Ameliorative impact of herbal formulation —Majoon-Dabeed-ul-ward and Sharbat-e-Deenar against CCl₄ induced liver toxicity via regulation of antioxidant enzymes and oxidative stress

Abstract

Polyherbal Unani formulations have been used in the treatment of liver diseases for a long time. (Ibrahim M, Khaja MN, Aara A, Khan AA, Habeeb MA, Devi YP, Narasu ML, Habibullah CM. Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: in vitro and in vivo studies. World J Gastroenterol. 2008:14:2566–2571.) The aim of the present study was to investigate comparative hepatoprotective potential of Majoon-e-Dabeed-ul-ward (MD) and Sharbat-e-Deenar (SD) against CCl₄ induced subchronic hepatic toxicity. In vivo study, albino rats were divided into 5 groups. Group I was control; Group II was experimental control treated with CCl₄ (0.15 mL/kg, i.p. for 21 days); Groups III–IV treated with SD (2 mL/kg, p.o.) and MD (1,000 mg/kg, p.o.) for 5 days following CCl₄ intoxication as in group 2 respectively; and Group V was positive control treated with silymarin (50 mg/kg, p.o.). In vitro hepatoprotective activity of SD and MD (25, 50, and 100 μg/mL) was assessed by SRB assay and flow cytometry analysis. CCl₄ exposure significantly elevated the release of hepatic enzymes i.e. AST, ALT, LDH, and SALP in serum and lipid peroxidation in liver tissue which all these parameters were reversed after SD and MD administration. Therapy for 5 days also normalized the levels of antioxidant enzymes i.e. catalase, SOD, GPx, GR, tissue GSH, and aniline hydroxylase in CCl₄ treated group. DNA damage and histological alterations caused by CCl₄ were restored towards normal group. In vitro study showed protective effect of SD and MD against CCl₄ treated HepG2 cell lines and rat hepatocytes. The results suggested that MD has a significant hepatoprotective potential and regulatory effect on oxidative stress than SD against CCl₄ induced hepatotoxicity, and that this effect may be related to its antioxidant activity.

Introduction

The liver is a central body’s organ that involves in energy metabolism and drug detoxification. It transforms various range of substances via phases I and II pathways of the cytochrome P-450 enzyme system by which it produces reactive oxygen radicals such as hydroxyl radical, superoxide radical anion, and nitric oxide.¹ It is believed that when the cellular antioxidant defense system is declined, it causes lipid peroxidation (LPO) and oxidative stress, which may further alter or damage essential biomolecules such as proteins, lipids, carbohydrates, and DNA.² Despite the fact that numerous synthetic chemicals, environmental pollutants, and hepatotoxicants (carbon tetrachloride, acetaminophen, and ethanol), microbial pathogens and unhealthy foods are responsible for causing liver diseases.³ Jaundice, necrosis, fatty liver, fibrosis, hepatitis, and cirrhosis are all health concerns around the world. In India, liver diseases account for the tenth most common cause of death. Every year, ~2 million people die as a result of liver disease. It is a new global health issue that has been reported to be much higher (8–30%) in India.⁴ Synthetic medications, on the other hand, are responsible for half of all acute liver disorders.

Traditional medicinal plants have been used since ages for the prevention and treatment of human diseases, including liver diseases. Almost 80% of the world’s population rely on traditional herbal medicines for primary health care.⁵ The traditional medicinal system is considered as an alternative and complimentary system of medicine for liver diseases. Many traditional medicinal plants and their combination in form of herbal formulations have been employed for liver protective drugs in the Ayurvedic as well as Unani system of medicine. Recent years, scientific community is showing great interest to rediscover medicinal plants as source of potential drug candidates. In view of this, 2 Unani polyherbal formulation—Sharbat-e-Deenar (SD) and Majoon-e-Dabeed-ul-ward (MD) are developed on the principles of unani system of medicine for the treatment of liver ailments. These herbal formulations were prepared after proper scrutiny of every ingredient of medicinal plants by the National Formulary of Unani Medicine (NFUM) and the preparation and standardization method of unani formulations were prescribed in Unani Pharmacopeia of India (UPI) for the treatment of chronic liver disease.⁶ SD contains 7 ingredients and MD 17 ingredients were prepared by mixing of useful parts of medicinal plants. Majoon-Dabeed-ul-ward is a semi-solid preparation of ~17 plant’s ingredients. Its main constituent is Gulab (Rosa damascene, 200 g). Sharbat-e-Deenar is a viscid preparation, the cichorium intybus (170 g) and cuscuta reflexa (100 g) are its main ingredient. Composition of plant constituents of both the Unani drugs are given in Table 1.

Table 1.

Plant ingredients of SD and MD.

