Rapid identification, isolation, and evaluation on anti-neuroinflammatory activity of limonoids derivatives from the root bark of Dictamnus dasycarpus

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Highlights

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    A total of 49 limonoids were rapidly identified by UNIFI software.

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    A new characteristic fragment ion, m/z 135.0803 ([C9H11O]+), of limonoids from Dictamnus dasycarpus was found.

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    A new compound, DAS, with a strong anti-neuroinflammatory activity through inhibiting NF-κB signaling pathways was obtained.

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    The structure-activity relationship between limonoids and their anti-neuroinflammatory was elucidated.

Abstract

A total of 49 limonoids derivatives were rapidly identified by UNIFI software and three new limonoids derivatives, named dasycarinone (1, DAS), isodictamdiol C (2) and dasycarinone A (3), along with nineteen known compounds, were isolated from the root bark of Dictamnus dasycarpus, named as “Baixianpi” in Chinese. Their structures were elucidated on the basis of spectroscopic data (UV, IR, HR-ESI-MS, NMR, CD spectra and OR). All the compounds were tested for anti-inflammatory activities by suppressing the nitric oxide (NO) production in lipopolysaccharide (LPS) induced BV-2 cells. DAS exhibited a strong anti-inflammatory activity with IC50 value of 1.8 μM. Nuclear Factor kappa B (NF-κB) luciferase assay and enzyme-linked immune sorbent assay indicated that DAS can suppress the release of inflammatory cytokines such as Tumor Necrosis Factor α (TNF-α), interleukin 6 (IL-6) via inactivating NF-κB signaling pathways. Moreover, we found that anti-inflammatory activities of obacunone-class are better than those of limonin-class by analyzing structure-activity relationship. Our results suggested that obacunone derivatives play an important role on anti-inflammation of Baixianpi. As a representative among them, DAS showed a strong anti-inflammatory activity via suppressing NF-κB signaling pathways.

Introduction

Dictamnus dasycarpus Turcz, a perennial herb with height of 100 cm belonging to the genus dictamnus of rutaceae, mainly grows in the northeast and Inner Mongolia in China, whose root bark (Baixianpi) is a traditional Chinese medicine with the effects of heat-clearing, dampness and detoxification. In clinical, it is mainly used in the treatment of skin inflammation, eczema, rubella, rheumatism, gynecologic inflammation [1]. Previous phytochemical studies have been reported for varied types of chemical constituents, including limonoids, alkaloids, sesquiterpenes, flavonoids, coumarins, terpenoids, steroidal. Among them, limonoids are considered as the main characteristic constituents [2]. However, it is not clear that which kind of limonoids is main active constitutes. What’s more, as a kind of triterpenoids, the characteristic chemical fragments of limonoids are ambiguous due to their complicated mass spectrum. According to reports, limonoids from the genus Dictamnus were rings A, D-seco limonoids and their degraded derivatives, which exhibited biological activities such as anti-cancer, anti-inflammation and neuroprotection [3,4]. Previous researches mainly aimed at skin inflammation rather than neuroinflammation. Above all, in this study we expected to identify limonoids rapidly as many as possible and obtained enough compounds of Baixianpi to research their anti-neuroinflammatory activities and pathway.

UPLC coupled with tandem mass spectrometry (MS/MS) has been a frequently-used method for the identification of complex and minor components due to its superior separation efficiency, detection sensitivity, and structural characterization potency [5]. Q-TOF-MS has been shown to be intrinsically capable of comprehensively acquiring accurate mass spectral data based on MS1 full scan and MSE-based data-independent acquisition (DIA), indicating promising potential for the global chemical profiling of complex composition [6,7]. UNIFI software, developed by Waters, can accomplish chromatographic peak detection, molecular formula prediction, database retrieval, MS/MS fragment matching and preliminary chemical characterization. UNIFI have been mostly applied for chemical and metabolic profile about Chinese traditional medicines (TCM) [[8], [9], [10]]. Provably, contacting UPLC/Q-TOF-MS and UNIFI can identify chemical components automatically, rapidly and accurately.

In total, we detected 49 limonoids derivatives with UPLC/Q-TOF-MS and UNIFI. And the characteristic of mass fragmentation was generalized. On the basis of the characteristic and guided by LC–MS, 13 limonoids and 9 degraded limonoids including three new compounds named as dasycarinone (1), isodictamdiol C (2) and dasycarinone A (3) were isolated from the root bark of D. dasycarpus. Among them, Compounds 11, 12, 14, and 15 were isolated from the genus Dictamnus for the first time. In addition, all of the isolates were tested for anti-neuroinflammatory activities by suppressing the NO production in LPS-induced BV-2 cells. Compound 1 exhibited strong inhibition of NO with IC50 value of 1.8 μM. Furthermore, dasycarinone (1) suppressed the release of inflammatory cytokines such as TNF-α, IL-6, and NF-κB signaling pathways. More importantly, we found that anti-neuroinflammatory activities of obacunone-class are better than those of limonin-class, which suggested that obacunone derivatives play an important role on anti-neuroinflammation of Baixianpi. In this paper, we discussed the details of the isolation, structural elucidation and bioactivity evaluation of these compounds.

Section snippets

Instruments and reagents

UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu Co., Tokyo, Japan). IR spectra were obtained on a Thermo Nicolet Nexus 470 FT-IR spectrometer (MA, USA). NMR spectra were recorded on a Varian INOVA-500 (Varian Co., USA) and Bruker Avance-400 FT NMR spectrometer (Bruker Co., Switzerland), and the chemical shifts were referenced to the solvent residual peak. HRESIMS data were determined on a Waters Xevo G2 Q-TOF mass spectrometer (Waters Co., Milford, MA, USA). The

Automated identification of known components

The representative chromatogram of Baixianpi and the sketch map of automated structural identification of components by UNIFI are shown in Fig. 1B and A, respectively. As shown in Table 1, 49 limonoids (U1-U49) were detected by UNIFI software, among them, 10 compounds were identified accurately compared with references. By analyzing MS/MS fragmentation of detected limonoids, we found that m/z 135.0803 and m/z 161.0598 were always existing ions in the MS/MS data of most limonoids (Table 1). As

Conclusion

In conclusion, a total of 49 limonoids were identified and three new limonoids along with nineteen known limonoids, were isolated from the root bark of Dictamnus dasycarpus. Our researches not only demonstrated that m/z 161.0598 ([C10H9O2]+) was a diagnostic fragment ion of limonoids also found another characteristic fragment ion m/z 135.0803 ([C9H11O]+) of limonoids. What’s more, a new compound, DAS, was obtained, which exhibited a strong anti-inflammatory activity with IC50 value of 1.8 μM by

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was financially supported by the National Key Technology R&D Programs “New Drug Innovation” of China (No. 2018ZX09711001-008-003), National Traditional Chinese Medicine Standardization Project (No. 2017-110108-83-03-001423) and National Key Research and Development Project (No. 2018YFC1707300).

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