Evaluation of Endophytic Fungal Fractions of Phyllanthus amarus schum and Thonn Leaves in Paracetamol and Ethanol Induced Hepatotoxicity

Introduction

Free radicals are highly reactive substances causing oxidative damage. These free radicals are generated during hepatic drug metabolism. Oxidative damage is a vital factor for several divergent diseases counting liver diseases, stroke, atherosclerosis, arthritis, cancer, and asthma. The liver is one such organ involved in different biochemical and physiological proceedings.¹ Various processes include homeostasis, immunity, metabolism, detoxification, and nutrient storage.²

Liver disorders are revolting owing to infinite detrimental elements such as industrial pollutants, dietary additives,free radicals, and prolonged consumption of alcoholic beverages and medications. Substances such as paracetamol and ethanol cause toxicity of the liver in higher doses. Paracetamol, a well-known analgesic, and antipyretic used in rat models to explore hepatoprotective action. It can fabricate reactive oxygen species and oxidative stress.³ Paracetamol is converted into a highly reactive metabolite, N-acetyl-p-benzoquinone imine by glucuronidation or sulfation of cytochrome P-450 system in therapeutic doses. The N-acetyl-p-benzoquinone imine gets converted to the nontoxic substance in lower doses but may react with liver proteins in the higher doses resulting in liver damage.⁴ 

One of the other causes leading to a degeneration of the liver is alcohol (Ethanol). Alcoholism is a threat to the human being as it may cause an imbalance of physiological functions. The literal acting mechanism is unknown, as some of them attributed to hepatotoxicity. One of the drugs causing liver cirrhosis is ethanol (alcohol). Severe alcoholic disorders are caused by a surplus intake of alcohol. Reports showed the increase in liver enzyme activity destroying hepatocytes.³ Alcohol toxicity is mainly caused by the trichloromethyl radical metabolite.⁴ Hence, there is an emerging focus on novel compounds that should be carried out to get a drug that is reliable and much more effective for the prevention of paracetamol and ethanol-induced hepatotoxicity.⁵

As liver diseases cause fatal consequences, numerous drugs are made available for these disorders, but they do not produce satisfactory results due to either adverse effects or high costs. Hence, there is an outlook in search of novel areas to cure liver diseases with quality, safety, and efficacy. Microorganisms that colonize the inside of the plant tissues are known as endophytes. They may be bacteria, fungi, or actinomycetes. Endophytes are plantassociated microbes where the microbial genotypes colonize specific plants. They also assist growing plants by fixing nitrogen, suppressing or out-competing pathogens, and producing hormones protecting from stress. Endophytes also show defensive mechanisms against herbivores and insects.⁶

Phyllanthus is an important genus of medicinal plants in India. Phyllanthus amarus Schum and Thonn (family Euphorbiaceae) is distributed as a weed in waste and cultivated lands.⁷ Because of its rich medicinal values, it is warned for a long time for the alleviation of human ailments. It is traditionally used in treating viral hepatitis in India.⁸ The plant contains secondary metabolites such as ellagitannin Phyllanthus, lignans, niranthin, and hypophyllanthin.⁹ Phytochemical review showed the appearance of triterpenoids including cycloeucalenyl acetate, ergosta-5,7,22-trien-3-ol acetate, macdougallin, 17-(1,5-Dimethylhexyl)-6- hydroxy-5-methylester9-en-3-yl acetate, steroids such as stigmasterol and β-sitosterol in the hexane fraction and 6,7- epoxypregn4-ene-9,11,18-triol-3,20-dione, 11,18- diacetate, bufalin, olean-13(18)-ene, methyl ursolate, barringenol R1 and 7,8-epoxylanostan-11-ol,3-acetoxy in the methanol fraction.¹⁰ Acinetobacter sp., an endophytic fungus, and Bacillus sp., endophytic bacteria, isolated from Phyllanthus amarus leaves, were recognized by 16s rRNA sequencing.11 Thirteen endophytic fungi were also isolated from the leaves, stems, and roots of Phyllanthus amarus.¹² The other endophytic fungi isolated from Phyllanthus amarus showed antibacterial activity against gram-positive and gram-negative bacteria.¹³

In literature, no work has been carried out /reported on paracetamol and ethanol-induced hepatotoxicity models of fungal endophytes isolated from Phyllanthus amarus. We have already published a paper on endophyte PALF2, obtained from Phyllanthus amarus leaves and screened for in vitro free radical scavenging activity and in vivo hepatoprotective activity in CCl4 induced model in our paper.¹⁴ The fractions of PALF-2 (P2EA & P2nB) were further analyzed for in vivo hepatoprotective activity in paracetamol and ethanol-induced hepatotoxicity.

