Introduction

The genus Ganoderma contains more than 80 species of wood decaying fungi mostly distributed in tropical and subtropical areas. Different species of Ganoderma have been used in Traditional Chinese Medicine for thousands of years for the treatment of many kinds of diseases, for example, hypertension, respiratory diseases, gastrointestinal disorders, autoimmune diseases [1]. The two species, G. lucidum and G. sinense were recorded in the recent five editions of Chinese Pharmacopoeia with the name “Lingzhi” from the year 2000. Numerous studies have shown that polysaccharides and the main secondary metabolites–triterpenoids, are responsible for the biological activities of Ganoderma, such as immunoregulatory, antiviral, hepatoprotective effects [2,3,4,5,6,7]. Besides, meroterpenes (farnesyl hydroquinones), the other main constituent in Ganoderma, have attracted much attention in recent years [8, 9]. Ganoderma triterpenes are always characterized by structural poly-oxygenations [6]. Among the reported structures, the popular positions been oxygenated are C-3, C-7, C-11, C-15, C-23, and C-26. The oxygenations in the side chain sometimes trigger the C-C bond cleavage by retro-aldol reaction or oxidative cleavage to yield nor-lanostane, a minor group of Ganoderema triterpenes, such as C24 (hexa-nor), C27 (tri-nor), and C-25 (penta-nor) lanostanes [5, 6].

Ganoderma australe is regarded as an alternative of the official-recognized species G. lucidum, and is used as a folk medicine in some ethnic minority areas of Yunnan Province, China. This fungus, however, was chemically under-investigated compared to other easily widely used Ganoderma species, such as G. lucidum, G. cochlear, and G. sinense. Previous studies on the secondary metabolites of G. australe have led to the isolation of some lanostane triterpenes [10,11,12,13], meroterpenoids [14, 15], and alkaloids [14]. In this study, we would like to report ten new nor-lanostane triterpenes isolated from G. australe, of which the structures are assigned by extensive NMR and HRESIMS spectroscopic analysis. The new structures are classified into tetra-nor-, penta-nor-, and hexa-nor-lanostanes, and are featured by C-6 oxygenation or α,β-unsaturated aldehyde groups, which are unusual modifications in the reported Ganoderma triterpenes, thereby inspiring a biosynthetic proposal of these compounds.

Results and discussion

Structure elucidation of compounds 1–10

Ganoaustratetraenone A (1) (Fig. 1), isolated as pale-yellow needles, has the molecular formula of C24H32O4, which was determined from the HRESIMS analysis (m/z 385.23724 [M + H]+, calcd. for C24H33O4, 385.23718) (Additional file: 1). The 1D NMR spectroscopic data of 1 (Tables 1 and 2) displayed six singlet methyls, six methylenes, two methines, four quaternary carbons (sp3 hybridized), a pair of non-protonated olefinic carbons, and four ketone carbonyls. The 1H and 13C NMR data of 1 showed high similarity to those of the previously described compound 4,4,14α-trimethyl-3,7,11,15,20-pentaoxo-5α-pregn-8-en [16], a semisynthetic product of methyl ganoderate O by alkaline treatment. There was only one difference between the structure of ganoaustratetraenone A and 4,4,14α-trimethyl-3,7,11,15,20-pentaoxo-5α-pregn-8-en, which was revealed by analyzing the NMR spectra. The pivotal HMBC correlation from H3-30 (δH 1.27, s) to a methylene at δC 32.3 (Fig. 2) suggested that C-15 of 1 is a methylene instead of being a ketone group in 4,4,14α-trimethyl-3,7,11,15,20-pentaoxo-5α-pregn-8-en. Therefore, compound 1 was elucidated to be a hexa-nor-lanostane derivative.

