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Epigynumgenane-type pregnane glycosides from Epigynum cochinchinensis and their immunosuppressive activity
Phytochemistry 168 (2019) 112127
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Epigynumgenane-type pregnane glycosides from Epigynum cochinchinensis and their immunosuppressive activity
T
Zengyuan Wanga,1, Mengjun Jianga,1, Afsar Khanb, Shengbao Caia, Xiaonian Lic, Junqiu Liud, Guoyin Kaid, Tianrui Zhaoa, Guiguang Chenga,∗, Jianxin Caoa,∗∗ a
Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, 650500, People's Republic of China Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, 22060, Pakistan c State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, People's Republic of China d College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, 310053, People's Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Epigynum cochinchinensis (Apocynaceae) Epigycoside E Pregnane glycosides Immunosuppressive Cytokines
Five undescribed C21 pregnane glycosides, epigycosides D−H, together with four known analogues, two lignans, and a flavonoid have been isolated from the stems of Epigynum cochinchinensis. The structures of pregnane glycosides were elucidated using spectroscopic techniques and acid hydrolysis. The in vitro immunological activities were assessed against Con A-stimulated proliferation of mice splenocytes. The C21 pregnane glycosides showed immunosuppressive activity in a concentration-dependent manner. Moreover, epigycoside E exhibited a potent immunosuppressive effect, and the IC50 value on Con A-stimulated mice splenocytes was 22.1 ± 6.4 μM. Epigycoside E also caused G0/G1 arrest, and inhibited TNF-α and IL-2 production.
1. Introduction Pregnanes are C21 steroidal compounds present in nature either in free state or as glycosides. In general, the sugar moiety is connected to the aglycone by an ether linkage, most frequently at C-20, C-3, or both (bisdesmosidic glycosides) (Deepak et al., 1997). Literature reveals that pregnane glycosides possess immunosuppressive (Ounaissia et al., 2016; Zhang et al., 2015), anti-inflammatory (Jeong et al., 2014), antioxidant (Ounaissia et al., 2016), anti-epileptic (Li et al., 2015), neuroprotective (Zhao et al., 2013), anti-microbial (Song et al., 2014), antihyperglycemic (Tsoukalas et al., 2016), cytotoxic (Liu et al., 2014; Zhang et al., 2013), antitumor (Wang et al., 2015), and slimming (Zhu et al., 2012) activities. Pregnane glycosides are an important class of specialised metabolites from medicinal plants in natural drugs, especially used as autoimmune disorder drugs. Clinical immunosuppressants, such as cortisone, rapamycin, and tacrolimus have limitations in the treatment of auto-immunological diseases with undesirable side effects including renal injury and liver toxicity. Hence, searching for natural immunosuppressants with high efficacy and low toxicity is urgent. Motivated by their efficacy, several immunosuppressive pregnane glycosides have been purified from
Cynanchum, Periploca, and Stephanotis plants. In our previous studies, some pregnane glycosides with potential immunosuppressive activity were isolated from Epigynum plants (Gao et al., 2017a, 2017b; Wan et al., 2017). These findings suggested that the exploration of pregnane glycosides is efficient to discover undescribed immunological substances. Epigynum cochinchinensis (Pierre) Mabb. (Apocynaceae) is a woody climber growing in the forest margins of Mainland Southeast Asia. Our previous phytochemical search on E. cochinchinensis led to the isolation of some pregnane glycosides (Shao et al., 2018a, 2018b; Wan et al., 2017). Among them, epigycoside A exhibited a significant inhibitory effect against Con-A induced proliferation of mice splenocytes. In our continuous investigation for structurally undescribed steroidal glycosides with bioactive potential, a further systematic phytochemical study on the stems of E. cochinchinensis was performed. As a result, five undescribed pregnane glycosides, epigycosides D−H (1–5), together with four known analogues (6–9), two lignans (10–11), and a flavonoid (12) were isolated (Fig. 1). Herein, we describe structural elucidation of the undescribed compounds based on 1D and 2D-NMR data, acid hydrolysis, enzymatic hydrolysis, and X-ray diffraction methods. Furthermore, the in vitro inhibitory activity of pregnane glycosides against the
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (G. Cheng), [email protected] (J. Cao). 1 The authors have contributed equally to this work. ∗∗
https://doi.org/10.1016/j.phytochem.2019.112127 Received 19 February 2019; Received in revised form 11 September 2019; Accepted 12 September 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
Phytochemistry 168 (2019) 112127
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Fig. 1. Chemical structures of compounds 1–12.
(δH 4.12 and 3.41) with C-17 (δC 91.3). The ROESY cross peaks of H-8 with H3-19 and H3-18 disclosed these groups as co-facial and β-oriented. The absence of ROESY correlations between H-8 and H-9/H-14/ H-17 established an α-orientation of these protons. Besides the aglycone unit, the NMR data also suggested a sugar moiety in 1, as indicated by an anomeric proton signal at δH 4.84 (br. s) and a secondary methyl signal at δH 1.26 (3H, d, J = 6.4 Hz, H3-6′) with corresponding carbon signals at δC 95.8 and 16.2, as well as four oxygenated methine signals at δC 69.4, 69.7, 70.9, and 71.3. Further analysis of the 1H–1H COSY correlations delineated a spin system of H-1′/H-2′/H-3′/H-4′/H-5′/H36′. Its 13C NMR resonances were assigned by the HSQC experiment and further confirmed with HSQC-TOCSY technique (Fig. S07). The monosaccharide unit in 1 was then identified as 6-deoxyhexose. The sugaraglycone O-linkage was determined by the HMBC correlations of δH 4.84 (1H, br. s, H-1′) with δC 77.5 (d, C-3) and of δH 3.63 (1H, m, H-3) with 95.8 (d, C-1′). The ambiguous coupling constants and ROESY correlations could not provide sufficient evidence for the relative configuration of the monosaccharide moiety. However, crystals were obtained in MeOH–H2O (4:1) mixture at 4 °C. The X-ray diffraction analysis was performed with Cu Kα radiation and confirmed the absolute configuration of 1, in which all the hydroxy groups at C-2′, C-3′, and C4′ of the sugar moiety were positioned axially (Fig. 3). Thus, the structure of 1 was established as 17-deoxy-epigynumgenane-20-one 3O-6′-deoxy-β-D-idopyranoside, and trivially named as epigycoside D. A molecular formula of C28H44O7 was assigned to epigycoside E (2) by HR-ESI-MS at m/z 515.2980 [M + Na]+, indicating one more CH2 group than that of 1. The NMR spectra of 2 were identical to those of 1 (Tables 1 and 2) apart from a methoxy group (δH 3.55; δC 60.3). The
proliferation of T lymphocytes is also discussed. 2. Results and discussion 2.1. Structure elucidation of compounds 1−5 Epigycoside D (1) was isolated as a white amorphous power and its molecular formula was determined as C27H42O7 based on the sodium adduct ion at m/z 501.2825 [M + Na]+ (calcd for 501.2823) in its positive HR-ESI-MS. In the 1H NMR spectrum of 1, three singlet signals at δH 0.91 (3H, s, H3-18), 0.94 (3H, s, H3-19), and 2.12 (3H, s, H3-21), one doublet signal for methyl protons at δH 1.26 (3H, d, J = 6.4 Hz, H36′), and one olefinic proton signal at δH 5.32 (1H, t, J = 2.5 Hz, H-6) were observed. The 13C NMR spectra displayed 27 carbon signals which were attributed to four methyls (δC 13.2, 16.2, 19.1, and 28.4), eight methylenes (19.4, 24.6, 29.1, 31.0, 34.2, 36.6, 38.3, and 68.5), eleven methines (δC 30.8, 49.2, 50.3, 69.4, 69.7, 70.9, 71.3, 77.5, 91.3, 95.8, and 121.2), and four quaternary carbons (δC 36.8, 37.2, 140.2, and 209.9) (Table 2). These data suggested that compound 1 was a C21 steroidal glycoside. The characteristic carbon signals of C-18 and C-19, a Δ5,6 double bond, two quaternary carbons at C-10 and C-13, and an oxygenated methylene at C-16 led to the establishment of an epigynumgenane-type pregnane aglycone with a six-membered tetrahydropyran D-ring (Wan et al., 2017). A distinguishing quaternary carbon signal was allocated to C-20 by a key HMBC correlation (Fig. 2) between δH 2.12 (H3-21) and δC 209.9 (C-20). The oxygenated secondary carbon signal at δC 91.3 was attributed to C-17, as evident from the HMBC correlations of H3-21 (δH 2.12), H3-18 (δH 0.91), and H2-16 2
Phytochemistry 168 (2019) 112127
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Fig. 2. Key HMBC ( ) and 1H–1H COSY (▬▬) correlations of compounds 1 and 4.
