I NTERNATIONAL J OURNAL OF R ESEARCH IN P HARMACEUTICAL S CIENCES

Fungal endophytes are the microbes residing in internal tissues of the plant forming symbiotic, mutualistic, communalistic and trophobiotic relationship. Endophytes from medicinal plants are considered as essential source of secondary metabolites accompanied by interesting biological/pharmacological activities. In this study, an effort was made to isolate, characterize endophytic fungi from leaves of Andrographis paniculata and to screen the fungal fractions for in-vitro antioxidant and hepatoprotective activity. Two fungal endophytes (APLF-1 and APLF-2) from Andrographis paniculata were isolated and fermented to get chloroform (A1C, A2C), ethyl acetate (A1EA, A2EA) and n butanol (A1nB, A2nB) extracts. All the endophytic fractions of APLF-1 and APLF-2 were assayed for free radical scavenging properties against 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical and reducing power. Fractions of APLF-2 showed good scavenging activity compared to APLF-1 extracts. Further, A2EA and A2nB were evaluated for hepatoprotective activity against CCl 4 induced hepatotoxicity. A2EA (50 mg/kg &100 mg/kg) and A2nB (50 mg/kg &100 mg/kg) reversed the elevated biochemical parameters with respect to CCl 4 treated group (p<0.001). The LPO, SOD and CAT levels were also restored by A2EA and A2nB (100 mg/kg p.o). APLF-1 and APLF-2 were studied for rDNA sequencing by PCR technique. The endophytic fungi, APLF-1 and APLF-2 were identified as Diaporthe sp. A25 and Preussia sp. PPV3.6 respectively based on their morphology and molecular characterization. The presence of polyvalent secondary metabolites in A2EA and A2nB were confirmed by HPTLC analysis.


INTRODUCTION
Liver diseases are the most serious ailments leading to mortality throughout the world.The reasons for such diseases are modern food styles, excessive medications, exposure to pollutants, toxic chemicals, surplus intake of alcohol, various infections and autoimmune disorders.(Elberry AA et al., 2010).
Lipid peroxidation and other oxidative parameters damages hepatocytes.No effective drug from synthetic origin is available till today for treating liver disorders.However, medicinal plants of various traditional systems of medicine and secondary metabolites are found to be effective in regeneration of hepatocytes by preventing lipid peroxidation.But still mortality is recorded due to liver disorders worldwide, thus the availability of drugs become limited and have demand for developing novel potent drugs (Eidi A et al., 2012).
Plant endophytes are group of microorganisms such as bacteria, fungiand actinomycetes which reside in the internal tissues or organs of the plants, showing no marked signs of disease in the medicinal plants (Gunatilaka AL et al., 2006, Schulz B et al., 2002).Endophytes have different types of associations such as symbiotic, mutualistic, trophobiotic and communalistic with the host plant.Endophytes are the chemical synthesizers, useful to their host by producing novel active compounds for applications in agriculture, medicine and in biotechnological industries (Sette LD et al., 2006).Many authors reported different endophytes and their isolated secondary metabolites from various medicinal plants and marine sources which found to exert anticancer (Moore A., 2009, Ding G., 2006), antibacterial (Liu JY et al., 2004, Wang FW et al., 2008) antifungal (YH Ye et al., 2005, Pongcharoen W et al., 2006) antimalarial (Masahiko I et al., 2007, Renee K et al., 2008) and immunomodulatory activities (Zhang AH et al., 2013).
Andrographis paniculata (Burm.f.) Wall.(Acanthaceae), an annual herbaceous plant, used from centuries in Indian tradition and Chinese herbal medicine.The aerial parts of plant contain the active constituents such as diterpenoids, stigmasterols, flavanoids, andrographolides (Koteswara et al., 2004).It possesses different pharmacological activities such as antimalarial (Dua VK et al., 2004), immunostimulant (Kumar RA et al., 2004), anticancer (Kumar RA et al., 2004), cardiovascular (Khalijah A et al., 2012), hepatoprotective (Soumendra D et al., 2009) activity have all been reported.The researchers also have concluded that the plant is the remedy for treatment for infective hepatitis (Raval et al., 2016).An endophytic bacterium was reported from Andrographis paniculata leaves and was screened for antimicrobial activity and antibiotic susceptibility (Arunachalam C et al., 2010).Five fungal endophytes were isolated from various parts of the Andrographis, two isolates obtained from the roots were identified as Aspergillus niger and Aspergillus flavus, other two isolates were obtained from the leaves as Alternaria and yeast like fungal species.Fusarium sp.was isolated from the internal tissue of the stem of the plant (Gajalakshmi et al., 2012).Also, an endophytic bacterium was also isolated from Andrographis paniculata as a plant growth promoter and regulators such as IAA and GA3.(Aziz SA et al., 2013).An endophytic fungus was also isolated from Andographis paniculata, for investigation of fungal lipid production as biodiesel precursors (Elfita E et al., 2015).
Data on fungal biodiversity and screening of fungal fractions isolated from Andrographis paniculata is limited in the literature and no work was found on hepatoprotective activity of Andrographis paniculata associated fungal fraction is reported.Hence, the present investigation aims to isolate, characterize endophytic fungi and to screen the fungal fractions for, free radical scavenging and hepatoprotective activity.

