A partial peroxisome proliferator-activated receptor gamma agonist isolated from the roots of Euphorbia sikkimensis

Abstract Chemical constituents of the Euphorbia sikkimensis roots was investigated and twelve known compounds were isolated, including three ent-atisane diterpenes: ent-(13S)-hydroxyatis-16-ene-3,14-dione (1), ent-(5β,8α,9β,10α,11α,12α)-11-hydroxyatis-16-ene-3,14-dione (2), ent-atisane-3-oxo-16α,17-diol (3); two kaurene diterpenes: ent-kaurane-3-oxo-16α,17-diol (4), ent-kaurane-3-oxo-16β,17-diol (5); one lathyane diterpene of latilagascene B (6); two flavonoids: quercetin (7), luteolin (8); one lignin d-pinoresinol (9); one coumarin scopoletin (10); together with ethyl gallate (11), p-hydroxybenzaldehyde (12). Their structures were identified based on the extensive spectroscopic analysis in comparison with the literature data. Compounds 1, 2, 4, 6 and 9 were isolated from Euphorbia sikkimensis for the first time. The agonistic activity of peroxisome proliferator-activated receptor gamma (PPARγ) for compounds 1, 7, 8, 9 and 11 was evaluated. Compound 1 exhibited moderate agonistic activity for PPARγ receptor with relative fluorescence intensity of 10.19 at 30.0 µM, in comparison with that of the positive control of rosiglitazone (28.50 at 2.0 µM).


Introduction
The genus Euphorbia, one of the largest genus belonging to the Euphorbiaceae family, is a well-known rich source for chemical scaffold diversity of isoprenoid metabolites (Singla and Pathak 1990;Shi et al. 2008).In particular, numerous phytochemical studies of this genus have led to isolation a lots of structurally interesting diterpenoids with different core skeletons and wide range of extensive bioactivities, such as antitumor, anti-inflammatory, cytotoxic, various vascular effects (Shi et al. 2008), antiangiogenic (Li et al. 2009;Zhao et al. 2022), antiviral (Ricigliano et al. 2020), antimalarial (Xu et al. 2021), inhibitory effects of α-glucosidase (Wei et al. 2018) and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) (Qi et al. 2014), which is a potential therapeutic target in the treatment of obesity, diabetics and cardiovascular diseases.Euphorbia sikkimensis Boiss are mainly distributed in southern region of mainland China, such as in the provinces of Guangxi, Yunnan and Tibet (Jiang et al. 2019).Its roots have long been applied as the herbal medicine for the treatment of jaundice, nephritis edema, poisoning, constipation, malaria, and rheumatism (Yang et al. 2013a;Jiang et al. 2016Jiang et al. , 2019)).Previous phytochemical investigation on the aerial parts of this species resulted in isolation of diverse structural classes of diterpenoids, triterpenoids, tocopherol derivatives (Yang et al. 2013a(Yang et al. , 2013b;;Jiang et al. 2019), flavonoids (Yang et al. 2013c;Jiang et al. 2019) and interesting carolignans with potential anti-HIV activity (Jiang et al. 2016).However, few studies have been carried out on its roots chemical composition.Aiming to explore new and bioactive metabolites from the genus Euphorbia, the chemical constituents of the ethyl acetate soluble fraction from the roots of E. sikkimensis collected from Yulong Mountain, Yunnan province has been investigated.As a result, twelve known compounds 1-12 were isolated as shown in Figure 1.Furthermore, compound 11 might be an artefactual ethyl ester of 3,4,5-trihydroxybenzoic acid during the extraction in ethanol (Venditti 2020).The peroxisome proliferator-activated receptor gamma (PPARγ) agonistic activity and cytotoxicity for compounds 1, 7, 8, 9 and 11 were also reported.