data, the glycosidic moiety of 4 was found similar to that of 3. The main difference of the aglycone in 4 was the replacement of a keto carbonyl carbon at C-20 by a hydroxy methine carbon, and the presence of a hemiketalic quaternary carbon instead of the oxymethine carbon at C17. The methyl signal at δH 1.20 (m, overlapped, H3-21) showed an HMBC interaction with δC 70.6 (C-20) and 100.3 (C-17), which further suggested the assignment. The relative configuration of C-17 was determined by ROESY correlations. The key cross peaks of H-8 (δH 1.55) with H3-19 (δH 0.98) and H3-18 (δH 1.00) were observed, which disclosed these groups as co-facial and β-oriented. The H3-18, in turn, showed correlations with H-20 (δH 3.84) and H3-21 (δH 1.20). Accordingly, an α-orientation was suggested for OH-17. However, the configuration of C-20 in the aglycone could not be established from ROESY spectrum due to free rotation of the C-17−C-20 bond. Enzymatic hydrolysis of compounds 4 and 6 was performed to yield the aglycone, which was confirmed the same for both the compounds by UHPLC-ESI-MS analysis with identical retention times (12.33 and 12.38 min, respectively) and mass data (see Supplementary Material, Figs. S35 and S37). Hence, the basic skeleton of the aglycone of 4 was established as epigynumgenane which was similar to that of epigynoside A (6) (Cao et al., 2005). The linkage of sugar moiety to C-20 of the aglycone via oxygen atom was deduced by an HMBC correlation of δH 4.77 (1H, br. s, H-1′) with δC 70.6, and of δH 3.84 (H-20) with 97.5 δC (C-1′) (Fig. 2). The positive HR-ESI-MS of epigycoside H (5) produced a sodium adduct ion at m/z 519.2933 [M + Na]+ (calcd 519.2928), representing the molecular formula C27H44O8. By comparing the 1D NMR spectroscopic data of 5 and 4, it was found that they shared the same aglycone moiety (Tables 1 and 2), but there were differences in sugar moieties. The sugar unit in 5 was deduced as 6-deoxy-β-D-idopyranose due to the absence of two methoxy groups and verified by the HMBC, 1H–1H COSY, and HSQC-TOCSY experiments. The NMR data of the sugar moiety in 5 was in line with that of 1. Acid hydrolysis of 5 and 1 and then the GC/MS analysis of trimethylsilylated thiazolidine derivatives of the sugars confirmed the same sugar moiety in both the compounds. Thus, the structure of compound 5 was characterized and named as epigycoside H. In addition to undescribed compounds, seven known compounds including four epigynumgenane-type steroids (6−9), two lignans (10–11), and a flavonoid (12) were also purified from Epigynum cochinchinensis. Their structures were determined as epigynosides A−C (6–8) (Cao et al., 2005), epigycoside A (9) (Wan et al., 2017), pinoresinol (10) (Brenes et al., 2000), syringaresinol (11) (Das et al., 1999), and santaflavone (12) (Goren et al., 2002) by comparing their spectroscopic data with those in the literature.
Fig. 3. X-ray ORTEP drawing of 1 with ellipsoids drawn at the 30% probability level. 1
H–1H COSY and HSQC-TOCSY spectra of 2 revealed the presence of a C–CH–CH–CH–CH–CH3 structural moiety. The structure of the sugar was deduced by the correlations of the anomeric proton signal at δH 4.77 (H-1′) with C-1′ (δC 97.0), C-2′ (δC 79.8), C-3′ (δC 71.6), C-4′ (δC 68.9), C-5′ (δC 70.3) and C-6′ (δC 16.4) in HSQC-TOCSY spectrum. The methoxy group was located at C-2′ of the monosaccharide unit due to HMBC correlations (Fig. S14). The monosaccharide unit was thus defined as 2-O-methyl-6-deoxy-β-D-idopyranose due to similar NMR data as that of epigynoside A isolated from Epigynum auritum (Cao et al., 2005). The connectivity of the sugar moiety was determined by an HMBC correlation of the anomeric proton signal at δH 4.77 to δC 78.1 (d, C-3). Thus, the structure of epigycoside E was established as 2. Epigycoside E (3) was obtained as colorless crystals, and its molecular formula C29H46O7 was established in regard to a sodiated HR-ESIMS ion (+ve) at m/z 529.3130 [M + Na]+ (calcd 529.3136), which differed from 1 by 28 more mass units. The IR and UV spectroscopic data of both the compounds were also similar. A detailed comparison of the 1H NMR spectra of 3 and 1 (Table 1) indicated that they had the similar basic skeleton, excluding two additional methoxy units in 3. The characteristic 13C NMR signals of a dioxygenated methine at δC 97.4, four oxymethines at δC 68.4, 70.6, 77.8, and 78.2, a methyl at δC 16.3, as well as two methoxy signals at δC 60.3 and 57.9 were almost identical to those of the sugar moiety in epigynoside E (i.e. 2,3-di-O-methyl6-deoxy-β-D-idopyranose) (Gao et al., 2017a). The absolute configuration of the sugar moiety in epigynoside E has already been established by X-ray diffraction analysis. The sugar-aglycone linkage was verified by an HMBC cross-peak from δH 4.77 (1H, br. s, H-1′) to δC 78.0 (d, C3). Thus, the structure of epigycoside F was characterized as 3. The molecular formula of epigynoside G (4) was determined as C29H48O8 by a sodiated ion peak at m/z 547.3232 [M + Na]+ (calcd 547.3241) in the HR-ESI-MS. By comparing the 1D NMR spectroscopic
2.2. Effects on splenocyte proliferation in vitro Furthermore, the cytotoxicity of the isolated steroids (1–9) was evaluated by the CCK-8 assay. For this purpose, the purified T-lymphocyte cells were treated with various concentrations of 1−9 (12.5, 3
Phytochemistry 168 (2019) 112127
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Table 1 1 H NMR spectroscopic data of compounds 1–5, (δ in ppm, J values in Hz)a. No.
1
2
1a 1b 2a 2b 3 4a 4b 6 7a 7b 8 9 11a 11b 12a 12b 14 15a 15b 16a 16b 17 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ 2′-OCH3 3′-OCH3
1.84 (dt, 13.4, 3.1) 1.01 (m) 1.93 (dt, 12.7, 3.2) 1.55 (m) 3.63 (m, W1/2 = 31.7) 2.34 (ddd, 13.0, 4.2, 1.9) 2.18 (m) 5.32 (t, 2.5) 2.05 (m) 1.41 (m) 1.41 (ovb) 1.01 (ov) 1.55 (ov) 1.32 (dd, 13.0, 3.1) 1.61 (dt, 13.0, 2.9) 1.22 (m) 1.01 (ov) 1.41 (ov) 1.01 (m) 4.12 (br. dd, 11.3, 4.0) 3.41 (m) 3.30 (s) 0.91(s) 0.94 (s)
1.86 1.03 1.98 1.60 3.57 2.35 2.23 5.33 2.06 1.44 1.40 1.03 1.57 1.33 1.63 1.24 1.03 1.44 1.03 4.14 3.44 3.32 0.91 0.96
(dt, 13.4, 3.2) (m) (m) (m) (tt, 11.4, 4.7) (ddd, 13.2, 4.8, 2.3) (m) (t, 2.5) (m) (m) (m) (ov) (m) (dd, 13.1, 2.8) (m) (d, 3.5) (ov) (ov) (ov) (ddd, 11.5, 4.8, 1.5) (td, 11.5, 3.3) (s) (s) (s)
1.87 1.04 2.00 1.62 3.57 2.34 2.24 5.35 2.15 1.55 1.46 1.04 1.58 1.34 1.60 1.23 1.04 1.42 1.04 4.16 3.45 3.33 0.93 0.98
(dt, 13.4, 3.3) (m) (m) (m) (m, W1/2 = 25.2) (ddd, 14.5, 4.7, 2.2) (m) (t, 2.4) (m) (m) (m) (ov) (m) (dd, 13.2, 3.4) (m) (m) (ov) (m) (ov) (br. dd, 11.4, 4.3) (td, 11.4, 3.0) (ov) (s) (s)
2.12 4.84 3.25 4.07 3.63 3.95 1.26
2.13 4.77 3.53 3.31 3.28 3.96 1.28 3.55
(s) (br. s) (m) (br. s) (m) (qd, 6.5, 1.5) (d, 6.5) (s)
2.15 4.77 3.33 3.66 3.36 3.84 1.29 3.57 3.42
(s) (br. s) (ov) (br. s) (m) (br. q, 6.6) (d, 6.6) (ov) (s)
a b
(s) (br. s) (d, 8.1) (br. s) (ov) (q, 6.4) (d, 6.4)
3
4
5
1.85 (dt, 13.3, 3.4) 1.05–1.01 (m, ov) 2.00 (m) 1.62 (m) 3.57 (m, W1/2 = 34.7) 2.34 (ddd, 12.9, 4.6, 2.0) 2.24 (m) 5.35 (t, 2.5) 2.08 (m) 1.42 (m) 1.55 (m) 1.05–1.01 (m, ov) 1.55 (ov) 1.34 (dd, 13.1, 5.1) 1.65 (m) 1.20 (m, ov) 1.65 (ov) 1.42 (ov) 1.34 (ov) 3.93 (ddd, 13.9, 11.1, 3.0) 3.70 (ddd, 11.1, 5.4, 0.9)
1.82 (dt, 13.4, 3.1) 1.01 (m) 1.92 (m) 1.58 (m) 3.59 (m, W1/2 = 34.6) 2.33 (ddd, 13.4, 4.9, 2.4) 2.16 (m) 5.31 (t, 2.4) 2.04 (m) 1.48 (m) 1.39 (m) 0.97 (ov) 1.49 (m) 1.30 (dd, 13.2, 2.6) 1.58 (m) 1.22 (m) 1.65 (m) 1.39 (ov) 1.28 (m) 3.90 (ddd, 13.5, 11.0, 3.1) 3.67 (br. dd, 11.0, 4.8)
1.00 (s) 0.98 (s) 3.84 (m) 1.20 (ov) 4.77 (br. s) 3.33 (m) 3.66 (t, 3.0) 3.36 (m) 3.85 (q, 6.5) 1.28 (d, 6.5) 3.58 (ov) 3.42 (s)
0.97 (s) 0.93 (s) 3.98 (m) 1.16 (d, 6.4) 4.84 (br. s) 3.30 (m) 3.99 (m) 3.56 (dd, 3.2, 1.0) 3.80 (q, 6.5) 1.25 (d, 6.5)
Measured for compounds 3 and 4 in MeOH-d4 while for 1, 2, and 5 in CDCl3. ov: overlapped.
