SECONDARY METABOLITE FROM ENDOPHYTIC FUNGI Aspergillus sp . THE LEAVE OF KUNYIT PUTIH ( Curcuma zedoaria ( BERG ) ROSCOE )

The compound from endophytic fungi of Aspergillus sp. from leaves of kunyit putih (Curcuma zedoaria (Berg.) Roscoe) has been isolated. Isolation begins with cultivation of Aspergillus sp. in 18 L PDB’s media (Potato Dextrose Broth) for 28 days. The liquid cultivation medium was extracted by partitioning method using ethylacetate and then evaporated. The extract was separated and purified by chromatography techniques. Elucidation stucture of the isolated compound was analysis by spectroscopic method NMR 1D and 2D. Antibacterial activity of isolated compound was tested using the disc diffusion method at concentrations 2500, 1000, 500, and 125 ppm. The isolated compounds obtained in the form of a yellow oil (24.30 mg). The C NMR spectrum indicated 24 signals of carbon and base on analysis spectrum DEPT 135 showed 5 signal methynes carbon, 1 signals methylene, 9 signals of methyl and 9 signals quarternary carbon. These signals from H and C-NMR suggested that this compound contained aromatic group and four carbonyl. The isolated compound show antibacterial activity at concentration 2500 ppm which inhibition zone for E. coli, S. dysenteriae, S. aureus, B. subtilis were 10.3 ; 8.3; 8.4; and 7.8 mm, respectively. Based on the analysis result of NMR 1D and 2D, the compound was methyl 6-(5'-(2"-acetoxy-2”-methylpropanoyl)-3"methyl-2'-(3"'-methylbutanoyl)phenyl)-3-methylbutanoate and has weak antibacterial activity. Keyword: Aspergillus sp., Curcuma zedoaria, endophytic fungi


INTRODUCTION
Endophytic fungi are defined as fungi which spend the whole or part of their lifecycle colonizing inter and intracellularly inside the healthy tissue of the host plants, typically causing no apparent symptoms of disease (Zhang, Song, & Tan, 2006;Rodriguez., White, Arnol, & Reman, 2009).Plant endophytic fungi have been recognized as an important and novel resource of natural bioactive products with potential application in agriculture, medicine and food industry (Gunatilaka, 2006;Verma, Kharmar, & Strobel, 2009).Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are rarely founded after the isolation and culturing of individual endophytes which followed by purification and characterization of some of their natural products (Strobel, Daisy, Castillo, & Harper, 2004).
Many endophytic bacterial live in association with their host and may play an important biological roles.Sulistiyani, Lisdiyanti, and Lestari, (2014) have been investigate the endophytic bacterial diversity associated with Curcuma zeodaria and total of 207 bacterial colonies were isolated from rhizomes, stems, and leaves and 73 endophytic bacteria were selected based on morphological characteristics.
The apparatus in the research were counter colony, autoclave, incubator, water bath, microscope, magnetic hotplate, UV lamp, column chromatography and generally apparatus in organic and microbiology laboratory, melting point was determined using Fisher John Apparatus.

Isolation of endophytic fungus
The study begins with the isolation of endophytic fungi from leaves Curcuma zedoaria plant.The procedure refers to Muharni el al. (2014) with slight modifications.The leaves sample were washed and sterilized in 70% ethanol for 5 min and 0.5% NaOCl for 5 min and then washed with sterile distilled water.The segment were placed on petri-plates containing potato dextrose agar medium (PDA).The plates were incubated at 25±2 o C. The plant segment observed every day to see the growth of endophytic fungus.Fungal colony that shows a different characteristic further purified by transferring it in the PDA medium other then some subculture to obtain pure fungal cultures (Aspergillus sp.) (Barik, Tayung, Jagadev, & Duta, 2010;Kour et al., 2008;Eyberger, Dondapati, & Porter, 2006)

Identification of the endophyte
The endophytic fungal strain was identified by the morphological method.The morphological examination was performed by scrutinizing the fungal culture, the mechanism of spore production, and the characteristics of the spores.All experiments and observations were repeated at twice (Guo et al., 2008) .

