Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 7, No. 2, April 2022 Research Paper Tritirachium oryzae and Other Endophytic Mediated Jambu Bol (Syzygium malaccense) are Potential as an Antioxidant Yustina Hapida1,2, Elfita3*, Hary Widjajanti4, Salni4 1Graduate School of Sciences, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Palembang, 30139, Indonesia2Universitas Islam Negeri Raden Fatah, Palembang, 30126, Indonesia3Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Palembang, 30862, Indonesia4Department of Biology, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Palembang, 30862, Indonesia *Corresponding author: elfita69@gmail.com AbstractNatural bioactive substances have been discovered produced of intracellular fungi. Intracellular fungi, as well as endophytic fungi, itcan be found in organs are leaves, stems, roots, fruits, flowers, and seeds. This study aimed to specify for antioxidant activity ofintracellular fungi Jambu Bol (Syzygium malaccense) mediated and identify secondary metabolites compounds. The liquid culture waspartitioned with ethyl acetate solvent. Using chromatographic techniques, extracts were separated from their secondary metaboliteswith antioxidant activity apply the DPPH procedure. Its chemical structure was determined using NMR spectroscopic research, andendophytic fungi were recognized using phenotypic characteristics and molecular classification. The endophytic fungus isolationyielded four isolates: YF11, YF12, YF13, and YF14. YF12, with an IC50 of 53.03 g/mL, was the fungus that exhibited good antioxidantactivity. Pure chemical secondary metabolites compounds were identified as 2-(4-hydroxyphenyl)-4-methoxytetrahydrofuran-3-ol. Tritirachium oryzae was identified as the endophytic fungus YF12 based on morphological studies and a phylogenetic tree. To boostits antioxidant activity, more study is needed to perform a semi-synthetic reaction on this pure molecule. KeywordsAntioxidant, Tritirachium oryzae, Syzygium malaccense, Endophytic Fungi Received: 8 December 2021, Accepted: 29 March 2022 https://doi.org/10.26554/sti.2022.7.2.220-227 1. INTRODUCTION The production of reactive oxygen species known as free radi- cals often occurs in all cells as part of normal metabolic pro- cesses (Goart et al., 2021; Phaniendra et al., 2015). Free rad- icals have been implicated in the pathogenesis of many chronic diseases such as cancer, hypertension, and diabetes (Liguori et al., 2018). An antioxidant is needed to inhibit cell damage (Forrester et al., 2018). The use of synthetic antioxidants in inhibiting the activity of free radicals in damaging body cells can cause toxic side eects. Therefore, the search for natural antioxidants is very necessary (Ibrahim et al., 2021). Endophytic fungi live in the internal tissues of all plant species without causing disease symptoms in their hosts (Nair and Padmavathy, 2014). These endophytic fungi can syn- thesize bioactive compounds that have proven useful for the process of new drug discovery. The production of bioactive secondary metabolites from endophytic fungi has become a focus of research in recent decades because endophytic fungi represent interesting microorganisms to explore due to their diverse biotechnological potential (Girón et al., 2021). Plants with ethnographic backgrounds for the treatment of various diseases are promising candidates for obtaining bioac- tive compounds from their endogenous fungi. Jambu Bol (S. malaccense) is found in Southern Sumatra, Indonesia as herbal medicine in treating diseases such as hypertension, diabetes, and diarrhea. The results of a literary study, Jambu Bol plants have been used as traditional medicine by humans in various parts of the world. In Brazil, it is used to treat diabetes (Freitas et al., 2015). In India and Nigeria, the fruit, seeds, and bark are used in treating dysentery, diabetes, gastric