Camphoratins A–J, Potent Cytotoxic and Anti-inflammatory Triterpenoids from the Fruiting Body of Taiwanofungus camphoratus

Abstract

Ten new triterpenoids, camphoratins A–J (1–10), along with 12 known compounds were isolated from the fruiting body of Taiwanofungus camphoratus. Their structures were established by spectroscopic analysis and chemical methods. Compound 10 is the first example of a naturally occurring ergosteroid with an unusual cis-C/D ring junction. Compounds 2–6 and 11 showed moderate to potent cytotoxicity with EC₅₀ values ranging from 0.3 to 3 μM against KB and KB-VIN human cancer cell lines. Compounds 6, 10, 11, 14–16, 18, and 21 exhibited anti-inflammatory NO-production inhibition activity with IC₅₀ values of less than 5 μM, which was more potent than the nonspecific NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME).

Taiwanofungus camphoratus (synonym: Ganoderma camphoratum; Antrodia cinnamomea; Antrodia camphorata) (Polyporaceae, Aphyllophorales), is a rare and precious medical fungus in Taiwan and is called a “national treasure of Taiwan”.¹ Its Chinese name is Zhan-Ku or Niu-Chang-Chih. The microorganism is parasitic to the inner heart-wood wall of old hollow trunks of Cinnamomum kanehirai Hay. (Lauraceae). The growth rate of natural T. camphoratus in the wild is very slow, and it is difficult to cultivate in a greenhouse, making fruiting bodies expensive to obtain. In traditional Taiwanese folk medicine T. camphoratus has been used as an important health food for treating food, alcohol, and drug intoxication, diarrhea, abdominal pain, hypertension, itching, and liver cancer.² Previous studies on the chemical constituents of the fruiting body of T. camphoratus showed that it is a rich source of triterpenoidic acids, some of which have shown anti-inflammatory,³ anticholinergic,⁴ and antiserotonergic activities.⁴ Furthermore, zhankuic acids A and C exhibited significant cytotoxicity against P-388 murine leukemia cells in vitro.⁴ The present study on the chemical constituents of an EtOH extract of the fruiting body of T. camphoratus has led to the isolation of 10 new triterpenoids, namely camphoratins A–J (1–10), and 12 known compounds, including zhankuic acids A–C (11–13),^4,5 zhankuic acid A methyl ester (14),⁴ antcin A (15),⁵ antcin C (16),⁵ antcin K (17),⁶ methyl antcinate H (18),⁷ eburicol (19),⁸ ergosterol D (20),⁹ methyl 4α-methylergost-8,24(28)-diene-3,11-dion-26-oate (21),¹⁰ and ergosterol peroxide (22).¹¹ Cytotoxic activity, inhibition of nitric oxide (NO) or reactive oxygen species (ROS) production, and free radical-scavenging activity of the isolates were evaluated in our study.

Results and Discussion

Camphoratin A (1) was obtained as a colorless powder. The HRESIMS of 1 showed a pseudomolecular ion peak at m/z 511.3038, consistent with a molecular formula of C₂₉H₄₄O₆Na, and eight degrees of unsaturation. The UV and IR absorption bands at 255 nm and 1709, 1660, and 3408 cm⁻¹, respectively, suggested the presence of α,β-unsaturated carbonyl and carboxylic acid functionalities. The former was corroborated by carbon resonances at δ 202.8 (qC), 154.3 (qC), and 141.2 (qC), and the latter was evidenced by the resonance at δ 176.9 (qC). An exocyclic double bond was also identified from the NMR signals at δ_C 150.7 (qC), 110.5 (CH₂) and δ_H 5.07, 5.23 (each 1H, s). The above data, coupled with the characteristic methyl signals at δ_H 0.90 (3H, s), 1.11 (3H, d, J = 7.6 Hz), 1.18 (3H, d, J = 6.8 Hz), 1.48 (3H, d, J = 7.2 Hz), and 1.57 (3H, s), suggested a 4-methyl-ergost-8-en-11-one skeleton similar to that of antcin C (16).⁵ The 3α-OH functionality was deduced by the correlations from H-3 (δ 3.89, 1H, d, J = 1.6 Hz, β-orientation) to C-4 and C-1 and from H₃-29 to C-3, C-4, and C-5 in the HMBC spectrum of 1. The hydroxy groups attached at C-7 and C-12 were further designated by the correlations from H-7 (δ 4.52, 1H, t, J = 8.4 Hz) to C-6 and C-8 and from H-12 (4.44, 1H, s) to C-13, C-14, and C-17, respectively. The C-25 carboxylic functionality was assigned due to the presence of an HMBC correlation from H-25 (δ 3.45, 1H, q, J = 6.8 Hz) to the carbon resonance at δ 176.9. A comparison of the NMR spectroscopic data of 1 with those of 16⁵ confirmed the above elucidation and unambiguously established the structure of 1. The relative configuration of 1 was determined by the NOE correlations observed in a NOESY experiment. In the NOESY spectrum of 1, H-7 showed NOE enhancements with both H-5 and H-14, suggesting that these protons had α-orientations. Furthermore, NOE correlations between H-3 and H-4 as well as between H-12 and H₃-18 disclosed that these protons are β-oriented.

Camphoratin B (2) was obtained as a colorless syrup. The HRESIMS of 2 gave an [M + Na]⁺ peak at m/z 495.3089 and established the molecular formula C₂₉H₄₄O₅Na, which is 16 mass units less than that of 1. The ¹H NMR and ¹³C NMR spectroscopic data of 2 were similar to those of 16 except for the presence of a 3α-OH in 2 rather than a carbonyl group in 16. The data were also similar to those of 1 except that the hydroxy-containing methine (C-12) in 1 was replaced by a methylene group in 2. This assignment was confirmed by the significant up-field shift of H-14 (δ 2.66, 1H, dd, J = 12.0, 6.0 Hz) in 2, relative to that in 1 (δ 3.57, 1H, dd, J = 12.0, 6.8 Hz), due to the absence of a quasi 1,3-diaxial interaction between H-14 and 12α-OH. In addition, the HMBC correlations from H₂-12 to C-11, C-13, C-14, and C-18 further demonstrated the above elucidations.

