Livogrit Ameliorates Thioacetamide-Induced Liver Fibrosis and Dyslipidemia in Rat Model by Regulating Oxidative Stress and Hepatic Collagen Levels, in a Non-Mutagenic Manner

Introduction

Chronic liver diseases are a significant global health concern, responsible for nearly two million deaths annually worldwide.¹ One critical aspect of liver diseases is hepatic fibrosis, a condition characterized by gradual buildup of excessive connective tissue within the liver, leading to formation of scar tissue. Liver fibrosis is reversible in its early stages; however, if left untreated, it may progress and ultimately result in liver failure.² This pathological process involves the abnormal accumulation of extracellular matrix component like collagen, within liver tissue. It arises from persistent response to injury and repair, causing significant alterations in liver structure and function.³

The process of collagen accumulation begins with activation of previously dormant stellate cells located in Disse’s space. These cells transform into myofibroblast-like cells under the influence of cytokines, produced by both liver and immune cells in bloodstream. This transformation leads to secretion of collagen and other components of the extracellular matrix. The rate of collagen accumulation exceeds the rate of breakdown, which is driven by enzymes called metalloproteinases released from various immune cells.⁴ Persistent inflammation in liver leads to fibrosis around specific areas, progressing into bridging fibrosis and ultimately culminating in cirrhosis. This results in significant changes in liver architecture, causing complications like portal hypertension, impaired hepatocellular function, and an increased risk of liver cancer.

Thioacetamide (TAA), earlier used as a fungicide and recognized as human carcinogen,⁵ is a well-established agent for inducing steatosis and fibrosis, ultimately leading to liver failure in experimental animals.^6–8 TAA rapidly metabolizes into free radical derivatives such as TAA sulfoxide and TAA-S-S-dioxide which lead to lipid peroxidation and eventually culminates in centrilobular damage and liver injury.⁹ This makes TAA-induced liver damage a valuable model for investigating therapeutic interventions targeting chronic liver diseases.

Addressing common liver ailments such as fatty liver, cirrhosis, and chronic hepatitis presents significant challenges when it comes to treatment options. While interventions like interferons, colchicines, penicillamine, and corticosteroids have been attempted, their effectiveness remains inconsistent.¹⁰

The primary focus of current treatment strategies revolves around mitigating factors that contribute to the development of fibrosis. Although laboratory and clinical studies have shown the potential for the reversibility of liver fibrosis, the effectiveness of existing interventions remains restricted. Moreover, several emerging therapies including antiviral medications for viral hepatitis, which typically act on a single anti-hepatic fibrotic target, can be associated with adverse effects. These limitations highlight the need for alternative therapeutic approaches involving combination of drugs that address multiple fibrotic pathways.^11–13

Among the complementary and herbal approaches investigated, silymarin, a flavonolignan derived from “milk thistle” (Silybum marianum), has gained prominence as a standard treatment for managing conditions such as hepatitis and advanced liver diseases.¹⁴ Consequently, the use of herb-based medicines as potential alternatives for managing chronic liver diseases and metabolic disturbances is gaining ground.¹⁵

Livogrit is an Ayurvedic medicine, specifically formulated by incorporating the traditional wisdom of Ayurveda for the treatment of liver ailments. It is formulated as a tablet which contains three botanicals, namely Boerhavia diffusa, Phyllanthus niruri, and Solanum nigrum traditionally used for liver ailments in Ayurveda. The formulation also contains excipients such as Gum acacia (Acacia arabica), Talcum (Hydrated magnesium silicate), Microcrystalline cellulose (MCC), and Croscarmellose sodium (Sodium carboxymethyl cellulose).

In the present study, we have evaluated pharmacological effects of Livogrit on TAA-induced hepatic fibrosis in male Sprague Dawley rat. The natural herbal interventions present promising avenues for exploration, potentially surpassing the current limitations in terms of both safety and effectiveness. However, scientific evidence on the safety of natural ingredients is also required since they are the mixture of various components. Therefore, mutagenic assay was performed according to the guidelines established by Organization for Economic co-operation and development (OECD).¹⁶

Livogrit has previously been shown to exert significant hepatoprotective effects using human hepatocyte-derived spheroids, primary rat hepatocytes, zebrafish, and rodent models. These studies have demonstrated Livogrit’s ability to prevent nonalcoholic steatohepatitis, by modulating steatosis, as well as its efficacy in reversing thioacetamide-induced hepatic damage, thereby strengthening its pharmacological relevance. In rodent model, Livogrit has been shown to mitigate ANIT-induced cholestasis-like symptoms by reducing hepatic inflammation and regulating key fibrosis and apoptosis-related genes (BAX, TGF-β, MMP-9, α-SMA), emphasizing its role in liver injury.^17–19 The current study extends this evidence by focusing on thioacetamide-induced liver fibrosis and dyslipidemia, two clinically relevant manifestations of chronic liver disease, further investigating Livogrit’s role in regulating oxidative stress and collagen deposition in rats. The impact of Livogrit on various factors, including changes in body weight gain, lipids, and triglycerides levels, nitrite levels, intra-hepatic collagen deposition, liver injury markers like ALT, AST, and ALP, as well as changes in liver histoarchitecture, was monitored. Additionally, an ultra-high performance liquid chromatography (UHPLC) was employed to detect the phytometabolites present in Livogrit.

Materials and Methods

Chemical Reagents

Livogrit was sourced from Divya Pharmacy, India (Internal Batch No.: CHIC/SARA/1019/069). Silymarin tablets (Silybon-140) were procured from Micro Labs Limited, India. TAA was purchased from HiMedia Laboratories, Mumbai, India (Catalog No. GRM1850). Hematoxylin, Eosin Yellow, and Masson’s Trichrome stain, were from Merck, Germany. ALP, AST and ALT, HDL, LDL, TC, and TG kits were from Randox Laboratories Ltd., United Kingdom. NBT, EDTA, and ammonium molybdate were purchased from Merck, Germany. Griess reagent was procured from Sigma Aldrich, USA. HPLC grade acetonitrile was procured from Finar, India. Methanol, orthophosphoric acid AR grade, diethylamine were purchased from Rankem, India. Standards gallic acid (Potency-97.9%), catechin (Potency-98.8%), ellagic acid (Potency-99.6%), caffeic acid (Potency-99.2%), rutin (Potency-94.2%), and methyl gallate (Potency-97.3%) were from Sigma Aldrich, USA. Corilagin (Potency-98.0%) was purchased from Cayman chemical company, USA; cinnamic acid (Potency-99.7%) and quercetin (Potency-99.9%) from SRL, India and boeravinone B (Potency-97.0%), was purchased from Natural remedies, India.

