The basic chemical substances of total alkaloids of Menispermi Rhizoma and their anti-inflammatory activities

Abstract Menispermi Rhizoma is the dried rhizome of Menispermum dauricum DC. (Menispermaceae), which commonly used to treat sore throat, enteritis, and dysentery in traditional Chinese medicine. To clarify the chemical basis of the total alkaloids of M. Rhizoma, HPLC was used to analyze total alkaloids, and then representative chemical constituents were separated by tracking. Nineteen compounds, including two new alkaloids (1R-methymenidaurine A-α-N-oxide (1) and 1R-7’-hydroxymethyl-menidaurine A (2)), thirteen known alkaloids, and four known flavonoids were isolated and identified using spectroscopic methods. Meanwhile, seven characteristic peaks were identified from the total alkaloids using HPLC analysis. Furthermore, compounds 1–18 were screened in vitro for their inhibitory effect against nitric oxide production in BV-2 microglia cells stimulated by lipopolysaccharide. Among them, six compounds showed weak inhibition, and the IC50 values of compounds 1 and 2 were 56.87 ± 1.61 and 53.67 ± 1.52 mM, respectively. GRAPHICAL ABSTRACT


Introduction
With the development of modern chromatographic and analytic techniques, research on the material basis of traditional Chinese medicine (TCM), including the chemical material basis, pharmacodynamic material basis, and quality markers (Q-markers) of TCM, has become a major research field of natural product chemistry (Tu et al. 2012;Liu et al. 2016;Xue et al. 2022).Recently, our research team used chromatographic separation and spectral techniques to track, isolate, and determine the structure of the representative principal components under the guidance of modern analytical methods, such as high performance liquid chromatography (HPLC) on TCM extracts.on this basis, the principal components chromatogram of extracts of was established, and representative principal components of TCM were confirmed quickly, efficiently, and accurately.For example, our research group studied the material basis of three different medicinal parts of Rhododendron Molle with the help of HPLC-evaporative light scattering detector (eLSD) analysis, and 12, 11, and 6 representative principal components were identified from the roots, flowers, and fruit, respectively.The results showed that the main chemical components of the three medicinal parts were different, which would inevitably lead to differences in their efficacy and toxicology (Song et al. 2022).As part of our series of studies, herein, a similar method was used to investigate the basic chemical substances of total alkaloids of Menispermi Rhizoma.M. Rhizoma is the dried rhizome of Menispermum dauricum DC. (Menispermaceae), which is distributed in Northeast and North China (Li et al. 2017).The Chinese Pharmacopoeia records that M. Rhizoma can be used to treat sore throat, enteritis, dysentery, and rheumatism (China Pharmacopoeia Committee 2020).In recent years, a variety of alkaloids have been isolated and identified from M. Rhizoma.According to their structural characteristics, they can be divided into dibenzyl tetrahydroisoquinoline, isoapomorphine oxide, morphiane, apomorphine, proberberine, isoquinoline, and isoindole-type alkaloids.These alkaloids are often used as indicators for quality control of M. Rhizoma (Wei et al. 2015;2016;2017).Studies have shown that alkaloids are the main active components of M. Rhizoma, which not only have antibacterial and anti-inflammatory activities, but also have good biological activities to protect the cardiovascular and cerebrovascular system, and anti-tumor, anti-depression, and anti-Alzheimer's disease activities (Li and Gong 2007;Li et al. 2012;Liu et al. 2012;Sun et al. 2014;Guo et al. 2015;Law et al. 2017;Pan et al. 2018;Zhang et al. 2018).In this study, the HPLC-ultraviolet (uV) method was used to analyze the total alkaloids of M. Rhizoma, and then modern chromatographic techniques were used to track, isolate, and identify the representative principal components of the total alkaloids.As a result, two new alkaloids, 1R-methymenidaurine A-β-N-oxide (1) and 1R-7′-hydroxymethyl-menidaurine A (2), together with thirteen known alkaloids (3-15), and four flavonoids (16-19) were obtained from the total alkaloids of M. Rhizoma.By comparison with the compounds obtained, seven characteristic peaks of HPLC-uV chromatograms, including six alkaloids and one flavonoid were determined.Furthermore, compounds 1-18 were tested in vitro tested for inhibition against lipopolysaccharide (LPS)-stimulated nitric oxide (No) release from microglia cells (BV-2) as well as their cytotoxicity against BV-2 cells.The results revealed the chemical basis of total alkaloids of M. Rhizoma, and provide a more scientific basis for its clinical application.

Plant material
The dried rhizome of Menispermum dauricum DC. (Menispermi Rhizoma) was collected in Hunan province on April 22, 2019, and identified by Prof. Baokang Huang of the Second Military Medical university (SMMu).A voucher specimen (20190422) was deposited at Shanghai university of Traditional Chinese Medicine.

Cell viability assay and nitric oxide inhibition activity assay
BV-2 microglial cells were bought from Shanghai Cell Bank, Chinese Academy of Sciences.The cell experimental procedures and data analysis for the inhibition of No production as well as the cytotoxic assay were consistent with previously described (Ren et al. 2020;Peng et al. 2022).Quercetin was used as a positive control.

Conclusions
In this study, a HPLC-uV method for the determination of the total alkaloids of M. Rhizoma was established.on this basis, the main components of the total alkaloids were separated by tracking.As a result, nineteen compounds from M. Rhizoma, including fifteen alkaloids and four flavonoids, were obtained and identified.Thereafter, the characteristic chemical chromatograms of the total alkaloids of M. Rhizoma were established for the first time, allowing us to preliminarily define the main chemical constituents of M. Rhizoma.According to the Chinese Pharmacopoeia in 2020, dauricine and daurisoline were taken as the index components in the medicinal materials and decoction parts of M. Rhizoma, and this study confirmed that both of them were main components of M. Rhizoma.However, it was worth mentioning that dauricine (7) and daurisoline (9) are bibienyl tetrahydroisoquinoline alkaloids, and the contents of the other three alkaloids (3, 4, and 5) were higher than that of daurisoline, so whether the other three alkaloids should be included in the index components as the quality control standard warrants further study.In addition, this study found four flavonoids among the total alkaloids of M. Rhizoma, including three flavonostilbenes (17, 18, and 19), which were first found in M. Rhizoma.Previous studies showed that flavonostilbenes are commonly found in Sophora alopecuroides (Leguminosae), and had anti-tumor, anti-inflammatory, antibacterial, and antiviral activities (Iinuma et al. 1995;Wan et al. 2015).Combined with the results of the in vitro activity assays, the cytotoxic activity of these flavonoids should be considered in further studies.
Figure 1. chemical structures of compounds 1-19.*Newcompounds have been isolated from M. Rhizoma.# compounds have not been reported in M. Rhizoma.