Lignans from Phyllanthus niruri L. and Their Antifusarium Properties

In this study, two lignan compounds were isolated from acetone extract of Phyllanthus niruri L. nirtetralin B ( 1 ) and phyllanthin ( 2 ) using several chromatographic methods followed by molecular structure elucidation mainly based on 1D and 2D of 1 H and 13 C NMR spectrum. The isolated compounds were tested for their antimicrobial properties against the plant pathogenic fungus, Fusarium oxysporum, using the agar plate well diffusion method. The microdilution method determined the minimum inhibitory concentration (MIC) and the minimum fungicide concentration (MFC). In addition, the microconidia germination inhibition test was carried out using the agar diffusion method. As a result, compound 1 had MIC and MFC values of 4 and 16 μg/mL, respectively. While compound 2 showed the same MIC and MFC values of 16 μg/mL. Further testing on the inhibition of germination of F. oxysporum microconidia showed that compound 2 inhibited microconidia germination 100% at a concentration of 2 × MIC. In comparison, compound 1 at the same concentration was only able to inhibit germination by 29%. This study revealed that compound 2 is a potential new fungicide derived from local medicinal plants. However, further research is needed to identify the interaction mechanism between the test compound and the fungal pathogen F. oxysporum to develop new antifungal agents.


INTRODUCTION
Indonesia has a variety of plants that can be used as medicinal plants (Saifudin et al., 2011;Miksusanti et al., 2009). One of the medicinal plants that have the potential to be developed is green meniran. This herbaceous plant with the scientific name Phyllanthus niruri Linn. is a member of the Euphorbiaceae family that grows wild in damp and rocky places, such as bushes and soil among grasses. P. niruri has been widely used as traditional medicine (Bana et al., 2016;Tjandrawinata et al., 2017;Ervina & Mulyono, 2019;Destryana & Ismawati, 2019;Qasrin et al., 2020). These plant species produce one or more bioactive compounds that are believed to have medicinal properties and are useful for treatment so that they are used as medicinal entities. In addition, it can contain many compounds that can serve as chemical models for new drug design, synthesis, or molecular semi-synthetics (Tjandrawinata et al., 2017;Baehaki et al., 2021). Bioactive compounds or secondary metabolites are standard compounds contained in a plant species (Alam et al., 2016). The compounds found in P. niruri include flavonoids, alkaloids, triterpenes, tannins, lignans, polyphenols, and sterols (Bagalkotkar et al., 2006;Paithankar et al., 2011;Murugaiyah & Chan, 2006;Rivai et al., 2013). These compounds play a role in various therapeutic activities, including antiviral, antimicrobial, anti-hepatic, antitumor, and antidiabetic (Paithankar et al., 2011).
In addition to being used for human health, in the last few decades, there has also been a tendency to use plant extracts or secondary metabolites in handling pests or diseases in food crops and livestock (Siva et al, 2008;Hidanah et al., 2017). One of the plant diseases that cause losses is the fungus Fusarium oxysporum (Matheron, n.d.) (Gordon, 2017). The fungus F. oxysporum is known to cause wilt disease in plants such as tomatoes, bananas, and other food crops (Ignjatov et al., 2012;Steinkellner et al., 2008;Walduck & Daly, 2006;Sutherland et al., 2013). Several plant extracts can inhibit F. oxysporum germination (Sutherland et al., 2013). Likewise, plant extracts of P. niruri showed inhibitory activity against F. oxysporum (Siva et al., 2008). However, until now, there have been no reports of testing the activity of compounds from P. niruri against F. oxysporum. Whereas compounds from the P. myrtifolius plant, the same genus as the secondary metabolites of the lignan group, have been reported to be able to inhibit the growth of F. oxysporum (Windayani et al., 2014). Therefore, this study was carried out to isolate secondary metabolites from the acetone extract of P. niruri and in vitro test its bioactivity against the fungal pathogen F. oxysporum.

Plant Material
The plants were collected from Bandung. The voucher specimens (P. niruri) were identified and deposited at the Herbarium Bandung, School of Life Sciences and Technology, Bandung Institute of Technology, Bandung, Indonesia under specimen number HBG-12183.

