Investigations on Screening of Endophytic Fungal Fractions of Andrographis Paniculata (Burm. F.) Wall Nees. Leaves in Paracetamol and Ethanol Induced Hepatotoxicity

INTRODUCTION

Acetaminophen (Paracetamol) is an analgesic and antipyretic drug leading to liver damage in therapeutic doses but when is used in higher doses results in dysfunctioning of the liver¹ . The World Health Organization (WHO) indicates the intensity of pain as it can be used along with non-steroidal analgesic drugs. It is contraindicated in cases of gastric ulcers, hypersensitivity, impairments in blood coagulation, during pregnancy, nursing mothers and children with increased body temperature² . Acetaminophen up to 90% gets metabolized to nontoxic glucuronide and sulfate and the remaining 10% is transformed to a toxic metabolite, N-acetyl-p-benzoquinone imine catalyzed by cytochrome P-450 under normal conditions3. Commonly, N-acetyl-pbenzoquinone imine detoxifies itself to different nonreactive molecules by glutathione peroxidase. But, paracetamol used in excess doses, produces much of N-acetyl-p-benzoquinone imine causing glutathione reduction leading to toxicity of the liver, due to the production of ROS and oxidative stress^1,4.

Ethanol (alcohol) is another hepatotoxic agent leading to severe liver damage. Consumption of alcohol is very common in different cultures causing alcohol abuse leading to morphological changes ranging from fatty liver to inflamed hepatic cells to progressive fibrosis and ultimately leads to cirrhosis. Various tissues in the body are being affected by alcohol consumptions⁵ . Hepatocytes get damaged first among these tissues as liver receives blood directly containing high concentrations of alcohol as a major organ where 90% of the metabolism of ethanol, produces toxic metabolites from alcohol⁶. Chronic intake of ethanol causes depletion in the major antioxidant factors in the liver, including enzymes and non enzymatic antioxidants. The depletion is due formation of ROS, which in excessive amount get generated resulting in cellular antioxidant defense system. Thus, excess alcohol consumption may accelerate an oxidative mechanism directly or indirectly, which produces cell death and tissue damage⁷ . As per the scientific literature, no synthetic drug is available for the treatment of liver disorders. Therefore, alternative treatments are needed for liver disorders to get replaced by existing synthetic drugs. 

Endophytes are microorganisms (including bacteria and fungi) residing inside the plants, few pathogens as such are favorable to the plant itself. These endophytes are present in the internal tissues of the plant without causing any adverse effects⁸. They interact with their hosts closely and are protected from adverse changes in the environment. The microbiota contributes to plant growth, productivity, carbon sequestration, and phytoremediation⁹ . The chief element of plant diversity is endophyte that influences the diversity. Endophytic fungi are being desired in the research sectors because of their benefits, such as growth promoting, biocontrol agents and production of secondary metabolites with good yield.

Andrographis paniculata (Burm. f) Nees, (Acanthaceae) known as “King of bitters,” is an herbaceous plant10, traditionally used in Indian and Chinese herbal medicine for various health benefits. The major chemical constituents reported are diterpenoids, stigmasterols, flavonoids, andrographolides¹¹. Andrographolide is a major bioactive phytoconstituent found in various parts of Andrographis paniculata, particularly in the leaves. The plant is mainly used in liver disorders. Many other biological activities have been reported such as antidepressant¹², antihyperglycemic¹³, antioxidant ¹³, immunostimulant¹⁴, anticancer^14, antimalarial¹⁵, cardiovascular¹⁶, hepatoprotective activity¹⁷, antiangiogenic¹⁸, antioxidant and anti-inflammatory¹⁹. Antimicrobial activity and antibiotic susceptibility were reported from the endophytes isolated from the leaves of Andrographis paniculata²⁰. Bacterial endophytes isolated from Andrographis paniculata were found to act as plant growth promoter and plant growth regulators²¹ and biodiesel precursors²². In our laboratory, we have isolated four bioactive fungi (APLF-1, APLF-2, APLF-3, and APLF-4) from leaves of Andrographis paniculata. Fractions prepared from these endophytes exhibited in vitro free radical scavenging activity^23,24. In the present study fractions of APLF-4 (A4EA & A4nB) were selected for in vivo hepatoprotective activity in paracetamol and ethanol-induced hepatotoxicity. 