25, and 50 μM), and the cell viability was measured. Results revealed that compounds 1–9 at the tested concentrations did not cause any significant cytotoxic effect in T-cells. Next, the immunosuppressive activity of 1−9 was assessed on mitogen-stimulated mice splenocytes, and dexamethasone (DXM) was used as a positive control. Fig. 4 shows the immunosuppressive results. In a concentration range from 12.5 to 50 μM, all the tested compounds displayed good inhibition on Con Ainduced spleen cell proliferation. When tested at 50 μM concentration, compound 2 had the strongest immunosuppressive activity, close to that of positive control (DXM), with similar stimulation indices of 1.24 and 1.17, respectively. However, DXM had a better immunosuppressive activity which was evident from its stimulation index at each tested concentration, significantly lower than all the compounds (p < 0.05). The median inhibitory concentration (IC50) values of compounds 1–9 are listed in Table 3. Compounds 4, 8, and 9 had a similar and relatively weaker inhibitory effect than 2, while compounds 3, 5, 6, and 7 possessed the weakest activity (IC50 > 80 μM).
Table 2 13 C NMR spectroscopic data of compounds 1–5, (δ in ppm)a. No.
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ 2′-OCH3 3′-OCH3
36.6 29.1 77.5 38.3 140.2 121.2 31.0 30.8 49.2 36.8 19.4 34.2 37.2 50.3 24.6 68.5 91.3 13.2 19.1 209.9 28.4 95.8 71.3 69.4 70.9 69.7 16.2
36.7 29.3 78.1 38.5 140.6 121.0 31.1 30.9 49.3 37.0 19.5 34.3 37.2 50.4 24.7 68.6 91.4 13.3 19.2 209.7 28.6 97.0 79.8 71.6 68.9 70.3 16.4 60.3
36.6 29.3 78.0 38.5 140.6 121.0 31.1 30.9 49.3 36.9 19.5 34.3 37.2 50.4 24.8 68.6 91.4 13.3 19.2 209.4 28.5 97.4 77.8 78.2 68.4 70.6 16.3 60.3 57.9
36.7 29.4 78.3 38.6 140.4 121.3 31.5 31.3 48.9 36.9 20.0 32.9 39.6 43.2 24.8 61.2 100.3 14.3 19.2 70.6 18.0 97.5 77.8 78.0 68.5 70.5 16.4 60.3 57.9
36.4 28.9 77.6 38.1 139.7 121.3 31.1 31.0 48.7 36.5 19.7 32.5 39.4 42.7 24.4 60.7 99.7 14.0 18.6 69.6 17.8 96.1 70.9 69.2 70.4 69.9 15.7
2.3. Epigycoside E induces G0/G1 phase arrest in Con A-induced T-cells To verify whether epigycoside E (2) treatment affects the Con Astimulated T-cells distribution, cell cycle was investigated in the presence of epigycoside E at 12.5, 25, and 50 μM. The population of T-cells in G0/G1, S, and G2/M phase was analyzed using Con A-stimulated Tcells by flow cytometry. As shown in Fig. 5, compared to that of the control group, the percentage of T cells in G0/G1 phase notably decreased from 74.21 to 65.81% in Con A treated group with a statistically significant difference (p < 0.01). The propidium iodide-stained T-cells showed that epigycoside E increased the cell frequency in G0/G1 phase (Fig. 5) in a concentration dependent manner. The percentage of
a
Measured for compounds 3 and 4 in MeOH-d4 while for 1, 2, and 5 in CDCl3. 4
Phytochemistry 168 (2019) 112127
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Fig. 4. Inhibitory effect of compounds 1–9 on Con A-stimulated T lymphocyte proliferation in vitro. DXM was used as a positive control. The values are presented as mean ± SD of triplicate replicates. Means (bar value) with different letters indicate significant differences (P < 0.05).
provide further evidence that epigycoside E (2) possesses good inhibitory activity on Con A-stimulated T-cells, ELISA was used to evaluate the effect of epigycoside E on the release of TNF-α and IL-2 cytokines. Compared with the control group, Con A treatment alone significantly induced the production of TNF-α and IL-2 cytokines (Fig. 6). Epigycoside E inhibited the production of TNF-α and IL-2, at concentrations ranging from 12.5 to 50 μM. For TNF-α production, epigycoside E had a similar and higher inhibitory effect at 25 and 50 μM with no significant difference (P > 0.05). Treatment of Con A-induced T-cells, with 2 at 50 μM concentration, showed a significant inhibition in the production of TNF-α, and there was no significant difference compared with the control group (Fig. 6A). Interestingly, the inhibition of IL-2 production was up to 58.2 ± 2.8 pg/mL when the con A-stimulated T-cells were exposed to 50 μM epigycoside E (Fig. 6B). Epigycoside E had a similar inhibitory effect on TNF-α and IL-2 production, and there was no significant difference in the concentrations of TNF-α in Con A-induced T-cells treated with 25 and 50 μM of epigycoside E. Hence, epigycoside E may have non-selective regulation on cytokines production. At present, we do not understand the mechanism of its effect, and we will further explore it in our subsequent studies.
Table 3 Immunosuppressive effect (IC50) of compounds 1–9 (mean ± SD, n = 3). Compound
IC50 ± SD (μM)
1 2 3 4 5 6 7 8 9 DXM (positive control)
51.7 ± 22.1 ± > 80 40.8 ± > 80 > 80 > 80 46.0 ± 45.5 ± 11.6 ±
17.5 6.4 12.1
16.1 15.3 0.7
T-cells in G0/G1 phase was 68.27, 70.64, and 73.39% when exposed to 12.5, 25, and 50 μM of epigycoside E, respectively. The G0/G1 phase arrest was induced by 7.58% after incubation with epigycoside E at a concentration of 50 μM compared to the Con A model group. The cell progression of epigycoside E treatment was markedly inhibited through G1 to S transition, which may influence the cell cycle regulatory molecules in G0/G1 phase. It was revealed in a study that propidium iodide-stained cells increased the percentage of G0/G1 cells according to the erlotinib concentration ranging from 1–15 μM (Luo et al., 2011). The cell cycle progression of 3-MCPD esters on Con A-induced T-lymphocytes caused G0/G1 arrest (Huang et al., 2018). These results provide a theoretical basis for the immunosuppressive mechanism of epigycoside E. The cell population at different stages may be involved by the expression levels of cell cycle regulatory proteins, such as cyclin A, D1, D3, and E, p-Rb, p21, p27, and so on (Luo et al., 2011; Wang et al., 2014). These findings may provide new insights to further understand the mechanism of cell-cycle arrest at molecular level.
3. Conclusions In our ongoing search for bioactive compounds from Epigynum plants, five undescribed pregnane glycosides named as epigycosides D−H (1–5), along with four known analogues, two lignans, and a flavonoid were isolated from the EtOAc fraction of E. cochinchinensis. Their structures were elucidated by extensive spectroscopic techniques and single-crystal X-ray diffraction analysis. Epigynosides A−C (6–8), epigycoside A (9), pinoresinol (10), syringaresinol (11), and santaflavone (12) are isolated for the first time from E. cochinchinensis, among which the pregnane glycosides with epigynumgenane skeleton are only reported from the plants of genus Epigynum. Compounds 1–9 showed good immunosuppressive activity against the proliferation of Tlymphocytes, among which epigycoside E (2) was the most effective immunosuppressant. Epigycoside E caused the G0/G1 phase arrest and the inhibition of multiple cytokines’ production, including TNF-α and IL-2. Therefore, the current study suggests that the plant E.
2.4. Inhibitory effect of epigycoside E on cytokines production Pro-inflammatory cytokines, predominantly interleukin (IL)-2 and tumor necrosis factor (TNF)-α have a prominent role in the regulation of autoimmune diseases (Gordon, 2003; Mesaik et al., 2010). Thus, to 5
Phytochemistry 168 (2019) 112127
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Fig. 5. Con A-induced T-lymphocyte cells treated with epigycoside E for 72 h. Cell cycle data are representative of three individual experiments. *P < 0.05, **P < 0.01, means significant difference from the G0/G1 phase group. #P < 0.05, means significant difference from the S phase group. &P < 0.05, means significant difference from the G2/M phase group.
packed glass columns (15 × 230 and 26 × 460 mm), was used for MPLC. Silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Chromatorex C18 (20–45 μm, Fuji Silysia Chemical Ltd., Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden) were used for column chromatography. GF254 silica gel plates (Qingdao Marine Chem. Ltd., Qingdao, China) were used for carrying out the TLC. Spots were visualized by spraying with 10% H2SO4 solution in EtOH and heating at 105 °C. The absorbance in bioassays was measured and recorded on a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cell proliferation rate was estimated by using a cell counting kit (CCK-8, Dojindo Molecular Technologies, Maryland, USA). Cell cycle assay kit and apoptosis analysis kit were purchased from Beyotime (Shanghai, China); mouse cytokines (TNF-α and IL-2) detecting ELISA kits were purchased from Beijing 4A Biotech Co., Ltd (Beijing, China).
cochinchinensis might be served as a potent immunosuppressive agent for the treatment of autoimmune diseases.
4. Experiments 4.1. General experimental procedures Optical rotations were determined with a JASCO P-1020 digital polarimeter. UV spectra were recorded with a Shimadzu UV-2401A spectrophotometer. IR spectra, with KBr pellets, were recorded with Bruker FT-IR Tensor 27 spectrometer. 1D and 2D NMR spectra were recorded on a Bruker DRX-500 MHz or an Avance III–600 MHz spectrometer with TMS as an internal standard. HRESIMS data were taken on a Thermo high resolution Q Exactive focus mass spectrometer. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph equipped with Zorbax SB-C18 columns (9.4 × 150 and 21.2 × 250 mm). Büchi pump system, equipped with C18 silica gel-
Fig. 6. A-B. Immunosuppressive effect of epigycoside E (2) on Con A induced T cells. The concentrations of TNF-α and IL-2 were detected in the supernatant in presence of 2 for 72 h at 12.5, 25 and 50 μM, respectively. The values are presented as mean ± SD of triplicate replicates. Different letters indicate significant differences (P < 0.05). 6
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and 13C NMR (150 MHz) data (MeOH-d4), see Tables 1 and 2; positive ion HRESIMS m/z 529.3130 [M + Na]+ (calcd for C29H46O7Na+, 529.3136).
