Six New Triterpene Derivatives from Aralia chinensis Var. dasyphylloides

Aralia chinensis var. dasyphylloides is widely distributed in China and used as a traditional herbal medicine for the treatment of digestive and immune system diseases. The present study aimed to search for novel oleanolic-type triterpenoids in low-polarity fractions. Six new triterpene derivatives (1–6), together with two known compounds were isolated from the barks of A. chinensis var. dasyphylloides. Their structures were elucidated by 1D- and 2D-NMR spectroscopic analysis and chemical methods. They were identified as 3-oxo-oleana-11,13(18)-dien-28,30-dioic acid (1), 30-hydroxy-3-oxo-oleana-11,13(18)-dien-28-oic acid (2), 3β-hydroxy-oleana-11,13(18)-dien-28-oic acid-28-O-β-d-glucopyranoside (3), 3β,30-dihydroxy-oleana-11,13(18)-dien-28-oic acid-28-O-β-d-glucopyranoside (4), 3β-hydroxy-oleana-11,13(18)-dien-28-oic acid-3-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (5), 3β,29-dihydroxy-oleana-9(11),12-dien-28-oic acid-28-O-β-d-glucopyranoside (6), namely, araliachinolic acids I and II and araliachinosides I–IV. The cytotoxicity of the isolated compounds was tested against HepG2, A549, SGC7901, and MCF7 cell lines, but no apparent activity was observed at a concentration of 50 μM.


Introduction
Aralia chinensis Linn. var. dasyphylloides Hand.-Mazz. Symb. (Araliaceae) is distributed in the Sichuan, Guizhou, Guangxi, and Hubei provinces of China [1]. It has been used as a traditional herbal medicine for the treatment of gastric ulcer, hepatitis rheumatic arthritis, and other diseases. Previous phytochemical investigations on this plant revealed the presence of essential oil [2] and oleanolic-type triterpenoid saponins [3,4]. Those saponins demonstrated inhibitory activities against α-glucosidase [5], moderate antioxidant effects and antiglycation activities [6], and cytotoxic activities against human nasopharyngeal carcinoma epithelial (CNE) cells [7].

Results and Discussion
The EtOH extract of the barks of A. chinensis var. dasyphylloides was fractionated by repeated medium-pressure liquid chromatography (MPLC) on normal and reversed-phase (RP) silica gel to yield the new derivatives 1-6 and two previously reported saponins (7 and 8). The structures of the new compounds were elucidated on the basis of extensive NMR spectroscopic analysis, including a series of 2D-NMR experiments (HSQC, HMBC, and NOESY), and mass spectrometry data. The known saponins (7 and 8) were identified by comparison of their spectral data with literature data [8,9].

Results and Discussion
The EtOH extract of the barks of A. chinensis var. dasyphylloides was fractionated by repeated medium-pressure liquid chromatography (MPLC) on normal and reversed-phase (RP) silica gel to yield the new derivatives 1-6 and two previously reported saponins (7 and 8). The structures of the new compounds were elucidated on the basis of extensive NMR spectroscopic analysis, including a series of 2D-NMR experiments (HSQC, HMBC, and NOESY), and mass spectrometry data. The known saponins (7 and 8) were identified by comparison of their spectral data with literature data [8,9].
