Diterpenoids isolated from the root of Salvia miltiorrhiza and their anti-inflammatory activity

Abstract Four new diterpene-type compounds normiltirone (3) and isosalviamides F-H (14–16) together with twelve known compounds (1, 2, 4–13) were isolated from the roots of Salvia miltiorrhiza. Their structures were mainly elucidated from detailed spectroscopic data. All isolated compounds were evaluated for their ability to inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages. Compound 11 showed a strong inhibitory effect, with an IC50 value of 3.4 ± 1.2 μM. Graphical Abstract


Introduction
The widespread Lamiaceae family (formerly Labiatae) comprises 220 genera and 4000 species distributed throughout most of the world as annual and perennial plants (Wu et al. 2012). This family includes the genus Salvia, which is the largest genus and consists of approximately 900 species. Many species of this genus are used in traditional medicine (Pan et al. 2010). In particular, the species Salvia miltiorrhiza Bunge has drawn the attention of natural product chemists and medicinal clinicians (Don et al. 2006). It has been used extensively as an important herbal drug for the treatment of coronary heart diseases, particularly angina pectoris and myocardial infarction (Wang et al. 2007). Moreover, it has been used to treat cerebrovascular disease, Alzheimer's disease, Parkinson's disease, renal deficiency, hepatocirrhosis, cancer, and bone loss (Su et al. 2015). Research into this plant revealed that phenolic acids and tanshinones were the main chemical components related to its bioactivities and pharmacological properties (Wang 2010;Zhang et al. 2018). Among the tanshinones, several were shown to exhibit antitumor (Xie et al. 2015;Mosaddik 2003), antimicrobial (Laila et al. 1993), anti-immunological (Kang et al. 2000), anti-inflammatory (Jang et al. 2003;Kim et al. 2002), anti-allergic (Choi and Kim 2004), and antioxidant (Weng and Gordon 1992) properties. In addition, the nitrogen-containing diterpenes were cytotoxic in HeLa, HepG2, and OVCAR-3 cell lines (Don et al. 2005).
In this study, a phytochemical investigation of the root of S. miltiorrhiza led to the isolation of four new diterpene-type compounds, normiltirone (3) and isosalviamide F-H (14-16), together with twelve known compounds (1, 2, 4-13). Their structural compounds were elucidated by combination analysis based on their NMR and mass spectra. The 1 H-and 13 C-NMR spectra were assigned based on homonuclear correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC). This paper reports the isolation and structural determination of the isolated compounds, as well as their inhibitory effect on nitric oxide (NO) production. Furthermore, all isolated compounds were screened for their cytotoxicity and ability to inhibit NO production.
Compound 3  The 13 C-NMR spectrum of compound 3 contained 19 signals, which were classified by DEPT experiments into two carbonyl groups, eight olefinic carbons (including five olefinic quaternary carbons), one aliphatic quaternary carbon, one aliphatic methine carbon, three aliphatic methylene groups, and four methyl groups (Table S1, supplementary material). The above findings accounted for three of the nine degrees of unsaturation, which indicated that 3 was apparently a tricyclic compound. The structure of 3 is analogous to resmariquinone, except for an additional oxygen in the molecular formula which has upfield-shifted signals of C-11 (162.0) and C-12 (160.7), compared with C-11 (182.4) and C-12 (181.5) of the ortho-quinone (Houlihan et al. 1985). Based on the signals of an anhydride moiety and key HMBC correlations ( Figure S5, supplementary material), one of three rings suggested the presence of a seven-membered-ring anhydride in the molecule. This type of 20-nor-abietane diterpenoids has been previously isolated from plants of the Salvia genus (Chang et al. 1990). In the HMBC spectrum, the long-range correlations of H-18/H-19 and C-3/C-4 and H-15 and C-12/C-13/C-14 led to the assignment of two methyl groups and the isopropyl group at C-4 and C-13. Thus, the structure of compound 3 (normiltirone) was conclusively determined.
Compound 14   J ¼ 9.2 Hz, H-7)], and two methyl groups [d H 1.40 (3H, d, J ¼ 6.9 Hz, H 3 -17) and 2.71 (3H, s, H 3 -18)]. The 13 C-NMR and DEPT spectra of compound 14 indicated the presence of 22 carbon signals including two methyl, six methine, the methylene, and 11 quaternary carbons. The positions of the methyl groups (CH 3 -17 and CH 3 -18) were indicated by the HBMC correlations of H 3 -18 with C-3, 4, and 5, and H-17 with C-13, 15, and 16. The COSY and HMBC spectra showed that compound 14 was a derivative of dihydrotanshinone I (11), except for the signal of H-1 (d H 10.74) and the presence of the ¼ CCH 2 CH 2 COONH 2 side chain ( Figure S2.6, S2.8, and S5, supplementary material). The chemical shift value of C-11 (d C 133.6) and C-12 (d C 147.0) indicated that ring E was an oxazole ring instead of an ortho-quinone group in 11. The place of the nitrogen and oxygen atoms in the oxazole ring (E ring) were deduced on the basic of chemical shift values and through comparison of the 1 H and 13 C NMR data with two known compounds of the same skeleton, salviamine D [the N atom at C-12 with H-1 (d H 9.19), C-11 (d C 144.1), and C-12 (d C 137.7)] and isosalviamine D [the N atom at C-11 with H-1 (d H 10.13), C-11 (d C 134.1), and C-12 (d C 143.0)], by the method of Don et al (Don et al. 2005). Thus, it was suggested that in compound 15, the N atom of the oxazole ring attached to C-11. The presence of CH 2 CH 2 COONH 2 chain was indicated by two methylene groups at d  (Tezuka et al. 1997) figures isolated from the Salvia genus. Therefore, the identity of compound 14 was established as isosalviamide F.
