Acyclic Triterpenoids from Alpinia katsumadai Inhibit IL-6-Induced STAT3 Activation

Abstract

The seeds of Alpinia katsumadai yielded two new acyclic triterpenoids, 2,3,6,22,23-pentahydroxy-2,6,11,15,19,23-hexamethyl-tetracosa-7,10,14,18-tetraene (3) and 2,3,6,22,23-pentahydroxy-2,10,15,19,23-hexamethyl-7-methylenetetracosa-10,14,18-triene (4), as well as two known compounds, 2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (1) and 2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (2). The absolute configurations of 2 and 3, which were determined by means of a modified Mosher’s method, are suggested as (3R; 5S; 22R) and (3R; 22R), respectively. Compounds 1–4 inhibited IL-6-induced JAK2/STAT3 activity in a dose-dependent fashion, with IC₅₀ values of 0.67, 0.71, 2.18, and 2.99 μM. Moreover, IL-6-stimulated phosphorylation of STAT3 was significantly suppressed in U266 cells by the administration of A. katsumadai EtOH extract and Compounds 1 and 2. These results suggest that major phytochemicals, Compounds 1 and 2, obtained from A. katsumadai may be useful candidates for designing new IL-6 inhibitors as anti-inflammatory agents.

1. Introduction

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that is secreted by immune and inflammatory-related cells (T cells and macrophages) to stimulate responses to viral infection, trauma, and other tissue damage [1,2]. Several studies have demonstrated that overproduction of IL-6 is relevant to many human diseases, such as cancer cachexia [3], rheumatoid arthritis [4], atherosclerosis [5], diabetes [6], and multiple myeloma [7]. IL-6 also impairs the endothelium-dependent dilatation of human veins in vivo [8]. Therefore, IL-6 is implicated in the pathogenesis of immune and inflammatory diseases, and blocking IL-6 would be an effective treatment for many of these human diseases. The seeds of Alpinia katsumadai Hayata (Zingiberaceae) are widely utilized as a traditional Chinese herbal medicine to treat inflammatory and digestive diseases [9]. The seeds contain a variety of major constituents, including diarylheptanoids [10,11,12], monoterpenes [13,14], sesquiterpenoids [15], flavonoids [16], and chalcones [15]. Additionally, the extracts and compounds isolated from this plant exhibit various biological properties, including antiemetic [12], antiproliferative [17], antiviral [18,19], antiasthmatic [20], antiseptic [21], and cytoprotective effects [22]. We have searched for IL-6 inhibitors from natural sources, and the EtOH extract of the seeds of A. katsumadai (AKEE) exhibited potent inhibitory effects on IL-6-induced STAT3 (signal transducer and activator of transcription 3) activity (see Table S1 in Supplementary Materials). Herein, we report the isolation and structural elucidation of new acyclic triterpenoid derivatives (3 and 4), and describe the biological properties of Compounds 1–4.

2. Results and Discussion

2.1. Isolation of Compounds

The EtOH extract was suspended in H₂O and partitioned with CHCl₃. CHCl₃-soluble materials, including active substances, were fractionated via open-column chromatography on silica gel and ODS (octadecylsilanized silica gel, C₁₈) and subjected to semi-preparative HPLC to yield two known acyclic triterpenoids (1 and 2) and two new compounds (3 and 4).

2.2. Determination of the Acyclic Triterpenoids Structure

Compound 1 was obtained as a yellow oil with [α]${}_{\mathrm{D}}^{20}$ +18.1 (CHCl₃, c 1.0). It exhibited a sodium adduct ion peak at m/z 501 [M + Na]⁺ in the ESIMS and a molecular formula of C₃₀H₅₄O₄. The ¹³C-NMR spectrum of 1 contained 15 peaks, which was half the number predicted from the ESIMS spectral data. A symmetrical structure with 30 carbons was suggested. The ¹H-NMR spectrum of 1 showed signals due to 10 methylene groups (δ_H 1.40, 1.58, 1.99~2.08 and 2.23), four methyl groups (δ_H 1.59 and 1.61), and four olefinic groups (δ_H 5.13 and 5.18). Two methine groups (δ_H 3.36, dd, J = 10.5, 2.0 Hz) attached to hydroxy groups, and four terminal methyl groups (δ_H 1.15 and 1.19) were also observed. In addition, signals at δ_C 73.0 and 78.2, attributed to oxygenated carbon, indicated the presence of a hydroxy group in the ¹³C-NMR spectrum of 1. The connectivity of proton and carbon atoms was assigned based on ¹H, ¹³C and HMQC spectra. Therefore, the structure of 1 was identified as 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene by spectroscopic methods (¹H-, ¹³C-NMR and MS) and by comparing the data with previously reported values (Figure 1) [23]. The absolute configuration was identified as 3R, 22R by comparison of the optical rotation value and ¹H-NMR spectra in previous reports [24].