Camphoratin C (3) was obtained as a colorless powder and was found to possess the molecular formula, C₂₉H₄₂O₆Na, as deduced from the HRESIMS and NMR data. Its IR spectrum showed the presence of hydroxy (3411 cm⁻¹), carboxylic acid (1707 cm⁻¹), and conjugated carbonyl (1674 cm⁻¹) groups. The UV spectrum showed similar absorption bands to those of 11–13,⁴ which indicated the presence of an 8(9)-en-7,11-dione moiety. This assignment was further corroborated by the carbon resonances at δ 203.05 (qC), 203.0 (qC), 155.0 (qC), and 144.2 (qC). A comparison of NMR spectroscopic data of 3 with those of 17⁶ revealed identical substitution at C-3 and C-4, while the C-7 hydroxy group in 17 was oxidized to a carbonyl moiety in 3.

Camphoratin D (4) was isolated as a colorless powder and exhibited an [M + Na]⁺ peak at m/z 509.2875, corresponding to a molecular formula of C₂₉H₄₂O₆Na as obtained from HRESIMS. Its IR spectrum showed the presence of hydroxy (3444 cm⁻¹), carbonyl (1708 cm⁻¹), and conjugated ketone (1674 cm⁻¹) groups. The UV spectrum showed absorption bands at 246 nm, suggesting the presence of an 8(9)-en-11-one moiety,⁵ which was corroborated by the carbon resonances at δ199.5 (qC), 154.3 (qC), and 141.2 (qC). The proton resonance at δ 4.98 (1H, t, J = 8.4 Hz) was attributed to the 7β hydroxy functionality. The other carbonyl carbon resonance at δ 211.0 was assigned to C-3 due to the observation of HMBC correlations from H₃-29 to C-3, C-4, and C-5. The side chain moiety was found to be the same as that in 1–3. Moreover, a hydroxy-containing quaternary carbon was assigned at C-14 according to the downfield shift of C-15 as compared to the corresponding carbon in 1–3. In the ¹³C NMR spectrum of 4, the significant upfield shift for C-12 (Δ = –9.4 ppm) and C-17 (Δ = –5.5 ppm) as well as the downfield shift for C-18 (Δ = +3.9 ppm), relative to those in 2 (C-12, δ 58.9; C-17, δ 55.0), suggested an α-orientation for 14-OH.^7,12

Camphoratin E (5), a colorless syrup, was found to possess the molecular formula C₃₀H₄₄O₅, as deduced from the HRESIMS and NMR spectroscopic data. Its IR spectrum showed the presence of hydroxy (3491 cm⁻¹), ester (1730 cm⁻¹), and conjugated carbonyl (1678 cm⁻¹) groups. The UV absorption band at ν_max 260 nm and carbon resonances at δ 203.1 (qC), 202.1 (qC), 153.7 (qC), and 144.7 (qC) indicated the presence of an 8(9)-ene-7,11-dione moiety. The NMR spectroscopic data of 5 were similar to those of 12⁴ except for additional methoxy signals at δ_H 3.66 and δ_C 51.9, which suggested that 5 is the methyl ester of 12. In the ¹³C NMR spectra of 5, several smaller signals at δ_C 31.0, 45.5, and 16.3 revealed the presence of an epimeric mixture at C-25.⁴

The HRESIMS of camphoratin F (6) established the same molecular formula, C₃₀H₄₄O₅, as that of 5. Its UV and IR spectra showed the presence of an 8(9)-en-11-one moiety (λ_max 251 nm and ν_max 1669 cm⁻¹), a carbonyl group (1711 cm⁻¹), and an ester moiety (1735 cm⁻¹). Comparison of the NMR data of 6 and 5 showed that the hydroxy-linked methine resonance of H-3 in 5 was absent from the NMR spectrum of 6, and replaced by a carbonyl functionality at δ_C 212.3. In addition, the carbon resonance for the C-7 carbonyl group in 5 was converted to a hydroxy-linked methine (δ_H 4.39, 1H, t, J = 8.0 Hz) in 6. A β-orientation of the hydroxy group at C-7 was deduced by the splitting pattern of H-7 as compared to those in 1 and 2. Similarly to 5, the NMR data of 6 showed the presence of an epimeric mixture at C-25 (Table 1).

Table 1.

¹³C NMR Data for Compounds 1–4 (in Pyridine-d₅) and 5–10 (in CDCl₃)