Table 1 1 H NMR spectroscopic data for compounds 15
Table 2 13C NMR spectroscopic data for compounds 110
Fig. 1
figure 1

Structures of compounds 110

Fig. 2
figure 2

Key 1H-1H COSY and HMBC correlations of compounds 110

Compound 2 (Fig. 1) was isolated as a white powder. The HRESIMS analysis of 2 gave a protonated ion peak at m/z 401.23224 [M + H]+, corresponding to the molecular formula of C24H32O5 (calcd. for C24H33O5, 401.23280) (Additional file: 1). The 1D NMR spectroscopic data of 2 (Tables 1 and 2) showed six singlet methyls, five methylenes, three methines including one oxygenated, four quaternary carbons (sp3 hybridized), a pair of non-protonated olefinic carbons, and four ketone carbonyls. Comparing the NMR data of 1 and 2 suggested that 2 was a structural congener of 1. Compound 2 differed from 1 by the presence of a hydroxy substituent at C-6, which was confirmed by the chemical shift of C-6 (δC 72.1), by the 1H-1H COSY correlation of H-5 (δH 2.30)/H-6 (δH 4.42), and by the key HMBC correlations from OH-6 (δH 3.68) to C-5 (δC 54.9), C-6, and C-7 (δC 202.6) (Fig. 2). The ROESY correlation of H3-19 (δH 1.24)/H-6 enabled the assignment of OH-6 as α orientation (Fig. 3). Thus, compound 2 was determined to be 4,4,14α-trimethyl-6α-hydroxy-5α-pregn-8-en-3,7,11,20-tetraone, and was trivially named ganoaustratetraenone B.

Fig. 3
figure 3

Key ROESY correlations of compounds 28, and 10

Ganoaustraldehyde A (3) (Fig. 1) was obtained as a white, amorphous powder. The 1D NMR spectroscopic data of 3 showed six methyl singlets, six methylenes, one methine, four quaternary carbons (sp3 hybridized), two pairs of non-protonated olefinic carbons, three ketone carbonyls, and an aldehyde carbon. The NMR data of 3 (Tables 1 and 2) showed similarity to those of ganodernoid A [17], a penta-nor-lanostane triterpenes isolated from Ganoderma lucidum. Analysis of the HMBC, 1H-1H COSY, and ROESY spectra of 3 allowed the elucidation of the chemical structure, and also revealed the differences between 3 and ganodernoid A. The key HMBC correlations from H3-30 (δH 1.25) to C-15 (δC 31.7), from H3-18 (δH 1.21) to C-17 (δC 170.2), and from H3-22 (δH 1.74) to C-17, C-20 (δC 133.0), and C-21 (δC 192.1) (Fig. 2) indicated that the C-15 in 3 was a methylene group, C-21 was an aldehyde group, and C-17 and C-20 was a double bond. Therefore, the 2D structure of 3 was elucidated as shown in Fig. 1. The key ROESY correlations between the aldehyde proton at δH 10.00 and H-12 (δH 3.11) (Fig. 3) suggested that the C-17-C-21 double bond was Z configuration. The above assignment is consistent with the molecular formula of 3, C25H32O4, which was determined from the HRESIMS analysis (ion peak at m/z 397.23721 [M + H]+, calcd. for C25H33O4, 397.23734) (Additional file: 1).

The white amorphous powder ganoaustraldehyde B (4) (Fig. 1) returned a protonated ion peak at m/z 413.23221 [M + H]+ in the HRESIMS analysis, suggesting the molecular formula of C25H32O5 (calcd. for C25H33O5, 413.23225) (Additional file: 1). The 1D NMR spectroscopic data of 4 (Tables 1 and 2) highly resembled to those of compound 3, indicating the analogous structures of 3 and 4. The signal of an oxymethine at δC 72.3 (C-6) in the 13C NMR spectra of 4 compared to those of 3, along with the 2D NMR correlations of 4, including the 1H-1H COSY correlations between H-5 (δH 2.34) and H-6 (δH 4.47), and HMBC correlations from 6-OH (δH 3.69) to C-6 (δC 72.3) and C-7 (δC 202.5) (Fig. 2) suggested that there was a hydroxy group substituted at C-6 in 4. The stereochemistry of the C-17-C-21 double bond was assigned as Z configuration according to the ROESY cross peaks between H-21 (δH 10.03) and H-12 (δH 3.08) (Fig. 3). The OH-6 was determined to be α configuration by the diagnostic ROESY signals of H-6/H3-19 (δH 1.25) (Fig. 3). Therefore, compound 4 was elucidated as shown in Fig. 1.

