Degraded Lignans and Phenylpropanoids from Styrax tonkinensis Resin with Anti-Inflammatory Activities

Phytochemistry 214 (2023) 113817

Available online 6 August 2023

0031-9422/© 2023 Elsevier Ltd. All rights reserved.

Nine pairs of undescribed enantiomers from the resin of Styrax tonkinensis (Pierre) Craib ex Hart with anti-inflammatory activity

Qun Li

^a,b

, Hong-Bin Fang

^b

, Bin-Yuan Hu

^b

, Yong-Ming Yan

^b

, Ya-Bin Jiao

^b

, Gan-Peng Li

^a,**

, Yong-Xian Cheng

^b,*,1

aKey Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, School of Ethnic Medicine, Yunnan Minzu University, Kunming, 650504, PR China

bInstitute for Inheritance-Based Innovation of Chinese Medicine, School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, 518060, PR China

A R T I C L E I N F O Keywords:

Styrax tonkinensis Plant resin Degraded lignans Phenylpropanoids Anti-inflammatory activities

A B S T R A C T

Nine pairs of undescribed enantiomers, (±)-styraxoids A− I (1–9), were isolated from the resin of Styrax tonki- nensis, and their structures were assigned by spectroscopic and computational methods. Compounds (±)-1 are a pair of degraded lignans, and the remaining compounds (±)-(2–9) are phenylpropanoid skeletons. Compounds (±)-8 and (±)-9 feature a 1,3-dioxolane moiety. The biological evaluation showed that both enantiomers of 1 could inhibit LPS-induced INOS and COX-2 in RAW264.7 cells in a dose-dependent manner.

1. Introduction

Styrax tonkinensis (Pierre) Craib ex Hart, which belongs to the genus Styrax of the family Styracaceae, is widely distributed throughout Asia, America, and the Mediterranean (Burger et al., 2016). The resin of S. tonkinensis has been used as a famous traditional medicine in China to treat various brain diseases for more than 1000 years (Wang et al., 2006a). Modern pharmacological studies have established its anti-inflammatory (Lee et al., 2016; Zhang et al., 2021), antitumor (Burger et al., 2016; In et al., 2013; Wang et al., 2021), antibacterial (De Oliveira et al., 2016; Debnath et al., 2022; Du et al., 2016), blood‒brain barrier protective, cerebral ischemic hypoxic injury protective proper- ties (In et al., 2013; Xie et al., 2021; Zhang et al., 2019) demonstrated by balsam acids (Castel et al., 2006; Wang et al., 2006b), triterpenoids (Wang et al., 2015, 2020), flavonoids (Braguine et al., 2012) and a few lignans (Li et al., 2005; Wang et al., 2021). Despite the above research, chemical and biological profiling in the resin of S. tonkinensis remains largely unknown. As a result, nine pairs of undescribed enantiomers were identified from the resin of S. tonkinensis (Fig. 1). In this study, the biological properties of these isolates were carried toward anti-inflammatory activity.

2. Results and discussion

2.1. Isolation and characterization of styraxoids A-I (1–9)

The extraction and separation protocols outlined in the Materials and Methods section gave rise to the isolation of nine undescribed enantio- mers compounds. All of them were obtained as racemic mixtures, which were separated through chiral HPLC to produce (+)- and (− )- antipodes.

Compound 1 was obtained as yellowish gum, possessing the molec- ular formula of C₂₀H₂₂O₆ based on the analysis of its HRESIMS (m/z 381.1300 [M +Na]⁺, calcd for 381.1309), with 10 degrees of unsatu- ration. The ¹H NMR spectrum of 1 (Table 1) displays one ABX system [δ_H 6.97 (1H, d, J =1.9 Hz, H-2), 6.87 (1H, d, J =8.0 Hz, H-5), and 6.86 (1H, dd, J =8.0, 1.9 Hz, H-6)], indicating the presence of one 1,2,4- trisubstituted phenyl group. Five olefinic/aromatic proton signals [δ_H 7.86 (2H, d, J =7.8 Hz, H-2

′

, 6

′

), 7.55 (1H, t, J =7.8 Hz, H-4

′

), and 7.40 (2H, t, J = 7.8 Hz, H-3

′

, 5

′

)], suggesting the presence of a mono- substituted benzene ring. The ¹³C NMR and DEPT spectra (Table 4) exhibit 20 carbons attributable to two methoxy groups, two sp³ meth- ylenes (including one oxygenated), eleven methine (including one oxygenated sp³), and five quaternary carbons. The structure of 1 was finally constructed by a detailed analysis of its 2D NMR data. The ¹H–¹H

* Corresponding author.

** Corresponding author.

E-mail addresses: ganpeng_@sina.com (G.-P. Li), yxcheng@szu.edu.cn (Y.-X. Cheng).

1 Lead contact.

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Phytochemistry

journal homepage: www.elsevier.com/locate/phytochem

https://doi.org/10.1016/j.phytochem.2023.113817

Received 4 April 2023; Received in revised form 31 July 2023; Accepted 1 August 2023

COSY spectrum displays the correlations of H-7/H-8/H-8

″

_{and H-8}

′′

_/H-

9

′′

. The HMBC correlations of H-9

′′

/C-7 and C-8 of 9

″

-OCH₃ (δ_H 3.49)/C- 9

″

and the chemical shifts of C-7 (δC 85.6), C-8 (δC 45.5), C-8’’ (δC 37.6), and C-9’’ (δ_C 105.0) indicated the presence of a tetrahydrofuran skeleton with oxygenated C-9

″

and C-9

″

replaced by methoxy. Furthermore, the HMBC correlations of H-7/C-1, C-2, and C-6 indicate that the benzene ring was connected to the tetrahydrofuran ring via C-7. The HMBC correlations of H-2

′

_{, H-6}’/C-7’ (δ_C 166.5), H-9/C-7

′

, and H-9/C-7, C-8, C- 8

″

suggest the presence of a benzoyloxy group connected to C-9 (δC

64.8). The chemical shifts of C-3 and C-4 indicate their oxygen-bearing nature. Furthermore, the HMBC correlation of H–OCH3/C-3 reveals the position of –OCH3 at C-3. Thus, the planar structure of compound 1 was deduced.

The relative configuration of 1 was deduced from the ROESY spec- trum (Fig. 2), which shows cross peaks of H-2/H-8/9

″

-OCH₃, indicating that H-2, H-8, and 9

″

-OCH3 are in the same orientation. In addition, the

observed correlations of H-2/Ha-8

′′

_/H-8/9

″

_{-OCH3 and H-7/Hb-8}

″

_suggest

that H-7/H-9

″

are in the same orientation. Compared with Sinkianli- gnans D (Li et al., 2022), the coupling constant between H-7 and H-8 (J7, 8 =9.2 Hz) in 1 indicated that H-7 and H-8 are in opposite orientations.

