A New Phenolic Glycoside from the Barks of Cinnamomum cassia

A new phenolic glycoside (1), named methyl 2-phenylpropanoate-2-O-β-d-apiofuranosyl-(1→6)-O-β-d–glucopyranoside, was isolated from the barks of Cinnamomum cassia, along with three known phenolic glycosides and four known lignan glycosides. The structure of 1 was elucidated by extensive interpretation of spectroscopic data and chemical method. Selected compounds were evaluated for their immunosuppressive activities against murine lymphocytes. Compounds 1, 2, 6 and 8 exhibited differential inhibition against ConA-induced T cells proliferation.


Introduction
The plant Cinnamomum cassia (Lauraceae), which originates in the south of China, is widely cultivated in tropical or subtropical areas, such as Fujian, Guangdong, Guangxi, Yunnan and Hainan

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Provinces of China, as well as Taiwan, India, Indonesia, Laos, Malysia, Thailand and Vietnam [1]. The bark of C. cassia, well known as Cinnamomi cortex in China, has long been used as spice and flavoring agents [2]. It also possesses various biological activities, such as insecticidal, anti-allergy, antipyretic, anticancer, anti-Alzheimer′s disease, antidiabetic [3] and immunosuppressive activity [4]. In our previous study [5], three diterpenoids, cinncassiols F and G, and 16-O-β-D-glucopyranosyl-19deoxycinncassiol G, with unprecedented carbon skeletons, were isolated from the barks of C. cassia. It is notable that two of them showed significant inhibitory effects on the proliferation of murine T cells induced by ConA. Recently, with the aim of discovering more bioactive compounds from C. cassia, the extracts of EtOAc and n-BuOH were further phytochemically studied, which led to the isolation of a new phenolic glycoside (1), together with three known phenolic glycosides (2)(3)(4) and four known lignan glycosides (5)(6)(7)(8). The immunomodulatory activities against murine lymphocytes of the selected compounds were reported in this work.
The HBMC correlations from H-1′ to C-2 and from H-1′′ to C-6′ revealed the linkages of the two sugars to be the same as those of 4. The sugars were finally confirmed by comparing the retention times of the three trimethylsilylthiazolidine derivatives obtained from GC analysis. On the basis of the above evidences, compound 1 was determined and named methyl 2 The crude extract of C. cassia has been reported to exhibit potent anti-complement [12] and immunosuppressive activities [4]. Thus, compounds 1, 2, and 5-8 were evaluated for their immunomodulatory activities against murine lymphocytes. The results ( Table 2) showed that compounds 1, 2, 6, and 8 inhibited the proliferation of ConA-induced murine T cells at a concentration of 50 μM, while the positive control at the concentration of 50 μM, resulted in an inhibitory ratio of 106.1% against T cells and an inhibitory ratio of 75.8% against B cells.

Plant Material
The

Acid Hydrolysis
A solution of 1 (2.0 mg), in 2 M aqueous CF3COOH (2.0 mL) was heated at 60 °C for 2 h in a water bath [13]. The reaction mixture was diluted in H2O (2.0 mL) and extracted with EtOAc (2.0 mL × 3), then the aqueous layer was concentrated to remove CF3COOH. The residue was dissolved in pyridine (1.0 mL), to which L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 mL) was added. Then, the mixture was kept at 60 °C for 30 min. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.4 mL) at 60 °C for 30 min in a water bath. Finally, the mixture was partitioned between n-hexane and H2O (1 mL each) and the n-hexane extract was analyzed by gas chromatography (GC) under the following conditions: column temperature, 250 °C; injection temperature, 250 °C; carrier N2 gas; flow rate 1.0 mL/min. In the acid hydrolysate of 1, D-glucose and D-apiose were confirmed by comparison of the retention times of their derivatives with those of D-glucose and D-apiose derivatives prepared in a similar way, which showed retention times of 10.03 and 15.62 min.

Lymphocyte Proliferation Test
Splenic lymphocytes were prepared as previously described by Kawaguchi et al. [14]. The lymphocyte proliferation was determined by WST-8 assay using Cell Counting Kit-8 (Dojindo) [15]. The prepared spleen cells (2.5 × 10 6 cells) were seeded into each well of a 96-well microplate, and various concentrations of compounds and 5 μg/mL of concanavalin A (Con A, from Canavaliaensiformis Type III), for selective stimuli on T cells were added, cyclosporine A (CsA) was used as a positive control. After being cultured at 37 °C with 5% CO2 for 48 h, 20 μL WST-8 was added to each well. The absorbance at 450 nm with a 600 nm reference was detected on a microtiter plate reader (Bio-Rad 680). All optical density (OD) values shown are the mean of triplicate sample ± SD. Controls with and without ConA and LPS were used to establish the baseline proliferation for stimulated and unstimulated cells. The lymphocyte proliferation rate was evaluated according to our reported procedure [5].

Conclusions
A new phenolic glycoside, methyl 2-phenylpropanoate-2-O-β-D-apiofuranosyl-(1→6)-O-β-Dglucopyranoside (1), was isolated from the barks of C. cassia, together with seven known compounds (2)(3)(4)(5)(6)(7)(8). The structure and relative configuration of the new isolate was elucidated by careful interpretation of spectroscopic data. This is the first report of samwiside (5) in the genus of Cinnamomum. Compounds 1, 2 and 5-8 were tested for their in vitro immunomodulatory activities. Compounds 2 and 6 showed weak inhibition against ConA-induced T cell proliferation at the dosage of 12.5-200 μM, and the inhibitory effects are not dose-dependent. Interestingly, at a concentration of 200 μM, compound 8 significantly inhibited ConA-induced T cell proliferation with an inhibition ratio of 80.1%, whilst at low concentrations of 25 and 12.5 μM, stimulated the proliferation of T cell.