver disease. This extract was found to significantly reduce the AST/ALT ratio by halting hepatic steatosis and inflammation.[11] Another systematic review done on Phyllanthus species in chronic HBV infection had shown a restoration in the levels of AST and ALT to normal in the Phyllanthus group favoring treatment with these herbal preparations.[12]

A recent study done in chronic HBV infection demonstrated that there was no significant improvement in the levels of either AST or ALT even after P. niruri administration for about 12 months which is in agreement with the results of the present study. However, it is rather tough to give conclusive evidence regarding its hepatoprotective action.[15] Hence, numerous larger trials of longer duration with an adequate sample size need to be conducted.

Our study showed that P. niruri administration helped to increase the appetite when compared to placebo. In general, liver injury is commonly associated with a loss of appetite. As there is an improvement in liver function parameters over time with P. niruri, this might be one of the reasons for increased appetite. Second, the extract might exert an effect on the mitochondria promoting their regeneration (or preventing their apoptosis), thereby increasing their efficiency and improving the appetite. A similar finding was observed in a study done by Oakes and Morris, in which an oral administration of hot water extract of P. niruri was found to improve the appetite.[20] Even traditionally, the entire plant extract is used for many years to treat poor appetite in adults by administering it either orally or in the form of a decoction.[21] A similar effect on appetite was seen even in children during postmarketing surveillance receiving a polyherbal preparation containing P. niruri for treating anorexia. It was found to act as a long-term therapy for appetite stimulation, and is considered to be safe with good tolerability. The author suggested that the appetite stimulant effect is due to the hepatoprotective potential of the plant.[22] In our study, an appetite stimulant activity was observed, suggesting that P. niruri might have a role in hepatic regeneration and thereby improving the liver functions and appetite. However, the levels of the hepatic transaminases were not significantly improved.

In our study, the administration of P. niruri did not improve the levels of other liver function parameters such as ALP, GGTP, albumin, bilirubin, and INR significantly. A study was done by Pucci et al. in patients with urolithiasis found that a daily administration of P. niruri for 12 weeks caused a reduction in ALP levels. The authors claimed it to be beneficial in kidney stones as ALP, a marker of bone metabolism when reduced might help in reducing the formation of calculi.[13] As our present study has a shorter treatment duration (4 weeks), this might be one of the reasons for not showing a significant improvement of ALP though it had gradually reduced over time when compared to placebo. Even in the case of bilirubin, its serum levels decreased over time in the P. niruri group when compared to placebo though not statistically significant. Previous studies with Phyllanthus species demonstrated their role in the treatment of jaundice. The underlying mechanism is due to a reduction in mRNA levels, thereby suppressing HBV and improving the liver functions to resolve jaundice, especially in HBV infections.[23] However, a majority of the conducted studies especially highlighted the role of Phyllanthus amarus in jaundice while the other studies have used the terms P. amarus and P. niruri interchangeably. Hence, the reliability of these results is not confirmatory as the species identification followed was not the same globally. It was also revealed that lignans such as phyllanthin and hypophyllanthin might be responsible for the hepatoprotective effect. Hence, to consider P. niruri as a treatment option, more randomized controlled trials should be conducted with proper authentication of species and considering its active phytoconstituents to determine its effect on serum bilirubin if any.

Renal function parameters such as uric acid and creatinine showed no improvement with the administration of P. niruri in the present study, and this finding was supported by a few other studies conducted previously. A preclinical study done in a rat model of urolithiasis demonstrated that there was no modification in the uric acid levels with P. niruri administration for 42 days.[24] Another study done in urolithiasis patients revealed that the administration of P. niruri (450 mg TDS) for 3 months showed no significance in the levels of uric acid and creatinine when compared to placebo.[25] A recent study done in patients with chronic HBV infection also failed to show any significant changes in the levels of serum creatinine when compared to a placebo.[15]

The study demonstrated that P. niruri administration showed a modest reduction in the levels of total cholesterol alone. Previous studies in streptozotocin- and alloxan-induced diabetic rodent models revealed that P. niruri extract caused a significant decrease in serum cholesterol and triglycerides. The authors claimed that the decrease in serum triglycerides is due to their decreased synthesis or inhibition of their release from the liver, and the decrease in cholesterol is due to the presence of active phytochemicals in P. niruri that promote the excretion of cholesterol through feces.[2627] To our knowledge, no well-designed trials are conducted to date to especially evaluate the hypolipidemic action of P. niruri in humans. Although our current study showed an improvement in the total cholesterol over time, a further increase in the daily dosing of P. niruri with a longer duration of administration might be beneficial to produce a significant benefit.

P. niruri extract showed a significant improvement in the level of total antioxidant levels (P = 0.034). Preclinical studies in rodents demonstrated that chronic ingestion of P. niruri extract could enhance the antioxidant systems, increasing the levels of superoxide dismutase (SOD), catalase, glutathione peroxidase, and Vitamin C. The authors inferred that the beneficial effect is result of the inhibition of lipid peroxidation by P. niruri.[28] Following this, another study was done to evaluate the antioxidant effects in healthy volunteers in which there was a modest increase in antioxidant markers such as ascorbic acid and gallic acid, but other antioxidants such as SOD and catalase and oxidants such as malondialdehyde were not altered. In the present study, the antioxidant level showed an improvement over time; a further increase in the dose of P. niruri with chronic administration might be of benefit to assess its actual effects as an antioxidant modulator.

P. niruri extract was found to be safe and was quite tolerable in most of the patients with alcoholic hepatitis. The most common adverse events recorded in our study were nausea, vomiting, gastritis, fatigue, and abdominal pain. Of these, nausea and vomiting were related to P. niruri treatment and were found to be significant when compared to placebo. However, these adverse events have already been reported with this herb in a systematic review comprising about 22 trials.[12] Furthermore, there was no treatment discontinuation in any of the patients due to adverse events in our study.

The limitations of our present study include (i) a shorter duration of treatment; warranting the confirmation of results with a trial of longer duration (at least 3 months) to translate these modifications into a clinical benefit; (ii) allocation of more number patients with AST levels >400 to the placebo group has led to baseline differences between the two study groups; stringent inclusion criteria recruiting patients with AST levels <400 might have been beneficial (as AST >400 is also suggestive of concomitant liver disease) to overcome the baseline variations; (iii) possibility of alcohol consumption by the patients during the trial period, though strictly advised on alcohol abstinence; and (iv) no adjustments were done for multiple comparisons.

Going forward, future studies can be conducted with active principles of P. niruri such as phyllanthin and hypophyllanthin as these have already demonstrated their hepatoprotective action. Generating robust evidence through such studies might help in coming up with new drugs for hepatoprotection and in the process might as well elucidate the mechanism of action of these active principles.

Conclusion

P. niruri extract at a dose of 500 mg twice a day for 4 weeks showed an improvement in the total antioxidant level with an appetite stimulant activity in mild–moderate alcoholic hepatitis patients compared to placebo. However, it failed in demonstrating a remarkable improvement in the liver and renal function parameters. It was relatively safe with a good tolerability profile and no SAEs attributed to P. niruri. However, long-term studies in a larger population should be conducted to confirm our findings in patients with mild–moderate alcoholic hepatitis.

Financial support

Financial support was provided by the Institutional Intramural Research Grant (JIP/Res/Intra-MD/phs2/2016-17), JIPMER, Puducherry.

Conflicts of interest

There are no conflicts of interest.