Compound 1 was isolated as a racemic mixture. This was confirmed by chiral HPLC analysis, which afforded (¡)-1 and (þ)-1 by a Chiralpak IC column (Fig. S42). Their absolute configurations were assigned as 7R, 8S, 9

″

_{S for (}¡)-1 and 7S, 8R, 9

″

_{R for (}þ)-1 by comparing their ECD spectra with the experimental ones (Fig. 3). In this way, the structure of compound 1 was finally identified and named (±)-styraxoid A.

Compound 2 was isolated as colorless gum. Its molecular formula was identified as C18H20O6 with 9 degrees of unsaturation based on its HRESIMS (m/z 355.1138 [M +Na]⁺, calcd for 355.1152), ¹³C NMR and DEPT spectra (Table 3). The ¹H NMR spectrum of 2 (Table 1) contains a typical ABX spin system [δ_H 6.90 (1H, d, J =1.9 Hz, H-2), 6.88 (1H, dd, J =8.0, 1.9 Hz, H-6), and 6.87 (1H, d, J =8.0 Hz, H-5)], suggesting the

Fig. 1. Structures of 1−9.

presence of 1,2,4-trisubstituted phenyl groups. Five aromatic protons [δH 7.96 (2H, d, J =7.8 Hz, H-2

′

,6

′

), 7.54 (1H, t, J =7.8 Hz, H-4

′

), and 7.41 (2H, t, J =7.8 Hz, H-3

′

,5

′

)] indicate the presence of one mono- substituted benzene ring. The ¹³C NMR and DEPT spectra (Table 4) exhibit 20 carbons attributed to two methoxy groups, one sp³ methylene (oxygenated), ten methines (including two oxygenated sp³), and five quaternary carbons. The structure of 2 was determined mainly by 2D NMR spectroscopic data (Fig. 2). The ¹H–¹H COSY (Fig. 2) correlations of H-7/H-8 and H-8/H-9 indicated the linkage of C-7 (δC 83.5)–C-8 (δC

77.2)–C-9 (δ_C 62.2). The chemical shifts of C-7, C-8, and C-9 indicate their oxygen-bearing nature. The HMBC correlations of H-7/C-1, C-2, C- 6, and H-8/C-1 indicate that a benzene ring was located at C-7 (Fig. 2).

The above data indicate the presence of a phenylpropanoid skeleton.

The key HMBC correlations of H-2

′

_{, H-6}’/C-7’ (δ_C 166.1), and H-8/C-7

′

show that a benzoyloxy was placed at C-8 (Fig. 2). The HMBC correla- tion of H–OCH3/C-3 reveals the position of –OCH3 at C-3. In this way, the planar structure of compound 2 was deduced.

The relative configuration of 2 was supposed to be threo by the coupling constant of H-7 and H-8 (J7,8 = 6.2 Hz) according to the reference (Weißbach et al., 2017; Xu et al., 2017). Compound 2 was also a racemic mixture, which was further purified by chiral HPLC to afford (+)-2 and (− )-2. Their absolute configurations were assigned as 7R,8S for (+)-2 and 7S,8R for (− )-2 by comparing the calculated and experi- mental ECD spectra (Fig. 4). As a result, the structure of 2, named (±)-styraxoid B, was determined as shown.

Compound 3 possesses a molecular formula of C17H18O5 (9 degrees of unsaturation), as deduced from the positive HRESIMS, ¹³C NMR, and DEPT spectra (Table 4). The ¹H NMR spectrum of 3 (Table 1) shows eight olefinic/aromatic protons [δH 8.06 (2H, d, J =7.8 Hz, H-2

′

, 6

′

), 7.59 (1H, t, J =7.8 Hz, H-4

′

), 7.46 (2H, t, J =7.8 Hz, H-3

′

_{, 5}

′

_); δ_H 6.76 (1H, br s, H-2), 6.75 (1H, d, J =8.0, H-5), and 6.87 (1H, dd, J =8.0, 1.9 Hz, H-6)]. The data indicate the presence of a 1,2,4-trisubstituted phenyl group and a monosubstituted benzene ring. The ¹³C NMR and DEPT spectra contain 17 carbons, corresponding to one methoxy group, two sp³ methylenes (one oxygenated), nine methines (including one oxygenated sp³), and five quaternary carbons. The observed ¹H–¹H COSY (Fig. 2) correlations of H-7/H-8 and the HMBC correlations of H- 9/C-7 and C-8 indicate the linkage of C-7 (δC 39.9)–C-8 (δC 71.2)–C-9 (δC

68.3). The HMBC (Fig. 2) correlations of H-7/C-1, C-2, C-6, and H-8/C-1 (δ_C 129.0) suggested the presence of a benzoate ring at C-7. These data indicate the presence of a phenylpropanoid skeleton. In addition, the HMBC correlations H-2

′

, H-6’/C-7’ (δC 166.9), and H-9/C-7

′

indicate the presence of a benzoyloxy at C-9. The HMBC correlation of H–OCH3/C-3 reveals the position of –OCH3 at C-3. Thus, the planar structure of 3 was

deduced to be that shown in Fig. 1.

The absolute configurations of compounds (±)-3 were assigned as 8R for (− )-3 and 8S for (+)-3 by comparing the ECD spectra (Fig. 4). In this way, the structure of compound 3 was finally identified and named (±)-styraxoid C.

Compound 4 was determined to have the molecular formula C18H20O6 (10 degrees of unsaturation) based on analysis of its positive HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR data (Table 2) of 4 contain eight aromatic signals at δH 8.03 (2H, d, J =7.8 Hz, H-2

′

, 6

′

), 7.59 (1H, t, J =7.8 Hz, H-4

′

), 7.44 (2H, t, J =7.8 Hz, H-3

′

, 5

′

), 6.97 (1H, d, J =1.9, H-2), 6.80 (1H, d, J =8.0, H-5), and 6.83 (1H, dd, J =8.0, 1.9 Hz, H-6). The ¹³C NMR and DEPT (Table 4) spectra show the presence of two methoxy groups, one sp³ methylene (oxygenated), ten methines (including two oxygenated sp³), and five quaternary carbons. These data are similar to those of 3, with only the differences being associated with the fact that the methylene at C-7 in 3 is replaced by a methoxy group in 4. This alteration is supported by the HMBC correlations of 7-OCH3/C-7 (δ_C 83.7). Thus, the planar structure of 4 was deduced to be that shown in Fig. 1.

In general, J7,8 =6.8 Hz means the configuration to be threo. How- ever, J_7,8 =6.8 Hz for 4 and J_7,8 =6.9 Hz for tonkinensisin C (Wang et al., 2021) is close to J =6.0 Hz. The main difference between 4 and tonkinensisin C is the chemical shift of H2-9 under the influence of the γ-gauche effect (Zhang et al., 2021). The shielded chemical shift of H2-9 and deshielded chemical shift of H-7 were observed for tonkinensisin C.

