INTRODUCTION:

The liver is that the fundamental detoxifying channel inside the human body. However, continuous exposure to sure chemotherapeutic operative, drugs, environmental toxins may lead to toxicity [2].

N-acetyl-para-aminophenol (paracetamol) was exposed in 1889[1]. As paracetamol is well facing, it is conventionally taken preferences as an over-the-counter (OTC) pain-relieving and antipyretic drug for cerebral pain and minor musculoskeletal movement [3, 4]. The hepatotoxic effect of paracetamol is participative with the toxic metabolite, N-acetyl-p benzoquinoneimine (NAPB), which forms in the liver. This metabolite is detoxicated by autogenous glutathione (GSH). However, if paracetamol is taken in high doses, it depletes GSH abundance and commands to an inability to detoxify NAPB sufficiently that leads to hepatic lethality [5, 6]. Liver damage is associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue GSH levels. In addition serum levels of many biochemical markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin, alkaline phosphatase are elevated [7]. This recommended that oxidative stress is a consequential factor in liver toxicity evoked by paracetamol [8].

A number of medicinal plants are used in traditional system of medicine for the management of liver disorders[9].Holarrhena antidysenterica L. (HA) is a small deciduous plant belongs to the family Apocynaceae. It has been utilized universally as a part of the numerous customary arrangement of medication. The stem bark of this plant, economically well-known as Kurchi has been used traditionally for the remedy of dysentery [10]. It has been extensively utilized for the treatment of gastrointestinal disorders such as amoebic dysentery from the time immemorial and also used as antibacterial, anti-diarrheal, and astringent properties [11,12]. The bark and seeds are used for asthma, bronchopneumonia, malaria and some other illness [13]. The seeds and leaves of the plant have disclosed to incorporate a number of steroid alkaloids Conessine (C24H40N2), Isoconessine (C24H40N2), Conessimine Isoconessimine (C23H38N2), Conarrhimine (C21H34N2), pyrrolidinebases, amino-glucosteroids, and amino-glum cardenolides [14,15]. Approximately 20% of the plants found in the world have been submit to pharmacological or biological test, furthermore a considerable number of new antibiotics introduce on the market are obtained from natural or else semi-synthetic resources [16]. Henceforth, the point of current assessment is to explain the anti-hepatotoxic influence of the standardized HA extract on a rat model of paracetamol induced hepatotoxicity which has not been reported till now.

MATERIALS AND METHODS:

Chemicals:

Silymarin powder (Silybon), Paracetamol (PCM), Standard kits for SGOT, SGPT and ALP, DPPH (1,1-diphenyl-2-picryl-hydrazyl), Dimethyl sulphoxide, Ethanol, Formic acid, Haemotoxylin dyes were obtained from Sigma-Aldrich, USA and all the other chemical and solvents were used of analytical grade.

Collection, Authentication and Characterization of HA:

The leaves and bark of the plant were collected from the nursery of CSIR–National Botanical Research Institute (NBRI), Lucknow, India in the month of May, 2016. The voucher specimen for the collected plant was deposited in the institute with the voucher no. 33428. The collected parts were dried, grounded, mixed and extracted with the suitable solvent using soxhlet apparatus. The extracted material was executed by a soxhlation technique in two steps. Firstly, the pulverized material was defatted to cover by soxhlet assembly exploitation 250 mL of 98% petroleum ether for 6 hours, which was followed by 9 hours of soxhlation by using ethanol (50%) as a solvent. Then the extract was refined and concentrated under reduced pressure in a rotary evaporator (Buchi R-200, USA) at 37°C and then freeze-dried in lyophilizer (Labconco, USA) to gain solid residual (HA extract, yield 10.3% w/w). The extract was completely dried in desiccator stored in refrigerator at 4^oC and protected from sunlight until the time for extract administration[17]. The dried extract was dissolved in 98% ethanol to obtain a stock solution of 10 mg/mL, which was used for application of spots on HPTLC plates.

