Hepatoprotective Potential of Endophytic Aspergillus Niger Strain A6 Fractions Isolated from Phyllanthus Amarus Schum and Thonn

Introduction

Endophytes are an undeveloped origin of pharmacologically active compounds having their species with rich in divergence. In the former years, numerous novel bioactive secondary metabolites from endophytes with therapeutic activities have been divulged.¹ These endophytes help in growth and survival of host plants by production of secondary metabolites.² Endophytic microbes are fungi or bacteria growing inside the plant tissues without causing any harm to the host. All the plant species in natural ecosystem are associated with endophytes. Endophytes also play an important role in improving stress tolerance to biotic and abiotic stresses. Most endophytes establish a symbiotic, mutualistic, or pathogenic relationship with the host plant.³ Endophytic fungi are mostly involved in the research sectors for their benefits, such as growth promoting, biocontrol agents and production of secondary metabolites with good yield.

Approximately 2 million deaths take place every year due to liver diseases. One of the causative factors for liver disorders are drugs. Although drug-induced liver diseases are rare, they still are serious problems to be considered. Almost 20% acute liver failure is due to these drugs.⁴ One such drug causing liver injury is paracetamol (acetaminophen), an analgesic and antipyretic drug. Overdose of the drug leads to severe liver injury, liver necrosis and kidney damage in humans.⁵ Oxidative stress may be one of the reasons causing the liver toxicity. In therapeutic doses, cytochrome P-450 enzymes metabolises paracetamol to N-acetyl-pbenzoquinoneimine (NAPQI), later this is metabolized and detoxified by glutathione (GSH). But in over dosage of the drug, cytochrome P450-catalyzed metabolises paracetamol, which leads to excessive formation of NAPQI, which in turn depletes cellular GSH, leading to toxicity of the liver due to the production of reactive oxygen species (ROS) and oxidative stress.⁶ According to the literature, no synthetic drug as such is available for treating liver disorders. Hence, drugs obtained from plants as well as microbial sources can be explored to manage liver toxicity.

Phyllanthus amarus (Euphorbiaceae) was used since many decades for treating various ailments in folk medicine. Phyllanthus species are rich in phytochemicals. The main bioactive principles are hypophyllanthin, phyllanthin, niranthin, kaempferol, nirtetralin, niranthin, epibubbialine, isobubbialine, nor-securinine, securinine, corilagingeraniin, dihydrosecurinine, isolated from different parts.^7,8 Several reports have shown that P. amarus exhibits numerous pharmacological properties such as antihepatitis, anti-malarial, antiviral, antibacterial, antidiarrheal.^9-13 It also possesses hepatoprotective, anti-carcinogenic, anti-inflammatory, anti-asthmatic, and anti-diabetic properties.^14-19 Phyllanthus amarus leaves have also been explored for the presence of bioactive endophytic microorganisms. Presence of endophytic bacteria like Bacillus sp and fungi like Acinetobacter sp. Nigrospora sp in the stem and leaves of Phyllanthus amarus has been reported.^20,21 Chromatographic studies of ethyl acetate of endophytic fungus Nigrospora sp advanced to the isolation of hypophyllanthin, phyllanthin, quercetin-3, 4-di-O-glucoside, and kaempferol-3-O-rutinoside.²¹ Considering these facts, the present investigation aimed to isolate and characterize endophytic fungus from leaves of Phyllanthus amarus and to screen the crude endophytic fractions for antioxidant and antihepatotoxic activity in rodents.

Materials and Methods

Plant Material

Leaves of Phyllanthus amarus were collected from surrounding areas of Dharwad, Karnataka, India. It was identified and authenticated by a Taxonomist, Department of Botany, Karnatak University, Dharwad, Karnataka, India. A voucher specimen (SETCPD/ Ph.cog/herb/86/12/2017) was deposited at the herbarium of postgraduate department of Pharmacognosy and Phytochemistry, SET’s College of Pharmacy, Dharwad, India.

