Two new isoquinolines from Corydalis saxicola

Abstract Two new isoquinolines (1 and 3), along with 4 known isoquinolines were obtained from the ethanol extract of Corydalis saxicola Bunting. Their structures were elucidated based on detailed spectroscopic data (NMR, HR-ESIMS) and comparison with literature data. The absolute configurations of the new compounds were assigned by comparing computed electronic circular dichroism (ECD). The anti-inflammatory effects of the isolates were assessed by inhibiting NO production in LPS-induced RAW264.7 macrophage cells, and the results showed that compounds 1–6 exhibited anti-inflammatory activities, with IC50 values ranged from 44.24 ± 1.16 to 69.00 ± 5.41 µM. Graphical Abstract


Introduction
Inflammation is the foundation of many pathological and physiological processes, which plays an important role in many diseases of the human body. Several studies have unveiled the association between inflammation and serious diseases, such as disorders diabetes, bronchial asthma, liver cancer, hypertension, atherosclerosis (Mantovani et al. 2008). Moreover, previous investigations revealed that natural products have good effects in the treatment of inflammation (Miyazawa et al. 1996;Liu et al. 2010;Muhammad et al. 2015;Qin et al. 2020Qin et al. , 2021. Therefore, it is an effective strategy to excavate lead compounds with outstanding anti-inflammatory activity from natural products. Corydalis saxicola Bunting, which belongs to the Papaveraceae family, is known as 'Yanhuanglian' in China. As a traditional medicinal herb, C. saxicola is mainly distributed in southwest of China , and is typically used to treat hepatitis, dysentery, diarrhoea, abdominal pain and bleeding haemorrhoids (Li et al. 2008). The research on C. saxicola showed that it contained a wide array of alkaloids and steroids (Wang et al. 2007;Wu et al. 2007), many of which revealed significant biological effects including anti-cancer, liver protection and anti-inflammation (Li 2009;Huang et al. 2012;Zhang et al. 2016;Yao et al. 2019;Liu et al. 2020;Xie et al. 2021). To explore the compounds with interesting structure and biological significance from C. saxicola, in this study, the 95% ethanol extract of C. saxicola have been investigated, leading to the discovery of two new isoquinolines (1 and 3) and four known analogues (2, and 4-6) ( Figure 1). Here, we describe the separation and structural identification of alkaloids, along with the evaluation of their anti-inflammatory activity.

Results and discussion
Corysaxicoline A (1) was purified as yellowish powder, whose molecular formula was determined as C 23 H 27 NO 6 based on the positive-ion HRESIMS peak at m/z 414.1908 [M þ H]   dd, J ¼ 11.0, 3.7 Hz) and 4.33 (1H, dd, J ¼ 11.0, 5.2 Hz)]; four methoxy groups [d H 3.72 (3H, s), 3.73 (3H, s), 3.76 (3H, s) and 3.77 (3H, s)]. Based on the 13 C NMR and HSQC data revealed 23 carbon signals, including 12 aromatic carbons (from d C 110.4 to 150.3), one carbonyl carbon (d C 173.1), four methylene carbons (d C 44.8, 38.2, 30.7 and 28.4), two methine carbons (d C 57.1 and 48.9), four methoxy carbons (d C 60.4, 56.0, 55.8 and 55.7). The 1 H and 13 C NMR data of 1 (Table S1) were similar to those of (8R, 13R, 14R)-8-methoxycarbonylmethylthalictrifoline (2) , except for the absence of the dioxymethylene and methyl signals and the appearance of two methoxy groups. The above deduction was confirmed by the HMBC correlation from H-OMe (d H 3.72) to C-9 and H-OMe (d H 3.77) to C-10. Therefore, the planar structure of 1 was determined by 2D NMR analysis. In the NOESY spectrum ( Figure S8), the correlation between H-14 (d H 4.33) and H-15a (d H 2.60) indicated that the relative configuration of the chiral carbons was 8a, 14a. The experimental ECD spectrum of 1 conformed to the calculation spectrum for (8R, 14S)-1 ( Figure S2). Finally, the absolute configuration of 1 was determined and was named corysaxicoline A.
Corysaxicoline B (3), yellowish powder, with the molecular formula C 21 H 23 NO 5 , which defined according to the HRESIMS peak at m/z 370.1674 [M þ H] þ (calcd. for 370.1654) and 13 C NMR data, and its possessed 11 degrees of unsaturation. The 1D NMR data analysis (Table S1) indicated that the chemical structure of 3 was closely related to thalictrifoline (Hughes et al. 1976), in addition to that presence of a hydroxyl group at nitrogen atom. Thus, the gross structure of 3 was defined by detailed analysis of 2D NMR information ( Figure S1). In the NOESY spectrum, the cross peak between H-15 (d H 1.43) and H-14 (d H 4.09) revealed the relative configuration of 3 was 13R', 14R', respectively. The absolute configuration of 3 was deduced as 13R, 14R by comparison of its CD spectrum with that of known (13R, 14R)-13-hydroxy-13-methyl-8oxosinactine . As a result, the structure of 3 was established and was named corysaxicoline B.
The anti-inflammatory activities of all the isolated compounds were assessed applying the Griess reaction in LPS induced RAW264.7 macrophage cells, when the compounds showed no cytotoxicity at a concentration of 100 lM. Compound 3 exerted inhibitory activity on NO production with IC 50 value of 44.24 mM as shown in Table S2.

Plant material
The whole plant of C saxicola was purchased from Guangxi Hefeng Pharmaceutical Co., Ltd. in June 2018. A voucher (No. TS-201808) was identified by Prof. Dr. Shao-Qing Tang form the School of Life Sciences, Guangxi Normal University, and deposited in the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University.

ECD calculations
As mentioned earlier, the ECD calculations were carried out for all new compounds. The method is described in detail in the supporting information.

Nitric oxide production measurements and cell viability assays
The inhibited effects of the isolates on nitric oxide production were evaluated by Griess reaction, and the cytotoxic effects of compounds on mouse macrophage RAW 264.7 cells were evaluated by an MTT kit, as described in previous study (Liu et al. 2010;Shen et al. 2016).

Conclusions
In conclusion, six isoquinoline alkaloids (1-6), including two new ones (1 and 3), were isolated from C. saxicola. These structures were established by analyzing spectroscopic data, comparing experimental and calculated ECD spectra. All the isolated compounds were evaluated for their anti-inflammatory activity, and compound 3 displayed inhibitory effects against lipopolysaccharide-induced NO release in macrophage RAW 264.7 cells, exhibiting IC 50 value of 44.24lM.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Guangxi innovation-driven development special project (Guike AA18118015), Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China (CMEMR2015-A03) and the National Natural Science Foundation of China (21462007).