4.2. Plant material The stems of Epigynum cochinchinensis (Pierre) Mabb. (Apocynaceae) were collected in May 2018 (summer season) from Kasi district (GPS coordinates: 19°14′N/102°17′E) of Vientiane province, Lao People's Democratic Republic, and identified by Prof. Yunhong Tan, Xishuangbanna Tropical Plant Garden. A voucher specimen (No. Cao20180510-01) is preserved at the Laboratory of Functional Foods, Yunnan Institute of Food Safety, Kunming University of Science and Technology.
4.3.4. Epigycoside G (4) White amorphous powder; [α]20 D -61.3 (c 0.03, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.5); IR (KBr) νmax: 3447, 2933, 1632, 1450, 1381, 1260, 1177, 1101, 1076, 1050 cm−1; 1H NMR (800 MHz) and 13C NMR (200 MHz) data (MeOH-d4), see Tables 1 and 2; positive ion HRESIMS m/z 547.3232 [M + Na]+ (calcd for C29H48O8Na+, 547.3241).
4.3. Extraction and isolation 4.3.5. Epigycoside H (5) White amorphous powder; [α]25 D -82.6 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 203 (3.2); IR (KBr) νmax: 3439, 2935, 2759, 1532, 1497, 1164, 1038, 612 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 519.2933 [M + Na]+ (calcd for C27H44O8Na+, 519.2928).
The air-dried and powdered stems of E. cochinchinensis (11.0 kg) were extracted with 70% aqueous MeOH under reflux condition and the solvent was evaporated in vacuo to obtain the MeOH extract, followed by suspension in H2O. The H2O layer was then successively partitioned with petroleum ether (10 L × 3) and EtOAc (10 L × 4). The EtOAc fraction (152 g) was fractioned over RP-18 column, eluted with a gradient of MeOH–H2O (20:80–100:0) to give six fractions (Frs. A-F). Fr. B (22 g) was then chromatographed on RP-18 column, eluting with MeOH–H2O (60:40–90:10) to yield five sub-fractions. Fr. B1 (220 mg) was subjected to semi-preparative reversed-phase C18-HPLC column eluting with a gradient of MeOH–H2O (60:40–80:20), followed by Sephadex LH-20 column eluting with MeOH–H2O (50:50, v/v), and further purified by semi-preparative HPLC (MeCN–H2O, 65:35) to obtain compounds 5 (5 mg, tR = 13 min) and 9 (4.6 mg, tR = 15 min). Fr. C (10.3 g) was fractionated by silica gel column (CHCl3-acetone, 20:1) to give four fractions, Frs. C1–C4. Fr. C2 was purified by semi-preparative HPLC (MeOH–H2O, 70:30) to yield compounds 7 (3 mg, tR = 9 min) and 11 (6 mg, tR = 11 min). Fr. C4 was purified by semipreparative HPLC (MeCN–H2O, 60:40) to give compound 6 (20 mg, tR = 15.5 min). Fr. D (23 g) was subjected to RP-18 column, eluting with MeOH–H2O (70:30–95:5) to afford Frs. D1-D5. Fr. D2 was subjected to silica gel CC and eluted with CHCl3-acetone (10:1–1:1) to yield compound 1 (11 mg). Fr. E (7 g) was subjected to an RP-18 column (MeOH–H2O, 70:30–90:10) to afford Frs. E1-E3. Fr. E2 (412 mg) was purified by silica gel CC and eluted with CHCl3-acetone (10:1) and then subjected to preparative TLC (petroleum ether: acetone, 3:1, Rf = 0.55) to yield compound 3 (5.8 mg). Fr. E3 (108 mg) was chromatographed over RP-18 silica gel (MeOH–H2O, 75:25–85:15) and further purified by semi-preparative HPLC (MeCN–H2O, 78:22) to give compounds 2 (5.8 mg, tR = 10.5 min) and 10 (2.7 mg, tR = 13.5 min). Fr. E5 (253 mg) was subjected to silica gel CC, eluting with CHCl3–MeOH (20:1), and further purified by semi-preparative HPLC (MeOH–H2O, 65:35) to yield compounds 4 (7 mg, tR = 9.5 min), 8 (3.8 mg, tR = 12 min), and 12 (2 mg, tR = 14 min).
4.4. X–ray crystallographic analysis of epigycoside D The Cu Kα radiation in Bruker APEX DUO diffractometer, equipped with an APEX II CCD, was used to get the X-ray data. The intensity data for epigycoside D (1) were collected at 100 K. Bruker integration program SAINT was used for cell refinement and data reduction. Structure was expanded by Fourier techniques, resolved by direct methods, and refined by the program and full-matrix least squares calculations. All the hydrogen atoms were fixed at calculated positions while all the nonhydrogen atoms were refined anisotropically (Cheng et al., 2016a). Crystallographic data for epigycoside D has been placed in Cambridge Crystallographic Data Center as supplementary publications (number: CCDC, 1880756). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ, U.K.; fax: (+44) 1223-336-033; or [email protected]). Crystallographic data for epigycoside D (1): 2(C27H42O7)•CH4O, M = 989.25, a = 7.66050(10) Å, b = 33.7814(6) Å, c = 10.0205(2) Å, α = 90°, β = 100.58°, γ = 90°, V = 2549.08(8) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.750 mm−1, 26,778 reflections measured, 8382 independent reflections (Rint = 0.0275). The final R1 values were 0.0366 (I > 2σ(I)). The final wR(F2) values were 0.1005 (I > 2σ(I)). The final R1 values were 0.0367 (all data). The final wR(F2) values were 0.1007 (all data). The goodness of fit on F2 was 1.043. Flack parameter = 0.06(3). 4.5. GC/MS analysis of sugars from compounds 1–5 Compounds 1–5 (each 2 mg) were dissolved in 2 mL HCl (2 M) and then hydrolyzed in a water bath at 90 °C for 2 h. After reaction, the mixture was partitioned with CHCl3 for three times. The aqueous layer was neutralized with 2 M NaOH and evaporated in vacuo to obtain a residue. The residue was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg) and stirred at 60 °C for 1 h. Trimethylsilylimidazole (1.5 mL) was added to the reaction mixture and incubated for 30 min at 60 °C. Then each sample was subjected to a Shimadzu GC/MS-QP2010 instrument, equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5 quartz capillary column and an H2 flame ionization detector under the conditions described elsewhere (Gao et al., 2017a; Cheng et al., 2016b). The configuration of sugar moieties was determined by comparing the retention times of their trimethylsilylated thiazolidine derivatives. The retention times of the sugar derivatives were 15.5 min for 1 and 5, 22.5 min for 3 and 4, and 17.5 min for 2, respectively. The retention times of 2-O-methyl-6deoxy-β-D-idopyranose and 2,3-di-O-methyl-6-deoxy-β-D-idopyranose derivatives were identical to those reported in the literature (Gao et al., 2017a).
4.3.1. Epigycoside D (1) White amorphous powder; [α]26 D -82.6 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 203 (3.6); IR (KBr) νmax: 3440, 1710, 1641, 1448, 1257, 1030, 960, 866, 661, 541 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 501.2825 [M + Na]+ (calcd for C27H42O7Na+, 501.2823). 4.3.2. Epigycoside E (2) White amorphous powder; [α]25 D -127.8 (c 0.3, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.1); IR (KBr) νmax: 3425, 2934, 1735, 1709, 1374, 1251, 1068 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 515.2980 [M + Na]+ (calcd for C28H44O7Na+, 515.2979). 4.3.3. Epigycoside F (3) White amorphous powder; [α]20 D -56.5 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.6); IR (KBr) νmax: 3439, 2934, 1711, 1633, 1446, 1381, 1257, 1177, 1102, 1072, 1049 cm−1; 1H NMR (600 MHz) 7
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4.6. Enzymatic hydrolysis and UHPLC-ESI-MS analysis of compounds 4–6
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4.7. Splenocyte proliferation assay A previously reported CCK-8 method was used to carry out the immunosuppressive activity (Gao et al., 2017a). 4.8. Cell cycle assay The purified T-cells from male BALB/c mice (2 × 106 cells/well) were cultured in 6-well plates and treated with or without compounds for 72 h in the presence of Con A (10 μg/mL). After incubation, the cell suspension was centrifuged for 5 min at 1000 g. Cells were collected, washed with cold PBS and fixed with 70% ice-cold EtOH at 4 °C overnight. Then the fixed cells were centrifuged and washed with cold PBS, and repeated twice. Stained with 10 μL propidium iodide (PI; 1 mg/mL) and 10 μL RNase A (10 mg/mL) for 30 min. Cell cycle was analyzed by guava® easyCyte 6-2L (Millipore, Billerica, MA, US) flow cytometry. 4.9. Determination of TNF-α and IL-2 cytokines production The concentrations of TNF-α and IL-2 from the collected supernatants were quantified by ELISA and the assay procedures were carried out as described in the kit manual. Briefly, freshly prepared mononuclear cells (1 × 105/well) were seeded in 96-well microtiter plate and treated with or without compounds at different concentrations in the presence of Con A (10 μg/mL). The plate was incubated at 37 °C for 72 h, then, the supernatants were collected and analyzed for TNF-α and IL-2 cytokines production. 4.10. Statistical analysis All the statistical tests were performed with the statistical program SPSS 16.0. The data were expressed as mean ± SD. Statistical significance was set at p < 0.05. All the analyses were performed using Origin 8.5 software (OriginLab, Northampton, MA, USA). Conflicts of interest The authors declare no conflict of interest. Acknowledgements This research was supported by the National Natural Science Foundation of China (grant numbers 31600274, 31960094 and 31460083), the Applied Basic Research Project of Yunnan Province (Grant No. 2018FB036), and the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University (Grant No. ZYAOX2018031). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112127. 8
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Epigynumgenane-type pregnane glycosides from Epigynum cochinchinensis and their immunosuppressive activity
T
Zengyuan Wanga,1, Mengjun Jianga,1, Afsar Khanb, Shengbao Caia, Xiaonian Lic, Junqiu Liud, Guoyin Kaid, Tianrui Zhaoa, Guiguang Chenga,∗, Jianxin Caoa,∗∗ a
Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, 650500, People's Republic of China Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, 22060, Pakistan c State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, People's Republic of China d College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, 310053, People's Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Epigynum cochinchinensis (Apocynaceae) Epigycoside E Pregnane glycosides Immunosuppressive Cytokines
Five undescribed C21 pregnane glycosides, epigycosides D−H, together with four known analogues, two lignans, and a flavonoid have been isolated from the stems of Epigynum cochinchinensis. The structures of pregnane glycosides were elucidated using spectroscopic techniques and acid hydrolysis. The in vitro immunological activities were assessed against Con A-stimulated proliferation of mice splenocytes. The C21 pregnane glycosides showed immunosuppressive activity in a concentration-dependent manner. Moreover, epigycoside E exhibited a potent immunosuppressive effect, and the IC50 value on Con A-stimulated mice splenocytes was 22.1 ± 6.4 μM. Epigycoside E also caused G0/G1 arrest, and inhibited TNF-α and IL-2 production.
