New diterpenoids and sesquiterpenoids from Siegesbeckia pubescens Makino and their anti-inflammatory activity

Abstract

Six undescribed terpenoids were isolated from the aboveground parts of Siegesbeckia pubescens Makino. These compounds include four diterpenoids (1–4) and two sesquiterpenoids (5–6), as well as thirteen known compounds (7–19). The structures of new compounds were determined through spectroscopic analysis, DP4+ Analysis and ECD calculation. The protective effect of all compounds against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells was tested. The result showed that compounds 2–9, 12–15 and 17–19 significantly inhibited the production of nitric oxide (NO) in LPS-induced RAW 264.7 cells, among which compound 18 demonstrated the best inhibitory activity against NO release. The potential anti-inflammatory mechanism was investigated through molecular docking. The molecular docking results showed that the active compounds had a strong binding affinity with the key targets. In conclusion, Siegesbeckia pubescens Makino has demonstrated multi-component, multi-target and multi-pathway therapeutic characteristics in the treatment of inflammation. These findings provide a valuable theoretical basis for the clinical application of this plant.

---

1 Introduction

Siegesbeckia herb, derived from the dried aerial parts of Siegesbeckia orientalis L., Siegesbeckia pubescens Makino or Siegesbeckia glabrescens Makino, is a traditional Chinese medicinal plant with documented therapeutic applications.¹ Modern pharmacological studies have identified multiple bioactive compounds in Siegesbeckia herb, including sesquiterpenes, diterpenes and flavonoids, which demonstrated promising anti-inflammatory, immune-modulatory and antitumor properties.^2–5

The medicinal value of this plant prompted us to conduct further phytochemical research on Siegesbeckia pubescens Makino, with the aim of discovering new active components. We isolated and identified four undescribed diterpenoids and two undescribed sesquiterpenoids from the plant (Fig. 1), along with 13 known compounds. The chemical structures of these compounds were determined through comprehensive spectroscopic methods such as HR-ESI-MS and 1D/2D NMR, and their absolute configurations were determined by ECD calculation. At the same time, in vitro evaluation was conducted to assess the protective effects of these compounds on the inflammatory response of RAW 264.7 cells induced by LPS. The interaction between active small molecules and key targets was studied by molecular docking technology, thereby revealing the potential application value of Siegesbeckia pubescens Makino in the treatment of inflammation.

Fig. 1 Chemical structures of compounds 1–6.

2 Results

2.1 Structure elucidation of compounds

Compound 1 was obtained as a white amorphous powder. HR-ESI-MS analysis showed a [M + NH₄]⁺ ion at m/z 356.2793 (calculated for C₂₀H₃₈O₄N, 356.2795) (Fig. S8). The molecular formula was established as C₂₀H₃₄O₄ (four degrees of unsaturation) based on combined ¹H-NMR and ¹³C-NMR data (Table 1). The ¹H-NMR spectrum revealed four singlet methyl groups δ_H 1.24 (3H, s), 1.41 (3H, s), 1.44 (3H, s), 1.76 (3H, s); one olefinic proton δ_H 5.61 (1H, s); three oxygenated methine protons δ_H 3.51 (dt, J = 11.4, 4.3 Hz), 4.24 (1H, m), 4.71 (1H, d, J = 4.1 Hz) and two oxygenated methylene protons δ_H 4.25 (1H, m), 4.06 (1H, m). The ¹³C-NMR spectrum displayed twenty carbon signals, including two olefinic carbons at δ_C 131.6 (C-8), 136.6 (C-14); four methyl carbons δ_C 23.7 (C-17), 29.3 (C-18), 18.3 (C-19), 18.6 (C-20), five methines, including three oxygenated carbons at δ_C 79.0 (C-3), 67.8 (C-6), 77.2 (C-15); six methylenes, including one oxygenated methylene at δ_C 64.3 (C-16); and three quaternary carbons as confirmed by HSQC and DEPT experiments. The NMR data of compound 1 resembled those of darutigenol,⁴ with the key difference being the presence of a hydroxyl group at C-6 in compound 1 instead of a methylene group, supported by ¹H–¹H COSY correlations between H-6 (δ_H 4.71)/H-5 (δ_H 1.18) and H-7 (δ_H 2.48, 2.63), as well as HMBC correlations from H₃-18 (δ_H 1.44), H₃-19 (δ_H 1.76), and H₃-20 (δ_H 1.41) to C-6 (δ_C 67.8). The planar structure of compound 1 was established through analysis of HMBC and ¹H–¹H COSY correlations (Fig. 2).

Table 1 ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) data of compounds 1–3

