RAPID SCREENING OF ANTIBACTERIAL AND ANTIOXIDANT METABOLITES FROM ENDOPHYTIC FUNGI ISOLATED FROM Papuacedrus papuana BY TLC-BIOAUTOGRAPHY

PurposeBalanophora polyandra Griff. is a holoparasitic medicinal plant that produces compounds with antibacterial and antioxidant activities. Plant endophytic fungi are an abundant reservoir of bioactive metabolites for medicinal exploitation, and an increasing number of novel bioactive compounds are being isolated from endophytic fungi. The present study investigated the diversity of culturable endophytic fungi from the roots of holoparasite B. polyandra to explore active strains and metabolites. In addition, the antibacterial and antioxidant activities of 22 strains cultured from B. polyandra were also evaluated.MethodsThe endophytic fungi were identified according to their colony morphology and ITS-5.8S rDNA sequencing. TLC-MTT-Bioautography assays and DPPH radical scavenging assays were employed to assess the antibacterial and antioxidant activities of ethyl acetate extracts of the endophytic fungi.ResultsOne hundred and twenty-five endophytic strains were isolated from the roots of B. polyandra, including 70 from female samples and 55 from male samples. Of them, twenty-two distinct isolates representing 15 genera and 22 species based on their ITS-rDNA genomic sequence were successfully identified from female and male samples of B. polyandra. The genus Calonectria was the most prevalent genus, with a CF% of 18.3, followed by the genera Clonostachys and Botryosphaeria, with CF% values of 13.4 and 10.0, respectively. Interestingly, the fungal extracts exhibited broad-spectrum antibacterial activities against gram-positive and gram-negative bacteria, as well as potential antioxidant activities with IC50 values ranging from 0.45 to 6.90 mg/mL. Among them, endophytes Bpf-10 (Diaporthe sp.) and Bpf-11 (Botryosphaeria sp.) showed the strongest biological activities and more abundant secondary metabolites.ConclusionsThis study reported the diversity of endophytic fungi from the roots of B. polyandra and the antibacterial and antioxidant activities of the crude extracts for the first time. The results revealed that B. polyandra contains diverse culturable endophytic fungi that potentially produce natural antibacterial and antioxidant compounds with great value to the agriculture and pharmaceutical industries.

Endophytes from an unexplored plant such as Spatterdock (SD) Nuphar lutea variegata could be a dependable producer of novel secondary metabolites active against drug resistance organisms. This study explored the diversity of fungal endophytes from mid-rib (MR) and leaf blade (LB) of Nuphar lutea variegata as well as the antimicrobial and antioxidant potentials of their secondary metabolites. Morphological features observed showed four species were isolated. A broad-spectrum antimicrobial activity observed to be concentration-dependent was observed for all the fungal crude extracts 1 mg/mL. Crude extract of SD-LB3 showed the best antibacterial activities against S. aureus and P. aeruginosa with a minimum inhibitory concentration of 0.5 and 0.25 µg/mL respectively. The antioxidant capacities of the fungal crude extracts showed a better neutralizing effect of free radicals compared to the standard Quercitin. Effective inhibition of free radicals was observed for all the fungal extracts from 20 to 100 mg/mL (96.7 – 99.3 %). The highest antioxidant activity was shown by the extracts of fungi isolated from the leaf blade with an average of 98 to 99.2 % at the tested concentrations of 20 to 100 mg/mL. Our study provides evidence of diverse endophytic fungi associated with Nuphar lutea variegata, and the antibacterial and antioxidant activities observed confirm their capacities as producers of bioactive secondary metabolites. This study is the first to isolate endophytic fungi form Nuphar lutea variegata and screen the antimicrobial and antioxidant activities of their secondary metabolites.