MATERIALS AND METHODS

2.1: Plant Material

Collection of leaves of Andrographis paniculata (Burm.f.) Wall. Nees was done from Dharwad district, Karnataka, India. The plant was authenticated by Dr. G R Hegde, Karnataka University, Dharwad (India). A specimen is stored in the herbarium, Postgraduate department of Pharmacognosy (SETCPD/Ph.cog/ herb/32/12/2015).

2.2: Isolation, Fermentation and Extraction

As reported earlier methods²⁴, endophytes were isolated using potato dextrose agar media and incubated at 25ºC-27ºC followed by fermentation by potato dextrose broth. These endophytes were then extracted with chloroform, ethyl acetate and n butanol solvents to get the respective extracts.

2.3: Hepatoprotective activity

A4EA and A4nB of APLF-4 were selected for in vivo hepatoprotective activity in paracetamol and ethanol induced hepatotoxicity ²⁴. Institutional Animal Ethics Committee (IAEC) permission was obtained (Animal House Reg. No.112/PO/ Re/S/99/CPCSEA, dated 26-10-2017 and IAEC letter dated 07.03.2016) for animal experimentation.

2.3.1: Animals

Albino Wister rats weighing 150–200 g were used. The inbred rats were collected from Venkateshwara Enterprises Bangalore, Karnataka. They were maintained in the animal house of SET’s College of Pharmacy, Dharwad for experimental purpose under controlled conditions of temperature (23±2°C), humidity (50±5%) and 12 h light-dark cycles. They were acclimatized for 7 days before the study. Animals were then randomized into groups and housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to pellets as basal diet and water ad libitum

2.3.2: Acute toxicity studies

Acute oral toxicity of A4EA and A4nB was determined using Swiss albino mice. The animals were fasted for 12 h prior to the experiment and were administered with single dose of fractions dissolved in 5% gum acacia and observed for mortality up to 48 hour (short term toxicity). Based on the short-term toxicity, the dose of next animal was determined as per OECD guideline 420.

2.3.3: Experimental design for Paracetamol induced hepatotoxicity^25,26

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

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

Group III: Rats received the standard drug silymarin 200 mg/kg b.w. p.o./5 days.

Group IV: Rats received with A4EA (50mg/kg) b.w. p.o./5 days.

Group V: Rats received with A4EA (100mg/kg) b.w. p.o./5 days.

Group VI: Rats received with A4nB (50mg/kg) b.w. p.o. /5 days.

Group VII:Rats received with A4nB (100 mg/kg) b.w. p.o./5 days.

On the 5th day of experimental period, after the drug administration of respective treatments, all animals except those in group I were administered with paracetamol 2 g/kg b.w. p.o. On the 6th day, after 2 h of respective drug treatments, animals were anaesthetized using diethyl ether inhalation jar. By puncturing retro-orbital bleeding under mild ether anesthesia, blood was collected, centrifuged (2500 rpm at 30°C for 15 min) and serum obtained was subjected to biochemical estimations. Livers were excised immediately, washed in ice cold normal saline and placed in 10% formalin solution for histopathological study. Liver homogenate was prepared to determine the levels of endogenous enzymes.

2.3.4: Experimental design for Ethanol induced hepatotoxicity^25,27

Animals were divided into following groups of six animals in each group (n=6). All animals except the group I was intoxicated with 20% ethanol (3.76 g/kg/day, p.o) for 18 days.

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 (200mg/ kg)

Group IV: Rats received with A4EA (50mg/kg) p.o. for 18 days

Group V: Rats received with A4EA (100mg/kg) p.o. for 18 days

Group VI: Rats received with A4nB (50 mg/kg) p.o. for 18 days

Group VII:Rats received with A4nB (100 mg/kg) p.o. for 18 days

All the animals were treated with respective drugs orally for a period of 18 days. On 19th day, animals were anaesthetized using diethyl ether inhalation jar. Blood was collected by puncturing retro-orbital plexus under mild ether anesthesia, centrifuged (2500 rpm at 30°C for 15 min) and serum was subjected to biochemical estimations. Livers were excised immediately and washed in ice cold normal saline and placed in 10% formalin solution for histopathological study. Liver homogenate was prepared to determine the levels of endogenous enzymes.

2.3.4: Biochemical parameters

In both models, after the treatment period, serum was separated and analyzed spectrophotometrically for AST, ALT, ALP, total and direct bilirubin, total triglyceride (TG), and total protein for both the models using diagnostic kits of ERBA diagnostics, Mannheim GMBH, Germany.

