Phthalide mono- and dimers from the rhizomes of Angelica sinensis and their anti-inflammatory activities

1 Introduction

The roots of Angelica sinensis (Oliv.) Diels (Umbelliferae family) is a well-known food and medicine homology herb, which has been widely used as therapeutic treatment for menstrual disorders and chronic constipation for thousands of years [1, 2]. Till now, there are more than ten types of secondary metabolisms have been identified from A. sinensis [3]. Among them, ligustilide and other structure intriguing dimeric phthalides were the representative components [4]. Their diverse structures and biological activities including anti-inflammatory, anticonvulsion, and platelet aggregation inhibition are long-standing attractive targets among natural product chemists and pharmacologists [5–8].

NO is synthesized and released into endothelial cells by nitric oxide synthase and plays vital role in the pathogenesis of inflammatory diseases [9]. Moreover, excessive production of NO is closely related to a variety of inflammatory disorders [10, 11]. Some phthalide dimers have been found to have significant anti-inflammation activity by inhibiting LPS-induced NO production [12]. Recently, an increasing number of studies have been reported on natural products with remarkable biological activities and novel structures from medicinal and dietary plants [13, 14]. To further explore anti-inflammatory natural products from medicinal plants, four phthalides including three pairs of enantiomeric dimers and a monomer (Fig. 1) were isolated and identified from the rhizomes of A. sinensis. Herein, we describe their structural elucidation and anti-inflammatory activities.

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2 Results and discussion

Angesicolide A (1) was isolated as colorless oil. The HR-ESI–MS of 1 (m/z 397.2015 [M + H]⁺, calcd for 397.2010) indicated its molecular formula of C₂₄H₂₈O₅, with 11 degrees of unsaturation. IR band at 1758 cm⁻¹ indicated the presence of an α,β-unsaturated lactone. Compound 1 demonstrated 24 carbon signals in ¹³C NMR, which was classified into two carbonyl carbons (δ_C 168.4, 168.2), six olefinic carbons, two oxygenated carbons (δ_C 89.9, 61.6), four methines, eight methylenes, two methyls (δ_C 13.8, 13.6) by DEPT experiments. The ¹H NMR (Table 1) demonstrated the presence of two butylidene side chains [δ_H 5.11 (1H), 2.33 (2H), 1.47 (2H), and 0.93 (3H); δ_H 3.27 (1H), 1.90, 1.78 (each 1H), 1.54 (2H), and 0.93 (3H)], four methine signals (δ_H 3.04, 3.58, 3.03, 3.57) and four methane signals [δ_H 2.35 (2H), 1.80 (2H), 2.17, 2.04 (each 1H), 1.86 (2H)]. These signals suggested 1 to be a phthalide dimer with the same basic skeleton as chaxiongnolide A [15]. Striking differences included an extra epoxy group based on same degree of unsaturation and the absence of a double bond. The crucial HMBC correlation between H-8ʹ (δ_H 3.27) with C-10ʹ (δ_C 19.2), C-9ʹ (δ_C 29.8), and C-3ʹa (δ_C 158.4) indicated that the epoxy group was situated at C-3ʹ and C-8ʹ. Further ¹H–¹H COSY, HSQC, and HMBC data verified the constructive planar structure (Fig. 2).

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The relative stereochemistry of 1 was elucidated based on interpretation of ROESY interactions. The butylidene side chain took Z-form by the ROESY correlation of H-8 and H-4a. Furthermore, the ROESY correlations of H-7′/H-5a and H-7/H-5′a indicated β-orientations for H-6 and H-7, and α-orientations for H-6′ and H-7′. Consequently, compound 1 was determined to possess the relative configurations of 6R*,7R*,6′R*, and 7′R* (Fig. 3). Although H-4′a showed a strong ROESY correlation with H-8′, it was insufficient to determine the relative configuration of C-3′ and C-8′. Therefore, two possible stereoisomers have been proposed: (6R*,7R*,3′R*,6′R*,7′R*,8′S*)-1A or (6R*,7R*,3′S*,6′R*,7′R*,8′R*)-1B. These configurations were subsequently subjected to NMR calculations by employing the GIAO approach at the wB97XD/6–31+G(d,p) level of theory [16]. The results indicated the whole relative configuration of 1 to be 6R*,7R*,3′R*,6′R*,7′R*,8′S* with a DP4 + MM probability of approximately 75% (Fig. 4B).