Plant Material
The leaves of Andrographis paniculata were collected from Dharwad district, Karnataka, India.Authentication of the plant was done by Dr. G R Hegde, Karnatak University, Dharwad (India).A specimen is stored in the herbarium, Postgraduate department of Pharmacognosy (SETCPD/Ph.cog/herb/32/12/2015).

Isolation of Endophytes
Andrographis paniculata (Burm.f.) Wall.Nees.leaves were washed thoroughly under tap water to take off any foreign particles adhering to it and were dried.They were then sterilized with 70% ethanol for 1 min, 1% sodium hypochlorite for 30 sec and further cleaned by passing through two sets of sterile distilled water.After surface sterilization, leaves were cut into small pieces, 1 cm each, the sterile samples were placed on a plate containing potato dextrose agar (PDA) media with 250 μg/mL streptomycin to suppress fungal contamination.The parafilm wrapped petri dishes were incubated at 25 ± 2°C for 7-14 days, till the fungal mycelia started growing on the samples.Fungal colonies were picked randomly from the dilution plates, checked for purity and grouped according to colony morphology (Katoch M et al., 2014).Two pure endophytic fungi namely APLF-1 and APLF-2 (endophytic fungi of Andrographis paniculata) leaves were selected for fermentation.

Fermentation and extraction
The Purified isolates of APLF-1 and APLF-2 were inoculated and fermented separately into a 3000 mL Erlenmeyer flask containing 600 mL of potato dextrose broth (Potato infusion from 200 g pota-toes+20 g of dextrose, pH 5.1± 0.2, 24 g/L).The flask was incubated at 27°C for 21 days under static culture condition without light.After 21 days of incubation, 500 mL of chloroform was added to the flask, mixed, and left overnight.Chloroform immersed fungus culture was homogenized at 4000 rpm to separate the mycelia from broth for 30 min and filtered by whatman filter paper under vacuum.The filtrate was collected and residual aqueous phase was partitioned twice with equal volumes of chloroform in a separator funnel.Aqueous phase obtained after chloroform extraction was further partitioned three times with equal volumes of ethyl acetate.Aqueous phase obtained after ethyl acetate extracting was further partitioned two times with equal volumes of n-butanol.The chloroform, ethyl acetate and n-butanol fractions of respective endophytic fungi were dried with vacuum rotary evaporator (Superfit Rotavap, PBU-6) and weighed to constitute the crude extract.(Kumar S et al., 2013;Wicklow DT.,1998).

PHENOTYPIC IDENTIFICATION Colony Morphology
The colonies grown on the agar slant were studied by the Lacto phenol cotton blue staining.The characteristics were compared with the known organisms from the literature (Astrid Leck., 1999).