Phytochemical Screening:

The HA extract was evaluated for identification of chemical constituents. Varied phytochemical tests were performed to screen the existence of active compounds described by Trease-Evans [18] and Harborne [19].

Experimental Section:

Ethics Statement:

All the experiments were performed in the morning time and agreement with the convention for the examinations on creatures were endorsed by CPCSEA, New Delhi (Approval number: 1732/GO/Re/S/13/CPCSEA).

Experimental Animals:

Male Wistar rats weighing 120–160 g were selected for the study obtained from animal house of the National Laboratory Animal Centre. They were conserved under controlled surroundings of temperature 25±26°C and relative humidity 44-56% with light/dark cycles of 12 h respectively, for one week. Food was withdrawn 18-24 hours before the experiment though water was given ad libitum.

Acute Oral Toxicity:

The acute oral lethality was determined according to Organization for Economic Cooperation and Development (OECD) guideline 423. The highest dose of HA extract (2000 mg/kg) was given to animals and observed for behavioral profile (readiness, fretfulness, fractiousness, and dreadfulness), autonomic profiles (poop and pee), neurologic profile (unconstrained action, reactivity, touch reaction, torment reaction, and step), physical states (lacrimation, loss of hunger, tremors, hair erection, salivation, loose bowels, and for grimness) and mortality. The observation was continuing for 14 days.

Pharmacological Evaluation:

Antioxidant Studies of HA Extract:

The antioxidant action of the extract was assessed on the basis of the radical scavenging effect of the stable DPPH (1, 1-diphenyl-2-picryl-hydrazyl) [20]. The mixture of 2 ml each of ethanol and DPPH solution (0.005%) was used as a control solution. The percent inhibition of HA extract was determined using the formula given below [21].

% inhibition of DPPH activity = (A–B/A) × 100

Where, A is optical density of the control and B is optical density of the sample (HA).

PCM Induced Hepatotoxicity:

The animals were divided into five different groups each containing six animals. Group I; control group (CG) 2% w/v acacia suspension, Group II; toxic group (TD) 2g/kg of PCM, Group III; standard group (SG) 100 mg/kg of Silymarin, Groups IV and V; test groups TG-1 (200 mg/kg HA) and TG-2 (400 mg/kg HA) respectively. All the animals were treated for 4 weeks through oral gavage [22].

Biochemical Analysis:

Blood samples of the fasted rats were collected from the retro-orbital plexus immediately with capillary tubes (Micro Hematocrit Capillaries, Mucaps) under ether anesthesia after completion of 4 weeks treatment. Then the blood was centrifuged at 3000 r/min for 15 minutes and serum was collected for different quantitative analysis of enzyme activities i.e. Serum glutamate pyruvate Transaminase (SGPT), Serum glutamic oxaloacetic transaminase (SGOT), Alkaline phosphatase (ALP), Total protein and Total bilirubin (TB) [23-24].

Enzymatic Evaluation:

Hepatic tissues of rats were homogenized (10%) in phosphate buffer (pH 7.4) with a Potter-homogenizer (Ultra-Turrax T25, made in Germany). The homogenate was centrifuged at 12,000 RPM for 20 min at 4°C to obtain post-mitochondrial supernatant (PMS). The supernatant was used for the estimation of Lipid peroxidation (LPO), Catalase (CAT), Superoxide dismutase (SOD), Malondialdehyde (MDA), Xanthine Oxidase (XOD) and lysosomal enzyme (Cathepsin D, β-Galactosidase) [25-30] and remaining hepatic flaps were utilized for the histopathology.

Determination of Hydroxyproline (Hyp) Content:

Hyp content was determined by the calorimetrically taking 0.2 g of liver tissue using a modified method of Jamall et al 1981 [31].

Histopathology:

Each formaldehyde-fixed sample was embedded in paraffin, cut into 5 μm thick sections and stained with hematoxylin-eosin (H-E). The slides were observed under a light microscope and photomicrographs were captured by using a camera (Olympus SZX 12, stereomicroscope system). These were observed for fibrosis, fatty aggression, and centrilobular necrosis and lymphocyte infiltration [32].