No.	1 (C5D5N)		2 (C5D5N)		3 (CD3OD)
	δC	δH	δC	δH	δC	δH
1	39.6	1.66 (m)	38.2	1.59 (m)	72.6	3.93 (t, 2.9)
	—	1.23 (m)	—	1.22 (m)	—	—
2	29.0	1.98 (m)	28.6	1.85 (m)	35.6	1.90 (m)
	—	1.92 (m)	—	1.85 (m)	—	1.78 (m)
3	79.0	3.51 (dt,11.4, 4.3)	79.1	3.59 (dd, 11.9, 4.2)	74.2	3.70 (dd, 12.2,4.6)
	—	—	—	—	—	—
4	40.3	—	40.6	—	40.0	—
5	56.9	1.18 (d, 2.0)	60.6	1.45 (dd, 10.5, 3.1)	44.8	1.62 (t, 12.6)
6	67.8	4.71 (d, 4.1)	69.6	4.19 (m)	19.5	1.77 (m)
	—	—	—	—	—	1.56 (m)
7	47.3	2.63 (dt, 14.4, 2.5)	48.4	2.93 (dt, 13.7, 4.1)	31.8	2.36 (m)
	—	2.48 (m)	—	2.57 (t, 12.2)	—	1.93 (m)
8	136.6	—	138.3	—	127.1	—
9	51.8	1.83 (m)	50.8	1.83 (m)	141.6	—
10	38.7	—	39.4	—	43.5	—
11	18.9	1.87 (m)	19.3	1.61 (m)	21.5	2.32 (m)
	—	1.63 (m)	—	1.55 (m)	—	2.08 (dd, 17.3, 2.8)
12	33.2	2.50 (m)	33.4	2.51 (dd, 12.7, 3.6)	30.6	1.34 (m)
	—	1.21 (m)	—	1.15 (m)	—	1.28 (dd, 12.3, 4.3)
13	40.9	—	38.5	—	44.3	—
14	131.6	5.61 (s)	130.2	5.53 (s)	84.3	3.56 (m)
15	77.2	4.24 (m)	76.8	4.11 (d, 9.1)	80.7	3.79 (dd, 2.4, 5.2)
16	64.3	4.25 (m)	64.4	4.21 (m)	74.7	4.20 (dd, 9.8, 5.2)
	—	4.06 (m)	—	4.05 (m)	—	3.57 (m)
17	23.7	1.24 (s)	23.6	1.20 (s)	14.9	0.96 (s)
18	29.3	1.44 (s)	32.7	1.98 (s)	28.7	1.04 (s)
19	18.3	1.76 (s)	17.1	1.49 (s)	16.3	0.82 (s)
20	18.6	1.41 (s)	16.6	0.82 (s)	20.8	1.01 (s)

---

Fig. 2 Key HMBC and ¹H–¹H COSY of compounds 1–6.

Furthermore, NOESY correlations between H-5/H-6, H-9 and H₃-17, as well as between H₃-18/H-3 and H-6, established that H-3, H-5, H-6, H-9, H₃-17, and H₃-18 were in the β-orientation. The observed NOESY cross-peaks of H₃-20/H-15 and H₃-19 indicated the α-orientation of H-15, H₃-19, and H₃-20 (Fig. 3). The chemical shift value of C-15 is 77.2 ppm, thus the configuration of C-15 is R configuration.³ Quantum chemical computational methods were employed to calculate the ¹³C NMR chemical shifts of four possible epimers of compound 1, namely (3R*,6S*,15R)-1, (3R*,6R*,15R)-1, (3S*,6S*,15R)-1, and (3S*,6R*,15R)-1. As shown in Fig. S48, the calculated ¹³C NMR chemical shifts of the (3R*,6S*,15R)-1 exhibited excellent agreement with the experimental data, with a correlation coefficient (R²) of 0.9946. Meanwhile, the mean absolute error (MAE) and corrected mean absolute error (CMAE) were 1.7 and 2.5, Furthermore, DP4+ probability analysis strongly confirmed this epimer as the true relative configuration, with a DP4+ probability of 100% (Fig. S49). Based on the comprehensive analysis, the relative configuration of compound 1 was determined. Through comparative analysis of experimental and calculated ECD spectra (Fig. 4), the absolute configuration of compound 1 was established as 3R,6S,15R. Finally, compound 1 was identified and named as ent-3α,6α,15,16-tetrahydroxypimar-8(14)-ene.

Fig. 3 NOESY correlations of compounds 1–6.

Fig. 4 Experimental and calculated ECD spectra of compounds 1 and 3–6.

Compound 2 was obtained as a white amorphous powder. HR-ESI-MS analysis showed a [M + NH₄]⁺ ion at m/z 356.2794 (calculated for C₂₀H₃₈O₄N, 356.2795), establishing the same molecular formula (C₂₀H₃₄O₄) as compound 1 (Fig. S16). Comparative analysis of 1D and 2D NMR data revealed that compound 1 and 2 share the same planar structure but differ in their C-6 configurations. NOESY correlations between H-6/H₃-19 and H₃-20 confirmed the β-orientation of H-6 (Fig. 3). By comparing the specific optical rotation of compound 2 and compound 1, it was found that the magnitudes of their specific optical rotations were equal while the signs were opposite.

Further support for the authentic configuration of this compound as 3R,6R,15R was provided by quantum chemical calculations and DP4+ probability analysis, with the DP4+ probability reaching 97.53% (Fig. S50 and S51). Finally, compound 2 was identified and named as ent-3α,6β,15,16-tetrahydroxypimar-8(14)-ene.