Quantification of Phytometabolites Present in Livogrit

The quantification of marker compounds was performed using Prominence-XR UHPLC system (Shimadzu, Japan) equipped with quaternary pump (Nexera XR LC-20AD XR), DAD detector (SPD-M20 A), auto-sampler (Nexera XR SIL-20 AC XR), degassing unit (DGU-20A 5R), and column oven (CTO-10 AS VP). Separation was achieved using Shimadzu shim-pack (3 µm, 3 × 100 mm) column subjected to binary gradient elution system. The two solvents used for analysis consisted of water containing 0.1% orthophosphoric acid; adjusted to pH 2.5 with diethyl amine (Mobile phase A) and acetonitrile (Mobile phase B). The gradient programming of solvent system was: 98% A for 0–4 min, 98–90% A from 4–8 min, 90–85% A from 8–15 min, 85% A from 15–20 min, 85–80% A from 20–25 min, 80–65% A from 25–35 min, 65–5% A from 35–45 min, 5–98% A from 45–46 and 98% A from 46 to 50 min with a flow rate of 0.5 mL/min. About 4 µL of standard and test solutions were injected and column temperature was maintained at 35°C. Wavelengths were set at 270 nm for gallic acid, methyl gallate, catechin, corilagin, ellagic acid, cinnamic acid, and boeravinone B; 325 nm for caffeic acid; and 365 nm for rutin and quercetin.

Preparation of Livogrit, Thioacetamide (TAA), and Silymarin

Livogrit was freshly triturated, weighed, and suspended in 0.25% sodium carboxymethyl cellulose (Na-CMC). TAA solution was prepared by dissolving in normal saline to obtain a solution of 20 mg/mL, which was injected intraperitoneally (i.p.) to animals. Silymarin (SLM) was dissolved in 0.25% Na-CMC and orally administered to rats at a dose of 100 mg/kg body weight on the basis of a previously published article.²⁰

Animals

Adult healthy male Sprague Dawley (SD) rat weighing 150–200 g was purchased from Hylasco Biotechnology Pvt. Ltd., India, a Charles River Laboratories licensed domestic animal supplier. All experimental procedures on laboratory animals were conducted according to the guidelines prescribed by the Committee for Control and Supervision of Experiments on Animals (CCSEA), Department of Animal Husbandry and Dairying, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India. The vivarium at Patanjali Research Foundation is duly registered with CCSEA (registration number: 1964/PO/Rc/S/17/CPCSEA). Prior to initiation of experiments, the animal ethics protocol was reviewed and subsequently approved by the Institutional Animal Ethics Committee of Patanjali Research Foundation vide approval number PRIAS/LAF/IAEC-073. Animals were kept in polypropylene cage at 22 ± 2°C, 60–70% humidity, and 12 hour light-dark cycle throughout whole experiment and were maintained under standard housing conditions with free access to standard diet and water ad libitum. The study is being reported according to ARRIVE guidelines.²¹

Experimental Design

The animal equivalent dose of Livogrit was computed on the basis of the differences in the body surface area of rats and humans. The recommended therapeutic dose of Livogrit in human is 2000 mg/day. Accordingly, the dose in mg/kg for a 60 kg individual will be 2000/60 which is 33.33 mg/kg/day. The rat equivalent dose was calculated by multiplying the human dose (mg/kg) by factor of 6.2.²² Rat equivalent dose was computed to be 206.67~ 207 mg/kg. Another dose for the study was selected, which is 1/3^rd of the therapeutic dose ie 69 mg/kg to capture a possible dose-response relationship.

For assessment of Livogrit’s pharmacological effects, a total of twenty five animals were quarantined for seven days. The animals were then randomized and divided into five treatment groups (n = 5 per group) on the basis of their respective body weights using a Microsoft Excel sheet. The sample size was decided on the basis of a preliminary study conducted in our laboratory. Group 1 served as Normal control (NC) and animals allocated to this group received 0.25% Na-CMC orally and normal saline intraperitoneally. Group 2 animals are Disease control (DC) and were administered twice a week with intra-peritoneal injection of TAA at a dose of 100 mg/kg.⁹

Additionally, they received 0.25% Na-CMC by gavage. Group 3 animals were administered with TAA and they received SLM, which served as method control to validate the experimental model, at a dose of 100 mg/kg. Group 4 animals were administered TAA and concurrent oral Livogrit treatment was given at 69 mg/kg daily dose. Group 5 animals were administered TAA and concurrent oral Livogrit treatment was given at 207 mg/kg daily dose.

The treatment was continued for up to 8 weeks. During this period, blood samples were collected from rats under transient isoflurane anesthesia on days 0, 14, 28, 42, and 56. The sera were separated and analyzed for a range of biomarkers, including ALT, AST, ALP, TC, TG, as well as cholesterol: HDL and LDL: HDL ratio. During the 8-weeks study, body weight of animals was recorded starting from day 0 and continued until the end of experiment. On day 56, 24 hours after final treatment, rats were euthanized by i.p. injection of sodium pentobarbital (150 mg/kg). Blood samples were collected, through retro-orbital plexus, centrifuged, and serum was separated for subsequent analysis.