Compound 1 was obtained as a white amorphous powder with the molecular formula determined to be C 30 H 42 O 5 on the basis of the molecular ion peak [M] + at m/z 482.3033 (calcd. 482.3032) observed in its HR-EI-MS and NMR spectroscopic data. The 1 H-NMR spectrum revealed six methyl group signals at δ (ppm) 1.09 (3H, s, Me-23), 1.04 (3H, s, Me-24), 1.04 (3H, s, Me-25), 0.86 (3H, s, Me-26), 0.93 (3H, s, Me-27), and 1.17 (3H, s, Me-29), as well as two cis olefinic protons at δ (ppm) 5.64 (1H, d, J = 11.0 Hz) and 6.73 (1H, dd, J = 11.0, 3.0 Hz). The 13 C-NMR and DEPT spectra revealed six methyls, nine methylenes, two sp 3 methines at δ (ppm) 55.6 (C-5) and 55.1 C-9), six sp 3 quaternary carbon signals, four olefinic signals at δ (ppm) 127.8 (C-11), 126.6 (C-12), 137.1 (C-13), and 133.7 (C-18), and three carbonyl signals at δ (ppm) 220.3 (C-3), 180.2 (C-28), and 181.9 (C-30). The 13 C-NMR signals, especially signals at δ (ppm) 55.6 C-5), 55.1 (C-9), 127.8 (C-11), 126.6 (C-12), 137.1 (C-13), and 133.7 (C-18), indicated the presence of an oleana-11,13(18)-diene-type triterpene, as confirmed by comparison with 3-oxo-11,13(18)-oleanadien-28-oic acid [10]. The main difference was the carboxyl group at C-30 and the down-field shift of C-20 at δ 45.7 ppm in 1. The carboxyl group also caused the downfield shift of C-29 at δ 29.0 ppm, as compared to δ 24.0 ppm in compounds with a C-30 methyl group [10][11][12]. In the HMBC spectrum (Figure 2), the correlation between the Me-24 protons at δ 1.04 ppm and C-3 (δ 220.3 ppm) revealed that one carbonyl group was located at C-3. The position of the second carboxyl group was determined by the HMBC correlation between Me-29 protons at δ 1.17 ppm and C-30 (δ 181.9 ppm), which could be confirmed by the NOESY correlations of H β -19 (δ 3.29 ppm) with H β -22 (δ 2.30 ppm), H α -22 (δ 1.34 ppm) with Me-29 (δ 1.17 ppm). The position of the third carboxyl group was determined by the HMBC correlation between H-16 at δ 1.96 ppm and C-28 (δ 180.2 ppm). Very recently, a compound has been reported as 3-oxooleana-11,13(18)-diene-28,30 dioic acid [13]. However, in this reference, the structure depicted corresponds to the 29-carboxylic acid derivative, and no evidence is provided for the location of the carboxylic group at C-29 or C-30. Thus, the structure of compound 1 is assigned here unambiguously for the first time (The NMR data was available at the Supplementary Materials), and the compound was named araliachinolic acid I. the structure depicted corresponds to the 29-carboxylic acid derivative, and no evidence is provided for the location of the carboxylic group at C-29 or C-30. Thus, the structure of compound 1 is assigned here unambiguously for the first time (The NMR data was available at the Supplementary Materials), and the compound was named araliachinolic acid I. Compound 2 was obtained as a white amorphous powder with the molecular formula determined to be C30H44O4 from the pseudo-molecular ion peak [M − H] − at m/z 467.3159 (calcd. 467.3161) observed in its HR-ESI-MS and its NMR spectroscopic data. The 1 H-and 13 C-NMR data (Table 1) of 2 were similar to those of 1. Careful comparison of the NMR data between compound 1 and 2 indicated that both compounds possessed the same carbon skeleton, but had different substitution at C-30. Compared with compound 1, the 1 H-NMR spectrum of 2 showed a pair of doublets at δ (ppm) 3.18 (1H, d, J = 10.0 Hz) and 3.38 (1H, t, J = 10.0 Hz), which were assigned to CH2-30. In the 13 C-NMR spectrum, C-30 was observed at δ 67.0 ppm and C-20 was downfield shifted at δ 37.5 ppm. In the HMBC spectrum, the correlation between Me-29 protons at δ 0.90 ppm and C-30 (δ 67.0 ppm) revealed the hydroxymethylene group to be located at C-30. The carbonyl group was positioned at C-3 based on the correlation between the Me-23 protons at δ 1.07 ppm and C-3 (δ 220.3 ppm). The diene structure was confirmed by the correlations between H-11 and H-12 protons (δ 6.50 ppm and 5.64 ppm) and C-13 (δ 138.0 ppm) and C-18 (δ 133.8 ppm), respectively. The position of the second carboxyl group was determined by the correlation between H-22 (δ 2.19 ppm) and C-28 (δ 180.2 ppm). Thus, compound 2 was a new compound, named araliachinolic acid II.