Compound 3 was obtained as a white amorphous powder. HR-ESI-MS analysis showed a [M + H]⁺ ion at m/z 337.2376 (calculated for C₂₀H₃₃O₄, 337.2373) (Fig. S24). Combined ¹H-NMR and ¹³C-NMR data established the molecular formula as C₂₀H₃₂O₄ with five degrees of unsaturation (Table 1). The ¹H-NMR spectrum exhibited four singlet methyl groups δ_H 0.82 (3H, s), 0.96 (3H, s), 1.01 (3H, s), 1.04 (3H, s), four oxygenated methine protons δ_H 3.56 (1H, m), 3.70 (1H, dd, J = 12.2, 4.6 Hz), 3.79 (1H, dd, J = 2.4, 5.2 Hz), 3.93 (1H, t, J = 2.9 Hz), and two oxygenated methylene protons δ_H 3.57 (1H, m), 4.20 (1H, dd, J = 9.8, 5.2 Hz). The HSQC and DEPT spectrum confirmed the existence of five quaternary carbons, including two alkenyl carbon signals at δ_C 127.1 (C-8) and 141.6 (C-9); four methyls at δ_C 14.9 (C-17), 28.7 (C-18), 16.3 (C-19), 20.8 (C-20); five methines, including four oxygenated carbons at δ_C 72.6 (C-1), 74.2 (C-3), 84.3 (C-14), 80.7 (C-15), and six methylenes, including one oxygenated methylene δ_C 74.7 (C-16). The spectroscopic data resembled those of ent-14β,16-epoxy-8-pimarene-3β,15α-diol,⁶ with the key difference being a hydroxyl group at C-1 in compound 3 instead of a methylene group supported by ¹H–¹H COSY correlations of H-1 (δ_H 3.93), H-3 (δ_H 3.70)/H-2 (δ_H 1.78, 1.90) and HMBC correlations from H₃-20 (δ_H 1.01) to C-1 (δ_C 72.6). The planar structure of compound 3 was established through HMBC and ¹H–¹H COSY correlations (Fig. 2).

NOESY correlations between H₃-18/H-3, H-5; H₃-17/H-14, H-15 confirmed the β-orientation of H-3, H-5, H-14, H-15, H₃-17 and H₃-18, while correlations H₃-20/H-1, H₃-19 established the α-orientation of H-1, H₃-19 and H₃-20 (Fig. 3). The chemical shift value of C-15 is 80.7 ppm, thus the configuration of C-15 is S configuration.³ Quantum chemical calculations predicted the ¹³C NMR chemical shifts of four stereoisomers of compound 3: (1R*,3R*,15S)-3, (1R*,3S*,15S)-3, (1S*,3S*,15S)-3, and (1S*, 3R*,15S)-3. As shown in Fig. S52, (1R*,3R*,15S)-3 exhibited excellent agreement between calculated and experimental ¹³C NMR data (R² = 0.9921, MAE = 2.1, CMAE = 0.7). DP4+ analysis further confirmed this as the authentic configuration (99.99% probability, Fig. S53), establishing the relative configuration. Comparative analysis of experimental and computed ECD spectra (Fig. 4) assigned the absolute configuration of compound 3 as 1R,3R,15S. Consequently, compound 3 was identified and designated as 14α,16-epoxy-ent-1β,3α,15,16-trihydroxypimar-8-ene.

Compound 4 was obtained as a white amorphous powder. HR-ESI-MS analysis exhibited a peak at m/z 399.2393 [M + HCOO]⁻ (calcd for C₂₁H₃₅O₇, 399.2388) (Fig. S32). Combined with ¹H-NMR and ¹³C-NMR data, the molecular formula was determined to be C₂₀H₃₄O₅, with four degrees of unsaturation (Table 2). The ¹H-NMR spectrum revealed three singlet methyl signals at δ_H 1.19 (3H, s), 1.29 (3H, s), and 1.29 (3H, s); three oxygenated methine proton signals at δ_H 4.31 (1H, m), 4.18 (1H, m), and 3.60 (1H, d, J = 4.4 Hz); and four oxygenated methylene proton signals at δ_H 4.30 (1H, m), 4.14 (1H, d, J = 10.2 Hz), 4.19 (1H, m), and 3.76 (1H, d, J = 11.5 Hz). The ¹³C-NMR spectrum displayed twenty carbon signals, and HSQC and DEPT experiments confirmed the presence of three methyl carbons at δ_C 21.8 (C-17), 28.7 (C-18), and 18.1 (C-20); five methine carbons, including three oxygenated methines at δ_C 63.9 (C-2), 82.3 (C-14), and 86.3 (C-15); eight methylene carbons, including two oxygenated methylenes at δ_C 62.3 (C-16) and 65.4 (C-14); and four quaternary carbons. The NMR data of compound 4 is similar to that of ent-8,15-epoxy-2α,16,19-trihydroxy-pimarane⁷ isolated in this study. The difference lies in that compound 4 has a hydroxyl group attached at the C-14 position, replacing the previous methylene signal. This is supported by the correlation in the HMBC spectrum from H-14 (δ_H 3.60) to C-7 (δ_C 37.7), C-8 (δ_C 82.0), C-9 (δ_C 47.7), C-12 (δ_C 26.7), C-17 (δ_C 21.8). The correlations of HMBC and ¹H–¹H COSY spectrum revealed the planar structure of compound 4 (Fig. 2).

Table 2 ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) data of compounds 4–6