Unani names	Botanical names	Part used	Weight (g)
Sharbat-e-Deenar (SD)
Post-e-bekh-e-Kasni	Cichorium intybus	Root bark	170
Tukm-e-Kasoos	Cuscuta reflexa	Seed	100
Tukm-e-Kasni	C. intybus	Seed	85
Guncha-e-Gul-e-Surkh	Rosa damascena	Flower bud	85
Rewand chini	Rheum emodi	Root	60
Gul-e-Nilofar	Nymphaea alba	Flower	45
Gaozan	Borago officinalis	Leaves	45
Majoon-Dabeed-ul-ward (MD)
Sumbul-ut-Teeb	Nardostachys Jatamansi	Whole plant	10
Mastagi	Pistacia lentiscus	Resin	10
Zafran	Crocus sativus	Flower (Stigma)	10
Tabasheer	Bambusa bambos	Resin	10
Darchini	Cinnamomum zeylanicum	Stem bark	10
Izkhar	Cymbopogon jwarancusa	Whole plant	10
Asaroon	Asarum europaeum u.	Root	10
Qust Shireen	Saussurea hypoleuca	Root	10
Gul-e-Ghafis	Gentiana olivieri	Root	10
Tukm-e-kasoos	C. reflexa	Seed	10
Majeeth	Rubia cordifolia	Root	10
Luk Maghsool	Coccus lacca	Usara	10
Tukm-e-Kasni	C. intybus	Seed	10
Tukm-e-Karafs	Apium graveolens	Seed	10
Zarawan Taweel	Aristolochia donga	Root	10
Habb-e- Balsan	Commiphora opobalsamum	Fruit	10
Ood-e-Hindi	Aquilaria agalocha	Stem	10
Qaranful	Syzygium aromaticum	Dried buds	10
Heel Khurd	Elettaria cardamomum	Dried fruit	10
Waaraq-e-Gul-e-Surkh	Rosa damascena	Petals	200

However, no scientific studies have been reported for comparative hepatoprotective potential of these formulations against CCl₄ induced subchronic liver injury. Thus, there is an emergent need to investigate the ameliorative effect of SD and MD scientifically at biochemical and molecular level so that this polyherbal formulations (PHFs) could attract the attention of a wide proportion of medical practitioners to be an excellent hepatoprotective agent against live ailments. Therefore, the aim of the present study was to evaluate comparative ameliorative impact of SD and MD against carbon tetrachloride (CCl₄) induced subchronic liver damage and to confirm earlier acute studies.

Materials and methods

Drug and chemicals

Sharbat-e-Deenar (SD) and Majoon-e-Dabeed-ul-ward (MD) were procured from Central Council for Research in Unani Medicine (CCRUM), New Delhi. Carbon tetrachloride (CCl₄; E-Merck), Silymarin, dimethyl sulfoxide, Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), gamma-glutamyl p-nitroanilide, glycylglycine, 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavine adenine dinucleotide (FAD), Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), sodium hydroxide, trichloroacetic acid (TCA), and perchloric acid (PCA) were purchased from Sigma Chemicals Co. United States. Dulbecco’s modified Eagle’s medium, fetal bovine serum, trypsin 0.25%, penicillin G, streptomycin, and phosphate-buffered saline were obtained from HiMedia Laboratories LLC, and all other chemicals and reagents were used of analytical grade. Kits for measurement of LDH and SALP activities were purchased from E-Merck. Human liver hepatoma cell lines (HepG2) was procured from the National Center for Cell Sciences (NCCS), Pune, India.

Animals

Female Sprague Dawley albino rats (160 g ± 10) between 6 and 8 months of age were used in the study. Animals were randomly selected from departmental animal facility where they were housed in polypropylene cages (3 animals/cage) under uniform husbandry conditions of light (14 h) and dark (10 h) with temperature (25 ± 2°C) and relative humidity (60–70%). Animals were fed on commercially available dry pellets of standard animal diet (Pranav Agro Industries Ltd, New Delhi, India) and drinking water ad libitum according to the study protocol approved by Institutional Animal Ethics Committee (Regd. no. CPCSEA/501/01/A). The experimental procedure was carried out as per guidelines set by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.

Dose selection and calculations of PHFs

The treatment dose 2 mL/kg, SD and 1,000 mg/kg, MD was selected from the acute effective doses of SD (1, 2, and 4 mL/kg) and MD (250, 500, and 1,000 mL/kg) against CCl₄ induced liver damage.^7,8 The toxicity of SD and MD at higher doses (4 mL/kg and 1,000 mg/kg) was also reported in previous studies.