The molecular formula of compounds 5 and 6 (Fig. 1) were same with those of 3 and 4, respectively. The 1D NMR data of 5 and 6 (Tables 1, 3 and 2) also exhibited high similarity to those of 3 and 4, respectively, thus indicating that 5 and 6 were the respective structure congeners of 3 and 4. Analysis of the 2D NMR spectra of 5 and 6 enabled us to identify the only difference between these two pairs of compounds, which was the configuration of C-17-C-21 double bond (Fig. 2). The diagnostic ROESY correlations of H3-21(δH 1.80)/H-12 (δH 3.05, 3.10), and H-22 (δH 10.00)/H-16 (δH 3.12 in 5; 3.19 in 6) (Fig. 3) suggested the E configuration of C-17-C-21 double bonds of 5 and 6 (Fig. 3). The configuration of OH-6 of 6 was determined as α orientation by the ROESY correlation of H-6/H3-19 (Fig. 3). Therefore, compounds 5 and 6 were identified as ganoaustraldehydes C and D, respectively.

Table 3 1H NMR spectroscopic data of compounds 610 (600 MHz, CDCl3)

Compound 7 (Fig. 1), a pale-yellow oil, had a protonated ion peak at m/z 399.25305 in the HRESIMS analysis (calcd. for C25H35O4, 399.25353) (Additional file: 1). Analysis of the 1H and 13C NMR spectroscopic data of 3 (Tables 3 and 2) revealed that this compound was a structural analogue of 5. The main difference between the two analogues was C-7. In the HMBC spectrum of compound 7, significant correlations from H-7 (δH 4.52) to C-8 (δC 158.1) and C-9 (δC 140.4) were observed (Fig. 2). This evidence together with the 1H-1H COSY correlation of H-5/H-6/H-7 (Fig. 2) suggested that C-7 in 7 was an oxygenated methine, instead of being a ketone group in 5. The exocyclic C-17-C-21 double bond was assigned as E configuration by the key ROESY correlations of H3-21 (δH 1.79)/H-12 (δH 3.04) (Fig. 3). The 7-OH was determined to be α configuration by the ROESY cross peak signals of H-15β (δH 2.14)/H-7 (δH 4.52) and the coupling constant of H-7 (t, 3.3 Hz) (Fig. 3) [18]. Therefore, the structure of 7 was assigned as ganoaustraldehyde E.

The 1D NMR data of the compounds 8 and 9 (Tables 3 and 2) each showed 26 carbon resonances with a high resemblance to those of compounds 2 and 1, respectively. Further analysis of the NMR spectroscopic data suggested that 8 and 9 differed from 2 to 1 by the existence of a trisubstituted double bond (C-20, C-22) and a COOH group (C-23), while by the absence of a ketone group (C-20), respectively. These changes suggested that 8 and 9 were two lanostane triterpenes with six carbon degradation (C-24, C-25, C-26, and C-27). The C-23 in 8 and 9 were carboxylic groups which were assigned by the HMBC correlations from H-22 (δH 5.77/5.78 in 8/9) and H3-21 (δH 2.17/2.18 in 8/9) to C-23 (δC 171.2/170.3 in 8/9) (Fig. 2), as well as the molecular formulas (C26H34O6 for 8, C26H34O5 for 9) which were designated by the HRESIMS analysis. The double bond of C-20(22) were assigned as E configuration by the key ROESY correlations between H-22 and H-17 (δH 2.87/2.85 in 8/9), H-16a, H-16b (Fig. 3). Therefore, compounds 8 and 9 were identified as ganoaustrenoic acids A and B (Fig. 1), respectively.

