Secobutanolides Isolated from Lindera obtusiloba Stem and Their Anti-Inflammatory Activity

Abstract

In this study, a new secobutanolide, named secosubamolide B (3), along with three previously known secobutanolides (1, 2, and 4), were successfully isolated from a methanol extract of the stem of Lindera obtusiloba. The chemical structures of these compounds were elucidated through the analysis of spectroscopic data, and then compared with the existing literature to confirm their identities. Furthermore, the anti-inflammatory effect of these isolated compounds on bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS) was evaluated. Compounds 1–3 showed the significant suppression of LPS-triggered IL-6 and IL-12 p40 production, with IC₅₀ values between 1.8 and 24.1 µM. These findings may provide a scientific foundation for developing anti-inflammatory agents from L. obtusiloba.

1. Introduction

Traditional Oriental medicine presents an abundant source of potential therapeutic agents that have yet to be fully explored by Western medicine. In Korea, decoctions prepared from the wood and bark of Lindera obtusiloba (Lauraceae) have been traditionally utilized for their anti-inflammatory properties, as well as for enhancing blood circulation and treating skin conditions such as dermatitis [1,2,3]. Additionally, herbal infusions made from L. obtusiloba have been historically used in the management of chronic liver diseases. The plant’s broad medicinal applications are largely due to its diverse range of bioactive compounds. Among the bioactive components identified in the leaves of L. obtusiloba are lignans and butanolides, which have been shown to possess significant anti-tumor, anti-allergic, and anti-inflammatory effects. Further research has also highlighted the plant’s antioxidant, hepatoprotective, and anti-coagulant properties, underscoring its considerable therapeutic potential [4,5,6]. In particular, secobutanolides from genus Lindera have shown promising anticancer effects against HepG2, P-388, A549 and HT-29 cancer cell lines, and also shown collagen-induced platelet aggregation inhibitory activity [7,8,9].

In our recent study, we set out to isolate and identify a novel butanolide that could expand the known spectrum of active compounds derived from L. obtusiloba. Through our investigation, we successfully isolated and elucidated the chemical structure of a new secobutanolide, in addition to three previously known secobutanolides, from a methanol extract of the plant’s stem. Moreover, we explored the anti-inflammatory potential of these isolated compounds by assessing their effects on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs).

Lipopolysaccharide (LPS), a predominant component of the outer membrane of gram-negative bacteria, is known for its ability to suppress inflammatory responses in vivo. However, LPS is also capable of inducing the release of various inflammatory cytokines in different cell types, leading to a robust acute inflammatory response to pathogens. Among the cytokines released, interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α are particularly important as key mediators of inflammation. IL-6 is especially noteworthy due to its dual role in promoting and regulating inflammation, making it a central player in many inflammatory diseases. IL-12 is crucial in the initiation and regulation of the cellular immune response, with its p40 and p80 subunits exhibiting distinct biological activities, independent of IL-12 and IL-23. Dendritic cells (DCs) are essential in orchestrating immune responses, primarily through their ability to modulate T-cell functions [10,11,12,13]. To uncover the anti-inflammatory components of L. obtusiloba, we tested the isolated compounds for their ability to modulate the LPS-induced expression of the pro-inflammatory cytokines IL-6 and IL-12 p40 in BMDCs. This investigation has contributed to a deeper understanding of the anti-inflammatory potential of compounds derived from L. obtusiloba, further supporting its use in traditional medicine and its potential use in therapeutic applications.

2. Results and Discussion

2.1. Isolation and Structural Characterization

Four secobutanolides were successfully isolated from a MeOH extract of L. obtusiloba stems. These compounds were identified as secomahubanolide (1) [14], secosubamolide A (2) [15], secosubamolide B (3), and secoisolitsealiicolide B (4) [16] (Figure 1). Notably, secosubamolide B (3) is a newly discovered compound, and all four compounds were identified in L. obtusiloba for the first time. This study represents the first thorough chemical investigation of secobutanolides in L. obtusiloba.

