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C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2
Accepted Manuscript C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2 Wanfang Zhu, Shengzhi Su, Yunhui Xu, Zijian Xie, Yidan Bai, Wenyuan Liu, Masahiko Abe, Toshihiro Akihisa, Feng Feng, Jie Zhang PII: DOI: Reference:
S0039-128X(18)30188-0 https://doi.org/10.1016/j.steroids.2018.09.016 STE 8323
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
24 May 2018 10 September 2018 29 September 2018
Please cite this article as: Zhu, W., Su, S., Xu, Y., Xie, Z., Bai, Y., Liu, W., Abe, M., Akihisa, T., Feng, F., Zhang, J., C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2, Steroids (2018), doi: https:// doi.org/10.1016/j.steroids.2018.09.016
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C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2
Wanfang Zhua, Shengzhi Sua, Yunhui Xud, Zijian Xied, Yidan Baie, Wenyuan Liue, Masahiko Abef, Toshihiro Akihisaf, Feng Feng, a,b,c,*, Jie Zhanga,b,*
aSchool
of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing
211198, PR China
bKey
Laboratory of Biomedical Functional Materials, China Pharmaceutical University,
Nanjing 211198, PR China
cJiangsu
Food and Pharmaceutical Science College, Huaian, Jiangsu, 223003, China
dMarshall
University, Marshall Institute for Interdisciplinary Research, Weisberg Engn Complex,
RM 4117,1628 Third Ave, Huntington, WV, 25703, USA
eDepartment
of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing
210009, China
fResearch
Institute for Science and Technology, Tokyo University of Science, 2641
Yamazaki, Noda, Chiba, 278-8510, Japan
*Corresponding
author.
Graphical abstract
ga1
Highlights
Isolation and identification three new and ten known C21 steroids from Streptocaulon juventas. Cytotoxicity activities against HepG2 and LO2 cells of isolated compounds. New compound 10 could induce apoptosis of HepG2 cells.
Abstract Three new C21 steroids, i.e., (3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-triol-3-O-β-Ddigitalopyranosyl-(1→4)-β-D-digitalopyranoside (4), (3β,17α,20S)- pregn-5(6)-ene-3, 17, 20-triol-20-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-Ddigital- opyranoside (8), (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-D-glucopyranoside (10), along with ten known C21 steroids were isolated from Streptocaulon juventas. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. All the isolated compounds were screened for their in vitro cytotoxicity against human liver cancer cells (HepG2) and the structure-activity relationships were also
analyzed. Moreover, compounds 1–3, 5, 10–12, which displayed cytotoxic activities in HepG2 cells, were tested for the selective index (SI) by the ratio of cytotoxic effect on human hepatocytes (LO2) to that on HepG2. As a result, new compound 10 exhibited a good inhibitory activity against HepG2 with IC50 value 11.7 μM as well as high SI value 3.5. Furthermore, compound 10 could induce HepG2 cells apoptosis by flow cytometry.
Keywords Streptocaulon juventas (Lour) Merr.
C21 steroid HepG2
Apoptosis
1 Introduction Apoptosis is a polygenic control process maintaining the stability of the environment in the body and the inhibition of this vital process may lead to the cancer occurrence [1,2]. Accumulated evidences have supported that anticancer drugs exert their
cytotoxic effects mainly by inducing tumor cell apoptosis [3]. C21 steroids is one species widely found in the plants of Asclepiadaceae family, which shows the possession of extensive pharmacological effects, such as anti-tumor effect [4]. Extensive evidences confirm that many C21 steroidal glycosides resist cancer by inducing apoptosis [5]. Streptocaulon juventas is a perennial herb belonging to the Asclepiadaceace family, which mainly distribute in the southern part of China, and it has long been used in China as a folk medicine for the treatment of dysentery, diarrhea, stomachache, fever, chronic nephritis and traumatic injury [6]. Moreover, traditional Chinese medicine takes the view that S. juventas could clear heat and remove toxin, which is believed to be related with the antitumor activity in traditional Chinese medicine [7]. In the present study, we describe the investigation on the C21 steroids from S. juventas as well as the cytotoxic effects of the isolated compounds against human liver cancer cells (HepG2), the active compounds (IC50 < 100) were also tested for the cytotoxicity activities against human hepatocytes (LO2) to acquire the selective index (SI). Furthermore, flow cytometry proved that new compound 10 could induce apoptosis of HepG2 cells. 2 Results and discussion 2.1 Structural elucidation of isolated compounds The n-BuOH fraction of S. juventas was subjected to column chromatography including silica gel, ODS and semipreparative RP-18 HPLC which led to the isolation of thirteen C21 steroids. They were three new steroids i.e., (3β,17α,20S)-pregn-5(6)ene-3, 17, 20-triol-3-O-β-D-digitalopyranosyl-(1→4)-β-D- digitalopyranoside (4),
(3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-triol-20-O-β-D- glucopyranosyl-(1→6)-β-Dglucopyranosyl-(1→2)-β-D-digitalopyranoside (8), and (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-D-glucopyranoside (10), and ten known steroids, i.e., pregnenediol (1) [8], hemindicusin (2) [9], periplocoside L (3) [10], periseoside C (5) [11], biondianoside D (6) [12], ∆5 pregnene-3β, 16α S-triol 20-O-β-D- glucopyranosyl(1→6)- β-D -glucopyranosyl-(1→2)- β-D -digitalopyranoside (7) [12], (Z)-3βhydroxy-5,17(20)-pregnadiene (9) [13], basikoside A (11) [14], carumbelloside II (12) [15], and stelmatocryptonoside D (13) [16]. (Figure 1 fig1). Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Compound 4 was obtained as white powder. Its molecular formula, C35H58O11, was deduced from the peak at m/z 677.3869 [M+Na]+ in the HRESIMS. For the aglycone part, The 1D NMR and HSQC spectra of compound 4 showed the presence of 3 methyl groups [δH 0.75 (3H, s), δC 14.79; δH 1.02 (3H, s), δC 19.82 and δH 1.16 (3H, d, J = 6.6 Hz), δC 18.74], 8 methylenes, 6 methines [including one olefinic methine at δH 5.37 (1H, br s), δC 122.74 together with two oxygenated methines at δH 3.54 (1H, m), δC 80.16 and δH 3.77 (1H, q, J = 6.0 Hz), δC 72.85], and 4 quaternary carbons [including an olefinic carbon at δC 141.94 and an oxygenated carbon at δC 86.99] (Table 1 tbl1). Combined analysis of spectral features in 1H-1H-COSY and HMBC (Figure 2 fig2), the aglycone of 4 was identified as same as that of compound 3. Furthermore, 1H NMR spectrum of 4 revealed signals for two methyl groups [δH 1.27 (3H, d, J = 3.0 Hz) and δH 1.26 (3H, d, J = 3.0 Hz)], two methoxy groups of deoxysugars [δH 3.49 (3H, s) and δH 3.44, (3H, s)] and two anomeric protons [δH 4.33