In the end, the configuration of 4 was assigned to 7,8-erythro. Compound 4 was divided into two enantiomers, (+)-4 and (− )-4, by chiral sepa- ration. Their absolute configurations were assigned as 7S, 8S for (+)-4 and 7R, 8R for (− )-4 by comparing its CD spectra with the experimental ones (Fig. 4). Thus, the structure of compound 4 was finally identified and named (±)-styraxoid D.

Compound 5 was obtained as yellowish gum. Its molecular formula was determined to be C17H18O6 (9 degrees of unsaturation) by its HRESIMS (m/z 341.0985 [M+Na]⁺, calcd for 341.0996), ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 5 (Table 2) contains eight ar- omatic signals at δ_H 8.03 (2H, d, J =7.8 Hz, H-2

′

, 6

′

), 7.56 (1H, t, J =7.8 Hz, H-4

′

), 7.44 (2H, t, J =7.8 Hz, H-3

′

_{, 5}

′

), 6.96 (1H, d, J =2.0, H-2), 6.90 (1H, d, J =8.0, H-5), and 6.87 (1H, dd, J =8.0, 2.0 Hz, H-6), suggesting the presence of a 1,2,4-trisubstituted benzene ring and a monosubstituted benzene ring. The ¹³C NMR and DEPT (Table 5) spectra show the presence of one methoxy group, one sp³ methylene (oxygen- ated), ten methines (including two oxygenated sp³), and five unproto- nated carbons. The data are similar to those of compound 4, with only the differences being associated with the fact that the methoxy group at C-7 in 4 is replaced by a hydroxyl in 5. This alteration is supported by their molecular weights differing by 14 units and compound 5 with the absence of a methoxy substitution at the C-7 position. Thus, the planar structure of compound 5 was deduced (Fig. 1). Detailed analyses of the

1H, ¹³C NMR, and HRESIMS data of compound 5 and compound 6 found their extreme similarities, suggesting that both of them share the same planar structure. Only the difference between 5 and 6 is the variation in relative configuration at two stereogenic carbons. Under the influence of the γ-gauche effect (Zhang et al., 2021), the shielded chemical shift of H2-9 and deshielded chemical shift of H-7 were observed for compound 6. Finally, the configuration of 5 is assigned to 7,8-erythro, and the configuration of 6 is assigned to 7,8-threo. Compounds 5 and 6 were also racemic mixtures, which were further purified by chiral HPLC to afford (+)-5, (− )-5, (− )-6, and (+)-6. Their absolute configurations were assigned as 7S, 8S for (+)-5 and 7R, 8R for (− )-5, 7R, 8S for (− )-6, 7S, 8R for (+)-6 by comparing the calculated and experimental ECD spectra (Figs. 4 and 5). Thus, the structures of compounds 5 and 6 were finally identified and named (±)-styraxoids E and F, respectively.

The planar structure of compound 6 was previously identified by GC‒MS (Burger et al., 2016), but its configuration is unknown. There are four possible stereo isomers, out of which separate two enantiomers, but siamyl benzoate remains unresolved. Tentatively, compound 6 is a Table 1

1H NMR data for 1−3 (δ_H in ppm, J in Hz, in CDCl3).

position 1^a 2^b 3^b

2 6.97 (d, 1.9) 6.90 (d, 1.9) 6.76 (br s)

5 6.87 (d, 8.0) 6.87 (d, 8.0) 6.75 (d, 8.0)

6 6.86 (dd, 8.0, 1.9) 6.88 (dd, 8.0, 1.9) 6.87 (dd, 8.0, 1.9) 7 4.75 (d, 9.2) 4.52 (d, 6.2) Ha: 2.88 (dd, 13.9, 5.5)

Hb: 2.80 (dd, 13.9, 7.7)

8 2.87 (m) 5.21 (m) 4.20 (br s)

9 4.38 (2H, m) Ha: 3.99 (m) Ha: 4.42 (m)

Hb: 3.94 (m) Hb: 4.30 (m) 2′,6′ 7.86 (d, 7.8) 7.96 (d, 7.8) 8.06 (d, 7.8) 3′,5′ 7.40 (t, 7.8) 7.41 (t, 7.8) 7.46 (t, 7.8) 4′ 7.55 (t, 7.8) 7.54 (t, 7.8) 7.59 (t, 7.8) 8″ Ha: 2.28 (m)

Hb: 2.05 (m) 9″ 5.10 (d, 4.9)

3-OCH3 3.86 (s) 3.87 (s) 3.88 (s)

7-OCH3 3.32 (s)

9″-OCH3 3.49 (s)

4-OH 5.59 (s) 5.60 (s) 5.53 (s)

aRecorded in 500 MHz.

b Recorded in 600 MHz.

previously undescribed compound with a definite configuration.

Compound 7 was isolated as colorless gum and found to have the molecular formula C₁₉H₂₀O₇ based on the analysis of its positive HRE- SIMS (m/z 383.1111 [M + Na]⁺, calcd for 381.1101). The ¹H NMR spectrum of 7 (Table 3) exhibits an aromatic ABX system [δ_H 6.95 (1H, d, J =2.0 Hz, H-2), 6.91 (1H, d, J =8.0 Hz, H-5), and 6.92 (1H, dd, J =8.0, 2.0 Hz, H-6)], and a monosubstituted benzene ring [δH 8.06 (2H, d, J = 7.8 Hz, H-2

′

, 6

′

), 7.56 (1H, t, J =7.8, 1.5 Hz, H-4

′

), and 7.44 (2H, t, J = 7.8 Hz, H-3

′

_{, 5}

′

_{)]. The} ¹³C NMR and DEPT spectra (Table 5) exhibit 19 carbons corresponding to one methoxy group, two sp³ methylenes, eleven methines (including three oxygenated sp³), and five quaternary carbons. The ¹H–¹H COSY spectrum (Fig. 2) showed correlations of H-7/

H-8/H₂-9, and the HMBC correlations (Fig. 2) of H-7/C-1, C-2, C-6, H-8/

C-1, and H-9/C-7 indicated the presence of a phenylpropanoid skeleton.

The HMBC correlations of H-8

′′

/C-7

′

(δ_C 166.4) and H-2

′

and H-6

′

/C-7

′

suggest the presence of a benzyloxy group connected to C-8

′′

(δ_C 65.0).

Two benzene rings and one carbonyl group account for 9 degrees of unsaturation. The linkage of C7–C8–C9, ¹H–¹H COSY correlations of H- 8’’/H-9

″

and the HMBC correlations of H-9’’ (δ_H 5.05)/C-7, C-9 led to the conclusion that C-7, C-8, C-9, and C-9’’ (δC 98.9) with two oxygen

atoms are connected to form a six-membered ring, which was further supported by the remaining one degree of unsaturation. The HMBC correlation of H–OCH₃/C-3 reveals the position of –OCH₃ at C-3. Finally, the planar structure of 7 was determined.

