Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 6, No. 3, July 2021 Research Paper Chemical Characterization of Secondary Metabolite from the Endophytic Fungus Trichordema reecei isolated from The Twig of Syzygium aqueum Ummi H. Habisukan1,2, Elfita3*, Hary Widjajanti4, Arum Setiawan4 1Universitas Islam Negeri Raden Fatah Palembang, South Sumatra, Indonesia2Graduate School of Sciences, Faculty of Mathematics and Natural Sciences, University of Sriwijaya,. Jl. Padang Selasa No. 524, Palembang 30139, South Sumatra, Indonesia3Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Jl. Palembang Prabumulih, Indralaya, South Sumatra, 30662 Indonesia4Department of Biology, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Jl. Palembang Prabumulih, Indralaya, South Sumatra, 30662 Indonesia *Corresponding author: elfita69@gmail.com AbstractEndophyticfungiaremicroorganismsthatliveinplants,withoutnegativeeffectsandaremutuallyrelatedtohostingplants. Explorationof bioactive compounds from Endophytic fungi is easier and cheaper because they do not require a large area, a short growingtime, and uncomplicated mixed compounds. Endophytic fungi are new and patent base secondary metabolites but they are notextensively characterized and investigated for the exploration of raw materials for drugs. The purpose of this study was to obtainantioxidant secondary metabolites from Endophytic fungi that live in the Syzygium aqueum medicinal plant. In this study, Endophyticfungi were isolated from S. aqueum twigs and molecular identification. The secondary metabolites were isolated by chromatographicmethod and chemical structure identified by spectroscopy. Antioxidant activity was evaluated by method 1,1diphenyl-2-picrylhydrazyl (DPPH). Phylogenetic analysis showed the Endophytic fungi of S. aqueum twig have a high similarity with the Trichordemareecei twig 19MSr.B2.3. The secondary metabolites from the ethyl acetate extract of the liquid culture of the fungus were identifiedas (4-hydroxy-3-(4-hydroxyphenyl)-5-oxotetrahydrofuran-2-yl) methyl acetate with IC50= 75.13 `g/mL. The secondary metabolitescan be developed into starting molecules for potential antioxidant agents. KeywordsEndophytic Fungus, Trichordema reecei, Syzygium aqueum, Secondary Metabolite, Antioxidant Activity Received: 6 March 2021, Accepted: 12 June 2021 https://doi.org/10.26554/sti.2021.6.3.137-143 1. INTRODUCTION The Endophytic fungi are microorganisms to colonize the host plant tissue (leaves, fruit, seeds, stems, and roots) without nega- tive eects and having a mutuallybenecial relationship. Plants have the function of protecting and providing nutrients for En- dophytic fungi. In turn, Endophytic fungi will produce bioac- tive compounds that help the host plants improve nutritional status for growth, resistance to herbivores and diseases, and physical stress tolerance (Specian et al., 2012; Mbilu et al., 2018; El-hawary et al., 2020). Endophytic fungi are the ba- sis of many promising natural products with varied chemi- cal structures, higher biodiversity, and attractive bioactivity. A few researchers focused on nding secondary metabolites from Endophytic Fungi because they have been identied as a storehouse of bioactive secondary metabolites such as antibi- otics, antiviral, antiprotozoal, anti-diabetic antiparasitic, anti- cancer, antioxidant, and immunosuppressant compounds that can be used in the agricultural, pharmaceutical and food indus- tries. These compounds are alkaloids, terpenoids, steroids, iso- coumarin derivatives, lactones, quinones, avonoids, phenols, indole derivatives, lignins, tannins, anthraquinones, xanthones, phenylpropanoids, phenolic acids, and peptides (Khan et al., 2019; Manganyi and Ateba, 2020). Natural products based on endophytic fungi are considered