Antifungal Assay
Testing antifungal properties by making a suspension of fungal inoculum F. oxysporum refers to the CLSI M38-A2 method (CLSI, 2003). Briefly, the fungus F. oxysporum was grown on PDA agar plates at 35 °C for seven days (Windayani et al., 2014). Seven-day fungal colonies were rinsed with 0.85% PBS (Phosphate Buffered Saline) solution to obtain a mixture of conidia, sporangiospores, and hyphae fragments. Then, the mixture was transferred into a sterile tube and left for three to five minutes until the heavier particles settle. The top (suspension) of the mixture was separated into a new tube and stirred using a vortex for fifteen seconds. Then, the density of the microconidia suspension was determined and adjusted by diluting the suspension using sterile distilled water to 1:50. Dilutions were carried out to obtain a colony density of 0.4 × 10 4 to 5 × 10 4 CFU/mL. Inoculum quantification was made by growing 0.01 mL of a suspension that had been diluted 1:100 on a PDA agar plate. Then aerobically incubated at 35 °C, the appearance of fungal colonies was observed every day. Fungal colonies after this double dilution were in the range of 5 × 10 4 CFU/mL.
Compound 1 was obtained as colourless needle crystals with a melting point of 55-56 °C. The 13 C NMR spectrum data for compound 1 showed 24 carbon signals. In DEPT and HSQC experiments on compound 1, five carbon methoxy signals were obtained (δ C 58.0 (C-2b), 58.0 (C-3b), 58.3 (C-8-OCH 3 ), 55.3 (C-OCH 3 ). 11a), 55.3 (C-12a)) and one methylenedioxy signal (δ C 100.7 (C-15)), while the other 18 carbon signals showed signals for the lignan framework, consist of twelve carbons. The aromatic sp 2 carbon signal consisted of four carbon signals methyne (102.59 (C-5), 111.7 (C-10), 112.8 (C-1), 119.9 (C-14) ppm) and eight quaternary carbon signals (δ C 124.9 ( C-4A), 132.2 (C-8A), 135.2 (C-6), 147.5 (C-7), 141.9 (C-8), 140.9 (C-9), 149.1 (C-11), 147.5 (C-12). In comparison, the other six carbon signals are three carbon signals of methylene (δ C 33.2, 73.0, 75.6) and aliphatic methyne (δ C 41.2, 45.2, 37.2), respectively. Furthermore, the COSY spectrum data for compound 1 showed a correlation between H-1↔H-2, H-2↔H-3, H-2↔H-2a, H-4 H-5, and H-13↔H-14. The positions of the methoxy and methylenedioxy substituents were determined based on the long-range correlation data 1 H↔ 13 C in the HMBC spectrum of compound 1, shown in Fig. 2. The information obtained from the 1D and 2D NMR experiments suggests that compound 1 has a 1-aryltetrahydronaphthalene or tetralin skeleton (Anjaneyulu et al., 1973;Chang et al., 2003). Comparing with previously reported data presented in Table 1, showing compound 1 has high compatibility with one of the anomers of nirtetralin (Wei et al., 2012). The coupling constant H-1 confirms it with H-2, J = 6.25 Hz, which means both protons are axially-axially oriented. In contrast, the other coupling constants that determine the structure of compound 1, namely H-2 and H-3 with J = 4.6 Hz, indicate both protons are axiallyequatorially oriented, thus compound 1 is suggested as nirtetralin B (Wei et al., 2012). Compound 2 was obtained as white crystals with a melting point of 96-97 °C. In the 13 C NMR spectrum data for this compound, 12 carbon signals appeared with the integration of each signal for two carbon atoms, so the total number of carbon atoms was 24. Based on the HSQC and DEPT experiments, of the twelve signals, three of them were methoxy (δ C 58.8; 56.0; 55.8), while the other nine carbon signals were six aromatic sp 2 carbon signals (δ C 133.7; 112.3; 147.2; 148.8; 111.1; 121.2, two methylene (δ C 35.0; 72.7), and one aliphatic methyne (δ C 40.8). These data point to the skeleton of the lignan compound dibenzylbutane. Further evidence of the molecular structure of compound 2 was obtained based on long-range correlation data on the HMBC spectrum and literature search. Further, it was shown that compound two had high similarity in its NMR parameters with the phyllanthin compound, the skeleton lignan dibenzylbutane. In addition, the data on the physical properties of compound 2 also showed similarities to the previously reported data (Krithika et al., 2009;Hanh et al., 2013). A comparison of the 1 H NMR chemical shift of compound 2 with the previously reported data is presented in Table 2.   Krithika et al., (2009) Furthermore, this section also presents the results of the antimicrobial activity of the two pure compounds isolated against fungal pathogens and suggestions for the relationship between structure and bioactivity. The isolated compounds from P. niruri, compounds 1-2, were tested as an antifungal against F. oxysporum, including sensitivity tests expressed by % inhibition values, MIC, and MFC, also microconidia germination inhibition tests. Both tests were carried out based on the standard CLSI M38-A2 protocol (CLSI, 2003). For the sensitivity test, the MIC and MFC values of each pure compound at a concentration of 1000 μg/mL are presented in Table 3.
