Antifungal Activity of Triterpenoids and Steroids Isolated from Pinus merkusii Bark Against Phanerochaete chrysosporium

The outer part of a tree, known as the bark, protects the internal tissues from external conditions and attacks from microorganisms. Its antifungal activities are due to the presence of chemicals in this bark. This study aimed to evaluating the toxicity of triterpenoids and steroids from the bark of Pinus merkusii against Phanerochaete chrysosporium (white-rot). The triterpenoids and steroids were isolated from n-hexane extract of the bark through column chromatography. Then, the antifungal activity was evaluated by measuring the growth rate of the fungus on potato dextrose agar (PDA) medium in a Petri dish. The isolation resulted in three triterpenoids (3βmethoxyserratt-14-en-21-one, serrate-14-en-3β,21β –diol, 3α,21βdimethoxy-14-serratene) and two steroids (β-sitosterol, Stigmast-4-en-3-one). There was more of antifungal activity for βsitosterol and serrate-14-en-3β,21β –diol, due to presence of hydroxyl bond and their hydrophobicity.


Introduction
Bark is an important part, covering almost the whole part of a tree. According to Wittstock and Gershenzon (2002); Alfredsen et al. (2008); and Pásztory et al. (2016), it protects the inner part of the tree from extreme and other external conditions such fire, cold, herbivore, pathogens, as well as detrimental insects and fungi. One of the ways it defends the tree is by storing secondary metabolites toxic to its enemies. Previous researches showed that its phenolics and lipophilics components possess bioactivity actions againts various pests, disease-causing organisms, and wood decaying fungi such as white and brown-rot fungi (Wijayanto et al. 2015;Shreaz et al. 2016;Lomelί-Ramίrez et al. 2016;Özgenç et al. 2017).
Pinus merkusii Jungh & de Vriese is a conifer of Pinaceae family, usually used as raw material in the production of pulp and paper, gum rosin as well as turpentine in Indonesia. Similarly, previous studies on P. merkusii bark showed the presence of phenolic and lipophilic extractives, also in its knots and stem wood (Wijayanto et al. 2015;Masendra et al. 2018a;Masendra et al. 2019;Masendra et al. 2020). In addition, Wijayanto et al. (2015) reported that the phenolic stilbenes of P. merkusii wood showed antifungal activity against Trametes versicolor (white-rot) and Poria placenta (brown-rot). Additionally, triterpenoids and steroids from Astilbe myriantha roots, Ficus drupacea bark and Pallavicinia lyellii have been reported to exhibit antifungal activity (Subhisha and Subramoniam 2005;Song et al. 2011;Yessoufoua et al. 2015). Furthermore, a previous work by Masendra et al. (2018b) showed the isolation of three triterpenoids and two steroids from the bark of P. merkusii. Based on this background, the aim of this study was to evaluate the toxicity of triterpenoids and steroids isolated from the bark of P. merkusii against P. chrysosporium. The use of the P. chrysosporium in this study due to the capability of this white-rot basidiomycete fungi to degrade lignin and also was used to test five species of woods in Brazil as their natural resistance (Oliveira et al. 2010).

GC-MS Analysis
Gas chromatography-mass spectrometry (GC-MS) data were collected with the instrument procured from Shimadzu, Kyoto, Japan, with the following conditions: column temperature from 50 °C (1 min) to 320 °C at 5 °C/min; injection temperature of 250 °C; detection temperature of 320 °C; acquisition mass range of 50 to 800 amu using helium as the carrier gas. The specification of column: DB-1 capillary column (30 × 0.25 mm inner diameter and 0.25 μm; GL Sciences, Tokyo, Japan). The 13 C and 1 H nuclear magnetic resonance (NMR) were determined by a JEOL ECZ-400 spectrometer (JEOL, Tokyo, Japan). Then, the NMR spectra were recorded using standard JEOL pulse sequences at 400 MHz and 100 MHz for 1 H and 13 C, respectively.

