A New Benzoyl Compound Isolated from the Endophytic Fungi of Kandis Gajah (Garcinia griffithii) and Asam Kandis (Garcinia cowa)

Garcinia griffithii and Garcinia cowa belong to the genus Garcinia. The genus Garcinia has been known to be a rich source of secondary metabolites, such as xanthones, benzophenones, flavonoids, steroids, terpenoids, and other phenolic derivatives. Previous investigations of endophytic fungi from G. griffithii revealed the presence of three compounds not found in the host. In order to the continue the phytochemical work on endophytic fungi of G. griffithii, the constituent of the endophytic fungi of G. griffithii was re-examined. In this study, a benzoyl compound similar to that found in the endophytic fungus of G. cowa was observed. The same benzoyl compound was also isolated from the endophytic fungus Acremonium sp of G. griffithii and Aspergillus sp of G. cowa with cultivation of eight weeks in static conditions at room temperature. The culture medium was partitioned using ethyl acetate and evaporated to obtain the concentrated extract. Isolation of compounds was performed using the chromatography method. The chemical structure was proposed on the basis of spectroscopic data, including ultraviolet (UV), infrared (IR), mass spectrometry (MS), proton nuclear magnetic resonance (H-NMR), carbon nuclear magnetic resonance (C-NMR), heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and correlation spectroscopy (COSY).


Introduction
Endophytic fungi are a new source of bioactive compounds being explored recently. They live in plant tissue in a certain period of time, can form colonies in plant tissues without harming the host, and usually provide symbiotic mutualism [1][2][3]. One of the interesting facts about endophytic fungi is their ability to produce bioactive compounds that can be similar or different with those produced by the host [4][5].
In the current study, we reported another secondary metabolite called benzoyl compound, which is extracted from Acremonium sp of G. griffithii and found in the endophytic fungus Aspergillus sp of G. cowa.

Materials and Methods
Source of endophytic fungi. Acremonium sp of G. griffithii obtained from stock fungus was stored in the Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences of Sriwijaya University. The fruit of G. cowa was collected in May 2012 from Lembah Arau, West Sumatra.
Isolation and identification of endophytic fungi. The isolation of Acremonium sp from twigs of G. griffithii was reported previously by Elfita et al. 2012 [16] and Debbab et al. [18]. The isolation method of Aspergillus sp from the fruit of G. cowa grown in Sarasah Bonta, Lembah Arau, Kabupaten Lima Puluh Kota, and West Sumatra was conducted according to the procedure by Elfita et al. 2011 [15]. The fungal strain was identified on the basis of the morphological method by the School of Hayati Science and Engineering, Bandung Institute of Technology, Indonesia. The voucher of specimen was stored in the Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences of Sriwijaya University.
Cultivation of endophytic fungi. Potato dextrose broth (PDB) medium of 300 mL was used for the cultivation of endophytic fungi and was placed into 30 flasks (1 L each). Fungal suspension containing 10 6 spores/mL was inoculated under sterile conditions to each 300 mL PDB medium (ratio 1:10). The cultures were incubated for eight weeks in static conditions at room temperature [19][20].
Extraction, exploration, and structure elucidation. Mycelia were removed from the endophytic fungus culture after eight weeks of incubation and the medium was filtered. The medium was extracted three times using ethyl acetate (1:1) followed by evaporation under vacuum to obtain the concentrated extract. The concentrated extract was separated by column chromatography over silica gel 60 (70-230 mesh) at the stationary phase (1:30) and eluent, which was previously determined by thin-layer chromatography silica gel 60 F 254 . The chosen eluent with increased polarity was n-hexane:EtOAc at a ratio of 10:0 to 0:10 (v/v). An eluate was collected and then combined using thin-layer chromatography into column fractions. Each fraction was evaporated and purified using the chromatography technique to obtain the purified compound.