Cultivation of pure fungal strain
The procedure for the cultivation refers to Muharni et al., 2014a with slight modifications.Cultivation of fungus that have been pure (Aspergillus sp), (a small park) were transferred into the medium under sterile conditions to the PDB medium.To isolate the secondary metabolites, the fungal strains were static cultivated in 30 flasks each containing 600 mL of PDB medium and incubated for 28 days at room temperature, so the metabolites sekunderrnya will enter into PDB medium.(Xu et al., 2008).Furthermore filtered to separate filtrate and biomass.The Filtrate containing secondary metabolites, then partitioned with ethylacetate solvent.Then the ethyl acetate phase was further concentrated by rotary vacuum evaporator at 40 o C and obtained ethyl acetate fraction of liquid cultures (5,0 g).

Concentrated ethylacetate from
Aspergillus sp (5.0 g) were isolated using column chromatography with silica gel 60 as stationary phase.The solvent system used for chromatography was n-hexane with increasing portion of EtOAc (gradient elution system).The ratio of the solvent between n-hexane and EtOAc were 100 : 0, 90 : 10, 10 : 90).Fraction were collected every 10 mL and each fraction was tested by TLC.The spot were detected by UV light (254 and 366 nm).Fraction having spots with the same Rf value were combined and treated as a group.Fraction 2 nd (0.9 g) was rechromatography using the same method to give pure compound form yellow oil (24.3 mg) (Hundley, 2005).The molecular structure of pure compound were determined on the basis of spectroscopic analysis ( 1 H-NMR, 13 C-NMR, DEPT, HMQC, HMBC, and COSY).

Antibacterial activity
The antibacterial activity assay used: Escherichia coli, Sigella dysentryae, Staphylococcus aureus and Bacillus subtilis.The antibacterial activity were determined by disc diffusion method were described previously for the preliminary of antibacterial activity (Lai, Chyau & Mau, 2004).A sterile paper disc was impregnated with test material and the disc was placed on the nutrient agar medium.Plates were then incubated at 37 o C for 72 h under anaerobic conditions.All disc diffusion tests were performed in three separated experiments and the antibacterial activity was expressed as the mean of inhibition diameters (mm).The test material was preparated in various concentration and as standard used ampicillin 10 ppm.