Compound 2 was isolated as a yellow oil with [α]${}_{\mathrm{D}}^{20}$ +4.8 (CHCl₃, c 1.0). It displayed a peak at m/z 493.3897 in the spectrum obtained by high-resolution electrospray ionization mass spectrometry (HRESIMS) corresponding to [M − H]⁻ (calcd. 493.3893), indicating a molecular formula of C₃₀H₅₄O₅. In the ¹H-NMR spectrum of 2 (

Table 1. ¹H- and ¹³C-NMR Spectroscopic data of Compounds 2–4.

Position	2 a		3 b		4 b
	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC
1	1.20, s	24.0	1.19, s	26.4	1.15, s	26.4
2	-	73.3	-	73.1	-	73.0
3	3.62, dd (8.4, 3.6)	78.5	3.35, dd (10.8, 1.8)	48.2	3.34, d (9.6)	78.1
4	1.63, m	36.1	2.03, m	22.9	1.40, 1.58, m	29.5
5	4.26, dd (7.6, 4.8)	78.6	1.55, m	42.4	2.01, m	24.3
6	-	134.9	-	73.0	4.09, m	75.1
7	5.43, t (6.4)	126.6	5.50, dd (15.6, 1.2)	136.7	-	151.3
8	2.13, m	26.6	5.58, dt (15.6, 6.0)	126.7	2.01, 2.19, m	31.0
9	2.05, m	39.4	2.74, t (6.6)	30.8	1.58, 1.65, m	35.3
10	-	135.1	5.14, td (7.2, 1.2)	122.2	-	135.9
11	5.14, m	124.7	-	135.0	5.22, t (6.6)	125.1
12	1.41, m	29.8	2.03, 2.10, m	39.5	1.58, m	29.6
13	1.59, m	39.8	1.40, 1.58, m	29.5	2.10, m	29.4
14	5.14, m	124.9	5.18, t (6.6)	124.9	5.14, q (6.6)	124.3
15	-	135.2	-	135.2	-	135.0
16	2.02, m	28.4	2.03, m	36.8	2.02, m	39.5
17	2.10, m	26.6	2.10, m	26.1	2.01, 2.10, m	26.4
18	5.19, t (6.4)	125.3	5.23, td (6.6, 1.2)	125.5	5.14, q (6.6)	124.9
19	-	137.3	-	135.9	-	135.1
20	2.23, m	37.0	2.02, 2.22, m	36.8	2.03, 2.21, m	36.7
21	2.09, m	26.2	1.58, m	29.6	1.40, 1.58, m	29.5
22	3.35, d (10.4)	78.9	3.35, dd (10.8, 1.8)	78.2	3.34, d (9.6)	78.1
23	-	72.8	-	73.1	-	73.0
24	1.19, s	23.5	1.15, s	23.4	1.19, s	23.4
25	1.17, s	26.4	1.15, s	23.3	1.19, s	23.3
26	1.63, s	11.9	1.26, s	28.1	4.87, 5.05, br s	109.9
27	1.62, s	16.1	1.60, s	16.0	1.60, s	15.9
28	1.61, s	16.2	1.61, s	15.9	1.62, s	16.0
29	1.60, s	16.2	1.61, s	15.9	1.61, s	15.9
30	1.15, s	26.6	1.19, s	26.5	1.15, s	26.4

^{a 1}H- and ¹³C-NMR spectra were recorded at 500 and 125 MHz, respectively, in CDCl₃; ^{b 1}H- and ¹³C-NMR spectra were recorded at 600 and 150 MHz, respectively, in CDCl₃.

