Metabolites characterisation of endophytic Phyllosticta fallopiae L67 isolated from Aloe vera with antimicrobial activity on diabetic wound microorganisms

Abstract This study aimed to assess the antimicrobial activity of endophytic Phyllosticta fallopiae L67 isolated from Aloe vera against diabetic wound microorganisms and characterise their active fraction for biologically important metabolites. The dichloromethane (DCM) extract exhibited the most significant activity with inhibition zones ranging from 11.33 to 38.33 mm. The minimal inhibitory and lethality concentrations of DCM extract ranged from 78.13 to 2500.00 µg/ml and 625.00 to 5000.00 µg/ml, respectively. The extract showed teratogenicity and lethality in the zebrafish model, where peritoneal and hepatic oedema occurred at 62.50 µg/ml, and no abnormality appeared at 31.25 µg/ml. The extract also inhibited more than 82% biofilm formation. Bioassay-guided fractionation on DCM extract yielded 18 fractions and the most active fraction was subjected to UPLC-QTOF-MS/MS analysis. Flavones, stilbenes, flavanonols, isoflavonoids, phenolic glycosides and phenol derivatives were detected. In conclusion, endophytic P. fallopiae possessed bioactive metabolites with significant antimicrobial activity against diabetic wound microorganisms. Graphical Abstract


Introduction
Diabetic wounds, particularly diabetic foot ulcers, have become a major public health concern. It is a long-term complication caused by tissue necrosis and polymicrobial infections after generalised trauma. It is reported that approximately 70% of limb amputations are due to diabetic wounds. Thus, it is vital to inhibit the growth of bacteria that leads to non-healing wounds at the early stage (Atosona and Larbie 2019;Patel et al. 2019). Endophytic fungi are a group of microorganisms inhabiting the plant tissues inter-or intra-cellularly without causing any apparent diseases to the host. They are a potential reservoir of bioactive metabolites because they can readily grow in large amounts, thus conserving plant resources (Yan et al. 2019). Literature has reported that approximately 20% of plant-derived metabolites can be obtained from its associated fungi (Adeleke and Babalola 2021). They can adapt to unusual and competitive environments such as extreme temperature, pressure and pH. These adverse conditions have triggered them to produce bioactive metabolites for survival (Toghueo 2020).
Genus Phyllosticta is a taxonomically diverse group of fungi occupying different ecological niches (Wikee et al. 2013). Phyllosticta fallopiae L67 is previously isolated as a fungal endophyte residing in a medicinal plant, Aloe vera. A. vera has long been used as a traditional medicinal plant for its various beneficial attributes (Taher et al. 2020). Several bioactive metabolites have been detected in A. vera, such as chromones, anthraquinones, flavonoids, coumarins and phytosterols (Kahramano glu et al. 2019). Nonetheless, there have been no detailed phytochemical and biological activity studies of endophytic P. fallopiae. Thus, the present study explores the antimicrobial activity of P. fallopiae on diabetic wound microorganisms and characterise its bioactive fraction for biologically important metabolites.