Objective: The aims of this study were to identify the endophytic fungi from Capsicum annuum L. and Allium cepa L., to determine antioxidant and antimicrobial activity of ethyl acetate extract of endophytic fungi isolated from C. annuum and A. cepa. Methods: Endophytic fungi was isolated with potato dextrose agar (PDA) from fruits of C. annuumand bulbs of A. cepa. Isolate of endophytic fungi was molecular identified to know the species or genus. Cultivation was carried out on rice media, 4 weeks on room temperature and the extraction by maceration using ethyl acetate. Antioxidant activity were tested by DPPH method. While antibacterial activity was tested by disk diffusion methods and microdilution methods. Results: Five isolates of endophytic fungi from red and green fruits of C. annuum and bulb of A. cepa have been isolated and the species or the genus have been confirmed. KCM 1 and KCM 2 isolates endophytic fungi from the red fruits of C. annuum were confirmed as Diaporthe sp and Chaetomium globosum. The KCH 1 isolate from green fruits of the C. annuum was confirmed as Trametes hirsuta. The KBM 1 and KBM 2 isolates from A. cepa were confirmed as Schizophyllum commune and Phlebia sp. The highest antioxidant and antibacterial activity was exposed by ethyl acetate extract of S. commune. Conclusion: Five isolates endophytic fungi from C. annuum and A. cepa were Diaporthe sp, C. globosum, T. hirsuta, S. commune and Phlebia sp. Ethyl acetate extract of S.commune gave highest antioxidant and antibacterial activity.

Abstract. Elfita, Oktiansyah R, Mardiyanto, Widjajanti H, Setiawan A. 2022. Antibacterial and antioxidant activity of endophytic fungi isolated from Peronema canescens leaves. Biodiversitas 23: 4783-4792. Peronema canescens Jack. or sungkai is a plant found in tropical rainforests. Also known as Peronema heterophyllum Miq., it belongs to the Verbenaceae family and grows widely in Thailand, Malaysia, and Indonesia. The local people believe that the antibacterial and antioxidant properties of its leaves increase immunity. Bioactivities of endophytic fungi associated with medicinal plants are currently of great interest due to their potential for development and use in the pharmaceutical, agricultural, and industrial fields. In this study, we investigated the antibacterial and antioxidant properties of an extract of leaves from P. canescens host plant and compared them with those of extracts from an endophytic fungus. Antibacterial and antioxidant compounds derived from active extracts of endophytic fungi have the capability to be developed as a source of antibacterial and antioxidant compounds equivalent to those of their host. In this study, endophytic fungi were isolated from the fresh tissue of P. canescens leaves and identified morphologically. Their antibacterial activity was tested using the Kirby-Bauer method, and their antioxidant activity was tested using the DPPH method. The potential antibacterial and antioxidant activities of the endophytic fungi extracts were identified molecularly, and the compounds were isolated using column chromatography. A structural determination of the antibacterial and antioxidant compounds was conducted by spectroscopy involving NMR 1D and 2D. Twelve of the endophytic fungi were gained from the P. canescens leaves, namely RD1-RD12. The RD6 isolate showed the greatest potential for antibacterial and antioxidant activity, and its molecular identification was expressed as Penicillium oxalicum. The pure compound produced was a yellowish white solid with strong antibacterial and antioxidant activity. Based on an analysis of spectroscopy NMR 1D and 2D, compound I was identified as 3-(2,6-dihydroxyphenyl)-2-hydroxyacrylic acid. The study demonstrated that endophytic fungi associated with medicinal plants and functioning as antibacterial and antioxidant agents have the potential to produce compounds as strong as those of the host plant. These compounds represent a potential new source for antibacterials and antioxidants.

Schinus terebinthifolius Raddi is a medicinal plant widely used for the treatment of various diseases. The secondary metabolites responsible for the pharmacological properties can be produced directly by the plant or by endophytic fungi. The objective of this study was to evaluate the diversity of endophytic fungi of different parts of S. terebinthifolius and to identify chemical compounds produced by endophytes and their antioxidant and antibacterial activities. For this, fruits, stem bark and roots were dried, ground and placed in fungal growth medium. The selected endophytes were grown and subjected to extraction with ethyl acetate. DPPH, FRAP, β-carotene bleaching and antimicrobial assays were performed. The phylogenetic tree was elaborated, encompassing 15 different species. The fungal extracts showed hydroxybenzoic acids and 1-dodecanol as predominant compounds. All fungal extracts exhibited antioxidant activity. The fungal extracts exhibited bactericidal and bacteriostatic activities against Gram-positive and Gram-negative bacterial ATCC strains and against methicillin-resistant nosocomial bacteria. Among the 10 endophytic fungi evaluated, the extract of the fungus Ochrocladosporium elatum showed higher phenolic content and exhibited higher antioxidant and antibacterial activities in all tests. Together, the results increase the known diversity of S. terebinthifolius endophytic fungi, secondary metabolites produced and their antioxidant and antibacterial activities.