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Angesicolide B (2) was obtained as colorless gum. 2 had the molecular formula of C₂₄H₂₈O₅ deduced by the HR-ESI–MS data (m/z 414.2271 [M + NH₄]⁺, calcd for C₂₄H₂₈O₅NH₄ 414.2275), with 11 degrees of unsaturation. The existence of hydroxy and α,β-unsaturated lactone moieties was evidenced by the IR absorption bands at 3437, 1772 and 1710 cm⁻¹. 2 showed 24 carbon signals including eight quaternary carbons, seven methines, seven methylenes and two methyls. The high similarity NMR data with that of levulanolide A indicated both compounds shared the same carbon skeleton [17], except for the existence of an additional hydroxyl group in 2 (δ_H 4.64; δ_C 68.1). The HMBC correlations of H-9 (δ_H 4.64) with C-8 (δ_C 113.0) and C-10 (δ_C 29.9) finally established the hydroxyl group was attached to C-9 (Fig. 2).

The α-orientations of H-6 and H-7, and the β-orientation of H-6′ were confirmed by the ROESY correlations of H-4′a/H-7, H-4′a/H-8′, H-7/H-8′, H-5/H-6′, and H-6/H-5′. Consequently, the relative configurations of C-6, C-7, C-3′a, and C-6′ were assigned as 6R*,7R*,3′aR*, and 6′R*, respectively (Fig. 3). The stereochemistry of the hydroxyl group at C-9 could not be determined through ROESY correlations. Consequently, two possible relative configurations were proposed for compound 2: (6R*,7R*,9R*,3′aR*,6′R*)-2A and (6R*,7R*,9S*,3′aR*,6′R*)-2B. The subsequent experimental and the calculated NMR data established the relative configuration of 2 as 6R*,7R*,9R*,3′aR*,6′R*, with a probability of approximately 85% for DP4 + MM (Fig. 4E).

Angesicolide C (3) was purified as yellow oil. It had the same molecular formula of C₁₂H₁₂O₃ with that of senkyunolide C [18], based on the HR-ESI–MS data at m/z 205.0861 [M + H]⁺ (calcd for 205.0859). Upon analysis of the 1D and 2D NMR data, 3 possessed the same planar structure as that of senkyunolide C. The most pronounced difference was the enolicdouble bond in 3 took E-configuration, as verified by the absence of ROESY correlation of H-8 and H-4 and the corresponding upfield chemical shifts at C-4 (Δδ_C − 8.2 ppm) and C-8 (Δδ_C − 3.7 ppm) in 3.

Based on the specific optical rotation and chiral HPLC data, compounds 1 and 2 were a mixture of enantiomers, which were further treated by chiral separation to give two pairs of enantiomers [(+)-1/(−)-1, (+)-2/(−)-2]. As shown in Fig. 4, the calculated ECD results finally established the absolute configurations of (+)-1 and (−)-1 as (6S,7R,3ʹS,6′S,7′R,8ʹR)-1 and (6R,7S,3ʹR,6′R,7′S,8ʹS)-1 (Fig. 4C), respectively. Similarly, the absolute configurations of (+)-2 and (−)-2 were assigned as (6R,7R,9R,3′aR,6′R)-2 and (6S,7S,9S,3′aS,6′S)-2 (Fig. 4F).

Compound 4 was firstly isolated from A. sinensis. Comparing its spectral data with the reported data, compound 4 was identified as (±)-lyocasuarolide A [19]. Compounds (+)-4 and (−)-4 were obtained by chiral HPLC analysis, see Supporting Information for details.

All the isolates (1–4) were evaluated in vitro for their inhibitory effects against NO production in LPS-induced RAW 246.7 mouse macrophages. As shown in Fig. 5 and Figure S1, compounds 2 and 4 and their enantiomers exhibit inhibitory activity against LPS-induced NO production without cytotoxicity. As shown in Table 2, compounds 2, (+)-2, (−)-2, 4, (+)-4, and (−)-4 exhibited significant NO inhibitory effects with IC₅₀ values between 1.23 and 5.55 μM, which were more potential than the positive control L-NMMA.

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3 Materials and methods

3.1 General experimental procedures

NMR, HR-ESI–MS, UV, and IR spectra were measured as described previously [20].

3.2 Plant materials

Details regarding the plant material, including collection and identification information, have been reported previously [20].

3.3 Extraction and isolation

Detailed information for compounds 1–4 isolation procedures please see the Supporting Information.

3.4 Chiral separation and characterization

Compounds 1 and 2 were analyzed and isolated with a CHIRALPAK AD-H chiral column to give (+)-1/(−)-1, (+)-2/(−)-2. Chiral separation details are shown in the Supporting Information.