The doses of PHFs for rat were calculated based on clinical human doses 10 mL SD and 5–10 g MD as mentioned by Unani pharmacopeia. Dose conversion (human to animal) was done by utilizing a standard conversion table.⁹

Preparation of doses

Carbon tetrachloride (CCl₄, 0.15/5 mL/kg, i.p.) was prepared with olive oil (1:1) and administered to animals through intraperitoneally (i.p.) for 21 days (3 weeks).¹⁰ The aqueous suspensions of SD (2/5 mL/kg) and MD (1,000 mg/5 mL/kg) were prepared with distilled water and administered orally to the experimental animals for 5 days after CCl₄ intoxication for 21 days. Silymarin was used as a positive control.¹¹

Experimental design

Female albino rats were divided into 5 groups of 6 animals each:

Group I received olive oil only (normal control);

Group II treated with CCl₄ for 21 days (0.15 mL/kg, i.p.; experimental control);

Groups III–IV (as in group II) were administered with the SD (2 mL/kg, p.o.) and the MD (1,000 mg/kg, p.o.) for 5 days respectively; and

Group V treated with the silymarin (50 mg/kg, p.o.) same as in groups 3–4 (Positive control).

After 24 h of last treatment, all experimental animals were euthanized. Blood was drawn from the retro orbital venous sinus just before the animals were euthanized. A blood sample was collected in nonheparinized centrifuged tubes and serum was isolated to assess biochemical variables. The rat liver organ was isolated for biochemical assay, microsome preparation, histopathological observation, and comet assay.

Isolation of serum and homogenate preparation

After keeping the blood for 1 h at room temperature, serum was isolated by centrifugation at 1,000 × g for 15 min and stored at −20°C until analyzed. Immediately after necropsy, tissues were excised, washed with normal saline, blotted, and stored frozen. For other enzymatic assays, the tissues were homogenized with a Remi Motor homogenizer (RQ-122) using glass tube and Teflon pestle, in different media according to the protocol of parameters. Tissue homogenates (5% w/v) were prepared in hypotonic solution (0.008% NaHCO₃). Liver homogenates (10% w/v) was prepared in 0.15-M KCl for determination of LPO and in 0.25-M sucrose for the estimation of glutathione content (GSH). Remaining tissues of liver, was homogenized with ice-cold 1.15% KCl for the determination of GR, GPx, and SOD activities.

Blood biochemistry

Liver functions tests i.e. Aspartate transaminase (AST) and Alanine transaminase (ALT) were estimated as per protocol of Reitman and Frankel¹² and absorbance of brown colored hydrozones measured at λ510 nm. Alkaline phosphatase (SALP) and lactate dehydrogenase (LDH) were estimated in serum by using standard E-Merck diagnostic kits.

Tissue biochemistry

Assay of LPO

LPO was determined spectrophotometrically by measuring the level of LPO product, malondialdehyde (MDA) as per protocol.¹³ Liver homogenates (10% w/v) was prepared in 0.15-M KCl for LPO. In this method, 1.0 mL of homogenate prepared in KCl solution was incubated at 37°C for 30 min. Proteins were precipitated by adding 1 mL of 10% TCA and then centrifuged at 2,000 rpm for 15 min. One-milliliter supernatant was taken as an aliquot in a separate tube to which 1 mL of TBA solution was added. The tubes were kept in boiling water bath for 10 min. After cooling the tubes, the optical density was read at λ 535 nm.

GSH level

GSH was measured by its reaction with 5–5′-dithio-bis 2-nitrobenzoic acid to give a yellow colored product.¹⁴ Liver homogenates (5% w/v) were prepared in 1% sucrose solution for the estimation of GSH. About 0.1 mL of homogenate was taken in a tube to which 0.9 mL of distilled water and 1.0-mL sulphosalicylic acid was added. The contents were mixed thoroughly and then centrifuged at 5,000 rpm for 10 min. About 0.5 mL of supernatant was taken in a tube and similarly blank and standards were prepared by taking 0.5 mL of distilled water and 0.5 mL of GSH standard respectively. To all the tubes, 4.5 mL of Tris-buffer and 0.5 mL of DTNB solution were added. After 6 min. OD was read at λ412 nm.¹⁵

Assessment of antioxidant enzyme activity (oxidative stress markers)

Catalase

The catalase was determined by assessing decomposition of hydrogen peroxide/min. In this method, 10% homogenate was prepared in 0.9% NaCl and centrifuged at 2,500 rpm for 15 min and the supernatant was used for assay. A typical reaction mixture containing 1 mL 50 mM potassium phosphate buffer (pH 7.4), add 25-μL sample. Reaction was initiated by addition of 1 mL H₂O₂. Amount of H₂O₂ consumed was determined by recording absorbance of solution at λ 240 nm and the Catalase activity was expressed as units/mg protein.¹⁵

Superoxide dismutase

Superoxide dismutase (SOD) was assayed by assessing inhibition of rate of adrenochrome formation.¹⁶ Homogenate mixture contained 0.5-mL bicarbonate buffer, 0.5-mL EDTA, 50-μL sample, 1-mL dH₂O and incubate 5 min. at room temperature. Reaction started by addition of 0.3-mL epinephrine and absorbance was recorded at λ480 nm for 3 min. The activity expressed in U/mg protein.

