Polyherbal Medicine Divya Sarva-Kalp-Kwath Ameliorates Persistent Carbon Tetrachloride Induced Biochemical and Pathological Liver Impairments in Wistar Rats and in HepG2 Cells

Abstract

Divya Sarva-Kalp-Kwath (SKK) is a poly-herbal ayurvedic medicine formulated using plant extracts of Boerhavia diffusa L. (Nyctaginaceae), Phyllanthus niruri L. (Euphorbiaceae), and Solanum nigrum L. (Solanaceae), described to improve liver function and general health. In the present study, we have explored the hepatoprotective effects of SKK in ameliorating carbon tetrachloride (CCl₄) induced liver toxicity using in-vitro and in-vivo test systems. Chemical analysis of SKK using Liquid Chromatography-Mass Spectroscopy (LC-MS-QToF) and High-Performance Liquid Chromatography (HPLC) revealed the presence of different bioactive plant metabolites, known to impart hepatoprotective effects. In human hepatocarcinoma (HepG2) cells, co-treatment of SKK with CCl₄ effectively reduced the hepatotoxicity induced by the latter. These effects were confirmed by studying parameters such as loss of cell viability; release of hepatic injury enzymatic biomarkers- aspartate aminotransferase (AST), and alkaline phosphatase (ALP); and changes in reactive oxygen species and in mitochondrial membrane potentials. In-vivo safety analysis in Wistar rats showed no loss in animal body weight, or change in feeding habits after repeated oral dosing of SKK up to 1,000 mg/kg/day for 28 days. Also, no injury-related histopathological changes were observed in the animal's blood, liver, kidney, heart, brain, and lung. Pharmacologically, SKK played a significant role in modulating CCl₄ induced hepatic injuries in the Wistar rats at a higher dose. In the 9 weeks' study, SKK (200 mg/kg) reduced the CCl₄ stimulated increase in the release of enzymes (ALT, AST, and ALP), bilirubin, total cholesterol, and uric acid levels in the Wistar rats. It also reduced the CCl₄ stimulated inflammatory lesions such as liver fibrosis, lymphocytic infiltration, and hyper-plasticity. In conclusion, SKK showed pharmacological effects in improving the CCl₄ stimulated liver injuries in HepG2 cells and in Wistar rats. Furthermore, no adverse effects were observed up to 10× higher human equivalent dose of SKK during 28-days repeated dose exposure in Wistar rats. Based on the literature search on the identified plant metabolites, SKK was found to act in multiple ways to ameliorate CCl₄ induced hepatotoxicity. Therefore, polyherbal SKK medicine has shown remarkable potentials as a possible alternative therapeutics for reducing liver toxicity induced by drugs, and other toxins.

Keywords: Divya Sarva-Kalp-Kwath; HepG2 cells; carbon tetrachloride; hepatoprotective effects; hepatotoxicity; safety.

Figure 1

Plant metabolite analysis of Divya Sarva-Kalp-Kwath (SKK): Aqueous extract of the SKK was screened for plant metabolites using liquid chromatography (LC)-based mass spectroscopy (MS). Spectral analysis revealed the presence of (A, i) 46 metabolites were detected in the positive mode and (A, ii) 48 metabolites were detected in the negative mode at different retention time (RT; min) (see Table 1). Using High Performance Liquid Chromatography (HPLC) analysis, SKK was analyzed and quantified for the metabolites, (B, i) Standards of caffeic acid, rutin, and quercetin, (B, ii) SKK contents of caffeic acid, rutin, and quercetin detected at 325 nm, (C, i) standards of gallic acid and catechin, (C, ii) SKK contents of gallic acid and catechin detected at 270 nm, and (D, i) standards of corilagin, (D, ii) SKK contents of corilagin detected at 270 nm. Gallic acid (RT: 5.10 min), catechin (RT: 13.14 min), caffeic acid (RT: 13.80 min), rutin (RT: 15.50 min), quercetin (RT: 19.56 min), and corilagin (RT: 21.13 min) were detected at their respective retention time (RT) in the developed and validated analytical HPLC method.

Figure 2

In-vitro analysis of cellular and biochemical effects of Divya Sarva-Kalp-Kwath (SKK): (A) significant loss of cell viability was observed following the stimulation of HepG2 cells with 10 mM of CCl₄. Co-treatment of the CCl₄ stimulated HepG2 cells with SKK led to a significant recovery of cell viability. Also, CCl₄ stimulation of the HepG2 cells led to an augmented release of liver injury serum-based biomarkers, (B) aspartate aminotransferase (AST), and (C) alkaline phosphatase (ALP). Release of these serum hepatotoxicity biomarkers was ameliorated following co-treatment of the CCl₄ stimulated HepG2 cells with SKK (0.5 and 1 mg/mL). Results are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. p-value *<0.05; **<0.01; ^#<0.01.

Figure 3

Reactive oxygen species and mitochondrial membrane potential measurement in Divya Sarva-Kalp-Kwath (SKK) and carbon tetrachloride (CCl₄) treated HepG2 cells: (A) Oxidative stress was induced in the HepG2 cells following stimulation with the CCl₄. Intracellular presence of reactive oxygen species (ROS; green color) and an increase in mitochondrial membrane potential (MMP; red color) were determined through epifluorescence microscope based imaging and HCS Studio software based analysis. Co-treatment of the HepG2 cells with SKK and CCl₄ significantly reduced the production of ROS and MMP. (B) Quantitatively, ROS levels in the CCl₄ stimulated HepG2 cells showed an upregulation. (C) Similarly, MMP in the CCl₄ stimulated HepG2 cells showed an upregulation. Both these parameters showed a reduction following the co-treatment of the HepG2 cells with CCl₄ and SKK. Results are expressed as Mean ± Standard Error of Means. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. p-value *<0.05; **<0.01. Bars represent 200 μm.