PPARγ agonistic activity
The PPARγ agonistic activity of compounds 1, 7, 8, 9 and 11 was evaluated by cell-based luciferase reporter gene assays and GW9662 block experiments in vitro.As shown in Table S1 (supplementary materials), the relative luciferase activity exhibited a concentration-dependent luciferase induction upon exposure to compound 1 in comparison with that of the control.While a dose-dependent luciferase decreased potency was also observed in the range of 100 to 10 µM compound 1 under the condition of extra addition of PPARγ antagonist GW9662.The results suggested that compound 1 is a moderate PPARγ receptor agonist in contrasting with its full agonist rosiglitazone with the relative luciferase activity of 28.5 at 2 µM.Noteworthy, the partial PPARγ agonists are highly desirable for the development of new-generation of natural insulin sensitizer in the treatment of metabolic diseases, because they are highly diverse with nontoxic properties and low reverse effects (Yi et al. 2017).In the last two decades, concerning the wide range of side effects of the full PPARγ agonists, such as edema, weight gain and increased incidence of heart attack of the current available clinical medicines, such as thiazolidinedione derivatives (TZDs), enormous increasing interests have been focused on development of natural antidiabetic pharmaceuticals from traditionally medical plants (Wang et al. 2014).Furthermore, Sullivan and co-works have evidenced that the partial agonists can play its due role to active physiological function of PPARγ (Sullivan et al. 2020).
In the last two decades, a wide range of structural diversity of natural products have been isolated as the PPARγ agonists from food and medicinal plants (Liu et al. 2014(Liu et al. , 2018;;Resetar et al. 2020;Wnok et al. 2021), as reviewed in literature (Wang et al. 2014).Interestingly, some natural products such as amorfrutins A and B, two isoprenoid-substituted benzoic acid isolated from two edible legumes Glycyrrhiza foetida and Amorpha fruticosa exhibit strong antidiabetics, lipid-lowering and inflammatory effects (Weidner et al. 2012;Gao et al. 2021).The physiological profiles studying indicated that these powerful pharmaceutical effects are mainly attributed to their highly selective agonistic activity of PPARγ through inhabiting the interaction of the nuclear receptor corepressor (NCoR) genes (Kharbanda et al. 2021), which is strikingly different from current synthetic PPARγ targeting full agonists like TZDs, without obvious increase of fat storage or other undesirable side effects.These outstanding researches demonstrate that exploration of natural PPARγ agonists, as the results of natural selection, is a promising strategy to identify new leads to conquer metabolic disease.

In vitro anticancer activity
Compounds 1, 7, 8, 9 and 11 were also selected for evaluating the cell growth capacities against human colon cancer cell line of HT29.Unfortunately, none of these compounds exhibited cytotoxic activity against HT29 cell line at 100 µM.In addition, the PPARγ agonistic activity and the cytotoxicity for the rest of the isolated compounds were not conducted due to their limited quantity.

Conclusion
In summary, chemical studying from the roots of E. sikkimensis have resulted in the isolation of twelve known compounds (1-12), containing three ent-atisane (1-3) and two ent-kaurane-type (4, 5) diterpenes, one lathyane diterpene (6), two flavonoids (7, 8), one lignin (9) and one coumarin (10), together with two phenols (11, 12).Based on us acknowledge, compounds 1, 2, 4, 6 and 9 are the first time isolated from E. sikkimensis.The cell-based luciferase reporter gene assays indicated that compound ent-(13S)-hydroxyatis-16-ene-3,14-dione (1) exhibited moderate PPARγ agonistic activity.Furthermore, this is the first time to report the PPARγ agonistic activity of compounds isolated from genus Euphorbia.In comparison with the above-mentioned undesirable side effects of the full PPARγ agonists, the natural products with moderate PPARγ agonistic activity are dire need as a rich source for exploring lead compounds for safe and effective treatment of diabetes mellitus.Therefore, this study affords scientific evidences that the genus Euphorbia might be a potential resource for exploring new natural drug leads to treat metabolic disease and further investigations are warranted.