1 Introduction

Liverworts are the largest group of pioneering land plants which arose during the adaptation of plants from marine to terrestrial environment [1]. They produced terpenoids and/or aromatic compounds as their major lipophilic constituents [2,3,4,5]. Many types of sesquiterpenoids from liverworts are the enantiomer to those metabolites from higher plants [6]. While, diterpenoids such as clerodanes, dolabellanes, fusicoccanes, kauranes, labdanes, pimaranes and others were found in numerous liverworts [6]. Recently several bioactive cyathane diterpenoids were discovered [7, 8]. Cyathane is precursor structure for biosynthesis of the verrucosane-type diterpenoid, a fused 3,6,6,5-tetracyclic carbon skeleton [9]. First verrucosane diterpenoid was isolated from Mylia verrucosa [10]. Later, some analogs such as neoverrucosane-, homoverrucosane-, epi-neoverrucosane- and epi-homoverrucosane-type were reported [11,12,13]. The latest epi-neoverrucosane analog was reported in 2013 [14]. Hereby, we report yet another new epi-neoverrucosane diterpenoid, 5β-acetoxy-13-epi-neoverrucosanic acid (1) was isolated, along with three known secondary metabolites, 13-epi-neoverrucosan-5β-ol (2), chelodane (3) and (E)-β-farnesene (4) from east Malaysia’s liverwort Pleurozia subinflata (Fig. 1).

Fig. 1
figure 1

Chemical structures of 1–4

Full size image

Besides, liverworts have long been used as traditional medicine by the indigenous people in some parts of China. In the past half-century, Prof. Yoshinori Asakawa (Tokushima Bunri University, Japan) has begun to study the chemical composition of liverworts collected from Asia, Europe and South America, many of which have reported to have diverse chemical structures and exhibited numerous biological activities [2, 6]. Thus, research focusing on the biological activity of liverwort and industrial use are significant. Our research examined the antifungal effects of the four isolated compounds against selected fungi separated marine organisms.

2 Results, Discussion and Conclusion

Compound 1 was isolated as colorless oil and analyzed for the molecular formula C22H34O4 by HR-ESI-MS [M − H]− ion at m/z 361.2391. The 13C and 1H NMR data (Table 1) indicated the presence of an isopropyl unit at δC 31.1, 23.2 and 22.3; δH 1.51–1.55, 1.00 and 0.78, one carboxylic carbon at δC 183.0, an acetoxy unit at δC 171.5, 21.5; δH 2.04, one oxygenated methine at δC 75.1; δH 5.28, two tertiary methyls at δC 25.5 and 16.4; δH 1.12 and 0.87, six methylenes, four methines, and three quaternary carbons which corresponding well to HSQC spectrum. Based on these findings, six degrees of unsaturation was calculated from HR-ESI-MS, and it attributed to two carbonyl units and one tetracyclic ring for 1.

Table 1 1H and 13C NMR (600 and 150 MHz) data of 1 (in CDCl3, δ in ppm, J in Hz).
Full size table

The 1H–1H COSY experiment revealed the spin systems as depicted by the bold lines in Fig. 2. In the HMBC spectrum, the three-bond correlations of H3–C(16) and H3–C(17) to the opposite carbons C(17) and C(16), and to C(13) and C(15), allowed the placement of isopropyl unit at C(13) which was further confirmed by 1H–1H COSY. The acetoxy unit at C(5) was confirmed by HMBC correlations between H–C(5) to 5-OAc. The downfield shifted of 13C and 1H NMR at C(5) further supported this deduction. The HMBC correlations of H2–C(11) to C(20); and H–C(14) to C(20) suggested the carboxylic carbon at C(10). These findings together with HMBC correlations of H3–C(18) to C(2), C(3), C(4) and C(5); and H3–C(19) to C(1), C(6), C(7) and C(8) permitted establishment for the planar structure of 1 (Fig. 2).