The relative configuration of compound 7 was confirmed by coupling constants and interpretation of the ROESY correlation. The ROESY correlations of H-7/H-9

″

indicate that H-7 and H-9

″

are in the same orientation. In addition, the relative configurations of H-7 and H-8 were supposed to be oppositely oriented by the coupling constant of H-7 and H-8 (J7,8 =9.0 Hz) according to the reference (Baciocchi et al., 2003).

Compound 7 was divided into two enantiomers, (− )-7 and (+)-7, by chiral HPLC separation. Their absolute configurations were assigned as 7R, 8S, 9

″

S for (− )-7 and 7S, 8R, 9

″

R for (+)-7 by comparing their ECD spectra with the experimental ones (Fig. 5). In this way, the structure of compound 7 was finally identified and named (±)-styraxoids G.

Compound 8 was obtained as colorless gum and has the same mo- lecular formula (C19H20O7) as that of 7, as deduced by analysis of their HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 8 (Table 3) contains eight aromatic signals at δ_H 8.06 (2H, d, J =7.8 Hz, H- 2

′

, 6

′

), 7.56 (1H, t, J =7.8 Hz, H-4

′

), 7.44 (2H, t, J =7.8 Hz, H-3

′

, 5

′

), 6.91 Fig. 2. ¹H–¹H COSY, Key HMBC, and ROESY correlations of compounds 1−5 and 7−9.

(1H, br s, H-2), 6.91 (1H, d, J =8.0, H-5), and 6.92 (1H, dd, J =8.0, 2.0 Hz, H-6). These data suggest the presence of a 1,2,4-trisubstituted benzene ring and a monosubstituted benzene ring. The ¹³C NMR and

DEPT spectra (Table 5) show the presence of one methoxy group, two sp³ methylenes (oxygenated), eleven methines (including two oxygen- ated sp³), and five unprotonated carbons. The ¹H–¹H COSY spectrum (Fig. 2) shows correlations of H-7/H-8/H₂-9, and the HMBC correlations of H-7/C-1, C-2, C-6, H-8/C-1, and H-9/C-7 indicate the presence of a phenylpropanoid skeleton. The HMBC (Fig. 2) correlations of H-8

′′

/C-7

′

(δ_C 166.7) and H-2

′

and H-6

′

/C-7

′

suggest the presence of a benzoyloxy group connected to C-8

′′

(δC 65.0). Two benzene rings and one carbonyl group account for 9 degrees of unsaturation. The ¹H–¹H COSY correla- tions of H-7/H-8/H-9, H-8’’/H-9

″

and the HMBC correlations of H-9’’

(δH 5.69)/C-7, C-8 led to the conclusion that C-7, C-8, C-9’’ (δC 102.0) and two oxygen atoms are connected to form a ring, which was further supported by the remaining one degree of unsaturation. Thus, the planar structure of 8 was deduced as shown in Fig. 1.

The relative configuration of 8 could be deduced by ROESY corre- lations of H-8 (δ_H 4.99)/H-9

′′

(δ_H 5.69), H2-8

′′

/H-7, and H-6/H-9

″

, which implied that H-8 and H-9

″

are in the same orientation and that H-7 and H-9

″

are in the opposite orientation. Compound 8 was divided into two enantiomers, (+)-8 and (− )-8, by chiral HPLC separation. Their absolute configurations were assigned as 7S, 8S, 9

″

R for (+)-8 and 7R, 8R, 9

″

S for (− )-8 by comparing its ECD spectra with the experimental ones (Fig. 5).

Thus, the structure of compound 8 was finally identified and named (±)-styraxoid H.

Compound 9 was determined to have the molecular formula C20H22O6 (10 degrees of unsaturation) based on analysis of its HRESIMS,

13C NMR, and DEPT spectral data (Table 5). The ¹H NMR data of 9 (Table 3) show eight olefinic/aromatic proton signals at δH 6.89 (1H, br s, H-2), 6.86 (1H, overlap, H-5), 6.86 (1H, overlap, H-6), 7.91 (2H, d, J

=7.8 Hz, H-2

′

, 6

′

), 7.56 (1H, t, J =7.8 Hz, H-4

′

), and 7.39 (2H, t, J =7.8 Hz, H-3

′

, 5

′

), which indicates the presence of one 1,2,4-trisubstituted phenyl group and a monosubstituted benzene ring. The ¹³C NMR and DEPT spectra (Table 3) indicate 20 carbons corresponding to three methyls, including one methoxy methyl, one sp³ methylene, ten methines (including two oxygenated sp³), and six quaternary carbons.

The ¹H–¹H COSY correlations (Fig. 2) of H-7/H-8/H2-9 and the HMBC correlations (Fig. 2) of H-7/C-1, C-2, C-6, H-8/C-1, and H-7/C-9 indi- cated the presence of a phenylpropanoid skeleton. The HMBC correla- tions of H-9/C-7

′

(δ_C 166.3) and H-2

′

and H-6

′

/C-7

′

suggest the presence of a benzoyloxy group at C-9 (δ_C 64.5). Two benzene rings and one carbonyl group account for 9 degrees of unsaturation. The ¹H–¹H COSY correlations of H-7/H-8 and the HMBC correlations of H-7 and H-8/C-8’’

(δ_C 108.9) led to the conclusion that C-7, C-8, C-8

″

and two oxygen atoms Fig. 3.Experimental and calculated ECD spectra of 1. Comparison of the

calculated ECD spectra of (7S,8R,9″R)-(−)-1, (7R,8S,9″S)-(+)-1, and (7R,8S) at the B3LYP/6-311G (d,p) level with the experimental spectra in MeOH.

Table 2

1H NMR data for 4−6 (δ_H in ppm, J in Hz, 5, 6 in CDCl3, and 4 in methanol-d4).

position 4^a 5^b 6^b

2 6.97 (d, 1.9) 6.96 (d, 2.0) 6.91 (d, 2.0)

5 6.80 (d, 8.0) 6.90 (d, 8.0) 6.88 (d, 8.0)

6 6.83 (dd, 8.0, 1.9) 6.87 (dd, 8.0, 2.0) 6.85 (dd, 8.0, 2.0)

7 4.18 (d, 6.6) 4.74 (d, 5.7) 4.66 (d, 7.0)

8 4.07 (m) 4.13 (m) 4.04 (m)

9 Ha: 4.47 (dd, 11.4,

3.3) Ha: 4.49 (dd, 11.8,

3.3) Ha: 4.36 (dd, 11.8,

3.6) Hb: 4.34 (dd, 11.4,

6.6) Hb: 4.41 (dd, 11.8,

6.4) Hb: 4.21 (dd, 11.8,

5.9) 2′,6′ 8.03 (d, 7.8) 8.03 (d, 7.8) 8.01 (d, 7.8) 3′,5′ 7.44 (t, 7.8) 7.44 (t, 7.8) 7.44 (t, 7.8) 4′ 7.59 (t, 7.8) 7.56 (t, 7.8) 7.57 (t, 7.8)

3-OCH3 3.85 (s) 3.87 (s) 3.85 (s)

7-OCH3 3.23 (s) aRecorded in 500 MHz.

b Recorded in 600 MHz.