ulcers, anti- inammatory, and antimicrobial (Bairy et al., 2005; Oyinlade, 2014). Endophytic fungus isolated from medicinal plants as an antioxidant, Syzygium aqueum has been reported to create secondary metabolites (Habisukan et al., 2022). Root bark, stem bark, and leaves of S. malaccense have been identied as endophytic fungus with antibacterial activity (Hapida et al., 2021). In this paper, we report the endophytic fungi that live in the fruit tissue of S. malaccense, their antioxidant activity, and the secondary metabolites contained in the active extract of the https://crossmark.crossref.org/dialog/?doi=10.26554/sti.2022.7.2.220-227&domain=pdf https://doi.org/10.26554/sti.2022.7.2.220-227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 endophytic fungus. 2. EXPERIMENTAL SECTION 2.1 Materials This study used PDA and PDB fungal growth media (Merck), alcohol 70%, aqua DM, sodium hypochlorite (NaOCl), DPPH (Sigma Alderich), organic solvents like ethyl acetate, n-hexane, chloroform, TLC silica gel, and chloramphenicol antibiotic. 2.2 Plant Material The organs used in this study were fresh and healthy fruit from Jambu Bol (S. malaccense) obtained in Palembang City, South Sumatra. The taxonomic examination of the Jambu Bol (S. malaccense) plant was carried out in the laboratory of the LIPI Botanical Garden Purwodadi. With number: B-302/III/KS. March 1, 2021. 2.3 Isolation of Endophytic Fungi Endophytic fungi to be isolated from fresh fruit washed under running water, and surface sterilized by soaking in 70% alcohol for ±180 seconds. After that rinsed with aseptic aqua DM in ±1 minute, then soaked with 3% sodium hypochlorite (NaOCl) for 60seconds, sodiumhypochlorite (NaOCl) for60seconds. The esh of the fruit used is cut aseptically. Then the samples were plated inPetridishescontainingPDAmedia, incubatedat room temperature for 3-14 days. Morphological observations on dierent colonies, then purication was carried out (Habisukan et al., 2021a). 2.4 Cultivation and Extraction Selected endophytic fungi were cultured on PDB (Potato Dex- trose Broth) media. A total of (±106 spores/mL) of pure culture was inoculated as much as 10% (v/v) in 50 mL, in a 1-liter bottle containing 250 mL of PDB medium. The fungi were then incubated for 3-4 weeks at 27◦C (room tempera- ture). The culture was ltered to lose from the mycelia. The liquid broth is composed and extracted with ethyl acetate, then shaken for 1 hour. The fungal extract was evaporated using an evaporator (Rumidatul et al., 2021; Syarifah et al., 2021). 2.5 Antioxidant Activity Test In methanol, a 0.05 mM DPPH solution was prepared. The standard liquid was made by dissolving the sample in 1000 g/mL dimethyl sulfoxide (DMSO). Diluting the reference liq- uid resulted in variations in sample concentration. 3.8 mL of 0.05 mM DPPH solution was added to 0.2 mL of various amounts of sample. The solution combination agreed and stored in an invisible place for 180 seconds to avoid oxidation and protect it from sunlight. A UV-Vis spectrophotometer was used to detect absorption at a _max of 517 nm. Ascorbic acid was used as a positive control and the same procedure was ad- ministered as a sample. The DPPH Radical Absorption Barrier Strength, namely percentage of inhibition and IC50 value was used to calculate antioxidant activity (Elta et al., 2021). %Inhibition = control absorbance − sample absorbace control absorbance x100% 2.6 Morphological and Molecular Identication of Endo- phytic Fungi Endophytic fungi thathavebeenpuried, identiedeachcolony based on the character formed, isolate fungi based on three main characteristics of the fungus, color and reverse colony characteristics, microscopic characteristics, and macroscopic characteristics. Thenthegroupwasdened(Huangetal.,2012; Nguyen et al., 2021). Endophytic isolates were identied using the method proposed by Metasari et al. (2020), which involves comparing the similarity of sample sequences to a web-based database known as BLAST (Basic Local Alignment Search Tool). 