Materials and Methods

Plant material

Leaves of Phyllanthus amarus were collected from the surroundings of Dharwad, Karnataka, India. The plant was identified and authenticated by a Taxonomist, Department of Botany, Government Science College, Dharwad, Karnataka, India, and a voucher specimen (SETCPD/Ph.cog/herb/86/12/2017) were stored at the herbarium of the postgraduate department of Pharmacognosy and Phytochemistry, SET’s College of Pharmacy, Dharwad, India.

Isolation, fermentation, and extraction

Phyllanthus amarus leaves were washed thoroughly under running tap water to remove adhered foreign particles followed by surface sterilization with 70% ethanol for 30 sec, 1% sodium hypochlorite (NaOCl) for 30 sec, and rinsed in sterile demineralised water. Small pieces of leaves were placed on potato dextrose agar (PDA) supplemented with antibiotic streptomycin (25 mg/100 ml) in Petri plates and incubated at 25◦C ± 27◦C until fungal growth was initiated. One pure colony namely, PALF-2 (endophytic fungi of Phyllanthus amarus) was selected for fermentation.¹⁴ The purified isolates of PALF-2 were inoculated and fermented in potato dextrose broth (PDB) followed by incubation at 25°C to 27°C for 21 days. After 21 days of incubation, extraction with ethyl acetate and n butanol was carried out to get P2EA (ethyl acetate) and P2nB (n butanol). PALF-2 was characterized by PCR sequential analysis as previously reported.14 Hepatoprotective activity.¹⁴

Hepatoprotective activity

Based on the previously reported data,¹⁴ P2EA and P2nB were further selected for activity in paracetamol and ethanol-induced hepatotoxicity.

Animals

Albino Wister rats (150-200 g) were collected from Venkateshwara Enterprises Bangalore, Karnataka. Animal house (Reg No.112/1999/CPCSEA, dated 19- 05-1999) of SET’s College of Pharmacy, Dharwad was used to maintain under required conditions. Grouping of the rats was done and accommodated in sanitized polypropylene cages containing sterile paddy husk. The animals were provided with a proper diet and water ad libitum. Ethical clearance for animal studies was obtained from the Institutional Animal Ethical Committee and standard operating procedures and protocols were followed as per The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Acute toxicity studies

Acute oral toxicity of P2EA and P2nB was determined using Swiss albino mice. Fasting for 12 hrs before the experiment was observed. A single dose of fractions was dissolved in 5% gum acacia and observed for mortality up to 48 h (short-term toxicity). Based on these results, the doses were selected according to OECD guidelines. 

Experimental design for paracetamol-induced hepatotoxicity³

Group I: Normal control, Rats received distilled water 5 ml/kg b.w. p.o./7 days

Group II: Paracetamol control, Rats received distilled water 5 ml/kg b.w. p.o./7 days (except 5th day)

Group III: Rats received the standard drug Silymarin 200 mg/kg b.w. p.o./7 days

Group IV: Rats received with P2EA (50 mg/kg) b.w. p.o./7 days

Group V: Rats received with P2EA (100 mg/kg) b.w. p.o./7 days

Group VI: Rats received with P2nB (50 mg/kg) b.w. p.o. /7 days

Group VII: Rats received with P2nB (100 mg/kg) b.w. p.o./7 days

For continuous 5 days, the rats in the group III to VII were treated with the standard drug and extracts. On the 5th day, following the extract treatments, all animals other than in group I were administered paracetamol 2 g/kg b.w. p.o., and on the 6th day, after 2 h of respective drug treatments, animals were anesthetized using a diethyl ether inhalation jar. The retro-orbital bleeding method was used to collect blood under mild anesthesia, followed by centrifugation for 15 min at 2500 rpm. Serum collected was levied to biochemical analysis. Excision of livers was carried out, washed in chilled normal saline, and kept ready for histopathology. Homogenate prepared were used for determining endogenous enzymes.

