Identification of 4-4 ’-( 1-methylethylidene )-bisphenol from an Endophytic Fungus Cladosporium oxysporum derived from Aglaia odorata

Endophytic fungi has an economic potential as raw material for biologically active compounds. Cladosporium oxysporum is one of the endophytic fungi isolated from Indonesian medicinal plant Aglaia odorata Lour (Local name: Pacarcina). This planth has been used for fever, cough, diarrhea, inflammation and injury. In our previous study, the ethyl acetate extract and several fractions of the extract of C. oxysporum showed antimicrobial activity against Candida albicans, Escherichia coli, and Staphylococcus aureus. The objective of the currents study is to investigate the chemical constituent of the active fraction. Purification of the metabolite was achieved by using column chromatography followed by preparative thin layer chromatography. Identification of the metabolite was conducted by using TLC densitometry, GC-FID and GCMS. Compound 1 was isolated from fraction 12. The purity of this compound was determined by 2D-TLC and GC-FID. The UV-Vis profile of compound 1 indicates a phenolic compound. Further analysis by using GCMS shows one peak at a retention time (Rt) of 23.80 minutes, predicted as 4-4’-(1-methylethylidene)bisphenol. The chemical constituent of the sub fraction 12.2.7 (fraction a) is identified as 4-4’-(1methylethylidene)-bisphenol.


INTRODUCTION
Fungi provide a large potential as a source for the discovery of new drugs such as various types of antibiotics (Schulz et al., 2002).Endophytic fungi are microbes that live in plant tissues without causing any symptoms in its host plant.Bills et al. (2002) reported that endophytic fungi from tropical region provide more active metabolites and vary significantly compared to those of subtropical region.Indonesian biodiversity, especially endophytic microbes have not been well studied, whereas its potency as rich sources of valuable active substances is very promising.Most of endophytic fungi are able to produce the bioactive metabolites.As continuation of our work on endophytic fungi, some endophytic fungi have one of the traditional herbs used as "jamu" has been trusted as fever therapy, cough, diarrhea, inflammation, and injury (Kinghorn et al., 2011).A. odorata Lour has a vascular tissue, living in a tropical climate, allowing their endophytic microbes that grow and live in it.
Previous study had reported the isolation of an endophytic fungus C. oxysporum from A. odorata Lour derived from Purwodadi Botanical Garden, East Java, Indonesia.The fungus was cultivated in liquid malt extract medium.Cultures were incubated in static condition at room temperature for 28 days, followed by extraction with ethyl acetate.The initial testing of the ethyl acetate extract showed antimicrobial activity (Sugijanto et al., 2005).Bioassay guided fractionation was further conducted.The ethyl acetate extract was fractionated by column chromatography using silica gel 60 with gradient elution n hexane, ethyl acetate, and methanol.The resulted 13 fractions were subjected to antimicrobial assay.
The results showed that several fractions C. oxysporum have antimicrobial activity, six of 13 fractions gave inhibition against S. aureus ATCC 6538, E. coli ATCC 8739, and C.

METHOD
Plant material and isolation of the endophytic fungi C. oxysporum was isolated from stem of the host plant A. odorata Lour collected from Purwodadi Botanical Garden, East Java, Indonesia as described by Sugijanto et al., 2009.Determination of the C. oxysporum was done by Dr. Arnulf Diesel, Institut für Pharmazeutische Biologie Universität Düsseldorf using biological molecular method (Sugijanto et al., 2008).

Cultivation and extraction of endophytic fungi
Cultivation of C. oxysporum was undertaken on 431 culture bottles each containing 40 mL malt extract liquid medium and was incubated for 28 days at room temperature (30±3 °C) with initial pH at 5.60 ± 0.05.The cultures broth and mycelia (13.2 L) were then extracted with ethyl acetate.The ethyl acetate layers were collected, combined, and concentrated in rotary evaporator at 35 °C to obtain a yellowish brown extract (5.52 g).