Measurement of Relative Liver Weight

The procedure involved isolation and dissection of rat livers, followed by rinsing them with normal saline. Subsequently, liver specimens were carefully dried with filter paper and their weight was recorded. A visual examination was conducted to identify any visible abnormalities in the organs. Determination of liver index was calculated by expressing liver weight as a percentage of body weight. Each liver was then divided into two parts: the right lobe was placed in a solution of isotonic 10% neutral buffered formalin for histological assessment, while the left lobe was washed with cold physiological saline and subsequently homogenized with cold phosphate buffered saline for biochemical assays.

Biochemical Analysis of Liver Injury Markers

Blood samples were collected and allowed to coagulate. After coagulation, the samples were centrifuged to separate the serum, which was then analyzed for various parameters, including ALT, AST, ALP, and lipid profiles. Standard automated techniques recommended by respective manufacturers were followed for these analyses. SOD and catalase levels were determined from liver tissue homogenate.^23,24 Nitrite levels from liver tissues were measured as described by Griess in 1879. Briefly, Griess reagent was added to de-proteinized sample. After 30 minutes of incubation at 37°C, the absorbance was measured at 548 nm using a microplate reader. Standard curve was generated with sodium nitrite in concentrations from 1 to 100 mmol/L.²⁵ For hydroxyproline measurement, rat liver tissues were homogenized and hydrolyzed in 6 N HCl for 16 hours at 96°C overnight. Hydroxyproline content was determined by a colorimetric method as described by Reddy & Enwemeka, 1996.²⁶ Total collagen content was calculated by assuming that the hydroxyproline averages 12.5% of the total collagen.²⁷

Histopathological Evaluation

The excised liver was rinsed with cold saline solution. Tissues were fixed in 10% neutral buffered formalin and processed by tissue processor (Histocore–TP1020, Leica Biosystems, Germany) and dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Further, sections of 3–5 µm thickness were obtained using a microtome (Leica Biosystems, Germany). The sections were stained with hematoxylin-eosin (H&E), Masson’s Trichrome (MT) and histological assessments were conducted using a microscope (Leica Biosystems, RM2245, Germany). Blinded evaluations of coded slides were performed by veterinary pathologist by employing a semi-quantitative scoring system. Briefly, samples of liver tissues were examined to identify and assess the distribution of focal, multifocal, and diffuse lesions. Lesions were scored on a scale from 0 (not present) to 4 (severe), with intermediate scores indicating varying degrees of severity (1 = minimal, 2 = mild, 3 = moderate). The scores for each lesion were averaged across all animals in a group to create individual lesion score. Ultimately, the mean of the lesion scores for the respective tissues was used for the final calculations. Images of histological slides were captured using Labomed Lx300i microscope with MICAPS Industrial Digital Camera (Model-HPSCMOS). Images were processed by MICAPS-MicroView software.

Bacterial Mutagenicity Assay (Ames assay)

S. typhimurium tester strains, namely, TA 98 (BAA 2720), TA 100 (BAA 2721), TA 1537 (29,630), TA1535 (29,629), and E. coli tester strain WP2 uvrA (49979) were procured from ATCC (American Type Culture Collection), USA. The test strains were grown overnight at 37°C by inoculating the freshly thawed strains in nutrient broth media. Mutagenicity was assayed by the plate incorporation method in the absence of metabolic activation, while the pre-incubation method was followed in the presence of metabolic activation as per Organization for Economic Co-operation and Development (OECD) guideline 471, and other publications.^16,28,29 The reverse mutation was assessed in four strains of S. typhimurium in addition to E. coli WP2 uvrA. The S9 metabolic activation mixture (S9 mix) was prepared according to the OECD guidelines and previously published protocols.^16,28,29 Briefly, 100 μL of test bacterial culture (1 × 10⁹ cells/mL) was incubated at 37°C with different concentrations of Livogrit or standard mutagen, in the absence or presence of S9 mix comprising 10% S9 fraction (procured from MOLTOX^®, Molecular Toxicology, Inc. NC, USA),

Subsequently, 2 mL of soft agar (0.6% bacto-agar, 0.5% NaCl, 50 μM biotin and 50 μM histidine for S. typhimurium strains or 50 μM tryptophan for E. coli strain) was added and poured immediately onto a plate of minimal agar (1.5% bacto-agar, Vogel-Bonner E medium, containing 0.4% glucose). Dimethyl sulfoxide (DMSO) was used as the vehicle control. Livogrit was completely soluble in DMSO up to a concentration of 50 mg/mL. The standard mutagens, namely, 4-nitroquinoline (Sigma Aldrich, USA) at concentration 0.15 µg/plate was used in TA 98; sodium azide (Sigma Aldrich, USA) at concentration 0.5 µg/plate was used in TA 100 and TA 1535; 9-aminoacridine (Tokyo Chemical Industries, India) at concentration 50 µg/plate was used in TA 1537 and 4-nitroquinoline (Sigma Aldrich, USA) at concentration 0.5 µg/plate was used in E. coli uvrA without S9 metabolic activation. While 2-aminoanthracene (Sigma Aldrich, USA) at a concentration 20 µg/plate was used with S9 metabolic activation, for all the strains tested.

The mutagenic effect of Livogrit was assessed at 0.05, 0.15, 0.5, 1.5, and 5.0 mg/plate concentrations, in triplicates, along with the vehicle control and respective positive controls, as per the OECD guidelines.¹⁶

The revertant colonies were counted after 64 to 72 hours of incubation period at 37°C. A positive result for mutagenicity was determined based on an increase in revertant colonies (Mutagenicity Ratio, MR ≥ 2.0), in at least one of the tester strains compared to the vehicle control.²⁹ The Mutagenicity Ratio (MR) was calculated by dividing the number of revertant (visible) colonies in Livogrit or the standard mutagen treated plates, by the number of revertant colonies in the vehicle control (DMSO) plates. To assess the statistical significance, a Student’s t-test was performed with p ≤ 0.05 considered significant.