1. Introduction Pregnanes are C21 steroidal compounds present in nature either in free state or as glycosides. In general, the sugar moiety is connected to the aglycone by an ether linkage, most frequently at C-20, C-3, or both (bisdesmosidic glycosides) (Deepak et al., 1997). Literature reveals that pregnane glycosides possess immunosuppressive (Ounaissia et al., 2016; Zhang et al., 2015), anti-inflammatory (Jeong et al., 2014), antioxidant (Ounaissia et al., 2016), anti-epileptic (Li et al., 2015), neuroprotective (Zhao et al., 2013), anti-microbial (Song et al., 2014), antihyperglycemic (Tsoukalas et al., 2016), cytotoxic (Liu et al., 2014; Zhang et al., 2013), antitumor (Wang et al., 2015), and slimming (Zhu et al., 2012) activities. Pregnane glycosides are an important class of specialised metabolites from medicinal plants in natural drugs, especially used as autoimmune disorder drugs. Clinical immunosuppressants, such as cortisone, rapamycin, and tacrolimus have limitations in the treatment of auto-immunological diseases with undesirable side effects including renal injury and liver toxicity. Hence, searching for natural immunosuppressants with high efficacy and low toxicity is urgent. Motivated by their efficacy, several immunosuppressive pregnane glycosides have been purified from
Cynanchum, Periploca, and Stephanotis plants. In our previous studies, some pregnane glycosides with potential immunosuppressive activity were isolated from Epigynum plants (Gao et al., 2017a, 2017b; Wan et al., 2017). These findings suggested that the exploration of pregnane glycosides is efficient to discover undescribed immunological substances. Epigynum cochinchinensis (Pierre) Mabb. (Apocynaceae) is a woody climber growing in the forest margins of Mainland Southeast Asia. Our previous phytochemical search on E. cochinchinensis led to the isolation of some pregnane glycosides (Shao et al., 2018a, 2018b; Wan et al., 2017). Among them, epigycoside A exhibited a significant inhibitory effect against Con-A induced proliferation of mice splenocytes. In our continuous investigation for structurally undescribed steroidal glycosides with bioactive potential, a further systematic phytochemical study on the stems of E. cochinchinensis was performed. As a result, five undescribed pregnane glycosides, epigycosides D−H (1–5), together with four known analogues (6–9), two lignans (10–11), and a flavonoid (12) were isolated (Fig. 1). Herein, we describe structural elucidation of the undescribed compounds based on 1D and 2D-NMR data, acid hydrolysis, enzymatic hydrolysis, and X-ray diffraction methods. Furthermore, the in vitro inhibitory activity of pregnane glycosides against the
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Corresponding author. Corresponding author. E-mail addresses: [email protected] (G. Cheng), [email protected] (J. Cao). 1 The authors have contributed equally to this work. ∗∗
https://doi.org/10.1016/j.phytochem.2019.112127 Received 19 February 2019; Received in revised form 11 September 2019; Accepted 12 September 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Chemical structures of compounds 1–12.
(δH 4.12 and 3.41) with C-17 (δC 91.3). The ROESY cross peaks of H-8 with H3-19 and H3-18 disclosed these groups as co-facial and β-oriented. The absence of ROESY correlations between H-8 and H-9/H-14/ H-17 established an α-orientation of these protons. Besides the aglycone unit, the NMR data also suggested a sugar moiety in 1, as indicated by an anomeric proton signal at δH 4.84 (br. s) and a secondary methyl signal at δH 1.26 (3H, d, J = 6.4 Hz, H3-6′) with corresponding carbon signals at δC 95.8 and 16.2, as well as four oxygenated methine signals at δC 69.4, 69.7, 70.9, and 71.3. Further analysis of the 1H–1H COSY correlations delineated a spin system of H-1′/H-2′/H-3′/H-4′/H-5′/H36′. Its 13C NMR resonances were assigned by the HSQC experiment and further confirmed with HSQC-TOCSY technique (Fig. S07). The monosaccharide unit in 1 was then identified as 6-deoxyhexose. The sugaraglycone O-linkage was determined by the HMBC correlations of δH 4.84 (1H, br. s, H-1′) with δC 77.5 (d, C-3) and of δH 3.63 (1H, m, H-3) with 95.8 (d, C-1′). The ambiguous coupling constants and ROESY correlations could not provide sufficient evidence for the relative configuration of the monosaccharide moiety. However, crystals were obtained in MeOH–H2O (4:1) mixture at 4 °C. The X-ray diffraction analysis was performed with Cu Kα radiation and confirmed the absolute configuration of 1, in which all the hydroxy groups at C-2′, C-3′, and C4′ of the sugar moiety were positioned axially (Fig. 3). Thus, the structure of 1 was established as 17-deoxy-epigynumgenane-20-one 3O-6′-deoxy-β-D-idopyranoside, and trivially named as epigycoside D. A molecular formula of C28H44O7 was assigned to epigycoside E (2) by HR-ESI-MS at m/z 515.2980 [M + Na]+, indicating one more CH2 group than that of 1. The NMR spectra of 2 were identical to those of 1 (Tables 1 and 2) apart from a methoxy group (δH 3.55; δC 60.3). The
proliferation of T lymphocytes is also discussed. 2. Results and discussion 2.1. Structure elucidation of compounds 1−5 Epigycoside D (1) was isolated as a white amorphous power and its molecular formula was determined as C27H42O7 based on the sodium adduct ion at m/z 501.2825 [M + Na]+ (calcd for 501.2823) in its positive HR-ESI-MS. In the 1H NMR spectrum of 1, three singlet signals at δH 0.91 (3H, s, H3-18), 0.94 (3H, s, H3-19), and 2.12 (3H, s, H3-21), one doublet signal for methyl protons at δH 1.26 (3H, d, J = 6.4 Hz, H36′), and one olefinic proton signal at δH 5.32 (1H, t, J = 2.5 Hz, H-6) were observed. The 13C NMR spectra displayed 27 carbon signals which were attributed to four methyls (δC 13.2, 16.2, 19.1, and 28.4), eight methylenes (19.4, 24.6, 29.1, 31.0, 34.2, 36.6, 38.3, and 68.5), eleven methines (δC 30.8, 49.2, 50.3, 69.4, 69.7, 70.9, 71.3, 77.5, 91.3, 95.8, and 121.2), and four quaternary carbons (δC 36.8, 37.2, 140.2, and 209.9) (Table 2). These data suggested that compound 1 was a C21 steroidal glycoside. The characteristic carbon signals of C-18 and C-19, a Δ5,6 double bond, two quaternary carbons at C-10 and C-13, and an oxygenated methylene at C-16 led to the establishment of an epigynumgenane-type pregnane aglycone with a six-membered tetrahydropyran D-ring (Wan et al., 2017). A distinguishing quaternary carbon signal was allocated to C-20 by a key HMBC correlation (Fig. 2) between δH 2.12 (H3-21) and δC 209.9 (C-20). The oxygenated secondary carbon signal at δC 91.3 was attributed to C-17, as evident from the HMBC correlations of H3-21 (δH 2.12), H3-18 (δH 0.91), and H2-16 2
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Fig. 2. Key HMBC ( ) and 1H–1H COSY (▬▬) correlations of compounds 1 and 4.
data, the glycosidic moiety of 4 was found similar to that of 3. The main difference of the aglycone in 4 was the replacement of a keto carbonyl carbon at C-20 by a hydroxy methine carbon, and the presence of a hemiketalic quaternary carbon instead of the oxymethine carbon at C17. The methyl signal at δH 1.20 (m, overlapped, H3-21) showed an HMBC interaction with δC 70.6 (C-20) and 100.3 (C-17), which further suggested the assignment. The relative configuration of C-17 was determined by ROESY correlations. The key cross peaks of H-8 (δH 1.55) with H3-19 (δH 0.98) and H3-18 (δH 1.00) were observed, which disclosed these groups as co-facial and β-oriented. The H3-18, in turn, showed correlations with H-20 (δH 3.84) and H3-21 (δH 1.20). Accordingly, an α-orientation was suggested for OH-17. However, the configuration of C-20 in the aglycone could not be established from ROESY spectrum due to free rotation of the C-17−C-20 bond. Enzymatic hydrolysis of compounds 4 and 6 was performed to yield the aglycone, which was confirmed the same for both the compounds by UHPLC-ESI-MS analysis with identical retention times (12.33 and 12.38 min, respectively) and mass data (see Supplementary Material, Figs. S35 and S37). Hence, the basic skeleton of the aglycone of 4 was established as epigynumgenane which was similar to that of epigynoside A (6) (Cao et al., 2005). The linkage of sugar moiety to C-20 of the aglycone via oxygen atom was deduced by an HMBC correlation of δH 4.77 (1H, br. s, H-1′) with δC 70.6, and of δH 3.84 (H-20) with 97.5 δC (C-1′) (Fig. 2). The positive HR-ESI-MS of epigycoside H (5) produced a sodium adduct ion at m/z 519.2933 [M + Na]+ (calcd 519.2928), representing the molecular formula C27H44O8. By comparing the 1D NMR spectroscopic data of 5 and 4, it was found that they shared the same aglycone moiety (Tables 1 and 2), but there were differences in sugar moieties. The sugar unit in 5 was deduced as 6-deoxy-β-D-idopyranose due to the absence of two methoxy groups and verified by the HMBC, 1H–1H COSY, and HSQC-TOCSY experiments. The NMR data of the sugar moiety in 5 was in line with that of 1. Acid hydrolysis of 5 and 1 and then the GC/MS analysis of trimethylsilylated thiazolidine derivatives of the sugars confirmed the same sugar moiety in both the compounds. Thus, the structure of compound 5 was characterized and named as epigycoside H. In addition to undescribed compounds, seven known compounds including four epigynumgenane-type steroids (6−9), two lignans (10–11), and a flavonoid (12) were also purified from Epigynum cochinchinensis. Their structures were determined as epigynosides A−C (6–8) (Cao et al., 2005), epigycoside A (9) (Wan et al., 2017), pinoresinol (10) (Brenes et al., 2000), syringaresinol (11) (Das et al., 1999), and santaflavone (12) (Goren et al., 2002) by comparing their spectroscopic data with those in the literature.