MOLECULAR CHARCTERIZATION BY PCR SE-QUENTIAL ANALYSIS
Using the genomic DNA extraction kit, genomic DNA was isolated from the given organism (Bhat Biotech Ltd. Bangalore, India) (Junichi K et al., 2015, Mathur SB et al., 2003).Amplification of the 16s rRNA gene was performed using the primers.Forward primer, 5'-TCCGTAGGTGAACCTGCGG-3', Reverse Primer, 5'-TCCTCCGCTTATTGATATGC-3'.PCR was performed with a total volume of 50 μL in a 0.2 mL thin walled PCR tube.The amplification was carried out in a Master cycler® Thermocycler (DNA-AMP Bhat Biotech) using the following program.Initial denaturation was carried out at 95 o C for 10 minutes followed by 35 cycles of denaturation at 94 o C for a one minute, annealing at 56 o C for one minute and extension at 72 o C for one minute.Final extension was carried out at 72 o C for 10 minutes.The PCR products from ITS gene PCR reactions were purified to remove unincorporated dNTPS and primers before sequencing using GE-NEASY GEL ELUTION KIT.Both strands of the rDNA region amplified by PCR were sequenced by automated DNA sequence -3037xl DNA analyzer from Applied Biosystems using BigDye® Terminator v3.1 cycle sequencing Kit (Applied Biosystems).Sequence data were aligned and dendrograms were generated using Sequence analysis software version 5.2 from applied biosystems.Appropriate software was used to align sequences acquired for plus and minus strands before to phylogenetic analysis.Sequences were compared to the non-redundant NCBI database using BLAST N. The expected value and e values were noted for the most similar sequences.Ten similar neighbors were aligned using CLUSTAL W2.The multiple-alignment file thus obtained was then used to create a Phylogram using the MEGA5 software (Saithou., 1987, Kim., 1993).

HIGH PERFORMANCE THIN LAYER CHROMA-TOGRAPHY OF A2EA AND A2NB
500mg each of A2EA and A2nB were dissolved in 1 mL of ethyl acetate and n-butanol respectively.3µL and 6µL of the above fractions was applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator.The plate was developed in Toluene and Ethyl acetate (1:1).The developed plates were visualized in short UV, long UV and then derivatised with vanillin sulphuric acid and scanned under UV 254nm, 366nm and 620nm.Rf value of the spots and densitometric scan were recorded (Harborne et al., 1998, Wagner et al., 1996).

Reaction with DPPH radical
All the fractions of APLF-1 and APLF-2 were used for DPPH free radical scavenging activity of at different concentrations.It was measured from bleaching of the purple colour of 2,2 Diphenyl -1picryl hydrazyl, where 1 mL solution of different concentration of fraction was added to 1.4 mL of DPPH and kept in dark for 30 min.The absorbance was measured at 517 nm and the percentage inhibition was calculated by using the following equation (Coruh N et al., 2007).

A1 =Absorbance of test
The results are expressed in terms of IC50 value which is the effective concentration at which the scavenging activity is 50%.

Reaction with hydroxyl radical
Hydroxyl radicals were generated by a Fenton reaction (Fe 3+-ascorbate-EDTA-H2O2 system), by using the fractions of APLF-1 and APLF-2.The scavenging capacity for the hydroxyl radicals was measured using deoxyribose method (Halliwell B et al., 1987).
The reaction mixture of 2.8 mM of 2-deoxy-2-ribose, 0.1 mM of phosphate buffer (pH 7.4), 20 μM of ferric chloride, 100 μM of EDTA, 500 μM hydrogen peroxide, 100 μM of ascorbic acid and different concentrations from (10-1000 μg/mL) of the test sample was made to a final volume of 1 mL.The mixture was incubated for 1 h at 37 °C.After the incubation, 0.8 mL of the reaction mixture was added to 1.5 mL of 2.8% TCA solution followed by addition of 1 mL of TBA solution (1% in 50 mM sodium hydroxide,) and 2mL of sodium dodecyl sulphate.The mixture was heated for 20 min at 90 °C to develop the colour.After cooling, the absorbance was measured at 532 nm against an appropriate blank solution.All experiments were performed in triplicate.The percentage of inhibition (IC50) was calculated.

Determination of reducing power
The reducing power of the fractions of APLF-1 and APLF-2 were determined by the method described by Oyaizu 1986. 1 mL of different concentrations of endophytic fractions ranging from 10-1000 μg/mL was mixed with 2.5 mL phosphate buffer (0.2 M, pH 6.6,) and 2.5 mL of 1% potassium ferricyanide.The mixture was incubated for 20 min.at 50°C.After addition of 2.5 mL 10% trichloroacetic acid, the mixture was centrifuged at 6500 rpm for 10 min.2.5 mL of the supernatant was mixed with 2.5 mL of distilled water to which 0.5 mL of 0.1% iron (III) chloride and the absorbance was measured at 700 nm using phosphate buffer as blank.These were done in triplicate and the mean values were given in the results.The percentage inhibition was calculated.