Fractionation and purification metabolites of endophytic fungi
Ethyl acetate extract of C. oxysporum (2.80 g) was separated by column chromatography over 200 g silica gel 60 (62.5 x 3.5 cm) as a stationary phase and eluted with mixture of n-hexane, ethyl acetate, and methanol in increasing polarity, which yielded 13 fractions.Fraction 12 (250 mg) showed antimicrobial activity against C. albicans using disk diffusion method (Sugijanto and Dorra, 2016).Fraction 12 was further purified using column chromatography on Sephadex LH 20 (25 g) with methanol as mobile phase, and obtained three sub-fractions, namely 12.1 -12.3.Sub-fraction 12.1 and 12.3 were not analysed due to low amount of samples.Sub-fraction 12.2 (52.3 mg) was then purified using preparative chromatography on silica gel 60 with dichloromethane and ethyl acetate (9:1v/v) as mobile phase.Eight sub-fractions were obtained, namely 12.2.1 to 12.2.8.Sub-fraction 12.2.7 (13.7 mg) was analyzed by TLC and obtained one spot which showed a red-purple spot with anisaldehyde-H2SO4 conc.reagent (Rf 0.34), whilst the other sub fractions the amount were too low to be analyzed and the spots were very weak.Purity of the fraction was analyzed by 2D-TLC.Sub fraction 12.2.7 (fraction a) was further analyzed using TLCdensitometry, GC-FID, and GC-MS.

Analytical condition
TLC analysis were performed on silica gel TLC plates 60 F254 aluminum sheets 20 x 20 cm (Merck).Visualization of the TLC spot were done with UV at a wavelength of 254 and 366 nm and anisaldehyde -sulfuric acid.GC-MS analysis was undertaken using GC-FID with Agilent Technologies 6890N and GC-MSD with the Agilent 6973 series equipped by Willey 7n.1 database ( 2004) on HP-5 column (30 m x 0,250 mm x 0,25 µm).Inlet temperature was set at 250 °C, split ratio 50:1, flow rate 1 mL/min (He), oven temperature was programmed from 100 °C -250 °C, 5 °C/min for 30 minutes analysis.Transfer line temperature from oven to detector was 280°C; energy of ionization was set at 70 eV.

Method of Identification
EI MS of peak 1 was compared to the Willey 7n.1, NIST and Mass Bank databases according to the method of Commission Decision 2002/657/EC, the identity of a compound can be verified if its MS spectrum showed at least 4 identical fragments compared to standard MS.

RESULT AND DISCUSSION
TLC of sub-fraction 12.2 was presented in Figure 1 showed 5 spots (UV 254) and 3 spots respectively: based on this separation on these spots, preparative TLC chromatography was performed.Sub-fraction 12.2 (52.3 mg) was selected to purified using preparative chromatography on silica gel 60 with dichloromethane and ethyl acetate (9:1 v/v) as mobile phase.Fraction 12.2 was purified based on consideration of the TLC profiles using preparative thin layer chromatography (stationary phase: silica gel 60; mobile phase: dichloromethane : ethyl acetate = 9:1); fractions that have identical TLC chromatograms were collected and evaporated into dryness; eight sub fractions, namely 12.2.2 until 12.2.8 were recovered.Only sub fraction 12.2.7 (13.7 mg; Rf 0.34) showed one spot by TLC, therefore it was selected for purity test using 2D TLC and was further analysis.

Purity test
Based on the results of the 2D TLC sub fraction 12.2.7 (fraction a) already pure or just a single spot, so that further analysis can be done by TLC-densitometry and GC.

Characterization of isolates
TLC-densitometry chromatogram profile of fraction a indicated there was a dominant peak at Rf 0.34, which was observed in the UV spectrum at ƛmax 273 nm.UV absorption at a wavelength of approximately 270 nm can be indicative of phenolic compound (Skoog et al., 2007).The result from Figure 2 showed that UV-visible absorption spectra profile from scanning spectrum fraction a were similarly to the UV visible absorption spectra of bisphenol A in previous publications (Zhuang et al., 2014).and 65 [M-163].Wiley data base predicted that peak 1 was 4-4'-(1-methylethylidene)bisphenol, with the highest quality score (87%).The EI-MS of peak 1 was identical with the EI-MS spectra of 4-4'-(1-methylethylidene)-bisphenol mass bank (www.massbank.jp)with a similarity score of 88.31%.Criteria resemblance to the database can be considered equal if > 80% (Odchimar et al., 2016).According to Commission Decision 2002/657/EC, the m/z 119

Figure 2 .
Figure 2. UV-visible absorption spectra of fraction a using TLC-densitometry.

Figure 3 .
Figure 3.Total Ion Chromatogram of fraction a

Figure 4 .
Figure 4. EI-MS of peak 1 of fraction a.