Cytotoxic Triterpenes From Trametes orientalis

Abstract

One new triterpene, 24-ene-2′-methyl carboxy acetylquercinicate, and 3 known compounds were isolated from the ethyl acetate extract of the fruiting bodies of Trametes orientalis (Yasuda) Imazeki. Their structures were elucidated by means of extensive spectroscopic analysis and compared with data reported in the literature. Furthermore, they were evaluated for their cytotoxicity against 5 human cancer cell lines in vitro. Compound 4 showed significant cytotoxicity, especially to the leukemic HL-60 cell line (IC₅₀ = 1.77 ± 0.07 μM), while compounds 1-3 showed significant cytotoxicity to SMMC-7721 (hepatoma cell line).

Trametes orientalis (Yasuda) Imazeki, family Polyporaceae, mainly grows on living, staked, and decaying trees. The fruiting bodies of this fungus have been used for the treatment of pulmonary tuberculosis, bronchitis, and rheumatic diseases in Chinese traditional medicine. ¹ Earlier studies showed that the ethyl acetate (EtOAc) extract of the fruiting bodies from this fungus exhibited antitumor ² and antimicrobial activities ³ and was also good for the lungs and other respiratory tissues. ⁴ However, only some sterols have been reported from phytochemical research in the past few years. ⁴ In order to search for effective cytotoxic constituents against tumor cell lines, a further phytochemical investigation was conducted on its EtOAc extract. As a result, a new compound and 3 known compounds were obtained (Figure 1). These were evaluated for cytotoxic activities against five human cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay.

Figure 1

Structures of compounds 1-4.

Results and Discussion

One new triterpene, 24-ene-2′-methyl carboxy acetylquercinicate (1), and 3 known triterpenoids (2-4) were isolated from the EtOAc extract of T. orientalis. The structures of the known compounds were determined to be 24- ene- carboxyacetylquercinic acid (2), ⁵ 24-ene-dimethyl carboxy acetylquercinicate (3), ⁵ and fomlactone B (4), ⁶ by comparing their spectroscopic data with those reported in the literature. Among them, compound 1 is new, and compounds 2-4 (Figure 1) are reported from this fungus for the first time.

Compound (1), as a white powder, $[\alpha {]}_{\mathrm{D}}^{20}$ = −58 (c 1.00, MeOH), had a molecular formula of C₃₅H₅₂O₇ on the basis of matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) ([M − H]⁺ m/z 583.3643, calcd. 583.3640), indicating 10 degrees of unsaturation. The IR spectrum showed absorptions for an ester carbonyl group (1738, 1730, 1715 cm⁻¹) and a conjugated carbonyl group (1687 cm⁻¹). The ¹³C-NMR spectrum (Table 1) revealed the presence of 3 ester carbonyl carbons (δ _C 166.0, 167.3, 179.5), a carbonyl carbon (δ _C 201.5), and 2 ethylenic linkages (δ _C 134.2, 134.7, 148.4, 125.3), one of which belongs to the terminal methylene signal (δ_C 125.3). Because of the larger chemical shift (δ _C 125.3), it is speculated that the alkenyl was attached to the electron-absorbing group. Six methyl protons (δ _H 0.72, 0.86, 0.87, 0.89, 0.91, 0.98, s) and a hydroxymethyl proton (δ _H 3.71, s) were also observed in the ¹H-NMR spectrum (Table 1). Combined with other ¹H-NMR signals in the high field, compound 1 was initially presumed as a triterpene. The NMR data for compound 1 were similar to that of 24-ene-carboxyacetylquercinic acid (2), ⁵ except for the presence of a methoxy signal (δ _C 52.4), presumed to be a mono-methyl esterification derivative of compound 2.

Table 1

Nuclear Magnetic Resonance (NMR) Data for Compound 1 (Trimethylsilane as the Internal Standard, Δ in ppm, J in Hz) ^a .

NO.	δ C	δ H
1	30.8 (t)	1.40-1.43 b , 1.69-1.71 b
2	23.2 (t)	1.40-1.46 b , 1.66-1.68 b
3	79.8 (d)	4.69 (1H, t, 2.5)
4	36.8 (s)
5	45.3 (d)	1.18-1.19 b
6	18.0 (t)	1.33-1.37 b , 1.66-1.68 b
7	26.0 (t)	2.00-2.03 b
8	134.1 (s)
9	134.6 (s)
10	36.8 (s)
11	20.9 (t)	2.03-2.05 b
12	30.7 (t)	1.48-1.52 b , 1.68-1.71 b
13	45.3 (s)
14	50.0 (s)
15	30.8 (t)	1.19-1.23 b , 1.54-1.57 b
16	28.4 (t)	1.86-1.89 b , 1.69-1.71 b
17	50.7 (d)	1.18-1.23 b
18	16.0(t)	0.72 (3H, s)
19	19.0 (t)	0.98 (3H, s)
20	33.9 (d)	1.90 (1H, m)
21	19.7 (t)	0.86 (3H, d, 6.0)
22	44.6 (d)	2.48 (dd, 10.0, 16.0),
		2.68 (dd, 2.8, 16.0)
23	201.5 (s)
24	148.1 (s)
25	40.2 (d)	3.65-3.66 (1H, m)
26	179.5 (s)
27	16.1 (t)	1.31 (3H, d, 7.2)
28	24.2 (t)	0.89 (3H, s)
29	27.6 (t)	0.87 (3H, s)
30	21.7 (t)	0.91 (3H, s)
31	125.3 (d)	5.91(br s)
		6.17(br s)
1′	166.0 (s)
2′	41.8 (d)	3.47 (2H, br s)
3′	167.3 (d)
OCH3	52.4(t)	3.71 (3H, s)