Statistical Analysis

Data were compiled from all the experimental groups and expressed as Mean ± Standard Error of Mean (SEM) for each group. Statistical analysis was performed using GraphPad Prism Version 7.4. software (San Diego, CA, United States). Percent change in body weight, hepatic injury markers, and lipid markers of different experimental groups were statistically analyzed by employing Two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of relative liver weight, oxidative stress markers, hydroxyproline, collagen content, and semi-quantitative histopathology data was analyzed by employing unpaired Student’s t-test for comparing NC and DC groups and One-way ANOVA followed by Dunnett’s multiple comparisons test for comparing treatment groups with DC group. p values <0.05 were considered statistically significant. The 95% confidence intervals were calculated, where applicable.

Results

Metabolite Analysis of Livogrit Through LC-MS-QToF and UHPLC

In our previously published study, Livogrit was subjected to LC-MS QToF analysis, which identified the presence of 68 metabolites. Of these metabolites, 46 were identified in positive ionization mode, 48 in negative ionization mode, while 26 phytometabolites were observed in both ionization modes (Figure 1A).³⁰ The major identified metabolites included gallic acid, methyl gallate, catechin, corilagin, caffeic acid, ellagic acid, rutin, cinnamic acid, quercetin, and boeravinone B. These metabolites originate from botanical sources such as Boerhavia diffusa L., Phyllanthus niruri L., and Solanum nigrum L. Therefore, with an objective of quantifying the selected metabolites, UHPLC-DAD analytical platform was employed, which revealed that each gram of Livogrit powder contained: 344.01 μg of gallic acid, 1199.47 μg of methyl gallate, 225.20 μg of catechin, 1165.27 μg of corilagin, 42.79 μg of caffeic acid, 3865.44 μg of ellagic acid, 1441.51 μg of rutin, 20.71 μg of cinnamic acid, 5.60 μg of quercetin, and 2.48 μg of boeravinone B (Figure 1B–D, Table 1). These metabolites were quantified using their respective standards (Table 1).

Table 1 Plant Metabolites Identified and Quantified in Livogrit by UHPLC-DAD Analysis, as Portrayed in Figure 1

Figure 1 Plant metabolite analysis of Livogrit. LC-MS-QToF analysis data of Livogrit has been reproduced from our previously reported study.30 Total Ion Chromatograms (TIC) of Livogrit tablet in both positive (Blue) and negative (Green) ionization mode with retention time (RT) of the identified compounds (A). Overlap UHPLC-DAD chromatograms of Blank (Purple), Livogrit tablet (Pink) and reference standard mix (Green) with respective RT and chemical structures. Gallic acid (RT: 4.23 min), methyl gallate (RT: 12.54 min), catechin (RT: 13.50 min), corilagin (RT: 16.14 min), ellagic acid (RT: 24.88 min), cinnamic acid (RT: 33.06 min), and boeravinone B (RT: 42.44 min) were quantified at 270 nm (quantification shown in Table 1). The solvent gradient program with respect to percentage change in mobile phase B is shown with dotted lines (B). Overlap UHPLC-DAD chromatograms of Blank (Purple), Livogrit tablet (Pink) and reference standard mix (Green). Caffeic acid (RT: 14.59 min) was quantified at 325 nm (C). Overlap UHPLC-DAD chromatograms of Blank (Purple), Livogrit tablet (Pink) and reference standard mix (Green). Rutin (RT: 25.67 min) and quercetin (RT: 34.77 min) were quantified at 365 nm (D).

Livogrit Improves Body Weight Gain and Decreases Relative Liver Weight in TAA-Administered Rats

As evident from the percent body weight data, a statistically significant decrease in body weight gain was observed following TAA treatment on days 14 (Figure 2A; p < 0.05), 42, and 56 compared to the NC group (Figure 2A; p < 0.01). However, Livogrit treatment significantly improved body weight gain at both the tested doses (69 mg/kg and 207 mg/kg), on day 56 (Figure 2A; p < 0.05) as compared to the animals allocated to the DC group. Additionally, the relative liver weight at the end of the study was computed. A significant increase in relative liver weight was observed in TAA-induced animals as compared to the NC group (Figure 2B; p < 0.05). Following Livogrit treatment at 207 mg/kg, there was a significant reduction in relative liver weight as compared to TAA-induced groups (Figure 2B; p < 0.05). Silymarin demonstrated tendency to ameliorate TAA-induced impaired body weight gain and relative liver weight. However, the effects were not statistically significant.

Figure 2 Change in body weight and relative liver weight of TAA-induced experimental animals in presence of Livogrit. Effect of Livogrit on percentage change in the body weight measured at day 0, 14, 28, 42 and 56 in TAA-induced rats (A). Relative liver weight to body weight on the day of termination (Day 56) was measured (B). Data is presented as Mean ± SEM (n = 5 animals per group). Percent change in body weight data was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. For relative liver weight, comparisons between NC and DC groups were performed by employing unpaired Student’s t-test and one-way ANOVA followed by Dunnett’s multiple comparisons test for comparing other groups with DC group. # p < 0.05 and ## p < 0.01 as compared to NC group; * p < 0.05 as compared to DC group.

Livogrit Mitigates TAA-Evoked Increase in Liver Injury Markers

When compared to NC group, TAA-administered animals demonstrated a statistically significant increase in serum ALT and AST activities (Figure 3A and B; p < 0.01 on days 28, 42, and 56). In addition, increased ALP activity was also noted on days 14, 28, 42, and 56 (Figure 3C; p < 0.01) as compared to NC group. Livogrit administered at both the doses of 69 and 207 mg/kg significantly decreased TAA-induced disruptions in these studied markers (Figure 3A and B; p < 0.01 for AST and ALT on days 28, 42, and 56 and Figure 3C; p < 0.01 on days 14, 28, 42, and 56). Similar effects were shown by the standard drug silymarin (Figure 3A–C; p < 0.01).

Figure 3 Livogrit normalizes liver injury markers in TAA induced liver fibrosis. Effect of Livogrit on serum ALT (A) AST (B) and ALP (C) in TAA-induced rats. Data is presented as Mean ± SEM (n = 5 animals per group) and was analyzed by using two-way ANOVA followed by Tukey’s multiple comparisons test. A p value < 0.05 was considered to be statistically significant. ## p < 0.01 as compared to NC group; ** p < 0.01 as compared to DC group.