Compound 1: Colorless oil; UV (MeOH) λ_max (log ε) 278 (3.56) nm; HR-ESI–MS m/z 397.2015 [M + H]⁺ (calcd for C₂₄H₂₈O₅ + H, 397.2010). IR (KBr) ν_max 2960, 1758, 1717, 1635, 679 cm⁻¹; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 1. (+)-(6R,7R,3′R,6′R,7′R,8′S)-1: [α]_D²⁵ + 17 (c 0.08, MeOH); ECD (MeOH) λ_max (Δε) 195 (−11.6), 226 (−0.6), 291 (+11.3) nm. (−)-(6S,7S,3′S,6′S,7′S,8′R)-1: [α]_D²⁵ − 21 (c 0.07, MeOH); ECD (MeOH) λ_max (Δε) 195 (+15.3), 219 (+2.0), 256 (−8.3) nm.

Compound 2: colorless gum; UV (MeOH) λ_max (log ε) 274 (4.40) nm; HR-ESI–MS m/z 414.2271 [M + NH₄]⁺ (calcd for C₂₄H₂₈O₅ + NH₄, 414.2275). IR (KBr) ν_max 3437, 2958, 2934, 2872, 1772, 1710, 1674, 1461 cm⁻¹; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 1. (+)-(6R,7R,9R,3ʹaR,6ʹR)-2: [α]_D²⁵ + 246 (c 0.18, MeOH); ECD (MeOH) λ_max (Δε) 202 (+24.8), 238 (−4.5), 261 (+2.7), 292 (−10.2) nm. (−)-(6S,7S,9S,3ʹaS,6ʹS)-2: [α]_D²⁵ − 346 (c 0.13, MeOH); ECD (MeOH) λ_max (Δε) 202 (−27.3), 238 (+6.7), 261 (−1.6), 292 (+12.7) nm.

Compound 3: yellow oil; HR-ESI–MS m/z 205.0861 [M + H]⁺ (calcd for C₂₄H₂₈O₅ + H, 205.0859). IR (KBr) ν_max 3182, 1721, 1670, 1620, 1589 cm⁻¹; ¹H (500 MHz, methanol-d₄) and ¹³C NMR (125 MHz, methanol-d₄) data see Table 1.

3.5 ECD calculation

The theoretical calculations of compounds 1 and 2 were calculated employing the Gaussian program package. Conformational analysis was performed using CONFLEX software. Conformations with a Boltzmann population exceeding 2% were chosen for optimization and spectral calculation. The theoretical calculation of ECD was performed using Time Dependent Density Functional Theory (TDDFT) at B3LYP/6-31G (d,p) (compound 1) and CAM-B3LYP/DGDZVP (compound 2) level. The ECD spectra of the compounds 1 and 2 were obtained by considering the Boltzmann distribution of each geometric conformation [21].

3.6 NO inhibitory assay

The NO inhibitory assay was carried out as described previously [20]. For details, see the Supporting Information.

3.7 Statistical analysis

The statistical analysis was carried out as described previously [20].

4 Plausible biosynthetic pathway for 1 and 2

Phthalide dimers with different linkages are generated by [4 + 2] or [2 + 2] cycloaddition of two monomeric phthalate units. As for 1 and 2, they might be formed by cycloaddition reaction between Z-ligustilide and the molecule of phthalide monomer (3,8-epoxyligustilide [22] and senkyunolide F [23]) (Scheme 1).

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5 Conclusion

In conclusion, three pairs of enantiomeric phthalides characterized as [2 + 2] and [4 + 2] cycloadducts and a phthalide monomer were obtained from the rhizomes of A. sinensis. The racemates were further separated by chiral column and all of them were evaluated for their inhibitory effects on LPS-induced NO production. Interestingly, the current results demonstrated that the phthalide dimers have more potential anti-inflammatory activity than that of monomeric phthalides. The discovery of 1–3 are enriching supplement for the diversity of the phthalides category and provides a new insight for their biosynthetic, synthetic, and pharmacological investigation.

Notes

Author contributions

Hongyan Wen, finished the isolation and identification studies; Sheng Li implemented the anti-inflammatory studies. Formal analysis and data compilation by Hongyan Wen and Sheng Li. Hongyan Wen wrote the first draft; Yu Zhang conceived the projects, revised manuscript, and provided financial support; All authors approved the final version of the manuscript.

Funding

This work was financially supported by National Key R&D Program of China (2022YFF1100301), Major Science and Technology Project of Henan Province (231100310200), Yunnan Province Science and Technology Department (202305AH340005).

Data availability

All data generated or analyzed during this study are included in this published article and its Additional file 1.

Declarations

Competing interests

The authors declare no competing financial interest.