Antimicrobial Activity of Fractions of Ethyl Acetate Extract of Cladosporium Oxysporum, an Endophytic Fungus Derived From Alyxia Reinwardtii

The endophytic fungi Cladosporium oxysporum was isolated from medicinal plants Alyxia reinwardtii. The antimicrobial activity of fractions of ethyl acetate extract and also antioxidant and antimicrobial activities of fraction 7 of ethyl acetate extract of (AR-7) were determined in this study. Antimicrobial activity was determined by TLC bioautography and disc diffusion methods. Antioxidant activity was determined by TLC-DPPH autography assay. The fractions of ethyl acetate extract of C. oxysporum at dose of 100 mg/ml were active as antimicrobial against S. aureus, E. coli and C. albicans in disc diffusion method test. Fraction AR-7 was active as antioxidant in TLC-DPPH autography assay. Fraction AR-7 was active as antimicrobial against S. aureus, but was not active against another tested microoorganisms in TLC-bioautography


INTRODUCTION
Endophytic fungi are those grow intra or intercelullarly in the tissues of higher plants without causing overt symptoms of disease. 1 Endophytic fungi have been known to produce various bioactive metabolites such as agrochemicals, antibiotics, immunosuppressants, antiparasitics, antioxidants, anticancer, antiviral, insecticidal and antidiabetic activity. 2,3,4 Alyxia reinwardtii (Apocynaceae) is widely used in jamu (Indonesian traditional herbal medicine. 5,6 It has been reported producing antimicrobial and antioxidant metabolites such as 5hydroxycoumarin, 8-hydroxycoumarin, pinoresinol, 9-.-hydroxypinoresinol, alyxialactone, pulosarioside and salisifoliol. [7][8][9][10] Hence, A. reinwardtiimeets the rationale for selection of host plant for endophytes isolation. 11 Previously, lecythomycin, a new antifungal metabolite, and other metabolites have been isolated from endophytic Lecythopora sp. residing in A. reinwardtii. 12,13 Endophytic fungi were also known for producing antioxidant metabolites. Antioxidant activity screening was performed as an early step to obtain antioxidant metabolites from endophytic fungi. [14][15][16][17] To the best of our knowledge, there is no report on antioxidant activity of metabolites produced by endophytic Cladosporium sp. to date. In our continuing studies on endophytic fungi, we now describe the bioactivity of another fungal species that we have isolated from the same host plant which was identified as Cladosporium oxysporum.The present study is aimed to determine the antimicrobial activity of the crude ethyl acetate extracts and the antioxidant and antimicrobial activities of fraction 7 of ethyl acetate extracts (fraction AR-7) of the C.oxysporum isolated from A. reinwardtii. AR-7 was the most abundant fraction and possessed relatively high antimicrobial activity among other fractions, so it was chosen to be studied further as early step of bioactive-guided isolation in order to obtain potent and save antioxidant and antimicrobial metabolites from the fungus.

General
This is a laboratory experimental study, conducted at Microbiology Laboratory of Assessment Service Unit, Faculty of Pharmacy, Airlangga University on July ± August 2014.

Isolation and Identification of endophytic fungi
Isolation of endophytic fungi was conducted as previously described. 12 The isolated fungus was identified by Dr. Arnulf Diesel (Heinrich -Heine -Universität Düsseldorf, Germany) as Cladosporium oxysporum based on its ITS-DNA sequences.

Culture of C. oxysporum
A small part of C. oxysporum was transferred under sterile conditions to the MEB medium (250 mL/flask), with pH of 5,6. The fungus was grown under static conditions at room temperature (approx. 30±3 o C) for four weeks.

Preparation of crude extract and fraction
The culture broth and mycelia were extracted with ethyl acetate. The organic layers were collected, and concentrated in vacuo to obtain a dried extract (5.49 g). The dried extract (3.0 g) was subjected to column chromatography on silica gel 60, and eluated with gradient mixtures of n-hexane, ethyl acetate and methanol. The eluates were collected and grouped based on their Thin Layer Chromatography (TLC) profile to yield 16 fractions, AR-1 to AR-16.

Determination of antioxidant activity
Thin Layer Chromatography2,2diphenyl-1-picrylhydrazyl (TLC-DPPH) autography was conducted as previously described. 18 In brief, AR-7 was diluted in methanol and applied on silica gel F 254 60 plates. Vitamin C (1000 ppm, 2 µL) was used as positive control. The plates were developed in presaturated solvent chamber with chloroform:methanol (9:1) as mobile phase and then sprayed with 0.04% DPPH solution in methanol.

