2. Results and Discussion
Compound
1 was isolated as oil. The HR-EI-MS spectrum gave a molecule ion [M]
+ at
m/z 215.0948, consistent with a molecular formula of C
13H
13NO
2. UV spectrum showed maximum absorption at 241 (4.20), 264 (4.09) and 310 (3.98) nm, indicating the presence of an indole skeleton [
7]. Its IR spectrum revealed NH absorption at 3320 cm
−1. Analysis of the
1H NMR spectrum of
1 revealed four typical mutually coupling aromatic protons of indole alkaloid at δ
H 7.08 (1H, td,
J = 8.0, 1.2 Hz, H-7), 7.11 (1H, td,
J = 8.0, 1.2 Hz, H-6), 7.26 (1H, dd,
J = 8.0, 1.2 Hz, H-8), 7.84 (1H, dd,
J = 8.0, 1.2 Hz, H-5) and one NH group at δ
H 7.66 (1H, br s, exchangeable with D
2O). According to the molecular formula, the degree of unsaturation can be calculated to be 8. After deducting the two rings and four double bonds of indole, there are also 2 remains. In addition to the carbon spectrum, there is a conjugated carbonyl signal at δ
C 183.99. It is speculated that the branched chain forms a ring (C ring). It can be confirmed from the fact that the methylene signal on the carbonyl α carbon is δ
H 2.63 (2H, s) in a lower magnetic field. In addition, the two carbon absorption signals on the pyrrole ring of indole, a particularly low magnetic field and a high magnetic field, are presumably affected by the electronic resonance of the carbonyl group. Therefore, one end of the branch is connected to a carbon with a higher magnetic field (δ
C 95.15) through a carbonyl carbon. The carbon with a lower magnetic field (δ
C 160.87) should also be connected to an oxygen and be affected by the carbonyl group to achieve such a low magnetic field. Observing the carbon spectrum, there is also a quaternary carbon connected to oxygen. It is obvious that the other end of the branch chain is connected to indole with this oxygen. There is also a single peak integrated into 6H in the
1H NMR, δ
H 1.57 (6H, s, CH
3-5′, 6′), which is presumed to be two methyl groups with the same signal, and the position is on the quaternary carbon (δ
C 86.2) connected to oxygen. The HMBC
3J-correlations (
Figure 2) from δ
H 2.63 (CH
2-3′) to δ
C 95.2 (C-3) and one weak
4J-correlations δ
H 1.57 (6H, s, CH
3-5′, 6′) to δ
C 160.9 (C-2), verify the junction of the 2,2-dimethyldihydropyrano ring to the indole moiety at C-2, and 3. The other key correlations of HMBC are illustrated in
Figure 2. Based on the above data, the structure of
1, named amycolataiwanensin A, was elucidated as 2,2-dimethyl-2,3-dihydropyrano[2,3-
b]indol-4(9
H)-one, which was further confirmed by
13C NMR, COSY, NOESY (
Figure 3), HSQC and HMBC (
Figure 2) experiments.
Compound
2, isolated as gum, showed a dehydrated molecular ion [M-H
2O]
+ peak at
m/z 262.1933 for C
17H
26O
2, corresponding to four indexes of hydrogen deficiency (IHD). The IR spectrum of
2 displayed an absorption for an OH group (3500 cm
−1) and a C = O group (1740 cm
−1). The
1H- and
13C-NMR, COSY, HMBC, and NOESY data (
Figure 3) established the structure of
2 as 12-acetoxy-11-hydroxyacora-3-ene.
The
1H-NMR spectrum exhibited signals for a trisubstituted olefinic proton [δ
H 5.30 (br s, H-3)], an oxymethylene at δ
H 3.93/4.05 (each d,
J = 11.0 Hz, CH
2-12), and the four methyl groups include three singlet methyl groups δ
H 1.24 (s), 1.58 (s), 2.08 (s) and a doublet methyl group at δ
H 0.82 (d, CH
3-14). From the carbon spectrum (
13C-NMR) and DEPT, because of the appearance of one quaternary carbon at δ
C 171.2 (ester) and one primary carbon at δ
C 20.9 (CH
3), it can be seen that there is an acetoxy group. In addition, there are two quaternary carbons δ
C 44.70 and 74.85 (of which δ
C 74.35 is the oxygen-containing quaternary carbon). Calculating the degree of unsaturation, subtracting a double bond and a carbonyl group, there is 2 left, so it can be determined that there are two more rings in this structure. According to the above characteristic spectrum information and reference data, [
8] it can be known that the NMR information of this structure are very similar to the spectrum of a known compound 12-acetoxy-11-hydroxyacora-4-ene, so it is inferred that this compound It is also belonged to acorane backbone compound. According to the signal of HMBC, the structure is similar to the known compound 12-acetoxy-11-hydroxyacora-4-ene, [
8] while the signal of NOESY explains the difference in stereo orientation: (1) The H-2 has NOE correlation signals with H-3 and H-14 in compound
2. (2) In the similar compound 12-acetoxy-11-hydroxyacora-4-ene, its H-6 has NOE correlation signal with H-13 and H-5. Therefore, the structure and steric orientation of compound
2 were proved. The above spectroscopic data proves that the structure is correct, and this new compound is named amycolataiwanensin B.
