Extract of a polyherbal formulation ameliorates experimental nonalcoholic steatohepatitis.

The objective of the present study is to evaluate the effect of the extract of a well-known hepatospecific polyherbal formulation, Liv.52, in an experimental model of high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) in rats. Feeding a HFD for 15 weeks resulted in significant impairment of the lipid profile, elevation of hepatic enzyme markers, and insulin resistance in rats. The histological examination of the liver furthermore indicated fibrotic changes and fat deposition in hepatic tissues. The treatment with Liv.52 extract [125 mg/kg body weight per os (b.wt. p.o.)], which was administered from week 9 onward, reversed the HFD-induced changes to a statistically significant extent, compared to the untreated positive control animals. The effect observed with Liv.52 extract was comparable to that of pioglitazone (4 mg/kg b.wt.), a standard drug that is useful in the management of NASH. The treatment with Liv.52 extract significantly reduced steatosis, collagen deposition, and necrosis in hepatic tissues, which indicates its antifibrotic and antinecrotic properties. The results obtained in the present set of experiments indicate that Liv.52 extract effectively reverses metabolic and histological changes associated with HFD-induced NASH.

Introduction

The liver, which is the key organ of metabolism and excretion, is constantly endowed with the task of detoxifying xenobiotics, environmental pollutants, and chemotherapeutic agents. Disorders associated with this organ are numerous and varied. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the most common liver diseases in the world. The mechanism involved in the pathogenesis of NAFLD/NASH has not been thoroughly investigated. Some studies show that insulin resistance has a key role in their pathogenesis. The exact mechanisms that mediate the transition from steatosis to NASH remain unknown, although oxidative stress and cytokine-mediated injuries may have a key role in NASH pathogenesis.1, 2 Under oxidative stress conditions, reactive oxygen species (ROS) lead to membrane lipid peroxidation, inflammatory responses, stimulation of stellate cells, and finally fibrosis.

Modern medicine has not yet found a promising curative agent. Hence, the current use of peroxisome proliferator-activated receptor gamma (PPAR-γ), corticosteroids, and immunosuppressive agents only produce symptomatic relief. Furthermore, their usage is associated with the risks of relapse and adverse effects.4, 5, 6

The indigenous system of medicine in India has a long tradition of treating liver disorders with plant drugs. Liv.52 is one such polyherbal proprietary formulations. It is approved by the Government of India's Drug Regulatory Authority, the department of Ayurveda, Yoga, Naturopathy, Unani, Siddha and Homoeopathy (AYUSH) of the Ministry of Health and Family Welfare (New Delhi, India). The usefulness of Liv.52 in liver disorders of various etiologies is confirmed by a substantial number of clinical trials conducted across the globe. It is also beneficial in treating viral hepatitis, drug-induced hepatic damage, and alcoholic liver disorders.7, 8, 9, 10 The present study aimed to evaluate the effect of Liv.52 extract in an experimental model of high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) in rats. In the present study, pioglitazone, a standard reference drug that further validates the experimental model/procedure, was used for comparative study. The HFD model is a very appropriate model to induce NASH, obesity, and insulin resistance. “Overnutrition” with carbohydrates or fats or both may lead to obesity-related NAFLD. Insulin resistance and obese diabetes do not solely initiate steatohepatitis, but they do contribute to the progression of the entire spectrum of pathology of steatohepatitis, at least partly, via the upregulation of genes involved in lipogenesis, inflammation, and fibrogenesis. Agonists of PPAR-γ reportedly prevent the progression of NASH in a dietary model of NASH associated with obese diabetes.

