Phytochemical constituents, chemotaxonomic significance and anti-arthritic effect of Eucommia ulmoides Oliver staminate flowers

Abstract Phytochemical investigation of Eucommia ulmoides Oliver staminate flowers provided twenty-seven compounds, including 11 triterpenes (1–11), 4 fatty acids (12–15), 1 coumarin (16), 1 chromone (17), 7 flavonoids (18–24) and 3 cyclopeptide alkaloids (25–27). Compounds 4–6, 8–9, 11–15, 17 and 25–27 were reported for the first time in the genus Eucommia. Their structures were determined by nuclear magnetic resonance, mass spectroscopy, optical rotation, circular dichroism spectroscopy analyses and comparison with literature data. The anti-arthritic activity of these compounds was also evaluated in this study. The result showed 1, 2, 4, 9 and 14 could significantly inhibit proliferation of fibroblast-like synovial cells from rats with IC50 values ranging from 11.83 to 50.12 μM. Furthermore, the chemotaxonomic significance of the isolated compounds has also been discussed. 12 and 25–27 might possess chemotaxonomic value for staminate flowers. Graphical Abstract


Introduction
As the sole species of Eucommiaceae, the barks, leaves, as well as staminate flowers of Eucommia ulmoides Oliver (E. ulmoides Oliver) are all used as medicinal remedies for osteoporosis and rheumatoid arthritis (Lu et al. 2013, Wang et al. 2019. However, the chemical compositions in the different medicinal parts distinguish each other a lot (Yan et al. 2018). The barks are rich in iridoids, lignans and phenylpropanoids (Jia et al. 2019). In leaves, the major components are iridoids (Bianco et al. 1974, Bianco et al. 1978, Bianco et al. 1982 and flavonoids (Zhang et al. 2020). The constituents reported from staminate flowers are mainly of flavonoids and triterpenes (He et al. 2014). Phytochemical taxonomy is the attempt to classify a category of plants in the form of the demonstrable differences and similarities in their small chemical compounds. The adoption of phytochemical taxonomy can deepen the understanding of species diversity (Wang and Chen 2010). Previous phytochemical investigations of E. ulmoides Oliver were extensively focused on its barks and leaves (Zhu and Sun 2018, He et al. 2014, Jia et al. 2019, while very little research on constituents from staminate flowers has been done. Therefore, we comprehensively studied the chemical constituents of E. ulmoides Oliver staminate flowers, as well as their chemotaxonomic significance and anti-arthritic effect herein.

Chemotaxonomic significance
Triterpenes from E. ulmoides Oliver were fewer reported. To the best of our knowledge, a total of ten triterpenes have been ever isolated from the staminate flowers and barks of E. ulmoides Oliver (He et al. 2014, Ding et al. 2014. In this study, eleven triterpenes were obtained from the staminate flowers. Among them, compounds 4-6, 8-9 and 11 were firstly reported from the genus Eucommia which further enriched the library of triterpenes in E. ulmoides Oliver. Some common fatty acid compounds, including oleic acid, linoleic acid and linolenic acid, have been reported in seeds and barks of E. ulmoides Oliver . Four fatty acid compounds (12-15) were isolated from the genus Eucommia for the first time. Compound 12 belongs to 16-membered macrocyclic lactone, which have showed a wide range of important biological activities including antibacterial, anti-inflammatory and anticancer effects (Janas and Przybylski 2019). It was the first report of this type constituents from the genus Eucommia, which might possess chemotaxonomic value for staminate flowers. Flavonoids are common in the leaves and staminate flowers of E. ulmoides Oliver (Zhang et al. 2020). This study provided seven compounds of this type, supporting that staminate flowers are rich in flavonoids.
It is worth mentioning that three cyclic peptide alkaloids (25-27) were isolated from staminate flowers for the first time, which was also the first report of alkaloid compounds from E. ulmoides Oliver. Compounds 26 and 27 were of cyclodipeptides. Cyclodipeptides, also called 2,5-diketopiperazines, are the smallest cyclic peptides but with higher stability. Because of their higher stability, protease resistance and conformation rigidity, cyclodipeptides exhibit better interaction with biological targets. Cyclodipeptides and their derivatives have a wide range of physiological activities, and they also can pass through the blood-brain barrier and thus protect the central nervous system (Mishra et al. 2017). The staminate flowers of E. ulmoides Oliver have shown an advantage in sedative and hypnotic effect over other parts of E. ulmoides Oliver (Chen et al. 2016), which may be associated with cyclodipeptides. Then, cyclopeptide alkaloids might sever as the chemotaxonomic makers for staminate flowers.

Anti-arthritic effect
Several studies have suggested that rheumatoid arthritis is related to inflammatory response (Aniss et al. 2020. Hence, all the isolated compounds were evaluated for the proliferation function of fibroblast-like synovial cells from rats by the in vitro Cell Titer-Glo (CTG) viability assay (Table S1). Then, the active compounds were screened out to determine its IC 50 (Table S2).
Compounds 1, 2, 4, 9 and 14 displayed obvious inhibitory activities against fibroblastlike synovial cells proliferation with IC 50 values from 11.83-50.12 lM (Table S2). Compounds 1À6 are of ursane-type triterpenes, while 8À11 belong to oleanane-type skeleton. When the carboxyl group at C-28 in 2 was reduced to the hydroxymethyl group as in 1, the activity was improved. On the contrary, the carboxyl group at C-28 was changed into the methyl group as in 3, the activity became poor. 2 and 4 showed equal inhibitory activities, indicating changement of the hydroxyl at C-3 into the keto group had little effect on activity. For these oleanane-type triterpenes (8À11), the hydroxyl group substituted at C-2 might be crucial for their anti-arthritic effect, considering 9 exhibited obvious inhibitory activity alone. The above structure-activity relationship discussion was preliminary, and remained to be examined further and verified.