Compound
3 had the dehydrated molecular ion peak [M-H
2O]
+ at
m/z 330.1831 (HR-EI-MS), as analyzed for C
20H
28O
5. The IR spectrum of
3 exhibited the presence of an OH group at 3400 cm
−1 and a CO moiety at 1695 cm
−1. The UV absorptions (λ
max 229.0 and 256.0 nm) confirmed an aromatic system. Seven IHD were determined from the molecular formula,
13C-NMR spectrum, and DEPT. Further spectral data (
Table 1 and
Table 2) and comparison with reference compounds [
9] established the structure of
3 as 6α,7α,11-trihydroxy-3-oxoferrugiol. The
1H-NMR spectrum of
3 indicated the presence of an iPr group (δ
H 1.27 (d) and 1.28 (d), and 2.99 (sep)) attached to the benzene ring, two phenol groups (δ 4.85 (s), 5.81 (s)), another three Me groups (δ
H 1.21 (s), 1.33 (s), and 1.45 (s)) attached to a quaternary carbon, two OCH groups (δ
H 4.96 (d, 5.0) and 4.44 (dd, 11.5, 5.0)), the signal of five substituted benzene ring at 6.81 (s, H-14). According to the
13C-NMR and DEPT, the benzene ring δ
C 125.33, 130.44, 139.96, 142.42, 132.91 and 119.70 composed of 6 olefinic carbons; there is a carbonyl group at δ
C 219.66, and two oxygen-containing tertiary carbons at δ
C 68.3 (C-7), and 74.3 (C-6). Based on the above information and combining the above characteristic spectra data, the compound
3 with the abietane skeleton can be identified. The H-1β at δ
H 3.03 (m) is the result of the displacement of the low magnetic field due to the influence of the hydroxyl group on C-11. In order to determine the structure and the position of each functional group, continue with two-dimensional nuclear magnetic resonance spectroscopy (HSQC, HMBC) and NOESY experiments. According to the key information of HMBC: (1) H-18 and H-19 are correlated to δ
C 219.66, so it is determined that the carbonyl group is located at the position of C-3; (2) H-14 is only related to δ
C 68.26, so the two tertiary hydroxyl carbons can be distinguished. (3) Because δ
H 5.81 (s, OH-12) is correlated to C-12, the two phenols can be distinguished and make sure that δ
H 5.81 (s) is connected to C-12. According to the signal from NOESY: (1) H-5 is connected with H-1α and H-18 respectively; (2) H-6 is connected with H-19 and H-20, so it is determined to be in the axial position; (3) H-7 is related to H-6β and H-14, and by its coupling constant (
J = 5.0 Hz), it can also be determined that it is in the equatorial position. Compound
3 is a previously undescribed diterpene and was named amycolataiwanensin C.
