Bioactivity-Guided Isolation and Identification of New and Immunosuppressive Monoterpenoid Indole Alkaloids from Rauvolfia yunnanensis Tsiang

Three new 11-hydroxyburnamine (1) and rauvoyunnanines A–B (2–3), and fourteen known (4–17) monoterpenoid indole alkaloids were isolated from the total alkaloids extract of Rauvolfia yunnanensis, which exhibited promising immunosuppressive activity on T cell proliferation in preliminary screening. Their structures were determined by analysis of high-resolution electrospray ionization mass (HRESIMS), ultraviolet (UV) and nuclear magnetic resonance (NMR) data, and by comparison with the literature. All the alkaloids were evaluated for inhibitory activity on T cell proliferation. Among them, one new compound (1) and reserpine (6) exhibited moderate immunosuppressive activity, with IC50 values of 5.9 μM and 5.0 μM, respectively.


Introduction
Abnormal T cell proliferation plays a very important role in the development of T-cell mediated organ transplantation rejection and autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [1,2]. It is crucial to find immunosuppressive agents on T cell proliferation for this kind of immunopathogenesis. Some clinical immunosuppressants inhibit T cell proliferation by different modes of action [3]. For example, cyclosporine and tacrolimus are calcineurin-inhibitors [4]. Sirolimus and everolimus (Target-of-Rapamycin Inhibitors) inhibit nucleotide synthesis and azathioprine [5,6], and mycophenolic acid inhibits inosine monophosphate dehydrogenase and de novo GTP biosynthesis [7]. However, their side effects, such as infections and toxicities, prompt scientists to look for new immunosuppressants constantly. Medicinal plants are also useful sources of immunosuppressive agents. Triptolide obtained from the Chinese herbal plant Tripterygium wilfordii Hook F is widely used in East Asia for the treatment of SLE, RA, etc. [8]. Sinomenine from Sinomenium acutum (Thunb.) Rehd. et Wils has been used for patients with autoimmune diseases as it possesses immunosuppressive activity [9].
Rauvolfia yunnanensis Tsiang (Apocynaceae) is a kind of shrub, mainly distributed in the south of China, such as Yunnan, Guizhou, and Guangxi Provinces. It is a medicinal plant of Dai Nationality in Yunnan Province, and its roots have been used for the treatment of hypertension, fever, sore throat, hepatitis, nephritis, and snakebite [10]. Until now, more than twenty indole alkaloids have been obtained from R. yunnanensis in previous studies [11][12][13]. The most famous one is reserpine, which possesses a significant antihypertension effect [14]. It was also reported that reserpine inhibited delayed hypersensitivity and contact sensitivity responses [15]. Yohimbine in combination with berberine has an immunoregulatory effect [16]. In our ongoing search for immunosuppressive compounds from medicinal plants [17], the total alkaloid extracts of whole R. yunnanensis plants exhibited promising immunosuppressive activity on T cell proliferation. Therefore, a comprehensive phytochemical investigation on the total alkaloids was carried out. The isolation, structural elucidation, and immunosuppressive activity of the isolated alkaloids are described herein.

Identification of New Compounds
Compound 1 was isolated as a yellowish, amorphous powder with [α]20D − 117.5 (MeOH, c 0.04). Its molecular formula was determined to be C 21 H 24 N 2 O 5 by positive HRESIMS at m/z 385.1766 [M + H] + (calcd 385.1758), corresponding to 11 degrees of unsaturation. Its UV spectrum showed absorption maxima at 207 and 293 nm, which is characteristic of a hydroindole/alkylaniline chromophore [18]. The 1 H NMR spectrum (Table 1) exhibited an ABX spin system at δ H 7.61 (1H, d, J = 8.1 Hz), 6.79 (1H, d, J = 1.8 Hz), and 6.71 (1H, dd, J = 8.1, 1.8 Hz), an ethylidene at δ H 5.28 (1H, m) and 1.64 (3H, d, J = 6.5 Hz), and a methoxyl group at δ H 3.70 (3H, s). The 13 C NMR spectrum (Table 1) displayed 21 carbon signals including one methyl, one methoxyl, four methylenes, seven methines (four sp 2 and three sp 3 ), and eight quaternary carbons (four sp 2 , three sp 3 , and one carbonyl). The above spectroscopic evidences, along with the previously isolated alkaloid structures from this plant indicated that compound 1 was a monoterpenoid alkaloid [11]. The key HMBC correlations ( Figure 1) between H-5 and C-2, H 2 -17 and C-7, H 2 -6 and C-16 suggested that compound 1 possessed a picraline-type skeleton, similar to 11-methoxyburnamine [19]. The HMBC correlations from H 2 -21 to C-19 indicated the presence of an ethylidine at C-20. In addition, the correlations of H-9 to C-7 and C-11 implied the location of a hydroxyl group at C-11. The presence of a methoxyl group at C-22 was also proved by the HMBC experiment of δ H 3.70 to δ C 175.6. The NOESY correlation between H 3 -18 and H-15 suggested that the configuration of C-19 should be E. Meanwhile, the NOESY correlations from H 2 -17 to H-14β indicated that the C-16 configuration is R ( Figure 2). Finally, compound 1 was elucidated as 11-hydroxyburnamine.

