Article pubs.acs.org/JAFC
Diterpenoids from Salvia miltiorrhiza and Their Immune-Modulating Activity Wen-Jun Wei,† Pan-Pan Zhou,† Chang-Jun Lin,‡ Wei-Feng Wang,‡ Ya Li,*,† and Kun Gao*,† †
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering and ‡School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, People’s Republic of China S Supporting Information *
ABSTRACT: Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), is one of the most popular traditional herbal medicines commonly used in China. Recently, danshen has been used as a health-promoting functional tea to prevent diseases by strengthening the human immunity in China. To search for secondary metabolites with immune-modulating activity, a phytochemical investigation was carried out on the roots of S. miltiorrhiza, which led to the isolation of 6 new diterpenoids (1−4, 16, and 20) along with 20 known diterpenoids. The structures and absolute configurations of these new compounds were elucidated on the basis of spectroscopic analysis, X-ray diffraction analysis, calculated optical rotation, and calculated electronic circular dichroism spectra. Among these isolates, compounds 3, 17, 19, and 23 promoted the proliferation of HMy2.CIR, exhibiting a protective effect on lymphocytes at the concentration from 2.50 to 40 μM, whereas compounds 2, 7, 8, 10, 14, 18, 22, and 25 inhibited the cell proliferation in a concentration-dependent manner. KEYWORDS: Salvia miltiorrhiza, diterpenoids, immune-modulating activity, human B lymphoblastoid cells
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measured on a PerkinElmer model 341 polarimeter. The ultraviolet (UV) spectra were obtained by a Shimadzu UV-260 spectrophotometer. Electronic circular dichroism (ECD) spectra were performed with an Olis DSM-1000 spectrometer, and the infrared (IR) spectra were acquired using a Bruker Tensor 27 spectrometer. High-resolution electrospray ionization mass spectrometry (HRESIMS) was carried out on a Bruker Daltonics APEX II spectrometer. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury-600BB or a Bruker AVANCE III-400 spectrometer with tetramethylsilane (TMS) as an internal standard. Semi-preparative high-performance liquid chromatography (HPLC) was carried out on Waters equipment (1525 pump and 2998 photodiode array detector) with a SunFre Prep C18 column (150 × 10 mm, 10 μm, with a flow rate of 2 mL/min). LiChroprep RP-C18 gel (40−63 μm, Merck, Germany) and Sephadex LH-20 were purchased from Amersham Pharmacia Biotech. Silica gel (200−300 mesh) used for column chromatography and silica GF254 (10−40 mm) used for thin-layer chromatography (TLC) were both supplied by the Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China. Plant Materials. The roots of S. miltiorrhiza were collected from Hebei, China, in June 2015 and identified by Zhang Guoliang, a professor at Lanzhou University. A voucher specimen (20150601) was stored at the Natural Product Laboratory of State Key Laboratory of Applied Organic Chemistry, Lanzhou University. Extraction, Isolation, and Purification Process. The dried roots of S. miltiorrhiza (10.0 kg) were extracted 3 times (every time for 7 days) with 95% EtOH (40 L) at room temperature, and the combined solvent was evaporated under reduced pressure to give the extract (401.3 g). This extract was then suspended in water (1 L), and the aqueous solution was extracted with EtOAc (1 L × 3) and n-butyl alcohol (1 L × 3), successively. The EtOAc extract (107 g) was chromatographed over macroporous resin, eluted with gradient
INTRODUCTION Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), is one of the most popular traditional herbal medicines commonly used in China. It has been used for the treatment of various diseases, including cardiovascular and cerebrovascular diseases, angina pectoris, myocardial infarction, chronic hepatitis, and liver fibrosis.1−3 Recently, danshen has been used as a healthpromoting functional tea to prevent diseases by strengthening the human immunity in China. Extensive studies on the chemical compositions of danshen have been carried out over the past few years. Numerous abietane-type diterpenoids have been obtained from this species, which exhibit diverse biological activities, such as antitumor, antibacterial, antifungal, antioxidant, anti-inflammatory, and antidiabetic activities.4,5 The lymphocyte is a kind of leukocyte produced by the lymphoid organs. The proliferative capacity of lymphocytes is often used to assess the level of immune response. The role of the immune system has become increasingly important in the understanding of the mechanisms involved in disease prevention.6 However, little is known regarding the immunemodulating activities of diterpeneoids from S. miltiorrhiza. For these reasons, we investigated the diterpeneoids from S. miltiorrhiza, and their activity on the proliferation of human B lymphoblastoid cells (HMy2.CIR) was also tested in vitro. Here, we reported the isolation, structure identification, and immune-modulating activity of the diterpenoids isolated from the roots of S. miltiorrhiza.