as one of the most relevant sources of discovery and molecular diversity for new drugs. Several studies show up to 51% of the bioactive metabolites produced from endophytic fungi have an unknown chemi- cal structure. They can produce metabolites similar to those produced by the plant itself or new compounds produced by plants. This group of microbes represents a great potential in biotechnology for the discovery of new drugs (Cruz et al., 2020; El-hawary et al., 2020). Syzygium aqueum, known as water guava, is native to Indone- sia and Malaysia. The parts of this plant are used for traditional medicine, such as drugs for gastrointestinal disorders, bleeding, diabetes, inammation, hypertension, antivirus, and analgesics. The use of this plant for medicine cannot be separated from https://crossmark.crossref.org/dialog/?doi=10.26554/sti.2021.6.3.137-143&domain=pdf https://doi.org/10.26554/sti.2021.6.3.137-143 Habisukan et. al. Science and Technology Indonesia, 6 (2021) 137-143 its phytochemical composition including avonoids, avonoid glycosides, chromone glycosides, terpenoids, steroids, tannins, phenols, phenyl glycosides, and acylphloroglucinol derivatives These compounds have activities as antioxidants, antidiabetic, anticancer, toxicity, antiviral, antibacterial, antifungal, anti- inammatory and anthelmintic which functions in traditional medicine. This S. aqueum plant is rich in bioactive compounds, so it is a potential host to obtain endophytic fungi that produce bioactive compounds (Aung et al., 2020; Palanisamy et al., 2011; Sobeh et al., 2018; Osman et al., 2009). Literature studies show that there are many bioactive com- ponents in this plant, but no research has been found on the isolation of endophytic fungi and their pure compounds from S. aqueum twigs. Therefore, this paper describes the isolation and identication of endophytic fungi from the twigs of S. aqueum, followed by the isolation, identication, and evaluation of the antioxidant activity of these secondary metabolites. In this study, it is known whether the secondary metabolites of this endophytic fungus have the potential to be developed as a source of antioxidants. 2. EXPERIMENTAL SECTION 2.1 Materials The chemicals used such as ethanol 70%, distilled H2O, sodium hypochlorite (NaOCl) (Merck), growth medium for fungi: PDA and PDB (Merck), silica gel for gravity column (Merck), TLC silica gel 60 F254 from Merck (Art.5554), DPPH (Sigma- Aldrich), and organic solvents (Merck). 2.2 Plant Material Collection The twig of S. aqueum was collected from Indralaya, Ogan Ilir Regency, South Sumatra, Indonesia. Plant identication was conducted at the Biosystematics Laboratory, Biology, FMIPA Sriwijaya University, with herbarium ID 237064. 2.3 Isolation of Endophytic Fungal Isolation of endophytic isolates followed the procedure by Mbilu et al., 2018; Habisukan et al., 2021 with a few mod- ications. S. aqueum twig is washed under running water to remove impurities. The surface of the twigs was sterilized by immersing the plant material in 75% ethanol for 2 minutes followed by 4% NaOCl for 1 minute and then rinsed twice using sterile distilled water. The plant material is allowed to dry on sterile lter paper and then cut to size 2 × 0.5 cm. Fur- thermore, three pieces were placed on a petri dish containing PDA (200 g potato, 20 g dextrose, and 15 g agar in 1000 mL of H2O, supplemented with 100 mg/L of chloramphenicol). Colonypuricationwasdonebyfurthersubculturing the fungal colonies in PDA until pure isolates were acquired. 2.4 Identication of Endophytic Fungal Identication of endophytic isolates followed the procedure by Yohandini et al., 2015 which is molecularly by comparing the similarity of sample sequences with a web-based database known as BLAST (Basic Local Alignment Search Tool). 