Compound 1 was isolated as a yellow oil. High-resolution electrospray ionization time-of-flight mass spectrometry (HR-ESI-TOF-MS) established its molecular formula as C₂₀H₃₆O₄, with an observed m/z of 341.2690 ([M + H]⁺). The ¹H-NMR spectrum of compound 1 (

Table 1. ¹H and ¹³C NMR spectroscopic data for compounds 1–3 in CDCl₃.

	1		2		3
	δH a (J/Hz)	δC b	δH a (J/Hz)	δC b	δH a (J/Hz)	δC b
1	-	166.6	-	166.2	-	166.6
2	-	129.7	-	132.2	-	129.7
3	4.90 (d, 5.0)	73.3	4.84 (d, 5.0)	72.2	4.89 (d, 5.0)	73.3
4	-	206.4	-	208.8	-	206.5
5	2.15 (s)	24.71	2.11 (s)	26.0	2.14 (s)	25.8
6	7.08 (t, 7.4)	149.1	6.91 (t, 7.2)	147.2	7.07 (t, 7.2)	149.2
7	2.35 (q, 7.4)	28.6	2.28 (q, 7.2)	28.0	2.34 (q, 7.2)	27.9
8	1.51 (m)	28.8	1.41 (m)	28.4	1.41 (m)	28.1
9	1.25 (m)	29.2	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
10	1.25 (m)	29.2	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
11	1.25 (m)	29.3	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
12	1.25 (m)	29.9	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
13	1.25 (m)	29.4	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
14	1.25 (m)	29.4	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
15	1.25 (m)	29.4	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
16	1.25 (m)	29.2	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
17	1.25 (m)	31.8	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
18	1.25 (m)	22.5	1.24 (m)	28.5–29.0	1.24 (m)	29.0–29.5
19	0.87 (t, 6.8)	13.9	1.24 (m)	31.5	1.24 (m)	29.0–29.5
20			1.24 (m)	22.5	1.24 (m)	29.0–29.5
21			0.86 (t, 6.7)	13.9	1.24 (m)	31.8
22					1.24 (m)	22.6
23					0.87 (t, 6.2)	13.9
1-OCH3	3.73 (s)	51.9	3.62 (s)	51.8	3.72 (s)	51.9

J values (Hz) are given in parentheses. ^a 600 MHz. ^b 150 MHz.

) displayed three characteristic signals: an olefinic proton at δ_H 7.08 (1H, t, J = 7.6 Hz), an oxymethine proton at δ_H 4.90 (1H, d, J = 5.0 Hz), and a methoxy group at δ_H 3.73 (3H, s). Correspondingly, the ¹³C-NMR spectrum (Table 1) revealed eight signals, including quaternary carbon atoms of a double bond at δ_C 129.7 (C-2) and 149.1 (C-6), a ketone group at δ_C 206.4 (C-4), and a carboxyl group at δ_C 166.6 (C-1), consistent with the structure of secomahubanolide, as reported by Cheng in 2005. These data indicated the presence of a secobutanolide skeleton. Compound 1 exhibited the E-form geometry of the trisubstituted double bond [δ_H 7.08 (1H, t, J = 7.6 Hz)] and demonstrated optical activity with ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −13.1° (c 0.32, CHCl₃), confirming a 3R configuration [15,16]. Based on these observations, the structure of compound 1 was identified as secomahubanolide.