Isolation of Endophytes

Leaves were washed rigorously in sterile water to remove adhered foreign materials, surface disinfected by soaking in 70% ethanol for three minutes and in 4% sodium hypochlorite (NaOCl) for one minute, later rinsed in sterile demineralized water and were stored aseptically. The washings were tested to ascertain proper surface sterilization. Small pieces of inner tissues (1 cm x 1 cm) were permeated on potato dextrose agar (PDA) added with streptomycin (125 µg/mL) in petri plates and incubated at 27ºC ± 2ºC until fungus was grown. Purity of fungal colonies was checked randomly and assembled according to colony morphology. Purified strains were stored on PDA slants at 4°C until further use.²²

Molecular characterization of endophytic fungi by PCR sequential analysis

Isolated fungal endophytes were identified using microscopic and molecular techniques. Using the genomic DNA fractionation kit (Bhat Biotech Ltd. Bangalore, India), genomic DNA was extracted from the said organism.²³ Phylogenetic analysis of endophytes was performed by the acquisition of ITS1-5.8S-ITS4 ribosomal gene sequencing. Internal transcribed spacer (ITS) region was amplified using the forward primer ITS1 5’-TCCGTAGGTGAACCTGCGG-3’ and reverse primer ITS4 5’-TCCTCCGCTTATTGATATGC-3’ using polymerase chain reaction (PCR) as per the previously described method.

Fermentation and preparation of endophytic fractions

The purified fungal isolate was injected and fermented into a 3L Erlenmeyer flask containing 800 mL of potato dextrose broth (PDB), incubated for 21 days at 25°C-27°C. After 21 days of incubation, chloroform was added to flask and was left overnight. Further, it was homogenized at 4000 rpm for 30 min to separate the mycelia from broth and filtered under vacuum. Aqueous phase acquired after chloroform extraction was further separated three times with equal volumes of ethyl acetate, followed by three times separation with n-butanol. All the extracts were dried using rotary flash evaporator (Superfit Rotavap, PBU-6) and weighed. The endophytic fractions were designated as P1C, P1EA and P1nB.

In vitro free radical scavenging activity

Reaction with DPPH radical

P1C, P1EA and P1nB were assayed for DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging activity following established method.²⁴

Reaction with hydroxyl radical

Hydroxyl radical scavenging capacity of P1C, P1EA and

P1nB was studied by deoxyribose method.²⁵

Determination of reducing power

Reducing power assay of P1C, P1EA and P1nB was carried out by the method described by Oyaizu 1986.²⁶

Hepatoprotective activity of endophytic fractions

Based on the results of in vitro antioxidant activity, P1EA and P1nB fractions were screened for in vivo liver protective activity in paracetamol induced hepatotoxicity.

Animals

Albino Wister rats weighing 180-200 gm were procured from Aditya biosys Pvt. Ltd. Tumukuru, Karnataka, India. They were accommodated in polypropylene cages containing sterile paddy husk as bedding, under controlled temperature of 23±2°C, controlled humidity of 60-70% and 12 hour light and dark cycles in a registered animal house (Reg No.112/1999/CPCSEA, dated 19-05-1999). They were climatized for seven days before the study. They had free access to standard, nutritionally balanced pellets diet (VRK nutritional solutions, Sangli, India) and water ad libitum. Ethical clearance for animal studies were secured from the IAEC and standard operating procedures and protocols were followed as per CPCSEA.

Acute toxicity studies

Acute toxicity of P1EA and P1nB were determined by using Swiss albino mice. The animals were abstained from food and water for 12 hours prior to the experiment, were dispensed with a single dose (2000 mg/kg) of extracts in 5% gum acacia and noted for mortality up to 48 hours (short term toxicity). Based on toxicity study, the next animal’s dose was determined as per OECD guideline 420.

