Anti-NLRP3 inflammasome abietane diterpenoids from <i>Callicarpa bodinieri</i> and their structure elucidation

Anti-NLRP3 inflammasome abietane diterpenoids from Callicarpa bodinieri and their structure elucidation

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Junbo Gao^a^,^b^,¹, Xingjie Zhang^a^,¹, Kun Shang^a^,¹, Weimao Zhong^b, Ruihan Zhang^a, Xiaochang Dai^a, Xiaoli Li^a^,^*, Qi Wang^a, Yan Zou^a, Weilie Xiao^a^,^*

Chinese Chemical Letters | 2020, 31(2) : 427 - 430

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Chinese Chemical Letters | 2020, 31(2): 427-430

• Communication •

Anti-NLRP3 inflammasome abietane diterpenoids from Callicarpa bodinieri and their structure elucidation

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Junbo Gao^a^,^b^,¹, Xingjie Zhang^a^,¹, Kun Shang^a^,¹, Weimao Zhong^b, Ruihan Zhang^a, Xiaochang Dai^a, Xiaoli Li^a^,^*, Qi Wang^a, Yan Zou^a, Weilie Xiao^a^,^*

Affiliations

^a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China

^b State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

Published: 2020-02-15 doi: 10.1016/j.cclet.2019.09.020

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Abstract

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Callicarpa bodinieri is a Chinese traditional medicine herbwith anti-inflammatory activity in clinic. Herein, we report two new 9, 10-seco and etherified abietane diterpenoids bodinieric acids J and K(1 and 2)and one known compound (3) isolated from the leaves and twigs of this plant. Their chemical structures were elucidated by detailed spectrometry data analysis and DP4+ NMR calculation methods. Hypothetical biosynthetic pathways of 1-3 were preliminarily speculated. Compound 3 inhibited inflammasome activation and exhibited blockage of NLRP3 inflammasome activation at non-cytotoxic concentrations in vitro.

Key words

Callicarpa bodinieri / Abietane-type diterpenoids / DP4+ NMR calculation / Anti-inflammation / Pyroptosis / NLRP3 inflammasome

Cite this Article

Junbo Gao, Xingjie Zhang, Kun Shang, Weimao Zhong, Ruihan Zhang, Xiaochang Dai, Xiaoli Li, Qi Wang, Yan Zou, Weilie Xiao. Anti-NLRP3 inflammasome abietane diterpenoids from Callicarpa bodinieri and their structure elucidation[J]. Chinese Chemical Letters, 2020 , 31 (2) : 427 -430 . DOI: 10.1016/j.cclet.2019.09.020

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Callicarpa bodinieri, belonging to the genus of Callicarpa in Verbenaceae family [1], has been widely used in traditional medicine for the treatment of inflammation [2], rheumatism, hematemesis, and gastrointestinal bleeding [3]. Previous phytochemical investigations of the leaves and twigs of C. bodinieri has led to the identification of flavonoids [4], lignans [5-7], glycosides [8-10], phenolic acids [11, 12], triterpenoids [13, 14], and diterpenoids [15-20], etc. In this study, two new 9, 10-seco and etherified abietane-type diterpenoids bodinieric acids J and K (1 and 2) and one known compound (3) were isolated and identified from Callicarpa bodinieri (Fig. 1). In order to clarify the biological source of 9, 10-seco and etherified abietane-type scaffold, the biosynthetic pathways of compounds 1-3 were preliminarily elucidated. Based on the medicinal utilization of this plant for the treatment of immunity-related diseases, inhibitory activities against NLRP3 inflammasome activation of 1-3 were evaluated.

Stereochemistry is a critical structural element for the presented physical and biological properties of an organic molecule. However, the incorrectly assigned structures are not rare, even with many new experimental techniques and methods emerged nowadays. DP4+ is one of the most sophisticated approaches that has been applied to the challenging task of stereo-chemical assignment using the probabilities from the differences between DFT-GIAO calculated and experimental NMR data [21, 22]. Along with the extensive application of DP4+ in NMR calculations last few years [23], it has been becoming an increasingly popular method for the structure elucidation in organic and natural product chemistry.