Determination of antimicrobial activity
Disc diffusion method to determine antimicrobial activity of the fractions of ethyl acetate extract was conducted as previously described. 19 Microorganisms used in this assay were obtained from the American typeculture collection (ATCC). They were Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Candida albicansATCC 10231. All microorganisms were stocked in appropriate conditions and regenerated before used. The tested microorganisms were cultured for 24 hours before the tests. Microbial suspensions in normal saline with 25%T were prepared. Ten µL of microbial suspension was added into 15 mL sterile agar media (fungi were grown on Saboroud dextrose agar and bacteria were grown on nutrient agar) and then homogenized and poured into steril petri dish. Sterile filter paper discs (diameter 6 mm) impregnated with 20µl of fractions at concentrations of 100000 ppm were applied over each of the culture plates previously seeded with the 10 6 cfu/ml cultures of bacteria and fungi respectively. Twenty µl of ketoconazole (2000 ppm) was used as positive control. Bacterial cultures and those of Candida albicans were then incubated at at 37 o C for 24 h. After incubation the diameter of the inhibition zone was measured in millimeter. The experiment was carried out in triplicates.
TLC-bioautography to evaluate antimicrobial activity of fraction AR-7 was conducted as previously described. 20 Sixteen microbial strains were obtained from the American typeculture collection Majority of the tested microorganisms were Gram negative ones, since the result of the previously performed disc diffusion method showed that Gram negative bacteria were more sensitive to AR-7 than Gram positive ones. AR-7 was diluted in methanol. Tetracyclin HCl (2 µg) were used as positive control. AR-7 and positive control were applied on silica gel F 254 60 TLC plate, and then developed in presaturated solvent chamber with chloroform-methanol (9:1). The plates were overlayed on medium containing tested microorganism. Petri dishes were kept on temperature 0-8 o C for 2 hours and then the TLC plates were removed aseptically. Petri dishes were then incubated at 37 o C for 24 hours. After incubation the diameter of the inhibition zone was measured in millimeter. The experiment was carried out in triplicates.

RESULTS AND DISCUSSIONS
One of the endophytic fungi isolated from host medicinal plant A. reinwardtii was identified as C. oxysporum based on its ITS-DNA gene sequences.
The occurence of isolation of C. oxysporum as endophyte is limited to date. The isolation of this species was reported previously from host Pinus densiflora and P. rigida growing in Daejeon, Korea and Euphorbia bupleuroides subsp. Luteola. 21,22 The previous study showed that ethyl acetate extract of C. oxysporum possessed the highest antimicrobial activity against S. aureus, S. thypi, E. coli, P. aeruginosa, B.subtilis and C. albicans, compared to methanolic and dichloromethane extracts. 23 Hence, we separated ethyl acetate extract of C. oxysporum.. The fractionation of the crude ethyl acetate of C. oxysporum yielded 16 fractions. The antimicrobial activity of the fractions against S. aureus, E. coli and C. albicans was determined by disc diffusion method. The diameter of inhibition zone of the fractions at dose of 2 mg/ disc is shown in table 1. Based on the amount and antimicrobial activity of the obtained fractions, fraction AR-7 was studied further for antioxidant and antimicrobial activities.
TLC-DPPH autography is a test based on scavenging the stable free radical DPPH. It is one of the techniques commonly applied for screening plant extracts for the presence of antiradical compounds. It is proved as a powerful tool for screening antioxidant compound in bioactive-guided isolation. 24 Fraction AR-7 was active as antioxidant in TLC-DPPH autography assay, shown by the bleached spots developed after spraying TLC plate with DPPH solution. Chromatogram of TLC plate after derivatization was shown in figure 1. We have not determined the metabolites responsible for antioxidant activity of AR-7 yet. In general, antioxidant phytochemicals are phenolic compounds such as flavonoids, catechines and carotenoids. 25 Endophytic fungi had been reported as the source of antioxidant metabolites. For example, graphislactone A is a well known antioxidant metabolite produced by Cephalosporium sp. IFB-E001 residing in Trachelospermum jasminoides (LINDL) LEM. 17  The antimicrobial activity of fraction AR-7 was determined by TLCbioautography. The result showed that AR-7 was active as antimicrobial against S. aureus FNCC 0047 and was not active against the rest of the tested microorganisms.
The diameter of inhibition zone of AR-7 was 3.45 mm, much lower than that of tetracycline HCl (18.25 mm) (figure 2). The antimicrobial activity of AR-7 determined by TLC bioautography was different from that of disc diffusion method previously performed. This difference activity was caused by the different amount of sample applied on each test. Two mg of AR-7 was used in disc diffusion method, while in TLC-bioautography only used 0.2 mg of AR-7. TLC-bioautography was excellent to determine antimicrobial activity of polar compounds with strong efficacy. Hence, the antimicrobial metabolites in fraction AR-7 might be non polar substances with a relative moderate activity.
The main advantage of TLCbioautography was its capability to analyze antimicrobial activity of each components in a mixture. 26 The result showed that there was one clear spot, indicating there was one compound that active as antibacterial agent. This spot needed to be separated and identified further.

CONCLUSIONS
The result of our study suggested that some fractions of ethyl acetate extract of C. oxysporum possessing antimicrobial activity, while fraction AR-7 at dose of 100 mg/ml having antimicrobial activity in disc diffusion method and moderate antioxidant activity.

AKNOWLEGMENT
We thank the Directorate General of Higher Education, Ministry of National Education, Indonesia for financial support, and Dr. Arnulf Diesel (Heinrich ±Heine-Universität Düsseldorf, Germany) for identifying the fungus.