Experimental design for hepatoprotective activity²⁷

Group I: Control, rats received distilled water (2 ml/kg) b.w. p.o. /5 days

Group II: Paracetamol control, rats received paracetamol

(2 g/kg b.w. p.o) /5 days

(Except 5^th day)

Group III: Rats received standard drug (Silymarin; 200 mg/kg) b.w. p.o./5 days

Group IV: Rats received P1EA (50 mg/kg) b.w. p.o./5 days

Group V: Rats received P1EA (100 mg/kg) b.w. p.o./5 days

Group VI: Rats received P1nB (50 mg/kg) b.w. p.o. /5 days

Group VII: Rats received P1nB (100 mg/kg) b.w. p.o./5 days

On the 5^th day of experiment, all the groups except Group I were administered with 2 g/kg b.w. p.o along with the respective drug treatment. On the 6^th day, after 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 thus obtained was subjected to biochemical estimations. Livers were removed immediately, cleaned in ice cold normal saline and were placed in 10% formalin solution for histopathological study. Liver homogenate was made ready to govern the levels of endogenous enzymes.

Biochemical parameters

After the treatment period, serum was taken out, analyzed for biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total and direct bilirubin, total triglyceride (TG), and total protein spectrophotometrically. ERBA Diagnostic’s diagnostic kit was used for determination.

Measurement of enzymatic and non-enzymatic antioxidant levels

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 ice-cold 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 was discarded. The supernatant was again centrifuged at 20,000 rpm for 1 hour at 4°C. Both the liver supernatants obtained were used for the estimation of non-enzymatic antioxidants (Lipid peroxidation) and enzymatic antioxidants (Catalase and superoxide dismutase).

Lipid peroxidation

Estimation of Thiobarbituric acid reactive substances (TBARS) was carried out in the liver homogenate by using standard protocol.²⁸ Lipid peroxidation was determined by using the formula €= 1.56 x 105M^-1 cm^-1 and expressed as TBARS (μm) per g of tissue.

SOD assay

Superoxide dismutase (SOD) activity was determined following established procedure and calculated as the amount of SOD required to inhibit the reduction of NBT by 50%. It was expressed in terms of units per mg of proteins.²⁹

Catalase assay

Catalase (CAT) activity was determined spectrophotometrically by using reported method. Catalase content in terms of U/mg of protein was estimated from the rate of decomposition of H₂O_2. A unit of catalase was defined as the quantity which decomposes 1 µm of H₂O₂ per min at pH=7.0 at 25°C, while H₂O₂ concentration falls from 10.3 to 9.2 mM.²⁹

Histopathological studies

Isolated liver of each animal was kept in 10% buffered neutral formalin and bovine solution. They were later fixed in paraffin by standard technique.³⁰ Sections of liver stained with alum-haematoxylin and eosin were observed photomicroscopically for histopathological changes.

Statistical evaluation

The data was expressed as Mean±SEM. Statistical comparisons were performed with one-way ANOVA followed by Tukey’s test using Graph Pad Prism version

6.0, USA.

Results

Isolation of endophytes

Endophytic fungus, PALF-1 was isolated from leaves of Phyllanthus amarus and was identified as Aspergillus niger strain A6 by PCR sequential analysis and phylogenetic studies (Figure 1).

Preliminary Phytochemical Analysis

The yield of P1C, P1EA and P1nB were found to be 3.5%, 5.7% and 5.5% respectively. Preliminary phytochemical analysis of P1C, P1EA and P1nB revealed the presence of tannins, triterpenoid, alkaloids and flavonoids as chief secondary metabolites.

PCR sequential analysis of PAlF-1

The sequencing for ITS gene for PALF-1 was aligned by Maximum likelihood method located on the TamuraNei model (Figure 2). It was matched with 10 nucleotide sequences. The tree showed highest log likelihood (-855.1369) for PALF-1 (Figure 3). Initial tree(s) for the heuristic search were obtained automatically. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; else, BIONJ method with MCL distance matrix was used. The analysis showed 11 nucleotide sequences. Codon positions included were 1^st +2^nd +3^rd +Non-coding. All positions containing gaps and missing data was eliminated. There was a total of 561 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. The DNA sequence of sample PALF-1 matches best with that of Aspergillus niger strain A6 (Figure 4). The sequence was compared with existing sequences in the National Center for Biotechnology Information (NCBI) database using the BLAST N program.