position	1a	2a	3a	4b	5a	6a	7b	8a	9a	10a
1	29.7	29.8	28.8	36.3	27.8	35.7	34.6	35.5	38.8	35.1
2	30.6	30.7	26.6	38.0	29.1	37.8	37.5	37.0	38.1	37.8
3	70.3	70.2	74.3	211.0	70.3	212.3	212.8	213.7	211.9	213.1
4	35.0	35.4	74.0	43.8	34.5	43.8	43.8	44.8	44.2	44.3
5	40.4	40.4	44.6	47.7	41.1	48.2	44.6	51.0	42.9	50.6
6	32.9	32.7	37.0	35.0	38.1	32.5	31.3	21.3	30.0	21.1
7	70.1	70.1	203.5	70.5	202.1	69.9	70.2	30.6	117.1	32.3
8	154.3	155.1	155.0	154.3	144.7	153.2	153.0	157.5	139.4	154.6
9	141.2	143.0	144.2	141.2	153.7	141.2	140.7	139.1	48.9	138.0
10	37.7	38.1	40.4	38.5	38.7	37.0	37.2	38.2	34.4	36.4
11	202.8	201.8	203.0	199.5	203.1	201.3	200.9	200.3	21.7	200.4
12	81.7	58.9	58.0	49.5	57.5	57.9	57.6	58.1	39.3	53.2
13	50.7	48.2	47.7	47.5	47.3	47.6	47.1	47.6	43.3	44.0
14	47.3	53.9	49.9	83.2	49.5	53.0	51.2	53.5	55.0	55.3
15	25.4	25.6	25.7	32.1	24.9	24.8	23.1	24.1	22.9	29.3
16	27.8	28.4	28.2	26.2	27.8	27.8	27.5	28.0	28.1	30.4
17	46.0	55.0	54.3	49.5	53.9	54.4	55.1	55.8	55.8	55.8
18	12.4	12.7	12.3	16.6	11.9	12.1	12.2	12.1	12.1	22.3
19	18.3	17.1	19.7	17.1	15.9	17.5	16.3	17.8	12.4	18.0
20	36.5	36.4	36.1	35.7	35.7	35.7	35.8	36.3	40.5	33.3
21	18.3	18.8	18.8	19.4	18.5	18.5	18.4	18.8	21.1	19.5
22	34.9	34.7	34.6	34.0	33.8	33.9	33.8	34.8	136.7	32.8
23	32.1	31.9	31.9	32.1	31.2	31.2	30.7	31.3	130.4	31.9
					31.0c	31.0c				31.8c
24	150.7	150.6	150.6	150.2	148.5	148.4	148.1	156.9	38.1	148.5
25	46.8	46.9	46.9	46.7	45.7	45.7	45.1	34.3	40.8	45.7
					45.5c	45.5c				45.5c
26	176.9	177.0	177.0	176.7	175.0	175.0	177.4	22.2	66.9	175.0
27	17.3	17.2	17.3	17.2	16.4	16.4	16.2	22.3	12.7	16.4
					16.3c	16.3c				16.3c
28	110.5	110.7	110.7	110.5	110.9	110.9	111.5	106.6	18.3	110.9
29	17.1	17.1	27.5	12.0	15.7	11.5	11.9	12.2		11.6
OMe					51.9	51.9				51.9

^aRecorded at 100 MHz at 25 °C.

^bRecorded at 125 MHz at 25 °C.

^cChemical shifts for 25-epimer.

Camphoratin G (7) was found to have the molecular formula C₂₉H₄₂O₅, 14 mass units less than 6, as deduced from the HRESIMS and NMR data. The UV and IR spectra of 7 were similar to those of 6, except that a carboxylic acid moiety was observed in 7 (ν_max 1707 cm⁻¹) rather than an ester group. Consequently, the structure of 7 might be structurally related to 6. Comparison of ¹H NMR data of 7 with those of 6, antcin F, and methyl antcinate G,⁷ disclosed that 7 might be a C-7 epimer of antcin F. Moreover, the splitting pattern of H-7 (d, J = 2.0 Hz) in 7 suggested a β-oriented proton⁷ rather than an α-proton like those in 1, 2, and 6.

The HRESIMS of camphoratin H (8) gave a pseudomolecular ion peak at m/z 447.3237 [M + Na]⁺, corresponding to the molecular formula C₂₉H₄₄O₂Na. Its UV and IR spectra showed the presence of an 8(9)-en-11-one moiety (λ_max 248 nm and ν_max 1678 cm⁻¹) and a carbonyl group (1711 cm⁻¹). Its ¹H NMR spectrum displayed signals for six methyl groups at δ_H 0.74 (s), 0.95 (d), 1.03 (d), 1.06 (d), 1.29 (d), and 1.34 (s). The above data coupled with the absence of the carbon resonance appropriate for a carboxylic acid or a methyl ester moiety suggested that 8 possesses a 4-methyl-ergost-8-en-11-one skeleton. The NMR spectra of 8 were similar to those of 15,⁵ except that the NMR signals appropriate for a carboxylic functionality were replaced by signals for a C-25 methyl group.

Camphoratin I (9) was found to have the molecular formula C₂₈H₄₄O₂ from HRESIMS analysis. The absence of UV absorption bands from 240 to 270 nm disclosed that 9 did not possess an 8(9)-en-7,11-dione or 8(9)-en-11-one moiety. The IR and ¹³C NMR data of 9 showed the presence of 28 carbon signals, including one carbonyl group (δ 211.9, ν_max 1716 cm⁻¹), two double bonds (δ 139.4, 117.1, 136.7, and 130.4), and one hydroxymethyl moiety (δ 66.9, ν_max 3336 cm⁻¹). The above data, coupled with the characteristic methyl signals at δ_H 0.57 (3H, s), 0.86 (3H, d, J = 6.8 Hz), 1.00 (3H, d, J = 6.8 Hz), 1.01 (3H, s), and 1.02 (3H, d, J = 7.2 Hz), suggested that 9 is similar to the known compound ergost-7-en-3-one.¹³ By comparison of the ¹³C NMR spectroscopic data of 9 with those of ergost-7-en-3-one, it was found that resonances for the nucleus of both compounds were superimposable, revealing that they have different substitution patterns in the side chain moiety. This was confirmed by the HMBC correlations from H₃-21 to C-17, C-20, and C-22, from H₃-28 to C-23, C-24, and C-25, and from H₃-27 to C-24, C-25, and C-26. Comparison of the NMR data of C-24, C27, and C-28 of 9 with those of synthetic compounds led to the establishment of an erythro configuration at C-24 and C-25 (24S, 25S or 24R, 25R). Furthermore, the 24S, 25S configuration for 9 was confirmed by the preparation of its (R)-MTPA ester, in which the proton resonance of H₂-26 showed a broad doublet at δ 4.17.¹⁴ In addition, the MTPA ester helped to establish the E geometry of C-22–C-23 double bond by a 15.0 Hz coupling constant.