Experimental design for ethanol-induced hepatotoxicity¹⁵

Group I: Normal control, Rats received only distilled water

Group II: Ethanol control, Rats received 20% ethanol (3.76 g/kg/ day, p.o. for 18 days)

Group III: Rats received with Silymarin (200 mg/kg)

Group IV: Rats received with P2EA (50 mg/kg p.o)

Group V: Rats received with P2EA (100 mg/kg p.o)

Group VI: Rats received with P2nB (50 mg/kg p.o)

Group VII: Rats received with P2nB (100 mg/kg p.o)

Group I-VII were treated with fractions/drugs orally for 18 days. On the 19th day, the procedure was followed as in the paracetamol model mentioned above.

Determination of biochemical parameters

Serum collected was analyzed spectrophotometrically for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total and direct bilirubin, total triglyceride (TG), and total protein in the above models using ERBA diagnostic kits, Germany.

Enzymatic and non-enzymatic antioxidant levels measurement

The liver homogenate was prepared with the help of a Teflon glass homogenizer (Yamato LSG LH-21, Japan) as reported previously.¹⁴ The prepared samples were estimated for non-enzymatic antioxidants (Lipid peroxidation (LPO)) and enzymatic antioxidants (Catalase (CAT) and Superoxide).

Lipid peroxidation: Liver homogenate containing thiobarbituric acid (TBA) reactive substances (TBARS) was estimated by using a standard procedure. Homogenate was mixed with 15% of trichloroacetic acid (TCA), 0.375% of TBA, and 5N HCl, followed by incubation for 10-15 min at 95°C. Then the absorbance was measured at 532 nm. Lipid peroxidation was determined by using the formula €= 1.56 x 105M-1 cm-1 and expressed as TBARS (μ moles)/g of tissue.¹⁶

Superoxide dismutase (SOD) assay: 1ml of homogenate was mixed with 1 ml of sodium carbonate (50 mM), 0.4 ml of nitroblue tetrazolium (NBT) (24 μm), 0.2 ml of EDTA (0.1 mM), and 0.4 ml of hydroxylamine HCl (1 mM). Absorbance was measured at 560 nm. Units were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50%. The specific activity was expressed in terms of units/mg of proteins.¹⁷ 

Catalase: To liver homogenate, 1.95 ml of 10 mM H2 O2 prepared in 60 mM phosphate buffer (pH=7.0) was added. The rate of degradation of hydrogen peroxide was measured at 240 nm/min. Catalase content in terms of U/mg of protein was estimated from the rate of decomposition of H2 O2 using the formula.¹⁸

k=2.303/ Δt x Log (A1/A2) S-1

Histopathological studies

Livers were excised quickly, placed in 10% buffered formalin followed by a bovine solution. Standard techniques were used for further paraffin-embedded as reported.¹⁹ Sections of liver were stained with alum hematoxylin and eosin which were then observed photo microscopically for histopathological changes.

Statistical evaluation

Statistical evaluation was done using one-way ANOVA followed by Tukey’s T-test using Graph Pad Prism version 8.0, USA. Data were expressed as Mean ± S.E.M

Results

Hepatoprotective activity PALF-2 endophyte identified as Nigrospora sp. CMH2_13 (KF 227831.1) respectively as reported previously in our paper.¹⁴

Acute toxicity (LD50) studies

Acute toxicity studies were performed according to OECD guidelines. No mortality was observed up to 1000 mg/kg b.w for P2EA and P2nB. The doses selected were 50 mg/kg and 100 mg/kg body weight of P2EA and P2nB fractions respectively.