In vitro free radical scavenging activity

DPPH assay

The IC₅₀ values for P1C, P1EA and P1nB were found to be 133.49 µg/mL, 73.54 µg/mL and 59.86 µg/mL respectively. P1nB strongly inhibited DPPH as compared to P1C, P1EA. A linear coefficient (r²=0.974, 0.962,

0.872) was obtained. The results are shown in Figure 5.

Hydroxyl scavenging assay

This assay exhibited the abilities of the endophytic fractions and standard mannitol to inhibit hydroxyl radical mediated by deoxyribose degradation in a Fe³⁺- EDTA-Mannitol and H2O2 reaction mixture. The IC50 values for P1C, P1EA, and P1nB were found to 194.44 µg/mL, 160.30 µg/mL, and 188.53 µg/mL respectively. P1EA strongly inhibited DPPH as compared to P1C, P1nB. A linear coefficient (r²=0.982, 0.981, 0.817) was obtained. The IC₅₀ value for standard mannitol was found to be 141.59 µg/mL with a linear correlation coefficient of r²=0.947. The results are depicted in Figure 6.

Reducing power Assay

This assay exhibited Fe (III) reduction as an indicator of electron donating activity, showing phenolic antioxidant action by fragmenting the radical chain by donation of hydrogen atom. Reducing power of P1C, P1EA, and P1nB (50 µg/mL - 450 µg/mL) increased with increase in concentration. P1EA and P1nB showed effective reductive ability as compared to P1C as shown in Figure 7.

Hepatoprotective activity

Acute toxicity (LD₅₀) studies

According to OECD guidelines, acute toxicity studies were carried by Up and Down method. Mortality was not seen up to 2000 mg/kg body weights were for P1EA and P1nB. Doses of 50 mg/kg and 100 mg/kg body weight were selected to screen the hepatoprotective activity.

Effect of P1EA and P1nB on serum biochemical parameters in paracetamol induced hepatotoxicity in rats

Administration of paracetamol (2 gm/kg b.w.) caused liver injury and necrosis of cells by raising the levels of AST, ALT, ALP, total and direct bilirubin, triglyceride and total proteins as compared to normal control. P1EA and P1nB (50 mg/kg and 100 mg/kg) reversed the elevated biochemical parameters as compared to paracetamol group (Table 1).

Effect of P1EA and P1nB on endogenous antioxidant enzymes in paracetamol induced hepatotoxicity

There was a marked increase in lipid peroxidation (LPO) levels (97.13±0.37) in PCT treated group. There was inhibition of in vivo lipid peroxidation by P1EA (50 mg/kg and 100 mg/kg), P1nB (50 mg/ kg) showed significance value (^***p<0.001). P1nB showed significance value at dose 50 mg/kg (^**p<0.01). The SOD level was decreased (12.18±0.32). P1EA (100 mg/kg), P1nB (50 mg/kg & 100 mg/kg) increased the SOD levels with significance value (^***p<0.001). There was a marked depletion of CAT levels (15.18±0.21). P1EA (100 mg/kg) and P1nB (100 mg/ kg) showed increase in CAT activity significantly (^***p<0.001) (Table 2).