Glutathione peroxidase

Glutathione peroxidase (GPx) enzyme activity was assessed by the measuring the reduction of NADP^{+ _t}o NADPH.¹⁷ Liver pieces (100 mg) were diced and 10% homogenate was prepared in 1.15% KCl and centrifuged at 10,000 rpm for 10 min. and supernatant was used for assay. Reaction mixture containing 0.3-mL sodium phosphate buffer, 0.1-mL reduced glutathione, 0.05-mL sodium azide, 0.05-mL suitable diluted homogenate, 0.01-mL NADPH, and 0.05-mL glutathione reductase was incubated at room temperature for 10 min. Reaction was initiated by addition of 0.05 mL H₂O₂ and absorbance change/minutes were recorded at λ340 nm for 5 min. Specific activity was expressed as μmol/min/mg protein.

Glutathione reductase

GR was estimated by measuring the rate of conversion of NADPH to NADP^+.18 Liver pieces (100 mg) were diced and 10% homogenate was prepared in 1.15% KCl and centrifuged at 10,000 rpm for 10 minute and the supernatant was used for assay. A typical reaction mixture containing 0.7-mL phosphate buffer, 0.1-mL oxidized glutathione (GSSG), and 0.1-mL suitably diluted homogenate was incubated at room temperature for 10 min. Reaction was initiated by the addition of 0.1-mL NADPH and absorbance change was recorded for 5 min. at λ 340 nm. Specific activity was expressed as μmol/min/mg protein.

Determination of drug-metabolizing activity (aniline hydroxylase)

Microsomal CYP2E1 activity was measured in terms of aniline hydroxylase (AH) by measuring the concentration of blue colored conjugate of phenol with p-amino phenol (PAP) as per method.¹⁹ In a tube containing 0.5-mL tris acetate buffer, 0.1 mL of NADPH, MgCl2 and aniline (0.1 mL), 0.2 mL microsomes were added. Mixed and incubated at 37°C for 20 min. Now 200-μL TCA (30%) were added, mixed and centrifuged at 3,000 rpm for 10 min. 750 μL of supernatant, p-aminophenol and TCA (5%) were taken for test, standard and blank respectively. Added 0.1 mL sodium carbonate and 0.5 mL phenol in all tubes and kept at room temperature for 30 min. before measuring optical density at λ630 nm.

Comet assay

The comet assay is a single cell gel electrophoresis used to measure single cell DNA damage.²⁰ About 0.2 g of liver tissues was homogenized in 1-mL chilled phosphate buffer saline (pH 7.2) and centrifuged at 400 × g for 10 min. Pellet contains nuclei fraction was taken for comet assay. 100-μL normal melting agarose was spread on frosted microscopic slide and allowed to solidify at 4°C for 10 min. Isolated nuclei and low melting agarose were taken in the ratio of 1:4 (100 μL) was cased on precoated slides and then kept at 4°C for 20 min. After solidification the slides were immersed into the chilled lysis buffer for 1 h in dark at 4°C (2.5-M NaCl, 100-mM EDTA, 10-mM Tris, pH 10, 1% Triton X-100, and 10% DMSO, Tris–HCl buffer [0.4 M, pH 7.5], and electrophoresis buffer [300-mM NaOH, 1-mM EDTA]). The slides were stained with coating 40-μL ethidium bromide (ETBr), rinsed with dH₂O twice and nucleus was observed under florescence microscope (LeitzAutoplan, excitation filter λ 595 nm, green filter). Observations were recorded by software Leica QWin V.

Histopathology

For electron microscopical studies, small pieces (1 mm³) of hepatic tissues were fixed in 3.2% glutaraldehyde (prepared in 0.1-M phosphate buffer) at 4°C for 18 h. This was followed by washing the tissue with phosphate buffer (pH 7.2). Post fixation was done with osmium tetraoxide (1%) and then dehydration was performed in acetone series and subsequently inclusion was done in eon embedding resin and polymerized at 70°C for 20 h. The tissue blocks were cut into ultra-thin sections using glass knives of ultra-microtome. The sections were placed on uncoated grids and stained with solution of uranyl acetate and lead citrate and analyzed with transmission electron microscope.