The molecular formula of camphoratin J (10) was assigned as C₃₀H₄₄O₄ based on its HRESIMS. Its UV and IR spectra showed the presence of an 8(9)-en-11-one moiety (λ_max 250 nm and ν_max 1669 cm⁻¹), a carbonyl group (1709 cm⁻¹), and an ester moiety (1738 cm⁻¹). The 8(9)-en-11-one moiety was deduced by the carbon resonances at δ 200.4, 138.0, and 154.6 and HMBC correlations from H₂-12 to the carbonyl carbon. The carbonyl and methyl ester functionalities were assigned at C-3 and C-25, respectively, based on the HMBC correlations from H₃-29 to C-3, C-4, and C-5 and from H-25 to the carbon resonance at δ 175.0, which was also correlated with the methoxy group at δ_H 3.66 (3H, s). The inspection of the 2D NMR spectra led to the establishment of the same planar structure as the known compound methyl 4α-methylergost-8,24(28)-diene-3,11-dion-26-oate.¹⁰ However, both compounds differed in carbon resonances for rings C and D, revealing the presence of epimers at the C/D ring junction. The relative configuration of the C/D ring junction in 10 was elucidated by the NOE enhancements observed in a selective 1D NOESY experiment. Irradiation of H₃-18 caused enhancements of H-14, H-20, and H₃-21, but not of H-17, suggesting a cis-fused C/D ring. The downfield shift (Δ = +10.4 ppm) of C-18 in the ¹³C NMR spectrum of 10, relative to that observed for methyl 4α-methylergost-8,24(28)-dien-3,11-dion-26-oate, was also in agreement with a rare cis C/D ring junction, which has been found in compounds from marine sponges.^15–17 Additional smaller carbon signals at δ_C 31.8, 45.5, and 16.3 were attributed to the other C-25 epimer. Compound 10 represents the first ergosteroid with cis-fused C/D ring junction.

The cytotoxicity of the new and known compounds (1–19) was evaluated in parallel with etoposide against the human cancer cell line KB and multidrug-resistant strain KB-VIN in vitro (Table 4). Several of the compounds, including 2–7, 11–13, and 15, showed moderate to potent cytotoxic activity with EC₅₀ values ranging from 0.3 to 15.5 μM. Compounds 3 and 5 showed the highest cytotoxicity against the KB cell line with EC₅₀ values of 0.3 and 0.45 μM, respectively. Compounds 4 and 6 also showed cytotoxicity against KB with EC₅₀ values of 1.0 and 2.0 μM, respectively. More importantly, compounds 4 and 6 retained their activity against multi-resistant strain KB-VIN with EC₅₀ values of 1.4 and 2.9 μM, respectively. It appeared that 4 and 6 were not substrates of P-glycoprotein (P-gp), a key transporter related to drug-resistance, based on the lack of cross-resistance shown by KB-VIN. By comparison, etoposide, a known P-gp substrate, was at least eight-fold less active in the KB-VIN cell line. Comparing the structures of compounds 3–6, we find that 3 and 5 contain the same C-3 α-hydroxy group and C-7 keto moiety, while 4 and 6 contain a C-3 keto and C-7 β-hydroxy group. It seems that the latter triterpene scaffold may overcome the resistance issue. Compound 3 has an additional C-4 β-hydroxy group and a free carboxylic acid tail in its R₅ moiety, which may contribute to the best activity profile of the series.

Table 4.

In vitro Cytotoxicity of Compounds 1–19.

Cmpd	EC50 (μM)
	KB	KB-VIN
1	NAa	NA
2	1.8	NA
3	0.3	2.3
4	1.0	1.4
5	0.45	2.7
6	2.0	2.9
7	15.0	17.5
8	NA	NA
9	NA	NA
10	NA	NA
11	3.0	6.2
12	7.3	8.5
13	15.5	6.4
14	>20 (21)b	>20 (25)
15	4.9	10.0
16	NA	NA
17	NA	NA
18	NA	NA
19	>20 (34)	>20 (18)
etoposide	4.5	>36

^aNA; Not active. No growth inhibition at 20 μM.

^bValues in parentheses are % inhibition at 20 μM.

The anti-inflammatory activities of 2, 6, 9, and 10–22 were evaluated by examining their effects on lipopolysaccharide (LPS)-induced inducible-nitric oxide synthase (iNOS)-dependent NO production and NADPH oxidase (NOX)-dependent ROS production in murine microglial cells (BV2) and peripheral human neutrophils (PMN). Compounds 6, 10, 11, 14–16, 18, and 21 significantly inhibited NOS activity with IC₅₀ values of 2.5, 1.6, 3.6, 0.6, 4.1, 4.2, 2.5, and 1.5 μM, respectively. They were more potent than N^ω-nitro-L-arginine methyl ester (L-NAME) (IC₅₀ 25.8 μM), a nonspecific NOS inhibitor, at inhibiting LPS-induced NO production (Table 5). The remaining compounds, except for 20, inhibited NOS activity with IC₅₀ values ranging from 6.3 to 22.3 μM (Table 5). NOX is the major ROS-producing enzyme in activated inflammatory cells.₁₈ We previously reported that drugs with anti-inflammatory activity also show potent NOX-inhibitory action._19,20 Therefore, we evaluated the isolates for effects on NOX activity in lysates of microglial cells and PMN. Our data suggest that none of the tested compounds were potent inhibitors of NOX in lysates of microglial cells and PMN, relative to the specific NOX inhibitor diphenyleneiodonium (DPI) (IC₅₀ 0.4 and 0.3 μM, respectively) (Table 5). In addition, the free radical-scavenging capacities of these compounds were examined in a cell-free 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution. None of the tested compounds showed significant free radical-scavenging activity.

Table 5.

