Chemical constituents from the seeds of Cullen corylifolium and their inhibitory activity on diacylglycerol acyltransferase

Abstract A large number of extracts of medicinal plants or natural products shows beneficial to combat obesity. In the present work, a new flavonoid named (2S,1″R,2″R)-4′-hydroxy-7-methoxy-6-(1,2,3-trihydroxy-3-methyl-butyl)-flavanone (1), along with seven known compounds (2–8) were isolated from the seeds of Cullen corylifolium. Their structures, including the absolute configurations, were determined by the analysis of comprehensive spectroscopic data and computational calculation methods. All isolates were evaluated for their diacylglycerol acyltransferase (DGAT) inhibitory activity. Compounds 1–4 exhibited different level of DGAT1 inhibitory activity with IC50 values ranging from 28.2 ± 1.1 to 127.3 ± 1.9 μM. In addition, 45 flavonoids which be evaluated for DGAT inhibitory activity were summarised and potential structure–activity relationships were discussed. Graphical Abstract


Introduction
Obesity is a chronic metabolic disease that people ignore and causes many serious public health concerns in the world (Rajan et al. 2020). The occurrence of obesity is associated with the excessive accumulation of fats which caused by various factors including lacking exercises, overeating, bad living habits, endocrine disorder and some other associated diseases (Rebello and Greenway 2020). Nowadays, researchers has made significant developments to treat obesity and star drugs such as orlistat and metformin were recognised as the potent anti-obesity drugs in recent years. Nonetheless, the side effects such as gastrointestinal reactions, vomiting and bloating could not be ignored. Diacylglycerol acyltransferase (DGAT) is a key enzyme in TG synthesis. Published articles have demonstrated that the isoform of DGAT called DGAT1 was the virtual enzyme which responsible for the synthesis of TG (Zhou et al. 2014). Therefore, inhibiting the activity of DGAT1 is a feasible therapeutic strategy to treat obesity.
Cullen corylifolium (L.) Medik. (syn. of Psoralea corylifolia L., Fabaceae) is a wellknown traditional medicinal plant which mainly distributes in Southeast Asia. In China, C. corylifolium is called 'Bu-Gu-Zhi', and the seeds of C. corylifolium are used to treat spermatorrhea, pollakiuria, asthma and nephritis . Recent investigations revealed that C. corylifolium also possesses potential to treat obesity, diabetes and osteoporosis (Tsai et al. 2007;Seo et al. 2014;Zaheer et al. 2016). Previous articles showed that not many types of chemical components were found in C. corylifolium, the flavonoids, coumestans and meroterpenes are three main chemical constituents (Won et al. 2015;Du et al. 2019;Zhu et al. 2019;Xu et al. 2020). Flavonoids contains a large number of polyphenolic grouping structure and have significant anti-obesity activity via inhibiting the activity of pancreatic lipase, DGAT or some other pathways (Rajan et al. 2020). Our previous works have isolated some bioactive compounds with DGAT1 inhibitory activity from Morus root bark, Acanthopanax senticosus and C. corylifolium (Qi et al. 2016;Li et al. 2017, Lin et al. 2018). In the present work, a new flavonoid named (2S,1 00 R,2 00 R)-4 0 -hydroxy-7methoxy-6-(1,2,3-trihydroxy-3-methyl-butyl)-flavanone (1), together with six known compounds (2-7), were isolated from the seeds of C. corylifolium ( Figure 1). Herein, we report the isolation, structural elucidation of these compounds, and the evaluation of their inhibitory activities on DGAT.
All isolates were evaluated for their inhibitory activity against DGAT1 and DGAT2 using the reported method (Qi et al. 2016). The known DGAT inhibitor, kuraridin, was used as the positive control. As shown in Table S2, compounds 1-4 exhibited different level of DGAT1 inhibitory activity with IC 50 values ranging from 28.2 ± 1.1 to 127.3 ± 1.9 lM, and 1, 3, 4 showed selective inhibitory activity against DGAT1. Among them, compound 1 showed high inhibitory activity on DGAT1 with IC 50 value of 28.2 ± 1.1 lM. In this study, no clear structure-activity relationship was obtained because of these various structures of compounds. Based on the potential DGAT1 inhibitory activity of compound 1, the drug-likeness of 1 were predicted by SwissADME software. The calculated results showed the log P o/w (iLOGP) and bioavailability scores of 1 were 2.41 and 0.55, respectively, and the lipinski of 1 was also a positive result. The result of pharmacokinetics showed GI absorption of 1 was high, which suggested that 1 could be better absorbed into bloodstream and exert its effects. The above results indicated that 1 has the values to be further studied.
To our knowledge, over 100 compounds have been isolated and reported from C. corylifolium. Flavonoids attracts the attention of researchers as the main chemical constituents of C. corylifolium and exhibited moderate DGAT inhibitory activity. Therefore, the present work also incidentally summarises the structures and DGAT inhibitory activity of flavonoids isolated from C. corylifolium with the expectation of clarifying the structure-activity relationship of flavonoids. As shown in Figure S8 and Table S3, 45 flavonoids which be evaluated for DGAT inhibitory activity were summarised from published articles in recent five years. In these published articles, all authors used kuraridine as the positive control and the IC 50 values of kuraridine in different articles are similar. Thus, it's possible to deduce some relationships about the structure of flavonoid and their DGAT inhibitory activity. The summarised data revealed that most flavonoids from C. corylifolium possess selective inhibitory activity on DGAT1, it's inspiring for developing anti-obesity drugs via specifically inhibiting the activity of DGAT1. Unfortunately, these compounds didn't exhibit remarkable inhibitory activity on DGAT1. In addition, the structures of 42 flavonoids contain prenyl group or the derivates of prenyl group, which indicated that prenyl group or the derivate of prenyl group might is the important moiety of flavonoids to exert DGAT1 inhibitory activity. Furthermore, the comparison of IC 50 values of 1-12 in Figure S8 showed the cyclic oxidation of prenyl groups are negative for the DGAT1 inhibitory activity compared with prenyl group. On the basis of the above data, searching for flavonoid with prenyl group might be the direction of developing DGAT1 inhibitor.