Compound 3 was obtained as a white amorphous powder with the molecular formula determined to be C36H56O8 from the pseudo-molecular ion peak [M − H] − at m/z 615.3895 (calcd. 615.3897) evident in its HR-ESI-MS and its NMR spectroscopic data. The NMR data of 3 (Table 1) were similar to those of 3β-hydroxy-11,13(18)-oleanedien-28-oic acid [14]. The main differences in the NMR data of these two compounds were the signals of a sugar moiety in the case of 3. Thus, in the 1 H-NMR spectrum of 3, the anomeric signal at δ 5.45 ppm (1H, d, J = 8.5 Hz) and further signals at δ (ppm) 3.27, 3.37, 3.30, 3.31, 3.66, and 3.85 revealed the presence of one sugar which could be identified as β-D-glucopyranoside by acid hydrolysis, derivatization, and HPLC analysis. In the HMBC spectrum, the β-D-glucose was linked to the carboxyl group at C-28, based on the up-field shift from δ 180.2 ppm to δ 176.9 ppm. The correlation between Me-24 protons at δ 0.77 ppm and C-3 (δ 79.7 ppm) confirmed the presence of one hydroxyl group at C-3. This hydroxyl group was β-oriented by the NOESY correlation of H-3 (δ 3.18 ppm) with H-5 (δ 0.83 ppm) (The NOESY data was available at the Supplementary Materials). Taken together, these data indicate compound 3 to be a new compound, which was named araliachinoside I.
Compound 4 was obtained as a white amorphous powder with the molecular formula determined to be C36H56O9 on the basis of the pseudo-molecular ion peak [M + Cl] − at m/z 667.3616 (calcd. 667.3613) observed in its HR-ESI-MS and its NMR spectroscopic data. A comparison of the NMR data between 4 and 3 indicated that the two compounds possessed the same structure, with the only difference  (Table 1) of 2 were similar to those of 1. Careful comparison of the NMR data between compound 1 and 2 indicated that both compounds possessed the same carbon skeleton, but had different substitution at C-30. Compared with compound 1, the 1 H-NMR spectrum of 2 showed a pair of doublets at δ (ppm) 3.18 (1H, d, J = 10.0 Hz) and 3.38 (1H, t, J = 10.0 Hz), which were assigned to CH 2 -30. In the 13 C-NMR spectrum, C-30 was observed at δ 67.0 ppm and C-20 was downfield shifted at δ 37.5 ppm. In the HMBC spectrum, the correlation between Me-29 protons at δ 0.90 ppm and C-30 (δ 67.0 ppm) revealed the hydroxymethylene group to be located at C-30. The carbonyl group was positioned at C-3 based on the correlation between the Me-23 protons at δ 1.07 ppm and C-3 (δ 220.3 ppm). The diene structure was confirmed by the correlations between H-11 and H-12 protons (δ 6.50 ppm and 5.64 ppm) and C-13 (δ 138.0 ppm) and C-18 (δ 133.8 ppm), respectively. The position of the second carboxyl group was determined by the correlation between H-22 (δ 2.19 ppm) and C-28 (δ 180.2 ppm). Thus, compound 2 was a new compound, named araliachinolic acid II.
Compound 3 was obtained as a white amorphous powder with the molecular formula determined to be C 36 H 56 O 8 from the pseudo-molecular ion peak [M − H] − at m/z 615.3895 (calcd. 615.3897) evident in its HR-ESI-MS and its NMR spectroscopic data. The NMR data of 3 (Table 1) were similar to those of 3β-hydroxy-11,13(18)-oleanedien-28-oic acid [14]. The main differences in the NMR data of these two compounds were the signals of a sugar moiety in the case of 3. Thus, in the 1 H-NMR spectrum of 3, the anomeric signal at δ 5.45 ppm (1H, d, J = 8.5 Hz) and further signals at δ (ppm) 3.27, 3.37, 3.30, 3.31, 3.66, and 3.85 revealed the presence of one sugar which could be identified as β-D-glucopyranoside by acid hydrolysis, derivatization, and HPLC analysis. In the HMBC spectrum, the β-D-glucose was linked to the carboxyl group at C-28, based on the up-field shift from δ 180.2 ppm to δ 176.9 ppm. The correlation between Me-24 protons at δ 0.77 ppm and C-3 (δ 79.7 ppm) confirmed the presence of one hydroxyl group at C-3. This hydroxyl group was β-oriented by the NOESY correlation of H-3 (δ 3.18 ppm) with H-5 (δ 0.83 ppm) (The NOESY data was available at the Supplementary Materials). Taken together, these data indicate compound 3 to be a new compound, which was named araliachinoside I.