BackgroundCentella asiatica L. (Apiaceae), a green leafy vegetable, has been used as a popular folk medicine in Bangladesh in the treatment of various ailments. The plant is reported to contain phenolics and flavonoids, but their bioactive potentials are not yet completely known. The present study was designed to investigate the role of the phenolic and flavonoids in the antioxidant, cytotoxicity, antibacterial and thrombolytic activities.MethodsFour solvent fractions viz. methanol (MSF), ethyl acetate (ESF), chloroform (CSF) and petroleum ether (PSF) were prepared from the dried powder of the whole plant by the modified Kupchan method. Total phenolic content and flavonoid content were determined by Folin Ciocalteu method and aluminum chloride colorimetric method, respectively. The antioxidant activity was assessed by the DPPH radical scavenging and total antioxidant capacity assays. The antibacterial activity was determined by the disc diffusion method and cytotoxicity was evaluated by the brine shrimp lethality bioassay. Thrombolytic activity was assayed using streptokinase as standard.ResultsQualitative analysis of phytochemical revealed the presence of phenolics and flavonoids along with other bioactive constituents. Among the extractives, CSF contained the highest content of phenolics (155.46 ± 0.52 mg GAE/g) and flavonoids (345.17 ± 1.12 mg QE/g) and exhibited the most potent antioxidant activity in terms of total antioxidant capacity (179.01 ± 0.89 mg AAE/g) and DPPH scavenging ability (IC50; 15.31 ± 0.32 µg/mL). Similarly, CSF showed the highest cytotoxicity with LC50 values of 13.80 ± 0.23 µg/mL, and thrombolytic activity with 43.94 ± 0.62% clot lysis. The fraction also exhibited broad spectrum antibacterial activity. A significant correlation was observed between the flavonoid content and total antioxidant activity (r2 = 0.894, p < 0.05), while high correlation was seen between phenolic and flavonoid content and DPPH radical scavenging, total antioxidant capacity and cytotoxicity (r2 = 0.612–0.928). Similarly, a positive correlation was found between phenolic and flavonoid content with thrombolytic and antibacterial activities.ConclusionThese results revealed that C. asiatica is a rich source of phenolics and flavonoids and correlated with antioxidant, cytotoxicity, antibacterial and thrombolytic activities. Hence isolation of phenolics and flavonoids from this plant may offer potential candidates which may be effective in the prevention of many chronic diseases.

The dried tuber of Alisma orientale (Sam.) Juzep. (AOJ) is a traditional Chinese medicine with high medicinal value. The endophytic fungi of medicinal plants are a treasure house of natural compounds. However, there is a lack of research on the diversity and biological activity of endophytic fungi of AOJ. In this study, high-throughput sequencing technology was used to study the diversity of endophytic fungi in the roots and stems of AOJ, and endophytic fungi with a high output of phenols and flavonoids were screened by chromogenic reaction, and the antioxidant and antibacterial activities and chemical constituents of crude extracts of their fermentation broth were studied. A total of 3,426 amplicon sequence variants (ASVs) belonging to 9 phyla, 27 classes, 64 orders, 152 families, and 277 genera were identified from AOJ. There were significant differences in the endophytic fungal communities of AOJ roots and stems, as well as in the endophytic fungal communities of triangular AOJ and circular AOJ. In addition, 31 strains of endophytic fungi were isolated from AOJ, of which 6 strains had good antioxidant and antibacterial activities. The crude extract of YG-2 had the strongest free radical scavenging ability and bacteriostatic ability, and its IC50DPPH, IC50ABTS, and IC50⋅OH values were 0.009 ± 0.000 mg/mL, 0.023 ± 0.002 mg/mL, and 0.081 ± 0.006 mg/mL, respectively. The results of LC-MS showed that the main component of the crude extract of YG-2 was caffeic acid (10.12 μmol/g). Overall, the results of this study preliminarily elucidated the diversity and community composition of endophytic fungi of AOJ, indicating that AOJ endophytic fungi have abundant secondary metabolites and good antioxidant and antibacterial activities. This study provides an important reference for further research, development and utilization of AOJ endophytic fungi and a theoretical basis for the further development of the endophytic fungus YG-2 (Chaetomium globosum) as a source of antioxidants.