The
1H-NMR signals of
4 at δ
H 0.84 (s), 1.02 (s) and 1.22 (s); 1.19 and 1.18 (each 3H, d,
J = 7.0 Hz, CH
3-16, 17), and 3.12 (H-15, COSY cross-peaks with δ
H 1.19 and 1.18) suggested that
5 has an iPr group and three Me groups attached to a quatenary C-atom. According to
13C-NMR and DEPT, in addition to isopropyl and three singlet methyl groups, there is a carbon at δ
C 78.24, which is a tertiary carbon connected to oxygen, and δ
C 183.25 and 187.31 show quinone group signals. In addition, there are four olefinic carbons, δ
C 123.85, 150.56, 145.74 and 145.97. Since the compound is yellow and the UV absorption spectrum shows, coupled with the above-mentioned spectral data (
1H-NMR and
13C-NMR data of known compounds in Reference [
10], it can be inferred that compound
4 is a derivative of hydroxybenzoquinone in the abietane skeleton. The C ring is a quinone ring, and H-1β (δ 2.79) is affected by the quinone group of C-11, so the magnetic field is relatively low. From the signal of δ
H 3.24 (dd, J = 10.6, 5.7 Hz), it can be seen that this H is in the axial position, and -OH is in the equatorial position. By heteronuclear correlation spectroscopy (HSQC, HMBC) and NOESY to analyze its structural correlation and stereo orientation. According to HMBC’s information as following: (1) δ 3.24 is related to C-18 and C-19 respectively, so it is determined that the hydroxyl group is connected to the position of C-3; (2) H-5 is connected to C-3, C-7, and C-3, respectively. C-10, C-18, C-19, C-20 are connected; (3) H-15 is connected with C-12, C-13, C-14 respectively; (4) H-20 is connected with C-1. C-5, C-9, C-10 are related. According to the signal from NOESY: (1) H-5 is related to H-1α, H-3, H-6α, H-7α, and H-18 respectively; (2) H-3 is related to H-5 and H-18 Therefore, it is determined that H-3 is in the axial direction; (3) H-20 is related to H-2β and H-6β; (4) H-2β is related to H-19 and H-20. The structure was proved to be correct, and the new compound was named amycolataiwanensin D.
Compound
5 was obtained as a colorless gum and had a molecular formula of C
19H
26O
3 by the HR-EI-MS (
m/z 302.1885 [M+H]
+, calcd for C
20H
30O
3 302.1882), requiring six degrees of unsaturation. The IR spectrum of
5 displayed absorption characteristic of a hydroxy (3396 cm
−1), carboxylic acid group (2700~3400 (OH) & 1697 (C = O) cm
−1), and benzene ring (1570 and 1498 cm
−1). The
13C NMR spectrum revealed the presence of 20 carbon signals, which were assigned with the assistance of DEPT spectrum as one sp
2 quaternary carbonyl carbon [δ
C 184.5 (C-18)], three sp
3 methyls [δ
C 24.8, 16.2, and 25.0 (C-17, 19 & 20)], five sp
3 methylenes [δ
C 37.8, 18.5, 36.6, 21.6, and 29.9 (C-1, 2, 3, 6 & 7)], two sp
3 methines [δ
C 44.5, and 70.1 (C-5 and 15)], three sp
2 methines [δ
C 124.4, 122.8, and 126.0 (C-11, 12, and 14)], two sp
3 quaternary carbons [δ
C 47.3 and 37.0 (C-4 and 10)], and five sp
2 quaternary carbons [δ
C 135.1, 148.6, and 142.6 (C-8, 9, and 13)]. The
1H-NMR and
13C-NMR spectra (
Table 1 and
Table 2) of
5 were similar to those of 12-methoxy-13-(1-hydroxy ethyl)podocarpa-8,11,13-trien-19-oic acid [
11,
12], except that an aromatic proton [δ
H 7.11 (d,
J = 8.2 Hz, H-12)] of
5 replaced a methoxyl group at C-12 [δ
H 3.65 (3 H, s, OCH
3)] of
5a. According to the DEPT-NMR spectrogram, there are three primary carbons, five secondary carbons, five tertiary carbons, and six tertiary carbons. The degree of unsaturation is estimated to be 7, which is consistent with the predicted structure. Viewed from the HMBC spectrum, δ
H 1.48 (H-16) is correlated to C-15 and C-13, and δ
H 4.80 (H-15) is correlated to C-16, C-14, C-13, and C-12, could be explained the position of the doublet methyl group (Me-15) and the hydroxyl group. The stereochemistry is determined from the NOESY spectrum. H-20 and H-19 have NOESY correlation, which is sufficient to show that -COOH is located in the equatorial. H-1β has NOESY association with H-11, and H-16 has NOESY association with H-12 and H-14, which are consistent with the speculated structure. Based on the above data, it can be determined that compound
5 is (1
R*,4a
S*)-7-(1-hydroxyethyl)-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid (15-hydroxyabieta-8,11,13-trien-18-oic acid) and named as amycolataiwanensin E.