Compound 4 was obtained as a white amorphous powder with the molecular formula determined to be C 36 H 56 O 9 on the basis of the pseudo-molecular ion peak [M + Cl] − at m/z 667.3616 (calcd. 667.3613) observed in its HR-ESI-MS and its NMR spectroscopic data. A comparison of the NMR data between 4 and 3 indicated that the two compounds possessed the same structure, with the only difference being the substitution of the methyl group at C-30 in 3 by a hydroxymethylene group in compound 4. This was confirmed by the correlation between Me-29 protons at δ 1.17 ppm and C-30 (δ 66.4 ppm) in the HMBC spectrum. β-D-Glucose was identified via HPLC analysis after acid hydrolysis and derivatization. These data indicated that compound 4 was a new compound, which was named as araliachinoside II.
Compound 5 was obtained as a white amorphous powder with the molecular formula determined to be C 41 H 64 O 12 from the pseudo-molecular ion peak [M + Cl] − at m/z 783.4086 (calcd. 783.4086) evident in its HR-ESI-MS and its NMR spectroscopic data. A comparison of the NMR data of 5 with 3β-hydroxy-oleane-11,13(18)-dien-28-oic acid-3-O-β-D-glucopyranosyl-(1 → 2)-β-D-xylopyranoside [7] showed that the two compounds were almost identical. After careful comparison, the sequence of the two monosaccharides in the two compounds was shown to be different. In the HMBC spectrum of compound 5, the anomeric signal at δ 4.98 ppm (β-D-glucose) was correlated with C-3 of the aglycon moiety (δ 89.3 ppm), which indicated the β-D-glucose to be directly linked to the aglycon. The interglycosidic linkage was established based on the correlation of the anomeric signal at δ 5.32 ppm (β-D-xylose) with C-2 of β-D-glucose (δ 84.2 ppm). Taken together, these data indicated compound 5 to be a new compound, named araliachinoside III.

General
Column chromatography (CC) was performed using silica gel (200-300 mesh, 300-400 mesh, Qingdao Haiyang Chemical Group Co., Qingdao, China). Thin-layer chromatography was performed on silica gel GF254 (Qingdao Haiyang Chemical Group Co., Qingdao, China). MCI was purchased from Mitsubishi Chemical Group Co. (Tokyo, Japan) Semi-preparative HPLC was performed on a DIONEX Ultimate 3000 system equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 µm, YMC Co. Ltd., Kyoto, Japan). HR-EI-MS was measured on a Waters Autospec Premier 776 mass spectrometer (Waters, Milford, MA, USA). HR-ESI-MS was recorded on an Agilent G6230 TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were obtained on a Bruker DMX-500 spectrometer (Bruker, Karlsruher, Germany) using TMS as an internal reference. L-cysteine methyl ester and standard monosaccharides (D-glucose and D-xylose) used in HPLC experiments were purchased from Aladdin industrial Co. Ltd. (Shanghai, China). O-Tolyl-isothiocyanate was obtained from Sigma-Aldrich Co. Ltd (Sigma-Aldrich China, Shanghai, China). Other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China.

Plant Material
The barks of A. chinensis var. dasyphylloides were collected in June 2016 from Li Chuan City, Hubei Province, China. They were identified by Dr. Xinqiao Liu from College of Pharmacy at South-Central University for Nationalities, China. A voucher specimen (No. EP-201606) was deposited at the herbarium of College of Pharmacy, South-Central University for Nationalities, China.