Livogrit Moderates TAA-Induced Dyslipidemia

Animals exposed to TAA exhibited elevated serum levels of TG and TC (Figure 4A and B; p < 0.01 on days 14, 28, 42, and 56); LDL (Figure 4C; p < 0.05 on day 14 and p < 0.01 on days 28, 42, and 56) with a concomitant reduction in HDL levels compared to NC group (Figure 4D; p < 0.01 on days 14, 28, 42, and 56). Livogrit significantly reduced TAA-induced hypertriglyceridemia at both 69 and 207 mg/kg (Figure 4A; p < 0.01 on days 14, 28, 42, and 56). Further, TAA-induced increase in TC levels was also decreased by Livogrit - 69 mg/kg (Figure 4B; p < 0.05 on day 42 and p < 0.01 on days 28 and 56). Similarly, Livogrit administered at the dose of 207 mg/kg demonstrated a significant diminution of increased TC levels (Figure 4B; p < 0.01 on days 28, 42, and 56). Livogrit administered at the dose of 207 mg/kg also decreased TAA-induced elevated LDL levels (Figure 4C; p < 0.05 on day 42 and p < 0.01 on day 56). Furthermore, Livogrit at both the tested doses significantly restored TAA-induced diminished HDL levels (Figure 4D; p < 0.01 on days 42 and 56). Silymarin significantly reduced hypertriglyceridemia (Figure 4A; p < 0.01 on days 14, 28, 42, and 56); hypercholesterolemia (Figure 4B; p < 0.01 on days 28, 42, and 56) and decreased LDL levels (Figure 4C; p < 0.05 on day 42, p < 0.01 on day 56). Similarly, it also restored HDL levels (Figure 4D; p < 0.01 on days 42 and 56).

Figure 4 Livogrit normalizes lipid profile in TAA induced liver fibrosis. Effect of Livogrit on (A) Triglyceride (TG); (B) Total cholesterol; (C) High-density lipoprotein (HDL); (D) Low-density lipoprotein (LDL); (E) LDL: HDL ratio; (F) Total Cholesterol: HDL ratio. Data is presented as Mean ± SEM (n = 5 animals per group) and was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. # p < 0.05 and ## p < 0.01 as compared to NC group; * p < 0.05 and ** p < 0.01 as compared to DC group.

The LDL to HDL and TC to HDL ratios significantly increased on days 28, 42, and 56 (Figure 4E and F; p < 0.01) in TAA-induced rats. However, Livogrit at both the tested doses significantly reduced these calculated ratios on days 42 and 56, respectively (Figure 4E and F; p < 0.01). The reference control drug silymarin showed a similar reduction (Figure 4E and F; p < 0.01 on days 42 and 56).

Livogrit Elevates SOD and Catalase Activity in Serum and Reduces Nitrite, Hydroxyproline Levels and Collagen Content in Liver of TAA-Induced Rats

The activities of antioxidant enzymes SOD (Figure 5A) and catalase (Figure 5B) were significantly decreased (p < 0.01 for SOD and p < 0.05 for catalase) upon TAA administration. These were significantly restored upon treatment with the therapeutically equivalent dose of Livogrit (207 mg/kg, Figure 5A and B; p < 0.05 for SOD; p < 0.01 for catalase). Silymarin treatment also demonstrated significant restoration of SOD and catalase activities when compared to DC group (Figure 5A and B; p < 0.01).

Figure 5 Livogrit decreases nitrite, hydroxyproline and collagen levels while elevating SOD and catalase levels in rats induced with TAA. Effect of Livogrit on Superoxide dismutase (SOD) (A); Catalase (B); Nitrite (C); Hydroxyproline (D); and collagen (E) in rats induced with TAA. Data is presented as Mean ± SEM (n = 5 animals per group). Comparisons between NC and DC groups were performed by employing unpaired Student’s t-test and one-way ANOVA followed by Dunnett’s multiple comparisons test was used for comparing other groups with DC group; # p < 0.05 and ## p < 0.01 as compared to NC group; * p < 0.05 and ** p < 0.01 as compared to DC.

When compared to NC group, TAA administration in animals assigned to DC group for 8 weeks, led to nitrosative stress in the liver, as demonstrated by a significant increase in hepatic nitrite levels (Figure 5C; p < 0.05). Livogrit treatment attenuated TAA-induced increased hepatic nitrite levels with significant effects noted at a dose of 69 mg/kg (Figure 5C; p < 0.05) The method control drug silymarin could also significantly reduce the observed increase in nitrite levels in the liver when compared to DC group (Figure 5C; p < 0.05).

Both hydroxyproline levels and collagen content were significantly elevated in the livers of rats allocated to DC group that received TAA, respectively, in comparison to NC group (Figure 5D and E; p < 0.01 and p < 0.05, respectively). Subsequent to the administration of Livogrit at doses of 69 and 207 mg/kg, a significant reduction in hydroxyproline levels was observed (Figure 5D; p < 0.05). Significant decrease in collagen levels was also observed in the group treated with 207 mg/kg (Figure 5E; p < 0.05). Silymarin also decreased the levels of hydroxyproline and collagen significantly (Figure 5D and E; p < 0.01 and p < 0.05, respectively).

Livogrit Attenuates Hepatic Fibrosis Induced by TAA

As depicted in Figures 6A and B, H&E staining revealed that, as compared to the NC group animals, the DC group demonstrated development of moderate multifocal fibrosis in the hepatic parenchyma along with an increase in collagen fiber deposition around the central vein (Figure 7A; p < 0.01). Additionally, minimal multi-focal necrosis and mononuclear cell infiltration were also observed in DC group, but the changes were non-significant (Figure 7B and C). The summation of the individual lesion scores in DC animals was significantly higher when compared to NC group (Figure 7D; p < 0.01). Oral administration of Livogrit inhibited TAA-induced fibrotic changes, wherein, significant effects were evident in Livogrit-207 mg/kg treated group (Figure 7A; p < 0.05), when compared to DC group. However, the total histopathological lesion score, was significantly lesser in both the tested doses of Livogrit (Figure 7D; p < 0.05). Silymarin, administered at the dose of 100 mg/kg decreased the observed fibrotic changes, however the effect was not significantly different when compared to the disease-control group (Figures 6C and 7A).