Each value represents Mean ± SEM (n=6) *p<0.05, ** p<0.01, ***p<0.001 compared to paracetamol treated group. One way ANOVA followed by Tukey’s multiple comparison tests

Histopathological studies

The effects of paracetamol, P1EA and P1nB on histomorphological changes in rat liver is shown in Figure 8. In normal control group, liver showed normal hepatic structure with normal hepatocytes, sinusoids and central vein. Degeneration of normal architecture of liver cells, infiltration of the lymphocytes, loss of cell boundaries and the collapse of epithelial tissues was seen in paracetamol treated group. Silymarin treated group showed restoration of hepatic structure, with normal hepatocytes, restored kupffer cells and central vein. In P1EA treated group at both the doses, mild congestion of central vein, regeneration of hepatocytes and mild dilated sinusoids were observed. Mild necrosis and less inflammation of hepatocytes, regeneration of hepatocytes were also observed in P1nB treated groups.

(I) Control group (II) Rats treated with Paracetamol showing hepatocyte vacuolization, ballooning degenerations of hepatocytes associated with neutrophilic infiltration (III) Rats treated only with Silymarin 150 mg/kg (IV) Rats treated only with P1EA; 50 mg/kg and Paracetamol (V) Rats treated with P1EA ;100 mg/kg and Paracetamol (VI) rats treated only with P1nB; 50 mg/ kg and Paracetamol. (VII) Rats treated only with P1nB; 100 mg/kg and Paracetamol. Liver sections were stained using hematoxylin-eosin method. Magnifications: 400

HPTLC fingerprinting

High performance thin layer chromatography (HPTLC) fingerprinting of P1EA and P1nB at 254 nm showed the presence of eleven and five multivalent secondary metabolites respectively. The range of Rf values was from 0.01 to 0.99 and high concentration of metabolite was found to be 32.09% and 33.69% with Rf values of 0.52 and 0.56 respectively. However, at 366 nm P1EA and P1nB revealed nine and six secondary metabolites at range of Rf values starting from 0.01 to 0.99 respectively. The highest concentration was found to be 23.97% with Rf value of 0.56 for P1EA and 24.87% with Rf value of 0.03 for P1nB respectively. After derivatisation at 620 nm, P1EA and P1nB showed the presence of seven and four polyvalent secondary metabolites respectively. The finger printing profile is shown in Figure 9.

Discussion

Endophytes are symbiotic microbes, especially bacteria and fungi which reside in internal plant tissues, not causing any disease to host plant.³¹ Endophytes impacts the physiology and development of the plant viz bioremediation, plant growth, symbiotic-mutualistic, commensalistic, trophobiotic interactions. They also support host plant defense against environmental stress.³² Phyllanthus amarus has been predominantly used in the treatment of liver diseases by traditional medicine practitioners. It was also reported that phyllanthin and hypophyllanthin, the two important constituents of Phyllanthus amarus are responsible for the hepatoprotective activity and also acted on antioxidant mechanism.³³ As far as endophytes are concerned, our previous study manifested the antioxidant and hepatoprotective activity of endophytic fractions of Nigrospora sp. CMH2_13 isolated from Phyllanthus amarus leaves.²¹ In this work, one more prominent culturable endophytic fungus was isolated from the leaves of Phyllanthus amarus and was identified as Aspergillus niger strain A6 by PCR sequential analysis. The fungus, Aspergillus niger is the most common known species in the genus Aspergillus. It is responsible for black mould diseases in certain fruits and vegetables such as grapes, apricots, onions, peanuts, and is a common contaminant of food.³⁴ Some secondary metabolites such as R(-)-glycerol monolinoleate, bis-dethio-(bis-methylthio)-gliotoxin, fumiquinazoline-F, fumiquinazoline-D, (Z,Z)-N,N’-[1-[(4-Hydroxy-phenyl)-methylene]-2- [(4-methoxy-phenyl)-methylene]-1,2-ethanediyl]- bis-formamide, pyrazoline-3-one trimer, tricho-9- ene-2a,3a,11a,16-tetraol, 2’-deoxy-thymidine, and cere- broside A were isolated from this fungus inhabited in Ipomoea batatas.^35,36