(1H, d, J = 7.8 Hz) and δH 4.51 (1H, d, J = 7.8 Hz)] suggesting the existence of two βD-digitalose which were confirmed by their 1D NMR and 2D NMR data and
comparison with the literature [10]. Their location and connection pattern were demonstrated by HMBC spectrum, in which obvious correlations from H-1'' to C-4' (δC 75.91) and H-1' to C-3 (δC 80.16) were observed. The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 3.77 (1H, q, J = 6.0 Hz)] to H-18 [δH 0.75 (3H, s)] indicating the configuration of C-17 is β as well as the correlations from H-21 [δH 1.16 (3H, d, J = 6.6 Hz)] to H-15β [δH 1.70 (1H, m)] and H-20 [δH 3.77 (1H, q, J = 6.0 Hz)] to H-16β [δH 2.01 (1H, m)] indicating that C-20 was S configuration [17]. Hence, compound 4 was determined to be (3β,17α,20S)- pregn- 5(6)-ene-3, 17, 20triol-3-O-β-D-digitalop- yranosyl-(1→4)-β-D-digitalopyranoside. Compound 8 was isolated as amorphous colorless crystal. The molecular formula was determined to be C40H66O17 by its HRESIMS (m/z: 841.4206 [M+Na]+). The 1H NMR,
13C
NMR and HSQC spectra could be assigned to the aglycone moiety of 21
carbon signals including, three methyl groups, eight methylenes, five methines, three quaternary and two olefinic carbons (Table 1). The NMR data of compound 8 indicated that the aglycone of compound 8 was similar to that of compound 7, except for the hydroxy group at C-16 changed to C-17, which was confirmed by the correlations of H-18 [δH 0.72 (3H, s)] with both C-16 (δC 31.83) and C-17 (δC 87.03) in the HMBC spectrum (Figure 2) The sugar moiety of 8 were determined to be β-Ddigitalose and β-D-glucose by detailed NMR analysis with the anomeric proton resonances at [δH 4.88 (1H, d, J = 7.8 Hz), δH 5.10 (1H, d, J = 7.2 Hz) and δH 5.00 (1H, d, J = 7.8 Hz)], respectively. The HMBC correlations from H-1''' to C-6'' (δC 69.86), H-1'' to C-2' (δC 78.87) and H-1' to C-20 (C 82.97) suggested that the
connections of the three sugars were through (1→6, 1→2) linkages and the sugar chain was located at C-20 of the aglycone. The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 4.08 (1H, q, J = 6.0 Hz)] to H-18 [δH 0.72 (3H, s)] indicating the configuration of C-17 is β. And the correlations from H-21 [δH 1.63 (3H, d, J = 6.0 Hz)] to H-15β [δH 1.74 (1H, m)] and H-20 [δH 4.08 (1H, q, J = 6.0 Hz)] to H-16β [δH 2.20 (1H, m)] revealing that C-20 was S configuration [17]. Thus, the structure of compound 8 was determined as (3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-
triol-20-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-D-
digitalopyranoside. Compound 10, obtained as white powder, had a molecular formula C27H44O8, as established by HRESIMS. The 1H NMR, 13C NMR and HSQC spectrum showed the presence of two methyl groups, one double bond, and a β-D-glucose (Tables 1 and 2 tbl2). Comparison of the NMR spectroscopic data of 10 with those of 4 revealed that one of the methyl groups which were always located in Me-18, -19, -21 was substituted by a hydroxy group, and the substituent position was determined to be C21, established by the correlations of H-17 [δH 2.30 (1H, m)] with both C-20 at δC and C-21 at δC 67.85. nThe location of the double bond was confirmed by the HMBC correlations of H-15 [δH 5.32 (1H, br s)] with both C-16 (δC 34.22) and C-17 (δC 56.18) and the sugar chain was located at C-3 of the aglycone out of the correlation of H-1' [δH 4.96 (1H, d, J = 7.8 Hz)] to C-3 (δC 78.85). The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 4.27 (1H, dd, J = 2.4, 8.1 Hz)] to H-18 [δH 1.12 (1H, s)] indicating the configuration of C-17 is β. And the correlations from H-20 to H-12β [δH 1.96 (1H, br d, J = 12 Hz)] demonstrating that C-20 was R configuration [17]. On the basis of the above analysis, the structure of
compound 10 was identified as (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-Dglucopyrano- side. 2.2 Cell cytotoxicity assay
2.2.1 MTT assay The cytotoxic activities of compounds 1–13 on HepG2 and LO2 cell lines were evaluated by MTT assays with cisplatin as the positive control (Table 3 tbl3). Compounds 3, 10, 12 exhibited potent cytotoxicities against HepG2 cells with IC50 values of 23.8, 11.7, 27.9 μM respectively. Compounds 1, 2, 5, 11 showed moderate inhibitory effects against HepG2 cells (IC50 42.6–73.0 μM) and other compounds with IC50 over 100 μM were regarded as inactive. The preliminary structure-activity relationships (SARs) suggested that hydroxy group substituent of compounds would strengthen the cytotoxic activity against HepG2 cells (1 > 9). When the number of substituted hydroxy group was the same, the position of substituent could also affect the activity (3 > 2). The sugar moiety is another important factor attributing to the activity [18]. The loss of sugar chain could enhance the cytotoxicity to HepG2 cells (3 > 4, 5 > 6). Furthermore, compounds 1–3, 5, 10–12, which displayed cytotoxic activities in HepG2 cells were tested for the selective index (SI) by the ratio of cytotoxic effect on normal human hepatocytes (LO2) to that on liver cancer cell line (HepG2). As a result, new compound 10 showed the highest selectivity with SI value of 3.5. At the same time, compound 10 also displayed the most significant activity against HepG2 cells (IC50 11.7 μM), while the SI and IC50 values of cisplatin were 1.3, 21.9,
indicating that compound 10 displayed the superior selectivity and cytotoxicity than the reference cisplatin. Therefore, compound 10 was selected for further study on HepG2 cell line. 2.2.2 Compound 10 induces apoptosis in HepG2 Cells To study whether the growth inhibition was related to cell apoptosis, HepG2 cells apoptosis mediated by compound 10 was determined by flow cytometry. Annexin V/PI staining was employed to discriminate among normal cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+), and necrosis cells (Annexin V-/PI+). As shown in Figure 3 fig3, compound 10 could increase the number of apoptosis cells in a concentration-dependent manner. The percentage of apoptotic cells was 38.4, 44.8, 51.7% respectively after adding compound 10 (3, 6, 12 μM), exhibiting significant differences with control (0.5% apoptotic cells). These results suggested that the growth inhibitory effect of compound 10 was partly related with the enhanced apoptosis in HepG2 cells. 3 Conclusion The present study showed that new compound 10 from S. juventas could significantly inhibit HepG2 cell growth proliferation via induction of apoptosis and showed high selectivity between HepG2 and LO2 in the meantime. These results demonstrated that compound 10 had an excellent potential to become lead for the development of safe drug for the treatment of liver cancer. 4 Experimental section