^{a 1}H-NMR and ¹³C-NMR data were recorded in deuterated chloroform at 500 MHz and 125 MHz, respectively.

^bSignals were overlapped.

Further analysis of compound 1 was performed with the aid of 2-dimensional (2D) NMR (Figure 2). The key heteronuclear multiple bond correlations from H-2′ (3.47, br s), CH ₃O (3.71, s) to C-3′ (δ _C 167.3) indicated that compound 1 was a mono-methyl esterification derivative of compound 2 at C-3′. The relative configuration of 1 was established by nuclear Overhauser effect spectroscopy analyses, where the cross-peak network observed between H-3/H-18/H-19/H-29, and H-18/H-20/H-27 confirmed that they were located on the same ring face. Based on these data, compound 1 was identified as, 24-ene-2′-methyl carboxy acetylquercinicate, a new compound.

Figure 2

Key nuclear Overhauser effect spectroscopy and heteronuclear multiple bond correlations of compound 1.

It was reported that compounds 2 and 3 had a significant inhibitory effect on Epstein-Barr (scar-rash virus), which is one of the main causes of a malignant tumor. ⁵ Therefore, the compounds indicated good antitumor activity. All of the isolates were evaluated for their cytotoxicity against human tumor cell lines, HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW-480 (colon cancer) using the MTS method, ⁷ using cisplatin as a positive control.

The primary screening results (Figure 3) of compounds 1-4 on 5 human tumor cell lines showed that when the sample concentration was 40 µM, compounds 1-3 had some toxicity to SMMC-7721, but not to the other test cells. Subsequently, the effective compounds 1-3 were further subjected to the measurement of half-maximal inhibitory concentration (IC₅₀) values. The results of rescreening (Table 2) showed that they exhibited significant cytotoxic activities to SMMC-7721, with IC₅₀ values of 13.3 ± 0.6, 18.9 ± 0.6, and 12.5 ± 1.2 µM, respectively, which were similar to that of cisplatin (Table 2). The structures of compounds 1-3 were very similar, except for the methyl esterification and, therefore, they may be allowed to be considered for structure–activity relationships. Because of the similar inhibition ratio, the degree of esterification of the molecular structure was speculated to have little effect on the cytotoxicity of hepatic cancer cells.

Figure 3

Cell inhibition of five indicated human cancer cell lines after treatment with compounds 1-4 at 40 µM. Data represent the mean ± standard deviation of 3 independent experiments.

Table 2

Cytotoxicities of Compounds 1-4 and the Positive Control Cisplatin.

Compound	HL-60	A-549	SMMC-7721	MCF-7	SW480
	IC50 ± SD a (μM)
1	—	—	13.3 ± 0.6	—	—
2	—	—	18.9 ± 0.6	—	—
3	—	—	12.5 ± 1.24	—	—
4	1.77 ± 0.07	15.7 ± 0.7	16.9 ± 0.4	15.9 ± 0.7	17.5 ± 0.6
Cisplatin b	10.0 ± 0.3	29.2 ± 2.	11.1 ± 0.6	29.8 ± 0.4	25.4 ± 1.9

IC₅₀, half-maximal inhibitory concentration; SD, standard deviation.

^aThe data represent the mean ± SD of 3 independent experiments.

^bPositive control.

However, compound 4 as a spiro triterpene is extremely rare. The cell inhibition of 4 was first tested at 40 µM, and the results showed that it exhibited significant cytotoxicity to the above 5 human tumor cells (Figure 3). Compound 4 displayed significant cytotoxic activities against HL-60, A-549, SMMC-7721, MCF-7, and SW480 cell lines with IC₅₀ values ranging from 1.8 to 17.5 µM (Table 2). Especially, the cytotoxicity to leukocyte HL-60 cell line (IC₅₀ = 1.77 ± 0.07 µM) was much higher than that of cisplatin (IC₅₀ = 10.0 ± 0.35 µM).