Result and Discussion
In this study, Acremonium sp was cultivated on 9 L of PDB medium for eight weeks at room temperature. The culture broth was extracted by solvent partition with EtOAc (1:1), followed by evaporation. The extract showed two major spots on TLC.
The fungal strain from the fruit of G. cowa was identified as Aspergillus sp by the School of Hayati Science and Engineering, Bandung Institute of Technology, Indonesia.
Aspergillus sp was cultivated on 9 L of PDB medium for eight weeks at room temperature. The culture broth was extracted by solvent partition with EtOAc (1:1), followed by evaporation. The extract showed one major spot on TLC. Figure 1 briefly illustrates the procedure for isolating the pure compound from the endophytic fungi Acremonium sp from the twigs of G. griffithii and Aspergillus sp from the fruit of G. cowa.   In the HMBC spectrum (Table 1.), correlations from H-3" (δ H 8.32) to C-1" ( C 195.6) , C-4" ( C 128.9), and C-5" ( C 135.0) suggested that C-1" was attached to the aromatic ring as a benzoyl group. Correlations from the hydroxyl proton at C-3' (δ H 8,46) to C-2' ( C 93.1) and C-4' ( C 73.1) and from 1'-OCH 3 (δ H 3.45) to C-1' ( C 90.6) demonstrated the presence of a cyclobuthene ring bearing methoxyl and hydroxyl groups. The methoxyl resonance connecting to C-1' in HMBC indicated C-1' with a high chemical shift ( C 90.6). Moreover, oxygenated carbon was attached to C-1 (carbonyl carbon). Correlations from the hydroxyl proton at C-3' (δ H 8,46) to C-2' with a low chemical shift as sp 2 C=C ( C 93.1) showed an anisotropy effect of carbonyl groups (C-1"). HMBC correlations from 2-CH 3 (δ H 1.66) to C-1 ( C 196.5), C-2 ( C 113.3), and C-3 ( C 186.2) indicated that C-1 and C-3 were located at the carbonyl group and hydroxyl group, respectively. Correlations from H-4 (δ H 4.59) to C-5 ( C 71.3) and H-5 (δ H 4.74) to C-4 ( C 70.8) revealed that C-4 and C-5 were attached to the hydroxyl groups. Figure 2 illustrates the HMBC correlation and δ-assignment of compound 1.    Figure 2.

Elucidation
Carbon C-1' of compound 1 had a similar position and chemical environment to C-1' of talaroflavone [21]. Carbon C-1'of the two compounds is C sp3 (quarterner), which binds two C sp2, one oxygenated C sp3, and one oxygen atom. Each carbon appeared at δ C 90.6 and 91.8 ppm. Furthermore, carbon C-2' of compound 1 had a position and chemical environment similar to those of C-3 of methyltriacetic lactone [21], each of which appeared on δ C 93.1 and 95.0 ppm. The chemical shift of C-1' (δC 90.6) was shown to approach the calculated data using Chem Draw Ultra 10 (δC 94.8) Combining the results obtained by the spectrometric methods (UV, IR, MS, 1 H-NMR, 13 C-NMR, HSQC, HMBC, and COSY) and comparing the results of the literature data [21] with the calculated data of Chem Draw Ultra 10, we determined C 22 H 26 O 8 as the molecular formula for compound 1 with a molecular weight of 418. The structure of compound 1 is 1-(2'-benzoyl-3,4-dihydroxy-1'-methoxycyclobut-2'-enyl)-3, 4, 5-trihydroxy-2-methylnona-2,6-dien-1-one. The confirmation structure of 1 will be followed by an X-ray single crystal, and this work is still in progress.
Elucidation of compound 1* from Aspergillus sp of G. cowa. The 1 H-NMR spectra recorded in CDCl 3 ( 1 H-500 MHz) indicated that the spectroscopy data of 1 and 1* are identical (see Table 1).
Based on the Dictionary Natural Products database (December 18, 2015), the benzoyl derivative could be a new compound. However, the identical benzoyl compounds (1 and 1*) were produced by two different endophyte fungi could further raise the question of whether it is related to the host of the same genus or assumed as a secondary metabolite typically produced by the fungi. Based on these phytochemical studies, further studies on the metabolite profiling of these endophytic fungi and plants should be performed for confirmation.

Conclusions
The endophytic fungi Acremonium sp from the twigs of G. griffithii produced the newly proposed benzoyl compound, which is identical to that found from the endophytic fungus Aspergillus sp from the fruit of G. cowa.