Bodinieric acid J (1) was isolated as a white powder; [α]_D²² -3.2 (c 0.28, MeOH). Its molecular formula was deduced to be C₁₉H₂₄O₄ from the sodium adduct molecular ion at m/z 339.1571 in the positive-ion HRESIMS spectrum. The IR spectrum exhibited absorptionbands at3386, 1740, 1672, 1595, 1279, 1133, 1042 cm^-1 (as shown in Fig. S8 in Supporting information), which reminded the presence of hydroxy, carboxyl, ether and aryl groups. These NMR data listed in Table 1 revealed that compound 1 was closely related to the abietane-type diterpenoid 3 that previously isolated from this plant by our group [24]. According to ¹H–¹H COSY spectrum, the key correlations of H-5/H₂-6/H₂-7 and H-11/H-12 were shown in Fig. 2. HMBC correlations of H₂-1 with C-2 and C-3;H-5 with C-6 and C-19; H₂-7 with C-5 and C-6;H₃-18 with C-3 and C-19; H₃-20 with C-1, C-5 and C-10; H-14 with C-7 and C-9 revealed the presence of a routine A-ring and part of B-ring structure in abietane-skeleton. The H-12 with C-11 and C-15; H-14 with C-12 and C-15; H₃-17 with C-15 correlations undoubtedly established its C-ring and isopropyl unit. In addition, C-9 and C-10 in B-ring were speculated to be disconnected and bridged by an oxygen atom according to the downfield ¹³C NMR chemical shift at δ_C 160.3 (C-9) and 85.0 (C-10), as well as its molecular formula. Thus, the planar structure of 1 was confirmed.

The relative configuration of 1 was preliminarily inferred from a ROESY experiment, which showed correlations between: H₃-20 and H-7α; H-7α and H₃-18 (Fig. 2). The configuration of 1 was further elucidated by the comparison of experimental and calculated NMR spectrum data. The NMR data of four different isomers that 4S, 5S, 10R (isomer 1), 4S, 5S, 10S (isomer 2), 4S, 5R, 10R (isomer 3) and 4S, 5R, 10S (isomer 4) were calculated using GIAO method [25]. The comparison of the computed results and experimental data were then processed by DP4+ probability statistical analysis [26, 27]. Details of the computational procedure for NMR calculation can be found in Supporting information. As shown in Table 2, the isomer of 4S, 5R, 10R was 100% consistent with experimental NMR data. Thus, the structure of 1 was consequently established and named as bodinieric acid J.

Bodinieric acid K (2) was purified as white microcrystalline solid, with [α]_D²² -14.9 (c 0.60, MeOH). Its positive ion HRESIMS revealed a peak for a sodium adduct ion at m/z 397.1622, corresponding to a molecular formula of C21H26O6Na. Compared the ¹H and ¹³C NMR data (Table 1) with that of compound 1, their only difference was the additional presence of a CH₃CO-group that bonded with C-18 hydroxyl to generate an ester in 2. This assignment could be confirmed by the HMBC correlations of H₂-18 with CH₃CO-, H₂-3 and C-19; H-5 with C-18 and C-19 (Fig. 3). The ROESY correlations were also similar to those of 1. Based on the biosynthetic considerations of natural products, the configuration of 2 was speculated to stay the same way. Consequently, the structure of 2 was then confirmed and named as bodinieric acid K.

To better understand the biosynthesis mechanism of 9, 10-seco and etherified B-ring of abietane-type scaffold, the biosynthetic pathways of compounds 1-3 were preliminarily elucidated as shown in Fig. 4. Compared with the synthesis of common abietaneskeleton in 3, the variant core structure of B-ring in compounds 1 and 2 were derived initially from the hydratization of 5, 10-alkenyl in GGPP to form intermediate A1. 9, 10-seco and etherified fragment in B1 was further generated by the cycloaddition of 10-hydroxyl and 8, 9-alkenyl. Herein, the unusual abietane-type diterpene of C1 was finally obtained through methyl rearrangement and dehydrogenation reactions.