Compound 6 was isolated as oil. Its molecular formula, C20H30O3, was determined on the basis of the positive HR-EI-MS at m/z 318.2197 [M]+ (calcd 318.2195) and was supported by the 1H, 13C, and DEPT data. The IR absorption bands of 6 revealed the presence of the COOH (3400 cm−1 for OH; 1699 cm−1 for CO) and a conjugated carbonyl (1670 cm−1) functions. According to DEPT plots, there are four primary carbons, seven secondary carbons, three tertiary carbons, and six quaternary carbons. The degree of unsaturation is estimated to be 6. In 13C-NMR spectrum, four signals at δ 29.1, 23.8, 16.5, and 14.6, and 1H-NMR at δ 0.67, 1.01, 1.13, & 1.14 (each 3H, s), showing the presence of four Me groups. The 13C-NMR spectrum at δ 183.8 (s) and the infrared absorption spectrum at 2700–3400, 1699 cm−1, it shows that this compound has a carboxylic acid group. In addition, δ 154.2 (s), 125.1 (d) and 1H-NMR spectrum δ 5.82 (1H, br s) show that there is a group of triple-substituted double bonds. The remaining 13C-NMR spectrum at δ 211.2 (s) shows the existence of carbonyl group (C = O). It can be inferred that 6 contains a carboxylic acid, a set of double bonds, and a carbonyl group. The remaining unsaturation is 3, which is inferred to be a tricyclic structure. HMBC correlations of δH 1.01 (H-17)/δC 49.5 (C-15), 211.2 (C-13); δ 1.13 (H-16) δ 49.5 (C-15), 211.2 (C-13); δ 5.82 (H-14)/δ 49.5 (C-15). From the HMBC spectrum, δ 1.01 (H-17) is correlated to δ 49.5 (C-15), 211.2 (C-13); δ 1.13 (H-16) is correlated to δ 49.5 (C-15), 211.2 (C-13), and the correlation between δ 5.82 (H-14) and δ 49.5 (C-15), and the double bond signal will shift to a low magnetic field. It is speculated that the double bond is conjugated to the carbonyl group, but δ 211.2 (C-13)) Unlike a conjugated carbonyl group, there may be one α carbon and three β carbons, which makes the carbonyl shift to a lower magnetic field. From the HMBC correlations between δ2.07 (H-9) and δ 39.7 (C-10), 14.6 (C-20); δ 1.98 (H-5) and δ 47.1 (C-4), 39.7 (C-10), 26.1 (C-6), 16.5 (C-19), 14.6 (C-20); δ 1.14 (H-19) and δ 183.8 (C-18), 47.1 (C-4), 36.9 (C -3); δ 5.82 (H-14) and δ 154.2 (C-8), 49.5 (C-15), 38.6 (C-7); δ 1.58 (H-12) and δ 211.2 (C -13), 59.7 (C-9), 23.8 (C-16); δ 1.13 (H-16), 1.01 (H-17) are related to δ 211.2 (C-13), 49.5 (C-15), 35.4 (C-12) respectively. From the above analysis of 1D and 2D spectra, compound 6 is 12(13→15)abeoabietane diterpenes, C-12 is not connected to C-13, and C-15 is reversed to cause the six-ring to seven-ring, geminal dimethyl group is substituted for isopropy. As for its proposal biosynthesis and the stereochemistry, it can be explained by the NOESY spectrum. H-20 and H-19 have a NOESY correlation, which is sufficient to show that -COOH is located in the equatorial direction. The spectra of HMQC and COSY confirm that the compound 6 is 12(13→15)abeo-13-oxo-8(14)-abietene-18-oic acid and designated as amycolataiwanensin F.
Compound
7 was obtained as oil. Its molecular formula C
13H
18O
5 was deduced from molecular ion at
m/z 254.1151 [M]
+ (calcd 254.1154) in the HR-EI mass spectrum. The presence of hydroxyl (3440 cm
−1), acetoxyl (1730 cm
−1), and benzene (1615 and 1518 cm
−1) groups were evident from the IR spectrum. The
1H (
Table 1) and
13C NMR (
Table 2) data of
7 were very similar to those of dihydrosyringenin [
13], except that an acetoxy group [δ
H 2.02 (3H, s); δ
C 20.9, 171.1 (OCOCH
3)] at C-9 in
7 replaced the hydroxy group of dihydrosyringenin. This was supported by HMBC correlation between OCH
2-9 (δ
H 3.76) and C-11 (δ
C 171.1) (
Figure 2). The full assignment of
1H and
13C NMR resonances was supported by
1H-
1H COSY, DEPT, HSQC, NOESY, and HMBC (
Figure 2) spectral analyses. Thus, the structure of
7 was established as shown in
Figure 1, and named amycolataiwanensin G.