2.5: Measurement of enzymatic and nonenzymatic antioxidant levels in Paracetamol and Ethanol induced models

2.5.1: Tissue preparation

Animals were sacrificed and perfused transcardially with ice-cold saline. The whole liver was perfused in situ with ice cold saline, dissected out, blotted dry and immediately weighed. A 10% liver homogenate was prepared separately with icecold saline-EDTA using Teflon-glass homogenizer (Yamato LSG LH-21, Japan). The homogenate was used for the estimation of proteins and lipid peroxidation. Liver homogenate was centrifuged at 10,000 rpm for 10 min and the pellet discarded. The supernatant was again centrifuged at 20,000 rpm for 1 h at 4°C. Both the liver supernatants obtained were used for the estimation of nonenzymatic antioxidants (Lipid peroxidation) and enzymatic antioxidants (Catalase and superoxide dismutase).

2.5.2: Lipid peroxidation

Thiobarbituric acid reactive substances (TBARS) in the liver homogenate were estimated by using standard protocol 28. The homogenate was incubated with 15% TCA, 0.375% TBA and 5N HCl at 95°C for 15 min, the mixture was cooled, centrifuged and the absorbance of the supernatant measured at 532 nm against appropriate blank. The amount of lipid peroxidation was determined by using the formula €= 1.56 x 105M-1 cm-1 and expressed as TBARS (μm) per g of tissue.

2.5.3: SOD assay

Liver homogenate (0.5 ml) was taken, and 1 ml of 50 mM sodium carbonate, 0.4 ml of 24μm NBT, and 0.2 ml of 0.1mM EDTA were added. The reaction was initiated by adding 0.4 ml of 1mM hydroxylamine hydrochloride. Zero time absorbance was taken at 560 nm followed by recording the absorbance after 5 min at 25º C. The control was simultaneously run without liver homogenate. SOD activity was determined as the amount of enzyme required to inhibit the reduction of NBT by 50%. The specific activity was expressed in terms of units per mg of proteins²⁹.

2.5.4: Catalase assay

Catalase activity was determined spectrophotometrically by using reported method, where 1.95 ml of 10 mM H2O2 in 60 mM phosphate buffer (pH=7.0), 0.05 ml of the liver homogenate was added and rate of degradation of H2O2 was followed at 240 nm/ min. Catalase content in terms of U/mg of protein was estimated from the rate of decomposition of H2O2 using the formula

k=2.303/ Δt x log (A1/A4) S-1

A unit of catalase is defined as the quantity which decomposes 1µm of H2O2 per min at pH=7.0 at 25°C, while H2O2 concentration falls from 10.3 to 9.2mM 30.

2.6: Protein Estimation Folin phenol reagent method was used to measure Total tissue protein content as reported previously^31. 

2.7: Histopathological studies

Isolated liver of each animal was kept in 10% buffered neutral formalin and bovine solution. They were further processed for paraffin embedding following standard microtechnique 32. Sections of liver stained with alum-haematoxylin and eosin, were observed photomicroscopically for histopathological changes.

2.8: Statistical evaluation

The data was expressed as Mean±S.E.M. Statistical comparisons were performed in one-way ANOVA followed by Tukey’s test using Graph Pad Prism version 6.0, USA.

RESULTS

Endophytic fungi isolated from the leaves of Andrographis paniculata were identified as Diaporthe sp. A25 (APLF-1) Preussia sp. PPV3 (APLF-2) Phyllosticta sp. ZLY-2010 isolate M1324 (APLF-3) and Aspergillus tubingensis strain Cs/7/2 (APLF-4) as previously reported.

3.1 Acute toxicity (LD50) studies

Acute toxicity studies were carried out according to OECD guidelines (Up and Down method). No mortality was observed upto 2000 mg/kg body weights were for A4EA and A4nB. Hence, doses of 50 mg/kg and 100 mg/kg body weight were selected to assess the hepatoprotective activity.

3.2 Effect of A4EA and A4nB on serum biochemical parameters in paracetamol and ethanol induced hepatotoxicity in rats

Administration of Paracetamol (2g/kg) and ethanol 20% ethanol (3.76 g/kg/day, p.o) significantly caused liver damage and necrosis of cells by elevating the levels of AST, ALT, ALP, total and direct bilirubin, triglyceride and total proteins as compared to normal control. A4 EA and A4nB, (50 mg/kg &100 mg/kg) reversed the elevated biochemical parameters as compared to paracetamol (Fig 1) and ethanol treated group (Fig 2).