Effects of Compounds 2, 6, 9, and 10–22 on NOX Activity^a in Murine Microglial Cells and PMN and on NOS Activity^b in Murine Microglial Cells

Compound	IC50 (μM) in NOX		IC50 (μM) in NOS
	activity from BV2 cell lysate	fMLP-induced NOX activation in PMN
2	NA	32.1 ± 3.5*	15.7 ± 0.9*
6	NA	11.2 ± 2.3*	2.5 ± 0.6*
9	NA	17.5 ± 3.9*	12.7 ± 2.2*
10	NA	15.8 ± 4.0*	1.6 ± 0.6*
11	NA	22.1 ± 6.7*	3.6 ± 0.8*
12	NA	NA	9.6 ± 0.7*
13	40.3 ± 3.5*	NA	16.2 ± 0.9*
14	NA	8.4 ± 2.1*	0.6 ± 0.3*
15	45.9 ± 7.9*	29.2 ± 6.7*	4.1 ± 0.5*
16	NA	22.6 ± 3.3*	4.2 ± 1.2*
17	NA	47.2 ± 8.4*	NA
18	16.0 ± 8.1*	18.1 ± 5.9*	2.5 ± 0.3*
19	NA	21.9 ± 6.3*	22.3 ± 2.9*
20	NA	27.9 ± 5.6*	30.6 ± 0.8*
21	NA	16.2 ± 4.3*	1.5 ± 0.7*
22	NA	20.3 ± 6.4*	6.3 ± 1.8*
DPI	0.4 ± 0.2	0.3 ± 0.1	–
L-NAME	–	–	25.8 ± 2.5

^aNOX activity was measured as ROS production triggered with NADPH (200 μM) or fMLP (2μM) in the presence of 1–50 μM of test drugs in BV2 cell lysate or peripheral human neutrophils (PMN). DPI, a NOX inhibitor, was included as a positive control for NOX inhibition.

^bNO production was measured in the presence of 1–50 μM of test drugs. L-NAME (a non-selective NOS inhibitor) was included as a positive control. Data were calculated as 50% inhibitory concentration (IC₅₀) and expressed as the mean ± S.E.M. from 3–6 experiments performed on different days using BV2 cell lysate or PMN from different passages or donors. NA: not active.“–”: samples not tested

^*P < 0.05 as compared with relative positive control, respectively.

Although it is now clear that proliferation of cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents, certainly potentiates and/or promotes neoplastic risk.²¹ In many circumstances, inflammation orchestrates the microenvironment around tumors, contributing to proliferation, survival, and migration. Cancer cells also use selectins, chemokines, and their receptors (involved in inflammatory response) for invasion, migration, and metastasis. Interestingly, Peng et al. found that a crude extract of Antrodia camphorata (Taiwanofungus camphoratus) significantly inhibited proliferation of three transitional cell carcinonoma (TCC) cell lines (RT4, a superficial cancer cell line and TSGH-8301, T24, two metastatic cancer cell lines), likely through different mechanisms.²² Combined with our observations in this study, it would be interesting to evaluate whether the antitumor invasion effect in the crude extract could be mediated through the anti-inflammatory properties of the contained triterpenoids. Thus, the newly identified camphoratin analogs with both potent cytotoxicity and anti-inflammatory activity merit further investigation as cancer chemotherapeutic agents or as anti-inflammatory drugs for the treatment of NO-dependent neurodegenerative disorders.

Experimental Section

General Experimental Procedures

Melting points were determined on a Yanagimoto MP-S3 micro-melting point apparatus. IR spectra were recorded on a Shimazu Prestige-21 FTIR spectrometer. Optical rotations were measured using a Jasco DIP-370 Polarimeter. UV spectra were obtained on a Hitachi UV-3210 spectrophotometer. ESI and HRESI mass spectra were recorded on a Bruker APEX II mass spectrometer. The NMR spectra, including ¹H NMR, ¹³C NMR, COSY, NOESY, HMBC, and HMQC experiments, were recorded on Bruker AVANCE-500 and AMX-400. Silica gel (E. Merck 70–230, 230–400 mesh) was used for column chromatography.

Fungal Material

Wild fruiting bodies of T. camphoratus, which grew in Ping-Tung County, Taiwan, were purchased from the Kaohsiung Society for Wildlife and Nature in 2003. The fungus was identified by Dr. Tun-Tschu Chang (Taiwan Forestry Research Institute). A voucher specimen (TSWu 2003005) was deposited in the Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.

Extraction and Isolation

The fruiting body of T. camphoratus (1.0 kg) was extracted with EtOH (4 × 10 L) under reflux for 8 h. The EtOH extract was concentrated to afford a brown syrup (161 g) and then partitioned between H₂O and n-hexane. The n-hexane layer (9.3 g) was chromatographed on silica gel and eluted with EtOAc in n-hexane (0–100% of EtOAc, gradient) to obtain 10 fractions. Fraction 4 was rechromatographed on a silica gel column using n-hexane–Me₂CO (19:1) as eluent to yield 8 (3.0 mg), 9 (6.0 mg), 10 (4.5 mg), 19 (22.0 mg), 20 (90.2 mg), 21 (22.1 mg), and 22 (16.5 mg). Compound 22 (41.1 mg) was obtained in the same way from fraction 8. The water layer (145 g) was filtered and concentrated under reduced pressure to give a brown syrup (55 g) and a water-insoluble portion (89 g). The water-insoluble portion was chromatographed on a silica gel column using CHCl₃–MeOH mixtures of increasing polarity for elution to obtain 10 fractions (WI-1–WI-10). Compounds 1 (2.2 mg), 5 (2.0 mg), 6 (14.2 mg), 9 (1.0 mg), 14 (1.29 g), 15 (53.8 mg), and 21 (62.2 mg) were obtained from a combined fraction (fractions WI-1 and WI-2) by silica gel column chromatography with gradient elution (CHCl₃–Me₂CO, 39:1 to 14:1). Fraction WI-3 was separated on a silica gel column using i-Pr₂O–MeOH (19:1) as the eluent to yield 11 (141.5 mg), 18 (11.0 mg), 16 (122.9 mg), and 12 (53.0 mg). Fraction WI-4 was chromatographed on a silica gel column with i-Pr₂O–MeOH (12:1) to give 7 (11.3 mg), 18 (38.0 mg), 16 (708.0 mg), and 12 (66.5 mg). Compounds 2 (5.0 mg), 4 (2.2 mg), 7 (3.4 mg), and 13 (286.2 mg) were obtained from fraction WI-5 using silica gel column chromatography (eluent, CHCl₃–MeOH, 12:1). Fractions WI-6 and WI-7 were combined and rechromatographed on a silica gel column with CHCl₃–MeOH (6:1) as the mobile phase to afford 3 (3.8 mg) and 13 (1.81 g). Compound 17 (1.16 g) was isolated from a combined fraction (fractions WI-8 and WI-9) by silica gel column chromatography using i-Pr₂O–MeOH (4:1) as the eluent.

