Introduction

The liver disease continues to be a public health problem. Unfortunately, drugs used to cure liver disease, whether conventional or synthetic, are ineffective and may have dangerous side effects (Larbie, Arthur, Woode, & Terlabi, 2012). In the absence of a reliable liver protection medication in western medicine, Ayurveda recommends a variety of herbal formulations for the treatment of liver disorders (Shahjahan, Sabitha, Jainu, & Devi, 2004). Because of the serious negative side effects of synthetic drugs, there is an increasing interest in using a rigorous testing approach to evaluate the scientific basis of conventional herbal medicines that appear to have hepatoprotective properties (Dhiman, 2012; Manokaran et al., 2008). Some herbal extracts and their chemical components have been shown in studies to greatly inhibit these pathological processes and protect hepatocytes from the aetiology of chronic liver damage. Due to the lack of effective liver safety medications in western medicine, a wide range of herbal preparations are prescribed for the treatment of liver diseases, with many claiming to provide substantial relief (Zhang et al., 2018). Attempts are being made around the world to gain clinical evidence for these herbal medicines that have long been published.

The plant kingdom has given a diversified range of bioactive molecules, which makes them medicinally a precious source. Due to enormous limitations in synthetic pharmaceutical products, very less or no harmful effects and increased awareness on natural products, there is a need of the hour to isolate the lead compounds from them (David, Wolfender, & Dias, 2015). One such plant that is currently under investigation for its potential hepatoprotective and antioxidant activity in our laboratory is Artabotrys hexapetalus (family: Annonaceae) (Mathew, V, & G, 2013). Commonly known as Hari Champa and South Climbing lang-lang in English (Jindal, 2017; Prabhukumar et al., 2017).

However, many medicinal plants used in remote villages and tribal villages of southern districts of Andhra Pradesh remain to be studied. A. hexapetalus is one such plant. This plant leaf is used in folklore medicine to treat liver diseases in the Kurnool and Ananthapuramu districts of Andhra Pradesh. In traditional medicine, its roots are used for treating Jaundice. Few studies have shown that the plant possesses anti-cancer and anti-microbial, anti oxidant, anti-diabetic activities. Furthermore, we also reported on the Phytochemical constituents of A. hexapetalus, which indicate the presence of flavonoids, tannins and triterpenes. The polyphenolic flavonoids, in particular, have proved to exhibit various pharmacological activities, including Hepatoprotective activity.

Our through literature survey shows that the hepatoprotective potential of A. hexapetalus ethanolic leaf extract has been proved by (Annapurna & Ganapathi, 2016) against paracetamol-induced hepatotoxicity only. No other eveidances were found to prove the hepatoprotective activity of AH against other hepatotoxicins like ethanol, Isoniazid and Rifampicin. Thus, this study was carried out to get insights into the utility of ethanolic extract of A. hexapetalus leaf against various hepatotoxic modles viz., paracetamol (PCT), ethanol (ETN) and Isoniazid and Rifampicin (IR) induced liver damage in rats as the animal model to develop a satisfactory hepatoprotective medicine.

Materials and Methods

Animals

The crude extracts were tested on Albino Wister rats of both sexes. The research proposal was approved by the Institute's Animal Ethics Committee (232 / a / 19 / CPCSEA). For one week before and after the trials, the animals were held at 27±2 °C, relative humidity 44-56 %, at a light and dark periods of 10 to 14 hours, respectively. The animals were fed a normal diet (Lipton, India) and were given water ad libitum 18 hours before the experiment. All the experiments were carried out in the morning, in accordance with existing laboratory animal treatment and ethical recommendations for the study of experimental pain in conscious animals (National Research Council, 2010).