Compound 10 (Fig. 1) returned a protonated ion peak at m/z 429.26361 in the HRESIMS analysis, implying it has the molecular formula of C26H36O5 (calcd. for C26H37O5, 429.26410). The molecular formula and the 1D NMR spectroscopic data of 10 (Tables 3 and 2) are reminiscent of those of 9, implying that 10 was also tetra-nor-lanostane triterpene. The only difference between 10 and 9 is located at C-7, which was indicated by a comparison of the NMR data. The key HMBC correlation from the proton at δH 4.48 (H-7) to C-5 (δC 45.2), C-6 (δC 29.4), C-8 (δC 159.5), C-9 (δC 140.2) (Fig. 2) suggested that C-7 was a hydroxymethine in 10 instead of being a ketone group in 9. The ROESY correlations of H3-18 (δH 0.68)/H-15β (δH 2.04)/H-7 (δH 4.48), and the coupling constant of H-7 (t, 3.0 Hz) facilitated to determine the orientation of 7-OH as α (Fig. 3). Therefore, compound 10 was identified as ganoaustrenoic acid C.

Since these nor-lanostane are characterized by unusual C-6 oxygenation, and α,β-aldehyde groups, a biosynthetic proposal is postulated as shown in Scheme 1. The key intermediate lanosterol is biosynthesized from squalene, the common precursor of triterpenes. Further oxygenation on multiple sites of the lanosterol leads to the key intermediate A [19, 20]. Abstraction of the proton of OH-20 by the retro-aldol reaction (route A) give compound 1, which has been further oxygenated at C-6 position to yield 2. Oxidative cleavage of C23-C24, followed by elimination of the C-20 hydroxy group through E1cb mechanism to produce 9, which is been further oxygenated or reduced to yield 8 and 10. Alternatively, A undergoes oxygenation at C-22, and oxidative cleavage of C22-C23, and further elimination of C-20 hydroxy group giving compounds 3 and 5. Late-stage oxygenation and reduction of 3 and 5 produce 4, 6, and 7.

Scheme 1
scheme 1

Postulated biosynthetic pathways to compounds 110

The anti-NO production activity of the isolates

All the isolates were subjected to screening their inhibition against the NO production in murine monocytic RAW 264.7 macrophages. As a result, only compounds 3 and 4 displayed moderate inhibitory activities with IC50 values of 32.5, 34.2 µM, respectively.

Conclusions

Ten previously undescribed nor-lanostane triterpenes were isolated from the fruiting bodies of Ganoderma australe. The chemical structures of the compounds were determined with the aid of extensive NMR and HRESIMS spectroscopic analysis. These compounds are featured by tetra-, penta-, and hexa-nor-lanostane scaffold, and by unusual oxidative modifications at C-6. These findings put the diversity of nor-lanostanes of Ganoderma origin one step forward.

Experimental section

General experimental procedures

An Autopol IV-T digital polarimeter (Rudolph, Hackettstown, USA) was used to measure the optical rotations. A Shimadzu UV-2401PC UV-vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) was used to record the UV spectra. The Bruker Ascend 500 MHz, Avance III 600 MHz, or Ascend 800 MHz spectrometers (Bruker Corporation, Karlsruhe, Germany) were used to record the one- and two-dimensional NMR spectra. HRESIMS spectra were measured on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA). Column chromatography (CC) were run on Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) and silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Medium pressure liquid chromatography (MPLC) was performed on an Interchim PuriFlash 450 chromatography system (Interchim Inc., Montlucon Cedex, France). The diameter and length of the column used for MPLC were 14 and 450 mm, respectively. The column was fill with Chromatorex C-18 silica gel (particle size: 40–75 µm, flow rate 40 mL/min, Fuji Silysia Chemical Ltd., Kasugai, Japan). Preparative high performance liquid chromatography (prep.-HPLC) was performed on an Agilent 1260 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA). The columns used for prep.-HPLC were Agilent Zorbax SB-C18 (5 µm of particle size, i.d. 9.4 mm × length 150 mm, flow rate 7 mL/min), and Agilent Zorbax SB C-8 column (5 µm of particle size, i.d. 9.4 mm × length 250 mm, flow rate 5 mL/min). RPMI 1640 medium (Hyclone, Logan, UT) was used in the anti-nitric oxide production assays. Griess reagent (reagent A and reagent B) was bought from Sigma (Sigma, St. Louis, MO). The plate reader was TECAN Spark 10 M (Tecan Trading AG, Switzerland).