Statistics:

All values were expressed as Mean ± SEM. The results were analyzed statistically using two way analysis of variance (ANOVA) followed by Bonferroni post-tests to calculate the level of significance. Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at *p<0.05, **p<0.01, ***p<0.001, when compared with the control group.

RESULTS:

Phytochemical Screening:

Preliminary phytochemical testing of HA extract demonstrated the presence of flavonoids, glycoside, amino acid, steroids, phenolic, polyesters, and saponins.

HPTLC Analysis of HA:

Investigation of HA extract revealed the presence of Flavonoids and the acquired quantitative information by utilizing HPTLC the presence of Gallic acid (Rf:0.31) and Quercetin (Rf:0.44) which are useful in the hepatic disease.

Antioxidant Studies of HA Extract:

As a kind of stable free radical DPPH indisputably that IC50 value for HA extract was found to be 175±1.41µg/ml, which demonstrates the significant antioxidant activity of HA (Figure – 1).

Figure – 1 DPPH free radical scavenging assay of HA extract

In Vivo Hepatoprotective Study:

Effect of HA extract of the Body weight, Liver weight and liver weight/body weight (LW/BW ratio) after induction with PCM:

The body weights of all the rats were measured weekly prior to scarification. The control group had shown normal body weight gains over 4 weeks. The administration of PCM caused decrease in the average body weight when compared to the normal control group. However, the group pretreatment with HA extract significantly increase in the average body weight when compared to the control group (p<0.05, p<0.01).

On the other hand, PCM administration caused increase in the average liver weight of toxic group when compared to the normal control group. However, the groups pretreatment with HA extract caused significant (𝑃< 0.05) reduction in the average liver weight of rats induced with PCM (Table – 1).

The mean relative liver weights (LW/BW ratio) of PCM-induced animals exhibited significant increase compared to the control normal group (𝑃< 0.05). However, the group that was pretreated with 400 mg/kg of HA extract showed decrease in the value of mean relative liver weights (Table – 1).

Table – 1 Effect of HA extract on percentage change of body and liver weight in PCM induced hepatic injury rats

Groups	Body weight, BW (g)	Liver weight, LW (g)	LW/BW
CG	164.93±4.34	4.20±0.089	2.54
TG	151.34±3.63	7.07±0.197	4.67
SG	160.43±3.34	4.40±0.179	2.74
TE-1	162.96±3.36	5.27±0.114	3.23
TE-2	159.98±4.40	4.49±0.179	2.80

Data are represented in mean ± SEM (n=6), CG = control group, TG = toxic group (PCM, 2 g/kg), SG = standard group (Silymarin, 100 mg/kg), TE-1 (HA extract, 200 mg/kg), TE-1 (HA extract, 400 mg/kg).

Quantitative analysis of enzyme activities:

In this study, the significant elevations of SGPT, SGOT, ALP and TB were recorded in treated groups as compared to respective control group animals. All parameters significantly reduced by administration of effective doses of HA. However, the results were more prominent with 400 mg/kg dose of HA against PCM induced hepatotoxicity (Figure – 2).

Effect on enzymes involved in oxidative stress:

MDA markers of lipid peroxidation were remarkably increased in the livers from PCM induced toxic group. Conversely, antioxidants SOD and CAT levels were marked decreased. However, all these changes induced by PCM were significantly suppressed by HA extract as shown in Figure – 3. Xanthine oxidase (XOD) produces reactive oxygen species (ROS) in catalyzing the reaction, and therefore, an increase in its activity leads to the oxidative stress. The extract of HA in appropriate dose was found effective in reducing oxidative stress. Moreover, HA extract also found effective in reducing raised enzymatic activity of β-Galactosidase and Cathepsin D against PCM induced hepatic damage.