Figure 6 Histopathological examination of rat liver sections stained with hematoxylin and eosin (H&E). The liver of control group rats shows a normal lobular architecture (A), accompanied by a normal distribution of hepatocytes around the central vein (CV). Liver section of TAA-exposed group displays thick fibrous strands that surrounds hepatic lobules and hepatocellular vacuolar degeneration (H&E) (B); Silymarin treated groups shows decrease in fibrosis (Fib) (C). Treatment with Livogrit at 69 mg/kg showing a moderate improvement (D) whereas treatment with Livogrit at 207 mg/kg shows a significant improvement in fibrosis score in the histopathological sections (E). The scale bar in the images represents 20 µm (left: ×100 magnification) 40 µm (right: ×400 magnification).

Figure 7 Lesion analysis of H&E stained histopathological liver sections. The total fibrosis score increased in TAA induced livers compared to control rat livers; A reduction was noted in Livogrit 207 mg/kg group (A) Total necrosis (B), mononuclear cell infiltrate (C) total lesion score (D) was calculated. Data is presented as Mean ± SEM (n = 5 animals per group) and was analyzed by employing unpaired Student’s t-test for comparing NC and DC groups and one-way ANOVA followed by Dunnett’s multiple comparisons test for comparing other groups with DC group. ## p < 0.01 as compared to NC group; * p < 0.05 as compared to DC group.

As there was a decrease in fibrosis score following Livogrit treatment, Masson’s Trichrome (MT) staining was additionally performed to specifically characterize degree of liver fibrosis. As observed in the H&E-stained liver sections, results obtained after staining the tissues with MT stain also demonstrated development of significant multifocal hepatic fibrosis as evidenced by increase in deposition of collagen fibers in the hepatic parenchyma of the DC group (Figure 8B; p < 0.01), in comparison with NC group. Livogrit inhibited TAA-induced fibrosis in dose-related manner (Figure 8A) with statistically significant inhibition evident at dose of 207 mg/kg (p < 0.05, Figure 8B). This increase in collagen formation was found to be reduced in silymarin treated group, which was nevertheless not statistically significant, when compared to DC group (Figure 8A and B).

Figure 8 Histopathological examination of rat liver sections stained with Masson’s Trichrome (MT). Control group showed normal lobular architecture accompanied by normal distribution of fine collagen fibers around central vein (CV); TAA-exposed group displayed thick fibrous strands that surrounded hepatic lobules with fibrosis (arrows) accompanied by abundant fibroplasia completely surrounding the hepatic lobules (blue with MT); Treatment with Silymarin shows apparent fibrosis; Treatment with Livogrit (69 mg/kg) showed a moderate improvement of the histopathological changes; Treatment with Livogrit (207 mg/kg) showed a marked improvement of histopathological changes, except for minor inflammatory cells infiltration (H&E) together with fine collagen deposition (MT), as indicated by arrows (A). Fibrosis score by MT staining was quantified (B). Data is presented as Mean ± SEM (n = 5 animals per group) and was analyzed by employing unpaired Student’s t-test for comparing NC and DC groups and one-way ANOVA followed by Dunnett’s multiple comparisons test for comparing other groups with DC group. ## p < 0.01 as compared to NC group; * p < 0.05 as compared to DC group. The scale bar in the images represents 20 µm (left: ×100 magnification) 40 µm (right: ×400 magnification).

Bacterial Mutagenicity Assay Demonstrate That Livogrit Has No Genotoxic Effects

Revertant colony numbers and their mutagenicity ratio (MR) >2 with respect to the vehicle (DMSO, here) are considered as an indicator of genotoxicity (OECD, 2020). Revertant bacteria are those that have undergone a mutational reversion to the prototrophic state, enabling continuous growth into visible colonies. The experiment was performed with vehicle control (DMSO), positive control (known mutagens) and with five concentrations of Livogrit. All five tester strains, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli uvrA were subjected to Livogrit from 0.05 mg/plate to 5 mg/plate without (Table 2) and with S9 metabolic activation (Table 3). Notably, mutagenicity ratio >2 was not observed in any of the tester strains, with or without metabolic activation.

Table 2 Bacterial Mutagenicity Analysis (Ames assay) of Livogrit Showing the Average Number of Revertants per Plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli uvrA Strains, in the Absence of Metabolic Activation (-S9)

Table 3 Bacterial Mutagenicity Analysis (Ames assay) of Livogrit Showing the Average Number of Revertants per Plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli uvrA Strains, in the Presence of Metabolic Activation (+S9)

Discussion

All liver diseases such as cirrhosis, hepatitis, drug-induced liver injury, and alcohol-induced liver injury cause severe hepato-cellular damage. This impairs blood coagulation systems and raises the risk of life-threatening liver problems such as hepatocellular cancer and liver failure.² Among various experimental models, thioacetamide-induced (TAA) liver fibrosis model is particularly useful for evaluating potential of anti-fibrotic drugs due to its well-established ability to induce a fibrotic response in rodents which closely resembles that observed in humans.³¹ TAA-induced toxicity is characterized by liver damage, which is indicated by release of cellular enzymes into the circulation as a result of abnormalities in liver cell transport mechanisms. It is a potent hepatotoxin which undergoes metabolic processing by the Cytochrome 450 enzyme, leading to the formation of toxic compounds like thioacetamide sulfoxide (TAAS) and thioacetamide dioxide (TAAD).^32,33 Additionally, TAA-induced liver fibrosis is associated with oxidative stress, resulting in free radical-mediated lipid peroxidation.³⁴ This fibrosis is characterized by regenerative nodule formation, development of portal hypertension and hyper-dynamic circulation,³⁵ as corroborated by histopathological examinations and plasma biochemical indicators of liver injury markers. Hepatic fibrosis treatment focuses on targeting factors involved in its pathogenesis, including anti-inflammation, liver protection, inhibition of stellate cell activation, and suppressing extracellular matrix production. Most anti-fibrotic drugs are in preclinical research, targeting various liver fibrosis etiologies in chronic liver diseases.