Immunosuppressive Activity Assay
All isolated compounds were evaluated for the inhibitory activity against human T cell proliferation according to reported protocols [17]. Compounds 1 and 6 showed immunosuppressive activity on human T cell proliferation, with IC 50 values of 5.9 µM and 5.0 µM, respectively. Other compounds with IC 50 > 50 µM were inactive (Table 2).
The n-BuOH (60.5 g) extraction was applied to macroporous resin D-101 CC eluted with a step gradient of EtOH-H 2 O (0 to 100%) solution. TLC analysis showed only the 20% EtOH fraction included alkaloids. Therefore, the 20% EtOH fraction (10.8 g) was submitted to NH-gel CC eluted with a gradient of CHCl 3 -MeOH (6:1 to 1:1) to give fractions F-I.

T Cell Preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors by density-gradient centrifugation using Lymphoprep. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS. The T cells were isolated using the Pan T Cell Isolation Kit II Human with negative selection. Then, the T cells were stained using the PE-anti-CD3 antibody (BD PharMingen, San Diego, CA, USA), and the number of stained cells was determined using flow cytometry (Acurri C6, Becton Dickinson, San Jose, CA, USA). The T cells were at 95% purity during the following experiments.

Cell Proliferation Assay
Flow cytometry was used to probe T cell proliferation using 5-carboxyfluorescein diacetate succinimide ester (CFSE, Molecular Probes, Eugene, OR, USA)-labeling. Briefly, human naïve T cells or PBMCs (10 6 cells/mL) were stained with 2.5 µM CFSE at 37 • C for 10 min, washed with PBS twice and re-suspended in RPMI 1640 medium containing 10% FBS. Then, the labeled naïve T cells (10 6 cells/mL) were activated by plate-bound anti-CD3 (2 µg/mL, HIT3a clone) and soluble anti-CD28 (1 µg/mL, CD28.2 clone, BD PharMingen). The labeled PBMCs (10 6 cells/mL) were activated with equal numbers of PBMCs irradiated with 3000 rad from another person. Subsequently, the proliferation of the T cells activated by the anti-CD3/anti-CD28 antibodies or alloantigen was measured by flow cytometry after stimulation for 72 h incubated with or without different concentrations of isolated compounds. The cells without a stimulation or drug served as the negative control, and the positive control was the cells with the stimulation but without the drug.

Statistical Analysis
The inhibitory concentrations of the compounds that reduced cell proliferation by 50% (IC 50 ) values were calculated using GraphPad Prism 6 (GraphPad, San Diego, CA, USA). One-way analysis of variance (ANOVA) with Dunnett comparisons on post-tests were used to analyze data and compare groups. The results are expressed as the mean ± S.E.M. P < 0.05 was considered to be statistically significant.

Conclusions
In this study, a new picraline-type alkaloid (1), a new sarpagine-type alkaloid (2), and a new serpentine-type alkaloid (3) were obtained from the whole plants of R. yunnanensis. Their structures were extensively elucidated by HRESIMS, 1D and 2D NMR, and UV analysis. Compounds 1 and 6 showed moderate immunosuppressive activity on T cell proliferation. Previous bioactivity studies of reserpine (6) mainly focused on antihypertension [14]. Although reserpine induced suppression on delayed hypersensitivity and contact sensitivity [15], its immunosuppressive activity on T cell proliferation was reported for the first time. The discovery of these new compounds enriched the chemical diversity of monoterpene indole alkaloids and also provided a potential immunosuppressant for further mechanism study.