Fig. 3. X-ray ORTEP drawing of 1 with ellipsoids drawn at the 30% probability level. 1
H–1H COSY and HSQC-TOCSY spectra of 2 revealed the presence of a C–CH–CH–CH–CH–CH3 structural moiety. The structure of the sugar was deduced by the correlations of the anomeric proton signal at δH 4.77 (H-1′) with C-1′ (δC 97.0), C-2′ (δC 79.8), C-3′ (δC 71.6), C-4′ (δC 68.9), C-5′ (δC 70.3) and C-6′ (δC 16.4) in HSQC-TOCSY spectrum. The methoxy group was located at C-2′ of the monosaccharide unit due to HMBC correlations (Fig. S14). The monosaccharide unit was thus defined as 2-O-methyl-6-deoxy-β-D-idopyranose due to similar NMR data as that of epigynoside A isolated from Epigynum auritum (Cao et al., 2005). The connectivity of the sugar moiety was determined by an HMBC correlation of the anomeric proton signal at δH 4.77 to δC 78.1 (d, C-3). Thus, the structure of epigycoside E was established as 2. Epigycoside E (3) was obtained as colorless crystals, and its molecular formula C29H46O7 was established in regard to a sodiated HR-ESIMS ion (+ve) at m/z 529.3130 [M + Na]+ (calcd 529.3136), which differed from 1 by 28 more mass units. The IR and UV spectroscopic data of both the compounds were also similar. A detailed comparison of the 1H NMR spectra of 3 and 1 (Table 1) indicated that they had the similar basic skeleton, excluding two additional methoxy units in 3. The characteristic 13C NMR signals of a dioxygenated methine at δC 97.4, four oxymethines at δC 68.4, 70.6, 77.8, and 78.2, a methyl at δC 16.3, as well as two methoxy signals at δC 60.3 and 57.9 were almost identical to those of the sugar moiety in epigynoside E (i.e. 2,3-di-O-methyl6-deoxy-β-D-idopyranose) (Gao et al., 2017a). The absolute configuration of the sugar moiety in epigynoside E has already been established by X-ray diffraction analysis. The sugar-aglycone linkage was verified by an HMBC cross-peak from δH 4.77 (1H, br. s, H-1′) to δC 78.0 (d, C3). Thus, the structure of epigycoside F was characterized as 3. The molecular formula of epigynoside G (4) was determined as C29H48O8 by a sodiated ion peak at m/z 547.3232 [M + Na]+ (calcd 547.3241) in the HR-ESI-MS. By comparing the 1D NMR spectroscopic
2.2. Effects on splenocyte proliferation in vitro Furthermore, the cytotoxicity of the isolated steroids (1–9) was evaluated by the CCK-8 assay. For this purpose, the purified T-lymphocyte cells were treated with various concentrations of 1−9 (12.5, 3
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Table 1 1 H NMR spectroscopic data of compounds 1–5, (δ in ppm, J values in Hz)a. No.
1
2
1a 1b 2a 2b 3 4a 4b 6 7a 7b 8 9 11a 11b 12a 12b 14 15a 15b 16a 16b 17 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ 2′-OCH3 3′-OCH3
1.84 (dt, 13.4, 3.1) 1.01 (m) 1.93 (dt, 12.7, 3.2) 1.55 (m) 3.63 (m, W1/2 = 31.7) 2.34 (ddd, 13.0, 4.2, 1.9) 2.18 (m) 5.32 (t, 2.5) 2.05 (m) 1.41 (m) 1.41 (ovb) 1.01 (ov) 1.55 (ov) 1.32 (dd, 13.0, 3.1) 1.61 (dt, 13.0, 2.9) 1.22 (m) 1.01 (ov) 1.41 (ov) 1.01 (m) 4.12 (br. dd, 11.3, 4.0) 3.41 (m) 3.30 (s) 0.91(s) 0.94 (s)
1.86 1.03 1.98 1.60 3.57 2.35 2.23 5.33 2.06 1.44 1.40 1.03 1.57 1.33 1.63 1.24 1.03 1.44 1.03 4.14 3.44 3.32 0.91 0.96
(dt, 13.4, 3.2) (m) (m) (m) (tt, 11.4, 4.7) (ddd, 13.2, 4.8, 2.3) (m) (t, 2.5) (m) (m) (m) (ov) (m) (dd, 13.1, 2.8) (m) (d, 3.5) (ov) (ov) (ov) (ddd, 11.5, 4.8, 1.5) (td, 11.5, 3.3) (s) (s) (s)
1.87 1.04 2.00 1.62 3.57 2.34 2.24 5.35 2.15 1.55 1.46 1.04 1.58 1.34 1.60 1.23 1.04 1.42 1.04 4.16 3.45 3.33 0.93 0.98
(dt, 13.4, 3.3) (m) (m) (m) (m, W1/2 = 25.2) (ddd, 14.5, 4.7, 2.2) (m) (t, 2.4) (m) (m) (m) (ov) (m) (dd, 13.2, 3.4) (m) (m) (ov) (m) (ov) (br. dd, 11.4, 4.3) (td, 11.4, 3.0) (ov) (s) (s)
2.12 4.84 3.25 4.07 3.63 3.95 1.26
2.13 4.77 3.53 3.31 3.28 3.96 1.28 3.55
(s) (br. s) (m) (br. s) (m) (qd, 6.5, 1.5) (d, 6.5) (s)
2.15 4.77 3.33 3.66 3.36 3.84 1.29 3.57 3.42
(s) (br. s) (ov) (br. s) (m) (br. q, 6.6) (d, 6.6) (ov) (s)
a b
(s) (br. s) (d, 8.1) (br. s) (ov) (q, 6.4) (d, 6.4)
3
4
5
1.85 (dt, 13.3, 3.4) 1.05–1.01 (m, ov) 2.00 (m) 1.62 (m) 3.57 (m, W1/2 = 34.7) 2.34 (ddd, 12.9, 4.6, 2.0) 2.24 (m) 5.35 (t, 2.5) 2.08 (m) 1.42 (m) 1.55 (m) 1.05–1.01 (m, ov) 1.55 (ov) 1.34 (dd, 13.1, 5.1) 1.65 (m) 1.20 (m, ov) 1.65 (ov) 1.42 (ov) 1.34 (ov) 3.93 (ddd, 13.9, 11.1, 3.0) 3.70 (ddd, 11.1, 5.4, 0.9)
1.82 (dt, 13.4, 3.1) 1.01 (m) 1.92 (m) 1.58 (m) 3.59 (m, W1/2 = 34.6) 2.33 (ddd, 13.4, 4.9, 2.4) 2.16 (m) 5.31 (t, 2.4) 2.04 (m) 1.48 (m) 1.39 (m) 0.97 (ov) 1.49 (m) 1.30 (dd, 13.2, 2.6) 1.58 (m) 1.22 (m) 1.65 (m) 1.39 (ov) 1.28 (m) 3.90 (ddd, 13.5, 11.0, 3.1) 3.67 (br. dd, 11.0, 4.8)
1.00 (s) 0.98 (s) 3.84 (m) 1.20 (ov) 4.77 (br. s) 3.33 (m) 3.66 (t, 3.0) 3.36 (m) 3.85 (q, 6.5) 1.28 (d, 6.5) 3.58 (ov) 3.42 (s)
0.97 (s) 0.93 (s) 3.98 (m) 1.16 (d, 6.4) 4.84 (br. s) 3.30 (m) 3.99 (m) 3.56 (dd, 3.2, 1.0) 3.80 (q, 6.5) 1.25 (d, 6.5)
Measured for compounds 3 and 4 in MeOH-d4 while for 1, 2, and 5 in CDCl3. ov: overlapped.
25, and 50 μM), and the cell viability was measured. Results revealed that compounds 1–9 at the tested concentrations did not cause any significant cytotoxic effect in T-cells. Next, the immunosuppressive activity of 1−9 was assessed on mitogen-stimulated mice splenocytes, and dexamethasone (DXM) was used as a positive control. Fig. 4 shows the immunosuppressive results. In a concentration range from 12.5 to 50 μM, all the tested compounds displayed good inhibition on Con Ainduced spleen cell proliferation. When tested at 50 μM concentration, compound 2 had the strongest immunosuppressive activity, close to that of positive control (DXM), with similar stimulation indices of 1.24 and 1.17, respectively. However, DXM had a better immunosuppressive activity which was evident from its stimulation index at each tested concentration, significantly lower than all the compounds (p < 0.05). The median inhibitory concentration (IC50) values of compounds 1–9 are listed in Table 3. Compounds 4, 8, and 9 had a similar and relatively weaker inhibitory effect than 2, while compounds 3, 5, 6, and 7 possessed the weakest activity (IC50 > 80 μM).