Despite positive results in experimental animal models, many anti-fibrotic candidate drugs show limited efficacy in clinical trials. This may be due to complex pathological mechanism of liver fibrosis. Most developed drugs target a single rather than multiple targets. Commonly used anti-hepatic fibrosis medications include immune-suppressants, glucocorticoids, and non-specific anti-inflammatory drugs; however, these medications tend to have incidence of undesirable reactions.^12,13 Therefore, traditional Ayurvedic medicine provides a promising alternative treatment approach. The current study aims to demonstrate the hepatoprotective potential of Livogrit, a polyherbal medicine, against liver fibrosis as an alternative treatment modality. Here, hepatic fibrosis was induced in rats by TAA and Livogrit was administered to rats for evaluation of its hepatoprotective effect.

Livogrit, has previously been evaluated for its hepatoprotective effects in various models of liver injury, including cholestasis, steatohepatitis, and hepatotoxicity, as reported in our earlier studies.^{17–19,30,36} It has already been tested in in vitro and in vivo systems,^19,30,36 including rat models, zebrafish,¹⁷ C. elegans,³⁷ human hepatocyte-derived spheroids, and primary rat hepatocytes,¹⁸ underscoring its broad applicability across experimental models of liver damage. Notably, its protective effect against TAA-induced hepatocellular toxicity was demonstrated in a zebrafish model.¹⁷ Our present study is in continuation with the previous investigation on zebrafish. Here, we have specifically assessed the antifibrotic potential of Livogrit in a rat model of thioacetamide-induced liver fibrosis.

In the present study, silymarin was employed as a reference control because of its well-documented antifibrotic and hepatoprotective properties. It is widely recognized for its ability to suppress the activation of hepatic stellate cells, which are the primary mediators of collagen production and liver fibrosis in disease models, including steatohepatitis.³⁸ Due to the absence of universally accepted standard treatments for liver fibrosis, silymarin is frequently utilized in preclinical research as a reference control to assess the effect of novel antifibrotic agents. Notably, several studies have reported the use of silymarin in models of thioacetamide (TAA)-induced chronic liver fibrosis, further supporting its relevance as a reference control to validate the experimental model in our study design.^39,40

In our study, TAA led to a marked impairment of body weight gain in animals, which was significantly reversed by Livogrit. This observed reduction in body weight gain in TAA-administered animals could be strongly attributed to toxic effect of TAA and is considered to be the most reliable and consistent symptoms of toxicity among the experimental animals.⁴¹ Additionally, relative liver weight (expressed as a percentage of terminal body weight) was observed to be increased in TAA group, which could be attributed to excessive collagen deposition in liver tissues in diseased animals due to increase in hydroxyproline,⁴² a major constituent of fibrillary collagen.⁴³ Treatment with Livogrit, apart from showing a discernable reduction in relative liver weight, hydroxyproline, and collagen content in liver tissues, also showed a remarkable improvement in histo-architecture of liver in TAA-induced rats. The presence of elevated hydroxyproline and collagen levels is closely connected to the occurrence and progression of liver fibrosis. Hydroxyproline is a specific amino acid found abundantly in collagen, the main structural protein in the liver’s connective tissue. As fibrosis advances, collagen accumulates, causing an increase in hydroxyproline levels. Therefore, hydroxyproline and collagen serve as a valuable indicator to assess the extent of liver fibrosis. TAA induction in rats led to multifocal liver fibrosis, evident upon histopathological evaluation with H&E staining, which was reconfirmed by MT staining. Notably, these effects were nearly normalized in Livogrit-treated rats, especially at the therapeutically relevant dose, wherein the nodules of hepatocytes were separated only by thin fibrous septa, surrounding degenerating hepatocytes induced by TAA. Since, portal tracts are where circulating lymphoid cells enter liver for first time, portal inflammation is widespread in many liver disorders. In this study, although there was no significant change in number of inflammatory mononuclear cells, numerous inflammatory cells and bile duct proliferation at portal site were observed primarily in TAA group while it appears less deteriorated in Livogrit treated animals. Livogrit treatment significantly reduces fibrosis, possibly by decreasing collagen deposition and lowering mononuclear cell infiltration. Previous studies in our lab demonstrated effect of Livogrit in reducing liver fibrosis, mononuclear cell infiltration, and hyperplasticity induced by carbon tetrachloride in Wistar rats.³⁰ Livogrit has also been shown to protect HepG2 cells from fatty acid-induced steatosis by reducing intracellular triglycerides and extracellular glycolic acid levels.³⁰

Enzymes such as Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) are frequently utilized as robust biochemical markers to assess hepatic injury. Liver cell damage releases hepatocyte enzymes into the bloodstream, elevating plasma AST and ALT activities. ALT, a more specific enzyme for hepatic injury, exhibits longer-lasting changes in activity than AST. In liver disorders, both enzyme activities have been reported to significantly increase.⁴⁴ Here, Livogrit treatment restored liver damage markers such as ALT, AST, and ALP to normal levels. Several other metabolic complications are common in chronic liver diseases. One of the common complication is dyslipidemia, which refers to the abnormal regulation and functioning of lipid. Liver diseases disrupt metabolism of lipids, leading to accumulation of lipids in hepatocytes which ultimately exacerbates liver fibrosis.⁴⁵ TAA-administered group exhibited significant increase in serum cholesterol, triglyceride, and LDL levels, which improved significantly with Livogrit therapy, thus demonstrating the preclinical potential of Livogrit to mitigate dyslipidemia. This was probably observed in chronic liver diseases because liver cannot remove LDL cholesterol from bloodstream when there is functional loss of LDL receptor.