Compound
8 was isolated as oil. Its molecular formula, C
17H
18O
3, was determined on the basis of the HR-EI-MS at
m/z 270.1252 [M]
+ (calcd 270.1256) and was supported by the
1H,
13C, and DEPT. The IR absorption bands of
8 revealed the presence of hydroxyl (3400 cm
−1) and benzene (1614 and 1520 cm
−1) functions. The
1H (
Table 1) and
13C NMR (
Table 2) data of
8 were similar to those of oblongifoliagarcinine A, [
14] except that a 4-methylpent-3-en-1-yl group [δ
H 5.08 (1H, t,
J = 7.1 Hz, H-3″), 1.59 (3H, s, CH
3-6″) 1.64 (3H, s, CH
3-5″), 2.09 (2H, m, H-2″), 1.75 (2H, m, H-1″); δ
C 22.8 (C-2″), 123.8 (C-3″), 131.9 (C-4″), 25.6 (C-6″), 17.6 (C-5″), 41.7 (C-1″)] at C-2 in
8 replaced the Me group at C-2 of oblongifoliagarcinines A [
14]. This was supported by NOESY correlations between Me-2 (δ
H 1.42) and H-2′′ (δ
H 2.09), and between H-3″ (δ
H 5.08) and both of CH
3-6″ (δ
H 1.64) and H-1′’ (δ
H 1.75) and by HMBC correlation between H-1′’ (δ
H 1.75) and C-3 & 9 (δ
C 129.8 & 138.5) (
Figure 2). The full assignment of
1H and
13C NMR resonances was further confirmed by DEPT,
1H-
1H COSY, NOESY, HSQC, and HMBC data (
Figure 2). Consequently, the structure of compound
8 was established as amycolataiwanensin H.
Compound
9 was obtained as oil and had the molecular formula C
22H
24O
3, as inferred from the HR-EI-MS showing the molecular-ion peak at
m/z 336.1728 [M]
+, indicating nine degrees of unsaturation. The IR spectrum of
9 showed absorption bands at 3395 cm
−1 for free OH groups, 1613, 1596, 1520, 1486 cm
−1 for aromatic moieties. The
1H-NMR and
13C-NMR spectra (
Table 1 and
Table 2) of
9 were similar to those of oblongifoliagarcinines A [
14], except that the single bond at C3–C4 [δ
H 2.78 (2H, t,
J = 6.7 Hz, H-4), 1.83 (2H, t,
J = 6.7 Hz, H-3)] of
9 replaced a pair of double bond [δ
H 5.65 (2H, d,
J = 9.8 Hz, H-4), 6.37 (2H, d,
J = 9.8 Hz, H-3)] of oblongifoliagarcinines A [
14]. The
1H-and
13C-NMR (
Table 1 and
Table 2) and HMBC data (
Figure 2) established the structure of amycolataiwanensin I (
9) as 6-(4-hydroxyphenyl)-2,2-dimethylchroman-8-ol.