Composition and preparation of Liv.52 extract

The Liv.52 extract constitutes a mixture of roots of Capparis spinosa (續隨子根 xù suí zǐ), seeds of Cichorium intybus (菊苣 jú jù), whole plant of Solanum nigrum (龍葵 lóng kuí), Terminalia arjuna (bark), seed of Cassia occidentalis (望江南 wàng jiāng nán), Achillea millefolium (洋蓍草 yáng shī cǎo; aerial plant), and Tamarix gallica (whole plant). These crude herbal materials were subjected to pulverization to obtain their coarse powders, and then blended. They were then soaked in pure water and boiled in a steam-jacketed stainless steel reactor until the extraction was complete. The liquid extract thus obtained was filtered through a muslin cloth and concentrated in the reactors to attain 30% total solids. The soft extract was then subjected to spray drying to obtain the dry extract powder. It was tested for its quality and consistency at each manufacturing step per the accepted principles of good manufacturing practice (GMP) and good laboratory practice (GLP). Its botanical identification, quality parameters, and Ayurvedic criteria complied with the international guidelines and pharmacopoeial standards.12, 13

High-performance liquid chromatography and liquid chromatography–mass spectrometry/mass spectroscopy fingerprinting of Liv.52 extract

Sample preparation and liquid chromatographic conditions

The concentration of the Liv.52 extract (DD-100) was 10 mg/mL in methanol. Liquid chromatography was performed by the Shimadzu LC-20AD series pump (Shimadzu Corporation, Kyoto, Japan) and DUG-20A3 series Shimadzu degasser (Shimadzu Corporation, Kyoto, Japan). Chromatographic separation was performed on the Luna C18 column (250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA). For the separation, the mobile phase gradient consisted of water (J.T. Baker brand; Avantor Performance Materials, Inc., Center Valley, PA, USA) with 10mM ammonium acetate and 0.1% formic acid in pump A and acetonitrile (J.T. Baker brand; Avantor Performance Materials, Inc.) in pump B. The linear gradient program was set as follows: 0–27 minutes of 20% of acetonitrile to 80% acetonitrile (linear); 27–30 minutes of 80% acetonitrile to 20% acetonitrile (linear); followed by 30 minutes of 20% acetonitrile (isocratic) equilibration period for 2 minutes, delivered at a flow rate of 0.6 mL/min and a run time of approximately 32 minutes. Peak elution was monitored at 245 nm and 360 nm through the Shimadzu SPD-20A UV/VIS detector (Shimadzu Corporation). The injection volume of 20 μL was injected through the SIL-HTC Shimadzu Autosampler (Shimadzu Corporation). The ambient temperature was achieved through a CTO-10 AS VP column oven (Shimadzu Corporation) at 25 °C.

Mass spectrometric conditions

The API 2000 mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada) was coupled with an electron spray ionization source and a chromatographic system. Batch acquisition and data processing were controlled by Analyst 1.5 version software.

Optimization of the mass spectroscopy (MS) parameters was performed with 2 mg/mL of test solutions that were prepared separately and diluted in methanol (J.T. Baker brand; Avantor Performance Materials, Inc.). The intensity response was checked in the positive ionization mode and negative ionization mode. The intense response was good in the positive mode. Interface conditions such as the declustering potential (40 V), ion spray voltage (3500 V), nebulizing gas [GS1 (55 psi) and GS2 (65 psi)], curtain gas (25 psi), focusing potential (400 V), entrance potential (10 V), and source temperature (420 °C) were optimized by multiple runs through liquid chromatography. Acquisition was performed by setting the mass of the analytes with the appropriate scan range. (Fig. 1).

Fig. 1

The chromatograms of Liv.52 extract by (A) high-performance liquid chromatography (LC) and (B) mass spectroscopy (MS).

Materials and methods

Drugs and chemicals

Liv.52 extract was procured from the Phytochemistry Department of The Himalaya Drug Company (Bangalore, India) and pioglitazone was procured from Sun Pharmaceuticals Industries Ltd. (Mumbai, India). Cholic acid and cholesterol were procured from HiMedia Laboratories Ltd. (Mumbai, India). All other chemicals used in the study were of reagent/analytical grade from reputed suppliers. Biochemical parameters such as glucose, cholesterol, triglycerides (TGs), serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and alkaline phosphatase (ALP) were estimated in a biochemistry autoanalyzer (EM-360; Erba Diagnostics Mannheim GmbH, Mannheim, Germany) using ready-made assay kits (Erba kits; Erba Diagnostics Mannheim GmbH). Insulin was estimated using a radioimmunoassay kit (RIAK-1) supplied by Bhabha Atomic Research Centre/Board of Radiation and Isotope Technology (BARC/BRIT; Mumbai, India).