Source of plants and Preparation of crude drug extract

A. Hexapetalus leaves were collected in Tirupati, Andhra Pradesh, India. Dr K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateshwara University, Tirupati, Andhra Pradesh, conducted the authentication. A specimen sample was preserved in the College's Pharmacognosy Department with the herbarium sample (voucher sample no-017 / C112 / Suresh-01). The leaves were degreased with petroleum ether and dried in the shade. Using a Soxhlet apparatus, the defatted substance was extracted with 95 % ethanol and then dried under a vacuum.

Phytochemical studies

All the extracts were subjected for Phytochemical study (Yadav & Agarwala, 2011).

Acute toxicity studies

Albino rats were used in an acute toxicity study for the ethanolic leaf extract of A. hexapetalus leaves. Before the trial, the animals were fasted overnight and held in normal conditions. The extract was given orally in increasing doses and were found to be healthy up to a dosage of 2000 mg/kg (Akhila, Manikkoth, Shyam, & Alwar, 2007).

Experimental animal and design

The experiment was conducted according to the modified procedures described previously (Dash et al., 2007). PCT (3g/kg), Ethanol 5mg/kg and IR (50+50 mg/kg) was dissolved in 0.5 % CMC for oral administration. Rats were randomly divided into six groups for each model and consisting of six rats. PCT intoxicated animals were grouped from P1-P6. E1-E6 represents a group of animals which were intoxicated by ethanol, and Group IR1-IR6 constitute animals intoxicated by IR. Table 1 shows the details of animal groupings for various hepato toxicity models.

The rats were given ether and then sacrificed after 48 hours of intoxication. SGOT, SGPT, ALP, and Bilirubin enzyme levels were measured using standard kits after blood was extracted via cardiac puncture into heparinized tubing. The liver was immediately removed and washed in ice-cold saline before being examined histologically. The animal grouping was shown in Table 1.

Biochemical determinations

Using test kits, biochemical parameters such as aspartate aminotransferase (AST), glutamate pyruvate transaminase (ALT) (Reitman & Frankel, 1957), serum alkaline phosphatase (ALP) (King, 1965), and gross bilirubin (Malloy & Evelyn, 1937) were determined. (Surat, Span Diagnostic).

In Vitro Anti Oxidant Activity

DPPH-scavenging activity

Hydrogen donation or radical scavenging ability using the stable radical DPPH was determined for the evaluation of the free radical scavenging activity of the extract. A 0.1 mM ethanol solution was prepared, and 1.0 ml of it was applied to 3.0 ml of the entire solution of extracts in water at various concentrations (10–100 g / ml). The absorbance was estimated after 30 mins at 517 nm. The reaction mixture's lower absorbance means a higher free radical removal activity. The standard drug was ascorbic acid (Marinova & Batchvarov, 2011).

Scavenging Of Hydrogen Peroxide (H₂O₂)

A 20 mM hydrogen peroxide solution in phosphate-buffered saline (pH 7.4) was prepared, and different amounts of extract or standard in methanol (1 ml) were added to 2 ml of peroxide solution buffer saline solution containing hydrogen. The absorbance was estimated at 230 nm after 10 minutes (Sroka & Cisowski, 2003).

Determination of Biochemical parameters

Various biochemical serum markers such as serum oxaloacetic glutamic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphate (ALP), bilirubin, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione (reduced) (GSH) were assessed using commercially available kits for each sample, and all analyses were carried out in triplicate (Acharya, Chatterjee, Biswas, Chatterjee, & saha, 2012; Dash et al., 2007; Haldar et al., 2011).

Histopathological studies

Dissected liver tissue was frozen in 10% formalin, dehydrated in 50% ethanol, eliminated in xylene, and embedded in paraffin. Photomicroscopic observations of cell necrosis, fat displacement, hyaline regeneration, and balloon degeneration were made using sections stained with hematoxylin and eosin dye (H-E).

Statistical analysis

The mean and standard deviation of the mean are used to express the data (SEM). Data were evaluated using one-way analysis of variance (ANOVA), and discrepancies between groups were calculated using Graph pad PRISM V5.02 software's Dunnett's post hoc test. The p<0.05 significance level was chosen.