No.	4 (C5D5N)		5 (CD3OD)		6 (CD3OD)
	δC	δH	δC	δH	δC	δH
1	51.0	2.37 (d, 11.4)	171.5	—	73.7	3.82 (dd, 11.5, 4.7)
	—	1.31 (m)	—	—	—	—
2	63.9	4.31 (m)	137.2	—	34.3	1.87 (m)
	—	—	—	—	—	1.87 (m)
3	46.2	2.92 (dd, 12.5, 3.6)	52.4	2.85 (m)	75.7	3.54 (t, 2.8)
	—	1.32 (m)	—	—	—	—
4	41.5	—	75.9	5.27 (t, 10.2)	75.4	—
5	56.1	1.26 (t, 7.3)	129.5	5.11 (d, 10.2)	51.7	1.88 (m)
6	20.1	1.82 (m)	142.6	—	71.8	4.20 (t, 10.1)
	—	1.82 (m)	—	—	—	—
7	37.7	2.12 (m)	33.1	2.83 (m)	61.1	1.74 (m)
	—	1.96 (dd, 13.8, 2.7)	—	2.01 (td, 12.8, 1.6)	—	—
8	82.0	—	27.9	2.66 (m)	78.0	4.05 (m)
	—	—	—	2.52 (m)	—	—
9	47.7	2.05 (m)	157.6	6.76 (dd, 10.2, 7.4)	44.6	2.32 (dd, 11.3, 3.8)
	—	—	—	—	—	1.35 (d, 11.8)
10	39.1	—	146.1	—	42.1	—
11	19.7	1.78 (m)	121.6	6.16 (d, 3.4)	42.6	2.66 (d, 12.0, 6.9)
	—	1.64 (m)	—	5.75 (d, 3.4)	—	—
12	26.7	2.09 (m)	60.5	4.45 (brd, 13.0)	182.0	—
	—	1.52 (m)	—	4.19 (brd, 13.0)	—	—
13	42.7	—	72.0	6.62 (dd, 8.6, 1.5)	14.9	1.30 (d, 6.9)
14	82.3	3.60 (d, 4.4)	70.0	4.13 (dd, 8.6, 1.9)	16.6	0.97 (s)
15	86.3	4.18 (m)	197.3	9.45 (d, 2.0)	23.3	1.32 (s)
16(1′)	62.3	4.30 (m)	168.6	—	—	—
	—	4.14 (d, 10.2)	—	—	—	—
17(2′)	21.8	1.19 (s)	129.1	—	—	—
18(3′)	28.7	1.29 (s)	139.5	6.13 (m)	—	—
19(4′)	65.4	4.19 (m)	21.0	1.91 (p, 1.5)	—	—
	—	3.76 (d, 11.5)	—	—	—	—
20(5′)	18.1	1.29 (s)	16.2	1.98 (dq, 1.5, 7.2)	—	—

---

Furthermore, the correlations of H-5/H-9, H₃-18; H-15/H₃-17 in the NOESY spectrum indicated that H-5, H-9, H-15, H₃-17 and H₃-18 were located on the same side of the ring and were in the β orientation. The correlations of H₂-19/H-14, H₃-20; H-2/H₃-20 suggested that H-2, H-14, H₂-19 and H₃-20 were in the α orientation (Fig. 3). The chemical shift value of C-15 is 86.3 ppm, thus the configuration of C-15 is S configuration.³ Quantum chemical calculations predicted the ¹³C NMR chemical shifts of four stereoisomers of compound 4: (2S*,14R*,15S)-4, (2R*,14R*,15S)-4, (2R*,14S*,15S)-4 and (2S*,14S*,15S)-4. As shown in Fig. S54, the calculated ¹³C NMR chemical shifts of (2S*,14R*,15S)-4 were in good agreement with experimental data, with R² = 0.9867, MAE = 1.9 and CMAE = 2.5. Further DP4+ probability analysis confirmed this as the authentic configuration (100.00% probability, Fig. S55), establishing the relative configuration of compound 4. Finally, comparative analysis of experimental and calculated ECD spectra (Fig. 4) determined the absolute configuration of compound 4 as 2S,14R,15S. Finally, compound 4 was identified and named as 8,15-epoxy-ent-2β,14β,16,19-tetrahydroxy-pimarane.

Compound 5 was isolated as a white amorphous powder. HR-ESI-MS analysis showed a peak at m/z 377.1591 [M + H]⁺ (calculated for C₂₀H₂₅O₇, 377.1595) (Fig. S40). The molecular formula was determined to be C₂₀H₂₄O₇ with seven degrees of unsaturation, based on combined ¹H-NMR and ¹³C-NMR data (Table 2). The ¹H-NMR spectrum exhibited characteristic signals including one aldehyde proton at δ_H 9.45 (1H, d, J = 2.0 Hz); two methyl groups at δ_H 1.91 (3H, p, J = 1.5 Hz) and 1.98 (3H, dq, J = 1.5, 7.2 Hz); five olefinic protons at δ_H 6.76 (1H, dd, J = 10.2, 7.4 Hz), 6.13 (1H, m), 6.16 (1H, d, J = 3.4 Hz), 5.75 (1H, d, J = 3.4 Hz), 5.11 (1H, d, J = 10.2 Hz); two oxygenated methine protons at δ_H 4.13 (1H, dd, J = 8.6, 1.9 Hz) and 6.62 (1H, dd, J = 8.6, 1.5 Hz); and two oxygenated methylene protons at δ_H 4.45 (1H, brd, J = 13.0 Hz) and 4.19 (1H, brd, J = 13.0 Hz). The ¹³C-NMR spectrum and DEPT spectrum revealed twenty carbon signals, including two methyl carbons at δ_C 21.0 (C-4′), 16.2 (C-5′); three carbonyl carbons at δ_C 171.5 (C-1), 197.3 (C-15), 168.6 (C-1′); eight olefinic carbons at δ_C 137.2 (C-2), 129.5 (C-5), 142.6 (C-6), 157.6 (C-9), 146.1 (C-10), 121.6 (C-11), 129.1 (C-2′), 139.5 (C-3′), three methylene carbons including one oxygenated methylene at δ_C 60.5 (C-12); and four methine carbons, including three oxygen-containing methylene signals δ_C 75.9 (C-4), 72.0 (C-13), 70.0 (C-14). The spectral data of compound 5 is similar to siegenolide A8 except for the replacement of a methoxy group with a hydroxyl group at C-14. This was supported by correlation of H-13 (δ_H 6.62)/H-14 (δ_H 4.13) in the ¹H–¹H COSY spectrum and the absence of the methoxy signal in the ¹H-NMR. The planar structure of compound 5 was established through HMBC and ¹H–¹H COSY correlations (Fig. 2).