Experimental animals

Inbred male Wistar rats (260–280 g) were housed under standard conditions of temperature (22 ± 3 °C) at a relative humidity of 55 ± 5% and 12-hour light/dark cycle prior to and during the study. The normal group animals were fed a standard pellet diet (Provimi Animal Nutrition India Pvt. Ltd., Bangalore, Karnataka, India), and water ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of The Himalaya Drug Company (Bangalore, India). The animals received humane care as prescribed by the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India (New Delhi, India).

Composition of HFD

The HFD consisted of 87.5% normal laboratory rodent feed, 10% animal fat, 0.5% cholic acid, and 2% cholesterol.

HFD-induced nonalcoholic steatohepatitis

Male Wistar rats (n = 36) that weighed 260–280 g were randomly divided into four groups consisting of nine animals each. Group I rats received a normal diet and all other groups received HFD for 60 days. After 60 days, blood was collected from the retro-orbital plexus under mild ether anesthesia to determine the lipid profile and to ensure that all rats in the HFD-fed groups were hyperlipidemic. After confirmation, the rats were segregated. All rats received an equal amount of water (10 mL/kg b.wt.) as a vehicle. Group I (i.e., animals fed a normal diet) served as normal control, Group II (i.e., animals fed a HFD) served as the positive control, and Group III rats received Liv.52 extract (125 mg/kg b.wt.) along with HFD. The dose was selected based on dose range finding studies performed in the laboratory to evaluate its hepatoprotective activity in rats; the dosing volume was determined by the weekly mean group body weight of the animals. Group IV rats ingested a HFD and received pioglitazone (4 mg/kg b.wt.).

Each group received its respective assigned treatment orally once daily for 42 days. Daily clinical observations and the weekly body weights of the animals were recorded for all groups. At the end of the treatment period, the animals underwent 12-hour fasting. Their blood was collected from the retro-orbital plexus under mild ether anesthesia to estimate biochemical parameters such as glucose, insulin, cholesterol, TG, SGPT, SGOT, and ALP. After collecting their blood, the rats were euthanized by deep ether anesthesia. A small portion of their liver was fixed in formalin, subjected to routine histological procedures, and stained with hematoxylin and eosin (H&E) and Masson trichrome.15, 16, 17

The serum insulin was estimated by standard radioimmunoassay technique using a kit (RIAK-1) supplied by BARIC/BRIT (Mumbai, India). The degree of insulin resistance was estimated by using the homeostasis model assessment (HOMA) as the index of insulin resistance. The formula for the HOMA index is:

Statistical analysis

All values are expressed as the mean ± standard error of the mean (SEM). The results were statistically analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's post test, using Prism GraphPad 4.03 software, Windows version (GraphPad Software Inc., San Diego, CA, USA). A p value <0.05 was considered statistically significant.

Results and discussion

The present study evaluated the effect of Liv.52 extract in an experimental model of HFD-induced NASH in rats. Feeding animals a HFD significantly impaired the lipid profile and elevated the level of serum insulin, glucose, and liver function marker enzymes SGPT, SGOT, and ALP. The changes were statistically significant, compared to the normal controls (i.e., animals fed a normal diet). Treatment with the Liv.52 extract was initiated at the end of 9th week of HFD supplementation. Group IV animals were treated with pioglitazone, which was used as the reference standard.

Forty-two days of treatment orally with Liv.52 extract (125 mg/kg) reversed the elevated serum markers SGOT, SGPT, ALP, and reversed the cholesterol and TG levels to a statistically significant extent, compared to the untreated HFD control animals. The oral administration of pioglitazone (4 mg/kg) also decreased the aforementioned serum parameters to a statistically significant level, compared to the positive untreated control (Table 1).