2.7 Measurement of Product Acidity YF12 endophytic fungi ethyl acetate extract sample (2 g) was preabsorbed with silica gel (70-230 mesh) in a ratio of 1:1. The samples were separated by column chromatography with the eluent system gradient n-hexane–ethyl acetate (10:0–0:10) and ethyl acetate–methanol (9:1–5:5). The eluent was collected every 10 mL in the vials and TLC analysis was performed on each vial to classify column fractions. The column fraction which shows the presence of a purple major stain is then con- tinued with column rechromatography until a pure compound is obtained. The chemical structure of the pure compound was analyzed based on NMR 1 and 2D spectroscopic data (Habisukan et al., 2021b). 3. RESULTS AND DISCUSSION 3.1 Endophytic Fungi of The Fruit of S. malaccense The results of the isolation of the endophytic fungus S. malac- cense obtained four isolates with codes YF11, YF12, YF13, and YF14 (Figure 1). Fungal identication was carried out based on the main characteristics of the fungus, namely macroscopic, microscopic, and another specic character. The success of identication and observation is inuenced by the ability to ob- serve individual morphology, or the technical ability to induce sporulation in agar cultures. The results of this identication were compared with the literature and the key fungal determi- nation (Ueda et al., 2010). 3.2 Morphological Identication of Endophytic Fungi Thefourendophytic fungi isolated fromS.malaccense fruitwere identied macroscopically with the following criteria: colony color, colony texture, topography, colony pattern, exudate drops, radial line, and concentric circle. Microscopic iden- tication based on criteria: spore type, spore form, hyphae, and other specic criteria. After observing the endophytic fun- gal isolate colonies, then microscopic observations were made on each colony. The characteristics of each isolate are based on microscopic observations shown in Tables 1 and 2. From the results of the identication of macroscopic and microscopic morphology, it was found that isolates from this © 2022 The Authors. Page 221 of 227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 Table 1. Macroscopic Characteristics of Endophytic Fungal Isolates from S. malaccense Fruit Code Colony complexion Inverse colony Type colony Model Elevation Exudate drop Radial line Concentric circle YF11 White Yellowish- white Velvety Zonate Flat - - √ YF12 White White and pink central Cottony Zonate Umbonate - √ √ YF13 White White, spreading Cottony Zonate Umbonate - √ √ YF14 Grayish white Cream Cottony Zonate Umbonate - √ √ Table 2. Microscopic Characteristics of Endophytic Fungi Isolates from S. malaccense Fruit Code Name spore Spore form Hyphae In character Genus / species YF11 Conidia Ellipsoidal Septate, aerial Conidiophores hyalin, simple, bearing spore masses on phialides at the apex: phialide verticillate Gliocladium sp YF12 Conidia ovoid Conidiofor Non septate Conidiophores upright, long, slender, simple, conidiogenous branches tapering to a rachislike, zigzag, fertile portion; conidia (sympodulo spores) apical on new growing points, hyaline, l-celled, globose or ovoid, saprophytic, conidiophores hyaline; conidia ovoid Tritirachium sp YF13 Zygospore Spora Septate, aerial Sporangiophores erect, branching sympodial, bearing terminal sporangia, rhizoids. Terminal sporangia, black, ovate Mucor sp YF14 Sporangiofor Spora Septate Sporangiophores, unbranched. Rhizoids the sporangiophores are terminate with a dark, round sporangium (40–275 mm in diameter) containing a columella and many oval, colorless or brown Rizopus sp Figure 1. Four Isolates of Endophytic Fungi from S. malaccense Fruit YF11 (Gliocladium sp), YF12 (Tritirachium sp), YF13 (Mu- cor sp), YF14 (Rhizopus sp) and Microscopic