Effect of P2EA and P2nB on biochemical markers in paracetamol and ethanol-induced models

Imposing paracetamol (2 g/kg) and ethanol (3.76 g/kg/ day, p.o) caused damage to the liver and cell necrosis by elevating the AST, ALT, ALP, total and direct bilirubin, and triglyceride when compared with normal control. P2EA and P2nB (50 mg/kg &100 mg/kg) inverted the increased biochemical levels in comparison to the paracetamol and ethanol-treated groups (Table 1 & 2).

Effect of P2EA and P2nB on LPO, SOD, and CAT in paracetamol and ethanol-induced models

There was a marked increase in LPO level in the paracetamol-treated group (97.13±0.37) and in the ethanol-treated group (79.97±0.69). P2EA (100 mg/kg) and P2nB (50 & 100 mg/kg) treated groups significantly inhibited in-vivo lipid peroxidation P2EA (50 mg/kg) showed a significant value as (**p<0.01), compared to silymarin. The SOD levels were decreased in paracetamol treated and ethanoltreated groups. P2EA and P2nB (50 & 100 mg/kg) showed significance value, as **p<0.01, when compared to silymarin. There was a lowering of CAT levels in paracetamol treated and ethanol-treated groups. P2EA (100 mg/kg), P2nB (100 mg/kg), increased the CAT activity significantly (***p<0.001) (Table 3 & 4).

Effect of P2EA and P2nB on histomorphological changes in the liver in paracetamol model

Normal liver showcased normal hepatocytes, central vein, and sinusoids. In the paracetamol-induced group, degenerative hepatocytes, lymphocyte infiltration, cell death, and epithelial tissues were observed. Silymarin (200 mg/kg) induced group reimposed hepatic layout, with normal structures of hepatic cells, kupffer cells, central vein. P2EA treated livers (50 mg/kg) showed necrosis characterized by loss of hepatocytes and replacement with erythrocytes. P2EA treated livers (100 mg/kg) showed dilated sinusoids, kupffer cells, and mild hyperplasia. P2nB (50 mg/kg) treated livers showed mild ballooning, necrosis, and sinusoidal congestion. P2nB (100 mg/kg) treated livers showed mildly congested sinusoids (Figure 1).

Effect of P2EA and P2nB on histomorphological changes in the liver in ethanol model

The normal liver was found to have normal hepatocytes, normal hepatic cells, normal globular, central vein, portal trait, and normal kupffer cells. In ethanol-treated group, extensive fatty degeneration, spotty necrosis, focal hemorrhage, central vein inflammation, degeneration of hepatic cells with centrilobular necrosis were observed. Silymarin treatment exhibited regeneration of hepatocytes, mild sinusoidal and central vein congestion. P2EA treated livers (50 mg/kg) also showed regeneration of hepatocytes. P2EA treated livers (100 mg/kg) showed mild ballooning of hepatocytes. P2nB (50 mg/kg) treated livers showed the appearance of normal hepatocytes around the fatty vacuoles and necrosis. P2nB (100 mg/kg) reduced the inflammation of sinusoids and hepatocytes. The injured liver returned to normal by showing reduced degeneration and mild hepatocyte ballooning (Figure 2)

Discussion

The liver is the main organ responsible for the metabolism, secretion, storage, and detoxification of exogenous and endogenous substances. It is also involved in the biochemical processes of growing, providing nutrients, supplying energy, and reproducing.²⁰ Because of these rational functions, liver disorders are causing threats to humans, thus remaining as major problems in this world. Even advancement in medicine has limited the effectiveness of the drugs in hepatic functions causing them to protect or regenerate hepatocytes. Thus, newer drug therapy must be introduced in curing these liver diseases worldwide without causing toxic/side effects.²¹ No synthetic drugs are available for proper detoxification of the liver; thus there is an immediate need for novel drugs for better efficacy of liver treatment^.22

Microbes growing innermost in the plant tissues exhibiting huge biodiversity in nature are called endophytes.²³ All the classes of vascular plants are discovered with endophytes. Different organisms such as fungi, bacteria, actinomycetes, and mycoplasma are reported as plant endophytes.²⁴ Endophytes show distinctive mechanisms, physiological or metabolic, empowering themselves to the environment inside the plants with various bioactive substances. They are also involved along the host to initiate the metabolic products with therapeutic value.25 Endophytes are novel discoveries providing methods for the fabrication of effective therapeutic compounds isolated from the plant tissues directly. These endophytes have overtaken the problems of resource shortages and ecological destruction due to slow growth or artificial exploitation.26 One beneficial effect of endophytes is that they are environment-friendly. 