Results

Phytochemical study

All extracts subjected for the phytochemical study showed the presence of alkaloids, proteins, amino acids, phenolic compounds, glycosides and flavonoids.

Acute toxicity studies

Up to doses of 2000 mg/kg, the ethanolic and aqueous extracts displayed no signs or symptoms of toxicity or mortality. The findings of the acute toxicity trials as seen in Table 2.

In vitro antioxidant study

Before proceeding for in vivo activity, the efficacy of the plants were tested in vitro. The in vitro antioxidant activity was performed by using DPPH free radical and Hydrogen Peroxide scavenging. Results were tabulated in Table 3.

Effect of the ethanolic extract of AH leaf on biochemical parameters against PCT induced hepatotoxicity

The liver markers SGOT, SGPT, ALP, Bilurubin, SOD, CAT, MDA, and GSH, are all very responsive, and their elevated levels indicate liver damage. The effects of the ethanolic extract of the HA leaf on different biochemical parameters are shown in Table 4. In standard control rats, there were no significant improvements in the levels of these parameters. PCT was injected into rats with mediated liver damage, resulting in significantly higher SGOT, SGPT, ALP, bilirubin, SOD, CAT, MDA, and GSH behaviours than the usual control group. However, as compared to the PCT-treated population, the AH treatment (400 mg/kg) showed a substantial reduction in the levels of elevated serum enzymes. The effect of HA on a dose-dependent basis is equal to that of silymarin therapy. These findings suggested that an ethanolic extract of HA leaves could protect rats from PCT-induced liver injury.

The non- PCM- intoxicated liver pretreated with 10% DMSO (normal) has normal lobular morphology and normal liver cells with well-preserved cytoplasm, a well-defined sinusoidal line, and a nucleus across the perivenular region (Figure 1 (a)). Figure 1 (b) reveals lymphocyte penetration, haemorrhage, and severe coagulative necrosis of the perivenular and midline regions with periportal preservation in a PCM-poisoned liver segment pretreated with 10% DMSO. The perivenular zone is primarily affected by coagulant necrosis of hepatocytes in PCM-induced liver toxicity (zone 3). With increasing AH dosage, these pathological improvements were found to be minimal, meaning that the extract would reverse PCM-induced intoxication (Figure 1 (d) - (f)). Pretreatment with the extract or silymarin greatly decreased the presence of marked necrosis, inflammation, and bleeding during PCM treatment (as seen in the negative control group).

Effect of the ethanolic extract of AH leaf on biochemical parameters against ETN induced hepatotoxicity

Increased amounts of liver biomarkers such as SGOT, SGPT, ALP, Bilurubin, SOD, CAT, MDA, and GSH revealed that the hepatotoxic agent ethanol-induced substantial liver harm. When compared to ETN-treated rats, rats given doses of 100, 200, and 400 mg/kg had slightly lower levels of biochemical markers. The maximum dose (400 mg/kg) had greater hepatoprotective efficacy than the lowest doses. The effect of the ethanolic extract of the HA leaf on biochemical parameters against ETN-induced hepatotoxicity is detailed in Table 5.

Histopathological examinations confirmed the hepatoprotective effect of the ethanolic extract of HA leaves on ETN-induced liver injury. Figure 2 (a) shows natural lobular morphology and regular liver cells with well-preserved cytoplasm, a well-defined sinusoidal axis, and a nucleus around the perivenular region in the non-intoxicated liver with ETN pretreated with 10% DMSO (standard). Figure 2 (b) shows normal histological structures in the livers of rats infected with silymarin (25 g / kg). In rats given ETN, abnormal liver cells, necrosis, and inflammation were observed (Figure 2 (c)). Rats given HA extract (100, 200, and 400 mg/kg) demonstrated a reduction in body weight. Inflammatory cells, artery congestion, cell degeneration, necrosis, and vacuoles were reduced or absent in rats treated with HA extract (100, 200, and 400 mg/kg). Figure 2 (d, e, and f) Lower doses of ethanolic extract of HA leaves (100 mg/kg) provided less safety than higher doses of 400 mg/kg.