Observations fruit belonged to the Ascomycota group, namely YF11 and YF12. YF13 and YF14 isolates belong to the basidiomycota group. Isolation of endophytic fungi that have been carried out, it was found that the number of fungi that could be isolated on the fruit of Jambu Bol (S. malaccense) had little and tended not to variety, this is presumably because this endophytic fungus is unique, presence is inuenced by place, tissue, environment, and plant ecology. The tissue in the fruit has a smaller amount of tissue compared to the leaves, in the fruit, there is also a phenolic contentdissolved in it. Tissues inplant organsprovide dierentmicro-habitats forfungalcoloniesandtissuespecicity provides dierent substrates for the survival of endophytic fungal colonies to give rise to dominant taxa (Huang et al., 2012; Nguyen et al., 2021). 3.3 Antioxidant Activity of Endophytic Fungi The ethyl acetate extract of endophytic fungi from S. malac- cense has been tested for antioxidant activity using the DPPH method as shown in Table 3. Based on its IC50 value, an ex- tract’s antioxidant activity can be classied as high (IC50 <100 © 2022 The Authors. Page 222 of 227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 g/mL), moderate (IC50 100-500 g/mL), or weak (IC50 500- 1000 g/mL) and inactive (IC50 >1000 g/mL) (Mbekou et al., 2021; Metasari et al., 2020). Only two extracts, the endo- phytic fungi extract YF12 (IC50 381.04±24.54 g/mL) and the endophytic fungi extract YF14 (IC50 482.83±64.85 g/mL), showed moderate antioxidant activity. Two endophytic fungus extracts, YF11 (IC50 2822.77±442.16 g/mL) and YF13 (IC50 1235.71±144.23 g/mL), were found to be in active as antioxi- dants. Rhizopus sp, isolates from the stems of the Toona sinensis plant, Fungus YF14 a member of the Rhizopus sp. species, was shown to exhibit substantial (moderate) antioxidant activity such as tannins (Rahmawati et al., 2016). Endophytic fungi extracts that have no antioxidant activity, such as isolates YF11 (Gliocladium sp) and YF13 (Mucor sp). The fungus Glocladium sp has been isolated from Canna indica showing high pheno- lic content but weak antioxidant activity (Eskandarighadikolaii et al., 2015). From the fungus Gliocladium sp, compounds ergosterol-5,8-peroxide and allitol were found which can in- hibit the activity of Mycobacterium tuberculosis (Uc-Cachón et al., 2019). Table 3. Antioxidant Activity of S. malaccense Endophytic Fungi was using The DPPH Method, with Ascorbic Acid as The Antioxidant Standard Sample Genus/species of identication Antioxidant activity IC50 (`g/mL) YF11 Gliocladium sp 2822.77±442.16 YF12 Tritirachium sp 381.04±24.54 YF13 Mucor sp 1235.71±144.23 YF14 Rhizopus sp 482.83±64.85 Compound 1 (isolated from YF12) 53.03±0.23 Ascorbic acid 16.03±0.96 Species in the genus Mucor sp that have been reported are Mucor racemosus, and Mucor circinelloides. Ethyl acetate extract of the endophytic fungus Mucorracemosus isolated from Hibiscus sabdaria was reported to contain no avonoids in its secondary metabolites (Khalil etal.,2020). However, theethanolicextract of Mucor circinelloides fungi can bind phenolic compounds, tan- nins and avonoids under nutritional stress conditions in the late exponential phase so it can be developed as nutraceuticals and natural antioxidants (Hameed et al., 2017). 