4.1 General methods
Optical rotations were measured with a Jasco P1020 polarimeter. IR spectra were acquired on a Bruker Tensor 27 spectrometer in KBr discs. A UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan) was applied to UV spectrum measurement. HR-ESI-MS spectra was obtained using a Bruker Micro TOF model mass spectrometer. Nuclear magnetic resonance (NMR) spectrum were measured on Bruker AV-300 or AV-500/600 spectrometers. Thin-layer chromatography (TLC) analyses were carried out using silica gel GF254 plates (EMD Millipore Corporation, Germany), and spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. Column chromatography separations were performed on silica gel (100-200 mesh and 200-300 mesh, Merck & Co., Inc.), and on ODS gel (50 μm, Fuji Silysia Chemical Ltd., Aichi, Japan). Semi-preparative HPLC was carried out on YMC-PACK ODS-AQ, and Shimadzu SCL-10A UV detector. 4.2 Plant material S. juventas was collected in November 2016, from Xishuangbanna, Yunnan Province, China and identified by Mr. Feng feng (China Pharmaceutical University). A voucher specimen (No. 201611) was deposited in the Department of Natural Medicinal Chemistry of China Pharmaceutical University, Nanjing, China. 4.3 Extraction and Isolation S. juventas (20.0 kg) were extracted with EtOH 95% (v/v) under reflux for three times (3 × 70.0 L, 2 h each). The extract was concentrated under reduced pressure to afford a crude residue (2.0 kg) which was then suspended in H2O (5.0 L) and partitioned with EtOAc (3 × 5.0 L) and n-BuOH (3 × 5.0 L) respectively. The concentrated n-BuOH soluble (250.0 g) was subjected to D101 CC and eluted with
MeOH–H2O (9:1→0:1 v/v) to afford six fractions, Frs. 1–6. Fr. 4 (76.7 g) from the MeOH–H2O (1:1) eluate, was divided into six parts (Fr. 4A–4F) by silica gel (100– 200 mesh) column chromatography (CC) eluted with gradient CHCl3–MeOH (1:0→0:1 v/v). Among them, Fr. 4B (11.5 g) was purified by column chromatography over reverse-phase C18 silica gel with MeOH–H2O (3:7→8:2 v/v) and silica gel (200– 300 mesh) using CHCl3–MeOH (55:1→15:1 v/v) as eluent, yielding compounds 1 (102.0 mg), 2 (3.0 mg), 3 (3.7 mg), 4 (4.0 mg), 11 (7.2 mg), 12 (18.0 mg). Fr. 4C (17.5 g) was subjected to reverse-phase C18 silica gel eluted with a gradient of MeOH–H2O (3:7→1:1, v/v) to afford compounds 5 (11.2 mg), 9 (17.3 mg), and 10 (18.3 mg). Fr. 4D (20.7 g) was applied to reverse-phase C18 silica gel eluting with a stepwise gradient of MeOH–H2O (4:6→6:4, v/v) to give Fr. 4D1–Fr. 4D3. Fr. 4D2 was further purified by repeated silica gel column chromatography to give compounds 6 (10.7 mg) and 13 (8.9 mg). Fr. 4D3 was separated by silica gel eluting with CHCl3– MeOH repeatedly, then subjected to preparative HPLC using MeOH–H2O (6:4, v/v) as the eluent to yield compounds 7 (21.7 mg, tR 25.0 min) and 8 (56.2 mg, tR 30.0 min). 4.3.1 Compound 4 25
Compound 4: white powder; [α]D -26.0 (c 0.342, MeOH); UV (MeOH) λmax: 204 nm; IR νmax cm-1: 3414, 2933, 1646, 1452, 1382, 1109, 1080; 1H NMR and
13C
NMR
(CD3OD) data are shown in Table 1 and 2; HRESIMS m/z: 677.3869 [M+Na]+ (C35H58NaO11, calcd. 677.3865). 4.3.2 Compound 8 25
Compound 8: colorless crystal; [α]D -36.0 (c 0.231, MeOH); UV (MeOH) λmax: 203
nm; IR νmax cm-1: 3383, 2934, 1648, 1379, 1165, 1073; 1H NMR and
13C
NMR
(C5D5N) data are shown in Table 1 and 2; HRESIMS m/z: 841.4206 [M+Na]+ (C40H66NaO17, calcd. 841.4196). 4.3.3 Compound 10 25
Compound 10: white powder; [α]D -6.3 (c 0.123, MeOH); UV (MeOH) λmax: 205 nm; IR νmax cm-1: 3421, 2932, 1645, 1454, 1376, 1161, 1076; 1H NMR and
13C
NMR
(C5D5N) data are shown in Table 1 and 2; HRESIMS m/z: 519.2921 [M+Na]+ (C27H44NaO8, calcd. 519.2915). 4.4 Acid hydrolysis of compounds 4, 8, 10 Compounds 4, 8, 10 (each 1.5 mg) were each reflux with 1 N HCl (2.0 mL) for 2 h at 90 ℃. After extracting with EtOAc (3 × 2 mL), the aqueous layers were neutralized with 1 N KOH and subjected to TLC analysis using the similar method as reference [19]. Identifications of digitalose from 4, 8 and glucose from 8, 10 presented in the aqueous were carried out by comparing their Rf values with those of authentic samples, Rf 0.59 (digitalose) and 0.47 (glucose). 4.5 Cell cytotoxicity assay
4.5.1 Cell lines and cell culture Liver cancer cell lines (HepG2) and human hepatocytes (LO2) were obtained from the cell bank of Chinese Academy of Sciences and cultured in RPMI medium 1640 (KeyGEN) with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), at 37 °C in an atmosphere of 5% CO2, 95% air and ˃ 95% humidity.
4.5.2 MTT assay Cytotoxicities of the compounds were assessed by MTT assay. The cells were seeded in 96-well plate at a density of 8 × 103 cells/well and allowed to adhere overnight incubation before addition of the compounds. Each cell line was exposed to the tested compounds at various concentrations in triplicate for 48 h, with cisplatin as a positive control. Followed by addition of MTT (5 mg/mL in PBS) for 4 h. After adding 150
μL DMSO, the OD value was measured at a wave length of 490nm. 4.5.3 Cell apoptosis analysis The Annexin V-FITC/PI kit was used to analysis the cell apoptosis according to the manufacturer instructions (KeyGEN). HepG2 cells were seeded into 24-well plate at a density of 1 × 105 cells/well. After overnight incubation, compound 10 (3, 6, 12 μM) was added to wells and stayed for 48 h before being analyzed by flow cytometry within 1 h (MACSQuant Analyzer 10, Miltenyi, Germany). 4.6 Statistical analysis Experimental data were expressed as mean ± S.D. triplicate. The results of bioactive assays were analyzed by program GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The statistical significance of the differences between the control and treatment groups were carried out with one-way analysis of variance (ANOVA) followed by the Newman-Keuls test.
Acknowledgements This work was supported by the Youth Science Fund Project of National Natural
Science Foundation of China (Grant No. 81703383), and the Natural Science Foundation of Jiangsu Province (Grant No. BK20170742).
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[17] X.Y. Li H.X. Sun Y.P. Ye F.Y. Chen J. Tu Y.J. Pana Four new immunomodulating steroidal glycosides from the stems of
Stephanotis mucronate Steroids 71 2006 683 690 18. X. Zhou, L.F. Zhou, B. Yang, H.J. Zhao, Y.Q. Wang, X.Y. Li, Y.P. Ye, F.Y. Chen, The loss of a sugar chain at C-3 position enhances Stemucronatoside K-induced apoptosis, cell cycle arrest and hedgehog pathway inhibition in HT-29 cells, Chem. Biodivers. 13 (2016) 1484–1492.