Fungal material

The fruiting bodies of Ganoderma australe were collected in Tongbiguan Natural Reserve, Dehong, Yunnan Province, China, in 2016, and identified by Prof. Yu-Cheng Dai, who is a mushroom research in Institute of Microbiology, Beijing Forestry University. A voucher specimen of G. australe was deposited in the Mushroom Bioactive Natural Products Research Group at South-Central Minzu University.

Extraction and isolation

10 L of mixed solvent CHCl3:MeOH (v/v 1:1) was used to extract the constituents from the grounded and dry fruiting bodies of Ganoderma australe (3.26 kg) (2.5 L × 4 times) at room temperature. The extract was further resuspended in distilled water and partitioned against ethyl acetate (EtOAc) to afford the EtOAc extract (130 g). The EtOAc extract was fractionated on MPLC by using a stepwise gradient of MeOH in H2O (20-100%) to afford seven fractions (A−G).

Fraction D (2.0 g) was separated by Sephadex LH-20 (CHCl3:MeOH = 1:1) to afford four subfractions (D1−D4). Subfraction D2 (800 g) was separated by silica gel column chromatography (CC) (petroleum ether-acetone from v/v 15:1 to 1:1) to obtain eleven subfractions (D2a−D2k). Compound 1 (5.2 mg, tR = 12.9 min) was purified from D2k (75 mg) by prep.-HPLC (MeCN-H2O: 30−50:50, 30 min, 4 mL/min). Subfraction D4 (610 mg) was subjected to silica gel CC (petroleum ether − acetone from v/v 6:1 to 1:1) and yielded eleven subfractions (D4a − D4k). Subfraction D4d (6.1 mg) was purified by prep.-HPLC (MeCN − H2O: 40:60−60:80, 25 min, 4 mL/min) to yield compound 2 (3.8 mg, tR = 16.5 min).

Fraction F (3.6 g) was separated by Sephadex LH-20 (MeOH) to afford twenty-six subfractions (F1-F26). Subfraction F26 (280 mg) was separated by column chromatography (CC) on silica gel (petroleum ether-acetone from v/v 15:1 to 1:1) to obtain four subfractions (F26a-F26d). Subfraction F26a (48 mg) was isolated on prep-HPLC (MeCN − H2O: 30:70 − 50:50, 30 min, 4 mL/min) to yield compounds 5 (tR = 7.80 min, 1.0 mg), 6 (tR = 6.50 min, 0.8 mg), and 4 (tR = 11.50 min, 1.3 mg). Compound 3 (0.9 mg, tR = 14.4 min) was purified from F26d (11 mg) by prep.-HPLC (MeCN − H2O: 30:70 − 50:50, 30 min, 4 mL/min).

Fraction E (1.6 g) was separated by Sephadex LH-20 (MeOH) to afford twelve subfractions (E1-E12). Subfraction E5 was separated by CC on silica gel (petroleum ether-acetone from v/v 15:1 to 1:1) to obtain four subfractions (E5a-E5d). Compound 8 (2.4 mg, tR = 29.4 min) was purified from E5b (23 mg) by prep.-HPLC [MeCN + MeOH (2:1) − H2O: 50:50, 40 min, 4 mL/min]. Compound 9 (2.1 mg, tR = 20.0 min) was purified from E5c (42 mg) by prep.-HPLC [MeCN + MeOH (2:1) − H2O: 40:60–60:40, 25 min, 4 mL/min]. Compound 10 (6.5 mg, tR = 13.5 min) was purified from E2e (210 mg) by prep-HPLC (MeCN-H2O: 30:70 − 50:50, 25 min, 5 mL/min). Compound 7 (1.8 mg, tR = 20.0 min) was purified from E2g (13 mg) by prep.-HPLC (MeCN-H2O: 50:50–70:30, 25 min, 4 mL/min).