ABSTRACTObjective: To investigate the antioxidant activity of methanolic extract of endophytic fungi isolated from Calotropis procera plant.Methods: The endophytic fungi were isolated from explants of leaf, stem, and flower of C. procera on potato dextrose broth (PDB) medium. The fungalisolates were mass cultured in PDB. The methanolic extracts of these fungi were prepared and evaluated for the antioxidant activity by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay and reducing power assay. The extracts were characterized for the presence of phenolics andflavonoids.Results: A total of 8 endophytic fungi were isolated from C. procera explants. Out of these 8 fungal isolates, the crude extracts of two fungi, i.e. CPIMR-2and CPIL-1 showed positive antioxidant activity. From the morphological characteristics, the isolates CPIMR-2 and CPIL-1 were identified as Penicilliumspp. and Aspergillus spp., respectively. Both the isolates were found to possess antioxidant potential with % inhibition value of 81.16% and 80.97%in the DPPH radical scavenging assay. The phytochemical screening of the methanolic extracts showed the presence of phenolics and flavonoids. Thetotal flavonoids content in Penicillium species and Aspergillus species were found to be 130.50 μg/mg and 94.91 μg/mg of extracts, respectively. Thetotal phenolic content was found to be 9.16 μg/mg and 12.13 μg/mg of extracts in Penicillium species and Aspergillus species, respectively.Conclusions: Endophtyic fungi were found to be present in medicinal value plant C. procera. The probable bioactive component for antioxidantactivity possessed by the fungi would be the flavonoids and phenolics. These metabolite produced by endophytic fungi from C. procera need to beexplored further as potential source of novel natural antioxidant compound.Keywords: Endophytes, Calotropis procera, Antioxidant, Radical scavenging, Reducing power, Flavonoids.

Phenolic and alkaloid extracts of Pangium edule Reinw (Flacourtiaceae) seed were investigated for their antioxidative activities using DPPH radical scavenging and I²-carotene bleaching assays. The extracts were evaluated for antibacterial activity against Salmonella typhimurium and Listeria monocytogenes. The acetone extract with higher phenolic content (22.22±0.05 mg GAE g-1) showed the most potent antioxidative activity in both DPPH radical scavenging and I²-carotene bleaching assays as compared to other extracts. The phenolic extract seems to have stronger inhibitory against L. monocytogenes than S. typhimurium. The free phenolic acid extract was found to have the highest Minimum Inhibition Concentration (MIC) among the seed extracts, indicates its weak antibacterial activity against both bacteria. Nevertheless, both tested pathogens were killed at the Minimum Bactericidal Concentration (MBC) of 30.3 and 55.5 mg mL-1, respectively, for the phenolic extracts. Significant correlation (p<0.05) was observed between the total phenolic content and its antioxidative activity (r = 0.878) as well as antibacterial (r = 0.840) activity suggesting that phenolics of the seed extract could be potential sources of natural antioxidant and antibacterial. © 2009 Asian Network for Scientific Information.

Avicennia marina, a mangrove species commonly found along coastal areas, plays both ecological and pharmacological roles, with its plant parts exhibiting antioxidant and antibacterial activities. This study aimed to investigate the diversity of endophytic fungi from various organs of A. marina collected from mangrove ecosystems and to explore and analyze their antioxidant and antibacterial activities. Endophytic fungi were isolated from the roots, stems, and fruits of A. marina using PDA medium and were morphologically identified. Each fungal isolate was cultivated in PDB medium for 4 weeks under static conditions, followed by extraction to obtain concentrated extracts. Antioxidant and antibacterial activities were assessed using the DPPH method and disk diffusion assay. A total of 23 fungal isolates were obtained from the roots, stems, and fruits of A. marina. The identification results showed that the root isolates had the highest genus diversity, followed by the stem and fruit isolates. The highest distribution of antioxidant and antibacterial activities was observed in the endophytic fungal extracts from fruits, followed by those from roots and stems. Notably, the majority of the 23 endophytic fungal extracts exhibited strong antioxidant and antibacterial activities. Isolates AMF3 and AMF6showed the most potent antioxidant activity, classified as very strong, with IC50 values below 20 ug/mL. Morphological identification revealed AMF3 as Neopestalotiopsis sp. and AMF6 as Aspergillus niger. This study highlights the potential of Neopestalotiopsis sp. and Aspergillus niger endophytic fungi from A. marina fruits as sources of natural antioxidant and antibacterial compounds, offering valuable insights for biotechnological applications of mangrove-associated endophytes.