3.4 Molecular Identication of Selected Endophytic Fungi The YF12 endophytic fungus has been chosen to proceed to the molecular identication stage and secondary metabolite isolation because it has a good IC50 value among the four other endophytic fungal isolates. The results of molecular data analysis of YF12 isolate using MEGA 11, with the Neighbor- joining method, bootstrap consensus tree with 1000 replica- tions, the evolutionary distance was calculated using the P- distance method to obtain a phylogenetic tree (Figure 2b). The results of a phylogenetic search using the Gene Bank, it was found that the YF12 isolate was a species of Tritirachium oryzae. The results of the nitrogen base examination and the phyloge- netic tree YF12 isolate can be seen in Figure 2. The its rDNA sequence of the YF12 isolate is shown as follows: TCACTAATGATCCTTCCGTAGGTGAACCTGCGGA AGGATCATTAGTGAATTTAAAAAATACAGAATTGGAT TGAAAAAAATCCAAATTCTTATTTCTTTATTCTTCTC TTCCACTGTGAAATTTTAAACTATTCGGGCGGTCTT TTGGCCGGTCGAGGTTTAGAGATGGGACTGAGTGA AAAAAAATTGTTGGGGAGTGCCTCCACTTTCAAGTG GAGCGGACGATCTGCAGTTTTAGTTCTTCTGTTCTC TGATCTAGCCGAATTACCCAATTTTTAGAGACAATGT TAAATTTGAATGTGTTTTTTTATTAAACAAATTAAAA CTTTCAGTGACGGATCTCTTGGCTCTCGCATCGATG AAGAACGCAGTAAATCGCGATACGTAATGTGAATTG CAGAAATATGTGAATCATCGAATCTTTGAACGCATCT TGCGCTCTGGGGTACTCCTCAGAGCATGCCTGTTTG AGTGTCTTTTAATTCTCATCTCATAATTTTTTATTAA TTTAAAATAATTATAGGTGGATCGTGGCTGTTTTGA CGACTTAACCTCGTCTCAGCTGAAATATAGAAAGCG ACGTCTAAAATTCAGAGTAATAAGATGTTAACGTCG GCGTGATAAGTAAATTACGCAGTCAGTTTTCTGTCT ATCTCTCTGGTGTTCGCTACGAATTACTATAGTTTT GTTTTCAACATTTGACCTCAAATCAGGTAGGACTAC CCGCTGAACTTAAGCATATCAATAAGCCGG. Tiraitirachium oryzae is a fungus that comes from the air, its interaction with humans was rst known to cause onychomyco- sis (Naseri et al., 2013). This fungus has thebiosynthetic ability of AgNPs because it contains a single carbon and nitrogen in culture media which can be synergized into nanoparticles to inhibit bacterial growth (Khlaifat et al., 2019). Onychomycosis is also reported as a producer of extracellular lipase enzymes that can hydrolyze waste oil (Al-limoun, 2020). This fungus belongs to the basidiomycota group and is often mistaken for Ascomycota, this species produces spores in the air and is often found as endophytes in certain plant species, its presence in mesophyll tissue (Agut, 2001). 3.5 Purication and Identication Bioactive Compound A total of 2 g of ethyl acetate extract of the endophytic fun- gus Tritirachium oryzae (YF12) was obtained using a gradi- ent (10:0-0:10) in ethyl acetate-methanol (9:1-5:5) to ob- tain ve fractions, namely F1-F5 made. Fraction F4 was re- chromatographed with an n-hexane-ethyl acetate gradient elu- ent (5:5-0:10) to produce a three-column fraction, F4.1-F4.3. Column fraction F4.2 is a chromatographic column with n- hexane-ethyl acetate (4:6) as eluent and the resulting solid is eluted with 8:2 n-hexane-ethyl acetate to obtain a pure yellow- ish color compound (compound 1) white solid 36 mg. The H-NMR spectrum of compound 1 (Figure 3) showed the presence of nine proton signals. There are two doublet signals with the integration of two protons in the aromatic region, namely at 𝛿H 7.56 and 8.20 ppm. This indicates that © 2022 The Authors. Page 223 of 227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 Figure 2. Molecular Examination Results on Selected Fungal YF12 Isolated from S. malaccense Fruit (a) The Composition of The Number of Nitrogen Bases Possessed by The YF12 Fungi Isolate was No. 1,753 bp, (b) The Phylogenetic Tree Showing The Species of YF12 Isolate Showed Tritirachium oryzae Species, The Scale used was 0.050 m Figure 3. The 1H-NMR Spectrum of Compound 1 (1H-500 MHz in CDCl3) compound 1 has two pairs of equivalence protons side by side. Thus it is known that compound 1 is an aromatic compound that has a para-substituted benzene ring. Then there are three oxymethine proton signals, namely at 𝛿H 4.16 (1H, m), 5.32 (1H, d, J= 2), and 5.78 ppm (1H, s), an oxymethylene proton signal at 𝛿H 3.99 ppm (2H, m), and a methoxy proton signal at 𝛿H 2.61 (3H, s). In addition, there are also two broad signals that are typical for hydroxyl protons, namely at H 3.55 and 7.19 ppm. The identication of protons in the 1H-NMR spectrum is supported by data on the 13C-NMR spectrum (Table 4) and HMQC (Figure 3). The 13C-NMR spectrum of compound 1 showed the presence of nine signals. 