Effect of the ethanolic extract of AH leaf on biochemical parameters against IR induced hepatotoxicity

The ethanolic extract of HA demonstrated significant hepatoprotective activity (p<0.05) against the toxicity caused by isoniazid and Rifampicin (IR) (50 mg/kg + 50 mg/kg) by enhancing liver function, as shown by lower liver enzyme levels relative to the control group. The full effects of hepatoprotective activity against the IR-induced hepatotoxicity model are seen in Table 6. The liver architecture of IR-induced rats pretreated with 10% DMSO was significantly damaged (p<0.05), with extreme hepatocyte necrosis, according to histopathological tests of liver removed from the rats. Regular lobular morphology and normal liver cells with non-IR intoxicated liver pretreated with 10% DMSO (normal). Figure 3 (a) shows natural lobular morphology and normal liver cells with well-preserved cytoplasm and well-defined sinusoidal line and nucleus across the perivenular region in non-IR intoxicated liver pretreated with 10% DMSO (normal). Figure 3 (b) shows normal histological structures in the livers of rats infected with silymarin (25 g / kg). In the IR-treated rats, changes in liver cells, necrosis, and inflammation were observed (Figure 3 (c)). Inflammatory cells, artery congestion, cell degeneration, necrosis, and vacuoles were reduced or absent in rats treated with HA extract (100, 200, and 400 mg/kg). (See Figure 2 (d, e, and f)). Lower doses, on the other hand, Inflammatory cells, artery congestion, cell degeneration, necrosis, and vacuoles were reduced or absent in rats treated with HA extract (100, 200, and 400 mg/kg). (See Figure 2 (d, e, and f)). Lower doses of ethanolic extract of HA leaves (100 mg/kg) provided less safety than higher doses of 400 mg/kg.

Discussion

Because of its metabolic and detoxifying capacities, the liver is an essential part of life. When people are exposed to a variety of endogenous and xenobiotic compounds, they develop a vast amount of intermediate and final products, which can induce hepatocellular death and are the leading causes of liver disease (Armeni & Principato, 2020; Meharie, Amare, & Belayneh, 2020). Traditional medicine relies on symptom control and liver transplantation in acute cases of liver failure in order to sustain liver function (Hoofnagle, Carithers, Shapiro, & Ascher, 1995). However, no medications are actually being used to improve the organ's detoxification ability. As a result, the use of botanical hepatoprotective agents is becoming increasingly common. Therefore, it would be absolutely imperative to demonstrate the efficacy of plant extracts in the presence of chemical-induced hepatotoxicity (Meharie et al., 2020).