Camphoratin A (3α,7β,11α-trihydroxy-11-oxo-4α-methylergosta-8,24(28)-dien-26-oic acid, 1)

colorless powder (MeOH); mp 117–119 °C; [α] _D ²⁵ +221 (c 0.001, MeOH); UV (MeOH) λ_max(log ε) 255 (3.49) nm; IR (KBr) ν_max 3408, 2959, 2930, 2875, 1709, 1660, 1215, 1059 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 2; ESIMS m/z 511 [M + Na]⁺; HRESIMS m/z 511.3038 (calcd for C₂₉H₄₄O₆Na 511.3035).

Table 2.

¹H NMR Data for Compounds 1–4 (in Pyridine-d₅)

position	1a	2a	3a	4b
1	1.93 m	1.85 m	2.10 td (13.2, 3.2)	1.50 m
	2.78 m	2.85 m	3.04 dt (13.2, 3.2)	3.28 m
2	1.86 m	1.86 m	1.92 m	2.40 m
	1.93 m	1.89 m	2.74 m	2.52 m
3	3.89 d (1.6)c	3.91 d (2.4)	4.02 br s
4	1.64 m	1.62 m		2.39 m
5	2.13 m	2.02 m	2.65 m	1.50 m
6	1.74 m	1.67 m	2.90 dd (13.2, 3.2)	2.23 m
	2.42 m	2.39 m	3.14 t (13.2)	2.51m
7	4.52 t (8.4)	4.50 t (8.4)		4.98 t (8.4)
12	4.44 s	2.43 d (13.2)	2.46 d (13.2)	2.74 d (15.8)
		2.95 d (13.2)	2.97 d (13.2)	2.89 d (15.8)
14	3.57 dd (12.0, 6.8)	2.66 dd (12.0, 6.0)	2.67 m
15	2.19 m	2.01 m	1.66 m	1.80 m
	2.50 m	2.49 m	2.74 m
16	1.42 m	1.45	1.44	1.60 m
				1.83 m
17	2.42 m	1.43 m	1.42 m	1.75 m
18	0.90 s	0.88 s	0.72 s	1.22 s
19	1.57 s	1.49 s	1.99 s	1.45 s
20	1.41 m	1.40 m	1.38	1.56 m
21	1.11 d (7.6)	0.89 d (7.6)	0.87 d (5.2)	1.01 d (6.5)
22	1.37 m	1.31 m	1.30 m	1.27 m
	1.81 m	1.75 m	1.75 m	1.88 m
	2.25 m	2.20 m	2.20 m	2.23 m
	2.44 m	2.39 m	2.38 m	2.42 m
25	3.45 q (6.8)	3.45 q (7.2)	3.45 q (7.2)	3.44 q (7.2)
27	1.48 d (7.2)	1.49 d (6.8)	1.49 d (7.2)	1.47 d (7.2)
28	5.07 s	5.06 s	5.06 s	5.06 s
	5.23 s	5.22 s	5.23 s	5.21 s
29	1.18 d (6.8)	1.18 d (6.4)	1.61 s	1.11 d (6.6)

^aRecorded at 400 MHz at 25 °C.

^bRecorded at 500 MHz at 25 °C.

^cJ values (in Hz) in parentheses.

Camphoratin B (3α,7β-dihydroxy-11-oxo-4α-methylergosta-8,24(28)-dien-26-oic acid, 2)

colorless syrup; [α]_D ²⁵ +54 (c 0.006, MeOH); UV (MeOH) λ_max (log ε) 255 (3.79) nm; IR (KBr) ν_max 3420, 2962, 2935, 2878, 1709, 1659, 1217, 1083 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 2; ESIMS m/z 495 [M + Na]⁺; HRESIMS m/z 495.3089 (calcd for C₂₉H₄₄O₅Na 495.3086).

Camphoratin C (3α,4β-dihydroxy-7,11-dioxo-4α-methylergosta-8,24(28)-dien-26-oic acid, 3)

colorless powder (MeOH); mp 186–188 °C; [α]_D ²⁵ +57 (c 0.067, MeOH); UV (MeOH) λ_max (log ε) 271 (3.80) nm; IR (KBr) ν_max 3411, 2966, 2936, 2878, 1709, 1674, 1230, 1062 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 2; ESIMS m/z 509 [M + Na]⁺; HRESIMS m/z 509.2874 (calcd for C₂₉H₄₂O₆Na 509.2879).

Camphoratin D (7β,14α-dihydroxy-3,11-dioxo-4α-methylergosta-8,24(28)-dien-26-oic acid 4)

colorless powder (MeOH); mp 175–177 °C; [α]_D ²⁵ +34° (c 0.004 MeOH); UV (MeOH) λ_max (log ε) 246 (3.97) nm; IR (KBr) ν_max 3444, 2971, 2936, 2878, 1708, 1670, 1229, 1187, 1068, cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 509 [M + Na]⁺; HRESIMS m/z 509.2875 (calcd for C₂₉H₄₂O₆Na 509.2879).