Hepatoprotective activity
Based on the results of free radical scavenging activity, A2EA and A2nB were further selected for in vivo hepatoprotective activity in CCl4 induced hepatotoxicity.

Animals
Albino Wister rats (150-200 g) were used and were collected from Venkateshwara Enterprises Bangalore, Karnataka.They were maintained in the animal house of SET's College of Pharmacy, Dharwad for experimental purpose.The animals were maintained under controlled temperature and humidity.All the animals were acclimatized for 7 days before the study.They were randomized into experimental and control groups and housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding.They had free access to standard pellets as basal diet and water ad libitum.

Acute toxicity studies
Acute oral toxicity of A2EA and A2nB 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 animal was determined as per OECD guideline 420.

Experimental design
Animals were divided into following groups of six animals in each group (n=6) Group I: Normal Control, rats treated with 0.9% NaCl (2 mL/kg day) Group II: Rats treated with CCl4 (2 mL/kg i.p. in olive oil) Group III: Rats treated with Silymarin (100mg/kg p.o) + CCl4 Group IV: Rats treated with A2EA (50mg/kg p.o) + CCl4 Group V: Rats treated with A2EA (100mg/kg p.o) + CCl4 Group VI: Rats treated with A2nB (50 mg/kg p.o) + CCl4 Group VII: Rats treated with A2nB (100 mg/kg p.o) + CCl4 During the treatment, rats were maintained under normal diet and water ad libitum.Animals of group II to VII received CCl4 (2 mL/kg i.p. in olive oil) subcutaneously, on 2 nd and 3 rd day after 30 min of administration of the treatment of silymarin and endophytic fractions.On the 6 th day, animals were sacrificed, blood was collected by retro-orbital bleeding under mild ether anesthesia, centrifuged (3000 rpm for 15 min) and serum was subjected to biochemical estimations.Liver was dissected out and placed in 10% formalin solution for histopathological study.Liver homogenate was prepared to determine the levels of endogenous enzymes (Suja SR et al., 2004, Qureshi et al., 2007).

Biochemical parameters
Serum was separated and analyzed spectrophotometrically for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphate (ALP), total and direct bilirubin, total triglyceride (TG), and total protein using diagnostic kits of Swemed diagnostics, Bengaluru, India.

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 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 non-enzymatic antioxidants (Lipid peroxidation) and enzymatic antioxidants (Catalase and Superoxide dismutase).

Lipid peroxidation
The concentration of thiobarbituric acid reactive substances (TBARS) in the liver homogenate was estimated using standard procedure.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 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 (μ moles) per gram of tissue (Banerjee et al., 2008).

Superoxide dismutase Assay (SOD)
In this assay, 0.5 mL of liver homogenate was taken, and to it, 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 started by addition of 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 run simultaneously without liver homogenate.Units of SOD activity 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 per mg of proteins (Flohe and Otting., 1984).

Catalase assay (CAT)
Here, 1.95 mL of 10 mM H2O2 in 60 mM phosphate buffer (pH=7.0), was added to 0.05 mL of the liver homogenate.The 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/A2) s-1 A unit of catalase is defined as the quantity which decomposes 1.0 µmole of H2O2 per min at pH=7.0 at 25°C, while H2O2 concentration falls from 10.3 to 9.2mM (Claiborne et al., 1985).

Estimation of Protein
Folin phenol reagent method was used to measure total tissue protein content as reported earlier (Lowry OH., 1951).

Histopathological studies
Liver of individual animal were excised, fixed in 10% buffered neutral formalin and fixed in bovine solution.They were further processed for paraffin embedding following standard microtechnique (Galigher AE et al., 1971).Sections of liver stained with alum-haematoxylin and eosin, were observed photomicroscopically for histopathological changes.

Statistical evaluation
The data were expressed as Mean±S.E.M. Statistical comparisons were performed in one-way ANOVA followed by Tukey's t-test using Graph Pad Prism version 5.0, USA.

RESULTS
Two prominent endophytic fungi, namely APLF-1 and APLF-2 were isolated from the leaves of Andrographis paniculata (Figure 1 and Figure 2).The percentage yield of fractions of APLF-1 and APLF-2 are illustrated in Table 1.

Preliminary Phytochemical Screening
Preliminary phytochemical investigations revealed the presence of flavonoids, alkaloids, triterpenoid and tannins for APLF-1 and APLF-2 fractions as important secondary metabolites (Table 2).