), four olefinic protons were observed at δ_H 5.14 (2H, m, H-11, H-14), 5.19 (1H, t, J = 6.4 Hz, H-18), and 5.43 (1H, t, J = 6.4 Hz, H-7), which indicated that 2 has a more asymmetrical structure than 1. In addition, six allylic methylene groups at δ_H 1.41, 1.59, 2.02, 2.05, 2.10, and 2.13 (each 2H, m, H₂-12, 13, 16, 9, 17, 8), four methyl groups at δ_H 1.60, 1.61, 1.62 and 1.63 (each 3H, s, H₃-29, 28, 27, 26), and two methylene groups at δ_H 2.09 and 2.23 (each 2H, m, H₂-21 and 20) were observed. A methylene group between two hydroxy groups was observed at δ_H 1.63 (2H, m, H₂-4). Additionally, the signals of three methine groups at δ_H 3.35 (1H, d, J = 10.4 Hz, H-22), 3.62 (1H, m, H-3), and 4.26 (1H, dd, J = 7.6, 4.8 Hz, H-5) and four terminal methyl groups at δ_H 1.15, 1.17, 1.19, and 1.20 (each 3H, s, H₃-30, 25, 24, 1) were observed. The ¹³C-NMR spectrum of 2 (Table 1) revealed the presence of 30 carbons, and the connectivity of the proton and carbon atoms was elucidated via DEPT and HMQC analyses. In the ¹³C-NMR spectrum, the signals at δ_C 78.5, 78.6, and 78.9 were attributed to methine carbon and suggested the presence of three hydroxy groups. In the HMBC experiment (Figure 2), long-range couplings were observed from H-7 (δ_H 5.43) to C-5 (δ_C 78.6), C-9 (δ_C 39.4) and C-26 (δ_C 11.9), from H-5 (δ_H 4.26) to C-3 (δ_C 78.5), C-7 (δ_C 126.6) and C-26 (δ_C 11.9), and from H-3 (δ_H 3.62) to C-5 (δ_C 78.6). The ¹H-¹H COSY spectrum of 2 showed correlated proton signals of H₂-4 (δ_H 1.63) with H-3 (δ_H 3.62) and H-5 (δ_H 4.26). Therefore, we confirmed that the hydroxy group was attached at the C-5 of 2. We also observed long-range correlations from H-18 (δ_H 5.43) to C-20 (δ_C 37.0) and C-29 (δ_C 16.2) and from H-22 (δ_H 3.35) to C-20 (δ_C 37.0), C-24 (δ_C 23.5), and C-30 (δ_C 26.6) in the HMBC spectrum of 2 (Figure 2). In addition, correlated proton signals at H₂-8 (δ_H 2.13), H-7 (δ_H 5.43), H₂-17 (δ_H 2.10), and H-18 (δ_H 5.19) were observed in the ¹H-¹H COSY spectrum of 2. Based on these results, 2 was identified as the acyclic triterpenoid, 2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (Figure 1) [25].

Compound 3 was isolated as a yellow oil with [α]${}_{\mathrm{D}}^{20}$ −0.4 (CHCl₃, c 0.1). It displayed a peak at m/z 517.3861 in the HRESIMS corresponding to [M + Na]⁺ (calcd. 517.3863), indicating a molecular formula of C₃₀H₅₄O₅. Comparison of the ¹H-NMR data with those of 2 (Table 1) indicated that 3 had two downfield-shifted olefinic protons at δ_H 5.50 (1H, dd, J = 15.6, 1.2 Hz, H-7) and 5.58 (1H, dt, J = 15.6, 6.0 Hz, H-8). The downfield effect indicates the presence of an electron-withdrawing group around these protons. In addition, six methylene groups at δ_H 1.58, 2.03, 2.10, 2.10, and 2.74 (each 2H, m, H₂-13, 16, 12, 17, 9), 2.02 and 2.22 (each 1H, m, H₂-20a, 20b) attached to an olefinic group, two oxymethine groups at δ_H 3.35 (1H, dd, J =10.8, 1.8 Hz, H-3, H-22), and four terminal methyl groups at δ_H 1.15, 1.15, 1.19, and 1.19 (each 3H, s, H₃-24, 25, 1, 30) were detected. The ¹³C-NMR spectrum of 3 (Table 1) revealed the presence of 30 carbons, and the connectivity of the proton and carbon atoms was elucidated via DEPT and HMQC analyses. In the ¹³C-NMR spectrum, the three oxygenated quaternary carbons C-6 (δ_C 73.0), C-2 (δ_C 73.1), and C-23 (δ_C 73.1) were elucidated via DEPT and HMQC. Detailed 2D correlation analysis identified long-range coupling from H-7 (δ_H 5.50) to C-6 (δ_C 73.0), C-8 (δ_C 126.7), and C-9 (δ_C 30.8), from H₂-5 (δ_H 1.55) to C-4 (δ_C 22.9), C-6 (δ_C 73.0), and C-7 (δ_C 136.7), and from H₃-26 (δ_H 1.26) to C-5 (δ_C 42.4), C-6 (δ_C 73.0), and C-7 (δ_C 136.7) (Figure 2). The ¹H-¹H COSY spectrum of 3 was from the H₂-9 (δ_H 2.74) sp³ methylene proton to H-8 (δ_H 5.58) and H-10 (δ_H 5.14). Therefore, the structure of 3 had a newly attached hydroxy group at the C-6 position. This hydroxy group influenced the shift of the double bond position from H-6 to H-7. Based on these data, 3 was identified as a new acyclic triterpenoid, 2,3,6,22,23-pentahydroxy-2,6,11,15,19,23-hexamethyl-tetracosa-7,10,14,18-tetraene (Figure 1).