Oxidative stress plays a crucial role in development of TAA-induced liver fibrosis. When TAA is metabolized by liver, it generates reactive oxygen species leading to liver cell necrosis. This damage plays a key role in progression of liver fibrosis. Antioxidants are necessary for destruction of free radicals in our bodies, and flavonoids have been recognized as potent antioxidants.^46,47 Moreover, several studies have demonstrated beneficial effects of antioxidant in protecting liver against TAA-induced injury.⁴⁸ Therefore, we sought to measure two major enzymatic antioxidants namely SOD and catalase in liver tissues along with hepatic nitrite levels. SOD and catalase are important enzymes that act as antioxidants in the body. They play a significant role in protecting the liver from damage caused by oxidative stress, which is a key factor in the development of TAA-induced hepatic fibrosis. SOD helps to convert harmful superoxide radicals into less damaging substances, while catalase plays a role in breaking down hydrogen peroxide and shielding tissues from highly reactive hydroxyl radicals. These enzymes help to counteract the harmful effects of oxidative stress in the liver, which is important in preventing inflammation and fibrosis caused by TAA exposure. TAA-challenged animals showed decrease in SOD and catalase activity, however, Livogrit treated rats showed significant enhancement in the activity of these enzymes, which reduced oxidative damage caused by reactive free radicals in liver.

Hence, a decrease in SOD and catalase activity may have detrimental consequences due to accumulation of superoxide radicals and hydrogen peroxide. These findings suggest that Livogrit’s hepato-protective effects may be attributed to its phyto-metabolites containing antioxidants.

Nitrite levels were higher in DC group and Livogrit treatment ameliorated TAA-induced increase in nitrite levels. Higher levels of nitrite in liver tissue are linked to development of liver fibrosis. It is an indicator for increased oxidative stress and inflammation, which are the key factors in progression of fibrosis in liver. Measuring nitrite levels indicates the severity of liver fibrosis and extent of oxidative damage.

Efficacy of herbal formulations in modulating diseases is significantly influenced by plant metabolites. Our prior investigation demonstrated effectiveness of Sarva-Kalp-Kwath (the decoction from which Livogrit has been derived) in alleviating liver damage induced by carbon tetrachloride. Some key plant metabolites, namely gallic acid, methyl gallate, cinnamic acid, caffeic acid, quercetin, catechin, rutin, and corilagin, were detected and these metabolites originated from plants Boerhavia diffusa L., Phyllanthus niruri L., and Solanum nigrum L.⁴⁹ The protective effect of Livogrit can be attributed to the presence of several active plant metabolites, which are known for their strong antioxidant, anti-inflammatory, and hepatoprotective properties based on prior research.^50–54 Gallic acid is known for its antioxidant and anti-inflammatory properties as it scavenges 2,2-diphenyl-1-picrylhydrazy (DPPH) radical by hydrogen-donating mechanism which is more efficient than Vitamin E.⁵⁵ Methyl gallate, another important plant metabolite, can modify mitochondria dependent apoptosis signaling pathway to rescue liver cells through inhibiting ROS production due to t-BHP induced oxidative stress.⁵⁶ Ellagic acid exerts its anti-fibrotic activity by inducing ferroptotic cell death of activated Hematopoietic Stem Cells (HSCs) in CCl₄/BDL mice.⁵⁷ An aqueous extract of thinner roots of B. diffusa exhibited remarkable protection of various enzymes such as AST and ALT against TAA-induced hepatic injury in rats.⁵⁸ Cinammic acid is known to mitigate non-alcoholic fatty liver disease (NAFLD) by suppressing intrahepatic triglyceride accumulation. This has been reported to be attributed to the inhibition of lipogenesis and fatty acid uptake, coupled with the promotion of hepatic fatty acid oxidation.⁵⁹ Quercetin is known to inhibit the activation and infiltration of liver macrophages and adjust M1-polarized macrophages through the Notch1 pathway thereby exhibiting anti-fibrotic activity.⁶⁰ Rutin is known to lower and attenuate hepatotoxicity induced by high cholesterol diet in rat model via restoration of key signaling molecules in TGF-β/Smad signaling pathway. Rutin has also been reported to ameliorate hepatic fibrosis induced by TAA in mice, accompanied by ameliorating serum transaminase, histopathological changes, the balance of ECM accumulations, and inflammation factors, such as IL-1, IL-18, and TNF-α.^61,62 Caffeic acid exerts its protective effect against high fat diet-induced NAFLD by means of integrative responses, including inhibition of gut dysbiosis, reduction in pro-inflammatory LPS release and subsequent suppression of lipid synthesis.⁶³ Catechin, derived from the Camellia sinensis plant, has received extensive attention due to its anti-inflammatory bioactivities in preclinical models of nonalcoholic steatohepatitis.⁶⁴

In mutagenicity assay, the spontaneous reversion for strains TA 98, TA 100, TA 1535, TA 1537 and E. coli uvrA in vehicle and with standard mutagens (positive controls) align with the previous reports.^28,65 Livogrit did not increase in the number of revertant colonies in tester strains with and without metabolic activation, at concentrations of 5.0, 1.5, 0.5, 0.15, and 0.05 mg/plate. Collectively, we demonstrate that Livogrit did not induce any frameshift or point mutations by base substitutions in any of the tester strains of S. typhimurium and E. coli uvrA. Thus, Livogrit was found to be non-mutagenic under the tested experimental conditions up to 5.0 mg/plate, with and without a metabolic activation. Previous studies conducted at our laboratory have additionally established that Sarva-Kalp-Kwath was found to be safe upto a dose of 1000 mg/kg/day in a 28-day oral non-clinical safety study in rats.³⁰

Conclusion

The present study revealed that Livogrit has the preclinical potential to regulate hepatic fibrosis and dyslipidemia by modulating liver enzymes, lipid markers, antioxidative enzymes, nitrite, and collagen levels in TAA-induced hepatic fibrosis. Livogrit also preserved the histopathological architecture of the hepatic tissue. Livogrit was found to be non-mutagenic under the tested experimental conditions with and without a metabolic activation system. Its diverse phytometabolites position Livogrit as a potential alternative therapeutic drug for preventing and treating chronic liver diseases, including hepatic fibrosis.