DPPH assay
The IC50 values for A1C, A1EA and A1nB were found to be 185.4µg/mL, 168.64 µg/mL and 391.2 µg /mL respectively where as IC50 value for ascorbic acid was found to be 32.53 μg/mL.The IC50 values for A2C, A2EA and A2nB extract were found to be 51.68 µg /mL, 43.70 µg /mL and 22.78 µg /mL respectively where as IC50 value for ascorbic acid was found to be 32.53 μg/mL (Figure 3).APLF-2 fractions showed significant scavenging activity as compared to APLF-1 extracts.

Hydroxyl Scavenging Assay
The assay showed the abilities of the extract and standard mannitol to inhibit hydroxyl radical-mediated deoxyribose degradation in a Fe 3+ -EDTA-Mannitol and H2O2 reaction mixture.The IC50 values for A1C, A1EA and A1nB were found to be 591.06µg/mL,453.09µg/mL and 458µg/mL respectively.The IC50 values for A2C, A2EA and A2nB were found to be 347.61µg /mL, 144.75 µg /mL and 220.24 µg /mL.APLF-2 fractions were more significant than APLF-1 fractions (Figure 4).

Reducing power Assay
Reducing power of fractions of APLF-1 and APLF-2 (50-450µg/mL) increased with increase in concentration.The A2EA and A2nB showed more effective reductive ability when compared to other extracts.In this assay, Fe (III) reduction is often used as a significant indicator of electron donating activity which is an important mechanism of phenolic antioxidant action by breaking the radical chain by donating a hydrogen atom.The A2EA and A2nB showed highest activity in a dose dependant manner as compared to the other fractions (Figure 5).

Acute toxicity (LD50) studies
Acute toxicity studies were carried out according to OECD guidelines (Up and Down method).No mortility was observed upto 2000 mg/kg body weights were for A2EA and A2nB.Hence, doses of 50 mg/kg and 100 mg/kg body weight were selected to assess the hepatoprotective activity of A2EA and A2nB fractions respectively.

Effect of APLF-2 on serum biochemical parameters in CCl4 induced hepatotoxicity in rats
Administration of CCl4 (2 mL/kg i.p.) significantly increased the levels of ALT, AST, ALP total and direct bilirubin, TG and total protein as compared to normal control.A2EA (50 mg/kg &100 mg/kg) and A2nB (50 mg/kg &100 mg/kg) reversed the elevated biochemical parameters as compared to CCl4 treated group (p<0.001)(Table3).

Effect of A2EA and A2nB on endogenous antioxidant enzymes
Rats treated with CCl4 group showed decreased SOD (p<0.0001) and CAT (p<0.0001)levels where as there There was a marked increase in LPO (p<0.0001) level in terms of TBARS was observed compared to control.A2EA (100mg/kg) and A2nB (50mg/kg &100 mg/kg) administration showed significantly reversed LPO levels.A2EA (50 &100 mg/kg) and A2nB (100mg/kg) increased the SOD levels showing significance values.A2EA (100mg/kg) and A2nB (100mg/kg) increased the levels of CAT significantly (Table 4).

Histopathology of liver
Histopathological characters of normal liver showed normal hepatic globular structure, inflammation of central vein, portal tract and normal kupffer cells were not seen (Figure 6A).Extensive fatty degeneration, central vein and sinusoidal congestion, spotty necrosis, focal hemorrhage, inflammation and ballooning of hepatocytes were found in CCl4 treated group (Figure 6B).Silymarin (100 mg/kg) treated group showed the hepatic globular architecture as normal.The central vein and sinusoidal congestion was observed.However, centrilobular degeneration, mild inflammation and

HPTLC fingerprint
HPTLC fingerprinting of A2EA and A2nB scanned at wavelength 254 nm showed the presence of nine and eleven polyvalent secondary metabolites respectively.The range of Rf values starts from 0.01 to 0.99 in which highest concentration of the metabolite was found to be 30.22% and 29.05 % with Rf values of 0.45 and 0.72 respectively.However, at 366 nm A2EA and A2nB reveled nine and thirteen secondary metabolites at range of Rf values starting from 0.02 to 0.99 respectively.The highest concentration was found to be 13.12 % with Rf value of 0.44 for A2EA and 10.69 % with Rf value of 0.24 for A2nB respectively After derivatisation at 620nm, A2EA and A2nB showed the presence of nine polyvalent secondary metabolites respectively.The range of Rf values starts from 0.02 to 0.94 in which highest concentration of the metabolite was found to be 31.54% and 52.03% with Rf values of 0.02 and 0.01 respectively (Figure 11 & 12).