Furthermore, NOESY correlations between H-13/H-3 and H-14 indicated that these protons are cofacial, adopting a β-orientation. Quantum chemical calculations were employed to predict the ¹³C NMR chemical shifts of two candidate isomers, (3R*,4S*,13R*,14R*)-5 and (3S*,4R*,13R*,14R*)-5. As shown in Fig. S56, the calculated ¹³C NMR data of (3R*,4S*, 13R*,14R*)-5 were in good agreement with the experimental results, with an R² value of 0.9776, a MAE of 4.4 and a CMAE of 2.3. Further DP4+ probability analysis corroborated this structure as the correct relative configuration, yielding a DP4+ probability of 99.53% (Fig. S57). Combined with the experimental and calculated ECD spectra comparison (Fig. 4), the absolute configuration of compound 5 was ultimately assigned as 3R,4S,13R,14R. Finally, compound 5 was identified and named siegenolide C.

Compound 6 was obtained as a white amorphous powder. HR-ESI-MS analysis showed a [M + H]⁺ ion at m/z 301.1647 (calcd for C₁₅H₂₅O₆, 301.1646) (Fig. S47). The molecular formula was established as C₁₅H₂₄O₆ (four degrees of unsaturation) based on ¹H and ¹³C NMR data (Table 2). The ¹H-NMR spectrum revealed three methyl signals at 0.97 (3H, s), 1.30 (3H, d, J = 6.9 Hz), 1.32 (3H, s), along with four oxygenated methine protons at δ_H 3.54 (t, J = 2.8 Hz), 3.82 (dd, J = 11.5, 4.7 Hz), 4.05 (m), and 4.20 (t, J = 10.1 Hz). The ¹³C NMR spectrum displayed fifteen carbon signals, including three methyls at δ_C 14.9 (C-13), 23.3 (C-14), 16.6 (C-15), seven methines included four oxygenated at δ_C 73.7(C-1), 75.7 (C-3), 71.8 (C-6), 78.0 (C-8), two methylene, and three quaternary carbons (one carbonyl at δ_C 182.0), as confirmed by HSQC experiments. The spectral data of compound 6 were similar to those of Inujaponin E,⁹ indicating that this compound is an eudesmane-type sesquiterpene lactone derivative. The key difference was the presence of hydroxyl groups at C-3 and C-6, replacing the original methylene groups. ¹H–¹H COSY correlations between H-2 (δ_H 1.87)/H-1 (δ_H 3.82), H-3 (δ_H 3.54); H-6 (δ_H 4.20)/H-5 (δ_H 1.88), H-7 (δ_H 1.74) confirmed the hydroxyl at C-3 and C-6. The planar structure of compound 6 was established through HMBC and ¹H–¹H COSY correlations (Fig. 2).

Furthermore, the correlations in the NOESY spectrum of H₃-14/H-6, H-8, H₃-15; H-2β/H-3, H₃-13, H₃-15 indicated that H-3, H-6, H-8, H₃-13, H₃-14, and H₃-15 were located on the same side of the ring, indicating a β orientation. The correlations of H-5/H-1 and H-7 indicated that H-1, H-5, and H-7 were in an α orientation (Fig. 3). Quantum chemical calculations were employed to predict the ¹³C NMR chemical shifts of eight candidate isomers: (3R*,6S*,8S*)-6, (3S*,6S*,8S*)-6, (3R*, 6R*,8S*)-6, (3R*,6S*,8R*)-6, (3S*,6S*,8R*)-6, (3S*,6R*,8S*)-6, (3R*,6R*,8R*)-6, and (3S*,6R*,8R*)-6. As shown in Fig. S58, the calculated ¹³C NMR chemical shifts of (3R*,6S*,8S*)-6 were in good agreement with the experimental data, with an R² value of 0.9975, a MAE of 1.6 and a CMAE of 0.5. Further DP4+ probability analysis corroborated this structure as the correct relative configuration, with a DP4+ probability of 100.00% (Fig. S59). Combined with the comparison of experimental and calculated ECD spectra (Fig. 4), the absolute configuration of compound 6 was assigned as 3R,6S,8S. Finally, compound 6 was identified and named as 1β,3α,4α,6α-tetrahydroxy-11aH-eudesma-12,8α-olide.

The other compounds were identified as siegeside A (7),¹⁰ siegesbeckianal (8),¹¹ ent-2α,15R,16,19-tetrahydroxypimar-8 (14)-ene (9),¹² 2-oxo-15,16,19-trihydroxy-ent-pimar-8(14)-ene (10),¹³ ent-12α,16-epoxy-2β,15α,19-trihydroxypimar-8-ene (11),¹³ ent-12α,16-epoxy-2β,15α,19-trihydroxypimar-8(14)-ene (12),¹³ siegesbeckia E (13),³ ent-3α15,16,19-tetrahydroxypimar-8(14)-ene(14),¹⁴ 14β,16-epoxy-ent-3β,15α,19-trihydroxypimar -7-ene(15),¹² (4R,5S,7S,8R,9S,10S,13R,16R)-7,17-dihydroxy kauran-19-oic acid (16),¹⁵ (4S,7R,8R,10S,11S)-11α,13-dihydro-4H-xanthalongi-4-O-β-D-glucopyranoside (17),¹⁶ siegenolide B (18),⁸ siegesbeckialide J (19)¹⁷ from their NMR data comparison with reported in the literature.