3.3 Effect of A4EA and A4nB on endogenous antioxidant enzymes in paracetamol and ethanol induced hepatotoxicity

An increase in LPO level was seen in paracetamol (46.79±1.35) and in ethanol (22.67±0.80) treated group. A4EA (100 mg/ kg) and A4nB (50 & 100mg/kg) significantly inhibited the increased susceptibility to lipid peroxidation (***p<0.001). A4EA (50 mg/kg) showed significance at (**p<0.001) value, these values were compared to silymarin. The SOD levels were decreased in paracetamol treated (26.09±0.73) and in ethanol treated (30.67±0.21) groups. A4EA and A4nB (50 & 100mg/kg) showed significance values when compared to silymarin. There was a marked depletion of CAT levels in paracetamol treated (44.85±0.41) and ethanol treated (65.31±0.33) groups. A4EA (100mg/kg), A4nB (100mg/kg), increased the CAT activity significantly (***p<0.001) (Table 1 & 2).

3.4 Histopathology of liver for paracetamol induced hepatotoxicity in rats

In normal group, arrangement of cells is seen normal; no inflammation was seen with respect to central vein, kuffer cells, hepatic vein and portal triads. In Paracetamol induced group, fatty acid degeneration was seen. Central vein dilation, ballooning and inflammation of hepatocytes were seen. In standard silymarin (100 mg/kg) treated group, hepatic regeneration was seen. Mild inflammation, portal triad, central vein and sinusoidal congestion were observed. All the extract treated groups showed mild inflammation of fibrous septae. Mild congestion of central vein observed. Decrease in the inflammation of hepatocytes was seen with mild inflammation and ballooning. No inflammation of kuffer cells was seen (Fig 3).

3.5 Histopathology of liver for ethanol induced hepatotoxicity in rats

In normal animals, liver showed normal hepatocytes, no inflammation of central vein and sinusoid congestion was seen. Hepatic structure was found to be normal. In ethanol treated group, disturbed architecture of hepatic cells with many enlarged degenerating cells having pale cytoplasm with fatty changes. In standard silymarin (100 mg/kg) treated group regeneration of hepatic cells was seen. Hepatic architecture restored to normal (Fig 4). All the extracts showed regeneration of hepatocytes around the necrosis and fatty vacuoles. No inflammation was found and injured liver cells were restored (Fig 4).

and pale cytoplasm with fatty changes (steatosis) disturbed architecture of the hepatic plates with many enlarged (cytomegaly) vacuolated degenerating cells having indistinct boundaries and pale cytoplasm with fatty changes (steatosis) disturbed architecture of the hepatic plates with many enlarged (cytomegaly) vacuolated degenerating cells having indistinct boundaries and pale cytoplasm with fatty changes (steatosis) disturbed architecture of the hepatic plates with many enlarged (cytomegaly) vacuolated degenerating cells having indistinct boundaries and pale cytoplasm with fatty changes (steatosis).

DISCUSSION

Endophytes are microorganisms, growing inside the tissues of living plants without causing apparent distress to the host. The endo-plant relationship mechanisms are yet under study, but they have shown symbiotic and mutualistic relation to the plant. Endophytes have been reported to exhibit functions as such stress tolerance, plant development, antibacterial, antiviral and antifungal activites ³³. Fungal endophytes reported from plants occurred in different environment conditions such as xerophytic, tropic, temperate and aquatic. Endophytes may be bacteria, fungi, or actinomycetes out of which endophytic fungi have been depicted as potent sources possessing novel chemical structure³⁴. Four prominent endophytic fungi, namely APLF-1, APLF-2, APLF-3 and APLF-4 were isolated from the leaves of Andrographis paniculata and identified as Diaporthe sp. A2523, Preussia sp. PPV323, Phyllosticta sp. ZLY-2010 isolate M13 24 and Aspergillus tubingensis strain Cs/7/224. Previously we have reported that A4EA and A4nB of leaf endophytic fungus, Aspergillus tubingensis strain Cs/7/2 isolated from Andrographis paniculata exhibited significant antioxidant and hepatoprotective activity in CCl4 induced hepatotoxicity²⁴.