The nirtetralin compound B (1) is a lignan of the aryltetralin type, showing a more active inhibition of the growth of F. oxysporum (MIC = 4.0 μg/mL) compared to the lignan dibenzylbutane, phyllanthin (2) (MIC = 16.0 μg/mL). Antifungal activity data on the two compounds suggested that the presence of a cyclohexane ring in compound 1 had a role in the activity of lignan compounds in inhibiting the growth of the fungus F. oxysporum. The effect of each compound on conidia germination of F. oxysporum was tested at concentrations of 0 × MIC to 4 × MIC for 72 hours at 35 ºC. The results of the F. oxysporum micronidia germination inhibition test are presented in Table 4.
In general, all tested pure compounds inhibited concentration-dependent F. oxysporum germination, where a sharp decrease began to occur at 0.5 × MIC. Compound 2 (phyllanthin) completely inhibited the germination of F. oxysporum at a concentration of 2 × MIC. These results are in agreement with the MFC values of the compounds ( Table 3). Meanwhile compound 1 (nirtetralin B), at a concentration of 4 × MIC did not completely inhibit F. oxysporum germination. This result can be understood for example compound 1 has an MIC value of 4.0 μg/mL while the MFC value of compound is 16.0 μg/mL (4 × MIC). In general, these results indicate that the isolated compounds from P. niruri plant can inhibit the germination of F. oxysporum conidia since the concentration of 0.5 × MIC.
Several other lignans have been evaluated as an antifungal. For example, three lignan compounds, aliterinate C, (+)pinoresinol, and (+)-medioresinol, exhibited better biologic effects on fungal organisms such as Pythium insidiosum than the antifungal drugs itraconazole and terbinafine (Sriphana et al., 2013). Two aryltetralin lignans isolated from Cordia exaltata were found to have weak antifungal properties against several Candida species (Nogueeria et al., 2013). Ecbolin A (furofuran lignan), a secondary metabolite of Exbolium viride, showed moderate antifungal activity against twelve fungi (Francina et al., 2012). (+)-Medioresinol is antifungal against Candida albicans through apoptotic mechanisms, namely oxidative stress and mitochondrial dysfunction (Hwang et al., 2012). In addition, justisidin B from P. myrtifolius, phyllanthin (2), a dibenzylbutane lignan isolated from P. niruri, also exhibited antifungal properties against C. albicans with an inhibitory level of 250 μg/mL (Windayani et al., 2014). The antifungal properties of compounds 1 and 2 in this work are consistent with previous studies (Gertsch et al., 2004). Therefore the results of this study add to the data on the beneficial effect of lignans of Phyllanthus species as antifungal agents. However, referring to the antifungal properties of lignans against Fusarium fungi, the data that have been reported are very limited. Pinoresinol (furanoid lignan) and secoisolariciresinol (dibenzylbutane lignan) have been shown to inhibit the growth and biosynthesis of trichothecene in F. graminearum (Kulik et al., 2014). Lyonisid, a glucoside derivative of the lignan aryltetraline isolated from Vaccinium myrtillus L., showed inhibition of the mycelia growth of F. oxysporum. However, eudesmin (a furanoid lignan) did not show antifungal activity against F. oxysporum (Szakiel et al., 2011). So the effect of lignans on Fusarium fungi depends on the type of lignan framework, and there is a tendency for lignans arylnaphthalene, aryltetralin, and dibenzylbutane to be potential antifungal compounds against F. oxysporum. The disclosure of conidia germination inhibitors is vital for overcoming infections caused by pathogenic organisms (Ebbole et al., 2004;Cantrell et al., 2005). Mycotic infections are difficult to treat since fungi are eukaryotic organisms with structures and metabolism similar to their hosts (Jin et al., 2004). Furthermore, long-term treatment with commonly used antifungals, such as amphotericin B, is toxic, whereas azole antifungals have a limited spectrum and efficacy, and their use may result in resistance to some strains of pathogenic fungi (Denning et al., 1997;Helmerhorst et al., 1999;Wiederhold, 2017).

CONCLUSIONS
In this study, two pure compounds from the lignan group of aryltetraline (nirtetralin, 1) and dibenzylbutane (phyllanthin, 2) have been isolated. The results of the antifusarium bioactivity test showed that nirtetralin B (1) could inhibit the growth of F. oxysporum at a concentration of 4 µg/mL (MIC). Both isolated compounds 1 and 2 were fungicidal at a concentration of 16 µg/mL. Further testing of the inhibition of germination of F. oxysporum microconidia showed that compound 2 inhibited microconidia germination 100% at a concentration of 2× MIC. In comparison, compound 1 at the same concentration was only able to inhibit germination by 29%. So compound 2 was estimated to have potential as the main compound antifusarium. However, further studies are needed to determine its in vivo activity and cytotoxicity to host plants.