Fig. 2
figure 2

1H–1H COSY, key HMBC and NOE correlations of 1

Full size image

The relative stereochemistry of 1 was deduced from the NOESY correlations (Fig. 2) and comparison of its chemical shift, coupling constants and NOE correlations with those of known analogs [10,11,12,13,14]. The NOE correlations of H–C(5) to H2–C(6α) (δH 1.76), H3–C(18) and H3–C(19); and H–C(14) to H3–C(19) have suggested H–C(5), H–C(14), H3–C(18) and H3–C(19) on α relative configuration. While, NOE correlations of H–C(1) to H2–C(3β) (δH 0.56) and H2–C(6β) (δH 0.84) showed H–C(1) must be on another face, β relative configuration. The earlier NOE cross peak of H–C(1) to H2–C(3) (δH 0.56) has led to the assignment of H2–C(3) (δH 0.56) on β configuration, therefore H2–C(3) (δH 0.69) must be on α configuration. With this finding, the configuration at H–C(2) can be assigned based on vicinal coupling constants of cyclopropane unit between H–C(2) and H2–C(3α) (3J2-3α = 8.3 Hz) and between H–C(2) and H2–C(3β) (3J2-3β = 4.3 Hz). These coupling constant values suggested H–C(2) has a cis relationship with H2–C(3α) within the cyclopropane unit, therefore α configuration was assigned for H–C(2). While, the carboxyl unit at C(10) was assigned on β configuration due to a trans-fused at C/D ring junction. Thus, the relative configurations of 1R*, 2S*, 4S*, 5R*, 7S*, 10S* and 14R* were determined as identical to those of known analogs of neoverrucosane and epi-neoverrucosane [10,11,12,13,14]. To distinguish epi-neoverrucosane from neoverrucosane, the NOE correlations of H–C(1) to H–C(15) and H3–C(17); and H–C(13) to H–C(14), showed 13-isopropyl unit to H–C(14) has a trans configuration, suggested a epi-neoverrucosane. Furthermore, similar NOE correlations of H–C(1) to H–C(15); and H–C(13) to H–C(14) were observed in 12-acetoxy-13-epi-neoverrucosann-5-one [14]. On the contrary, these NOE were not observed in neoverrucosane-type, neoverrucosan-5β,9β-diol, instead H–C(13) to H–C(20) was detected [15]. Thus, the structure 1 was established without confusion. The configuration of isopropyl unit at C(13) generated during formation of tricyclic system (Fig. 3) determined the biosynthesis of neoverrucosane or epi-neoverrucosane [9]. To the best of our knowledge, compound 1 was considered as first discovery of 13-epi-neoverrucosane that containing a carboxyl moiety or even among related skeletons such as verrucosane and neoverrucosane. The methyl at C(20) of 1 might have followed a three-step oxidation, via a hydroxyl and carbonyl, to the corresponding carboxylic acid [16], as shown in the purposed biosynthetic pathway (Fig. 3).

Fig. 3
figure 3

Proposed biosynthetic pathway of 1

Full size image

The known compounds were identified as 13-epi-neoverrucosan-5β-ol (2) [11], chelodane (3) [17], and (E)- β-farnesene (4) [18], after compared its spectroscopic data with published literatures. The tetracyclic diterpenes are relatively rare in nature, and mainly found in the species of Plagiochila, Jamesoniella and Fossombronia [6]. However, we found epi-neoverrucosane-type diterpene derivatives (1 and 2) from east Malaysia’s liverwort, Pleurozia subinflata. These secondary metabolites seem to be the good chemosystematic markers for P. subinflata in Borneo. Compound 3 was widespread in marine sponges such as Chelonaplysilla erecta, Raspailia sp. and even in Sigmosceptrella sp. [17, 19, 20]. However, this is the first record for 3 found in liverwort. Compound 4 was the most common farnesane-type sesquiterpene in liverworts. It was distributed throughout more than 20 Jungermanniales and Pleuroziales species including Pleurozia [6].

Compounds 1–4 were evaluated its biological potentials against fungal strains isolated from the Bornean ocean, Lagenidium thermophilum IPMB 1401, Haliphthoros sabahensis IPMB 1402, Fusarium moniliforme NJM 8995, Fusarium oxysporum NJM 0179, Fusarium solani NJM 8996 and Ochroconis humicola NJM 1503 (Table 2). The minimum inhibition concentration (MIC) values of compound 1 against L. thermophilum and H. sabahensis were 12.5 and 50 μg/mL, respectively. While, compounds 2 and 3 showed MIC values of 100 μg/mL against L. thermophilum. Compound 4 was inactive (> 100 μg/mL) against the tested fungi.

Table 2 The MIC (μg/mL) of 1–4 against six fungal strains
Full size table

In conclusion, this is the first time ever since 2013 of the last epi-neoverrucosane being discovered from nature. A new 13-epi-neoverrucosane diterpenoid, 5β-acetoxy-13-epi-neoverrucosanic acid (1) along with three known secondary metabolites, 13-epi-neoverrucosan-5β-ol (2), chelodane (3) and (E)-β-farnesene (4) were found in east Malaysia’s liverwort Pleurozia subinflata. Compound 1 exhibited effective antifungal activity (MIC values of 12.5 μg/mL) against Lagenidium thermophilum.

3 Experimental Section

3.1 General Experimental Procedures

Optical rotation was taken on the automatic polarimeter (AUTOPOL IV automatic polarimeter) in chloroform solutions at 28 °C. IR spectrum was recorded on the FTIR spectroscopy (Perkin Elmer). NMR spectra were recorded on the 600 MHz FT-NMR (Jeol) using deuterated chloroform (CDCl3) with tetramethylsilane (TMS) as the internal standard. MS spectra were obtained using LC-ESI-IT-TOF-MS (Shimadzu). For preparative TLC, Merck Kieselgel 60 F254 was used and Kieselgel 60 was used for column chromatography. Purification was performed using high performance liquid chromatography (LC-10 AT, Shimadzu) equipped with UV detector.