Table 3.

¹H NMR Data for Compounds 5–10 (in CDCl₃)

Position	5a	6a	7b	8a	9a	10 a
1	1.40 m	1.25 m	1.26 m	1.33 m	1.49m	1.33 m
	2.50 m	2.95 m	2.95 m	3.18 m	2.13 m	2.88 m
2	1.72 m	2.35 m	2.40 m	2.35 m	2.30 m	2.37 m
	1.94 m	2.49 m	2.49 m	2.51 m	2.42 td (14.4, 8.8)	2.50 m
3	3.79 br s
4	1.74 m	2.35 m	2.40 m	2.36 m	2.24 m	2.38 m
5	2.12 m	1.39 m	1.46 m	1.39 m	1.83 m	1.41 m
6	2.25 t (15.1)c	1.56 m	1.57 m	1.43 m	1.27 m	1.42 m
	2.41 dd (15.1, 3.0)	2.49 m	1.89 m	1.78 m	1.83 m	1.78 m
7		4.39 t (8.0)	4.26 d (2.0)	2.18 m	5.18 br s	2.11 m
				2.37 m		2.30 m
9					1.75 m
11					1.54 m
					1.64 m
12	2.40 d (13.6)	2.32 d (14.0)	2.38 d (14.5)	2.33 d (14.4)	1.27 m	2.18 d (13.9)
	2.89 d (13.6)	2.83 d (14.0)	2.84 d (14.5)	2.80 d (14.4)	2.04 m	2.47 d (13.9)
14	2.62 dd (12.4, 7.0)	2.71 m	2.78 m	2.64 dd (12.0, 7.6) 1.81 m		2.09 m
15	1.47 m	1.90 m	1.90 m	1.52 m	1.41 m	1.37 m
	2.55 m	2.09 m	2.07 m	1.81 m	1.52 m	1.93 m
16	1.25 m	1.44 m	1.40 m	1.42 m	1.29 m	1.47 m
	1.98 m	1.96 m	1.90 m	1.81 m	1.73 m	2.08 m
17	1.42 m	1.38 m	1.46 m	1.48 m	1.28 m	1.39 m
18	0.67 s	0.77 s	0.72 s	0.74 s	0.57 s	1.02 s
19	1.31 s	1.44 s	1.26 s	1.34 s	1.01 s	1.37 s
20	1.42 m	1.41 m	1.43 m	1.47 m	2.05 m	1.46 m
21	0.93 d (5.6)	0.92 d (5.5)	0.93 d (6.0)	0.95 d (5.6)	1.02 d (7.2)	0.90 d (6.4)
22	1.18 m	1.25 m	1.32 m	1.22 m	5.25 m	1.17 m
	1.57 m	1.58 m	1.59 m	1.53 m		1.47 m
23	1.95 m	1.98 m	2.00 m	1.89 m	5.25 m	1.95 m
	2.16 m	2.15 m	2.17 m	2.10 m		2.14 m
24					2.23 m
25	3.13 q (7.0)	3.12 q (6.8)	3.16 q (7.0)	2.24 m	1.58 m	3.13 q (7.0)
26				1.06 d (6.8)	3.45 dd (10.4, 6.4)
					3.56 dd (10.4, 6.4)
27	1.28 d (7.0)	1.27 d (6.8)	1.30 d (7.5)	1.03 d (6.8)	0.86 d (6.8)	1.28 d (7.0)
28	4.92 s, 4.88 s	4.91 s, 4.89 s	4.94 s	4.67 s	1.00 d (6.8)	4.92 s, 4.88 s
	4.90 s, 4.86 sd	4.87 s, 4.85 sd	4.99 s	4.74 s		4.90 s, 4.87 sd
29	0.96 d (6.4)		1.03 d (7.0)	1.29 d (6.8)		1.04 d (6.6)
OMe	3.66 s	3.66 s				3.66 s

^aRecorded at 400 MHz at 25 °C.

^bRecorded at 500 MHz at 25 °C.

^cJ values (in Hz) in parentheses.

^dChemical shifts for 25-epimer.

Camphoratin E (methyl 3α-hydroxy-7,11-dioxo-4α-methylergosta-8,24(28)-dien-26-oate, 5)

colorless syrup; [α]_D ²⁵ +166 (c 0.007, MeOH); UV (MeOH) λ_max (logε) 260 (3.68) nm; IR (KBr) ν_max 3491, 2959, 2936, 2877, 1730, 1678, 1235, 1202, 1169 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 507 [M + Na]⁺; HRESIMS m/z 507.3088 (calcd for C₃₀H₄₄O₅Na 507.3086).

Camphoratin F (methyl 7β-hydroxy-3,11-dioxo-4α-methylergosta-8,24(28)-dien-26-oate, 6)

colorless powder (MeOH); mp 100–101 °C; [α]_D ²⁵ +174 (c 0.008, MeOH); UV(MeOH) λ_max (log ε) 251 (4.05) nm; IR (KBr) ν_max 3386, 2967, 2877, 1732, 1711, 1669, 1235, 1197, 1167, 1083 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 507 [M + Na]⁺; HRESIMS m/z 507.3083 (calcd for C₃₀H₄₄O₅Na 507.3086)

Camphoratin G (7α-hydroxy-3,11-dioxo-4α-methylergosta-8,24(28)-dien-26-oic acid, 7)

colorless powder (MeOH); mp 196–198 °C; [α]_D ²⁵ +139 (c 0.007, MeOH); UV (MeOH) λ_max (log ε) 247 (4.33) nm; IR (KBr) ν_max 3420, 2964, 2930, 2875, 1707, 1659, 1171 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 493 [M + Na]⁺; HRESIMS m/z 493.2929 (calcd for C₂₉H₄₂O₅Na 493.2930).