Endophytic fungi produce bioactive metabolites that exhibit antibacterial, antioxidant, and anti-cancer activity. Rui (Harrisonia perforata (Blanco) Merr.) is a traditional medicinal plant proven to be an antibacterial and anticancer. Endophytic fungi live in plant tissues without damaging or producing chemicals that infect the host cell. Endophytes produce the same and similar compound as an original plant. This study aimed to isolate the endophytic fungi from Rui and determine their antibacterial, antioxidant, and cytotoxic activity. 13 fungal isolates were successfully isolated using PDA for seven days at 30°C. Molecular identification using the ITS1 and ITS4 DNA sequences only revealed six species: Diaporthe sp., Phomopsis sp., Aspergillus tubingensis, Aspergillus viridinutans, Pseudofusicoccum sp., and Daldinia eschscholtz. Seven isolates showed antibacterial activity based on plate assay. Aspergillus tubingensis and RA-1 exhibited antibacterial activity against Staphylococcus aureus and Salmonella typhi, with the MIC and MBC starting from 24 to 32 mg/ml. The DPPH assay showed the most increased antioxidant activity in Daldinia eschscholtzii with an IC50 of 98.14±2.39 µg/ml. The Brine Shrimp Lethality Test (BSLT) showed the highest potential cytotoxic activity, as shown by Aspergillus tubingensis and Phomopsis sp. with LC50 of 7.78±7.48 and 30.83±0.39 µg/ml. In conclusion, our study demonstrates that the fungal extract from Rui could be a source of antibacterial, antioxidant, and cytotoxic agents.

Abstract. Syarifah, Elfita, Widjajanti H, Setiawan A, Kurniawati AR. 2021. Diversity of endophytic fungi from the root bark of Syzygium zeylanicum, and the antibacterial activity of fungal extracts, and secondary metabolite. Biodiversitas 22: 4572-4582. The decoction of the root bark of Syzygium zeylanicum has been used as traditional medicine, such as for treating pathogenic bacterial infections. Endophytic fungi that live in medicinal plant tissues have a high species diversity and biological activities correlate with their host. Therefore, this study aimed to explore the diversity of endophytic fungi from the root bark of S. zeylanicum and to determine the antibacterial activity of endophytic fungi and their secondary metabolites. In this study, we isolate and identify the endophytic fungi from the root bark of S. zeylanicum, continued by screening their antibacterial activity against two Gram-negative bacteria (Escherichia coli InaCCB5 and Salmonella thypi ATCC1048 and two Gram-positive bacteria (Staphylococcus aureus InaCCB4 and Bacillus subtilis InaCCB1204) by the Kirby-Bauer method. The fungal extract with the highest antibacterial activity proceeded with the isolation and determination of the structure of their bioactive compounds. The isolates were morphologically identified. Isolates that showed strong antibacterial activity were identified by molecular identification. Isolation of bioactive compounds was carried out by chromatographic techniques and the determination of the structure of pure chemical compounds was performed by the spectroscopic analysis. In total, there were 8 isolates of endophytic fungi were obtained from the root bark of S. zeylanicum, namely SZR1 – SZR8. SZR2 isolate has the highest antibacterial activity. Molecular identification through phylogenetic analysis showed that SZR2 isolate had high similarity with Penicillium brefeldianum. Isolation of bioactive compounds from SZR2 produced compound 1 in the form of light yellow crystals which showed strong antibacterial activity against S. typhi, E. coli, and B. subtilis with MIC values of 64 g/mL. Compound 1 was identified as p-hydroxybenzaldehyde, which was also obtained in its host. In conclusion, the endophytic fungus Penicillium brefeldianum produces similar secondary metabolites and antibacterial activity as its host plant.