2.2 Evaluation of anti-inflammatory activities

The inhibitory effect of compounds 1 to 19 on the production of NO by LPS-activated RAW 264.7 cells was tested. Within the concentration range of 50 µM, no significant cytotoxicity of these compounds was detected by the MTT assay. Subsequently, the evaluation was conducted at the above-mentioned maximum safe concentration. The result showed that compounds 2–9, 12–15 and 17–19 could significantly inhibit the production of NO at 50 µM, among which compound 18 demonstrated the best inhibitory activity against NO release (Fig. 5).

Fig. 5 The NO production induced by LPS in RAW 264.7 cells of 1–19.

2.3 Predicted binding modes of compounds and iNOS, COX-2 using molecular docking analysis

iNOS and COX-2 are key enzymes involved in inflammatory processes. iNOS catalyzes the production of nitric oxide from L-arginine and is implicated in the development of various inflammatory diseases. COX-2 plays a significant role in initiating inflammatory responses and contributing to tissue damage. Consequently, inhibiting the activity of iNOS and COX- 2 represents a strategic approach for treating inflammatory disorders.²

The compounds (3, 5, 7, 17, 18) were subjected to molecular docking. The results of the molecular docking study indicated that compounds 3, 5, 17, and 18 had strong interactions with the iNOS protein, and compounds 3, 5, 7, 17, and 18 had strong interactions with the COX-2 protein (S82–S90). Detailed information on the specific binding residues and the logarithms of free binding energies was collated in Tables S3 and S4.

3 Materials and methods

3.1 General experimental procedures

Silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), MCI gel (CHP20P, Mitsubishi Chemical Corporation, Tokyo, Japan) and ODS (50 µm, YMC, Kyoto, Japan) were used for column chromatography. Semi-preparative HPLC was performed on a Shimadzu LC-6AD liquid chromatography with a C18 column (10 mm × 250 mm, 5 µm, YMC). NMR spectra were determined by Bruker Avance Ⅲ 600 (Germany, Bruke), HR-ESI-MS data were obtained from Agilent 6546 LC-Q-TOF (USA, Agilent); optical rotation was recorded by JASCO P-2000 polarimeter (Japan, JASCO); ECD spectra were determined by Biologic MOS-450 (France, Biologic). RAW 264.7 cells (STCC20020P-1, Wuhan Servicebio Technology Co., Ltd, China).

3.2 Plant material

The dried aerial parts of Siegesbeckia pubescens Makino were purchased from Bozhou Medicinal Materials Market in Anhui Province and it was identified by Professor Guo Shenglei of Heilongjiang University of Chinese Medicine. The voucher specimen (no. 2023712-1) is stored in the same institution.

3.3 Separation

Dried Siegesbeckia pubescens Makino (5.0 kg) was extracted three times with 75% ethanol for 2 hours each time. The combined filtrate was concentrated under reduced pressure to obtain the ethanol extract. The extract was suspended in water and extracted with petroleum ether, yielding 150.0 g of petroleum ether-soluble fraction and 450.0 g of water-soluble fraction.

The water layer (450.0 g) was fractionated by silica gel column chromatography using a gradient elution of CH₂Cl₂–MeOH (100 : 0 to 100 : 50), yielding nine fractions (Fr. A–Fr. I). Fr. B (20.5 g) was further purified by silica gel column chromatography with a gradient of CH₂Cl₂–MeOH (100 : 10 to 100 : 20), affording four subfractions (Fr. B-1 to Fr. B-4). Fr. B-2 was subjected to ODS reverse-phase column chromatography using a MeOH–water gradient (100 : 20 to 100 : 0), yielding five fractions (Fr. B2-1 to Fr. B2-5). Fr. B2-3 was isolated by semi-preparative HPLC with methanol–water solvent system (70 : 30), yielding compounds 8 (4.9 mg), 10 (6.4 mg), 16 (7.0 mg), and 18 (2.3 mg).

Fraction D (20.5 g) was separated by ODS reverse-phase column chromatography using a MeOH–water gradient (10 : 90 to 100 : 0), yielding five subfractions (Fr. D-1 to Fr. D-5). Fr. D-3 was further purified by semi-preparative HPLC with MeOH–water (32 : 68), affording compounds 13 (7.8 mg), 14 (5.0 mg), and 15 (8.7 mg). Fr. D-4 (3.3 g) was fractionated via MCI reverse-phase column chromatography with a MeOH–water gradient (30 : 70 to 100 : 0), resulting in four subfractions (Fr. D4-1 to Fr. D4-4). Fr. D4-3 was isolated by semi-preparative HPLC using MeOH–water system (60 : 40), yielding compounds 3 (4.5 mg), 6 (3.0 mg), and 19 (6.0 mg).

Fraction E (40.0 g) was subjected to ODS reverse-phase column chromatography with a MeOH–water gradient (10 : 90 to 100 : 0), yielding six fractions (Fr. E-1 to Fr. E-6). Fr. E-2 was purified by semi-preparative HPLC eluted with MeOH–water (50 : 50), obtaining compounds 1 (4.5 mg), 2 (4.2 mg), 7 (7.8 mg), 9 (10.4 mg), and 11 (8.7 mg). Further purification of Fr. E-3 by semi-preparative HPLC with MeOH–water (58 : 42) yielded compounds 4 (3.0 mg), 5 (4.0 mg), 12 (5.2 mg), and 17 (5.0 mg).

3.3.1 ent-3α,6α,15,16-tetrahydroxypimar-8(14)-ene. White amorphous powder; [α]_D25 = +20.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 356.2793 [M + NH₄]⁺ (calcd for 356.2795), ¹H-NMR and ¹³C-NMR (C₅D₅N, 600, 150 MHz) data, see Table 1.