The leaves and twigs of Callicarpa bodinieri have been used as traditional Chinese medicine for thousands of years in the treatment of inflammation-related diseases, especially pyroptosis-related diseases, such as rheumatoid arthritis and gout [28]. Inflammasome plays a key role in autoinflammatory and autoimmune diseases, such as gout, rheumatoid arthritis etc. [29]. Its activation causes an inflammatory form of programmed cell death of macrophages called pyroptosis, which leads to maturation of Caspase-1 and release of inflammatory mediators, including IL-1β and IL-18. Excessive IL-1β production is harmful and causes immune dysfunctions [30]. Thus, the isolated compounds were firstly evaluated for the inhibition of pyroptosis, which was induced by NLRP3 inflammasome activation.

The effects of compound 1-3 on pyroptosis inhibition were tested in mouse macrophage J774A.1 cell. Among the three compounds, compound 3 inhibited LDH (Lactate dehydrogenase) release in J774A.1 cell rather than compounds 1 and 2 (Fig. 5a). To furtherly detect whether compound 3 inhibits inflammasome activation, the IL-1β secretion and maturation as well as Caspase-1 maturation were evaluated. As shown in Fig. 5b, compound 3 significantly inhibited IL-1β secretion. Besides, MTT assay showed that cell viabilities were not affected by compound 3 at concentrations 12.5 -50 μmol/L (Fig. 5c), suggesting that the effects of compound 3 on J774A.1 cell were not attributable to its cytotoxicity. Furthermore, compound 3 blocked IL-1β and Caspase-1 maturation in Western blot assays (Figs. 5d and e). Compound 3 also inhibited ASC (apoptosis-associated speck-like protein containing a CARD) specks formation in macrophages (Fig. 5f). These results suggest that compound 3 inhibits LPS and Nigericin induced NLRP3 inflammasome activation in mouse macrophages.

In summary, two new 9, 10-seco and etherified abietane diterpenoids bodinieric acids J and K (1 and 2) and one known compound (3) were isolated from the leaves and twigs of Callicarpa bodinieri. Their chemical structures were elucidated by detailed spectrometry data analysis as well as DP4+ NMR calculation. Hypothetical biosynthetic pathways of 1-3 were also preliminarily speculated. Compound 3 inhibited macrophage pyroptosis and exhibited blockage of NLRP3 inflammasome activation in vitro.

Acknowledgments

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This project was financially supported by the National Natural Science Foundation of China (Nos. 81422046 and 21762048), the Yunnan Applicative and Basic Research Program (Nos. 2015BC002, 2018FY001 and 2018FA048), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_17R94), the Key Program of Natural Science of Yunnan Province to W.L. Xiao, the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education (No. 2017KF02), the Natural Science Foundation of Yunnan University (No. 2017YDQN03) and the Foundation of Yunnan Educational Committee (No. 2018JS002). We thank the high-performance computing center of Yunnan University and KIB (CAS) for providing the computational resources.

Appendix A. Supplementary data

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Supplementary material related to this article can befound, in the online version, at doi:https://doi.org/10.1016/j.cclet.2019.09.020.

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Year 2020 volume 31 Issue 2

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Article Info

doi: 10.1016/j.cclet.2019.09.020

Receive Date：2019-08-07
Online Date：2026-01-31
Published：2020-02-15

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Received：2019-08-07
Revised：2019-09-07
Accepted：2019-09-10

Affiliations

^a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China

^b State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Fig. 1 Structure of compounds 1-3 isolated from Callicarpa bodinieri.

Fig. 2 Key ¹H-¹H COSY, HMBC, and ROESY correlations of 1.

Fig. 3 Key ¹H-¹H COSY, HMBC, and ROESY correlations of 2.

Fig. 4 Hypothetical biosynthetic pathways of compounds 1-3.

Fig. 5 Compounds from Callicarpa bodinieri inhibits NLRP3 inflammasome activation. (a) LDH release assay. MCC950 was used as a positive control. (b) Supernatant IL-1β inhibition. (c) Cytotoxicity of compound 3. (d, e) Western blot assays for supernatant IL-1β and Caspase-1 treated by compound 3. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01. ns, not significant. (f) ASC formation treated by compound 3. Blue = DAPI, red = ASC, white arrows indicated ASC specks, scale bar = 20 μm.

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