Paracetamol, an analgesic and antipyretic, when used in large doses leads to liver damage. Excessive initiation of NAPQI in paracetamol induced hepatotoxicity binds covalently to lipids, proteins and DNA molecules.³⁷ NAPQI also leads to lowering of glutathione concentration in liver, causing depletion of antioxidant agents. Paracetamol toxicity may be due to oxidative stress and imbalance of reactive oxygen species (ROS) and the antioxidant capacity of cells.³⁸ Hepatic diseases are recognized by high levels of serum indicators like ALT, AST, total and direct bilirubin, triglycerides and total proteins. Administration of paracetamol increased activities of hepatic sensitive indicators, reflecting the damage or changes in the cell membrane permeability paramounting to leakage of enzymes from cells to the circulation. In the present study, increased levels of serum hepatic markers suggested that an extensive liver injury was due to paracetamol leading to membrane damage by the the process of lipid peroxidation. The improvement of the functional status of the liver was due to stabilization of enzyme levels after administration of P1EA and P1nB fractions of endophytic Aspergillus niger strain A6. Reduction in biochemical parameters indicates the regeneration process of hepatocytes. The liver architecture showed hydropic degeneration, necrotic changes, congestion and dilatation of central veins. These results complemented with our earlier report where pretreated rats with ethyl acetate and n-butanol fractions of endophytic Nigrospora sp. CMH2_13 decreased the CCl4 induced hepatotoxicity.²¹ However, in another study, ethyl acetate fraction of endophytic Achaetomium sp., isolated from Euphorbia hirta exhibited significant in vitro antioxidant and hepatoprotective potential.³⁹

ROS plays an indispensable role in lipid peroxidation. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in the cells. Increased free radicals may lead to overproduction of MDA. In our study, paracetamol induction increased the hepatic lipid peroxidation (LPO) in treated rats. Treatment with P1EA and P1nB exhibited a significant inhibitory role against TBARS formation in rats and thereby, decreased the paracetamol induced liver damage. This LPO reduction may be due to capability of P1EA and P1nB to scavenge ROS. Free radical scavengers and antioxidant enzymes such as SOD, CAT and GPx system protects the biological systems against the damaging effects.⁴⁰ Detoxification of superoxide anion and H₂O₂ in cells will be by the coordinative effect of these enzymes. A significant decrease in the levels of SOD and CAT was observed in paracetamol group as compared to normal control group. P1EA and P1nB at different doses maintained the SOD and CAT activities, which was comparable to control group, thereby reducing the stress. This probably may be due to removal of ROS from the tissue.

In histopathological studies, the early changes after paracetamol administration were dilated central vein, necrosis and fatty degeneration due to oxidative stress. Necrosis and fatty vacuoles were regenerated around the hepatic cells by P1EA and P1nB, showing mild inflammation and congestion of sinusoid and central vein. High performance thin layer chromatography (HPTLC) is one of the rapid and accurate chromatographic techniques for many phytoconstituent assays and quality control of natural extracts. HPTLC fingerprinting of P1EA and P1nB scanned at wavelength 254 nm, 366 nm and after derivatisation at 620 nm respectively, showed the presence of polyvalent compounds. It might be assumed that the antioxidant and hepatoprotective activity of P1EA and P1nB may be due to the synergistic effect of these constituents.

Conclusion

A culturable endophytic fungus Aspergillus niger strain A6 was isolated from the leaves of Phyllanthus amarus. Ethyl acetate and n-butanol fractions of Aspergillus niger strain A6 at a dose of 50 mg/kg and 100 mg/ kg body weight exhibited antihepatotoxic activity as evidenced by biochemical and histological parameters against paracetamol induced hepatotoxicity. This may be due to free radical scavenging properties and may also be attributed to antioxidant components in P1EA and P1nB. Hence, we can conclude that endophytes also exhibit same therapeutic properties as that of the plant and may also secrete known or novel metabolites. Further, explorations of biomolecules from endophytic fractions are needed along with studies on mechanism of action of bioactive endophytes.