[18] X. Zhou L.F. Zhou B. Yang H.J. Zhao Y.Q. Wang X.Y. Li Y.P. Ye F.Y. Chen The loss of a sugar chain at C-3 position enhances Stemucronatoside K-induced apoptosis, cell cycle arrest and hedgehog pathway inhibition in HT-29 cells Chem. Biodivers. 13 2016 1484 1492 19. R. Xue, N. Han, C. Ye, H.B. Wang, J. Yin, Cardenolide glycosides from root of Streptocaulon juventas, Phytochemistry 88 (2013) 105–111.
[19] R. Xue N. Han C. Ye H.B. Wang J. Yin Cardenolide glycosides from root of Streptocaulon juventas Phytochemistry 88 2013 105 111
Fig. 1. Chemical structures of compounds (1–13) from Streptocaulon juventas. Fig. 2. Key 1H-1H COSY, HMBC and selected ROESY correlations of compounds 4, 8 and 10. Fig. 3. Compound 10 induces apoptosis in HepG2 cells. (A) Flow cytometric analysis of annexin V-FITC/PI staining in HepG2 cells treated with compound 10 (0, 3, 6, 12 μM) for 48 h. (B) Histogram for early plus late apoptosis rate in HepG2 cells. ***p < 0.001 compared with control group in three separate experiments.
Table 1. 1H and 13C NMR data for aglycone of compounds 4 (CD3OD, J in Hz), 8 (C5D5N, J in Hz) and 10 (C5D5N, J in Hz). Position
4 δC
1
8 a
38.56
δH
b
1.07 (m)
δC
a
38.23
1.86 (m) 2
30.75
1.58 (m)
δH
10 b
0.98 (m)
δC
a
31.23
1.76 (m) 33.11
1.47 (m)
δH b 1.30 (m) 1.75 (m)
27.57
1.57 (br s)
1.90 (m)
1.79 (br t, 11.4),
2.02 (m)
3
80.16
3.54 (m)
72.05
3.77 (m)
78.85
3.98 (m)
4
39.75
2.25 (br t, 13.0)
43.98
2.61 (br s),
30.86
1.53 (m)
2.40 (br d, 13.2)
2.62 (br s)
1.78 (m)
5
141.94
/
142.31
/
35.64
2.06 (m)
6
122.74
5.37 (br s)
121.85
5.38 (br s)
27.27
1.21 (br d, 11.4) 1.87 (m)
7
33.23
1.75 (m)
32.83
1.95 (m)
1.90 (m)
24.95
2.05 (m)
1.47 (m), 1.60 (br s)
8
33.18
1.45 (m)
32.79
1.61 (m)
35.84
2.12 (m)
9
51.45
0.94 (m)
50.49
1.09 (m)
40.70
1.41 (br d, 12.0)
10
37.87
/
37.36
/
37.38
/
11
21.71
1.43 (m)
21.45
1.40 (m)
22.49
1.25 (br d, 12.0)
1.63 (m) 12
38.00
1.54 (m)
1.70 (m)
38.98
2.0 (m)
1.43 (br d, 12.0)
2.18 (m)
42.58
2.57 (m)
1.27 (br d, 12.0) 1.96 (br d, 12.0)
13
46.99
/
46.40
/
47.27
/
14
52.54
1.76 (m)
51.80
2.13 (m)
155.50
/
15
24.48
1.15 (m)
24.55
1.12 (m)
118.09
5.32 (br s)
34.22
2.77 (br s)
1.70 (m) 16
32.40
1.74 (br s)
1.56 (m)
31.83
2.01 (m)
1.69 (br d, 12.6), 2.20 (m)
2.88 (br s)
17
86.99
/
87.03
/
56.18
2.30 (m)
18
14.79
0.75 (s)
14.48
0.72 (s)
18.67
1.12 (s)
19
19.82
1.02 (s)
20.16
1.05 (s)
24.05
0.86 (s)
20
72.85
3.77 (q, 6.0)
82. 97
4.08 (q, 6.0)
73.46
4.27 (dd, 2.4, 8.1)
21
18.74
1.16 (d, 6.6)
19.40
1.63 (d, 6.0)
67.65
3.94 (dd, 8.4, 10.2) 4.16 (dd, 2.4, 10.8)
a Measured
at 150 MHz
b Measured
at 600 MHz
Table 2. 1H and 13C NMR data for sugars of compounds 4 (CD3OD, J in Hz), 8 (C5D5N, J in Hz) and 10 (C5D5N, J in Hz). Position
4 δC a
8 δH b
δC a
δH b
10 δC a
δH b
β -D-Digitalose
β-D-Digitalose
β-D-Glucose
1'
102.94
4.33 (d, 7.8)
104.32
4.88 (d, 7.8)
103.65
4.96 (d, 7.8)
2'
71.43
3.55 (m)
78.87
4.55 (t, 9.0)
74.97
4.37 (m)
3'
85.73
3.24 (m)
85.24
3.60 (m)
75.87
4.09 (t, 7.8)
4'
75.91
4.05 (br s)
68.58
4.06 (br s)
72.33
4.30 (m)
5'
71.69
3.57 (m)
71.42
3.75 (m)
79.22
4.28 (m)
6'
17.41
1.27 (d, 3.0)
17.92
1.58 (d, 6.0)
63.40
4.43 (dd, 5.0, 11.7) 4.58 (br d, 11.4)
-OCH3
58.58
3.49(s)
57.30
3.57 (s)
β -D-Digitalose
β-D-Glucose
1''
105.10
4.51 (d, 7.8)
106.42
5.10 (d, 7.2)
2''
71.95
3.58 (m)
76.45
4.10 (m)
3''
84.35
3.15 (m)
78.92
4.12 (m)
4''
68.80
3.80 (br s)
71.76
4.12 (m)
5''
72.27
3.62 (m)
77.37
3.77 (m)
6''
16.81
1.26 (d, 3.0)
69.86
4.36 (dd, 6, 11.1) 4.68 (br d, 10.8)
-OCH3
57.18
3.44 (s)
β-D-Glucose 1'''
105.53
5.00 (d, 7.8)
2'''
75.95
4.14 (m)
3'''
78.27
4.20 (t, 9.0)
4'''
71.71
4.28 (t, 9.0)
5'''
78.79
3.90 (m)
6'''
62.96
4.39 (dd, 4.2, 12.3) 4.47 (br d, 12.0)
a Measured
at 150 MHz
b Measured
at 600 MHz
Table 3. Cytotoxicity of compounds 1–13 on HepG2 and LO2 cells. Cytotoxicity IC50 a (μM) NO. HepG2
LO2
SI b
1
72.5 ± 1.0
112.8 ± 1.0
1.6
2
73.0 ± 1.0
108.0 ± 1.0
1.5
3
23.8 ± 1.1
13.6 ± 1.0
0.6
4
>100
N.D. c
N.D. c
5
42.6 ± 1.1
35.9 ± 1.1
0.8
6
>100
N.D. c
N.D. c
7
>100
N.D. c
N.D. c
8
>100
N.D. c
N.D. c
9
>100
N.D. c
N.D. c
10
11.7 ± 1.1
40.4 ± 1.1
3.5
11
48.9 ± 1.1
36.3 ± 1.1
0.7
12
27.9 ± 1.1
34.7 ± 1.1
1.2
13
>100
N.D. c
N.D. c
Cisplatin d
21.9 ± 0.9
27.5 ± 1.1
1.3
50:
a IC
b SI:
50% inhibition concentration, the values based on quintuple points.