DISCUSSION
Endophytic microbes were reported from various plants existing in different ecosystems.All classes of vascular plants and grasses are found to endow with endophytic organisms.They also possess chemical structures and are evolved in biological activities with roles as defensive compounds against competitors/parasites/predators, growth and reproduction Endophytes have the ability to produce a wide range of secondary metabolites with unique structure, synthesized through different metabolic pathways e.g.isoprenoid, polyketide, amino acid derivation (Tan RX et al., 2001).Thus, researchers are provided with numerous leads for compounds with possible development as new drugs for their accessibility and large scale production by the pharmaceutical industry.The diversity of Endophytic fungi is significantly higher in the leaf than stem (Strobel G et al., 2003).This can be assigned to the similarity of endophytes in specific tissues and the availability of nutrients to nourish the plant (Soussi A et al., 2015).
Diaporthe (Phomopsis) species are isolated from various hosts and are assigned as plant pathogens, non-pathogenic endophytes or saprobes.Diaporthe is one of the most frequently confronted genera of endophytic fungi in various plant hosts (Murali et al., 2006, Botella & Diez., 2011).Diaporthe sp was also isolated from a well-known medicinal plant in China, Cephalotaxus hainanensis Li which reported the antimicrobial activity (Yang HR et al., 2015).Many reports reveled the presenec of secondary metabolites such as quinoline alkaloids such as quinine, quinidine, cinchonidine, and cinchonine from Diaporthe sp. which was isolated from Cinchona ledgeriana.(Maehara S et al., 2012).
Preussia is a genus of fungi belonging to family Sporormiaceae.(Lumbsch TH., 2007).This extensive genus has the species that grows on dung or in the soil (Kirk PM et al, 2008).Nine different plants were used to isolate a total of 18 Preussia species from Australian dry rainforests.13 out of 18 Preussia sp revealed antimicrobial activity showing the importance of potentially significant bioactive compounds (Mapperson RR et al., 2014).
The important secondary metabolites identified by preliminary phytochemical investigation of APLF-1 and APLF-2 fractions were flavonoids, alkaloids, triterpenoid and tannins.The fractions of APLF-1 and APLF-2 were studied for in vitro antioxidant activity by DPPH radical, hydroxyl free radical and  by reducing power.Among all the fractions of APLF 1 and APLF-2, fractions of APLF-2 exhibited good scavenging activity with minimum IC50 values and were further set for hepatoprotective studies.The acute toxicity was conducted for as per OECD guidelines which showed that APLF-2 fractions are as non-toxic.Hence, 50mg/kg and 100 mg/kg doses were selected for the in vivo studies.
Hepatoprotective activity was carried out for A2EA (50mg/kg and 100mg/kg) and A2nB (50mg/kg and 100mg/kg) in CCl4 induced hepatotoxicity.The alteration in serum biochemical parameters like ALT, AST and ALP is caused due to CCl4 as it produce free radicals such as CCl3 -and CCl3COO -, affecting the cellular permeability of hepatocytes leading to elevated levels.Cytochrome P-450 is majorly involved enzyme in the drug metabolism.These radicals covalently get bound to molecules causing lipid degradation of the adipose tissue.Hence, CCl4 treated rats showed significant hepatic damage, by elevation in the serum AST, ALT, ALP, total and direct bilirubin, TG and total protein levels.High levels of AST and ALT are released from the liver into the blood during necrosis of hepatocytes (Nelson et al., 2005, Emeka, et al., 2011).The AST and ALT levels were slightly decreased to normal after the administration of the fractions of APLF-2 as compared to controls.
In this study, administration of A2EA (50 mg/kg &100 mg/kg) and A2nB (50mg/kg &100 mg/kg) and silymarin showed a decline in AST, ALT and ALP levels.The increased hepatocytic functioning leads to elevated biliary pressure of ALP.Regeneration of hepatocytes can be done due to healing of hepatic parenchyma in serum levels of transaminases.
Antioxidants act by blocking the free radical action as associated in the pathological process of various disorders and aging process (Aruoma OI et al., 2003).Free radicals also are necessary for the body as they regulate the numerous biological processe.