Compound 4 was isolated as a yellow oil with [α]${}_{\mathrm{D}}^{20}$ +13.3 (CHCl₃, c 0.1). It displayed a peak at m/z 493.3898 in the HRESIMS corresponding to [M − H]⁻ (calcd. 493.3898), indicating a molecular formula of C₃₀H₅₄O_5. Comparison of the ¹H-NMR data with those of 3 (Table 1) indicated that 4 had three olefinic groups at δ_H 5.14 (1H, q, J = 6.6 Hz, H-14, -18), and 5.22 (1H, t, J = 6.6 Hz, H-11) and two terminal olefinic protons at δ_H 4.87 (1H, brs, H₂-26a) and 5.05 (1H, brs, H₂-26b), which were supported by the DEPT and COSY spectra. In addition, two methylene group signals between two hydroxy groups were observed at δ_H 1.58 (2H, m, H₂-4) and 2.01 (2H, m, H₂-5), three oxymethine groups were observed at δ_H 3.34 (1H, d, J = 9.6 Hz, H-3, -22), and 4.09 (1H, m, H-6), and four terminal methyl groups were observed at δ_H 1.15, 1.15, 1.19, and 1.19 (each 3H, s, H₃-1, 30, 24, 25). In the ¹³C-NMR spectrum (Table 1), three oxymethine carbons, C-6 (δ_C 75.1), C-3 (δ_C 78.1), and C-22 (δ_C 78.1), were elucidated via DEPT and HMQC analyses. Detailed 2D correlation analysis between C-6 and terminal olefinic protons revealed long-range couplings from H₂-5 (δ_H 2.01) to C-6 (δ_C 75.1) and C-7 (δ_C 151.3), from H-6 (δ_H 4.09) to C-5 (δ_C 24.3) and C-26 (δ_C 109.9), from H₂-8 (δ_H 2.19) to C-6 (δ_C 75.1), C-26 (δ_C 109.9), and C-7 (δ_C 151.3), and from H₂-26 (δ_H 4.87 and 5.05) to C-6 (δ_C 75.1) and C-8 (δ_C 31.0). Therefore, Compound 4 had a secondary alcohol group (C-6) instead of the methyl group at the C-6 of 3. This hydroxy group influenced the movement of the double bond position, and terminal olefinic protons appeared at the C-7 position. Based on these results, Compound 4 was identified as a new acyclic triterpenoid, 2,3,6,22,23-pentahydroxy-2,10,15,19,23-hexamethyl-7-methylenetetracosa-10,14,18-triene.

We applied the modified Mosher’s method to determine the absolute configuration of the three chiral centers at C-3, C-5, and C-22 in 2, and corresponding to the Δδ_H pattern for the stereochemistry of secondary diols reported by Freire et al. [26,27,28]. The mono- or tris-(S)-MTPA esters (2a, 2c) and -(R)-MTPA esters (2b, 2d) were prepared by treating 2 with (R)-, and (S)-MTPA chloride, respectively. The difference in the chemical shift values (Δδ_H = δ_S − δ_R) of (S)-MTPA (2a, 2c), and (R)-MTPA ester derivatives (2b, 2d) was calculated to assign the absolute configuration of 2 (Figure 3). Based on the results summarized in Figure 3, the absolute configurations of C-3, C-5, and C-22 in 2 were determined as R, S and R, respectively. To elucidate absolute configuration of Compound 3, a pair of MTPA esters (3a and 3b) were prepared in the same manner. As shown in Figure 3, the absolute configurations of C-3 and C-22 in 3 were assigned as R and R, respectively. Unfortunately, we were unable to prepare the relevant MTPA ester derivatives to determine the absolute configuration of 4, and further studies are required to elucidate stereostructure of acyclic triterpenoid (4).

2.3. IL-6/STAT3 Inhibitory Effects of Acyclic Triterpenoids 1–4

Compounds 1–4 were tested for their inhibitory effects on STAT3-dependent luciferase activity induced by IL-6. Hep3B cells stably transformed with the pStat3-Luc plasmid were stimulated with IL-6 (10 ng/mL) for 12 h in the presence or absence of Compounds 1–4, and STAT3-dependent promoter activity was measured [29]. Compounds 1–4 inhibited IL-6-induced STAT3 activity in a dose-dependent fashion, with IC₅₀ values of 0.67, 0.71, 2.18 and 2.99 μM (Figure 4). These four acyclic triterpenoids showed more potent inhibitory activity on STAT3-dependent luciferase activity than that of genistein as the positive control (IC₅₀ value, 15.0 µM in this assay system) [29]. Additionally, Compounds 1–4 were non-cytotoxic at the IC₅₀ dose indicated in this study (see Figure S34 in Supplementary Materials).