Aspergillus tubingensis, Aspergillus niger and Aspergillus awamori are some of the species belonging to the genus Aspergillus nigri. Aspergillus tubingensis is a darkly pigmented member³⁵. Lack of production of mycotoxin in Aspergillus tubingensis, has been led it into applications in industry and biotechnology. American food and drug administration (FDA) has conceded Aspergillus tubingensis as safe³⁶. it is capable of producing enzymes such amylase, lipase, glucose oxidase, phytase, xylanase, acid and phosphatase. Amylase produced by A. tubingensis has potential use in the manufacture of bioethanol from distilled waste water and molasses residues³⁷. Four new dimeric naphtho-γ-pyrones, named rubasperone D, rubasperone E, rubasperone F and its atropisomer rubasperone G, together with four known monomeric naphtho-γ-pyrones, TMC 256 A1, rubrofusarin B, fonsecin, and flavasperone were isolated from the mangrove endophytic fungus Aspergillus tubingensis (GX1-5E) cultivated in solid rice medium38. Rubrofusarin B displayed inhibitory activities against tumor cell lines of MCF-7, MDA-MB-435, Hep3B, Huh7, SNB19, and U87 MG with IC50 values between 19.928μM and 47.98μM. Authors have reported that endophytic fungi, Aspergillus tubingensis and other Aspergillus species showed activity against 5 bacterial and 2 fungal strains. These endophytic fungi were isolated from Eugenia jambolana Lam. (Syzygium cumini)³⁹. A pyrone named rubrofusarin was isolated from fermentation of Aspergillus tubingensis in Lycium ruthenicum. It showed strong inhibitory activity against Escherichia coli⁴⁰.  

Alcohol is mainly examined for the development and progression of alcoholic diseases, leading to multiple organs injuries. The enzymes responsible for alcohol metabolism are chiefly cytochrome P-450 and alcohol dehydrogenase in vivo. Excessive alcohol intake elevates the reactive oxygen species (ROS) or cellular lipid peroxidation, causing liver disorders. The oxidative stress bestows hepatic cell destruction by various molecule targets such as lipids, proteins and DNA. Above normal limits, consumption of alcohol triggers liver lesions amid which hepatic steatosis are seen in all of them consuming in excess. Along with the fatty liver inflammation, fibrosis and cirrhosis is also noticed ^41.  

The very common, popular and safe analgesic and antipyretic drug used worldwide is paracetamol. The overdose of paracetamol when exceeding to more than 4 g/day results in dysfunctioning of liver. In overdoses, it is metabolized and converted into a toxic compound, N-acetyl p-benzoqinone overwhelming glutathione (GSH) leading to lipid peroxidation, DNA fragmentation, and liver damage^42. One of the other mechanisms involved in paracetamol toxicity is oxidative stress; imbalance between the level of reactive oxygen species (ROS) and the antioxidant capacity of cells ^43.

In the present study, hepatoprotective activity of A4EA and A4nB extracts were carried out against paracetamol and ethanol induced hepatotoxicity. The rats treated with paracetamol and ethanol showed significantly elevated activity of liver function markers such as AST, ALT, AST, total and direct bilirubin and triglycerides. This may be due to damaged liver leading to depletion in cell coherence and leakage, causing enzyme release into the bloodstream⁴⁴. Administration of A4EA and A4nB at the dose of 50 and 100 mg/ kg significantly lowered the biochemical levels. Reduction in biochemical parameters shows indication of the regeneration process of hepatocytes.

ROS are majorly responsible for oxidative degradation of lipids. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in the cells. Increased free radical may lead to overproduction of MDA. A4EA and A4nB treated rats significantly decreased the elevated LPO level. The enzymes increase the oxidation of proteins during liver damage. The endogenous antioxidant enzyme levels of SOD and CAT were significantly decreased in A4EA and A4nB as compared to those in the paracetamol and ethanol treated group by reactive oxygen species. Western blotting method was used to determine the protein levels of liver tissues. A significant increase of protein level was observed by administration of A4EA and A4nB (50 and 100 mg/kg).

The necrosis and fatty vacuoles around the hepatocytes were able to regenerate by A4EA and A4nB (50 mg/kg and 100 mg/kg), showing mild congestion of central vein and sinusoid. The inflammation also was reduced. Thus, A4EA and A4nB (50 and 100 mg/kg) altered the effects of excess dose of paracetamol and ethanol on the hepatic morphology and architecture.

CONCLUSION

APLF-4 was isolated from the leaves of Andrographis paniculata and was identifies as Aspergillus tubingensis strain Cs/7/2 by PCR sequential analysis. A4EA and A4nB exhibited significant hepatoprotective activity in paracetamol and ethanol induced models. Antioxidant activity and Membrane stabilization mechanisms may be responsible for hepatoprotective activity of A4EA and A4nB as it can be used in treatment of liver disorders.

DECLARATION OF INTEREST: None.