Table 3

1H NMR data for 7−9 (δH in ppm, J in Hz, in CDCl3).

position 7^a 8^b 9^b

2 6.95 (d, 2.0) 6.91 (br s) 6.89 (br s)

5 6.91 (d, 8.0) 6.91 (d, 8.0) 6.86 (overlap)

6 6.92 (dd, 8.0, 2.0) 6.92 (dd, 8.0, 2.0) 6.86 (overlap)

7 4.28 (d, 9.0) 4.99 (d, 7.8) 5.31 (d, 6.8)

8 3.71 (m) 3.98 (m) 4.65 (m)

9 Ha: 4.31 (dd, 10.9, 5.0) Ha: 3.94 (m) Ha: 4.00 (m) Hb: 3.59 (dd, 10.9, 3.3) Ha: 3.64 (m) Hb: 4.00 (m) 2′_,6′ 8.06 (d, 7.8) 8.06 (d, 7.8) 7.91 (d, 7.8) 3′_,5′ 7.44 (t, 7.8) 7.44 (t, 7.8) 7.39 (t, 7.8)

4′ 7.56 (t, 7.8) 7.56 (t, 7.8) 7.56 (t, 7.8)

8″ 4.45 (dd, 4.5, 1.1) Ha: 4.60 (dd, 11.8, 4.6) Hb: 4.41 (dd, 11.8, 3.3)

9″ 5.05 (t, 4.5) 5.69 (t, 4.6) 1.50 (s)

10″ 1.67 (s)

3-OCH3 3.88 (s) 3.90 (s) 3.87 (s)

7-OCH3

4-OH 5.72 (s) 5.71 (s)

aRecorded in 400 MHz.

b Recorded in 500 MHz.

Table 4

13C NMR data for 1− 4 in CDCl3 (δ_C in ppm).

position 1^b 2^b 3^b 4^a

1 133.8 C 129.6 C 129.0 C 131.1 C

2 109.6 CH 109.3 CH 112.0 CH 112.1 CH

3 146.9 C 146.9 C 146.7 C 149.1 C

4 145.5 C 145.7 C 144.7 C 147.5 C

5 114.2 CH 114.3 CH 114.7 CH 115.9 CH

6 120.6 CH 120.9 CH 122.2 CH 121.9 CH

7 85.6 CH 83.5 CH 39.9 CH2 85.7 CH

8 45.5 CH 77.2 CH 71.2 CH 73.7 CH

9 64.8 CH2 62.2 CH2 68.3 CH2 67.3 CH2

1′ 129.9 C 130.0 C 130.0 C 131.4 C

2′_{, 6}′ _{129.7 CH 129.8 CH 129.8 CH 130.6 CH}

3′_{, 5}′ _{128.5 CH 128.5 CH 128.6 CH 129.5 CH}

4′ _{133.2 CH 133.3 CH 133.4 CH 134.2 CH}

7′ 166.5 C 166.1 C 166.9 C 168.1 C

8″ 37.6 CH2

9″ 105.0 CH

10″

3-OCH3 55.9 CH3 56.1 CH3 56.0 CH3 56.3 CH3

7-OCH3 57.5 CH3 57.1 CH3

9″_{-OCH3 55.1 CH3}

a Recorded in 125 MHz.

b Recorded in 150 MHz.

are connected to form a five-membered ring, which was further sup- ported by the remaining one degree of unsaturation. The two methyl groups were placed at C-8

′′

(δ_C 108.9), which was supported by the HMBC correlations of H-9

″

, H-10

″

, H-7, and H-8/C-8

′′

(δ_C 108.9). The HMBC correlation of H–OCH3/C-3 reveals the position of –OCH3 at C-3.

Finally, the planar structure of 9 was determined.

The relative configuration of 9 was deduced from the NOE cross- peaks observed between H-7/H3-9

″

and H-8/H3-9

″

, suggesting that H-8

and H-7 are in the same orientation. Compound 9 was divided into two enantiomers, (− )-9 and (+)-9, by chiral separation. Their absolute configurations were assigned as 7R,8S for (− )-9 and 7S,8R for (+)-9 by comparing their ECD spectra with the experimental ones (Fig. 5). In this way, the structure of compound 9 was finally identified and named (±)-styraxoid I.

2.2. Biological evaluation of 1− 9

Modern pharmacological studies have shown that the resin of S.

tonkinensishas certain antipyretic and anti-inflammatory effects (Li et al., 2022; Zhang et al., 2019). However, the material basis for efficacy is unknown. More detailed exploration is required to clarify the effective anti-inflammatory components of the resin of S. tonkinensis. This study evaluated the anti-inflammatory activity of nine pairs of undescribed enantiomers, (±)-styraxoids A− I (1–9), isolated from the resin of S. tonkinensis. First, the CCK8 assay results (Fig. 6) showed that the enantiomeric pairs of 1 and 6− 9 had no obvious cytotoxicity up to 20 μM, and the enantiomeric pairs of 2− 5 exhibited moderate toxicity at 20 μM. Second, the anti-inflammatory activity of enantiomeric pairs 1− 9 on iNOS and COX-2 enzymes was evaluated by Western blot analysis at a fixed concentration (20 μM), which showed that LPS-induced iNOS and COX-2 in RAW264.7 cells were inhibited by (±)-1. Furthermore, a dose-dependent experiment carried out with (±)-1 at 10, 20, and 40 μM (Fig. 7) revealed that both enantiomers exhibited dose-dependent anti-inflammatory potential. In addition, (±)-2 also showed certain activities. These results indicate that degraded lignans have better anti-inflammatory activity than phenylpropanoid derivatives, which are studied with a benzoate backbone.

Fig. 4.Experimental and calculated ECD spectra of 2−5. Comparison of the calculated ECD spectra of (7R,8S)-(+)-2, (7S,8R)-(−)-2; (8R)-(−)-3, (8S)-(+)-3; (7S,8S)- (+)-4, (7R,8R)-(−)-4; and (7S,8S)-(+)-5, (7R,8R)-(−)-5 at the B3LYP/6-311G (d,p) level with the experimental spectra in MeOH.