2.5 Cultivation of Endophytic Fungal Pure isolates of Endophytic fungi were cultured at 6 asks con- taining 200 mL of PDB medium (consisting of 20 g dextrose monohydrate, 200 g potatoes, and 1 L (water). Six mycelia agar plugs (5 × 5 mm2) were inoculated into each erlenmeyer and then incubated at room temperature under static condi- tions for four weeks. After the incubation was complete, the media was ltered and extracted three times using ethyl acetate. Ethyl acetate extract was evaporated with a rotary evaporator until a concentrated extract (6.4 g) and stored in a glass bottle (Gustianingtyas et al., 2020; Elta et al., 2019). 2.6 Antioxidant activity test by DPPH method Antioxidant activity test of ethyl acetate viscous extract using the DPPH method following the procedure by Budiono et al, 2019 with modication. The sample concentrations were prepared with variations of 200, 100, 50, 25, 12.5, 6.25, 0.0 `g/mL, and gallic acid as a positive control. The percentage of DPPH absorption inhibition with the following formula: % Inhibition = Control Absorbance - Sample Absorbance Control Absorbance ×100% 2.7 Isolation and Identication of Secondary Metabolite The crude extracts were separated using column chromatog- raphy (stationary phase: silica gel) and eluted with a graded eluent (n-hexane: ethyl acetate: methanol). Eluate droplets were collected every 10 ml in the vials and monitored by TLC analysis to group the vials into subfractions. The subfraction was identied as having secondarymetabolites, then puried by column chromatography until a pure compound was obtained. The structure was identied by NMR 1D and 2D (Budiono et al., 2019). 3. RESULTS AND DISCUSSION 3.1 Isolation of Endophytic Fungal S. aqueum twig has isolated EF labeled with B11. The molecu- lar identication yields sequence assembly 577 bp endophytic fungus B11 is as follows: CTCCCAAACCCCAATGTGAACGTTACCAATCTGTTG CCTCGGCGGGATTCTCTGCCCCGGGCGCGTCGCAG CCCCGGATCCCATGGCGCCCGCCGGAGGACCAACTC AAACTCTTTTTTCTCTCCGTCGCGGCTTCCGTCGCG GCTCTGTTTTACCTTTGCTCTGAGCCTTTCTCGGCG ACCCTAGCGGGCGTCTCGAAAATGAATCAAAACTTT CAACAACGGATCTCTTGGTTCTGGCATCGATGAAGA ACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGA ATTCAGTGAATCATCGAATCTTTGAACGCACATTGC GCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAG CGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCG GCGTTGGGGATCGGCCCCTCACCGGGCCGCCCCCG AAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGC GCAGTAGTTTGCACACTCGCACCGGGAGCGCGGCG CGGCCACAGCCGTAAAACACCCCAAACTCTGAAATG TTGACCTCGGATCAGGTAGGAATACCCGCTGAACTT © 2021 The Authors. Page 138 of 143 Habisukan et. al. Science and Technology Indonesia, 6 (2021) 137-143 Figure 1. Phylogenetic Tree of the Endophytic Fungal B11 AAGCATA. The strain B11 has the accession number MW52 7423. The endophytic fungal B11 had the highest homology with Trichordemareecei twig 19MSr.B2.3. Phylogenetic analysis follows the method described shown in Figure 1. 3.2 Isolation and Identication of Secondary Metabolite Separation of concentrated EtOAc extracts 3.2 g (silica gel: 70-230 mesh; 50 g) obtained subfraction B (0.9 g) with a purple stain on TLC analysis. Purication was done by column chromatography (silica gel 70-230 mesh; 20 g) with eluent n-Hexane - EtOAc (7:3 → 0:10) yielded two fractions (B1– B2). The B2 fraction (426 mg) was puried again by column chromatography until compound 1 (244 mg) was obtained. Figure 2 shows the 1H-NMR spectrum of the isolated com- pound 1. The special characteristic of the para-substituted aromatic ring in compound 1 can be identied in the H-NMR spectrum by the presence of a double pair of aromatic proton signals at 𝛿H 7.74 and 8.20 ppm each of which has a value of J= 8.5 Hz with two integration. Another characteristic that can be identied from the