The IL-6-induced JAK2/STAT3 signaling pathway plays a positive role in inflammation and neoplasia [30]. The phosphorylation of JAK2 and STAT3 leads to dimerization of STAT3 and translocation to the nucleus, where it transcribes pro-inflammatory cytokine genes, such as IFN-γ, IL-17, and IL-1β [31]. To determine whether the inhibitory effects on IL-6/STAT3 are dependent on JAK2/STAT3 phosphorylation, we evaluated the protein level by Western blotting analysis. As shown in Figure 5, A. katsumadai EtOH extract and Compounds 1 and 2 inhibited IL-6-stimulated phosphorylation of JAK2 and STAT3 in a concentrations-dependent manner. In particular, Compounds 1 and 2, which were major constituents of the A. katsumadai EtOH extract (see Figure S35 in Supplementary Materials), were significantly responsible for inhibiting IL-6/STAT3 activation.

3. Materials and Methods

3.1. General Experimental Procedures

¹H-NMR (300, 500 and 600 MHz), ¹³C-NMR (75, 125 and 150 MHz), HMQC, and HMBC spectra were obtained on a Varian Unity 300, Bruker Biospin Avance 500, and JEOL JNM-ECA600 spectrometer, with CDCl₃ as a solvent. ESI-MS was conducted using a Shimadzu LCMS-IT-TOF mass spectrometer. Optical rotations were determined on a JASCO DIP-370 polarimeter. The HPLC system consisted of a Hitachi L-2130 pump, Hitachi UV detector L-2400, and Capcell Pak C18 column (20 × 250 mm, Shiseido, Tokyo, Japan). Reversed-phase column chromatography was conducted using RP-C₁₈ silica gel (ODS-A, 250 × 20 mm, YMC Co. Ltd., Kyoto, Japan), and silica gel column chromatography was conducted using Kieselgel 60 (70–230 and 200–400 mesh, Merck, Darmstadt, Germany). TLC was conducted using Kieselgel 60 F₂₅₄ plates (Merck).

3.2. Extraction and Isolation

The seeds of A. katsumadai were purchased at a herbal market in Daejeon, Korea. The authenticity of the plants was confirmed by Prof. Y. H. Kim, at the College of Pharmacy of Chungnam National University (Daejeon, Korea). A voucher specimen (PBC-386A) was deposited in the Korea Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology. Dried seeds of A. katsumadai (1.8 kg) were extracted with EtOH (10 L) for 7 days at room temperature. The ethanol extract was evaporated in vacuo to yield a residue (180 g). The residue was suspended in distilled H₂O (3 L) and extracted with CHCl₃ (10 L). The CHCl₃-soluble components were then evaporated in vacuo, and the resulting extract (85 g) was subjected to silica gel (Kieselgel 60, 230–400 mesh, 150 g, Merck, Darmstadt, Germany) column chromatography using a gradient of CHCl₃–CH₃OH (100:0, 90:1, 70:1, 50:1, 30:1, 15:1, 5:1 and 1:1; each 3 L, v/v) as eluent to yield 22 fractions (F1-22) on the TLC profile. F8 (2.4 g) was subjected to reverse-phase column chromatography (100 g) and was eluted with CH₃OH–H₂O (60:1, 70:1, 80:1, 90:1 and 100:0; each 2 L, v/v), to yield six sub-fractions (F8-1, -6) based on the TLC profile. F8-3 (1.0 g) was separated by semi-preparative HPLC (YMC ODS–H80, flow rate: 6 mL/min) eluted with CH₃CN–H₂O (80:1, v/v) to yield 1 (300 mg, t_R 40 min). F9 (700 mg) was subjected to reverse-phase column chromatography (100 g) eluted with CH₃OH–H₂O (50:1, 60:1, 70:1, 80:1, 90:1, and 100:0; each 2 L, v/v) to yield seven sub-fractions (F9-1, -7). F9-3 (300 mg) was successively separated by preparative HPLC (CH₃CN–H₂O, 70:1, v/v) to yield 2 (100 mg, t_R 30 min). F16 (8.4 g) was subjected to reverse-phase column chromatography eluted with CH₃OH–H₂O (2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 100:0; each 500 m L, v/v), to yield seven sub-fractions (F16-1, -7). F16-4 was subjected to reverse-phase column chromatography eluted with CH₃CN–H₂O (2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 100:0; each 300 mL, v/v) to yield seven sub-fractions (F16-4-1, -7). F16-4-6 (538 mg) was eluted with CH₃OH–H₂O (4:1, v/v) and was subjected to further separation by preparative HPLC (Capcell Pak C₁₈, flow rate: 5 mL/min) to obtain five fractions. F16-4-6-3 was purified by semi-preparative HPLC (CH₃CN–H₂O, 11:9, flow rate: 5 mL/min) to yield 3 (30 mg, t_R 38 min). F16-4-6-5 was purified by preparative HPLC (CH₃CN–H₂O, 3:2, flow rate: 5 mL/min) to yield 4 (32 mg, t_R 45 min).