Table 1

The serum biochemical parameters of the rats.

	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)	Cholesterol (mg/dL)	TG (mg/dL)	Fasting glucose (mg/dL)	Insulin (μU/mL)	HOMA index
Normal control	152.3 ± 5.7	69.8 ± 1.7	164.3 ± 10.3	97.88 ± 3.7	117.4 ± 14.3	64.33 ± 3.3	27.93 ± 3.5	3.857 ± 0.44
Positive control (untreated)	199.6 ± 6.7b	88.5 ± 5.1a	272.4 ± 25.4b	146 ± 10a	174.3 ± 12.4a	78.5 ± 5.2a	52.71 ± 3.6b	7.678 ± 0.44a
Liv.52 extract (125 mg/kg p.o.)	163.6 ± 7.5c	71.7 ± 6.2c	190.5 ± 12.6d	113.1 ± 8d	106.9 ± 12d	48.43 ± 2.8d	35.33 ± 2.5c	4.686 ± 0.43d
Pioglitazone (4 mg/kg p.o.)	163.0 ± 18.0c	69.4 ± 2.6d	201.2 ± 13.2c	116.8 ± 6.3c	144.5 ± 21.6c	48 ± 3.1d	34.00 ± 6.23c	4.671 ± 0.43d

All values are expressed as the mean ± the standard error of the mean (SEM).

ALP = alkaline phosphatase; HOMA = homeostasis model assessment; p.o. = per os; SGOT = serum glutamic oxaloacetic transaminase; SGPT = serum glutamate pyruvate transaminase; TG = triglyceride.

Indicates significance at p < 0.05, compared to the normal control.

Indicates significance at p < 0.01, compared to the normal control.

Indicates significance at p < 0.05, compared to the positive control.

Indicates significance at p < 0.01, compared to the positive control.

Insulin resistance that developed because of the intake of a HFD was also decreased in animals treated with Liv.52 extract or pioglitazone. This difference was statistically significant, compared to the positive untreated HFD animals. Liv.52 extract and pioglitazone both decreased the fasting serum glucose, the insulin level, and the HOMA index, which are measures of insulin resistance (Table 1).

The histopathological examination and scoring of the liver sections (stained by H&E and Masson trichome) also revealed the beneficial effect of Liv.52 extract and pioglitazone in reversing many of the pathological changes associated with a HFD in rats. Most notably, the drugs significantly reduced collagen deposition, necrosis, and steatosis in hepatic tissues, which are hallmarks of NASH (Table 2 and Fig. 2, Fig. 3). Nonalcoholic steatohepatitis refers to a spectrum of hepatic pathology that resembles alcoholic liver disease, but appears in individuals who have low or negligible alcohol consumption. Nonalcoholic fatty liver disease ranges from a fatty liver alone to NASH. Increasing evidence suggests that NASH is associated with progressive fibrosis, cirrhosis, and eventually hepatocellular cancer. It has been suggested that the term “nonalcoholic fatty liver disease” should be used only for the more severe forms of NAFLD that correspond to types 3 and 4 with alcoholic-like histological findings. The medical conditions most frequently associated with NAFLD and NASH are obesity, diabetes mellitus, and dyslipidemia.

Table 2

Histopathological findings of the rat liver samples among the four groups.