Compound 2 was obtained as a yellow oil. The molecular formula was established as C₂₂H₄₀O₄ by high-resolution electrospray ionization time-of-flight mass spectrometry (HR-ESI-TOF-MS; m/z 369.2996 ([M + H]⁺)). Both the ¹H-NMR spectrum [δ_H 6.91 (1H, t, J = 7.2 Hz), δ_H 4.84 (1H, d, J = 5.0 Hz), and δ_H 3.62 (3H, s)] and ¹³C-NMR spectrum [δ_C 132.2 (C-2), 147.2 (C-6), 166.2 (C-1), and 208.8 (C-4)] were similar to that of secomahubanolide (1) (Table 1). Compound 2 exhibited the E-form geometry of the trisubstituted double bond, as indicated by the ¹H NMR signal at δ_H 6.91 (1H, t, J = 7.6 Hz). Its optical rotation was measured at ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −11.7° (c 0.15, CHCl₃), suggesting a 3R configuration [15,16]. Additionally, compound 2 featured two extra methylene units at δ_H 1.24 in its side chain compared to secomahubanolide (1). Based on these characteristics, the structure of compound 2 was determined to be secosubamolide A.

Compound 3 was also isolated as a yellow oil, with its molecular formula established as C₂₄H₄₄O₄ through high-resolution electrospray ionization time-of-flight mass spectrometry (HR-ESI-TOF-MS), yielding an m/z of 397.3319 ([M + H]⁺). The HR-MS spectrum clearly shows that compound 3 has two more methylene groups than compound 2, just as compound 2 has two more methylene groups than compound 1. The HR-MS data for all three compounds exhibit a consistent increasing trend. Therefore, we can conclude that compound 3 is compound 2 with two additional methylene groups on the side chain, and this inference is further confirmed by the NMR spectra. The ¹H NMR (Table 1) spectrum revealed signals at δ_H 7.07 (1H, t, J = 7.2 Hz), δ_H 4.89 (1H, d, J = 5.0 Hz), and δ_H 3.72 (3H, s), while the ¹³C NMR spectrum showed resonances at δ_C 132.0 (C-2), 146.8 (C-6), 166.2 (C-1), and 208.9 (C-4), closely resembling those of secomahubanolide (1) and secosubamolide A (2). Compound 3 maintained the E-form geometry of the trisubstituted double bond, as seen in the signal at δ_H 6.82 (1H, t, J = 7.6 Hz), and exhibited an optical rotation of ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −21.7° (c 0.52, CHCl₃), confirming the 3R configuration [15,16]. Moreover, compound 3 displayed two additional methylene units at δ_H 1.24 in its side chain compared to secosubamolide A (2). Based on these observations, the structure of compound 3 was identified as secosubamolide B.

2.2. Anti-Inflammatory Activity of Compounds 1–3

Prior to evaluating the anti-inflammatory potential of secobutanolides isolated from the methanol extract of L. obtusiloba, we initially examined the effects of compounds 1–3 (at a 50 μM concentration) on BMDC cell viability using an MTT colorimetric assay. The results showed that these compounds did not exhibit any cytotoxicity at the concentrations tested (data not shown).

Following this, the effects of compounds 1–3 on the production of IL-6 and IL-12 p40 were assessed at various concentrations ranging from 1 to 50 μM. In these experiments, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) was utilized as a positive control [14]. SB203580 demonstrated a marked ability to inhibit the production of both IL-6 and IL-12 p40, with calculated IC₅₀ values of 3.5 μM and 5.0 μM, respectively (Figure 2). The tested secobutanolides, identified as compounds 1–3, also showed significant inhibitory effects on IL-6 production, with IC₅₀ values determined to be 1.8 μM, 3.4 μM, and 24.1 μM, respectively. Similarly, these compounds exerted strong inhibitory activity against IL-12 p40 production, with IC₅₀ values of 3.1 μM, 2.9 μM, and 16.3 μM, as shown in

Table 2. Anti-inflammatory effects of compounds 1–3.

Compound	IC50 (µM) a
	IL-12 p40	IL-6
1	3.1 ± 0.4	1.8 ± 0.1
2	2.9 ± 0.6	3.4 ± 0.2
3	16.3 ± 0.6	24.1 ± 1.2
SB203580 b	5.0 ± 0.1	3.5 ± 0.2

The ^a IC₅₀ values for selected compounds are given in column IL-12 p40 and IL-6. ^b Positive control.

.