Selective Index.
c N.D.:
Not determined.
d Reference
compound.
S0039-128X(18)30188-0 https://doi.org/10.1016/j.steroids.2018.09.016 STE 8323
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
24 May 2018 10 September 2018 29 September 2018
Please cite this article as: Zhu, W., Su, S., Xu, Y., Xie, Z., Bai, Y., Liu, W., Abe, M., Akihisa, T., Feng, F., Zhang, J., C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2, Steroids (2018), doi: https:// doi.org/10.1016/j.steroids.2018.09.016
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C21 steroids from Streptocaulon juventas (Lour) Merr. induce apoptosis in HepG2
Wanfang Zhua, Shengzhi Sua, Yunhui Xud, Zijian Xied, Yidan Baie, Wenyuan Liue, Masahiko Abef, Toshihiro Akihisaf, Feng Feng, a,b,c,*, Jie Zhanga,b,*
aSchool
of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing
211198, PR China
bKey
Laboratory of Biomedical Functional Materials, China Pharmaceutical University,
Nanjing 211198, PR China
cJiangsu
Food and Pharmaceutical Science College, Huaian, Jiangsu, 223003, China
dMarshall
University, Marshall Institute for Interdisciplinary Research, Weisberg Engn Complex,
RM 4117,1628 Third Ave, Huntington, WV, 25703, USA
eDepartment
of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing
210009, China
fResearch
Institute for Science and Technology, Tokyo University of Science, 2641
Yamazaki, Noda, Chiba, 278-8510, Japan
*Corresponding
author.
Graphical abstract
ga1
Highlights
Isolation and identification three new and ten known C21 steroids from Streptocaulon juventas. Cytotoxicity activities against HepG2 and LO2 cells of isolated compounds. New compound 10 could induce apoptosis of HepG2 cells.
Abstract Three new C21 steroids, i.e., (3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-triol-3-O-β-Ddigitalopyranosyl-(1→4)-β-D-digitalopyranoside (4), (3β,17α,20S)- pregn-5(6)-ene-3, 17, 20-triol-20-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-Ddigital- opyranoside (8), (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-D-glucopyranoside (10), along with ten known C21 steroids were isolated from Streptocaulon juventas. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. All the isolated compounds were screened for their in vitro cytotoxicity against human liver cancer cells (HepG2) and the structure-activity relationships were also
analyzed. Moreover, compounds 1–3, 5, 10–12, which displayed cytotoxic activities in HepG2 cells, were tested for the selective index (SI) by the ratio of cytotoxic effect on human hepatocytes (LO2) to that on HepG2. As a result, new compound 10 exhibited a good inhibitory activity against HepG2 with IC50 value 11.7 μM as well as high SI value 3.5. Furthermore, compound 10 could induce HepG2 cells apoptosis by flow cytometry.
Keywords Streptocaulon juventas (Lour) Merr.
C21 steroid HepG2
Apoptosis
1 Introduction Apoptosis is a polygenic control process maintaining the stability of the environment in the body and the inhibition of this vital process may lead to the cancer occurrence [1,2]. Accumulated evidences have supported that anticancer drugs exert their
cytotoxic effects mainly by inducing tumor cell apoptosis [3]. C21 steroids is one species widely found in the plants of Asclepiadaceae family, which shows the possession of extensive pharmacological effects, such as anti-tumor effect [4]. Extensive evidences confirm that many C21 steroidal glycosides resist cancer by inducing apoptosis [5]. Streptocaulon juventas is a perennial herb belonging to the Asclepiadaceace family, which mainly distribute in the southern part of China, and it has long been used in China as a folk medicine for the treatment of dysentery, diarrhea, stomachache, fever, chronic nephritis and traumatic injury [6]. Moreover, traditional Chinese medicine takes the view that S. juventas could clear heat and remove toxin, which is believed to be related with the antitumor activity in traditional Chinese medicine [7]. In the present study, we describe the investigation on the C21 steroids from S. juventas as well as the cytotoxic effects of the isolated compounds against human liver cancer cells (HepG2), the active compounds (IC50 < 100) were also tested for the cytotoxicity activities against human hepatocytes (LO2) to acquire the selective index (SI). Furthermore, flow cytometry proved that new compound 10 could induce apoptosis of HepG2 cells. 2 Results and discussion 2.1 Structural elucidation of isolated compounds The n-BuOH fraction of S. juventas was subjected to column chromatography including silica gel, ODS and semipreparative RP-18 HPLC which led to the isolation of thirteen C21 steroids. They were three new steroids i.e., (3β,17α,20S)-pregn-5(6)ene-3, 17, 20-triol-3-O-β-D-digitalopyranosyl-(1→4)-β-D- digitalopyranoside (4),
(3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-triol-20-O-β-D- glucopyranosyl-(1→6)-β-Dglucopyranosyl-(1→2)-β-D-digitalopyranoside (8), and (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-D-glucopyranoside (10), and ten known steroids, i.e., pregnenediol (1) [8], hemindicusin (2) [9], periplocoside L (3) [10], periseoside C (5) [11], biondianoside D (6) [12], ∆5 pregnene-3β, 16α S-triol 20-O-β-D- glucopyranosyl(1→6)- β-D -glucopyranosyl-(1→2)- β-D -digitalopyranoside (7) [12], (Z)-3βhydroxy-5,17(20)-pregnadiene (9) [13], basikoside A (11) [14], carumbelloside II (12) [15], and stelmatocryptonoside D (13) [16]. (Figure 1 fig1). Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Compound 4 was obtained as white powder. Its molecular formula, C35H58O11, was deduced from the peak at m/z 677.3869 [M+Na]+ in the HRESIMS. For the aglycone part, The 1D NMR and HSQC spectra of compound 4 showed the presence of 3 methyl groups [δH 0.75 (3H, s), δC 14.79; δH 1.02 (3H, s), δC 19.82 and δH 1.16 (3H, d, J = 6.6 Hz), δC 18.74], 8 methylenes, 6 methines [including one olefinic methine at δH 5.37 (1H, br s), δC 122.74 together with two oxygenated methines at δH 3.54 (1H, m), δC 80.16 and δH 3.77 (1H, q, J = 6.0 Hz), δC 72.85], and 4 quaternary carbons [including an olefinic carbon at δC 141.94 and an oxygenated carbon at δC 86.99] (Table 1 tbl1). Combined analysis of spectral features in 1H-1H-COSY and HMBC (Figure 2 fig2), the aglycone of 4 was identified as same as that of compound 3. Furthermore, 1H NMR spectrum of 4 revealed signals for two methyl groups [δH 1.27 (3H, d, J = 3.0 Hz) and δH 1.26 (3H, d, J = 3.0 Hz)], two methoxy groups of deoxysugars [δH 3.49 (3H, s) and δH 3.44, (3H, s)] and two anomeric protons [δH 4.33