Fifteen
13C-NMR signals and the HR-EI-MS confirmed the molecular formula C
15H
26O
2 of
10. Analysis of its IR spectrum suggested that
10 contained OH (3299 cm
−1) moiety. The three IHD (from the DEPT experiment), the
13C-NMR data, and the molecular formula indicated that
10 is a sesquiterpene. Further spectral data established the structure of
10 as (2
S*,3
S*,6
R*)-3,6,8,8-tetramethyloctahydro-1
H-3a,7-methanoazulene-2,6-diol (3α-hydroxycedrol). The
1H-NMR shows that δ
H 0.94 (d) is a doublet methyl group attached to a tertiary carbon. At δ
H 1.00 (s), 1.23 (s), and 1.32 (s), there are three singlet methyl groups on the quaternary carbon. Among them, δ
H 1.23 (s) and 1.32 (s) are located in the lower magnetic field because they are connected to the hydroxyl group. From
13C-NMR, DEPT, and HSQC plots, there are two carbons attached to oxygen at δ
C 72.84 and 81.43, 72.84 belongs to the quaternary carbon, and δ
C 81.43 belongs to the tertiary carbon. Calculating the degree of unsaturation, because there is no double bond or carbonyl carbon, it can be proposed this compound is a tricyclic ring. Based on the above spectral data, it is speculated that the compound should be a cedrane skeleton. After comparing with the reference data, and comparing with the
1H-NMR and
13C-NMR of the known compound cedrol [
15], there is only one more oxygen-containing tertiary carbon. It can be confirmed by the signal (δ
H 3.58 (1H, ddd,
J = 15.5, 10.5, 5.5 Hz, H-3); δ
C 81.4) appeared on the
1H-NMR &
13C-NMR. Continue to perform two-dimensional heteronuclear correlation spectroscopy (HSQC, HMBC) and NOESY to further determine the structural relevance and stereo orientation of
10. The
1H signal at δ
H 1.45 (H-2)/δ
H 1.36 (CH
2-4) and δ
H 0.94 (CH
3-12) showed a two and three-bond connectivities with C-3 (δ
C 81.4) in the HMBC plot (
Figure 2), which suggested that the second OH group at C-3. According to the signals from NOESY, determine the relative configuration of the C-3 and C-8 hydroxyl groups: (1) Compound
3 exhibited the HMBC correlation: H-3/12-Me, and judging from the split pattern (ddd), H-3 is located in the β-axial position; (2) 15-CH
3 is correlated to H-9α and H-9β, so it is judged that 15-CH
3 is equatorial. The structure was proved to be correct, and compared with the literature, it was confirmed that this was a compound discovered for the first time in nature, named amycolataiwanensin J.
NO is a mediator in the inflammatory response involved in host defense. In the course of our search for potential diverse secondary metabolites from natural fungal sources, and to further understanding of the bioactive metabolites of the genus
Amycolatopsis, we examined the EtOAc extract of
A.
taiwanensis, which showed inhibitory activity on LPS-induced NO release production in RAW 264.7 murine macrophages, as determined by our primary screening (approximately 95% inhibition at a concentration of 10 μg/mL). Investigation of the bioactive metabolites of the active EtOAc extract from the titled material
A.
taiwanensis, led to the isolation of ten new compounds. Due to the small quantity of isolated compound (
1), we evaluated the inhibitory effects of amycolataiwanensins B–J (
2–
10, resp.) on the production of NO induced by LPS. The inhibitory activity data of the 10 isolated compounds on NO generation by macrophages are shown in
Table 3. From the results of our anti-inflammatory tests, the following conclusions can be drawn: (
a) They showed potent inhibition with IC
50 values between 12.8 to 34.2 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. (
b)The high cell viability (>80%) indicated that the inhibitory activity of LPS-induced nitrite production by compounds
3,
5,
8, and
9 (IC
50 value: 17.52, 12.31, 17.81 and 13.32
μM) did not result from its cytotoxicity. (
c) Compounds
6 &
7 (IC
50 value: 24.83 and 12.78
μM) also showed inhibition of NO production of macrophages, but the low cell viability (<80%) suggested the possibility of cytotoxicity. (
d) The sesquiterpene derivative, compound
2 exhibited less effective NO inhibition. (
e) Among the abietane diterpene analogues, compound
3 (with (abietane with 3-isopropylbenzene-1,2-diol unit in C ring) exhibited more effective inhibition than its analogue, compound
4 (abietane with hydroxybenzoquinone unit in C ring), compound
5 (with 1-phenylethan-1-ol moiety in C ring) and compound
6 (with 7,7-dimethylcyclohept-2-en-1-one moiety in C ring. (
f) Among the aromatics analogues, compound 7 (simple aromatic with 9-acetoxydihydrosyringenin) displayed better inhibition than its analogue, compound
8 (abietane with hydroxybenzoquinone unit in C ring), compound
5 (with 1-phenylethan-1-ol moiety in C ring) and compound
9 (with 7,7-dimethylcyclohept-2-en-1-one moiety in C ring. (
g) Furthermore, the RT-PCR analysis in the present study indicated that LPS treatment increased the level of iNOS mRNA expression, and that compounds
3,
5,
8 and
9 inhibited this increase in a concentration-dependent manner. At the highest concentration, none of the compounds tested showed any obvious cytotoxicity toward RAW 264.7 cells. (
h) Cytotoxic effects were measured using MTT assay. The high cell viability (>95%) indicated that the inhibitory activities of LPS-induced NO production by active compounds
3,
5,
8, and
9 were not resulted from its cytotoxicity.