2,3,22,23-Tertrahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (1). Yellow oil; C₃₀H₅₄O₄; [α]${}_{\mathrm{D}}^{20}$ +18.1 (c 1.0, CHCl₃); IR (neat) ν_max 3400, 2950 cm⁻¹; ESI-MS: m/z 501 [M + Na]⁺; ¹H-NMR (300 MHz, CDCl₃) δ_H 5.18 (2H, m, H-7, H-18), 5.13 (2H, m, H-11, H-14), 3.36 (2H, dd, J = 2.0, 10.5 Hz, H-3, H-22), 2.23 (2H, m, H₂-5a, H₂-20a), 2.08 (2H, m, H-8, H-17), 2.06 (2H, m, H₂-5b, H₂-20b), 2.01 (2H, m, H₂-9, H₂-16), 1.99 (2H, m, H₂-12, H₂-13), 1.59 (3H, s, H₃-27, H₃-28), 1.58, (2H, m, H₂-4a, H₂-21a), 1.61 (3H, s, H₃-26, H₃-29), 1.40 (2H, m, H₂-4b, H₂-21b), 1.19 (3H, s, H₃-1, H₃-24), 1.15 (3H, s, H₃-25, H₃-30); ¹³C-NMR (75 MHz, CDCl₃) δ_C 15.88 (C-27, C-28), 15.95 (C-26, C-29), 23.23 (C-25, C-30), 26.36 (C-1, C-24), 26.47 (C-8, C-17), 28.17 (C-9, C-16), 29.64 (C-4, C-21), 36.77 (C-5, C-20), 39.61 (C-12, C-13), 72.99 (C-2, C-23), 78.23 (C-3, C-22), 124.40 (C-11, C-14), 125.01 (C-7, C-18), 134.79 (C-10, C-15), 134.91 (C-6, C-19).

2,3,5,22,23-Pentahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (2). Yellow oil; C₃₀H₅₄O₅; [α]${}_{\mathrm{D}}^{20}$ +4.8 (c 1.0, CHCl₃); IR (neat): ν_max 3400, 2900 cm⁻¹; UV(MeOH) λ_max (logε) nm: 210 (2.51); ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) spectra, see Table 1; HRESI-MS: m/z 493.3897 [M − H]⁻ (calcd. for C₃₀H₅₃O₅, 493.3893).

2,3,6,22,23-Pentahydroxy-2,6,11,15,19,23-hexamethyl-tetracosa-7,10,14,18-tetraene (3). Yellow oil; C₃₀H₅₄O₅; [α]${}_{\mathrm{D}}^{20}$ −0.4 (c 0.1, CHCl₃); IR (MeOH): ν_max 3300, 2950, 1450, 1050 cm⁻¹; UV(MeOH) λ_max (logε) nm: 205 (2.42); ¹H-NMR (600 MHz, CDCl₃) and ¹³C-NMR (150 MHz, CDCl₃) spectra, see Table 1; HRESI-MS: m/z 517.3861 [M + Na]⁺ (calcd. for C₃₀H₅₄O₅Na, 517.3863).

2,3,6,22,23-Pentahydroxy-2,10,15,19,23-hexamethyl-7-methylenetetracosa-10,14,18-triene (4). Yellow oil; C₃₀H₅₄O₅; [α]${}_{\mathrm{D}}^{20}$ +13.3 (c 0.1, CHCl₃); IR (MeOH): ν_max 3300, 2950, 1050 cm⁻¹; UV(MeOH) λ_max (logε) nm: 205 (2.34); ¹H-NMR (600 MHz, CDCl₃) and ¹³C-NMR (150 MHz, CDCl₃) spectra, see Table 1; HRESI-MS: m/z 493.3899 [M − H]⁻ (calcd. for C₃₀H₅₃O₅, 493.3898).

3.3. (R)- and (S)-MTPA Ester of Compounds 2–3

Compounds 2–3 (4 mg) were dissolved in pyridine-d₅ (10 mL), and (R)- or (S)-MTPA-Cl (10 μL) and DMAP (2 mg) were added. The mixture was transferred vials under an N₂ gas stream. The each vials were incubated in a water bath for 4 h (40 °C). The reactions afforded (S)- or (R)-MTPA ester derivatives 2S, 3S, and 2R, 3R, and each residues were purified by preparative HPLC using a C₁₈ column (Phenomenex kinetex C₁₈, 150 × 21.0 mm), eluting with a gradient solvent system composed of H₂O/CH₃OH (40:60 → 0:100, v/v). The ¹H-NMR spectra of each derivative were obtained from the reaction.