In vitro study (SRB assay)

Cell culture: HepG2 human liver hepatoma cells were grown (1 × 10⁵ cells) in standard conditions. With Dulbecco’s modified Eagle medium (DMEM) added with 10% Fetal Bovine serum (FBS) and antibiotics like penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified atmosphere of 5% Carbon dioxide (CO₂) at 37°C until confluent. The SRB assay was performed in triplicates (n = 3) to assess the cell viability of treated cells. The stock solutions of MD (5 mg/mL) and silymarin were prepared and treated cells at 3 concentration 25, 50, and 100 μg/mL in CCl₄ treated -HepG2 cell lines.

Cytotoxic effect of MD

Sulforhodamine B (SRB) testing was performed to assess cytotoxic effect and protective effect of the test formulation.²¹ Three doses of the test formulation SD and MD, i.e. 25, 50, and 100 μg/mL, were used alone and in CCl₄ (3 mM) treated HepG2 cell lines. After 24 h of the last treatment, cytotoxicity was determined by SRB assay. Silymarin was used as a positive control.

Effect of MD on CCl₄ induced toxicity

CCl₄ at concentration (3 mM), SD, MD, and silymarin at 3 concentrations 25, 50, and 100 μg/mL were used in this study. The CCl₄ were added along with SD, MD, and silymarin (positive control) as a conjoined treatment to the cell culture 96 Elisa wellplates. Group 1 treated with DMEM Medium (Normal control). Group 2 cells treated with CCl₄ (100 μL). Groups 3–5 (as in group 2): treated with SD along with CCl₄. Groups 6–8 (as in group 2): treated with MD along with CCl_4. Groups 9–11 (as in group 2) treated with silymarin, and Elisa well plate was allowed to grow cells in the presence of test material by further incubating the plates for 48 h. After incubation, TCA was added to stop the cell growth and then washed the plate after 1 h, and thereafter 100-μL SRB dye (0.4%) was added into each well and plate was allowed to stand at 30 min. and washed plates with 1% acetic acid. The plates were dried and Tris-buffer (100 μL/well) was added to each well to solubilize the bound dye. The plates were shaken gently for 10 min on a shaker and the optical density of color product was recorded on ELISA reader of Robotic liquid handling system at 540 nm.

Cell cycle analysis

The protective effect of test formulation was further determined by flow cytometry. Freshly isolated rat liver hepatocytes were seeded at a density of 3 × 10⁶ viable cells/60 mm pre collagen coated plate / 3 mL. Williams medium A with 10% fetal calf serum and 0.1-μM insulin at 37°C in a humidified atmosphere of 5% CO₂. After 3 h, cells adhere to the surface, displaying epithelial morphology.

In the presence of 3% FCS, the cells were incubated in 3-mL fresh Williams medium E containing 0.1-M insulin, 0.1-M dexamethasone, 5-nM epidermal growth factor, and 1-nM glucagons. For 24 h, monolayer cultures were treated with 3-mM CCl₄ in dimethyl sulfoxide with or without SD and MD (100-μg/mL effective dose selected from SRB assay).

The SD & MD was dissolved in DMSO at a concentration of 20 mg/mL (DMSO 0.3%, v/v) and added to the culture plates alongside the CCl₄ toxicant. The cells were treated with propidium iodide (PI), and then flow cytometry (ModFit Software, Topsham, ME) was used to analyze cells growth.²²

Statistical analysis

The data were presented as a mean S.D. The statistical significance was determined using 1-way ANOVA and the student’s t-test at P ≤ 0.05.²³ The percentage protection (%) was also used to determine the protective activity of treatment groups. The protective activity of treatment groups was expressed as a percentage of protection calculated by the following formula:

Percent protection (%) = 1 − (D − C/T − C) × 100.

where, D = Drug, C = Control, and T = Toxicant.

Results

In vivo study

Effect of SD and MD on hepatic biomarkers

Figure 1 showed the effect of CCl_4, SD, and MD on liver functions tests. CCl₄ subchronic exposure for 21 days significantly increased the levels of hepatic markers enzymes i.e. AST, ALT LDH, and SALP in serum (P ≤ 0.05). Five-day administration of SD (2 mL/kg) and MD (1,000 mg/kg) significantly reversed the level of AST, ALT, LDH, and SALP in treatment group when compared to the control group (P ≤ 0.05). The maximum percent recovery was observed with MD therapy in AST (92%), ALT (96%), SALP (89%), and LDH ( 63%), followed by SD therapy.

Fig. 1.

Values are mean ± S.E., N = 6; ANOVA ^@ = significant. ^#P ≤ 0.05 vs control, ^*P ≤ 0.05 vs CCl₄, CCl₄ = carbon tetrachloride, SD = Sharbat-e-Deenar (2 mL/kg), MD = Majoon-e-Dabeed-ul-ward (1,000 mg/kg), and S = Silymarin (50 mg/kg).