Natural products from endophytic fungi have a wide range of medicinal applications, including antibacterial and antioxidant agents. The research aimed to evaluate the antibacterial and antioxidant activities of compounds derived from endophytic fungi isolated from medicinal plants collected from Simeulue Island by TLC dot-blot and microdilution assays. Eighty-one fungal extracts were screened for antibacterial and antioxidant activities. Antibacterial activity was evaluated against Gram-positive and negative bacteria, i.e., Staphylococcus aureus InaCC B-4 and Escherichia coli InaCC B-5, respectively. Antioxidant activity was carried out by using free radical scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH). The results of the TLC bioassay revealed that the fungal extracts have activities as antibacterial and antioxidant are 65 (against S. aureus), 57 (against E. coli), and 48 extracts (DPPH free radical scavenger). Two extracts, Phomopsis sp. 48BtSi-2.1 and Xylaria sp. 55DnSi-1.1, showed strong antibacterial activity against S. aureus with MIC value of 32 μg/ml. Furthermore, nine extracts (Schizophyllum sp. 20DnSi-1; Hyphomycetes 36BhSi-1.1; Phomopsis sp. 36DnSi-2.1; Schizophyllum sp. 39DnSi-1.1; Xylaria sp. 39RpSi-2.1; Phomopsis sp. 41BtSi-1.1; Phomopsis sp. 48BtSi-2.1; Lasiodiplodia sp. 48BtSi-3.1; Schizophyllum sp. 50DnSi-3) were strong activity against E. coli with MIC value ranges from 32 to 64 μg/ml. One extract, Xylaria sp. 04BtSi-2.2 has strong antioxidant activity as DPPH free radical scavenger with an IC50 value of 21.66 μg/ml (AAI value of &gt;1).

As a medicinal plant, Conyza blinii is known to contain a wealth of bioactive constituents, including flavonoids, terpenes, and triterpenoid saponins, which contribute to its anti-inflammatory and anticancer properties. Endophytic fungi, which symbiotically inhabit plant tissues, are recognized for their ability to synthesize bioactive metabolites analogous to those of their hosts. However, the potential of C. blinii-associated endophytes remains underexplored. This study aims to isolate and characterize phenols-producing endophytic fungi from C. blinii, evaluate their biological activities, and analyze their chemical components to provide new insights for drug development. During the study, 20 endophytic fungi were isolated from C. blinii. The Folin-Ciocalteu method was used to screen for strains capable of producing phenolic compounds. To assess their bioactivity, ethyl acetate extracts of different concentrations were tested for antibacterial and antioxidant activities. Antibacterial activity was evaluated using minimum inhibitory concentration (MIC) determinations, while antioxidant activity was assessed through 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical, hydroxyl radical, and superoxide anion radical scavenging assays. Additionally, liquid chromatography-mass spectrometry analysis was conducted to quantify the active components in the extracts. Among the isolated 20 endophytic fungi, four strains successfully produced phenolic compounds, with the highest total phenolic content of 77.17 ± 1.93 mg milligrams of gallic acid equivalents per gram of extract (GAE/g). All ethyl acetate extracts from the endophytic fungi exhibited good antibacterial and antioxidant properties. Notably, Fusarium circinatum demonstrated exceptional antioxidant activity, with scavenging rates for DPPH and ABTS radicals reaching 94.28 ± 0.042% and 96.60 ± 0.017%, respectively. The ethyl acetate extract of F. foetens showed remarkable antibacterial effects against Escherichia coli and Staphylococcus aureus, with MIC values as low as 0.5 mg/mL. Furthermore, liquid chromatography-mass spectrometry (LC-MS) analysis revealed that F. foetens could produce various high-value phenolic compounds, including tyrosol (626.1884 ng/mL) and homovanillic acid (369.15486 ng/mL), which hold potential pharmaceutical value. This study isolated 20 endophytic fungi from C. blinii, discovering that four strains, produced phenolic compounds with strong antioxidant and antimicrobial properties. Among them, F. circinatum exhibited the highest antioxidant activity. Additionally, the fungi produced bioactive metabolites with potential applications in health care, medicine, and agriculture. These findings highlight the potential of C. blinii endophytes for sustainable bioactive compound production.

Antileishmanial activity of Ptilostemon chamaepeuce subsp. cyprius.