Spectroscopic data of compounds

Ganoaustratetraenone A (1)

Pale-yellow needles; [α] 24D  +164.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 255.0 (3.94); 1H NMR (600 MHz, CDCl3) data, see Table 1, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 385.23724 [M + H]+, calcd. for C24H33O4, 385.23718.

Ganoaustratetraenone B (2)

White powder. [α] 24D  +259.4 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 260.0 (3.82); 1H NMR (600 MHz, CDCl3) data, see Table 1, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 401.23224 [M + H]+, calcd. for C24H33O5, 401.23280.

Ganoaustraldehyde A (3)

White, amorphous powder; [α] 24D  +10.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 250.0 (3.73); 1H NMR (500 MHz, CDCl3) data, see Table 1, 13C NMR (125 MHz, CDCl3) data, see Table 2; HRESIMS m/z 397.23721 [M + H]+, calcd. for C25H33O4, 397.23734.

Ganoaustraldehyde B (4)

White, amorphous powder; [α] 24D  +55.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 250.0 (3.88); 1H NMR (600 MHz, CDCl3) data, see Table 1, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 413.23221 [M + H]+, calcd. for C25H33O5, 413.23225.

Ganoaustraldehyde C (5)

White, amorphous powder; [α] 24D  +33.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 250.0 (4.10); 1H NMR (600 MHz, CDCl3) data, see Table 1, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 397.23743[M + H]+, calcd. for C25H33O4, 397.23734.

Ganoaustraldehyde D (6)

White, amorphous powder; [α] 24D  +68.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 250.0 (3.82); 1H NMR (600 MHz, CDCl3) data, see Table 2, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 413.23224[M + H]+, calcd. for C25H33O5, 413.23225.

Ganoaustraldehyde E (7)

Pale-yellow oil. [α] 24D  +156.8 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 250.0 (4.62); 1H NMR (600 MHz, CDCl3) data, see Table 2, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 399.25305 [M + H]+, calcd. for C25H35O4, 399.25353.

Ganoaustrenoic acid A (8)

White powder. [α] 24D  +162.7 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 220.0 (4.08); 1H NMR (600 MHz, CDCl3) data, see Table 2, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 443.24299 [M + H]+, calcd. for C26H35O6, 443.24336.

Ganoaustrenoic acid B (9)

White powder. [α] 24D  +10.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 225.0 (4.23); 1H NMR (600 MHz, CDCl3) data, see Table 2, 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS m/z 427.24808 [M + H]+, calcd. for C26H35O5, 427.24845.

10 ganoaustrenoic acid C (10)

Yellowish oil. [α] 24D  +84.3 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 225.0 (3.90); 1H NMR (600 MHz, CDCl3) data, see Table 2, 13C NMR (200 MHz, CDCl3) data, see Table 2; HRESIMS m/z 429.26361 [M + H]+, calcd. for C26H37O5, 429.26410.

Nitric oxide production in RAW 264.7 macrophages

The murine monocytic RAW 264.7 macrophages were cultured in RPMI 1640 medium 10% Fetal Bovine Serum (FBS). Firstly, the DMSO stocks of the compounds were made, and further been serially diluted in media to make different concentrations of stocks. The cell mixtures were distributed into 96-well plates (2 × 105 cells/well) and preincubated in a humid atmosphere with 5% CO2 for 24 h at 37 °C. Then different concentrations of stocks of the test compounds were added into each well, the maximum concentration of the test compound was 25 µM. Then the lipopolysaccharides (LPS) were added to each cell (final concentration 1 µg/mL), and continued to incubate for another 18 h. After adding 100 µL of Griess reagent to 100 µL of each supernatant from the LPS-treated or LPS- and compound-treated cells in triplicates, and incubated for another 5 min, the nitric oxide (NO) production of each cell was assessed by measuring the absorbance at 570 nm. Pyrrolidine dithiocarbamate (PDTC) was used as the positive control (IC50 20.0 µM).