Table 5

13C NMR data of 6−9 in CDCl3 and 5 methanol-d4 (δ_C in ppm).

position 5^b 6^b 7^a 8^a 9^b

1 129.8 C 129.7 C 129.6 C 129.8 C 129.8 C

2 109.0 CH 109.2 CH 109.6 CH 109.6 CH 109.6 CH

3 146.9 C 146.9 C 147.0 C 147.0 C 146.7 C

4 145.7 C 145.8 C 146.2 C 146.1 C 145.7 C

5 114.6 CH 114.6 CH 114.4 CH 114.7 CH 114.5 CH

6 119.8 CH 119.9 CH 120.8 CH 120.0 CH 119.8 CH

7 74.0 CH 74.8 CH 84.3 CH 78.8 CH 78.7 CH

8 74.5 CH 74.8 CH 67.3 CH 84.8 CH 77.0 CH

9 65.8 CH2 65.9 CH2 70.7 CH2 60.4 CH2 64.5 CH2

1′ 131.8 C 132.0 C 129.9 C 129.8 C 130.0 C

2′, 6′ 129.9 CH 129.8 CH 130.0 CH 130.0 CH 129.8 CH 3′_{, 5}′ _{128.6 CH 128.6 CH 128.5 CH 128.6 CH 128.4 CH}

4′ _{133.4 CH 133.4 CH 133.3 CH 133.5 CH 133.1 CH}

7′ _{167.4 C 167.0 C 166.4 C 166.7 CH 166.3 C}

8″ 65.0 CH2 65.0 CH2 108.9 C

9″ 98.9 CH 102.0 CH 27.7 CH3

10″ 25.2 CH3

3-OCH3 56.1 CH3 56.0 CH3 56.1 CH3 56.2 CH3 56.0 CH3

7-OCH3

9″_-OCH3

aRecorded in 125 MHz.

b Recorded in 150 MHz.

Fig. 5.Experimental and calculated ECD spectra of 6− 9. Comparison of the calculated ECD spectra of (7R,8S)-(−)-6, (7S,8R)-(+)-6; (7R,8S,9″_S)-(−)-7, (7S,8R,9″_R)- (+)-7; (7S,8S,9″R)-(+)-8, (7R,8R,9″S)-(−)-8; (7R,8S)-(− )-9 and (7S,8R)-(+)-9 at the B3LYP/6-311G (d,p) level with the experimental spectra in MeOH.

Fig. 6. RAW264.7 cell proliferation in response to compounds (±)-1–(±)-9 at the same doses assayed by CCK-8 assay. Data represent the mean ±SEM values of three experiments. *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001 compared with DMSO alone. Dexamethasone (DEX) was used as a positive control. B-D, The protein levels of INOS and COX-2 were determined by Western blotting, and GAPDH was used as a control.

3. Conclusions

Herein, nine previously undescribed enantiomeric pairs, (±)-styr- axoids A− I, from S. tonkinensis resins, and their structures were fully characterized, including their absolute configurations, by spectroscopic and quantum chemical computational methods. Compound 7 was con- structed with a 1,3-dioxane moiety, and compounds 8 and 9 contain a 1,3-dioxolane moiety. The results showed that both enantiomers of 1 could inhibit LPS-induced iNOS and COX-2 in RAW264.7 cells in a dose- dependent manner. These isolations provide previously undescribed insight into the chemical profiling of S. tonkinensis resins beyond well- investigated structural types, such as lignans and triterpene flavo- noids, and suggest their potential role in improving LPS-induced inflammation.

4. Experimental 4.1. General

A UV-2401PC spectrometer from Shimadzu was used to record UV spectra. Utilizing an Anton Paar MCP-100 digital polarimeter, optical rotations were measured. On a Chirascan instrument, CD spectra were obtained (Applied Photophysics). Using TMS (tetramethyl silane) as an internal standard, 1D and 2D NMR spectra were captured on Bruker Avance 400 MHz, 500 MHz, and 600 MHz spectrometers (Bruker, Karlsruhe, Germany). A Shimadzu LC-20AD AB SCIEX triple TOF X500R MS spectrometer was used to acquire HRESIMS (Shimadzu Corporation, Tokyo, Japan). C18 silica gel (40–60 μm; Daiso Co., Japan), MCI gel CHP 20 P (75–150 μm; Mitsubishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 were used in column chromatography (CC) (Amersham Pharmacia, Uppsala, Sweden). Semipreparative HPLC was performed using a Saipuruisi (SEP) chromatograph and a YMC-Pack ODS-A column (250 ×10 mm, i.d., 5 μm). Preparative HPLC was performed on a SEP chromatograph with a YMC-Actus ODS-A column (250 ×20 mm, i.d., 5 μm). For the purification of racemic substances, chiral HPLC was used using a Daicel Chiralpak column (IC, 250 mm ×4.6 mm, i.d., and 5 μm).

4.2. Resin material

Resins of S. tonkinensis were collected from Laos in August 2021. A voucher specimen (CHYX0668), identified by Prof. Qingqian Zeng (Guangdong Institute of Traditional Chinese Medicine), was deposited at the School of Pharmaceutical Sciences, Shenzhen University, China.

4.3. Extraction and isolation

Powdered resins of S. tonkinensis (18 kg) were extracted with 95%

EtOH (3 ×120 L), each for 24 h at room temperature, and concentrated in vacuo. The crude extract (15.6 kg) was divided into 5 parts (Fr. A–Fr.

E) by using an MCI gel CHP 20 P column eluted with aqueous MeOH (50%–100%). Fr. B (379.0 g) was first subjected to a MCI gel CHP 20 P column (aqueous MeOH, 50%–100%) to provide eleven fractions (Fr.

B.1–Fr.B.11) based on TLC analysis. Fr.B.8 (15 g) was separated by silica gel (petroleum ether–Me2CO, 10:1–1:1) to afford five parts (Fr.

B.8.1− Fr.B.8.5), and Fr.B.8.3 (8.2 g) was separated by Sephadex LH-20 (MeOH) to afford six parts (Fr.B.8.3.1− Fr.B.8.3.6). Fr.B.8.3.3 (4.26 g) was further divided into eight portions (Fr.B.8.3.3.1–Fr.B.8.3.3.8) by using silica gel (petroleum ether–Me2CO, 100:1–1:1). Fr.B.8.3.3.5 (310 mg) was submitted to a preparative HPLC column eluted with aqueous MeCN with 0.1% FA, 30%–100%, flow rate: 7.0 mL/min to yield five parts (Fr.B.8.3.3.5.1− Fr.B.8.3.3.5.5). Compound 1 (3.6 mg, tR =19.1 min) was acquired from the fifth subfraction isolated by semipreparative HPLC (aqueous MeCN with 0.1% FA, 50%, flow rate: 3.0 mL/min). Fr.

B.9 (4.5 g) was separated by Sephadex LH-20 (MeOH) to afford three parts (Fr.B.9.1− Fr.B.9.3). Fr.B.9.2 (2.0 g) was also separated by a Sephadex LH-20 (MeOH) column to afford five parts (Fr.B.9.2.1− Fr.

B.9.2.5). Fr.B.9.2.3 was further divided into six portions (Fr.