Isolation and Identification The Secondary Metabolits
The fungus strain was identified as Aspergillus Fungus Aspergillus sp after cultivation in media 18L PDB then extracted with ethyl acetate and obtained 5 g of concentrated ethyl acetate extract.5.0 g of ethylacetate extract after being separated by column chromatography techniques to yield pure compound in the form of yellow oil (24.3 mg) (Figure 1).The 1 H NMR data (Table 1) disclosed the presence of two protons at δH 5.93 and 5.56 ppm (1H, s) were characteristic for vinyl proton.The proton signal at δH 3.80 ppm (3H, s) was assigned to a methoxy group.Furthermore, the presence proton signals at δH 2.46 each (3H, m) for one proton methylene and one proton methine, signals at 1.67 (1H, m), and 1.70 (1H, m) for two methine groups.The 1 H NMR data also indicated the presence of eight methylprotons at δH 1.94 ppm, δH 1.65 δH 1.63 ppm and 1.61 ppm, δH 1.18 (3H, d, J = 7.75), 0,93 (3H, d, J = 7.75), 1.17 ppm (3H, d, J = 7.15), and 0.85 ppm (3H, d, J = 7.15.).
The 13 C NMR spectrum (Figure 2) indicated 24 signal carbon consist that 10 signals as C sp 2 and 14 signals as C sp 3 .Base on analysis spectrum DEPT 135 showed 5 signal methines carbon at δC 99.1; 98.5; 38.9; 27.4 ppm and δC 26.7 ppm , 1 signals methylene carbon at δC 40.4 ppm, 9 signals methyl carbon at δC 57.1; 26.7; 25.3; 25.2; 17.9; 16.5; 11.7; 11.5 and δC 8.3 ppm and 9 signal quarternary carbon at δC 200.9; 196.7; 175.6; 174.5; 167.3; 167.1; 165.1, 98.7 and δC 77.3 ppm.Signals carbon at δC 196.7 ppm and 200.9 ppm indicated these compound have two carbonyl groups and 175.6 and 174.5 caracteristic for ester carbonyl.Signal methyl carbon at δC 57.1 ppm characteristic for signal methoxy carbon.These signals from 1 H and 13 C-NMR suggested that this compound contained aromatic group and four carbonyl group.The presences of the funcional groups above were suggested by the long range coupling HMBC and correlation of the chemical H and C shift for all protonnated carbons was determined based on the HMQC spectrum as summarized in Table 1.
NMR 2D analysis for HMQC spectrum showed the proton signal at δH 5.93 ppm and δH 5.56 ppm attached to carbon signal at δC 99.1 ppm and 98,5 respectively and proton at δH 3.80 attached to carbon at δC 57.1 ppm.HMBC spectrum showed proton at δH 5.93 was correlated to carbon at δc 98.5 and δc 167.1 ppm, while proton at δH 5.56 showed correlation to carbon at δC 174.6 and 196.7 ppm.This data indicated that two proton vinilic was not place at the carbon besides it.Further HMBC spectrum showed correlation proton of δH 3.80 ppm (3H, s) to carbon at δC 174.6 ppm indicated as proton methoxy from ester group.
HMQC spectrum showed that proton at δH 1.61 attached to carbon at δc 25.3 and HMBC spectrum showed that proton at δH 1.61 was correlated to carbon δc 77.3 dan δc 196.7 ppm.While proton at δH 1.63 ppm at HMQC spectrum showed attached to carbon at δC 26.7 ppm and HMBC spectrum showed correlation to carbon at 77.3 ppm.This data indicated that two proton methyl were bounded at the same carbon that δC 77.3 ppm.The proton at δH 1.94 at HMQC spectrum showed attached to carbon at δC 8.3 ppm and HMBC spectrum showed correlated to carbon at δC 98.7.Proton at δH 2.46 attached to carbon at 39.8 and 40.4 ppm (Figure 3).This data supported that proton at δH 2.46 ppm were one signal methine proton and one as signal methylene proton.Furthermore at HMBC spectrum showed correlation proton at δH 1.94 ppm to carbon δc 165.2; δc 167.1; and δc 98.7 ppm indicated the proton attached at aromatic ring.HMQC spectrum also showed that proton at δH 1.18 and 0.93 ppm attached to carbon at δc 17.9 ppm, and δc 11.5 ppm, while HMBC spectrum (Figure 4) showed both of this proton was correlation to carbon δc 27.4 dan 39.8.Proton at δH 1.17 and 0.85 ppm at HMQC spectrum showed attached to carbon at δC 16.5 and 11.7 ppm at HMBC spectrum showed both of this proton correlation to carbon δc 26.7 ppm 40.4 ppm.
COSY spectrum (Figure 4) showed proton at δH 1.17 ppm and 0.85 ppm each was correlated to proton at δH 1.67 ppm and 2.46, while that proton at δH 0.93 ppm and 1.18 correlated to proton at δH 1.70 ppm.The presences of the functional groups above were suggested by the long range coupling HMBC experiment.
Exploration of secondary metabolites research needs to be done in order to get the profile of organic compounds produced by endophytic fungus of Curcuma zedoaria.
Based on the literature study, the biosynthetic pathways of secondary metabolites produced from endophytic fungus has not been found clearly.The substances isolated from endophytic have different biosynthetic pathways: isoprenoid, polyketide, amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols, isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines (Barbara, Christine, Anne, & Kristen, 2002).Literature survey also showed these compounds have never found either of Curcuma zeodaria or host plants of other plants.This compound also not yet been found of other endophytic fungi, but the compounds proposed are similar to compounds that are reported of fungal endophytic dothiorelon B and dothiorelon C, were isolated from microbial Dothiorella sp who live on the leaves of the species Cynodon dactylon (L) (Poaceae) (Radji, 2005).Other similar compounds ever discovered was 2-{4methyl-2-[(2-methylpropanoyl)oxy] phenyl}oxiran-2-yl)methyl-3-methylbutanoic (Yannai, 2004) (Figure 6).

Antibacterial Activity
The antibactetrial activity of this compound was evaluated according to the method previously described.
The antibacteiral properties of isolated compound was evaluated according to the method described previously (Lai, 2004 ).This compound showed inactive antibacterial for all bacterial test until concentration test 1000 ppm.This compound will show activity by concentration 2500 ppm with the mean of inhibition diameters (mm) for E. coli, S. dysenteriae, S. aureus, B. subtilis 10,3 ; 8.3, 8.4; and 8.8 mm respectively and standard antibacterial ampicillin at concentration 10 ppm showed inhibition diameters 7.5; 8.5; 7.0; and 9.5 mm respectively.Base on this data the compound show weak activity.

Figure 1 .
Figure 1. Isolation of the compound from ethyl acetate extract of Aspergillus sp from the leaves of Curcuma zedoaria

Table 1 .
The NMR data of isolated compound, recorded at 1 H -500 MHz; 13 C-125 MHz, spectral data of recorded in CDCl3