Results and discussion
In this study, P. fallopiae L67 isolated from A. vera was ultrasonicated using n-hexane (n-Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and butanol (BtOH). Table S1 depicts the antimicrobial activity shown by different extracts of P. fallopiae against a broad spectrum of microorganisms. The DCM extract exhibited the most significant inhibitory activity, with inhibition zones ranging from 11.33 to 38.33 mm. Xu et al. (2021) have previously reported the antimicrobial activity of crude EtOAc extract from P. capitalensis against P. aeruginosa (6.49 mm) and MRSA (12.06 mm). Nevertheless, the inhibition zones were significantly smaller than P. fallopiae. The bioactive DCM extract was further evaluated for its MIC and MLC using a broth microdilution assay (Table  S2). At concentration 625.00 mg/ml, DCM extract was able to inhibit the growth of most of the microorganisms, particularly, Gram-positive bacteria. Based on the results, MLC was quadruple (2500.00 mg/ml) of MIC to kill most of the test microorganisms. A microbial kill curve study was performed to assess the dynamic interactions between the DCM extract of P. fallopiae and the susceptible test microorganisms. Figures S1-S5 demonstrate the kill curves of DCM extract against B. subtilis, MRSA, S. boydii, Yersinia spp. and C. albicans in terms of bacterial growth (A) and the growth reduction % (B) at various treatments over the period. The DCM extract at double MIC concentration caused approximately 95% growth reduction to all test microorganisms, except MRSA, within 24 h of treatment. It was noticed that DCM extract at MLC was sufficient to exhibit a bactericidal effect where microbial cells were significantly alleviated after 24 h of incubation. This might be due to the antimicrobial metabolites present in the DCM extract along with the depletion of nutrients and accumulation of wastes in the medium, making the extract unfavourable to microbial growth (Llorens et al. 2010). The kill curves study demonstrated that the antimicrobial activity of DCM extract was concentration-dependent.
A zebrafish model of acute toxicity was performed to assess the toxicity of the bioactive DCM extract. Figure S6 shows the microscopic observation of various toxicity effects on zebrafish in the presence of DCM extract. The death of all animals occurred at a concentration of 125.00 mg/ml or above. Meanwhile, peritoneal and hepatic oedema occurred at 62.50 mg/ml, and no abnormality appeared at 31.25 mg/ml or below. In detail, the acute toxicity of DCM extract on zebrafish embryonic development was shown in Figure S7. Various scenarios were noticed while hatching from the embryonic sac, i.e., 100% of embryos were hatched at 15.63 lg/ml while 33.33% were found unhatched in the untreated group. The results revealed that the spectrum of toxicity is proportional to the increasing concentrations at certain levels (31.25-125.00 lg/ml), after which cell death is mandatory. A curve ( Figure S8) was drawn from the % of mortality at different concentrations. The LC 50 obtained was 3.16 lg/ml per equation derived from the standard curve, indicating a concentration of 3.16 lg/ml DCM extract could kill 50% of the test animals. According to Chahardehi et al. (2020), the toxicity of samples against zebrafish was categorised into harmful (10 lg/ml < LC 50 < 100 lg/ml), toxic (1 lg/ml < LC 50 < 10 lg/ml), and highly toxic (LC 50 < 1 lg/ml). Previous studies on different species of Phyllosticta found that the metabolites produced are significantly toxic to several cancerous cell lines. Golias et al. (2020) performed the toxicity test of crude extract of P. capitalensis in vitro, and the cytotoxic concentration (CC 50 ) was 135.95 lg/ml in the fibroblast cells. Nonetheless, further toxicity tests with in vitro and in vivo models are warranted to confirm the toxicity of P. fallopiae.
Biofilm is an accumulation of microorganisms in the extracellular polymeric matrix composed mainly of extracellular polysaccharides, nucleic acids, enzymes, and proteins (Flemming et al. 2007). Biofilm formation is an important virulence strategy in the pathogenesis that causes significant medical problems (Rajamani et al. 2019). Figure  S9 shows the effect of DCM extract on the inhibition of biofilm formation in Grampositive and negative bacteria. Higher concentrations of DCM extract caused lower biofilm formation and more significant inhibition of cell adhesion. More than 82% of biofilm formation was inhibited at the highest concentration (1.25 mg/ml) on Grampositive bacteria. Regarding Gram-negative bacteria, P. aeruginosa showed less inhibition on the biofilm formation. It might be due to Gram-negative bacteria being used to form more biofilm than Gram-positive bacteria. In addition, P. aeruginosa is more persistent to environmental stresses due to profound modification of the cell envelope and overproduction of several protein molecules under biofilm-forming conditions (Crouzet et al. 2017). It is worth to note that this is the first study that reported the biofilm formation or inhibition from Phyllosticta sp. The biofilm eradication test was also conducted to determine the DCM extract's ability to eradicate the pre-formed biofilm ( Figure S10). On average, the data implied that DCM extract was less efficient in removing biofilms formed by Gram-positive bacteria. Among the Gram-negative bacteria, the biofilm formed by Yersinia spp. was subjected to the most eradication at all concentrations, followed by K. pneumonia, E. coli and P. aeruginosa.
The bioactive DCM extract of P. fallopiae was then subjected to column chromatography to yield 18 fractions (C1-C18) after pooling. The fractions were subjected to antimicrobial assay (Table S3), revealing the most active fraction as C6. The accurate masses and retention times of major metabolites in fraction C6 were measured in positive ionisation mode using an autosampler-equipped UPLC-QTOF-MS/MS system. The total ion chromatograms (TIC) obtained in the positive ion mode is shown in Figure S11. In total, 17 secondary metabolites were detected, including three flavones, three stilbenes, two flavanonols, two isoflavonoids, two phenolic glycosides, two phenol derivatives and others. The proposed chemical identity of each metabolite is based on a qualified match within ±5 mDa error by the UNIFI Scientific Library (Table S4 and Figure S12-S13). Flavonoid and phenolic constituents, kushenol I (15), kushenol M (17), kuwanon A (4), moracin C (11), ophiopogonanone B (7), 4 0 -methylpinosylvin (8) and flavokawain A (9) are among the detected antimicrobial metabolites in the bioactive fraction of P. fallopiae (Park et al. 2003;Kostecki et al. 2004;Abegaz et al. 2007;Kim et al. 2012;Al-Madhagi et al. 2019;Li et al. 2021;Ya'acob et al. 2022). Notably, the detected fungi metabolites in this study are closely related to the reported secondary metabolites of the host plant, A. vera

Conclusions
Endophytic fungi associated with medicinal plants are one of the renewable sources of biologically active metabolites. In this study, endophytic P. fallopiae was subjected to extensive investigations on diabetic wound healing activity. Chromatographic separation on the active DCM extract led to 18 fractions with varied activities. The most active fraction against diabetic wound microorganisms was characterised for its metabolites using UPLC-QTOF-MS/MS analysis. This bioactive fraction warrants indepth investigations on the isolation, bioactivity study, and quantitation of the purified bioactive metabolites with diabetic wound healing potential.

Disclosure statement
The authors reported no potential conflict of interest.

Funding
The authors would like to acknowledge Universiti Kuala Lumpur Short Term Research Grant (STRG 17003) for the funding.