	Normal control	Positive control (untreated)	Liv.52 extract (125 mg/kg b.wt.)	Pioglitazone (4 mg/kg b.wt.)
H&E stain
Inflammatory reaction	0.778 ± 0.32	2.000 ± 0.29a	1.444 ± 0.24	1.333 ± 0.33
Biliary hyperplasia	0.556 ± 0.29	2.444 ± 0.34b	0.222 ± 0.22d	0.667 ± 0.33d
Fat vacuolating accumulation in parenchyma	0.000 ± 0.00	3.333 ± 0.21b	2.857 ± 0.50	1.714 ± 0.47c
Necrosis in parenchyma	0.000 ± 0.00	1.571 ± 0.28b	0.428 ± 0.29d	0.222 ± 0.15d
Masson’s trichrome stain
Collagen deposition	0.667 ± 0.29	3.500 ± 0.18b	1.714 ± 0.29d	1.875 ± 0.44d
Fat vacuolating accumulation	0.000 ± 0.00	3.333 ± 0.21b	2.857 ± 0.50	1.714 ± 0.47c

H&E Stain (score):

Inflammatory reaction (score of 1–3)

Biliary hyperplasia (score of 1–3)

Fat vacuolating accumulation (score of 1–4)

Necrosis in parenchyma [score-focal (1) and multi-focal (2)]

Masson's trichrome Stain (score):

Collagen deposition (score of 1–4)

Fat vacuolating accumulation in the parenchyma (score of 1–4)

All values are expressed as the mean ± the standard error of the mean (SEM).

b.wt. = body weight; H&E = hematoxylin and eosin.

Indicates significance at p < 0.05, compared to the normal control.

Indicates significance at p < 0.01, compared to the normal control.

Indicates significance at p < 0.05, compared to the positive control.

Indicates significance at p < 0.01, compared to the positive control.

Fig. 2

Photomicrographs of the liver sections show (A) normal structure and architecture in the normal control animals fed a normal diet; (B) severe microvesicular and macrovesicular steatosis in the positive control animals fed a high-fat diet; (C) Grade 2 steatosis in animals fed a high-fat diet and treated with Liv.52 extract at a dose of 125 mg/kg body weight; and (D) Grade 2 steatosis in animals fed a high-fat diet and treated with piglitazone at a dose of 4 mg/kg body weight (hematoxylin and eosin; magnification, 100×).

Fig. 3

Photomicrographs of liver sections show(A) no collagen deposition in the normal control animals fed a normal diet; (B) severe perivascular edema and collagen deposition in the positive control animals fed a high-fat diet; (C) Grade 2 collagen deposition around the periportal zone and perivascular necrosis and edema in animals treated with Liv.52 extract (12 mg/kg body weight); and (D) mild steatosis and collagen deposition in animals treated with picoglitazone (4 mg/kg body weight) (Masson's trichome; magnification, 100×).

The HFD model is useful in reproducing the human condition because it contains predominantly saturated fat intake, which is positively correlated with the insulin sensitivity index, and alanine aminotransferase levels in patients with NASH. Peripheral insulin resistance can induce hepatic steatosis, which then causes insulin resistance in the liver and is characterized by a reduced insulin-suppressing effect in hepatic glucose production; this aggravates peripheral insulin resistance and contributes to hepatic lipogenesis.

Insulin resistance is a complex metabolic abnormality that affects the ability of peripheral tissues to respond to insulin. It is a prominent feature of metabolic syndrome and type 2 diabetes, and is a major risk factor for cardiovascular disease. There may be a close interplay between increased tumor necrosis factor (TNF)-α expression, insulin resistance, and lipolysis in adipose tissue that results in the increased delivery of free fatty acids, TNF-α, and cortisol to the liver, which initially lead to the development of steatosis. In patients with NAFLD, increased fat accumulation eventually results in hepatic insulin resistance. The insulin resistance increases fat oxidation, which in association with TNF-α, generates oxidative stress and mitochondrial uncoupling. The expression of TNF-α and mitochondrial dysfunction leads to hepatocyte death, inflammation, and ultimately fibrosis.22, 23

In the present study, the effect of Liv.52 extract was compared with pioglitazone, which was used as the reference standard in a rat model of NASH. Previous reports indicate the usefulness of pioglitazone in an experimental model of NASH in rats. Pioglitazone is also a reasonable option for treating NASH, particularly in patients with type 2 diabetes mellitus or impaired glucose tolerance. Pioglitazone therapy was associated with reduced hepatocellular injury and fibrosis in patients with NASH. However, the cardiovascular benefit–risk ratio of this drug must at least be considered.