3.3.2 ent-3α,6β,15,16-tetrahydroxypimar-8(14)-ene. White amorphous powder; [α]_D²⁵ = −20.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 356.2794 [M + NH₄]⁺ (calcd for 356.2795), ¹H-NMR and ¹³C-NMR (C₅D₅N, 600, 150 MHz) data, see Table 1.

3.3.3 14α,16-epoxy-ent-1β,3α,15,16-trihydroxypimar-8-ene. White amorphous powder; [α]_D²⁵ = −10.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 337.2376 [M + H]⁺ (calcd for 337.2373), ¹H-NMR and ¹³C-NMR (CD₃OD, 600, 150 MHz) data, see Table 1.

3.3.4 8,15-epoxy-ent-2β,14β,16,19-tetrahydroxy-pimarane. White amorphous powder; [α]_D²⁵ = −10.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 399.2393 [M + HCOO]⁻ (calcd for 399.2388), ¹H-NMR and ¹³C-NMR (C₅D₅N, 600, 150 MHz) data, see Table 2.

3.3.5 Siegenolide C. White amorphous powder; [α]_D²⁵ = −20.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 377.1591 [M + H]⁺ (calcd for 377.1595), ¹H-NMR and ¹³C-NMR (CD₃OD, 600, 150 MHz) data, see Table 2.

3.3.6 1β,3α,4α,6α-tetrahydroxy-11aH-eudesma-12,8α-olide. White amorphous powder; [α]_D²⁵ = −20.0, (c = 0.1, MeOH); HR-ESI-MS: m/z 301.1647 [M + H]⁺ (calcd for 301.1646), ¹H-NMR and ¹³C-NMR (CD₃OD, 600, 150 MHz) data, see Table 2.

3.4 NMR chemical shift calculation and DP4+ probability analysis

Initially, conformational space exploration was performed using SPARTAN software, where semi-empirical PM6 methodology was employed to conduct a global conformational search for potential isomers of the target compound. Subsequently, density functional theory (DFT) geometry optimization was executed for all low-energy conformers in Gaussian 09,¹⁸ utilizing the B3LYP functional and 6-31G(d) basis set as the computational benchmark. The reliability of potential energy minima was verified via vibrational frequency analysis (absence of imaginary frequencies). To enhance energy evaluation accuracy, single-point Gibbs free energy calculations were performed for each conformer using the M062X functional combined with the 6-311+G(2d,p) basis set, and dominant conformers (>5% population at 298.15 K) were selected based on Boltzmann distribution.

NMR parameters were computed using the gauge-independent atomic orbital (GIAO) method at the mPW1PW91//6-31+G(d,p) theoretical level. To simulate solvation effects, a self-consistent reaction field (SCRF) implicit solvent model was incorporated, specifically parameterized for the dielectric environment of MeOH. The agreement between calculated and experimental chemical shifts was systematically evaluated through multi-dimensional metrics, including linear correlation coefficient (R²), mean absolute error (MAE), corrected mean absolute error (CMAE), and DP4+ probability analysis.¹⁹

3.5 ECD calculation

The theoretical calculations of 1–6 were performed using Gaussian 09. The possible conformations were initially obtained from the program Spartan 14 and then optimized at B3LYP/6-31G(d) level in the gas phase. Room-temperature equilibrium populations were calculated according to the Boltzmann distribution law. The ECD calculations were performed using Time Dependent Density Functional Theory (TDDFT) at wB97xd/6-311G(d,p) level in methanol with PCM model. The calculated ECD spectra were obtained by weighing the Boltzmann distribution rate of each geometric conformation, and the sigma/gamma value for processing the calculated ECD was 0.3 eV. All calculated curves were shifted +25 nm to better simulate experimental spectra.

3.6 Anti-inflammatory activity assay

The anti-inflammatory effects of the compounds were evaluated in vitro by measuring nitric oxide (NO) production in LPS-induced RAW 264.7 cells. RAW 264.7 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 2 × 10⁵ cells per well and cultured for 24 h. The experiment included three groups: blank control, LPS group (1 µg mL⁻¹), and LPS (1 µg mL⁻¹) + compound group (12.5, 25, 50 µM). Dexamethasone was as the positive control. After 24 h of incubation, the cell supernatant was collected for further analysis. NO levels in RAW 264.7 cells were measured using the Griess reagent assay. Briefly, 50 µL of supernatant was mixed with 50 µL of Griess Reagent I and II in 96-well plate. Absorbance at 540 nm was measured at room temperature, and NO concentrations were calculated based on a standard curve.^11,20

3.7 Molecular docking studies

The three-dimensional crystal structures of iNOS (PDB ID: 3EJ8) and COX-2 (PDB ID: 1CX2) were obtained from the RCSB Protein Data Bank. Candidate compounds underwent structural optimization using Chem 3D software and were subsequently exported as PDB files for molecular docking. Molecular docking was performed using AutoDock to identify the most favorable binding conformation for each compound. Three-dimensional visualization of docking results was conducted using PyMOL.

4 Conclusion

In summary, we isolated 19 terpenoids from the aerial parts of Siegesbeckia pubescens Makino, including six novel compounds. Notably, compounds 2–9, 12–15, and 17–19 exhibited significant anti-inflammatory activity. The results of molecular docking revealed the initial anti-inflammatory mechanism of bioactive terpenoids binding to iNOS/COX-2. Our study expanded the material basis of the terpenoid chemical components of this plant species, revealed the potential application of Siegesbeckia pubescens Makino as a therapeutic agent for inflammation.

