Terpenoids as anti-inflammatory substances inhibiting COX-2 isolated from the fibrous roots of Alangium chinense (Lour.) Harms

Abstract A new sesquiterpene, 1-carbonyl-2,8-dihydroxy-11-oxabicyclo [4,4,1] germacra- 2(3),4(5),6(7),8(9)-tetraene (1) and four known compounds (3E, 23E)-3-caffeoyl-23-coumaroylhederagenin (2), (3E, 23E)-dicoumaroylhederagenin (3), morettinone (4), 24-ehylcholesta-3,6-dione (5) were isolated from the ethyl acetate layer of the fibrous root of Alangium chinense (Lour.) Harms. The structure of compound 1 was characterized by its 1H-NMR, 13C-NMR, DEPT, HMBC, HSQC spectrums, and the structures of the known compounds were determined by comparison of their spectroscopic data with those reported by the literatures. The obtained compounds were evaluated for their anti-inflammatory against cyclooxygenase (COX-2). Compound 1 has a good inhibitory effect against COX-2 with IC50 20.43 ± 4.72 μM. The compounds 2–5 have inhibitory effect against COX-2 with IC50 49.19 ± 0.76, 23.29 ± 0.99, 47.78 ± 1.33, and 44.44 ± 0.12 μM, respectively. Graphical Abstract


Introduction
Alangium chinense (Lour.) Harms, which belongs to the Alangium genus in the Alangiaceae family, has been used in Miao medicine for hunders of years, and recorded in the quality standard of traditional Chinese medicine and ethnic medicinal materials in Guizhou Province (Guizhou Medical Products Administration 2003).
A.chinense has excellent anti-inflammatory, anti-tumor, antibacterial and analgesic effects (Ahad et al. 2012;Zhang et al. 2017). And it is primarily used for the treatment of rheumatism, numbness of limbs and schizophrenia (Wu et al. 2010). Previous chemical investigations have shown that the main active compounds were triterpenoids, phenols, alkaloids, steroids, tannins, etc. (Simone et al. 2009), and its ethanol extract and total alkali significantly reduced the foot swelling of SD rats, lower the arthritis index and improve the pathological changes of joints Pu et al. 2016). However, this fork medicine has certain toxicity, and the anabasine is the main component for relaxing muscle (Zhai et al. 2012).
Cyclooxygenase (COX-2) is an irreducible synthase, which is a key enzyme for initiating inflammation and fever (Cui and Jia 2021). Furthermore, pathological prostaglandins synthesized by COX-2 can mediate the pathological processes of inflammation and fever (Miguel et al. 2018). Under normal conditions, COX-2 is hardly expressed in tissues. However, when the body is stimulated by inflammatory factors, monocyte macrophages and fibroblasts will induce the expression of COX-2 and cause the synthesis and aggregation of pathological prostaglandins at the injury site, which accelerates the progress of inflammation and fever (Katori and Majima 2000). The mechanism of non-steroidal antipyretic, analgesic and anti-inflammatory drugs widely used in clinic is to inhibit COX-2 and reduce the production of prostaglandins (Kang et al. 2017). In order to further discover the anti-inflammatory active components, especially the COX-2 inhibitors, from the A. chinense, five compounds (1-5), including a new gemarane sesquiterpene (1) and four known compounds, (3E, 23E) À3-caffeoyl-23-coumaroylhederagenin (2), (3E, 23E) -dicoumaroylhederagenin (3), morettinone (4), 24-ehylcholesta À3,6dione (5) were isolated from the ethyl acetate layer of the fibrous root of A.chinense, and then identified by NMR data. In addition, these compounds were evaluated for the effect of anti-COX-2 in this study.
Their structures are shown in Figure 1. The aim of this study is to find the active ingredients that inhibit COX-2 from the ethnic medicine A. chinense. In this study, these ingredients all had certain inhibitory activity against COX-2 by screening the anti-inflammatory activities in vitro. The new germacrane sesquiterpenoid showed the excellent inhibitory activity with IC 50 20.43 ± 4.72 lM. The oleanane tetracyclic triterpene compounds 2 and 3 exhibited inhibitory effect with IC 50 49.19 ± 0.76 and 23.29 ± 0.99 lM, which suggested that the substituent group on C-23 paly an important role in the inhibitory effect. The triterpene compound 4 and the steroid compound 5 manifested inhibition with IC 50 47.78 ± 1.33 and 44.44 ± 0.12 lM. The compounds 1 and 3 may be the anti-inflammatory components of the fibrous root of A. chinense. The specific mechanism research will be carry out in the follow-up work.

Plant material
The fibrous root of Alangium chinense (Lour.) Harms was purchased from Nayong town, Guizhou Province, China. The morphological characteristics of the plant were compared with those recorded in the flora of China (Editorial Committee of flora of China, Chinese Academy of Sciences, 1983) and identified by Qingde Long (Guizhou Medical University), with the number of 20191010.

Study of the anti-inflammatory activity of COX-2 inhibitors
COX-2 assay buffer, COX-2 cofactor working solution, COX-2 working solution, celecoxib solution (6.25, 25, 50, 100, 200, 400, 800 lM), and tested compounds (6.25, 25, 50, 100, 200, 400, 800 lM) were prepared. 80 mL COX-2 assay buffer, 5 mL cofactor working solution and 5 mL tested compounds were added to blank control. 75 mL COX-2 assay buffer, 5 mL cofactor working solution, and 5 mL working solution were added to 100% enzyme activity control, positive control and sample groups. In addition, 5 mL tested compounds, 5 mL celecoxib solution and 5 mL tested compounds were added to 100% enzyme activity control, positive control and sample groups, respectively. These groups were incubated at 37 C for 10 min. Then 5 mL COX-2 probe and 5 mL COX-2 substrate working solution were added to each well. The fluorometric assay was performed after incubation at 37 C for 5 min. The excitation wavelength was 560 nm and the emission wavelength was 590 nm. The average fluorescence values were calculated for each sample well and blank control well, which can be recorded as RFU Blank Control, RFU 100% Enzyme Activity Control, RFU Positive Inhibitor Control and RFU Sample, RFU Relative Fluorescence Unit, respectively. Inhibition (%) ¼ (RFU 100% Enzyme Activity Control -RFU Sample)/(RFU 100% Enzyme Activity Control -RFU Blank Control) Â 100%.

Conclusions
In summary, one new sesquiterpene (1) and four known compounds were obtained from the roots of A. chinense. The new compound 1 and oleanane-type triterpenoids showed strong inhibiting effects to COX-2, which may revealed the material basis of anti-inflammatory.