B.9.2.3.1–Fr.B.9.2.3.6) by using silica gel (petroleum ether–Me2CO, 10:1–1:1). Fr.B.9.2.3.2 was purified by semipreparative HPLC (aqueous MeCN with 0.1% FA, 30%, flow rate: 3.0 mL/min) to yield 2 (5.0 mg, tR

=22.9 min). Compound 4 (26.3 mg, tR =18.3 min) was obtained from Fr.B.9.2.2.3 (52.3 mg), which was purified by semipreparative HPLC (aqueous MeCN with 0.1% FA, 40%, flow rate: 3.0 mL/min). Compound 3 (3.65 mg, tR = 20.5 min) was purified by semipreparative HPLC (aqueous MeCN with 0.1% FA, 30%, flow rate: 3.0 mL/min). Fr.B.6 (13.4 g) was divided into six portions (Fr.B.6.1–Fr.B.6.6) by using silica gel (petroleum ether–Me2CO, 10:1–1:1). While Fr.B.6.3 (2.9 g) was divided into three portions of gel filtrated through Sephadex LH-20 (MeOH). Fr.B.6.3.2 (521.0 mg) was filtered by using preparative HPLC (aqueous MeCN with 0.05% FA, 30%–100%, flow rate: 7.0 mL/min) to generate seven fractions (Fr.B.6.3.2.1− Fr B.6.3.2.7). Fr.B.6.3.2.6 (89 mg) was further separated by semipreparative HPLC (aqueous MeCN with 0.05% FA, 35%, flow rate: 3.0 mL/min) to give 5 (15.2 mg, tR = 17.3 min). In addition, compound 6 (41.0 mg, tR =24.9 min) was ob- tained from the fourth part (Fr.B.6.3.2.7, -72.0 mg) that was purified by semipreparative HPLC (aqueous MeCN with 0.1% FA, 30%, flow rate:

3.0 mL/min). Fr.B.6.2 (1.5 g) was gel filtered through Sephadex LH-20 (MeOH) to afford two parts (Fr.B.6.2.1 and Fr.B.6.2.2). The first part (305 mg) was separated by thin-layer chromatography (petroleum ether–Me2CO, 2:1) to afford Fr.B.6.2.1.1-Fr.B.6.2.1.4. Among these, Fr.

Fig. 7.Compounds (− )-1 and (+)-1 suppressed proinflammatory expression in LPS-induced RAW 264.7 cells. Cells were incubated in different concentrations of compounds (¡)-1 and (+)-1 for 2 h and then exposed to 1 μg/mL LPS for 12 h. A-B, The protein levels of INOS and COX2 were determined by Western blotting, and GAPDH was used as a control. Dexamethansone (DEX) was used as a positive control.

B.6.2.1.1 (82.0 mg) was subjected to semipreparative HPLC (MeCN/H2O containing 0.05% FA in water, 35%, flow rate: 3 mL/min) to afford 7 (8.9 mg, tR =28.3 min). Fr.B.6.2.1.2 (68.0 mg) was subjected to semi- preparative HPLC (MeCN/H2O containing 0.05% FA in water, 35%, flow rate: 3 mL/min) to afford 8 (13.1 mg, t_R =26.5 min) and 9 (2.6 mg, t_R = 27 min).

Interestingly, chiral HPLC analysis indicated that compounds 1− 9 all contain their enantiomers, corresponding to the small value of optical rotation. For this reason, all of the compounds were subjected to further purification by chiral HPLC (flow rate: 1 mL/min). These separations gave (− )-1 (0.6 mg, tR =8.5 min) and (+)-1 (0.7 mg, tR =13.0 min) (n- hexane/EtOH with 0.01% FA, 80:20) (Fig. S88); (+)-2 (1.0 mg, tR =9.5 min) and (− )-2 (0.9 mg, tR = 19.8 min) (n-hexane/EtOH containing 0.01% FA, 85:15) (Fig. S91); (− )-3 (tR =13.8 min, 0.9 mg) and (+)-3 (tR

= 20.4 min, 0.9 mg) (n-hexane/EtOH containing 0.01% FA, 85:15) (Fig. S94); (+)-4 (2.1 mg, tR =6.7 min) and (− )-4 (2.2 mg, tR =12.9 min) (n-hexane/EtOH with 0.01% FA, 75:25) (Fig. S97); (+)-5 (tR =9.7 min, 1.2 mg) and (− )-5 (t_R =13.8 min, 1.3 mg) (n-hexane/EtOH con- taining 0.01% FA, 80:20) (Fig. S100); (− )-6 (2.0 mg, tR =11.6 min) and (+)-6 (2.0 mg, tR =12.7 min) (n-hexane/EtOH containing 0.01% FA, 80:20) (Fig. S103); (− )-7 (1.3 mg, t_R =13.0 min) and (+)-7 (1.4 mg, t_R

= 17.0 min) (n-hexane/ethanol containing 0.05% TFA in ethanol, 85:15) (Fig. S106); (+)-8 (1.5 mg, tR =17.7 min) and (− )-8 (1.3 mg, tR

= 19.9 min) (n-hexane/ethanol containing 0.05% TFA in ethanol, 80:20) (Fig. S109); (− )-9 (0.9 mg, tR =9.7 min) and (+)-9 (0.9 mg, tR = 14.8 min) (n-hexane/ethanol containing 0.05% TFA in ethanol, 90:10) (Fig. S112).

4.4. Data for characterization of 1–9 are given below 4.4.1. (±)-styraxoid A (1)

Yellowish gum; UV (MeOH) λmax (logε) 281 (1.71), 256 (1.59), 228 (2.28), 215 (2.16) nm; [α]²⁵D − 12.5 (c 0.03, MeOH); CD (MeOH) Δε₂₄₃

− 0.26, Δε₂₁₉ − 0.48; (− )-1; [α]²⁵_D +10.0 (c 0.03, MeOH); CD (MeOH) Δε₂₄₂ –0.64, Δε₂₁₈ − 0.38; (+)-1; HRESIMS (positive) m/z 381.1304 [M +Na]⁺(calcd for C20H22O6Na, 381.1309); ¹H and ¹³C NMR data, see Tables 1 and 4.

4.4.2. (±)-styraxoid B (2)

Colorless gum; UV (MeOH) λ_max (logε) 254 (0.93), 229 (2.2), 215 (1.96) nm; [α]²⁵D +17.6 (c 0.03, MeOH); CD (MeOH) Δε₂₄₉ +0.93, Δε₂₃₃ +5.37, Δε₂₁₅ +4.10; (+)-2; [α]²⁵D − 11.8 (c 0.03, MeOH); CD (MeOH) Δε₂₅₄ –1.26, Δε₂₃₄ − 4.94, Δε₂₁₉ − 4.40; (− )-2; HRESIMS (positive) m/z 355.1138 [M + Na]⁺(calcd for C₁₈H₂₀O₆Na, 355.1152); ¹H and ¹³C NMR data, see Tables 1 and 4.