Author contributions

L. X. Qu: writing–original draft, investigation, formal analysis, Y. J. Guan: investigation, formal analysis, data curation. X. Y. Li: visualization, investigation, data curation. X. N. Li: data curation. H. X. Kuang: project administration. L. Yang: editing, supervision, project administration, funding acquisition. H. Jiang: editing, supervision, project administration, funding acquisition, conceptualization.

Conflicts of interest

There are no conflicts to declare.

Data availability

The raw data supporting the conclusions will be made available by the authors upon request.

Supplementary information (SI): original NMR data of all compounds, DP4+ analysis results, cellular activity data, and molecular docking results. See DOI: https://doi.org/10.1039/d5ra08807k.

Acknowledgements

This work was supported by Heilongjiang Province Traditional Chinese Medicine Innovation Team and Talent Support Program Project (2025); Heilongjiang Province 'Double First Class' Discipline Collaborative Innovation Achievement Construction Project (LJGXCG2025-F30); Heilongjiang Province Young Qihuang Scholars Cultivation Project (2023); National Famous Elderly Traditional Chinese Medicine Experts Inheritance Studio Construction Project ([2022] No. 75); Seventh Batch of National Old Chinese Medicine Experts Academic Experience Inheritance Project ([2022] No. 76); Heilongjiang University of Traditional Chinese Medicine Postgraduate Innovation Fund Project (2023yjscx034); the National Training Program for Traditional Chinese Medicine Characteristic Technology Inheritance Talents (NO. [2023]), the Construction of key disciplines of Chinese medicine processing (NO. [2023]) and the Traditional Chinese medicine Processing technology inheritance base project. The authors would like to thank Stork (https://www.storkapp.me/) for their invaluable technical support.

References

1. The State Commission of Chinese Pharmacopoeia, Chinese Pharmacopoeia, China Medical Science Press, 2025.
2. Y. Y. Zheng, Z. F. Guo, H. Chen, T. R. G. Bao, X. X. Gao, A. H. Wang and J. M. Jia, Phytochemistry, 2023, 205, 113503.
3. X. X. Gao, Z. J. Rong, G. Q. Long, G. S. Hu, T. Yan, N. Li, J. M. Jia and A. H. Wang, Bioorg. Chem., 2020, 99, 103854.
4. L. F. Ding, H. Y. Wang, D. S. Wang, Z. Q. Xie, W. Nie, L. D. Song and X. D. Wu, Chin. Tradit. Pat. Med., 2019, 41, 840–843.
5. T. Y. Wang, M. Y. Zhao, X. N. Yue, Y. Chen, C. Q. Lu, H. Wang and K. F. Fan, Chin. J. Exp. Tradit. Med. Formulae, 2025, 31, 106–114.
6. F. Wang, X. L. Cheng, Y. J. Li, S. Shi and J. K. Liu, J. Nat. Prod., 2009, 72, 2005–2008.
7. J. B. Wang, M. H. Wu, C. Gao and H. Z. Fu, Bioorg. Med. Chem., 2019, 27, 1320–1326.
8. Q. Wu, H. Li, S. Y. Lee, H. J. Lee and J. H. Ryu, Molecules, 2015, 20, 2850–2856.
9. X. D. Wu, L. F. Ding, W. C. Tu, H. Yang, J. Su, L. Y. Peng, Y. Li and Q. S. Zhao, Phytochemistry, 2016, 129, 68–76.
10. J. B. Wang, K. H. Xie, H. Q. Duan, Y. Wang, H. Ma and H. Z. Fu, Bioorg. Med. Chem. Lett., 2017, 27, 1815–1819.
11. H. Jang, J. W. Lee, J. G. Kim, H. R. Hong, T. P. L. Le, J. T. Hong, Y. Kim, M. K. Lee and B. Y. Hwang, Bioorg. Chem., 2018, 80, 81–85.
12. S. Schwikkard, A. Alqahtani, W. Knirsch, W. Wetschnig, A. Jaksevicius, E. I. Opara, M. K. Langat, J. L. Andriantiana and D. A. Mulholland, J. Nat. Prod., 2017, 80, 30–37.
13. Y. Xiang, H. Zhang, C. Q. Fan and J. M. Yue, J. Nat. Prod., 2004, 67, 1517–1521.
14. R. Wang, W. H. Chen and Y. P. Shi, J. Nat. Prod., 2010, 73, 17–21.
15. Q. Q. Yuan, W. B. Song, W. Q. Wang, J. J. Wei, J. Li, Y. Liu and L. J. Xuan, Phytochem. Lett., 2017, 21, 273–277.
16. Z. P. Yu, J. S. Zhang, Q. Q. Zhang, S. J. Yu, Y. Y. Zhang, J. H. Yu and H. Zhang, Fitoterapia, 2019, 138, 104292.
17. X. X. Gao, X. F. Shen, Y. Y. Zheng, L. Y. Yang, X. Y. Zhang, G. S. Hu, J. M. Jia and A. H. Wang, J. Nat. Prod., 2021, 84, 2808–2821.
18. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Gaussian, Inc., Wallingford CT, 2013.
19. B. A. Franco, E. R. Luciano, A. M. Sarotti and M. M. Zanardi, J. Nat. Prod., 2023, 86, 2360–2367.
20. L. Li, H. Z. Qin, S. Lin, H. Zhu and A. D. Wei, Chin. Tradit. Pat. Med., 2023, 45, 2525–2531.

---