Camphoratin H (4α-methylergosta-8,24(28)-dien-3,11-dione, 8)

colorless syrup; [α]_D²⁵ +41 (c 0.008, MeOH); UV (MeOH) λ_max (log ε) 248 (3.94) nm; IR(KBr) ν_max 2965, 2940, 2877, 1711, 1678 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 447 [M + Na]⁺; HRESIMS m/z 447.3237 (calcd for C₂₉H₄₄O₂Na 447.3239).

Camphoratin I [(25S)-26-hydroxy-ergosta-7,22-dien-3-one, 9]

colorless powder (MeOH); mp 192–193 °C; [α]_D ²⁵ +128 (c 0.003, MeOH); IR (KBr) ν_max 3336, 2956, 2873, 1716, 1024 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 435 [M + Na]⁺; HRESIMS m/z 435.3242 (calcd for C₂₈H₄₄O₂Na 435.3239).

Camphoratin J (methyl 3,11-dioxo-4α-methyl-14β-ergosta-8,24(28)-dien-26-oate, 10)

colorless needles (MeOH); mp 100–102 °C; [α]_D ²⁵ +164 (c 0.005, MeOH); UV (MeOH) λ_max (logε) 250 (4.35) nm; IR (KBr) ν_max 2953, 2873, 2856, 1738, 1709, 1669,1460, 1453, 1375, 1077 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1 and 3; ESIMS m/z 491 [M+Na]⁺; HRESIMS m/z 491.3135 (calcd for C₃₀H₄₄O₄Na 491.3137).

Preparation of (R)-MTPA Ester of 9

To a solution of 9 (0.5 mg) in pyridine (0.4 mL) was added (S)-MTPA chloride (25 μL), and the mixture was allowed to stand for 3 h at room temperature. The reaction was quenched by the addition of 1.0 mL of H₂O, and the mixture was subsequently extracted with EtOAc (3 × 1.0 mL). The EtOAc-soluble layers were combined, dried over anhydrous MgSO₄, and evaporated. The residue was subjected to silica gel column chromatography using n-hexane–EtOAc (4:1) as the eluent to yield the (R)-MTPA ester, 9a (0.7 mg). Selective ¹H NMR (500 MHz, CDCl₃) of 9a: δ 7.38–7.53 (5H, m, Ph), 5.21 (1H, dd, J = 15.0, 7.5 Hz, H-23), 5.16 (1H, dd, J = 15.0, 7.5 Hz, H-22), 4.17 (2H, br d, J = 6.4 Hz, H₂-26), 3.54 (3H, s, OMe), 1.02 (3H, d, J = 6.5Hz, H₃-21), 1.01(3H, s, H₃-19), 0.95 (3H, d, J = 7.0 Hz, H₃-28), 0.85 (3H, d, J =7.0 Hz, H₃-27), 0.57 (3H, s, H₃-18).

In vitro Cytotoxicity Assay

The KB-MDR cell line system was a generous gift from Professor Y.-C. Cheng, Yale University and was developed using step-wise vincristine selection as reported.²³ The MDR cell-line KB-VIN and the parent KB nasopharyngeal were propagated in RPMI-1640 medium supplemented with 25mM HEPES, 10% fetal bovine serum, and 100 U/mL penicillin G, 100 μg/mL streptomycin, 250 ng/mL amphotericin B and cultured at 37 °C in a humidified atmosphere of 95% air/5% CO_2. KB-VIN cells were challenged with 0.5 μg/mL vincristine every two weeks and drug was removed at least one day prior to using the cells. For the anti-proliferation assay, trypsinized cells were seeded in 96-well microplates at densities of 5000 cells per well with compounds added from DMSO-diluted stock to give a dose-range of 20 down to 0.16 μM. After 3 days in culture, attached cells were fixed in situ with 10% trichloroacetic acid and then stained with 0.4% sulforhodamine B (SRB) in 1% HOAc.²⁴ The absorbance at 562 nm was measured using a microplate reader after solubilizing the bound dye. Results were plotted using Prizm software (GraphPad, San Diego CA). The mean EC₅₀ is the concentration of agent interpolated from graphical results that caused a 50% reduction in the cell number of treated versus untreated cells and is an average from at least two independent determinations; variation was less than 5% of the mean.

Microglial Cell Culture and Measurements of NO²⁴

The murine microglial cell line (BV2) was cultured in Dulbecco’s modified Eagle medium (Gibco, USA) supplemented with 5% fetal bovine serum (Hyclone). Production of NO was measured by the accumulation of nitrite in the culture medium 24 h after stimulation with LPS (0.5 μg/mL) by the Griess reagent. NO production was measured in the presence of 1–50 μM of test drugs. L-NAME (a non-selective NOS inhibitor) was included as a positive control. Data were calculated as 50% inhibitory concentration (IC₅₀) and expressed as the mean ± S.E.M. from 3–6 experiments performed on different days.

Measurement of NOX Activity

Activity was measured as ROS production triggered with NADPH (200 μM) or fMLP (2 μM) in the presence of 1–50 μM of test drugs in BV2 cell lysate or PMN. DPI, a NOX inhibitor, was included as a positive control for NOX inhibition. Methodology was been previously described.²⁵

Measurement of DPPH Radical-Scavenging Capacity

DPPH radical-scavenging capacity assay was performed as in our previous report.¹⁹ Briefly, drugs were diluted in MeOH into a range of concentrations (10–50 μM). DPPH solution (200 μL; 200 μM in MeOH) was added to 10 μL of each diluted sample in a 96-well microplate, and the resulting solution was allowed to react for 30 min in the dark at ambient temperature. The absorbance caused by the DPPH·radical at 517 nm was determined by a microplate-spectrophotometer. The radical scavenging capacity was expressed as delta OD₅₁₇ (ΔOD₅₁₇), and values were the means of three replicates. Trolox, an antioxidant, was included as a reference.