Antifungal Activity
The toxicity of the isolated compounds against the growth of P. chrysosporium were investigated through literature of Lukmandaru (2013). Exactly 1 mg/ml concentration of each sample was prepared and a solution of 300 µl was placed on the surface of 20 ml PDA medium with 12.3 mg/ cm 2 concentration in a 9 cm diameter petri dish. The extract solution was allowed to stand for 1 h air dry before inoculation. The blank was performed to the sample solvent without extract and each sample was conducted in three replications. The lapachol analytical standard (Aldrich, Germany) was used in this assay as a positive control. Then, the growth rate of sample was calculated using equation 1: Growth rate (%) = A1 /A0 x 100% (1) Where A = π x (d/2) 2 , d = diameter of sample growth, A0 is growth rate of blank and A1 is growth rate of sample.

Statistical Analyses
One-way analysis of variance (ANOVA) were performed on the results of the growth rate using SPSS version 20 (IBM, New York, USA) with 95% confidence level. Significant results were further subjected to post-test Tukey HSD.

Antifungal Activity of Isolated Compounds
The antifungal activity of isolated compounds was measured with reference to the growth rate of P. chrysosporium. The fungus growth on PDA medium as represented in Figure 3, showed a cycle shape and fungus spread from the center of petri dish to the surface of PDA. Also, the results of the one-way ANOVA showed significant difference (p=<0.01) among the compound. The growth rates of C1-C3, as shown in Figure 4, were 100% with no antifungal activity. However, there were significantly higher growth inhibition rates in β-sitosterol (C4) and serrate-14-en-3β,21β-diol (C5) with inhibition of 45.5% and 60.2%, respectively, but still low compared with lapachol standard with 26.7%. Also, the positive control with lapachol was two times higher than β-sitosterol and serrate-14-en-3β,21β-diol.  In this research, no literature was found on the antifungal activity of triterpenoids and steroids from P. merkusii bark against P. chrysosporium. However, the antifungal activity of other compounds have been reported from other species such as 3b,6b,24-trihydroxyurs-12-en-27-oic acid from Astilbe myriantha showing strong antifungal activity against Colletotrichum gloeosporioides (Song et al. 2011), epilupeol acetate from Ficus drupace against Aspegillus versicolor (Yessoufoua et al. 2015), and βsitosterol from Senecio lyratus against Fusarium spp. (Kiprono et al. 2000).

Correlation between Antifungal Activity and Chemical Structure of Isolated Compounds
It was observed that β-sitosterol and serrate-14-en-3β,21β-diol affected the growth rate of P. chrysosporium. Also, among the isolated samples, only β-sitosterol and serrate-14-en-3β,21β-diol have hydroxyl bond (-OH) in their structures as shown in Figure 1. This is an indication that the hydroxyl bond, which determine the lipophilicity and hydrophobicity of the compounds, affect the antifungal activity.
The hydroxyl bond was located in A and B rings in βsitosterol, but in A and E rings in serrate-14-en-3β,21β-diol. Hence, with β-sitosterol, the fungal growth rate was lower compared with serrate-14-en-3β,21β-diol. This means that the hydroxyl position and hydrophobicity of A and B ring were stronger in inhibiting the growth of P. chrysosporium compared with the bond in A and E rings. In line with these findings, Sekine et al. (2009), andZengin andBaysal (2014) also reported that the hydrophobicity and hydroxyl position of terpenes, latifolin, and the derivative products affected the antibacterial and antifungal activity by inhibiting the growth of brown-and white-rot. Additionally, a recent study by Konuk and Ergüden (2020) reported that the -OH position and hydrophobicity of phenolic terpenoids affected the disruption of cell membrane integrity of Saccharomyces cerevisiae.

Ecological Role and the Potential Use of Triterpenoids and Steroids
The terpenoid compounds in P. merkusii bark was reported to play a vital role in preventing living tissues from chemical deterioration (Seki et al. 2012). In addition, the presence of triterpenoids and steroids in this bark, especially those with hydroxyl bond in their ring are more effective against P. chrysosporium (white-rot). Therefore, high concentration of triterpenoids and steroids in the bark of a tree protect its living tissues from damage due to fungal attacks.
Due to the impact of microorganisms such as bacterial and white-rot fungus attacks, there is need for further research on natural wood preservatives. The use of natural preservatives could reduce the negative effects of chemicals in the air. According to Tascioglu et al. (2013);Smith et al. (1989); and Lin et al. (2007), some plants extracts could be processed into alternative wood preservatives due to their actions against microorganisms' attacks. Hence, the accumulation of triterpenoids and steroids in the bark of P. merkusii, could be used as alternative wood preservatives.