4 carbon signals ap- pear in the aromatic region, namely two equivalent aromatic methine carbons (𝛿H 123.8 and 126.8 ppm), one aromatic quaternary carbon at 𝛿C 147.6, and one aromatic oxyaryl car- bon at low eld 𝛿C 164.5 ppm. In addition, ve oxygenated carbon signals appear in the 𝛿C region of 40.0–75.0 ppm. The analysis of the proton and carbon NMRspectrawere conrmed by the data on the HMQC spectrum shown in Figure 4a and Table 4, namely the 1H-13C correlation through one bond. The HMQC spectrum showed seven correlations consisting of two correlations on the aromatic ring and ve correlations on oxygenated 1H-13C on aromatic substituents. The other two proton signals at 𝛿H 3.55 and 7.19 ppm do not correlate with the HSQC spectrum. This indicates that the two protons are hydroxyl protons. Figure 4. The NMR 2D Spectrum of Compound 1 (1H-500 MHz; 13C-125 MHz in CDCl3 (a) HMQC Spectrum, (b) HMBC Spectrum, (c) The HMBC Correlation and 𝛿-Assig- nment of Compound 1 The HMBC spectrum (Figure 4) shows the 1H-13C cor- relation through two or three bonds. The aromatic proton signal at 𝛿H 8.20 ppm is correlated through three bonds with its equivalent aromatic carbon (𝛿C 123.8 ppm). The aromatic proton at 𝛿H 7.56 ppm is correlated through three bonds with equivalent aromatic carbon (𝛿C 126.8 ppm) and oxygenated carbon (𝛿C 73.5 ppm). Furthermore, the oxygenated methine proton at 𝛿H 5.32 ppm was correlated via three bonds with the equivalent aromatic carbon (𝛿C 126.6 ppm) indicating that the oxygenated methine group is directly attached to the aro- matic ring and is para-substituted with the hydroxyl group. The HMBC spectrum in Figure 4b does not provide all the information for the 1H-13C correlation through two or three bonds. However, the substituent para hydroxyl group is a furan ring-substituted for hydroxyl and methoxyl groups which are indicated by the presence of a hydroxyl proton signal at 𝛿H 3.55 (1H, broad) and a methoxyl proton at 𝛿H 2.61 (3H, s). © 2022 The Authors. Page 224 of 227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 The 1D and 2D NMR spectral data for compound 1 are shown in Table 4. Table 4. NMR Data for Compound 1 No. C 𝛿C ppm 𝛿H ppm (ΣH. multiplicity. J (Hz)) HMBC 2 73.5 5.32 (1H, d, J = 2) 126.8 3 66.2 5.78 (1H, s) 4 55.6 4.16 (1H, m) 5 64.1 3.99 (2H, m) 6 41.0 2.61 (3H, s) 1’ 147.6 2’ 126.8 7.56 (1H, d, J = 8.5) 126.8; 73.5 3’ 123.8 8.20 (1H, d, J = 8.5) 123.8 4’ 164.5 5’ 123.8 8.20 (1H, d, J = 8.5) 123.8 6’ 126.8 7.56 (1H, d, J = 8.5) 126.8; 73.5 3- OH 3.55 (1H, broad) 4’- OH 7.19 (1H, broad) Basedonspectralanalysisof 1H-NMR, 13C-NMR,HMQC, and HMBC, it can be explained that compound 1 has a para- substituted benzene ring between the hydroxyl group and the furan ring. The oxygenated methine carbon of this furan ring bonds directly to the aromatic carbon. The hydroxyl and methoxyl substituents are bonded to the furan ring. Thus, the proposed chemical structure of compound 1 is 2-(4-hydroxy- phenyl)-4-methoxytetrahydrofuran-3-ol as shown in Figure 5. Figure 5. Chemical Structure of Compound 1: 2-(4-Hydroxy- phenyl)-4-Methoxytetrahydrofuran-3-ol The results of the antioxidant test using the DPPH method, the IC50 value was 53.03±0.23 with moderate criteria. Chemi- cal structure 2-(4-hydroxyphenyl)-4-methoxytetrahydrofuran- 3-ol, contains simple phenolic compounds, phenolic com- poundsareknownasantioxidantsandantibacterials (Zeb,2020). The group of antioxidant compounds present in the host, es- pecially in S. malaccense fruit contains more complex com- pounds such as anthocyanins such as cyanidin 3-glucoside, followed by cyaniding 3,5-glucoside and peonidin 3-glucoside (Batista et al., 2017; Nunes et al., 2016), because anthocyanins are phenol derivatives other than avonoids (Zeb, 2020). In the essential oil of guava fruit S. malaccense 2-phenyl ethanol and its esters (2-phenylethyl acetate, 2-phenylethyl isopen- tanoate, 2-phenylethyl benzoate and 2-phenylethyl phenylac- etate) and herbs (1-octen-3-ol) contributes