Figure 4

In-vivo Safety Analysis of Divya Sarva-Kalp-Kwath (SKK): (A) Male Wistar rats were randomized and divided into four treatment groups (n = 5): Group 1: Normal control (NC) animals were administered with distilled water only; Group 2: SKK doses of 100 mg/kg; Group 3: SKK dose of 500 mg/kg; and Group 4: SKK dose of 1,000 mg/kg. Water extract of SKK was dissolved in distilled water and administered daily by gavage for 28 days. The animals were observed for physiological, histopathological, and biochemical changes. (B) Body weight change in the SKK exposed animals was measured daily and no changes were detected throughout the study period. (C) Feed consumption of the SKK treated animals was determined daily and averaged per week. The animals did not show any change in their feed consumption. Histopathological analysis of the (D) Liver, kidney, heart, brain, and lung tissue samples obtained from the Wistar rats, did not show any pathological lesions following exposure- Control, SKK 100 mg/kg, SKK 500 mg/ml, and SKK 1,000 mg/kg treatment. Results are expressed as a Mean ± Standard Error of Means. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. No statistical significance was detected.

Figure 5

In-vivo pharmacological effect study of Divya Sarva-Kalp-Kwath (SKK) in carbon tetrachloride (CCl₄) treated wistar Rats: (A) Wistar rats were divided randomly into four groups (n = 5): Group 1 (NC): Animals were administered with olive oil (intraperitoneal injection; 0.25 ml/kg; every 3rd day for 9 weeks) and 0.25% Na-CMC, Group 2 (DC): Animals were administered with CCl₄ in olive oil v/v (intraperitoneal injection; 0.5 ml/kg; every 3rd day for 9 weeks), Group 3 (PC): Animals were administered with CCl₄ in olive oil v/v (intraperitoneal injection; 0.5 ml/kg; every 3rd day for 9 weeks) with the concurrent oral treatment of SLM (100 mg/kg; once daily for 9 weeks), Group 4 (SKK-treated): Animals were administered with CCl₄ in olive oil v/v (intraperitoneal injection; 0.5 ml/kg; every 3rd day for 9 weeks) with the concurrent oral treatment of SKK (100 and 200 mg/kg; once daily for 9 weeks). The animals were observed for physiological, histopathological, and biochemical changes during and after completion of the study period. (B) Substantial loss of body weight was detected in the CCl₄ stimulated Wistar rats. Treatment of the CCl₄ stimulated animals with human equivalent and higher dose of SKK (100 and 200 mg/kg) resulted in the minor recovery in body weight loss. (C) Wistar rats treated with CCl₄ showed loss of food habits over a period of 28 days. Recovery was detected in the CCl₄ stimulated animals following treatment with the high dose of SKK (200 mg/kg). SLM (100 mg/kg) was used as a positive control in the study and did not induce any changes in the feed habits. Results are expressed as Mean ± Standard Error of Means. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. p-value ^#<0.01; *<0.05; **<0.01.

Figure 6

In-vivo biochemical analysis of CCl₄ stimulated wistar rats treated with Divya Sarva-Kalp-Kwath (SKK): Wistar rats stimulated with CCl₄ showed a significant upsurge in their serum enzyme levels–(A) alanine transaminase (ALT), (B) aspartate aminotransferase (AST), (C) alkaline phosphatase (ALP), (D) total bilirubin, (E) total cholesterol, and (F) uric acid. Co-treatment of the CCl₄ stimulated rats with SKK (100 and 200 mg/kg) showed a significant reduction in their serum liver injury biomarkers. SLM (100 mg/kg) was used as a positive control in the study. Results are expressed as Mean ± Standard Error Mean. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. p-value ^#<0.01; *<0.05; **<0.01.

Figure 7

Histopathological Analysis of Wistar Rats Treated with CCl₄ and Co-Treated with Divya Sarva-Kalp-Kwath (SKK): (A) Histopathological investigation was performed in the liver tissue samples obtained from the Wistar rats treated for 9 weeks with CCl₄, reference drug: SLM (100 mg/kg) and SKK (100 and 200 mg/kg). Results indicated- (i) Untreated animal liver tissue represents normal histology of liver tissue; portal triad (PT), central vein (CV), hepatocyte, (ii) CCl₄ treated animals showed the presence of severe fibrosis and lymphocytic infiltration (FL), hyperplastic bile duct in the hepatic regions, (iii) Wistar rats treated with SLM (100 mg/kg) showed the development of moderate fibrosis and lymphocytic infiltration (FL), hyperplastic bile duct (BD), vacuolation (VC) in the hepatic tissues; (iv) CCl₄ and SKK (100 mg/kg) co-treated animals showed moderate levels of fibrosis and lymphocytic infiltration (FL), hyperplastic bile duct (BD), vacuolation (VC) in their hepatic region; (v) Wistar rats treated with CCl₄ and higher dose of SKK (200 mg/kg) showed the presence of mild fibrosis and lymphocytic infiltration (FL), hyperplastic bile duct (BD) in their hepatic regions. Finally, based on individual lesion scoring, the CCl₄ treated animals showed marked increase in- (B) hyperplastic bile duct (BDH), (C) Hepatocellular vacuolation (HCV), (D) interlobular fibrosis and lymphocytic infiltration (IF & LI), and (E) Total Lesion Score (TLS). The positive control drug, SLM (100 mg/kg) significantly ameliorated the hepatic injuries induced by CCl₄ treatment. CCl₄ stimulated animals co-treated with SKK at 100 and 200 mg/kg also showed significant amelioration in their hepatic lesion score. Results are expressed as Mean ± Standard Error of Means. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test was used to calculate the statistical difference. p-value ^#<0.01; **<0.01.