SECONDARY METABOLITE OF Aspergillus fumigatus , ENDOPHYTIC FUNGI OF THE MEDICINAL PLANT Garcinia griffithii

The endophytic fungi Aspergillus fumigatus was isolated from the tissues of the fruits of Garcinia griffithii. The fungal strain was identified from the colony, and it was characteristic of cell morphology. The ethyl acetate extracts derived from fungus cultures showed major spots on TLC under UV light, which was continued to the isolation of the secondary metabolites. The structure of the isolated compound was elucidated on the basis of NMR analyses (H-NMR, C-NMR, HMQC, HMBC and H-H COSY). The compounds were identified as: 4,6-dihydroxy, 3,8a-dimethyl-1-oxo5-(3’-oxobutan-2’-yl)-1,4,4a,5,6,8a-hexahydronaphthalen-2-yl-1”,2”-dimethyl-5”-(2”’-methylprop-1”’-enyl)cyclopentanecarboxylate.


Introduction
Endophytes are microbes that colonize living, internal tissues of plants without causing any immediate negative effects [1].They reside inside the tissues of nearly all healthy plants.The relationship that they establish with the plant varies from symbiotic to bordering on pathogenic [2].They are synergistic to their host, and at least some of them are thought to be useful to the plant by producing special substances, such as secondary metabolites, that prevent the host from being attacked successfully by fungi and pests [3].Via what appears to be their contribution to the host plant, the endophytes may produce a plethora of substances of potential use to modern medicine, agriculture, and pharmaceutical industry.The potential prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great [4].
The present study is focused on isolation and determination of endophytic fungi from fruits of G. griffithii growing wildly in Lembah Arau, West Sumatra, which has allowed us to explore and to elucidate the structure of natural products produced by the endophytic fungi Aspergillus fumigatus.

Methods
Plant material and fungal isolation.Isolation of endophytic fungi was standardized and modified based on the method described by Debbab et al. [13].Fruits of G. griffithii were collected in April 2010 from Sarasah Bonta, Lembah Arau, Kabupaten Lima Puluh Kota, West Sumatra.Voucher specimens have been deposited in the Laboratory of Herbarium, Universitas Andalas (ANDA), Padang.The fruits were washed in sterilized distilled water twice.Surface sterilization was done by immersing the stems in 70% ethanol for 2 min (twice) followed by rinsing again twice in sterilized distilled water.Then, the fruit were cleaved aseptically into small segments (≈1 cm in length).The material was placed on a petri dish containing potato dextrose agar medium (PDA) supplemented with 100 µg/mL of chloramphenicol to suppress bacterial growth and then incubated at room temperature (25 °C).After several days hyphae growing from the plant material were then transferred to other plates, incubated again for 10 days, and periodically checked for culture purity.The fungal strain was identified based on the colony and the characteristic of cell morphology [14].
Potato dextrose broth (PDB) culture of isolated fungi.Ten flasks (1 L each) containing 500 mL of PDB medium were autoclaved.A small part of the medium from a petri dish containing the purified fungi was transferred under sterile conditions to the PDB medium.The fungi strain was grown on PDB medium at room temperature for two months.

Extraction, exploration and structure elucidation.
The culture was extracted with 5 L ethyl acetate (twice).Evaporation of the extract resulted a dry extract, which was applied to chromatograph over a silica gel column with n-hexane : ethyl acetate as solvent (gradient elution).Based on detection by TLC (SiO gel F254, Merck, Darmstadt, Germany) using n-hexane : EtOAc (4 : 6) as a solvent system, the collected fractions were combined, and subjected to recolumn over a silica gel with n-hexane : ethyl acetate (4 : 6) to obtain pure compound.The molecular structure is identified by spectroscopic methods including 1 H-NMR, 13 C-NMR, HMQC, HMBC, and COSY.

Results and Discussion
Characterization of endophytic fungi.Characterization of the macroscopic was analyzed by colony morphology and the characteristic of cell morphology.The colony morphology was determined by growing the isolates of the fungus in three different mediums, namely Czapez dox agar (CDA), malt extract agar (MEA), and potato dekstrose agar (PDA).The cell morphology was determined by observing the nonreproductive structures, reproductive structures, conidia, and conidiophores, and it was compared with literature, that the fungi is A. fumigatus.The results of characterization are shown in Figure 1 and 2.

Extraction and exploration.
Ethyl acetate extract containing secondary metabolite was concentrated under reduced pressure to give crude extract 6,42 g.TLC results of EtOAc extract indicates that there is one  The HMBC correlations from the methyl protons indicated that the methyl groups were attached to C-3, C-8a, C-2', C-3', C-1'', C-2'', and two methyl groups were attached to C-2'''.
Furthermore, correlation at δ H 2.12 ppm (H- The COSY spectrum also revealed the presence of two methylene groups in cyclopenthane with correlation H-2" to H-3" and H-3" to H-4" ( 3 J).The COSY correlation of compound 1 is shown in Figure 4.
Compound 1 was not found in the host plant G. griffithii.The structure which has A and B rings similar to rings A and B in the xanthone, while ring C in xanthone is the result of this cyclizationand aromatization of compound 1.Search by ScinceFinder found that this compound is new.