Structural characterization and immunosuppressive activity of a new pregnane glycoside from Epigynum cochinchinensis

Abstract Phytochemical investigation on the ethyl acetate fraction of the leaves of Epigynum cochinchinensis led to the isolation of a new C21 pregnane glycoside, epigycoside B (1), together with three known analogues. Their structures were elucidated on the basis of extensive spectroscopic techniques, including UV, MS, and NMR experiments, as well as the chemical methods. Compound 1 displayed in vitro immunosuppressive activity against concanavalin A (Con A)/Lipopolysaccharides (LPS)-stimulated proliferation of mice splenocyte. The activity was significant as compared with control group at 50 μM concentration.


Introduction
Pregnanes and their glycosides are an important class of natural products with unique structural features and diverse bioactivities (Li et al. 2013). These molecules are structurally comprised of an aglycone and sugar moieties; and are mainly classified into polyhydroxypregnane-type and secopregnane-type steroidal glycosides. In pregnane glycosides, the sugar moiety is linked to an alcoholic hydroxy group of the pregnane aglycone through an acetal linkage, typically at C-3 or C-20 position or both. Previous pharmacological investigations on pregnane glycosides have demonstrated promising immunosuppressive , anti-inflammatory (Seeka et al. 2017), anti-epileptic (Li et al. 2015), neuroprotective (Zhao et al. 2013), anti-hyperglycemic (Tsoukalas et al. 2016) and cytotoxic (Wang et al. 2017) activities. A number of pregnane glycosides have been isolated from plants of the family Apocynaceae (Cao et al. 2005;Lei et al. 2011;Gao et al. 2017).
The members of the genus Epigynum (Apocynaceae) are widely distributed in forest margins of Thailand, Vietnam, Burma, Laos, and Cambodia (Middleton et al. 2005). Previous investigations on the genus Epigynum resulted into diverse secondary metabolites, including pregnane glycosides, triterpenoid saponins, and phenolic derivatives (Cao et al. 2005;Gao et al. 2016Gao et al. , 2017Wan et al. 2017). Many of them, such as epigynosides E-G and epigycoside A showed a significant immunosuppressive activity against concanavalin A-induced proliferation of mice splenocytes Wan et al. 2017). In our ongoing search for novel and immunosuppressive pregnane glycosides, investigation of the EtOAc-soluble fraction from the leaves of Epigynum cochinchinensis led to the isolation of a new pregnane glycoside, epigycoside B (1), along with three known analogues (2-4) ( Figure 1). The structure of new 1 was elucidated by means of spectroscopic techniques, including HRMS, 1D and 2D NMR experiments, and chemical methods. The known compounds were identified as epigynoside E (2), and epigunosides A (3) and B (4) (Cao et al. 2005;Gao et al. 2017) by comparison with the reported data. Herein, we report the isolation and structure elucidation of epigycoside B. All the isolated compounds were tested for their in vitro cytotoxic activity by MTT assay. Furthermore, the in vitro immunosuppressive activity of epigycoside B was investigated by CKK-8 assay.
Similar to compound 1, compound 2 had a pregnane skeleton with the same aglycone (rel. 3β,17α, 20S)-pregn-5-ene-3,17,20-triol, and comprised of a monosaccharide sugar unit linked at C-3. Based on detailed comparison of the reported physical and spectroscopic data, compound 2 was identified as epigynoside E . Compounds 3 and 4 were readily identified as epogynumgenane-type pregnane glycosides with a tetrahydropyran D-ring. By comparing their 1 H and 13 C NMR spectroscopic date with reported data, the known compounds were identified as epigynosides A (3) and B (4) (Cao et al. 2005).
All the isolated compounds were tested for their in vitro cytotoxicity against SMMC-7721, MCF-7, HL-60, SW480, and A-549 human cancer cell lines. None of them showed cytotoxic activity (IC 50 > 10 μM). Furthermore, the immunosuppressive activity of compound 1 was evaluated on mitogen-stimulated mice splenocyte proliferation with dexamethasone as a positive control. Compound 1 significantly inhibited LPS-induced B lymphocyte and ConAinduced T lymphocyte proliferation, which was close to the efficacy of dexamethasone 50 μM concentration (Figures 2 and 3).
However, compared with the control group, there were no significant differences of compound 1 in inhibitory effect on mitogen-stimulated splenocyte proliferation, at a concentration of 12.5 μM.  Figure 2. In vitro effect of compound 1 on con a-stimulated splenocyte proliferation. dXM was used as a positive control. the values were presented as mean ± sd of triplicate replicates. significant difference was determined by aNoVa and tukey test (*p < 0.05, compared with the control group).  Figure 3. In vitro effect of compound 1 on lPs-stimulated splenocyte proliferation. dXM was used as a positive control. the values were presented as mean ± sd of triplicate replicates. significant difference was determined by aNoVa and tukey test (*p < 0.05, compared with the control group).

Conclusion
In our ongoing immunosuppressive screening program from Epigynum plants, a new pregnane glycoside, epigycoside B (1), was isolated from the ethyl acetate fraction of the leaves of E. cochinchinensis together with three known analogues (2-4). The structure of 1 was elucidated by extensive spectroscopic analyses, including 1D and 2D NMR, HRESIMS, UV and IR techniques. Epigynoside E (2) is isolated from E. Cochinchinensis for the first time, while epigynosides A (3) and B (4) were previously reported from the stem of E. cochinchinensis. All the isolated compounds were test for their in vitro cytotoxicity against SMMC-7721, MCF-7, HL-60, SW480, and A-549 human cancer cell lines. None of them showed cytotoxic activity. The in vitro immunosuppressive experiment showed that epigycoside B (1) significantly inhibited the proliferation of LPS-induced B lymphocytes and ConA-induced T lymphocytes.

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

Funding
This work was financially supported by the National Natural