General experimental procedures
Optical rotation was measured with a AUTOPOL IV automatic polarimeter (Rudolph Research Analytical, USA). UV spectra were measured on Shimadzu UV-2401A spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were made by FTIR-8400S spectrometer with KBr discus (Shimadzu, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were obtained from a Bruker AV-500 spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal standard. Mass spectra were obtained on a Bruker micro-TOF-Q mass spectrometer (Bruker, Karlsruhe, Germany). Column chromatography was performed using silica gel (200-300 lm particle size, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and RP-18 (150-63 lm particle size, Merck, Darmstadt, Germany). TLC was performed with precoated silica gel GF 254 glass plates (Qingdao Marine Chemical Co., Ltd). HPLC was carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector (Shimadzu, Tokyo, Japan) using a YMC Pack ODS-A column (5 lm, 20 Â 250 mm).

Plant material
The seeds of C. corylifolium were collected in Jilin, Jilin province, China, and authenticated by Associate Prof. Jiu-Zhi Yuan (School of Traditional Chinese Medicine, Shenyang Pharmaceutical University). A voucher specimen of this plant (No. BZ-20210825) was deposited at the College of Pharmacy, Binzhou Medical University, Binzhou, China.

Dgat1 and DGAT2 assays
The DGAT1 and DGAT2 inhibitory activities of compounds 1-8 were evaluated by the same procedures in our previous articles (Qi et al. 2016).

Conclusion
In conclusion, chemical investigation of the seeds of C. corylifolium led to the isolation of eight isolates including a new compound named (2S,1 00 R,2 00 R)-4 0 -hydroxy-7methoxy-6-(1,2,3-trihydroxy-3-methyl-butyl)-flavanone. Their structures were elucidated by detailed spectroscopic analysis and and computational calculation methods. All isolates were evaluated for their DGAT inhibitory activity and compounds 1-4 exhibited moderate DGAT1 inhibitory activity. Based on the published articles, some potential relationships between the structure of flavonoid in C. corylifolium and DGAT inhibitory activity were deduced and discussed. The present work and summary provide a basis for the development of DGAT1 inhibitors from C. corylifolium.

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