Our study was undertaken to screen the liver protection of endophytic fungal extracts against paracetamol and ethanol-induced hepatotoxicity.

Paracetamol (PCM) induced liver damage is a very commonly used model for screening hepatoprotection. PCM gets bound with the cellular proteins causing dysfunctioning of mitochondria followed by massive cell death and necrosis of the liver. Due to this the serum biomarkers levels are elevated and altered.¹ Alcohol consumption worldwide causes three million deaths every year. It is a high-risk factor associated with liver disorders. The mechanism of ethanol induction is unknown. But according to the hypothesis, it may lead to steatosis development, alcoholic hepatitis, and cirrhosis as it causes changes in weight and volume.²⁷ Oxidative stress in the liver causes changes in the normal metabolism of the liver leading to the production of ROS and escalate in the ratio of NADH/NAD+. An oxidative alteration in mitochondria has been seen after the exposure of endogenic substances.²⁸

In our experiments, the biochemical variables such as AST, ALT, ASP, total bilirubin, direct bilirubin, and total triglycerides showed significantly increased levels in paracetamol and ethanol-treated groups. Treating with P2EA and P2nB (50 mg/kg &100 mg/kg) decreased the levels to normal significantly showing good hepatoprotective activity. Lipid peroxidation was investigated by the measurement of malondialdehyde (MDA) and antioxidant enzymes such as catalase and superoxide dismutase. The protection of P2EA and P2nB (50 mg/kg &100 mg/kg) reinstated the activity of the antioxidant enzymes (CAT and SOD) and also increased the LPO levels. The values were compared with control group. 

In our study, paracetamol and ethanol caused a significant elevation in AST, ALT, ALP, total and direct bilirubin, and triglycerides levels. Pretreatment with P2EA and P2nB (50 mg/kg &100 mg/kg) fractions exhibited a significant depletion in the elevated levels caused by paracetamol and ethanol-induced changes. These fractions showed membrane stabilizing activity as there was a decrease in serum levels. The regeneration has started taking place when there are depleted biochemical variables. A decrease in AST and ALT to the normal value indicates the regeneration process. Lipid peroxidation (LPO) is the oxidative degradation of lipids. The free radicals take the electrons from the lipids in cell membranes, resulting in cell damage. There is a significant rise in LPO in both the models. Malondialdehyde (MDA) is the marker component to generate free radicals in liver damage. The enzymes increase the oxidation of proteins during liver damage, thus altering the polyunsaturated fatty acids to other fatty acids and causing diminished fluid, leading to cell death. Results of P2EA and P2nB treatment in paracetamol and ethanol administration significantly decreased these changes. The antioxidant mechanism may be responsible for hepatoprotection by P2EA and P2nB. SOD and CAT enzyme levels were decreased by P2EA and P2nB at doses of 50 and 100 mg/kg as a raise by ROS.

Normal hepatocytes cell morphology was observed in the normal liver compared to induced groups where they showed normal hepatic structure whereas degeneration of normal liver cells, lymphocyte infiltration, loss of cell boundaries, and the collapse of epithelial tissues were found in paracetamol induced group. Treated livers showed necrosis characterized by loss of hepatocytes and replacement with erythrocytes, dilated sinusoids, kupffer cells, and mild hyperplasia with mild ballooning, necrosis, and sinusoidal congestion. Thus, P2EA and P2nB at both doses altered the virulent effects of an excess dose of paracetamol and ethanol on the hepatic morphology and architecture.

Conclusion

P2EA and P2nB showed significant hepatoprotection in paracetamol and ethanol-induced models from the isolated endophytic fungi PALF-2 from Phyllanthus amarus leaves. Membrane stabilization mechanisms and antioxidant activity may be responsible for potent hepatoprotective activity by P2EA and P2nB used in treating liver diseases.