Table 2 13 C NMR spectroscopic data of compounds 1–5, (δ in ppm)a. No.
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ 2′-OCH3 3′-OCH3
36.6 29.1 77.5 38.3 140.2 121.2 31.0 30.8 49.2 36.8 19.4 34.2 37.2 50.3 24.6 68.5 91.3 13.2 19.1 209.9 28.4 95.8 71.3 69.4 70.9 69.7 16.2
36.7 29.3 78.1 38.5 140.6 121.0 31.1 30.9 49.3 37.0 19.5 34.3 37.2 50.4 24.7 68.6 91.4 13.3 19.2 209.7 28.6 97.0 79.8 71.6 68.9 70.3 16.4 60.3
36.6 29.3 78.0 38.5 140.6 121.0 31.1 30.9 49.3 36.9 19.5 34.3 37.2 50.4 24.8 68.6 91.4 13.3 19.2 209.4 28.5 97.4 77.8 78.2 68.4 70.6 16.3 60.3 57.9
36.7 29.4 78.3 38.6 140.4 121.3 31.5 31.3 48.9 36.9 20.0 32.9 39.6 43.2 24.8 61.2 100.3 14.3 19.2 70.6 18.0 97.5 77.8 78.0 68.5 70.5 16.4 60.3 57.9
36.4 28.9 77.6 38.1 139.7 121.3 31.1 31.0 48.7 36.5 19.7 32.5 39.4 42.7 24.4 60.7 99.7 14.0 18.6 69.6 17.8 96.1 70.9 69.2 70.4 69.9 15.7
2.3. Epigycoside E induces G0/G1 phase arrest in Con A-induced T-cells To verify whether epigycoside E (2) treatment affects the Con Astimulated T-cells distribution, cell cycle was investigated in the presence of epigycoside E at 12.5, 25, and 50 μM. The population of T-cells in G0/G1, S, and G2/M phase was analyzed using Con A-stimulated Tcells by flow cytometry. As shown in Fig. 5, compared to that of the control group, the percentage of T cells in G0/G1 phase notably decreased from 74.21 to 65.81% in Con A treated group with a statistically significant difference (p < 0.01). The propidium iodide-stained T-cells showed that epigycoside E increased the cell frequency in G0/G1 phase (Fig. 5) in a concentration dependent manner. The percentage of
a
Measured for compounds 3 and 4 in MeOH-d4 while for 1, 2, and 5 in CDCl3. 4
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Fig. 4. Inhibitory effect of compounds 1–9 on Con A-stimulated T lymphocyte proliferation in vitro. DXM was used as a positive control. The values are presented as mean ± SD of triplicate replicates. Means (bar value) with different letters indicate significant differences (P < 0.05).
provide further evidence that epigycoside E (2) possesses good inhibitory activity on Con A-stimulated T-cells, ELISA was used to evaluate the effect of epigycoside E on the release of TNF-α and IL-2 cytokines. Compared with the control group, Con A treatment alone significantly induced the production of TNF-α and IL-2 cytokines (Fig. 6). Epigycoside E inhibited the production of TNF-α and IL-2, at concentrations ranging from 12.5 to 50 μM. For TNF-α production, epigycoside E had a similar and higher inhibitory effect at 25 and 50 μM with no significant difference (P > 0.05). Treatment of Con A-induced T-cells, with 2 at 50 μM concentration, showed a significant inhibition in the production of TNF-α, and there was no significant difference compared with the control group (Fig. 6A). Interestingly, the inhibition of IL-2 production was up to 58.2 ± 2.8 pg/mL when the con A-stimulated T-cells were exposed to 50 μM epigycoside E (Fig. 6B). Epigycoside E had a similar inhibitory effect on TNF-α and IL-2 production, and there was no significant difference in the concentrations of TNF-α in Con A-induced T-cells treated with 25 and 50 μM of epigycoside E. Hence, epigycoside E may have non-selective regulation on cytokines production. At present, we do not understand the mechanism of its effect, and we will further explore it in our subsequent studies.
Table 3 Immunosuppressive effect (IC50) of compounds 1–9 (mean ± SD, n = 3). Compound
IC50 ± SD (μM)
1 2 3 4 5 6 7 8 9 DXM (positive control)
51.7 ± 22.1 ± > 80 40.8 ± > 80 > 80 > 80 46.0 ± 45.5 ± 11.6 ±
17.5 6.4 12.1
16.1 15.3 0.7
T-cells in G0/G1 phase was 68.27, 70.64, and 73.39% when exposed to 12.5, 25, and 50 μM of epigycoside E, respectively. The G0/G1 phase arrest was induced by 7.58% after incubation with epigycoside E at a concentration of 50 μM compared to the Con A model group. The cell progression of epigycoside E treatment was markedly inhibited through G1 to S transition, which may influence the cell cycle regulatory molecules in G0/G1 phase. It was revealed in a study that propidium iodide-stained cells increased the percentage of G0/G1 cells according to the erlotinib concentration ranging from 1–15 μM (Luo et al., 2011). The cell cycle progression of 3-MCPD esters on Con A-induced T-lymphocytes caused G0/G1 arrest (Huang et al., 2018). These results provide a theoretical basis for the immunosuppressive mechanism of epigycoside E. The cell population at different stages may be involved by the expression levels of cell cycle regulatory proteins, such as cyclin A, D1, D3, and E, p-Rb, p21, p27, and so on (Luo et al., 2011; Wang et al., 2014). These findings may provide new insights to further understand the mechanism of cell-cycle arrest at molecular level.
3. Conclusions In our ongoing search for bioactive compounds from Epigynum plants, five undescribed pregnane glycosides named as epigycosides D−H (1–5), along with four known analogues, two lignans, and a flavonoid were isolated from the EtOAc fraction of E. cochinchinensis. Their structures were elucidated by extensive spectroscopic techniques and single-crystal X-ray diffraction analysis. Epigynosides A−C (6–8), epigycoside A (9), pinoresinol (10), syringaresinol (11), and santaflavone (12) are isolated for the first time from E. cochinchinensis, among which the pregnane glycosides with epigynumgenane skeleton are only reported from the plants of genus Epigynum. Compounds 1–9 showed good immunosuppressive activity against the proliferation of Tlymphocytes, among which epigycoside E (2) was the most effective immunosuppressant. Epigycoside E caused the G0/G1 phase arrest and the inhibition of multiple cytokines’ production, including TNF-α and IL-2. Therefore, the current study suggests that the plant E.
2.4. Inhibitory effect of epigycoside E on cytokines production Pro-inflammatory cytokines, predominantly interleukin (IL)-2 and tumor necrosis factor (TNF)-α have a prominent role in the regulation of autoimmune diseases (Gordon, 2003; Mesaik et al., 2010). Thus, to 5
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Fig. 5. Con A-induced T-lymphocyte cells treated with epigycoside E for 72 h. Cell cycle data are representative of three individual experiments. *P < 0.05, **P < 0.01, means significant difference from the G0/G1 phase group. #P < 0.05, means significant difference from the S phase group. &P < 0.05, means significant difference from the G2/M phase group.
packed glass columns (15 × 230 and 26 × 460 mm), was used for MPLC. Silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Chromatorex C18 (20–45 μm, Fuji Silysia Chemical Ltd., Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden) were used for column chromatography. GF254 silica gel plates (Qingdao Marine Chem. Ltd., Qingdao, China) were used for carrying out the TLC. Spots were visualized by spraying with 10% H2SO4 solution in EtOH and heating at 105 °C. The absorbance in bioassays was measured and recorded on a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cell proliferation rate was estimated by using a cell counting kit (CCK-8, Dojindo Molecular Technologies, Maryland, USA). Cell cycle assay kit and apoptosis analysis kit were purchased from Beyotime (Shanghai, China); mouse cytokines (TNF-α and IL-2) detecting ELISA kits were purchased from Beijing 4A Biotech Co., Ltd (Beijing, China).
cochinchinensis might be served as a potent immunosuppressive agent for the treatment of autoimmune diseases.
4. Experiments 4.1. General experimental procedures Optical rotations were determined with a JASCO P-1020 digital polarimeter. UV spectra were recorded with a Shimadzu UV-2401A spectrophotometer. IR spectra, with KBr pellets, were recorded with Bruker FT-IR Tensor 27 spectrometer. 1D and 2D NMR spectra were recorded on a Bruker DRX-500 MHz or an Avance III–600 MHz spectrometer with TMS as an internal standard. HRESIMS data were taken on a Thermo high resolution Q Exactive focus mass spectrometer. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph equipped with Zorbax SB-C18 columns (9.4 × 150 and 21.2 × 250 mm). Büchi pump system, equipped with C18 silica gel-
Fig. 6. A-B. Immunosuppressive effect of epigycoside E (2) on Con A induced T cells. The concentrations of TNF-α and IL-2 were detected in the supernatant in presence of 2 for 72 h at 12.5, 25 and 50 μM, respectively. The values are presented as mean ± SD of triplicate replicates. Different letters indicate significant differences (P < 0.05). 6
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and 13C NMR (150 MHz) data (MeOH-d4), see Tables 1 and 2; positive ion HRESIMS m/z 529.3130 [M + Na]+ (calcd for C29H46O7Na+, 529.3136).
4.2. Plant material The stems of Epigynum cochinchinensis (Pierre) Mabb. (Apocynaceae) were collected in May 2018 (summer season) from Kasi district (GPS coordinates: 19°14′N/102°17′E) of Vientiane province, Lao People's Democratic Republic, and identified by Prof. Yunhong Tan, Xishuangbanna Tropical Plant Garden. A voucher specimen (No. Cao20180510-01) is preserved at the Laboratory of Functional Foods, Yunnan Institute of Food Safety, Kunming University of Science and Technology.