5-Mono-MTPA Ester of 2 (2a). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 6.41 (1H, dd, J = 10.8, 4.2 Hz, H-5), 5.94 (1H, t, J = 6.0 Hz, H-7), 5.39 (1H, t, J = 7.2, H-18), 5.31 (1H, br s, H-11), 5.26 (1H, br s, H-14), 3.77 (1H, m, H-3), 3.73 (1H, m, H-22), 2.70 (1H, m, H₂-20a), 2.51 (1H, m, H₂-4a), 2.40 (5H, m, H₂-4b, -8a, -13a, -17a, -20b), 2.11 (8H, m, H₂-8b, -9, 12a, -13b, -16, -17b), 1.83 (3H, s, H₃-26), 1.82 (2H, m, H₂-21), 1.71, (3H, s, H₃-27), 1.66, (3H, s, H₃-28), 1.62 (3H, s, H₃-29), 1.54 (3H, s, H₃-1), 1.52 (3H, s, H₃-24), 1.51 (3H, s, H₃-25), 1.48 (3H, s, H₃-30).

5-Mono-MTPA Ester of 2 (2b). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 6.36 (1H, dd, J = 10.2, 4.2 Hz, H-5), 5.90 (1H, t, J = 5.4 Hz, H-7), 5.39 (1H, t, J = 7.2, H-18), 5.31 (1H, br s, H-11), 5.27 (1H, br s, H-14), 3.78 (2H, m, H-3, -22), 2.71 (1H, m, H₂-20a), 2.56 (1H, m, H₂-4a), 2.36 (5H, m, H₂-4b, -8a, -13a, -17a, -20b), 2.08 (8H, m, H₂-8b, -9, 12a, -13b, -16, -17b), 1.82 (2H, m, H₂-21), 1.71 (3H, s, H₃-26), 1.66, (3H, s, H₃-27), 1.62, (3H, s, H₃-28), 1.61 (3H, s, H₃-29), 1.55 (6H, s, H₃-1, -24), 1.52 (3H, s, H₃-25), 1.51 (3H, s, H₃-30).

3,5,22-Tris-MTPA Ester of 2 (2c). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 6.06 (1H, dd, J = 11.4, 4.2 Hz, H-5), 6.00 (1H, t, J = 6.6 Hz, H-7), 5.53 (1H, d, J = 10.2, H-3), 5.39 (1H, d, J = 10.2 Hz, H-22), 5.36 (1H, t, J = 6.6 Hz, H-18), 5.30 (2H, m, H-11, -14), 2.75 (1H, m, H₂-20a), 2.36 (1H, m, H₂-4a), 2.30 (5H, m, H₂-4b, -8a, -13a, -17a, -20b), 2.18 (8H, m, H₂-8b, -9, 12a, -13b, -16, -17b), 1.95 (2H, m, H₂-21), 1.81 (3H, s, H₃-26), 1.66, (3H, s, H₃-27), 1.65, (3H, s, H₃-28), 1.62 (3H, s, H₃-29), 1.40 (3H, s, H₃-1), 1.39 (3H, s, H₃-24), 1.33 (3H, s, H₃-25), 1.32 (3H, s, H₃-30).

3,5,22-Tris-MTPA Ester of 2 (2d). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 6.36 (1H, dd, J = 10.2, 4.2 Hz, H-5), 5.90 (1H, m, H-7), 5.55 (1H, d, J = 10.8, H-3), 5.32 (1H, m, H-22), 5.27 (1H, m, H-18), 5.20 (2H, m, H-11, -14), 2.56 (1H, m, H₂-20a), 2.44 (1H, m, H₂-4a), 2.38 (5H, m, H₂-4b, -8a, -13a, -17a, -20b), 2.11 (8H, m, H₂-8b, -9, 12a, -13b, -16, -17b), 1.77 (2H, m, H₂-21), 1.67 (3H, s, H₃-26), 1.62, (3H, s, H₃-27, -28), 1.60 (3H, s, H₃-29), 1.56 (3H, s, H₃-1), 1.51 (3H, s, H₃-24), 1.45 (3H, s, H₃-25), 1.44 (3H, s, H₃-30).

3,22-Bis-MTPA Ester of 3 (3a). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 5.90 (1H, m, H-7), 5.87 (1H, m, H-8), 5.70 (1H, m, H-18), 5.52 (1H, m, H-14), 5.45 (2H, m, H-3, -22), 5.38 (1H, m, H-10), 2.90 (2H, m, H₂-9), 2.56 (1H, m, H₂-20a), 2.01 (1H, m, H₂-4a), 1.96 (13H, m, H₂-8, -9, -12, -13, -16, -17, -20b), 1.88 (2H, m, H₂-21), 1.83 (2H, m, H₂-5), 1.70 (3H, s, H₃-28), 1.66, (3H, s, H₃-29), 1.62, (3H, s, H₃-27), 1.54 (3H, s, H₃-1), 1.53 (3H, s, H₃-30), 1.52 (3H, s, H₃-26), 1.51 (3H, s, H₃-24), 1.50 (3H, s, H₃-25).