(1H, d, J = 7.8 Hz) and δH 4.51 (1H, d, J = 7.8 Hz)] suggesting the existence of two βD-digitalose which were confirmed by their 1D NMR and 2D NMR data and
comparison with the literature [10]. Their location and connection pattern were demonstrated by HMBC spectrum, in which obvious correlations from H-1'' to C-4' (δC 75.91) and H-1' to C-3 (δC 80.16) were observed. The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 3.77 (1H, q, J = 6.0 Hz)] to H-18 [δH 0.75 (3H, s)] indicating the configuration of C-17 is β as well as the correlations from H-21 [δH 1.16 (3H, d, J = 6.6 Hz)] to H-15β [δH 1.70 (1H, m)] and H-20 [δH 3.77 (1H, q, J = 6.0 Hz)] to H-16β [δH 2.01 (1H, m)] indicating that C-20 was S configuration [17]. Hence, compound 4 was determined to be (3β,17α,20S)- pregn- 5(6)-ene-3, 17, 20triol-3-O-β-D-digitalop- yranosyl-(1→4)-β-D-digitalopyranoside. Compound 8 was isolated as amorphous colorless crystal. The molecular formula was determined to be C40H66O17 by its HRESIMS (m/z: 841.4206 [M+Na]+). The 1H NMR,
13C
NMR and HSQC spectra could be assigned to the aglycone moiety of 21
carbon signals including, three methyl groups, eight methylenes, five methines, three quaternary and two olefinic carbons (Table 1). The NMR data of compound 8 indicated that the aglycone of compound 8 was similar to that of compound 7, except for the hydroxy group at C-16 changed to C-17, which was confirmed by the correlations of H-18 [δH 0.72 (3H, s)] with both C-16 (δC 31.83) and C-17 (δC 87.03) in the HMBC spectrum (Figure 2) The sugar moiety of 8 were determined to be β-Ddigitalose and β-D-glucose by detailed NMR analysis with the anomeric proton resonances at [δH 4.88 (1H, d, J = 7.8 Hz), δH 5.10 (1H, d, J = 7.2 Hz) and δH 5.00 (1H, d, J = 7.8 Hz)], respectively. The HMBC correlations from H-1''' to C-6'' (δC 69.86), H-1'' to C-2' (δC 78.87) and H-1' to C-20 (C 82.97) suggested that the
connections of the three sugars were through (1→6, 1→2) linkages and the sugar chain was located at C-20 of the aglycone. The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 4.08 (1H, q, J = 6.0 Hz)] to H-18 [δH 0.72 (3H, s)] indicating the configuration of C-17 is β. And the correlations from H-21 [δH 1.63 (3H, d, J = 6.0 Hz)] to H-15β [δH 1.74 (1H, m)] and H-20 [δH 4.08 (1H, q, J = 6.0 Hz)] to H-16β [δH 2.20 (1H, m)] revealing that C-20 was S configuration [17]. Thus, the structure of compound 8 was determined as (3β,17α,20S)-pregn-5(6)-ene-3, 17, 20-
triol-20-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-D-
digitalopyranoside. Compound 10, obtained as white powder, had a molecular formula C27H44O8, as established by HRESIMS. The 1H NMR, 13C NMR and HSQC spectrum showed the presence of two methyl groups, one double bond, and a β-D-glucose (Tables 1 and 2 tbl2). Comparison of the NMR spectroscopic data of 10 with those of 4 revealed that one of the methyl groups which were always located in Me-18, -19, -21 was substituted by a hydroxy group, and the substituent position was determined to be C21, established by the correlations of H-17 [δH 2.30 (1H, m)] with both C-20 at δC and C-21 at δC 67.85. nThe location of the double bond was confirmed by the HMBC correlations of H-15 [δH 5.32 (1H, br s)] with both C-16 (δC 34.22) and C-17 (δC 56.18) and the sugar chain was located at C-3 of the aglycone out of the correlation of H-1' [δH 4.96 (1H, d, J = 7.8 Hz)] to C-3 (δC 78.85). The ROESY spectrum (Figure 2) showed correlations from H-20 [δH 4.27 (1H, dd, J = 2.4, 8.1 Hz)] to H-18 [δH 1.12 (1H, s)] indicating the configuration of C-17 is β. And the correlations from H-20 to H-12β [δH 1.96 (1H, br d, J = 12 Hz)] demonstrating that C-20 was R configuration [17]. On the basis of the above analysis, the structure of
compound 10 was identified as (3β, 20R)-pregn-14(15)-ene-3, 20, 21-triol-3-O-β-Dglucopyrano- side. 2.2 Cell cytotoxicity assay
2.2.1 MTT assay The cytotoxic activities of compounds 1–13 on HepG2 and LO2 cell lines were evaluated by MTT assays with cisplatin as the positive control (Table 3 tbl3). Compounds 3, 10, 12 exhibited potent cytotoxicities against HepG2 cells with IC50 values of 23.8, 11.7, 27.9 μM respectively. Compounds 1, 2, 5, 11 showed moderate inhibitory effects against HepG2 cells (IC50 42.6–73.0 μM) and other compounds with IC50 over 100 μM were regarded as inactive. The preliminary structure-activity relationships (SARs) suggested that hydroxy group substituent of compounds would strengthen the cytotoxic activity against HepG2 cells (1 > 9). When the number of substituted hydroxy group was the same, the position of substituent could also affect the activity (3 > 2). The sugar moiety is another important factor attributing to the activity [18]. The loss of sugar chain could enhance the cytotoxicity to HepG2 cells (3 > 4, 5 > 6). Furthermore, compounds 1–3, 5, 10–12, which displayed cytotoxic activities in HepG2 cells were tested for the selective index (SI) by the ratio of cytotoxic effect on normal human hepatocytes (LO2) to that on liver cancer cell line (HepG2). As a result, new compound 10 showed the highest selectivity with SI value of 3.5. At the same time, compound 10 also displayed the most significant activity against HepG2 cells (IC50 11.7 μM), while the SI and IC50 values of cisplatin were 1.3, 21.9,
indicating that compound 10 displayed the superior selectivity and cytotoxicity than the reference cisplatin. Therefore, compound 10 was selected for further study on HepG2 cell line. 2.2.2 Compound 10 induces apoptosis in HepG2 Cells To study whether the growth inhibition was related to cell apoptosis, HepG2 cells apoptosis mediated by compound 10 was determined by flow cytometry. Annexin V/PI staining was employed to discriminate among normal cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+), and necrosis cells (Annexin V-/PI+). As shown in Figure 3 fig3, compound 10 could increase the number of apoptosis cells in a concentration-dependent manner. The percentage of apoptotic cells was 38.4, 44.8, 51.7% respectively after adding compound 10 (3, 6, 12 μM), exhibiting significant differences with control (0.5% apoptotic cells). These results suggested that the growth inhibitory effect of compound 10 was partly related with the enhanced apoptosis in HepG2 cells. 3 Conclusion The present study showed that new compound 10 from S. juventas could significantly inhibit HepG2 cell growth proliferation via induction of apoptosis and showed high selectivity between HepG2 and LO2 in the meantime. These results demonstrated that compound 10 had an excellent potential to become lead for the development of safe drug for the treatment of liver cancer. 4 Experimental section