Effect of SD and MD on tissue biochemistry

Effect of CCl₄, hepatic LPO, reduced glutathione, and AH was represented in Fig. 2. CCl₄ induced oxidative stress significantly increased MDA content (lipid peroxidation) and decreased GSH content in liver tissue compared to the control group (P ≤ 0.05). An enzyme activity of AH is significantly diminished by CCl₄ administration. Therapy with SD (2 mL/kg) and MD (1,000 mg/kg) restored the level of AH, GSH, and LPO towards control group. Percent protection showed maximum ameliorative effect in MD at 1,000 mg/kg in the level of LPO (91 %), GSH (67 %), and AH activity (92%) when compared with the SD formulation.

Fig. 2.

Values are mean ± S.E., N = 6; ANOVA ^@ = significant. ^#P ≤ 0.05 vs control, ^*P ≤ 0.05 vs CCl₄, CCl₄ = carbon tetrachloride, SD = Sharbat-e-Deenar (2 mL/kg), MD = Majoon-e-Dabeed-ul-ward (1,000 mg/kg), and S = Silymarin (50 mg/kg).

Effect of SD and MD on oxidative stress markers

Figure 3 depicts protective effect of SD and MD against CCl₄-subchronic toxicity. CCl₄ treated animal significantly reduced the activities of antioxidant enzymes i.e. SOD, catalase, GR, and GPx when compared with the control group. Administration of MD for 5 days in CCl₄-treated animals significantly improved the level (54–62%) of antioxidant enzyme activities followed by SD.

Fig. 3.

Values are mean ± S.E., N = 6; ANOVA ^@ = significant. ^#P ≤ 0.05 vs control, ^*P ≤ 0.05 vs CCl₄, CCl₄ = carbon tetrachloride, SD = Sharbat-e-Deenar (2 mL/kg), MD = Majoon-e-Dabeed-ul-ward (1,000 mg/kg), and S = Silymarin (50 mg/kg).

Effect of SD and MD on CCl₄ induced DNA damage

The effect of CCl₄, SD, and MD on hepatocytes DNA was shown in Fig. 4. The damage was expressed as a percentage of DNA migration in the tail, and each slide contained 20–25 nuclei. In the CCl₄-treated group, we observed higher DNA damage (20.0%) and comet tail length (18.40 μm) compared with the control group. In the treatment groups, we observed decreased comet length (MD; 1,000 mg/kg) and (SD; 2 mL/kg) compared to the CCl₄-treated group.

Fig. 4.

Values are mean ± S.E., N = 6. ^#P ≤ 0.05 vs control, ^*P ≤ 0.05 vs CCl₄, ANOVA, ^@ = significant at 5%level. CCl₄ = carbon tetrachloride; SD = Sharbat-e-Deenar ((2 mL/kg); MD = Majoon-e-Dabeed-ul-ward (1,000 mg/kg); % percent protection.

Histological examination

Figure 5 demonstrated the histological effect of SD and MD in CCl₄-treated groups. A control rat liver electron micrograph revealed a well-formed nucleus, endoplasmic reticulum, and abundant mitochondria in the cytoplasm (Fig. 5A). The most notable findings in the CCl₄-exposed group demonstrated extensive cellular damage, including hepatocyte disorganization, swelling in mitochondria, endoplasmic reticulum disorganization and degranulation, and damaged nuclei. The nuclei had a deformed appearance and karryohexis, with a clear scalloping nuclear envelop (Fig. 5B).

Fig. 5.

Protective effect of SD and MD on hepatocyte damage caused by CCl₄: A) Control liver, normal hepatocyte architecture. B) CCl₄ (0.15 mL/kg) showed hepatic necrosis, cellular degeneration and loss of cellular organelles and sinusoidal space. C) SD (2 mL/kg + CCl₄) with mild recovery of hepatocyte structure contain minimum mitochondria and ER. D) MD (1,000 mg/kg, + CCl₄) showed the well-formed the nucleus with nuclear boundaries along with the presence of mitochondria and endoplasmic reticulum. E) Silymarin (50 mg/kg + CCl₄) restored the normal cellular morphology.

Figure 5C showed SD-treated liver sections revealed well-defined hepatic cell boundaries. The nucleus appeared normal, the nuclei envelopes were intact, and there were fewer mitochondria and endoplasmic reticulum, indicating less improvement in cellular morphology compared to the control group. On the other hand, Fig. 5D demonstrated the effect of MD therapy on CCl₄-treated rats. MD-treated CCl₄ group showed improvement in hepatocyte architecture with intact nuclear envelopes, well-arranged endoplasmic reticulum, and abundant mitochondria, indicating the start of the regeneration process. Fat droplets were extremely rare to see. The mitochondria were spherical with well-formed crests and were densely packed near the nucleus. Endoplasmic reticulum was significantly increased. These histological findings were also seen in the positive control group, with silymarin indicating normal architecture of the liver section, and that was similar to the control group (Fig. 5E). The histological effect of herbal formulations was also linked to biochemical results.