Conclusion

In this study, a new compound, 24-ene-3′-methylcarboxyacetylquercinicate, and 3 known homolanostanoid triterpenes were isolated from the fruiting bodies of T. orientalis by column chromatography (CC). The bioassay in vitro indicated that fomlactone B exhibited strong cytotoxicity against the HL-60 cell line. All compounds exhibited strong cytotoxicity against SMMC-7721 in vitro, with IC₅₀ values ranging from 11.1 to 18.9 µM, which have the potential for further development into antilung cancer drugs.

Experimental

General

The optical rotations of the compounds were measured with a JASCO P-1020 DIP digital polarimeter (Jasco, Tokyo, Japan). The IR spectra were recorded on a Bruker Tensor-27 spectrometer with KBr pellets (Bruker, Bremen, Germany), the UV spectra on a Shimadzu UV-2401 PC spectrometer (Shimadzu, Suzhou, China), and the circular dichromism spectra on a Jasco J-725. The 1D-NMR and 2D-NMR spectra were obtained using an Avance III-500 instrument (Bruker) with trimethylsilane as the internal standard. The electrospray ionization (ESI)-MS experiment was performed on a Bruker HTC/Esquire mass spectrometer and the MALDI-TOF/TOF-MS on a Bruker Daltonics Flex Analysis spectrometer (Bruker). CC was performed using silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Fairfield, CT, USA), and reversed-phase C8 silica gel (50 mm, Merck, Darmstadt, Germany). Analytical thin-layer chromatography (TLC) was performed using silica gel GF254 plates (0.25 mm thickness, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). The detection was performed by spraying the plates with 5% sulfuric acid followed by heating.

Fungal Material

The fruiting bodies of T. orientalis (Yasuda) Imazeki were collected in Changbai Mountain area of Jilin Province, China, in September 2015 and identified by Dr Zhang Ying of Southwest Forestry University. A voucher specimen (TO01) was deposited in the Forestry College of Southwest Forestry University.

Extraction and Isolation

The fruiting bodies of T. orientalis (5.0 kg) were dried in the sun and then cut with pliers. Small pieces of the material were extracted 3 times (15 L × 2 hours × 3) with ethyl acetate at water bath temperature (70°C), and the combined solution was concentrated to produce a crude extract (200 g). This was subjected to silica gel CC, eluting with light petroleum–ethyl acetate (10:1 to 4:1), and then chloroform–methanol (10:1 to 4:1) to give 5 fractions (fractions 1-5) based on TLC analysis.

Fraction 4 (5.0 g) was further chromatographed on silica gel using light petroleum–acetone (10:1 to 1:1) to afford 2 fractions (Fr. 4-1-2). Fr.4-2 was crystallized from chloroform:methanol (1:9), resulting in compound 4 (8.1 mg).

Fraction 5 (100.0 g) was subjected to further silica gel CC, eluting with light petroleum–acetone (15:1 to 1:2) to give 4 fractions (Fr. 5-1-Fr. 5-4). Fr. 5-3 was then separated by silica gel CC eluting with chloroform–acetone (30:1 to 5:1) to obtain 3 fractions (Fr. E-3A-Fr. E-3C). Fr. E-3A was further treated by repeated silica gel CC using chloroform–acetone (30:1 to 5:1) to produce compound 3 (5.0 mg). Fraction E-3C was further purified by repeated silica gel CC eluting with chloroform–acetone (20:1 to 5:1) to give subfractions Fr. E-3-3-1 and Fr. E-3-3-2. Compound 1 (10.0 mg) was isolated from Fr. E-3-3-2 by Sephadex LH-20 (chloroform–methanol, 7:3) CC. Fr. E-4 was separated over a silica gel column eluting with chloroform–acetone (10:1 to 1:1) to obtain 3 fractions (Fr. E-4A-Fr. E-4C). Fr. E-4C was further treated by silica gel CC using chloroform–methanol (15:1 to 3:1) to yield compound 2 (5.3 mg).

24-Ene-2′-methyl carboxy acetylquercinicate (1)

White powder. $[\alpha {]}_{\mathrm{D}}^{20}$ = −58 (c 1.00, MeOH); UV (MeOH) λ _max (log ϵ): 206 (0.39) nm, 242 (0.14) nm; ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) data, see Table 1; ESI-MS m/z 607 [M + Na]⁺; MALDI-TOF/TOF-MS m/z 583.3643 [M − H]⁺ (calcd. 583.3640).

Cytotoxicity Assay

Lung cancer (A-549), human myeloid leukemia (HL-60), breast cancer (MCF-7), hepatocellular carcinoma (SMMC-7721) and colon cancer (SW-480) were obtained from the American Type Culture Collection. Proliferation inhibition of compounds 1-4 against these 5 cell lines was measured by the MTS method. ⁷ Cells were plated in 96-well plates for 24 hours before treatment and continuously exposed to different concentrations of compounds for 72 hours. Dimethyl sulfoxide (0.1% v/v) was used as the negative control and cisplatin as positive control.