4.1 General methods
Optical rotations were measured with a Jasco P1020 polarimeter. IR spectra were acquired on a Bruker Tensor 27 spectrometer in KBr discs. A UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan) was applied to UV spectrum measurement. HR-ESI-MS spectra was obtained using a Bruker Micro TOF model mass spectrometer. Nuclear magnetic resonance (NMR) spectrum were measured on Bruker AV-300 or AV-500/600 spectrometers. Thin-layer chromatography (TLC) analyses were carried out using silica gel GF254 plates (EMD Millipore Corporation, Germany), and spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. Column chromatography separations were performed on silica gel (100-200 mesh and 200-300 mesh, Merck & Co., Inc.), and on ODS gel (50 μm, Fuji Silysia Chemical Ltd., Aichi, Japan). Semi-preparative HPLC was carried out on YMC-PACK ODS-AQ, and Shimadzu SCL-10A UV detector. 4.2 Plant material S. juventas was collected in November 2016, from Xishuangbanna, Yunnan Province, China and identified by Mr. Feng feng (China Pharmaceutical University). A voucher specimen (No. 201611) was deposited in the Department of Natural Medicinal Chemistry of China Pharmaceutical University, Nanjing, China. 4.3 Extraction and Isolation S. juventas (20.0 kg) were extracted with EtOH 95% (v/v) under reflux for three times (3 × 70.0 L, 2 h each). The extract was concentrated under reduced pressure to afford a crude residue (2.0 kg) which was then suspended in H2O (5.0 L) and partitioned with EtOAc (3 × 5.0 L) and n-BuOH (3 × 5.0 L) respectively. The concentrated n-BuOH soluble (250.0 g) was subjected to D101 CC and eluted with
MeOH–H2O (9:1→0:1 v/v) to afford six fractions, Frs. 1–6. Fr. 4 (76.7 g) from the MeOH–H2O (1:1) eluate, was divided into six parts (Fr. 4A–4F) by silica gel (100– 200 mesh) column chromatography (CC) eluted with gradient CHCl3–MeOH (1:0→0:1 v/v). Among them, Fr. 4B (11.5 g) was purified by column chromatography over reverse-phase C18 silica gel with MeOH–H2O (3:7→8:2 v/v) and silica gel (200– 300 mesh) using CHCl3–MeOH (55:1→15:1 v/v) as eluent, yielding compounds 1 (102.0 mg), 2 (3.0 mg), 3 (3.7 mg), 4 (4.0 mg), 11 (7.2 mg), 12 (18.0 mg). Fr. 4C (17.5 g) was subjected to reverse-phase C18 silica gel eluted with a gradient of MeOH–H2O (3:7→1:1, v/v) to afford compounds 5 (11.2 mg), 9 (17.3 mg), and 10 (18.3 mg). Fr. 4D (20.7 g) was applied to reverse-phase C18 silica gel eluting with a stepwise gradient of MeOH–H2O (4:6→6:4, v/v) to give Fr. 4D1–Fr. 4D3. Fr. 4D2 was further purified by repeated silica gel column chromatography to give compounds 6 (10.7 mg) and 13 (8.9 mg). Fr. 4D3 was separated by silica gel eluting with CHCl3– MeOH repeatedly, then subjected to preparative HPLC using MeOH–H2O (6:4, v/v) as the eluent to yield compounds 7 (21.7 mg, tR 25.0 min) and 8 (56.2 mg, tR 30.0 min). 4.3.1 Compound 4 25
Compound 4: white powder; [α]D -26.0 (c 0.342, MeOH); UV (MeOH) λmax: 204 nm; IR νmax cm-1: 3414, 2933, 1646, 1452, 1382, 1109, 1080; 1H NMR and
13C
NMR
(CD3OD) data are shown in Table 1 and 2; HRESIMS m/z: 677.3869 [M+Na]+ (C35H58NaO11, calcd. 677.3865). 4.3.2 Compound 8 25
Compound 8: colorless crystal; [α]D -36.0 (c 0.231, MeOH); UV (MeOH) λmax: 203
nm; IR νmax cm-1: 3383, 2934, 1648, 1379, 1165, 1073; 1H NMR and
13C
NMR
(C5D5N) data are shown in Table 1 and 2; HRESIMS m/z: 841.4206 [M+Na]+ (C40H66NaO17, calcd. 841.4196). 4.3.3 Compound 10 25
Compound 10: white powder; [α]D -6.3 (c 0.123, MeOH); UV (MeOH) λmax: 205 nm; IR νmax cm-1: 3421, 2932, 1645, 1454, 1376, 1161, 1076; 1H NMR and
13C
NMR
(C5D5N) data are shown in Table 1 and 2; HRESIMS m/z: 519.2921 [M+Na]+ (C27H44NaO8, calcd. 519.2915). 4.4 Acid hydrolysis of compounds 4, 8, 10 Compounds 4, 8, 10 (each 1.5 mg) were each reflux with 1 N HCl (2.0 mL) for 2 h at 90 ℃. After extracting with EtOAc (3 × 2 mL), the aqueous layers were neutralized with 1 N KOH and subjected to TLC analysis using the similar method as reference [19]. Identifications of digitalose from 4, 8 and glucose from 8, 10 presented in the aqueous were carried out by comparing their Rf values with those of authentic samples, Rf 0.59 (digitalose) and 0.47 (glucose). 4.5 Cell cytotoxicity assay
4.5.1 Cell lines and cell culture Liver cancer cell lines (HepG2) and human hepatocytes (LO2) were obtained from the cell bank of Chinese Academy of Sciences and cultured in RPMI medium 1640 (KeyGEN) with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), at 37 °C in an atmosphere of 5% CO2, 95% air and ˃ 95% humidity.
4.5.2 MTT assay Cytotoxicities of the compounds were assessed by MTT assay. The cells were seeded in 96-well plate at a density of 8 × 103 cells/well and allowed to adhere overnight incubation before addition of the compounds. Each cell line was exposed to the tested compounds at various concentrations in triplicate for 48 h, with cisplatin as a positive control. Followed by addition of MTT (5 mg/mL in PBS) for 4 h. After adding 150
μL DMSO, the OD value was measured at a wave length of 490nm. 4.5.3 Cell apoptosis analysis The Annexin V-FITC/PI kit was used to analysis the cell apoptosis according to the manufacturer instructions (KeyGEN). HepG2 cells were seeded into 24-well plate at a density of 1 × 105 cells/well. After overnight incubation, compound 10 (3, 6, 12 μM) was added to wells and stayed for 48 h before being analyzed by flow cytometry within 1 h (MACSQuant Analyzer 10, Miltenyi, Germany). 4.6 Statistical analysis Experimental data were expressed as mean ± S.D. triplicate. The results of bioactive assays were analyzed by program GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The statistical significance of the differences between the control and treatment groups were carried out with one-way analysis of variance (ANOVA) followed by the Newman-Keuls test.