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MATERIALS AND METHODS Received: Revised: Accepted: Published:
General Experimental Procedures. X-ray crystallography was performed on a Bruker Smart charge-coupled device (CCD) diffractometer (Bruker, Ltd., Karlsruhe, Germany) using graphicmonochromated Mo Kα radiation. Optical rotations were then © 2017 American Chemical Society
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May 22, 2017 June 23, 2017 July 5, 2017 July 5, 2017 DOI: 10.1021/acs.jafc.7b02384 J. Agric. Food Chem. 2017, 65, 5985−5993
Article
Journal of Agricultural and Food Chemistry Table 1. 1H NMR Data of Compounds 1,a 2,b 3,b 4,c 16,b and 20b δH multi (J in Hz) number
1
2
1
8.83 d (8.4)
9.57 d (8.0)
2 3
7.66 t (8.4) 7.56 d (8.4)
7.51 t (8.0) 7.43 d (8.0)
3.15 3.45 2.20 6.09
8.52 d (9.0) 7.84 d (9.0)
7.81 d (9.2) 7.68 d (9.2)
7.30 d (8.0) 7.56 d (8.0)
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
3 2.99 3.65 1.86 1.70 2.33
16
m m m m m
9.24 d (8.8)
7.28 d (8.4) 7.53 d (8.4)
20
7.57 t (8.8) 7.39 m
8.29 d (8.4) 7.38 m
2.48 1.61 5.23 1.89 1.33
d (10.0) t (12.0) m dd (10.0, 2.4) m
2.13 t 5.86 dd (9.6, 2.4) 6.54 dd (9.6, 2.8)
6.57 s 3.34 6.16 2.27 4.40 3.79 0.57 2.79
m d (2.4) m t (9.0) t (9.0) d (6.6) s
7.31 3.85 4.90 4.30 1.49 2.76
s m t (8.4) t (8.0) d (6.8) s
3.60 4.91 4.32 1.40 2.81
21 22 23 11-OH a1
m m m t (7.6)
4
H NMR (600 MHz), TMS, measured in CD3OD. CDCl3.
m t (9.6) t (8.4) d (6.8) d (6.8)
m t (10.0) t (8.8) d (6.4) s
3.08 d (12.8) 2.97 d (12.8)
2.93 d (12.8) 3.11 d (12.8)
2.04 s
2.04 s 3.67 s 4.53 s
4.41 brs b1
3.58 4.92 4.32 1.40 1.57
6.91 s 3.16 m 1.27 d (6.8)
3.17 m 3.78 m 1.30 d (7.2) 2.69 s
1.24 1.04 1.13 1.10
d (6.8) s s s
2.10 s
H NMR (400 MHz), TMS, measured in CDCl3. c1H NMR (600 MHz), TMS, measured in (150 MHz, CD3OD), see Tables 1 and 2; and HRESIMS m/z, 319.0932 [M + Na]+ (calcd for C18H16O4Na, 319.0941). Salmiltiorin B (2): red oil; [α]25 D −74 (c 0.5, CHCl3); IR (KBr) νmax, 3409, 2924, 2373, 1588, 1459, 1357, 1133, 1024, 765 cm−1; ECD Δε, 222 + 3.03, 245 − 4.44, 258 + 4.76, 272 − 3.59; UV (MeOH) λmax (log ε), 219.0 (0.313), 283.5 (0.313) nm; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), see Tables 1 and 2; and HRESIMS m/z, 265.1220 [M + H]+ (calcd for C18H16O2H, 265.1223). Salmiltiorin C (3): yellow oil; [α]25 D −128 (c 0.5, CHCl3); IR (KBr) νmax, 3412, 2965, 2928, 1705, 1620, 1423, 1357, 1301, 1186, 935, 755 cm−1; ECD Δε, 247 − 29.92, 326 + 22.80; UV (MeOH) λmax (log ε), 229.0 (0.693), 297.5 (0.233), 341.5 (0.245) nm; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), see Tables 1 and 2; and HRESIMS m/z, 361.1413 [M + H]+ (calcd for C21H22O4H, 361.1410). Salmiltiorin D (4): yellow oil; [α]25 D −50 (c 0.2, CHCl3); IR (KBr) νmax, 3420, 2938, 1726, 1623, 1421, 1364, 1216, 1113, 757 cm−1; ECD Δε, 249 − 17.52, 320 + 9.34, 354 − 9.66; UV (MeOH) λmax (log ε), 324.0 (0.409) nm; 1H NMR (400 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3), see Tables 1 and 2; and HRESIMS m/z, 399.1794 [M + H]+ (calcd for C23H26O6H, 399.1802). Salmiltiorin E (16): red powder; [α]25 D −30 (c 0.2, CHCl3); IR (KBr) νmax, 3395, 2926, 2372, 1656, 1588, 1459, 1218, 1113, 1031, 756 cm−1; ECD Δε, 208 + 1.73, 257 − 1.45, 295 + 0.66; UV (MeOH) λmax (log ε), 235.0 (0.560), 286.5 (0.234) nm; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), see Tables 1 and 2; and HRESIMS m/z, 303.0986 [M + Na]+ (calcd for C18H16O3Na, 303.0992). Salmiltiorin F (20): yellow oil; [α]25 D −20 (c 0.1, CHCl3); IR (KBr) νmax, 3371, 2961, 1735, 1603, 1423, 1365, 1263, 1028, 757 cm−1; ECD