C-NMR spectrum is the presence of a high-intensity carbon signal at 𝛿C 128.3 and 123.9 ppm for two carbons in the same chemical shift respectively. HMQC spectrum analysis showed that the two high-intensity carbons bind to aromatic protons equivalent at 𝛿H 7.74 and 8.20 ppm respectively. A methyl acetate-substituted cyclic lactone ring- side group was identied by the presence of two carbonyl ester Figure 2. The 1H-NMR spectra of compound 1 signals at 𝛿C 164.7 and 170.9 ppm respectively and a singlet signal at 𝛿H 2.00 ppm with triple integration for the methyl proton. Two multiplet proton signals at 𝛿H 4.35 and 4.24 ppm are the methylene groups of the methyl ester which are strengthened by DEPT 135 and HMQC spectrum, which are bound to 𝛿C 63.9 ppm. The position of the phenol hydroxyl group was determined based on the 13C-NMR spectrum analy- sis. oxyaryl carbon at 𝛿C 150.6 ppm while the other hydroxyl groups in the lactone ring were identied as bound to carbon 𝛿C 67.4 ppm. 1H and 13C NMR spectrum shows that the basic structure of compound 1 is a para-substituted aromatic ring of which the hydroxyl group and ester. In acetone, the number of hy- droxyl groups can be seen from the proton signal that is not bound to carbon. The HMQC spectrum (Figure 3) shows the presence of seven single-bonded 1H-13C correlations, two correlations for aromatic rings and ve correlations for ester substituents. There are two uncorrelated protons 1H-13C one bond identied as aromatic hydroxy protons (𝛿H 7.68 ppm) and aliphatic hydroxyl protons (𝛿H 5.48 ppm). The HMBC spectrum in Figure 4 shows that the ester group substituent is a pentacyclic lactone ring. Methine carbon at 𝛿C 67.4 ppm (C-4’) binds a hydroxyl group that correlates 1H-13C three bonds with signal C-3’ and a methine proton which correlates two bonds with carbonyl carbon (C-5’). Methine carbon at 𝛿C 71.2 ppm (C-2’) binds a methyl ester group is indicated by the 1H-13C correlation of two methylene proton bonds with C-2’ and the triple bond correlation with C-3’ and C carbonyl methyl ester. The COSY spectrum in Figure 5 shows the aromatic pro- tons at 𝛿H 7.74 ppm 1H-1H correlated three bonds with aro- © 2021 The Authors. Page 139 of 143 Habisukan et. al. Science and Technology Indonesia, 6 (2021) 137-143 Figure 3. The 13C-NMR (A) DEPT 135 (B), HMQC (D) Spectra of Compound 1 and HMQC Correlation (D) matic protons at 𝛿H 8.20 ppm and correlated long distances with four bonds with methine protons at 𝛿H 4.45 ppm. Fur- thermore, it was identied the correlation of 1H-1H three methine proton bonds at 𝛿H 4.45 ppm with methine pro- tons at 5.26 ppm. This shows the two equivalent aromatic protons are in the ortho position. These results support the proposed structure that compound 1 is a para-substituted ben- zene ring that is directly attached to the lactone substituent methine carbon. Compound 1 was identied as (4-hydroxy- 3- (4-hydroxyphenyl)-5-oxotetra hydrofuran-2yl) methyl acetate. The molecular structure with the numbering of carbon atoms and the chemical shift placement of the carbon and protons is shown in Figure 5. Based on a literature study, compound 1 has a dierent phenyls structure with Phenyls of S. aqueum and other Syzygium spp. This shows that compound 1 is produced by endophytic fungi and the host does not have the gene to promote the compound. there is no genetic evolution between fungal genes and host genes in promoting their secondary metabolites. The Trichodermaspp inhabiting healthytissues of host plants asendophytic fungi (Eltaetal.,2014). Endophytic fungi from Trichoderma family can can produce a broad variety of bioac- tive compounds. Ding et al., 2019 isolated an antibacterial compound derived from isocumarin, chromone derivatives, and lichexanthone from the endophytic fungus