Acknowledgements This work was supported by the Youth Science Fund Project of National Natural
Science Foundation of China (Grant No. 81703383), and the Natural Science Foundation of Jiangsu Province (Grant No. BK20170742).
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Fig. 1. Chemical structures of compounds (1–13) from Streptocaulon juventas. Fig. 2. Key 1H-1H COSY, HMBC and selected ROESY correlations of compounds 4, 8 and 10. Fig. 3. Compound 10 induces apoptosis in HepG2 cells. (A) Flow cytometric analysis of annexin V-FITC/PI staining in HepG2 cells treated with compound 10 (0, 3, 6, 12 μM) for 48 h. (B) Histogram for early plus late apoptosis rate in HepG2 cells. ***p < 0.001 compared with control group in three separate experiments.
Table 1. 1H and 13C NMR data for aglycone of compounds 4 (CD3OD, J in Hz), 8 (C5D5N, J in Hz) and 10 (C5D5N, J in Hz). Position
4 δC
1
8 a
38.56
δH
b
1.07 (m)
δC
a
38.23
1.86 (m) 2
30.75
1.58 (m)
δH
10 b
0.98 (m)
δC
a
31.23
1.76 (m) 33.11
1.47 (m)
δH b 1.30 (m) 1.75 (m)
27.57
1.57 (br s)
1.90 (m)
1.79 (br t, 11.4),
2.02 (m)
3
80.16
3.54 (m)
72.05
3.77 (m)
78.85
3.98 (m)
4
39.75
2.25 (br t, 13.0)
43.98
2.61 (br s),
30.86
1.53 (m)
2.40 (br d, 13.2)
2.62 (br s)
1.78 (m)
5
141.94
/
142.31
/
35.64
2.06 (m)
6
122.74
5.37 (br s)
121.85
5.38 (br s)
27.27
1.21 (br d, 11.4) 1.87 (m)
7
33.23
1.75 (m)
32.83
1.95 (m)
1.90 (m)
24.95
2.05 (m)
1.47 (m), 1.60 (br s)
8
33.18
1.45 (m)
32.79
1.61 (m)
35.84
2.12 (m)
9
51.45
0.94 (m)
50.49
1.09 (m)
40.70
1.41 (br d, 12.0)
10
37.87
/
37.36
/
37.38
/
11
21.71
1.43 (m)
21.45
1.40 (m)
22.49
1.25 (br d, 12.0)
1.63 (m) 12
38.00
1.54 (m)
1.70 (m)
38.98
2.0 (m)
1.43 (br d, 12.0)
2.18 (m)
42.58
2.57 (m)
1.27 (br d, 12.0) 1.96 (br d, 12.0)
13
46.99
/
46.40
/
47.27
/
14
52.54
1.76 (m)
51.80
2.13 (m)
155.50
/
15
24.48
1.15 (m)
24.55
1.12 (m)
118.09
5.32 (br s)
34.22
2.77 (br s)
1.70 (m) 16
32.40
1.74 (br s)
1.56 (m)
31.83
2.01 (m)
1.69 (br d, 12.6), 2.20 (m)
2.88 (br s)
17
86.99
/
87.03
/
56.18
2.30 (m)
18
14.79
0.75 (s)
14.48
0.72 (s)
18.67
1.12 (s)
19
19.82
1.02 (s)
20.16
1.05 (s)
24.05
0.86 (s)
20
72.85
3.77 (q, 6.0)
82. 97
4.08 (q, 6.0)
73.46
4.27 (dd, 2.4, 8.1)
21
18.74
1.16 (d, 6.6)
19.40
1.63 (d, 6.0)
67.65
3.94 (dd, 8.4, 10.2) 4.16 (dd, 2.4, 10.8)
a Measured
at 150 MHz
b Measured
at 600 MHz
Table 2. 1H and 13C NMR data for sugars of compounds 4 (CD3OD, J in Hz), 8 (C5D5N, J in Hz) and 10 (C5D5N, J in Hz). Position
4 δC a
8 δH b
δC a
δH b
10 δC a
δH b
β -D-Digitalose
β-D-Digitalose
β-D-Glucose
1'
102.94
4.33 (d, 7.8)
104.32
4.88 (d, 7.8)
103.65
4.96 (d, 7.8)
2'
71.43
3.55 (m)
78.87
4.55 (t, 9.0)
74.97
4.37 (m)
3'
85.73
3.24 (m)
85.24
3.60 (m)
75.87
4.09 (t, 7.8)
4'
75.91
4.05 (br s)
68.58
4.06 (br s)
72.33
4.30 (m)
5'
71.69
3.57 (m)
71.42
3.75 (m)
79.22
4.28 (m)
6'
17.41
1.27 (d, 3.0)
17.92
1.58 (d, 6.0)
63.40
4.43 (dd, 5.0, 11.7) 4.58 (br d, 11.4)
-OCH3
58.58
3.49(s)
57.30
3.57 (s)
β -D-Digitalose
β-D-Glucose
1''
105.10
4.51 (d, 7.8)
106.42
5.10 (d, 7.2)
2''
71.95
3.58 (m)
76.45
4.10 (m)
3''
84.35
3.15 (m)
78.92
4.12 (m)
4''
68.80
3.80 (br s)
71.76
4.12 (m)
5''
72.27
3.62 (m)
77.37
3.77 (m)
6''
16.81
1.26 (d, 3.0)
69.86
4.36 (dd, 6, 11.1) 4.68 (br d, 10.8)
-OCH3
57.18
3.44 (s)
β-D-Glucose 1'''
105.53
5.00 (d, 7.8)
2'''
75.95
4.14 (m)
3'''
78.27
4.20 (t, 9.0)
4'''
71.71
4.28 (t, 9.0)
5'''
78.79
3.90 (m)
6'''
62.96
4.39 (dd, 4.2, 12.3) 4.47 (br d, 12.0)
a Measured
at 150 MHz
b Measured
at 600 MHz
Table 3. Cytotoxicity of compounds 1–13 on HepG2 and LO2 cells. Cytotoxicity IC50 a (μM) NO. HepG2
LO2
SI b
1
72.5 ± 1.0
112.8 ± 1.0
1.6
2
73.0 ± 1.0
108.0 ± 1.0
1.5
3
23.8 ± 1.1
13.6 ± 1.0
0.6
4
>100
N.D. c
N.D. c
5
42.6 ± 1.1
35.9 ± 1.1
0.8
6
>100
N.D. c
N.D. c
7
>100
N.D. c
N.D. c
8
>100
N.D. c
N.D. c
9
>100
N.D. c
N.D. c
10
11.7 ± 1.1
40.4 ± 1.1
3.5
11
48.9 ± 1.1
36.3 ± 1.1
0.7
12
27.9 ± 1.1
34.7 ± 1.1
1.2
13
>100
N.D. c
N.D. c
Cisplatin d
21.9 ± 0.9
27.5 ± 1.1
1.3
50:
a IC
b SI:
50% inhibition concentration, the values based on quintuple points.
Selective Index.
c N.D.:
Not determined.
d Reference
compound.