Paracetamol (PCT) and ethanol (ETN) were generally consumed by a human for the reasons like pyrexia and those who have a habit of taking alcohol, respectively. Isoniazid and Rifampicin (IR) are the most widely used drugs to treat tuberculosis. All these agents were known to induce hepatotoxicity. So, the same hepato toxins were chosen to induce hepatotoxicity in rats and evaluate the hepatoprotective activity of Artabotrys hexapetalus. The rats were given an ethanolic extract of the leaves of A. hexapetalus. In humans and laboratory animals, PCT, ETN, and IR have been shown to cause hemorrhagic liver necrosis in many trials. In this study, rats treated with PCT, ETN, and IR developed infiltration, vacuolation, and inflammation in the liver, resulting in increased rat liver weight (Figure 1 b, Figure 2 b and Figure 3 b). The hepatoprotective ability of plant extracts in different animal models was evaluated using PCT, ETN, and IR mediated hepatotoxicity. Bioactivation of these hepatotoxins by cytochrome P450 results in strongly unstable reactive free radicals. These can kill cells by peroxiding membrane lipids and binding covalently with other macromolecules in hepatocytes. When the membrane is damaged, cytosolic and endoplasmic enzymes are released, indicating that the liver's structure and function have been compromised. Elevated amounts of SGOT, SGPT, ALP, Bilurubin, SOD, CAT, MDA, and GSH are signs of this. As a result, measuring the amounts of these biomarkers of liver injury will show the plant extract's and solvent fractions' hepatoprotective function. The ethanolic extract reduced the levels of SGOT, SGPT, ALP, Bilurubin, SOD, CAT, MDA, and GSH in a dose-dependent manner in the current sample. At the lowest dosage, 100 mg/kg ethanolic extract of HA leaves had little effect on all biomarkers of liver damage, but medium and high doses resulted in substantial reductions in AST, ALT, and ALP levels (Table 6; Table 5; Table 4). This may indicate that the lower dose is smaller than the minimal effective dose and cannot induce a substantial decrease in liver enzyme levels, whereas the other two doses are high enough to do so. Percent reduction in hepatic injury biomarkers revealed that 200 mg/kg and 400 mg/kg of ethanolic extract had an effect that was almost identical to the normal (Table 6; Table 5; Table 4). With the exception of the 100 mg/kg dosage, pre-and post-treatment with ethanolic extract in all doses (200 mg/kg and 400 mg/kg) significantly reduced the severity of the liver injury. The ethanolic extract can stabilise liver cell membranes and avoid enzyme degradation, as shown by the return of enzyme levels to near-normal levels in ethanolic rats before and after surgery.

Other possible explanations for the therapeutic activity of A. hexapetalus leaf extract include preventing the formation of free radicals and neutralising them, as well as the plant's ability to defend against hepatotoxins. The crude ethanolic extract was fractionated to concentrate or isolate the active ingredients. The majority of the polar components of the plant leaf may be attributed to the available flavonoids material, according to this report. Since the active theory or ingredients responsible for the hepatoprotective behaviour of the ethanolic extract and solvent fractions of A. hexapetalus are unclear, it is impossible to pinpoint the compounds are responsible for the antioxidant and hepatoprotective effects. Alkaloids and flavonoids have been found to have antioxidant properties in previous research. The crude ethanolic extract and the solvent fractions were subjected to preliminary phytochemical analysis, which showed a number of secondary metabolites that seemed to be dispersed differently in the extract. It is fair to believe that the phytochemicals found in the plant work individually or in concert to create A. hexapetalus hepatoprotective function. It's likely that the flavonoids and alkaloids in the raw leaf extract have a hepatoprotective impact by scavenging free radicals and preventing lipid peroxidation and cell injury, as has been proposed with some other plants. Alkaloids and flavonoids are sometimes classified as natural antioxidants because of their ability to scavenge free radicals.

In conclusion, this analysis added to the growing body of evidence that the ethanolic extract has hepatoprotective properties comparable to the regular treatment. Both biomarkers of liver damage were reduced in a dose-dependent manner before and after surgery, according to the findings. As a result, these findings suggest that the plant's hepatoprotective effect is spread to the polar bioactive concepts contained in the ethanolic fraction. While the plant extract's hepatoprotective function is yet to be discovered, one of the expected mechanisms is its antioxidant activity. Overall, according to the findings of the acute oral toxicity report, the ethanolic extract of the leaf of A. hexapetalus is considered safe. In addition, future experiments will use HPLC / LC-MS / MS strategies to isolate and characterise new antioxidants.

Conclusions

The current study's experimental evidence showed that the leaf of A. hexapetalus has hepatoprotective function against PCT, ETN, and IR-induced liver toxicity. The presence of flavonoids and other components in the plant may be responsible for this behaviour. To confirm the mechanism underlying this hepatoprotective effect, additional in vitro and in vivo studies will be needed.