Oxidative stress may contribute to the development of several age-related and chronic diseases such as cancer, diabetes, neurodegenerative diseases, and cardiovascular diseases.26, 27, 28 In particular, ROS and reactive nitrogen species have a crucial role in disease induction and in the progression of liver diseases such as hepatocellular carcinoma, viral and alcoholic hepatitis, and nonalcoholic steatosis.29, 30 In our previous studies, Liv.52 extract exhibited a potent beneficial effect in regulating oxidative stress induced by various liver toxicants. It abrogates copper (II)-induced cytotoxicity in HepG2 cells by inhibiting lipid peroxidation, increasing the glutathione content, and increasing endogenous antioxidant enzyme activities. By preventing intracellular glutathione depletion and lipid peroxidation, it also reportedly inhibits hepatic cell death evoked by tertiary-butyl hydroperoxide.

There are other reports that indicate the usefulness of plant-based antioxidants in the management of NASH. Genistein is a strong antioxidant agent that is capable of decreasing the plasma TNF-α level and attenuating the incidence of NASH. A Chinese polyherbal blend comprising Panax pseudoginseng (三七 sān qī), Eucommia ulmoides (杜仲 dù zhòng), Polygonati rhizoma (黃精 huáng jīng), and licorice root (甘草根 gān cǎo gēn) reportedly markedly reduces macrosteatosis while significantly improving histological markers of inflammation in HFD-fed experimental animals. These effects have also been associated with a marked reduction in serum aminotransferases and hepatic lipoperoxide and glutathione content, which implies that the herbal complex has noticeable antioxidant activity.

Nonalcoholic steatohepatitis is histologically characterized by diffuse fatty infiltration, lobular inflammation, ballooning degeneration, and fibrosis in the liver.35, 36 In the present study, many liver histopathological features of animals fed a HFD were similar to the findings of other published reports, and the treatment by Liv.52 extract or pioglitazone significantly reversed these histopathological anomalies (Fig. 2, Fig. 3). The Liv.52 extract treatment significantly reduced steatosis, collagen deposition, and necrosis of hepatic tissues, which indicate its antifibrotic and antinecrotic properties. Increased mean body weight occurred in the animals treated a HFD, compared to the normal control group; however, this difference was not statistically significant. The treated groups showed a marginal decrease in mean body weight in comparison to the positive untreated controls; however, this finding was similarly insignificant (Fig. 4).

Fig. 4

The mean body weight change in the different groups.

The results of the present study showed that the treatment with the Liv.52 extract was comparable to pioglitazone at the selected dose, by the method employed, and by the mode of administration. The mechanism behind the beneficial action of Liv.52 extract in the present experimental model of NASH could be because of its potent antioxidant and other hepatospecific actions, as reported previously.

Conclusion

From the biochemical and histopathological data obtained in the present study, the treatment by Liv.52 extract administered orally at a daily dose of 125 mg/kg b.wt. exhibited a beneficial effect by reversing serum biochemical parameters in rats with NASH induced by a HFD. The Liv.52 extract treatment also significantly reduced steatosis, collagen deposition, and necrosis in hepatic tissues, which indicate its antifibrotic and antinecrotic properties. The effect of Liv.52 extract was comparable to that of pioglitazone at the selected dose, by the method employed, and by the mode of administration. The mechanism behind the beneficial action of Liv.52 extract in the present experimental model of NASH could be because of its potent antioxidant and other hepatospecific actions, as previously reported in the literature.

Conflicts of interest

The authors declare no conflicts of interest and guarantee no ethical conflicts among the authors or the experimental methodology. The test drug (i.e., Liv.52 extract) was received in a coded form from the Phytochemistry Department of The Himalaya Drug Company (Bangalore, India) and all experiments were performed in the Department of Pharmacology at the Research & Development Center of The Himalaya Drug Company (Bangalore, India). No formal funding from any agency was used for this project.