Trichoderma harzianum isolated from the leaves of Ficus elastic. Harwoko et al., 2019 reported antifungal and cytotoxic compounds, namely Dithiodiketopiperazines produced by the endophytic fungi Trichoderma harzianum isolated from Zingiber ocinale Figure 4. The HMBC spectra (A) and HMBC Correlation (B) of Compound 1 Figure 5. The COSY Spectra (A) and COSY Correlation (B) of Compound 1 © 2021 The Authors. Page 140 of 143 Habisukan et. al. Science and Technology Indonesia, 6 (2021) 137-143 Table 1. The NMR Data of Compound 1 No. C 𝛿C ppm 𝛿H ppm (ΣH. multiplicity. J (Hz)) HMBC COSY 1 148.2 - - - 2 128.3 7.74 (1H, d, 8.5) 71.2; 128.3; 148.2 8.20; 4.45 3 123.9 8.20 (1H, d, 8.5) 123.9; 148.2; 150.6 7.74 4 150.6 - - - 5 123.9 8.20 (1H, d, 8.5) 123.9; 148.2; 150.6 7.74 6 128.3 7.74 (1H, d, 8.5) 71.2; 128.3; 148.2 8.20 2’ 71.2 5.26 (1H, m) 128.3; (150.6k) 4.45 3’ 55.1 4.45 (1H, d, m) 63.9 5.26 4’ 67.4 6.35 (1H, d, 1) 164.7 - 5’ 164.7 - - - 1” 170.9 - - - 2” 20.7 2.00 (3H, s) 170.9 - 3” 63.9 4.35 (1H, m) 55.1; 71.2; 170.9 - 4.24 (1H, m) 55.1; 71.2; 170.9 4’-OH - 5.48 (1H, d, 4.5) 55.1 - 4-OH - 7.68 (1H, br) - - Figure 6. Compound 1 as (4-hydroxy-3-(4-hydroxyphenyl)-5- oxotetrahydrofuran-2-yl)methyl acetate (Palanisamyet al., 2011)). Besides that, taxol compounds from Trichoderma sp. isolated from Taxus chinensis (Liu et al., 2009). Five sesquiterpene compounds from Trichoderma sp. from the stem of Paeonia delavayi (Wu et al., 2011), Trichodermin com- pounds (4b-acetoxy-12, 13-epoxytrichothec-9-ene) from T. koningiopsis VM115 isolated from Vinca plants have antifungal activity (Leylaie and Zafari, 2018). This trichodermin com- pound is also produced by the endophytic compound Tricho- dermabrevicompactum isolated from Alliumsativum (Shentu et al., 2014). Compound 1 has not been found in the endophytic fungus Trichoderma sp. so that it can complete the chemical prole of the genus. Compound 1 has moderate antioxidant activitywith a value of IC50 = 75.13 `g/mL, while gallic acid as a standard antioxi- dant has a value of IC50 = 11.45 µg / mL. However, higher antioxidant activity can be obtained by semisynthesis of com- pound 1 to produce phenyl similar to gallic acid. The OH group in the aromatic ring of compound 1 is ortho-directing Figure 7. Phenyls of S. aqueum and other emph Syzygium spp (Aung et al., 2020) Figure 8. Estimates Semisynthesis Reaction of Compounds 1 to Produce a New Compound which is more Active with the Standard like Structure Antioxidant Gallic Acid © 2021 The Authors. Page 141 of 143 Habisukan et. al. Science and Technology Indonesia, 6 (2021) 137-143 so that the electrophilic reaction of Nitration (2 mol HNO3 / H2SO4) is followed by reduction of the nitro group (Fe/HCl) will produce two amine groups on both ortho sides of the hy- droxyl group. The next step is the transformation of the amine func- tional group by reaction with cold nitrous acid (HONO) in HCl solution to yield aryldiazonium chlorides (ArN2+CI). Dis- placement by -OH may be prepared from diazonium salts by reaction with hot aqueous acid. This series of reactions is esti- mated to produce the compound (4-hydroxy-5-oxo-3- (3,4,5- trihydroxyphenyl) tetrahydrofuran-2-yl) methyl acetate (Fig- ure 8) (Fessenden and Fessenden, 1982). 4. 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Hayati Journal of Biosciences, 22(3); 143–148 © 2021 The Authors. Page 143 of 143 INTRODUCTION EXPERIMENTAL SECTION Materials Plant Material Collection Isolation of Endophytic Fungal Identification of Endophytic Fungal Cultivation of Endophytic Fungal Antioxidant activity test by DPPH method Isolation and Identification of Secondary Metabolite RESULTS AND DISCUSSION Isolation of Endophytic Fungal Isolation and Identification of Secondary Metabolite CONCLUSIONS ACKNOWLEDGEMENT