Effect HA on hydroxyproline (Hyp) content:

Collagen contended of liver tissue was quantified by the assurance of Hyp content shown in Figure – 4. The Hyp content was significantly reduced by HA extract in both doses as compared to control group. However, the reduction in Hyp content was more prominent with 400 mg/kg of HA extract. The livers in PCM induced injury model groups were puffy, stiff and acquired an irregular and granular surface.

Figure – 4 Effect of HA extract on hydroxyproline content in PCM induced hepatotoxicity

Data are represented in mean ± SEM (n=6), CG = control group, TG = toxic group (PCM, 2 g/kg), SG = standard group (Silymarin, 100 mg/kg), TE-1 (HA extract, 200 mg/kg), TE-1 (HA extract, 400 mg/kg).

Histopathology:

Histopathological evaluation performed in this study demonstrated those normal control groups were devoid of estimable transformations in liver histology shown in Figure – 5A. This group exhibited normal lobular architecture and normal hepatic cell with intact cytoplasm. However, PCM induced degeneracy and necrosis of liver cells, the existence of pycnotic nuclei, granular cytoplasm, presence of hemorrhage and an increase in intracellular gaps with inflammatory bands and loss of cellular boundaries has shown in Figure – 5B. Silymarin demonstrated the normal structure of hepatic lobules, radish dark dots are glycogen, and white dots demonstrate vacuoles, and prominent blood vein (Figure – 5C). Interestingly, these pathological changes were found to be reduced with the increasing doses of HA extract indicating the ability to reduce PCM induced intoxication. The HA extract in 200 mg/kg dose exhibited recuperation of hepatic parenchyma, mellow congestion and miniaturized scale vesicular changes as shown in Figure – 5D. However, in higher dose of 400 mg/kg indicated stamped recuperation in hepatic cells with nuclei, cytoplasm, focal vein and portal triad (Figure – 5E). Histological examination of liver tissues in rats supplemented with HA extract at the dose of 400 mg/kg screening nearly normal tissue formation, the absence of inflammatory cells in the central areas revealing hepatoprotective assets. Table – 2 exhibits the histopathological scoring of the liver tissues pretreated with the respective test solution.

Figure – 5(A,B,C,D,E) Effect of HA extract against PCM induced histopathological changes

(A) Liver section of Normal control rats, (B) Liver section of PCM induced rats (C) Liver section of Silymarin treated rats (D) Liver section of rats treated with HA extract(200 mg/kg) and (E) Liver section of rats treated with HA extract (400 mg/kg).

Table – 2 Histopathological scoring of the tissue of PCM-induced hepatic injury rats after pretreatment with HA extract

Groups	Steatosis	Necrosis	Inflamma-tion	Hemorrhage
CG	–	–	–	–
TG	–	+++	++	++
SG	–	+	+	–
TE-1	–	++	+	–
TE-2	–	+	+	–

The severity of various features of hepatic injury was evaluated based on those following scoring schemes: − normal, + mild effect, ++ moderate effect, and +++ severe effect.

DISCUSSION:

The main finding of this investigation was that the administration of HA extract in rats was related to a halfway abatement in paracetamol actuated hepatotoxicity. The excess measurements of the agony diminishing and antipyretic, acetaminophen address an emerge among the most generally perceived pharmaceutical thing poisonings in the United States today [33]. Although PCM considered safe at restorative measurements; however, in overdose acetaminophen creates a centrilobular hepatic necrosis that can be lethal [34]. The examination was finished to evaluate antioxidant and hepatoprotective impact of HA and the preparatory phytochemical compound, for instance, alkaloids, flavonoids, tannins, phenolics, saponins, terpenoids and other fragrant compound are discretionary metabolites that are made in a plant. The role of theses metabolites and reactive oxygen species (ROS) in the pathogenesis of human diseases like cancer, aging, liver diseases, respiratory diseases has been widely recognized [35]. The DPPH assay indisputably delineated that IC50 value of HA extract was 175±1.41µg/ml that demonstrates the significant antioxidant activity [36]. Hematological parameters revealed the degree of injurious impact of outside compound including plant extract on the blood constituents of animals. Such poisonous quality testing is applicable to chance investigation as changes in the hematological framework have a higher prescient incentive for human lethality [37]. The outcomes exhibited that HA extract was neither lethal to the circulating red blood cell, white blood cell and platelets, nor it interferes with their production. Obliteration to the anatomical trustworthiness of the liver has reflected by an addition in the levels of serum transaminase [38] because these are cytoplasmic in the area and are discharged into the dissemination after cellular damage and increment in the level of γ-GT additionally represented the serious liver harm actuated by paracetamol. γ-GT is a membrane-bound compound, that is for all intents and purposes missing in non-hepatic tissues [39]. Silymarin has liver formative impacts by stimulating the protein known as RNA polymerase in the core of liver cells. These outcomes in an expansion of ribosomal protein combination which recovers hepatocytes [40]. The SGPT is an enzyme present in hepatocytes (liver cells), when a cell is damaged, it leaks the enzyme into the blood, where it is measured. SGPT rises dramatically in active liver damage. SGOT is similar to SGPT in that it is another enzyme associated with liver parenchyma cells. It is raised in acute liver damage but also present in red cells, cardiac and skeletal muscle and is therefore not specific to the liver.ALP is present in cells lining the biliary ducts [41]. Correlate with normal controls, the PCM organization essentially constricted serum levels of SGPT, SGOT, ALP and TB (p<0.05, p<0.01) which are adversely influenced by HA extract defending the doses picked.

Various intracellular compounds such as CAT, SOD provide protection against oxidation [42]. MDA a breakdown product is a measure of lipid hydroperoxides, causing lipid peroxidation. The HA extract caused increase in the level of MDA and a significant decrease in SOD, and CAT in hepatotoxic rats when compared to the respective control group animals, which are the signs of oxidative stress due to excessive formation of free radicals in the experimental animals [43,44]. XOD is a Mo–Fe–S flavin-containing hydroxylase and it contributes in different types of damage [45]. These chemicals create ROS in catalysing the response, which leads to expansion in the oxidative anxiety and the assay of lysosomal enzyme. Increased action of β-Galactosidase, Cathepsin- D (m units/mg of protein) get resolved after HA and the more likely sources of the elevated activities of cathepsin-D and β-galactosidase in the liver supernatant fraction of rats treated with the hepatotoxin. The secretion of proteases and other hydrolases from phagocytic cells is important because the lyric process of necrotic liver tissues occurs extracellularly [46]. Collagen is the major structural protein of the cellular matrix of the liver. Collagen deposition on hepatic injury as markers was determined by the hepatic hydroxyproline content. Hyp content in the liver increased significantly after PCM administration for 4 weeks. HA treatment promoted collagen degradation by increasing Hyp level. The assessing of liver histology revealed that there were macro lump separated by fibrous septa, as well as statues, inflammation, hepatocyte ballooning, and necrotic tissue as predicted by Haemotoxylin-eosin staining and widespread alterations of normal liver architecture, and increases in connective tissue septa. It was ameliorated by HA treatment, it also reduced the severity of liver damage and formation of fibrous septa induced by PCM.

ACKNOWLEDGEMENT:

The authors are thankful to our Honorable Director CSIR-NBRI Lucknow for their assistance and providing laboratory facilities. Further, we extend our gratitude to University Grant Commission (UGC-RGNSRF), New Delhi, India for providing grant to the perform the study (Grant Number 201213-RGNF-2012-13-SC-UTT-24384). I am so much thankful to my colleague Mr. Shravan Kumar Paswan (SRF) for their constant support during the experiment and Animal work.

ETHICAL STATEMENT:

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were approved by CPCSEA, New Delhi, India. (Approval number: 1732/GO/Re/S/13/CPCSEA).

CONFLICT OF INTEREST STATEMENT:

The authors declare that they have no conflict of interest.

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Received on 10.01.2018 Modified on 18.02.2018

Research J. Pharm. and Tech 2018; 11(4): 1633-1639.

DOI: 10.5958/0974-360X.2018.00304.9