In vitro study

In vitro hepatoprotective effect of MD in CCl₄-treated HepG2 cell lines

Figure 6 showed the cytotoxic effect of SD, MD, and the positive control silymarin were tested at concentration of 25, 50, and 100 μg/mL for cytotoxicity in HepG2 cell lines and were found to be non-toxic to the cells.

Fig. 6.

Values are mean ± S.E. of 6 wells in each group of 3 repeated experiments.

Figure 7 showed the hepatoprotective effect of MD on CCl₄-treated HepG2 cell lines. The administration of CCl₄ (3 mM) significantly reduced cell viability (51%) in HepG2 cell lines. Following CC_l4 exposure, the cell culture was incubated with MD extract at different concentrations of 25, 50, and 100 μg/mL. MD-treated cells increased HepG2 cell line proliferation in culture media in a concentration-dependent manner. MD demonstrated a significant dose-dependent protective effect against CCl₄ induced cell viability loss. The results showed that the maximum cell viability was 90% at 100 μg/mL, followed by 78% (50 μg/mL) and 77% (25 μg/mL), which was similar to the positive control silymarin.

Fig. 7.

Values are mean ± S.E. of 6 wells in each group of 3 repeated experiments.

Fig. 8.

Effect of MD on cell cycle progression (S phase) in CCl₄ treated cultured hepatocytes. A) Normal hepatocyte S phase. B) CCl₄ (3 mM) treated rat hepatocyte decline the cell viability (DNA synthesis) indicating loss of cell population. C) SD (2 mL/kg + CCl₄) improved moderately the DNA replication in S phase indicating improve cell viability. D) MD (1,000 mg/kg + CCl₄) restored the DNA replication in S phase indicating improve cell viability. E) Silymarin (50 mg/kg + CCl₄) showed the normal S phase indicating DNA protection toward normal DNA synthesis against toxicity caused by CCl₄.

Effect of MD on cell cycle progression in CCl₄-treated rat hepatocytes

The effect of MD on the S phase of the cell cycle in CCl₄-treated cells is depicted inFig. 4 Fig.8.Flow cytometric analysis was used to determine the DNA content profile in the cell cycle-S phase of cultured hepatocyte cells. In CCl₄-treated culture hepatocytes, the percentage of cells in the cell cycle (S phase, DNA content) declined substantially, whereas SD and MD administration at 100 μg/mL increased cell density, similar to silymarin.

Discussion

The hepatotoxic effect of CCl₄ in experimental animals is known to be free radical mediated processes.²⁴ The free radicals produced by hepatic CYP450-mediated CCl₄ metabolism are trichloromethyl (CCl₃•) and trichloromethyl peroxy (CCl₃O₂•) radicals reported to initiate LPO damage leading the hepatic damage.^25,26 The liver is the primary target organ for metabolic action of CCl₄.

In the present study, intraperitoneal administration of CCl₄ for 21 days has caused significantly increased level the AST, ALT, SALP, and LDH enzyme activities in serum. The elevated serum level of AST and ALT showed one AST; ALT ratio that indicates necrosis of the hepatocytes. The elevated serum ALP activity might be due to the intrahepatic cholestasis. The leakage of hepatic enzymes indicate liver damage has occurred due to LPO membrane damage. LPO level was high in CCl₄ treated group compared to the normal group animals. This suggests that LPO is the initial marker of liver damage, which could be the result of free radical (CCl₃•) induced membrane damage during metabolism of CCl₄. This damage also indicates that formation of free radicals in higher quantities in experimental treated group (CCl₄ alone) that causes oxidative stress. Overwhelming of oxidative stress causes failure of antioxidant defense system and hepatic damage.²⁶–28 Microsomal LPO also decreases the activity of drug-metabolizing enzyme AH after CCl₄ administration, which clearly demonstrated damage in endoplasmic reticulum.²⁹ Therapy with herbal formulations restored AH enzymatic activity similar to positive control silymarin. This restoration might be due to the improving membrane integrity of cells after SD and MD administration.²⁹ We further studied the non-enzymatic antioxidant such as GSH and endogenous antioxidant enzymes knowns as oxidative stress markers i.e. SOD, CAT, GPx, and GR that involve in neutralization of free radicals and mitigation of cellular oxidative stress.³⁰ Decreasing the level of these enzymes are responsible for increasing lipid peroxidative damage and causes free radical induced oxidative stress, which leads to loss of membrane integrity, and finally loss of hepatocyte functions. We find in our studies that CCl₄ administration causes decline levels in all these hepatic antioxidant enzymes when compared with the normal control animals. The similar findings also find in herbal formulation like Liv-52 and Livomyn.^28,31

We also assessed DNA damage using single-cell gel electrophoresis. Known as the COMET test, this test analyses comet tail length to assess the extent of DNA damage.^32,33 Our data confirmed that CCl₄-mediated oxidative stress induced DNA damage. We also observed that SD and MD treatment reduced DNA damage. These results demonstrate that herbal formulations may protect DNA from oxidative damage produced by subchronic CCl₄ exposure. In groups treated with MD, we observed a larger reduction in DNA damage than in groups treated with SD. The observations in these groups were comparable to those treated with silymarin.