mixtures of H2O/EtOH (70:30, 50:50, 20:80, and 5:95) to afford four fractions (A−D). Fraction C (37.4 g) was chromatographed on a silica gel column with a gradient of petroleum/acetone (15:1, 5:1, 2:1, 1:1, and 1:2) as the eluent, and then nine subfractions (C1−C9) were collected according to TLC analysis. Fraction C2 (3 g) was applied to reversed-phase column chromatography (from 30:70 to 100:0 MeOH/H2O) to give eight fractions (C2a−C2h). Fraction C2d was separated on a silica gel column (25:1, 15:1, and 5:1 PE/EtOAc) to afford compounds 20 (20.4 mg), 22 (14.7 mg), 24 (10.0 mg), 25 (6.4 mg), and 26 (16.0 mg). Fraction C2f was further purified on Sephadex LH-20 (MeOH), followed by silica gel column chromatography to afford compounds 9 (12.2 mg), 10 (10.4 mg), and 23 (9.4 mg). Fraction C3 (10 g) was separated on a silica gel column with a gradient of PE/EtOAc (15:1, 5:1, 2:1, and 1:2) as the eluent, to yield compounds 2 (17.0 mg), 11 (15.1 mg), 14 (510.6 mg), and 21 (11.0 mg). Fraction C4 (6 g) was separated on Sephadex LH-20 chromatography (1:1 MeOH/CHCl3) to afford compounds 4 (4.0 mg), 12 (3.9 mg), 13 (823.4 mg), and 16 (4.1 mg). Compounds 12 (3.9 mg), 15 (103.5 mg), 17 (5.4 mg), and 18 (20.5 mg) were obtained from fraction C5 (9 g) by silica gel column chromatography with gradient mixtures of PE/CHCl3/EtOAc (15:1:1, 5:1:1, 2:1:1, and 1:1:1) as the eluent. Fraction C6 (13 g) gave compounds 5 (13.1 mg), 6 (8.0 mg), 7 (20.7 mg), and 8 (6.4 mg) over reversed-phase column chromatography (from 30:70 to 100:0 MeOH/H2O). Fraction C7 was purified with the aid of reversed-phase semi-preparative HPLC, eluted with MeOH/H2O (1:2, 2 mL/min) to obtain compounds 1 (3.4 mg), 3 (16.0 mg), and 19 (7.3 mg). Salmiltiorin A (1): colorless crystal; melting point (mp), 201−204 °C; [α]20 D −67 (c 0.1 CHCl3); IR (KBr) νmax, 2962, 2368, 1756, 1586, 1260, 1097, 797 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR 5986
DOI: 10.1021/acs.jafc.7b02384 J. Agric. Food Chem. 2017, 65, 5985−5993
Article
Journal of Agricultural and Food Chemistry Table 2. 13C NMR Data of Compounds 1,a 2,b 3,b 4,c 16,b and 20b
various concentrations of these compounds and (three wells without drugs) fixed with 10 μL of 50% trichloroacetic acid (TCA) to represent the cell population at the time of drug addition (T0). After additional incubation for 48 h, cells were fixed with 50 μL of 50% TCA. Then, the cells were stained with SRB at 0.4% (v/v) in 1% acetic acid. Unbound SRB was washed out by 1% acetic acid, while SRBbound cells were solubilized with 10 mM Trizma base. The absorbance at 540 nm was measured using a Microplate Reader. Using the following absorbance measurements, such as time zero (T0), control growth (C), and cell growth in the presence of these compounds (Tx), the percentage growth was calculated at each of the compound concentration levels. The cell proliferation was calculated as (Tx − T0)/(C − T0). Statistical Analysis. All data presented were obtained from three independent experiments and were presented as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). The mean values were compared using Student’s t test, and p values of