to the complexity of the aroma(Pino et al., 2004). Similarcompounds, such as 2- (4-Hydroxyphenyl)-5-(3-hydroxypropenyl)-7-methoxyben- zofuran, which is a derivative of ailanthoidol, have been re- ported to have been isolated from a neolignan from Zanthoxy- lum ailanthoides and Salvia miltiorrhiza Bunge. This compound has a low IC50 value, by activating protein kinases with the help of mitogens which can make this compound an eective func- tional chemical candidate for the prevention of inammatory diseases (Kim et al., 2013). Chemical constituents that have been reported are tetrahy- droxanthone and oxyanthrone from Tritirachium sp associated with Pseudoceratina purpurea isolated from marine sponge col- lected from oshore sites in Sakuraguchi, Ishigaki Island, Oki- nawa Prefecture, Japan (Ueda et al., 2010). There are three an- thraquinone compounds from Tritirachium sp by water-soluble Tetrazolium-8 (WST-8) colorimetric assay, were shown to be antiproliferative against Hela cells (IC50: 11, 17, and 17 M respectively) (Ueda et al., 2010). There are 4,5-dihydroxy-10- oxo-9,10-dihydroanthracen-9-yl acetate, and 2-(2-methoxy- 4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium compounds (Zhang et al., 2017). The Pinus wallichiana plant from the Western Himalayas, has been isolated from the endophytic fungus Tritirichium oryzae and has high antifungal activity on Candida albicans and a broad spectrum as an antimicrobial (Qadri et al., 2014). In addition, this species is known to cause several infections in hu- mans such as corneal ulcers, otomycosis, onychomycosis, and dermatomycosis, but the genus Tritirachium also has potential in biotechnology to produce several enzymes such as proteases, amylase, glucanase, xylanase, pectinases, lipase, and proteinase K, Phylogenetically, it is known that the fungus Tritirachium oryzae is closely related to T. dependents (Bezerra et al., 2020). Another host plant known to host Tritirachium oryzae is the bark of the Hancornia Speciosa Gomes plant, which has bio- logical activity as an antibacterial against B. subtilis, E. coli, and P. aeruginosa bacteria (Chagas et al., 2017). Isolation of fungi of the Tritirachium genus from the soil of various gardens in Shiraz, Iran was found as much as 0.24%, at a pH between 7-8, which are referred to as keratinophilic fungi (Pakshir et al., 2013). There is a diversity of species of Tritirachium oryzae isolated from transgenic and non-transgenic “Bt” cotton plants (the commonly farmed cotton plant that has been genetically engineered) on leaves and stems in Brazil (Vieira et al., 2011). © 2022 The Authors. Page 225 of 227 Hapida et. al. Science and Technology Indonesia, 7 (2022) 220-227 Mangrove plants on the southwest coast of India, the genus Tri- tirachium has antimicrobial activity as well (Maria et al., 2005). 4. CONCLUSIONS Code YF12 was Tritirachium oryzae from S. malaccense pro- duced secondarymetabolites compound. Compound 1 as 2-(4- hydroxyphenyl)-4-methoxytetrahydrofuran-3-ol with mod- erate of antioxidant activity. For increasing the antioxidant activity of compound 1, further research is needed, namely a semisynthetic reaction to add a hydroxyl group at the C-3 and/or C-5 positions. So this compound 1 can be used as a basic ingredient of antioxidant compounds. 5. 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Page 227 of 227 INTRODUCTION EXPERIMENTAL SECTION Materials Plant Material Isolation of Endophytic Fungi Cultivation and Extraction Antioxidant Activity Test Morphological and Molecular Identification of Endophytic Fungi Measurement of Product Acidity RESULTS AND DISCUSSION Endophytic Fungi of The Fruit of S. malaccense Morphological Identification of Endophytic Fungi Antioxidant Activity of Endophytic Fungi Molecular Identification of Selected Endophytic Fungi Purification and Identification Bioactive Compound CONCLUSIONS ACKNOWLEDGMENT