The biochemical observations were supplemented with histopathological examination of rat liver sections. Although, the degree of CCl₄ caused hepatotoxicity, which consequently seen as hepatic steatosis (e.g. fatty infiltration), centrilobular necrosis, fatty liver, fibrosis, and ultimately cell death.^34,35 Our histological findings pointed out that group II (CCl₄ treated) showed vacuolization of cells, loss of intracellular organelles (mitochondria, endoplasmic reticulum, nucleus etc.), necrosis, and fatty acid degeneration and disturbed the cord arrangement when compared to the normal control group. These cellular changes were probably due to oxidative stress and membrane damage. Treatment of SD and MD for 5 days markedly reduced the cellular damage by reversal of intracellular integrity when compared to CCl₄ treated group. Our finding revealed that MD formulation showed better recovery in cellular morphology than SD-treated group against toxicity. Thus, it indicates that the SD and MD both have free radical scavenging activities and hepatoprotective effect against CCl₄ induced subchronic liver toxicity.

Research studies reported that the hepatoprotective herbal formulations are in great demand due to their safety and ameliorative efficacy against liver diseases.^36,37 The statistical analysis also confirmed that MD has shown significant recovery of LPO, GSH level, antioxidant and AH enzyme activity compared to the SD formulation.

Considering the above results, the comparison of the potency of the 2 herbal formulation in protecting HepG2 further investigated by SRB assay and flow cytometry analysis.^21,38 The possible protective activity of herbal formulation towards loss of cell viability induced by CCl₄ was also tested at 3 doses 25, 50, and 100 μg/mL. In vitro study confirmed the both formulations (SD and MD) increase HepG2 cell viability and rat hepatocytes against CCl₄ toxicity indicating their protective and regenerative action. The SD and MD did not show any adverse effect on HepG2 cell lines. The percent protection confirms that MD is more effective at higher dose (100 μg/mL) followed by SD against CCl₄ and it provides pronounced cell viability effect similar to the positive control silymarin. Thus, it can be assumed that the observed protective effect in present findings of herbal formulation may be responsible for their ameliorative effect in vivo and in vitro. The protection might be due to free radical scavenging effect and synergistic effect of individual ingredients of herbal drugs.^3,39

Treatment with SD and MD for 5 days restores the antioxidant levels, which is a positive sign of liver protection. The similar observation was found with the positive control, silymarin. It has been reported in previous studies that SD and MD formulation possess potent antioxidant activity determined by DPPH assay (2, 2-diphenyl-1-picrylhydrazyl) and hydrogen peroxide (H₂O₂) assay. The significant total phenolic content was also to be found in MD formulation followed by SD [7a,8a].

The flavonoids such as emodin (Rheum emodi) gallic acid,⁴⁰ kaempferol (Rosa damascene),⁴¹ esculetin and thymoquinone (Cinchorium intybus)⁴² etc. limonon, terpinolene, and myrcene (Elettaria cardamomum)⁴³ cuscutin, rutin, gallic acid (Cuscuta reflexa)⁴⁴ are reported to have antioxidant and hepatoprotective efficacy. Therefore, the Unani drugs- Sharbat-e-Deenar (SD) and Majoon-Dabeed-ul-ward (MD) enhance the recovery of liver function as well as the restore enzymatic levels with improvement of cellular morphology against CCl₄ induced subchronic liver damage.

Conclusion

The result of this study indicates that the SD and MD are capable to improve liver function and antioxidant defense system against CCl₄ induced subchronic liver damage. However, the pronounced ameliorative effect was observed in MD formulation followed by the SD and these herbal drugs showed hepatoprotective potential due to combined effect of all ingredients present in the formulation, which can be used for the treatment of hepatic diseases.

Authors’ contributions

AKS was the primary investigator for this study, specifically performing the experiments, collecting data/evidence, and writing the research manuscript. NS assisted in carrying out the research. SS helped in histological observations of rat liver. MB assisted in the statistical analysis of study data. SKN contributed to data curation. SS was the project investigator who provided lab facility for carrying out research experiments and assisted in the design of methodology as well as conceptualizing the research’s goal.