Framing cell-signaling is one of the important mechanism that played by free radicals.At the same time they are also harmful to the body.The damage caused by free radicals can be repaired by various enzymes such as SOD, CAT, glutathione, glutathione reductase and peroxidase.Thus, antioxidants play a major role in such defense mechanisms (Bahar E et al., 2013).
The antioxidant effect on DPPH is due to the ability for hydrogen donation.Reaction with DPPH radicals of A1C, A1EA and A1nB showed no significant scavenging activity where as the activity was increased with increasing concentration of A2C, A2EA and A2nB.The reducing power of fractions of APLF-1, APLF-2 and ascorbic acid (50-450µg/mL), increased with increase in concentration.The A2EA showed more effective reduction as compared to other fractions.The reducing power may be due to mono and dihydroxyl substitutions in the aromatic ring, possessing potent hydrogen donating ability.The fractions of APLF-1 and APLF-2 exhibited hydroxyl radical scavenging activity in a dose dependent manner.Molecules inhibiting deoxyribose degradation and chelating the iron ions, prevents complexation with the deoxyribose and imparting them inactive in a Fenton reaction.A2C, A2EA and A2nB showed chelating effect on ferrous ions, as they minimize the concentration of metal in the Fenton reaction compared to EDTA.
Lipid peroxidation elevation is one of major results of CCl4 induced hepatotoxicity (Brattin & Glende., 1985).In CCl4 treated group, the TBARS concentration was elevated, which was dose dependent, inhibited by A2EA and A2nB.The data imparted that A2EA and A2nB may exert antioxidant effects by inhibiting the lipid peroxidation.ROS production was increased on administration of CCl4 as a significant decrease in the endogenous antioxidant enzymes activity such as SOD and CAT.A2EA and A2nB (50 and 100 mg/kg) administration exhibited significant elevation in the SOD and CAT enzymatic activity.Also, the protein expression levels were evaluated by Western blotting analysis.CCl4 treatment resulted in significant down-regulations of the protein expression levels of SOD and CAT compared with the normal control.A2EA and A2nB (50 and 100 mg/kg) administration significantly increased protein levels of the two antioxidant enzymes in the liver.Our results suggested that A2EA and A2nB (50 and 100 mg/kg) may diminish oxidative stress by augmenting the activity and protein expression of the antioxidant enzymes in vivo.Histopathology of normal liver revealed normal hepatic globular architecture, where as in CCl4 treated group hepatocytes showed extensive fatty degeneration, central vein and sinusoidal congestion, spotty necrosis, focal hemorrhage, inflammation and ballooning of hepatocytes.A2EA (50 mg/kg and 100 mg/kg) showed normal architecture of hepatocytes round the central vein and sinusoid.Cells around necrosis and fatty vacuoles were regenerated, no degeneration and ballooning of hepatocytes was seen.A2nB (50 mg/kg and 100 mg/kg) also showed reduced inflammation and protection against hepatocytes.These fractions returned the injured liver to quite normal by showing reduced degeneration and mild hepatocyte ballooning.These observations showed that A2EA and A2nB (50 and 100 mg/kg) decreased the toxic effects of CCl4 on hepatic architecture.HPTLC fingerprints profile is an important and powerful procedure for identification of presence of phytoconstituents.It is a liner, precise and accurate method for identification, authentication and characterization of the medicinal plant extracts.Newer drugs can be formulated by isolating and identifiying the bioactive compounds used for treating various diseases by HPTLC (Elangovan NM et al., 2015).The development of HPTLC fingerprint for A2EA and A2nB shows the direction for the quality control of phytoconstituents from extracts.
Further, investigation is required to characterize the secondary metabolites and quantitative estimation with marker compounds.

CONCLUSION
The ethyl acetate and n butanol endophytic fungal fractions of APLF-2 from leaves of Andrographis paniculata exhibited hepatoprotective activity in CCl4 induced model.The results showed that the antioxidant effects of A2EA and A2nB may be responsible for their hepatoprotective activity.HPTLC fingerprinting analysis exhibited the chemical diversity of A2EA and A2nB of APLF-2.Further investigations are empowered to isolate and identify marker compound(s) responsible for hepatoprotective activity with reference to endophytes.