3.2 Biological Materials

Specimens of P. subinflata (M. Suleiman & S.-Y. Ng 5946) were collected from Mount Trus Madi (5° 33′ 13.1″ N, 116° 30′ 41.9″ E), Sabah, Malaysia in August 2015. The specimens were identified based on external morphology by the fifth author. A voucher specimen (BORHB0026) is deposited in the BORNEENSIS Herbarium at Institute for Tropical Biology and Conservation (ITBC), Univeristi Malaysia Sabah (UMS).

3.3 Extraction and Isolation

The air-dried liverwort specimens (42 g) were extracted using 100% methanol (MeOH) (1.0 L × 3 each for two days). The crude extract was partitioned between distilled water (150 mL) and ethyl acetate (EtOAc) (50 mL × 3). After removal of the organic solvent, the EtOAc fraction (500 mg) was chromatographed on a Si gel column using hexane (Hex) and EtOAc system as eluent with increasing polarity (Hex/EtOAc: 9:1, 8:2, 7:3, 5:5, 100% EtOAc) to yield five fractions, 1–5. Fraction 2 (76 mg) was subjected to repeated preparative TLC with toluene to yield 2 (8.8 mg), 3 (7.4 mg) and 4 (15.4 mg). Fraction 3 (60 mg) was subjected to repeated preparative TLC with hexane/EtOAc: 7:3, and the resulted sub-fraction was further purified by semi-preparative high performance liquid chromatography (HPLC) to yield 1 (12.8 mg). The isolation was operated using a reverse phase C18 column (5 μm, 10 mm × 250 mm) measured at UV wavelength of 210 nm under gradient elution with the following conditions: 40–100% acetonitrile (MeCN)/H2O.

3.3.1 5β-Acetoxy-13-Epi-neoverrucosanic Acid (1)

Colorless oil; [α]28.0D + 67.8 (c = 0.5, CHCl3); IR νmax 3488, 3060, 1735 and 1712 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.28 (1H, dd, J = 7.6, 10.7 Hz, H-5), 2.45 (1H, ddd, J = 1.4, 8.9, 11.6 Hz, H-11α), 2.20 (1H, dd, J = 4.3, 13.1 Hz, H-1), 2.04 (3H, s, OAc), 1.88 (1H, m, H-13), 1.81–1.84 (1H, m, H-12α), 1.81–1.84 (1H, m, H-9α), 1.76 (1H, dd, J = 7.6, 12.5 Hz, H-6α), 1.67 (1H, dd, J = 7.6, 13.1 Hz, H-14), 1.59–1.62 (1H, m, H-9β), 1.51–1.55 (1H, m, H-8α), 1.51–1.55 (1H, m, H-12β), 1.51–1.55 (1H, m, H-15), 1.21–1.23 (1H, m, H-11β), 1.12 (3H, s, H-18), 1.09–1.11 (1H, m, H-8β), 1.00 (3H, d, J = 6.9 Hz, H-17), 0.87 (3H, s, H-19), 0.84 (1H, m, H-6β), 0.78 (3H, d, J = 6.9 Hz, H-16), 0.69 (1H, dd, J = 4.3, 8.3 Hz, H-3α), 0.65 (1H, ddd, J = 4.1, 4.1, 8.3, H-2), 0.56 (1H, dd, J = 4.3, 4.3 Hz, H-3β); 13C NMR (CDCl3, 150 MHz) δ 183.0 (C, C-20), 171.5 (C, OAc), 75.1 (CH, C-5), 52.0 (C, C-10), 51.7 (CH, C-14), 45.3 (CH, C-13), 44.1 (CH, C-1), 42.3 (CH2, C-6), 36.8 (C, C-7), 36.3 (CH2, C-11), 34.9 (CH2, C-8), 32.3 (CH2, C-9), 31.1 (CH, C-15), 27.5 (CH2, C-12), 26.7 (CH, C-2), 25.5 (CH3, C-18), 23.2 (CH3, C-17), 22.3 (CH3, C-16), 21.8 (CH2, C-3), 21.5 (CH3, OAc), 20.1 (C, C-4), 16.4 (CH3, C-19); negative ion HRESIMS: m/z 361.2391 (calcd for C22H33O4 [M–H]−, 361.2384).

3.4 Antifungal Assay

The minimum inhibitory concentration (MIC) of the fungistatic on hyphae was performed by incorporating the pure compound solutions (12.5, 25.0, 50.0, 100.0 μg/mL) onto PYGS agar in a petri dish followed by inoculation of six tested fungal strains [21,22,23]. The MIC was determined visually as the lowest concentration showing no hyphal growth after they were incubated at 25 °C for 7 days.