This study not only highlights the robust anti-inflammatory properties of these secobutanolides, but also provides important insights into their structure–activity relationships. Notably, the inhibitory effects were significantly amplified by the presence of unsaturated aliphatic substituents on the acylglycerol framework. Moreover, the activity of these compounds appeared to increase proportionally with the length of the fatty acid side chains, indicating that these structural characteristics are critical for their bioactivity. These findings could be pivotal in informing the design and synthesis of new anti-inflammatory agents derived from the secobutanolide structure, offering a promising direction for future drug development efforts.

3. Materials and Methods

3.1. General Information

Column chromatography: silica gel (Kieselgel 60, 70−230, and 230−400 mesh, Merck, Darmstadt, Germany); YMC RP-18 resins, and thin-layer chromatography (TLC): silica-gel 60 F₂₅₄ and RP-18 F₂₅₄S plates (both 0.25 mm, Merck, Darmstadt, Germany); optical rotation: Jasco DIP-370 automatic polarimeter (Easton, MD, USA); NMR spectra: JEOL ECA 600 spectrometer (Tokyo, Japan); High-resolution electrospray ionization mass spectra (HR-MS): Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Santa Clara, CA, USA).

3.2. Plant Material

In 2001, dried stems of L. obtusiloba were provided by Bomyeong Herbal Market (Seoul, Republic of Korea), Korea, and the voucher specimen (KM-000101) was deposited with the Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine, Korea.

3.3. Extraction and Isolation

The air-dried and chopped stems of L. obtusiloba (3.0 kg) were extracted three times with MeOH for 10 h in a water bath. The MeOH extract obtained was evaporated to dryness in a rotary evaporator, affording crude extract (185.3 g, 6.1% yield). The extract was dissolved in distilled water (1.5 L), and 1.2 L of the resulting solution was consecutively partitioned using CHCl₃, EtOAc, and n-BuOH; CHCl₃ (48.5 g), EtOAc (20.4 g), n-BuOH (36.8g), and H₂O fractions were obtained.

A portion of the CHCl₃ layer (48.5 g) was subjected to column chromatography using silica gel column chromatography and eluted with a stepwise gradient of EtOAc:MeOH (20:1–10:1–5:1–2:1–1:1, v/v) to yield three fractions (1a–c). Fraction 1a was chromatographed on a silica gel column using a gradient of n-Hexane:EtOAc (8:1–3:1 v/v) to afford sub-fractions (2a–c). Fraction 2b was further chromatographed on a silica gel chromatography column with CHCl₃:MeOH (90:1–70:1 v/v) to afford subfractions (3a–b). Fraction 3d was further chromatographed on a silica gel chromatography column with CHCl₃:MeOH (60:1 v/v) to yield compound 1 (12.1 mg). Fraction 3b was chromatographed on a silica gel column n-Hexane:acetone (8:1–7:1 v/v) to give compounds 2 (24.1 mg), 3 (2.4 mg), and 4 (49.9 mg).

Secomahubanolide (1): yellow oil; UV (MeCN): λ_max 215 nm; HR-ESI-TOF-MS m/z 341.2690 [M + H]⁺; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −13.1° (c = 0.32, CHCl₃); IR (KBr, cm⁻¹) 3450, 1735, 1710 cm⁻¹; ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz): see Table 1.

Secosubamolide A (2): yellow oil; UV (MeCN): λ_max 215 nm; HR-ESI-TOF-MS m/z 369.2996 [M + H]⁺; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −11.7° (c = 0.15, CHCl₃); IR (KBr, cm⁻¹) 3450, 1735, 1710 cm⁻¹; ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz): see Table 1.

Secosubamolide B (3): yellow oil; UV (MeCN): λ_max 215 nm; HR-ESI-TOF-MS m/z 397.3319 [M + H]⁺; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$: −21.7° (c = 0.52, CHCl₃); IR (KBr, cm⁻¹) 3450, 1735, 1710 cm⁻¹; ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz): see Table 1. HR-MS, UV, and NMR data of compound 3 can be found in the Supplementary Materials.