4.3.4. Epigycoside G (4) White amorphous powder; [α]20 D -61.3 (c 0.03, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.5); IR (KBr) νmax: 3447, 2933, 1632, 1450, 1381, 1260, 1177, 1101, 1076, 1050 cm−1; 1H NMR (800 MHz) and 13C NMR (200 MHz) data (MeOH-d4), see Tables 1 and 2; positive ion HRESIMS m/z 547.3232 [M + Na]+ (calcd for C29H48O8Na+, 547.3241).
4.3. Extraction and isolation 4.3.5. Epigycoside H (5) White amorphous powder; [α]25 D -82.6 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 203 (3.2); IR (KBr) νmax: 3439, 2935, 2759, 1532, 1497, 1164, 1038, 612 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 519.2933 [M + Na]+ (calcd for C27H44O8Na+, 519.2928).
The air-dried and powdered stems of E. cochinchinensis (11.0 kg) were extracted with 70% aqueous MeOH under reflux condition and the solvent was evaporated in vacuo to obtain the MeOH extract, followed by suspension in H2O. The H2O layer was then successively partitioned with petroleum ether (10 L × 3) and EtOAc (10 L × 4). The EtOAc fraction (152 g) was fractioned over RP-18 column, eluted with a gradient of MeOH–H2O (20:80–100:0) to give six fractions (Frs. A-F). Fr. B (22 g) was then chromatographed on RP-18 column, eluting with MeOH–H2O (60:40–90:10) to yield five sub-fractions. Fr. B1 (220 mg) was subjected to semi-preparative reversed-phase C18-HPLC column eluting with a gradient of MeOH–H2O (60:40–80:20), followed by Sephadex LH-20 column eluting with MeOH–H2O (50:50, v/v), and further purified by semi-preparative HPLC (MeCN–H2O, 65:35) to obtain compounds 5 (5 mg, tR = 13 min) and 9 (4.6 mg, tR = 15 min). Fr. C (10.3 g) was fractionated by silica gel column (CHCl3-acetone, 20:1) to give four fractions, Frs. C1–C4. Fr. C2 was purified by semi-preparative HPLC (MeOH–H2O, 70:30) to yield compounds 7 (3 mg, tR = 9 min) and 11 (6 mg, tR = 11 min). Fr. C4 was purified by semipreparative HPLC (MeCN–H2O, 60:40) to give compound 6 (20 mg, tR = 15.5 min). Fr. D (23 g) was subjected to RP-18 column, eluting with MeOH–H2O (70:30–95:5) to afford Frs. D1-D5. Fr. D2 was subjected to silica gel CC and eluted with CHCl3-acetone (10:1–1:1) to yield compound 1 (11 mg). Fr. E (7 g) was subjected to an RP-18 column (MeOH–H2O, 70:30–90:10) to afford Frs. E1-E3. Fr. E2 (412 mg) was purified by silica gel CC and eluted with CHCl3-acetone (10:1) and then subjected to preparative TLC (petroleum ether: acetone, 3:1, Rf = 0.55) to yield compound 3 (5.8 mg). Fr. E3 (108 mg) was chromatographed over RP-18 silica gel (MeOH–H2O, 75:25–85:15) and further purified by semi-preparative HPLC (MeCN–H2O, 78:22) to give compounds 2 (5.8 mg, tR = 10.5 min) and 10 (2.7 mg, tR = 13.5 min). Fr. E5 (253 mg) was subjected to silica gel CC, eluting with CHCl3–MeOH (20:1), and further purified by semi-preparative HPLC (MeOH–H2O, 65:35) to yield compounds 4 (7 mg, tR = 9.5 min), 8 (3.8 mg, tR = 12 min), and 12 (2 mg, tR = 14 min).
4.4. X–ray crystallographic analysis of epigycoside D The Cu Kα radiation in Bruker APEX DUO diffractometer, equipped with an APEX II CCD, was used to get the X-ray data. The intensity data for epigycoside D (1) were collected at 100 K. Bruker integration program SAINT was used for cell refinement and data reduction. Structure was expanded by Fourier techniques, resolved by direct methods, and refined by the program and full-matrix least squares calculations. All the hydrogen atoms were fixed at calculated positions while all the nonhydrogen atoms were refined anisotropically (Cheng et al., 2016a). Crystallographic data for epigycoside D has been placed in Cambridge Crystallographic Data Center as supplementary publications (number: CCDC, 1880756). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ, U.K.; fax: (+44) 1223-336-033; or [email protected]). Crystallographic data for epigycoside D (1): 2(C27H42O7)•CH4O, M = 989.25, a = 7.66050(10) Å, b = 33.7814(6) Å, c = 10.0205(2) Å, α = 90°, β = 100.58°, γ = 90°, V = 2549.08(8) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.750 mm−1, 26,778 reflections measured, 8382 independent reflections (Rint = 0.0275). The final R1 values were 0.0366 (I > 2σ(I)). The final wR(F2) values were 0.1005 (I > 2σ(I)). The final R1 values were 0.0367 (all data). The final wR(F2) values were 0.1007 (all data). The goodness of fit on F2 was 1.043. Flack parameter = 0.06(3). 4.5. GC/MS analysis of sugars from compounds 1–5 Compounds 1–5 (each 2 mg) were dissolved in 2 mL HCl (2 M) and then hydrolyzed in a water bath at 90 °C for 2 h. After reaction, the mixture was partitioned with CHCl3 for three times. The aqueous layer was neutralized with 2 M NaOH and evaporated in vacuo to obtain a residue. The residue was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg) and stirred at 60 °C for 1 h. Trimethylsilylimidazole (1.5 mL) was added to the reaction mixture and incubated for 30 min at 60 °C. Then each sample was subjected to a Shimadzu GC/MS-QP2010 instrument, equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5 quartz capillary column and an H2 flame ionization detector under the conditions described elsewhere (Gao et al., 2017a; Cheng et al., 2016b). The configuration of sugar moieties was determined by comparing the retention times of their trimethylsilylated thiazolidine derivatives. The retention times of the sugar derivatives were 15.5 min for 1 and 5, 22.5 min for 3 and 4, and 17.5 min for 2, respectively. The retention times of 2-O-methyl-6deoxy-β-D-idopyranose and 2,3-di-O-methyl-6-deoxy-β-D-idopyranose derivatives were identical to those reported in the literature (Gao et al., 2017a).
4.3.1. Epigycoside D (1) White amorphous powder; [α]26 D -82.6 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 203 (3.6); IR (KBr) νmax: 3440, 1710, 1641, 1448, 1257, 1030, 960, 866, 661, 541 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 501.2825 [M + Na]+ (calcd for C27H42O7Na+, 501.2823). 4.3.2. Epigycoside E (2) White amorphous powder; [α]25 D -127.8 (c 0.3, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.1); IR (KBr) νmax: 3425, 2934, 1735, 1709, 1374, 1251, 1068 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data (CDCl3), see Tables 1 and 2; positive ion HRESIMS m/z 515.2980 [M + Na]+ (calcd for C28H44O7Na+, 515.2979). 4.3.3. Epigycoside F (3) White amorphous powder; [α]20 D -56.5 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 202 (3.6); IR (KBr) νmax: 3439, 2934, 1711, 1633, 1446, 1381, 1257, 1177, 1102, 1072, 1049 cm−1; 1H NMR (600 MHz) 7
Phytochemistry 168 (2019) 112127
Z. Wang, et al.
4.6. Enzymatic hydrolysis and UHPLC-ESI-MS analysis of compounds 4–6
References
Compounds 4–6 (each 2 mg) in MeOH–H2O (1:100, 10 mL) were hydrolyzed with β-glucosidase (100 mg) at 37 °C for 4 days. The reaction mixture was subjected to reversed-phase C18-HPLC column eluting with MeOH–H2O (70:30) to yield the aglycone (Arthan et al., 2002). In order to confirm that compounds 4–6 had the same aglycone, an UHPLC-ESI-MS experiment was carried out. The UHPLC-ESI-MS TIC chromatograms of aglycones from compounds 4–6 are shown in Supplementary Material (Figs. S35–S37).
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4.7. Splenocyte proliferation assay A previously reported CCK-8 method was used to carry out the immunosuppressive activity (Gao et al., 2017a). 4.8. Cell cycle assay The purified T-cells from male BALB/c mice (2 × 106 cells/well) were cultured in 6-well plates and treated with or without compounds for 72 h in the presence of Con A (10 μg/mL). After incubation, the cell suspension was centrifuged for 5 min at 1000 g. Cells were collected, washed with cold PBS and fixed with 70% ice-cold EtOH at 4 °C overnight. Then the fixed cells were centrifuged and washed with cold PBS, and repeated twice. Stained with 10 μL propidium iodide (PI; 1 mg/mL) and 10 μL RNase A (10 mg/mL) for 30 min. Cell cycle was analyzed by guava® easyCyte 6-2L (Millipore, Billerica, MA, US) flow cytometry. 4.9. Determination of TNF-α and IL-2 cytokines production The concentrations of TNF-α and IL-2 from the collected supernatants were quantified by ELISA and the assay procedures were carried out as described in the kit manual. Briefly, freshly prepared mononuclear cells (1 × 105/well) were seeded in 96-well microtiter plate and treated with or without compounds at different concentrations in the presence of Con A (10 μg/mL). The plate was incubated at 37 °C for 72 h, then, the supernatants were collected and analyzed for TNF-α and IL-2 cytokines production. 4.10. Statistical analysis All the statistical tests were performed with the statistical program SPSS 16.0. The data were expressed as mean ± SD. Statistical significance was set at p < 0.05. All the analyses were performed using Origin 8.5 software (OriginLab, Northampton, MA, USA). Conflicts of interest The authors declare no conflict of interest. Acknowledgements This research was supported by the National Natural Science Foundation of China (grant numbers 31600274, 31960094 and 31460083), the Applied Basic Research Project of Yunnan Province (Grant No. 2018FB036), and the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University (Grant No. ZYAOX2018031). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112127. 8