4.4.3. (±)-styraxoid C (3)

Colorless gum; UV (MeOH) λmax (logε) 280 (1.35), 253 (0.87), 229 (2.11) nm; [α]²⁵D − 5.9 (c 0.03, MeOH); CD (MeOH) Δε₂₄₉–1.23, Δε₂₃₅ +0.20, Δε₂₁₉ –0.48; (− )-3; {[α]²⁵D +6.0 (c 0.03, MeOH); CD (MeOH) Δε₂₅₅ +0.63, Δε₂₃₈ − 1.21, Δε₂₂₁ +0.44; (+)-3; HRESIMS (positive) m/z 325.1058 [M + Na]⁺(calcd for C17H18O5Na, 325.1046); ¹H and ¹³C NMR data, see Tables 1 and 4.

4.4.4. (±)-styraxoid D (4)

Yellowish gum; UV (MeOH) λ_max (logε) 281 (1.47), 253 (1.06), 230 (2.19) nm; [α]²⁵_D +8.8 (c 0.03, MeOH); CD (MeOH) Δε₂₂₃+1.57, Δε₂₁₃ +0.31, ; (+)-4; [α]²⁵D − 25.0 (c 0.03, MeOH); CD (MeOH) Δε₂₂₂− 1.09, Δε₂₁₄ − 0.33; (− )-4; HRESIMS (positive) m/z 355.1152 [M +Na]⁺(calcd for C₁₈H₂₀O₆Na, 355.1152); ¹H and ¹³C NMR data, see Tables 2 and 4.

4.4.5. (±)-styraxoid E (5)

Yellowish gum; UV (MeOH) λ_max (logε) 253 (0.93), 229 (2.2), 214 (1.96) nm; [α]²⁵D +16.1 (c 0.03, MeOH); CD (MeOH) Δε₂₃₂+3.63, Δε₂₁₄ +1.21, ; (+)-5; [α]²⁵D − 6.5 (c 0.03, MeOH); CD (MeOH) Δε₂₂₈− 3.34, Δε₂₁₈ − 1.63; (− )-5; HRESIMS (positive) m/z 341.0985 [M +Na]⁺(calcd

for C17H18O6Na, 341.0996); ¹H and ¹³C NMR data, see Tables 2 and 5.

4.4.6. (±)-styraxoid F (6)

Yellowish gum; UV (MeOH) λ_max (logε) 256 (1.02), 229 (2.28), 214 (2.03) nm; [α]²⁵_D − 17.7 (c 0.03 MeOH); CD (MeOH) Δε₂₂₃− 3.24, Δε₂₁₆

− 1.24, ; (− )-6; [α]²⁵D +28.1 (c 0.03, MeOH); CD (MeOH) Δε₂₃₄+6.07, Δε₂₁₃ +1.08; (+)-6; HRESIMS (positive) m/z 341.0986 [M +Na]⁺(calcd for C17H18O6Na, 341.0996); ¹H and ¹³C NMR data, see Tables 2 and 5.

4.4.7. (±)-styraxoid G (7)

Colorless gum; UV (MeOH) λ_max (logε) 255 (1.02), 229 (2.32), 214 (2.07) nm; [α]²⁵D − 5.5 (c 0.03, MeOH); CD (MeOH) Δε₂₅₇+0.46, Δε₂₃₆

− 2.37, (− )-7; [α]²⁵D +5.5 (c 0.03, MeOH); CD (MeOH) Δε₂₅₃+0.63, Δε₂₃₆ +2.52; (+)-7; HRESIMS (positive) m/z 383.1111 [M +Na]⁺(calcd for C₁₉H₂₀O₇Na, 383.1101). ¹H and ¹³C NMR data, see Tables 3 and 5.

4.4.8. (±)-styraxoid H (8)

Colorless gum; UV (MeOH) λ_max (logε) 255 (0.95), 230 (2.31), 214 (2.07) nm; [α]²⁵D +8.3 (c 0.03, MeOH); CD (MeOH) Δε₂₅₇+0.46, Δε₂₃₆

− 2.37, (+)-8; [α]²⁵D − 19.4 (c 0.03, MeOH); CD (MeOH) Δε₂₅₃+0.63, Δε₂₃₆ +2.52; (− )-8; HRESIMS (positive) m/z 383.1111 [M +Na]⁺(calcd for C19H20O7Na, 383.1101). ¹H and ¹³C NMR data, see Tables 3 and 5.

4.4.9. (±)-styraxoid I (9)

Colorless gum; UV (MeOH) λmax (logε) 255 (1.17), 230 (2.26), 214 (2.08) nm; [α]²⁵D − 19.4 (c 0.03, MeOH); CD (MeOH) Δε₂₅₃+0.22, Δε₂₃₉ +0.30, (− )-9; [α]²⁵D +13.3 (c 0.03, MeOH); CD (MeOH) Δε₂₄₉+0.30, Δε₂₃₈ − 0.20; (+)-9; HRESIMS (positive) m/z 381.1317 [M +Na]⁺(calcd for C20H22O6Na, 381.1333). ¹H and ¹³C NMR data, see Tables 3 and 5.

4.5. Biological assay

4.5.1. Cell culture and treatment

RAW264.7, a mouse macrophage line (Procell Life Science & Tech- nology Co., Wuhan, China), was cultured in high-glucose DMEM (C11995500BT, Gibco) supplemented with 10% fetal bovine serum (FBS) (2094468CP, Gibco), 100 U/ml penicillin and 100 μg/mL strep- tomycin at 37 ^◦C in a humidified environment containing 5% CO2.

4.5.2. Cell viability assay

CRAW 264.7 cells (5 ×10⁵ cells/mL) were seeded into 96-well plates with complete DMEM. After culturing overnight, cells were treated with compounds (20 μM) or DMSO for 24 h. Then, a Cell Count Kit-8 (CCK-8, Beyotime, Shanghai, People’s Republic of China) was added into each well for 1 h at 37 ^◦C. The absorbance of each well was determined at 450 nm using a microplate reader (BioTek, USA).

4.5.3. Western blot analysis

After lipopolysaccharide (LPS) treatment, total protein was extracted from the cell lines using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing protease inhibitor cocktail (Roche, Ger- many), phosphatase inhibitor cocktail (Roche, Germany), and PMSF (Beyotime, China), and protein samples were quantified using the BCA assay (Thermo Scientific, USA). Equal amounts of protein extracts were separated by 8% SDS‒PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA, incubated with the indicated antibodies overnight at 4 ^◦C, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature.

The bands were visualized and measured via the ECL kit (Pierce, USA) and analysis system (Bio-Rad, CA, USA). Densitometry analysis of the immunoblot results was performed using ImageJ software (NIH, USA).

4.5.4. Statistical analysis

All experiments used to obtain data were performed in triplicate. The results are represented as the mean ±SEM. Statistical analyses were performed by using GraphPad Prism 6 (GraphPad Software, San Diego,

CA, USA) with one-way ANOVA. Differences were considered significant when *P ≤0.05, **P ≤0.01, and ***P ≤0.001.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All required data available in Supporting Information Acknowledgments

This study was supported financially by the Shenzhen Fundamental Research Program (No. JCYJ20200109114003921) and the National Natural Science Fund for Distinguished Young Scholars (81525026).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.phytochem.2023.113817.

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