Secoisolitsealiicolide B (4): yellow oil; UV (MeOH): λ_max 213 nm; ¹H-NMR (400 MHz, CDCl₃, δ_H): 7.08 (1H, t, J = 7.5, H-6), 5.81 (1H, ddt, J = 17.5, 10.6, 6.8 Hz, H-16), 4.99 (1H, dd, J = 17.2, 2.1 Hz, H-17a), 4.93 (1H, dd, J = 10.3, 1.4 Hz, H-17b), 4.90 (1H, s, H-3), 4.03 (1H, s, 3-OH), 3.73 (3H, s, 1-OCH₃), 2.35 (2H, quartet, J = 7.6 Hz, H-7), 2.16 (3H, s, H-5), 2.04 (2H, quartet, J = 6.9 Hz, H-15), 1.28 (2H, br s, H-9), 1.28 (2H, br s, H-10), 1.28 (2H, br s, H-11), 1.28 (2H, br s, H-12), 1.28 (2H, br s, H-13), 1.28 (2H, br s, H-14); ¹³C-NMR (100 MHz, CDCl₃, δ_C): 206.5 (C-4), 166.6 (C-1), 149.2 (C-6), 139.2 (C-16), 129.8 (C-2), 114.2 (C-17), 73.4 (C-3), 52.0 (1-OCH₃), 33.7 (C-15), 28.8 (C-7), 28.6 (C-8), 28.7–29.5 (C-9), 28.7–29.5 (C-10), 28.7–29.5 (C-11), 28.7–29.5 (C-12), 28.7–29.5 (C-13), 28.7–29.5 (C-14), 24.9 (C-5).

3.4. Cell Culture

The BMDCs were derived from wild-type C57BL/6 mice. Briefly, bone marrow cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 50 μM of 2-ME, and 2 mM of glutamine supplemented with 3% J558L hybridoma cell culture supernatant containing granulocyte macrophage colony-stimulating factor for dendritic cell generation. The culture medium was replaced with fresh medium every second day. On day 6 of culture, non-adherent cells and loosely adherent DC aggregates were harvested, washed and resuspended in RPMI 1640 supplemented with 5% FBS. All animal procedures were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee of Jeju National University (#2010−0028).

3.5. Cytokine Production Measurements

The effect of isolated compounds on cytokine production by LPS-stimulated cells was determined using ELISA (BD PharMingen, San Diego, CA, USA). The final concentration of chemical solvents did not exceed 0.1%. BMDCs were cultured in DMEM medium containing macrophage colony-stimulating factor. On day 6 of incubation, BMDMs and BMDCs were harvested and seeded in 48-well plates at a density of 1 × 10⁵ cells/0.5 mL, and then treated with the PTE for 1 h before stimulation with LPS (10 ng/mL). At the end of the incubation period, the IL-12 p40 and IL-6 levels in the medium were determined by ELISA. All experiments were performed in triplicate.

3.6. Statistical Analysis

The student’s t-test and one-way ANOVA were used to determine the statistical significance of the differences between values for a variety of experimental and control groups. Data were expressed as the mean ± SD (at least in triplicate). The statistical significance was determined by one-way analysis of variance followed by Dunnett’s multiple comparison tests, p < 0.05.

4. Conclusions

In this study, four compounds, including a newly discovered secobutanolide (compound 3), were successfully isolated from a methanol extract of Lindera obtusiloba. The inhibitory effects of the isolated compounds (1–3) were assessed in terms of their ability to suppress the production of cytokines IL-12p40 and IL-6, which are stimulated by lipopolysaccharide (LPS) in bone marrow-derived dendritic cells (BMDCs). Notably, this research represents the first investigation into the secobutanolide components of L. obtusiloba and their potential anti-inflammatory properties. The compounds isolated in this study were identified from the plant for the first time, indicating their novelty. These findings suggest that secobutanolides are active constituents of L. obtusiloba and may serve as a scientific foundation for developing new anti-inflammatory agents derived from this plant.