Compound 15 produced a molecular ion peak at m/z 358.1317 [M] þ (calcd. for C 22 H 18 N 2 O 3 , 358.1317) in HR-EI-MS, with two hydrogen atoms fewer than compound 14. The 1 H-and 13 C-NMR spectra of 15 also displayed signals for an AMX system, a pair of ortho-coupled doublets, H-1 resonance shifted downfield at d H 10.83 (d, J ¼ 8.5 Hz), the position of methyl group CH 3 -18 at d H 2.74 (3H, s) on C-4, and an oxazole ring with a -CH 2 CH 2 COONH 2 unit at C-20. Comparison of the NMR data (Table S1, supplementary material) between 15 and 14 showed that these compounds have the same skeleton, with the exception of the ring D. The HMBC correlations of a methyl singlet (d H 2.44, H-7) with C-13, 15, and 16 (d C 113.4, 143.2, and 115.6, respectively) and an oxygenated olefinic methine singlet (d H 7.85, H-15) with C-14 and C-16 (d C 151.1 and 115.6) showed the ring D was a 3-methylfuran ( Figure S5, supplementary material). As mentioned above, the H-1 d H 10.65 (d, J ¼ 8.5 Hz) and H 3 -17 d H 2.44 (s) resonance was typically shifted to downfield and upfield, respectively, suggesting that a nitrogen in the oxazole ring (E ring) was instead attached to C-11 (Don et al. 2005). Therefore, the N-bearing carbon C-11 was assigned as d C 134.3. The structure of 15 was therefore proposed as isosalviamide G.
Compound 16 was obtained as a pale-yellow, amorphous powder. The molecular formula was deduced from its HR-EI-MS (m/z 362.1267 [M] þ , calcd. for C 21 H 18 N 2 O 4 , 362.1267). The EI-MS of 16 exhibited a significant base peak at m/z 318 [M-CONH 2 ] þ , which was the feature of aliphatic primary amides group. The 1 H-and 13 C-NMR spectra were very similar to those of compound 14, except for the absence of signals typical of a furan ring (ring D) (Table S1, supplementary material). In the 1 H-and 13 C-NMR spectra of 16, a hydrogen-bonded of the hydroxyl group was evident at d H 14.47 (in pyridine-d 5 ) and signals for acetyl group were at d H 2.81 (3H, s) and d C 31.4 and 202.8. In the HMBC spectrum, the correlations of the aromatic proton at d H 8.66 (H-7) with the oxygenated aromatic carbon at d C 160.9 (C-14), and the methyl group H-16 with the two carbon signals at C-13 and C-16 (d C 104.5, 202.8) demonstrated that the acetyl group was located at C-13 ( Figure S5, supplementary material). Nevertheless, in this molecule, the H-1 resonance was shifted downfield to d H 10.65 (d, J ¼ 8.5 Hz) and H 3 -17 was shifted upfield to d H 2.81, with respect to a nitrogen in the oxazole ring attached to C-12 in the known compound isoalviamine D (Don et al. 2005). Thus, the structure of compound 16 was established as isosalviamide H.
To assess the effects of all isolated compounds (1-16) on LPS-induced production of NO in RAW 264.7 cells, cell culture media were harvested and assayed by using the Griess reaction (Lee et al. 2010). The amount of NO produced was determined based on the amount of nitrite, a stable metabolite of NO. Among the tested compounds, 30 lM 15,16-dihydrotanshinone I (11) completely abolished NO secretion. Compound 11 showed the strongest inhibitory effects, with an IC 50 value of 3.4 ± 1.2 lM, comparable with that of the positive control, celastrol. Tanshinol B (7), tanshinone IIB (8), methyl tanshinonate (10), and dehydrodanshenol A (13) showed moderate effects, with IC 50 values of 15.5, 20.8, 23.8, and 21.7 lM, respectively (Table S2, supplementary material). The other isolated compounds were weakly active or inactive, with IC 50 values greater than 30 lM. To determine whether compound 11 suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 cells, cells were stimulated with LPS in the presence of these compounds at various concentrations (3-30 lM), and iNOS and COX-2 expression was determined by western blotting. Compound 11 downregulated the expression of iNOS protein in a concentration-dependent manner ( Figure S6, supplementary material).
The cytotoxic effects of all isolated compounds were also evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Jin et al. 2002), to determine whether their inhibition of NO production resulted from the cytotoxicity effects of the compounds. The viability of RAW264.7 cells treated with compounds at the highest concentration (i.e., 30 lM) was 43.1%, 58.0%, 44.3%, and 44.9% for 15,16dihydrotanshinone I (11) and salviamide A-C (14-16), respectively, whereas the other compounds did not affect cell viability (data not shown). In this study, three new abietane diterpene alkaloids (14-16) were cytotoxic to RAW 264.7 cells, but showed little efficacy on the inhibition of NO production in these cells. However, the inhibitory effect of dihydrotanshinone I (11) on NO production may be a result of its cytotoxicity to RAW 264.7 cells.

Disclosure statement
No potential conflict of interest was reported by the authors.