3,22-Bis-MTPA Ester of 3 (3b). ¹H-NMR (pyridine-d₅, 600 MHz) δ_H 5.89 (1H, m, H-7), 5.86 (1H, m, H-8), 5.72 (1H, m, H-18), 5.53 (1H, m, H-14), 5.44 (2H, m, H-3, -22), 5.38 (1H, m, H-10), 2.90 (2H, m, H₂-9), 2.55 (1H, m, H₂-20a), 2.08 (1H, m, H₂-4a), 1.91 (13H, m, H₂-8, -9, -12, -13, -16, -17, -20b), 1.80 (2H, m, H₂-21), 1.79 (2H, m, H₂-5), 1.70 (3H, s, H₃-28), 1.66, (3H, s, H₃-29), 1.62, (3H, s, H₃-27), 1.53 (3H, s, H₃-1, -30), 1.52 (3H, s, H₃-26), 1.51 (3H, s, H₃-24), 1.50 (3H, s, H₃-25).

3.4. Biological Materials and Cell Culture

Recombinant human IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-phospho Stat3 (Tyr⁷⁰⁵) IgG was purchased from Calbiochem (Darmstadt, Germany). All reagents, including genistein, were obtained from Sigma-Aldrich Ltd. (St. Louis, MO, USA). Human hepatoma Hep3B and U266 cells were obtained from the American Type Culture Collection (ATCC No. HB-8064 and TIB-196TM, Rockville, MD, USA) and were maintained in DMEM and RPMI1640 media supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C in a 5% CO₂ incubator. All cell culture reagents were obtained from GibcoBRL (Life Technologies, Cergy-Pontoise, France).

3.5. Luciferase Assay

Hep3B cells stably expressing pStat3-Luc, which were previously established by Chang et al. [32], were seeded onto 96-well culture plates at 2 × 10⁴ cells/well. After 24 h, the cells were starved for 12 h and were then treated with IL-6 (10 ng/mL) with or without compounds for 12 h. The luciferase assay was performed with a Promega kit according to the manufacturer’s protocol (Madison, WI, USA).

3.6. Cell Viability

Hep3B cells were seeded at a plating density of 2 × 10⁴ cells/well and were cultured for 24 h to allow them to adhere to the plate. After 24 h, the culture medium was changed to serum-free medium supplemented with samples at the indicated dose. MTT (0.5 mg/mL) was added after a 48 h culture, and 200 μL of DMSO was then added to each well after a 4 h incubation at 37 °C. The absorbance of the samples at 540 nm was measured against a background control using a 96-well plate reader. The percentage of viable cells under each treatment condition was determined relative to the negative control.

3.7. Western Blotting Analysis

U266 cells were stimulated with IL-6 (10 ng/mL) for 20 min in the presence or absence of compounds. Western blot analysis was performed to evaluate STAT3 and JAK2 protein expression in the U266 cell line, as described in previous studies [33]. The phosphorylation status of STAT3 and JAK2 was examined using anti-phospho-Stat3 (1:1000), anti-Stat3 (1:1000), anti-phospho-Jak2 (1:1000), and anti-Jak2 (1:1000) antibodies (Cell Signaling, Beverly, MA, USA) and then were incubated with the appropriate horseradish peroxide-conjugated secondary antibody (1:5000) at RT. The optical densities of antibody-specific bands were quantified using ImageJ software.

3.8. Statistical Analyses

Data are expressed as the means ± standard error of the mean (S.E.), and the statistical analyses were performed using Student’s t-test in Prism 5 software (GraphPad software, San Diego, CA, USA). A probability value of 0.05 (p < 0.05) was considered significant.

4. Conclusions

In conclusion, four acyclic triterpenoid derivatives (1–4) were isolated from the EtOH extract of A. katsumadai. Compounds 3 and 4 were identified as new acyclic triterpenoids 2,3,6,22,23-pentahydroxy-2,6,11,15,19,23-hexamethyl-tetracosa-7,10,14,18-tetraene and 2,3,6,22,23-pentahydroxy-2,10,15,19,23-hexamethyl-7-methylenetetracosa-10,14,18-triene, respectively. In addition, major constituents, Compounds 1 and 2, in A. katsumadai showed more potent inhibitory activity on IL-6-induced STAT3 activation. Based on our results, these triterpenoids could be useful candidates for designing new IL-6 inhibitors as anti-inflammatory agents.