A new flavone C-glycoside and a new bibenzyl from Bulbophyllum retusiusculum

Abstract A new flavone C-glycoside, apigenin 6-C-α-arabinofuranosyl 8-C-α-arabinopyranoside (1) and a new bibenzyl, bulbotetusine (2), were isolated from the tubers of Bulbophyllum retusiusculum. Their structures were established on the basis of extensive spectroscopic analyses. The absolute configuration of 2 was determined by the comparison of experimental and calculated electronic circular dichroism. Compounds 1 and 2 showed no obvious cytotoxic activity against any five human tumour cell lines with IC50 values >40 μM.


Introduction
Plants of the Orchidaceae are known to be a rich source of stilbenoids such as bibenzyls, phenanthrenes and 9,10-dihydrophenanthrenes (Majumder & Kar 1987;Leong et al. 1999;Zhang et al. 2007). The genus Bulbophyllum, belonging to the subtribe Bulbophyllinae of the Orchidaceae consists of over 1000 species found in Africa and Asia (Leong et al. 1997). Some Bulbophyllum plants were used as folk medicines to treat tuberculosis, chronic inflammation and fracture (Xu et al. 2009). The phytochemical research showed that phenanthrene derivatives and bibenzyls are the predominant constituents of this genus , Zhao et al. 2015. Bulbophyllum retusiusculum, a species from genus Bulbophyllum, is widely distributed in the Yunnan Province of China growing at an elevation of 500-2800 m. To the best of our knowledge, there has been no phytochemical research on this plant. To explore the new and bioactive constituents from genus Bulbophyllum, phytochemical investigation on the tubers of B. retusiusculum was carried out to afford a new flavone C-glycoside, which was rare in Bulbophyllum (Majumder & Sen 1991;Sun et al. 2014), and a new bibenzyl (Figure 1). Their cytotoxic activities were also investigated. This paper deals with the isolation and structural elucidation of these two new compounds from B. retusiusculum.
The absolute configuration of C-7 was determined by the comparison of experimental and calculated electronic circular dichroism (eCD) in Figure S3. The results showed that the theoretical eCD data for 7R-isomer were in good agreement with the experimental spectrum. Thus, the absolute configuration of C-7 was determined as R, which was same as that of dendrocandin A (Li et al. 2008). Therefore, compound 2 was assigned as [R]-3,4-methylenedioxy-5,3′,7-trihydroxy bibenzyl and named bulbotetusine.
Compounds 1 and 2 were examined in a panel of cancer cell lines, including HL-60, SMMC-7721, A-549, MCF-7 and SW-480 human tumour cell lines in vitro with cisplatin and paclitaxel as the positive controls. The cytotoxic activity was estimated by a method described previously (Wu et al. 2014). The result showed that both compounds 1 and 2 exhibited no obvious cytotoxic activities against any tested cell lines (IC 50 > 40 μM). Many bibenzyl compounds were reported to exhibit cytotoxic activities ). The absence of cytotoxic activity for 2 indicated that the presence of OH-7 had negative effect on cytotoxic activity, which was consistent with literatures ).

General procedure
Melting point was determined on a XRC-1 melting point apparatus and uncorrected. Optical rotation was measured with a Jasco P-1020 digital polarimeter. A nicolet Magna-IR 550 spectrometer was used for scanning IR spectroscopy with KBr pellets. nMR spectra were acquired with either a Bruker AM-400 spectrometer (400 MHz for 1 H nMR and 100 MHz for 13 C nMR) or a Bruker DRX-600 (600 MHz for 1 H nMR and 150 MHz for 13 C nMR) spectrometer. HR-eSI-MS analyses were recorded with Agilent g3250AA and Auto Spec Premier P776 spectrometer. Silica gel (200-300 mesh or 300-400 mesh) and Sephadex LH-20 were used for column chromatography.

Plant material
The tubers of B. retusiusculum were collected in Yangbi County, Dali City, in Yunnan Province of China, in June 2014, and identified by associate researcher Jiang-Miao Hu from the Kunming Institute of Botany, The Chinese Academy of Sciences, PR China. The voucher specimen (2014-JBL-1) has been deposited in the school of Chemical Science and Technology of Yunnan university.

Calculation
The theoretical calculations of compound 2 were performed using gaussian Program by Yunnan electronic Computing Center. Two possible R and S geometries of compound 2 were previously optimised by Density Functional Theory methods at the B3LYP/6-31g(d) level (Ding et al. 2009) and excitation energies and rotational strengths were calculated using time-dependent Density Functional Theory at the B3LYP/6-31g(d,p) level in acetonitrile with PCM model (Liao et al. 2015). The eCD spectrum is simulated from electronic excitation energies and velocity rotational strengths.

Conclusion
The chemical investigation of the tubers of B. retusiusculum resulted in the isolation of a new flavone C-glycoside, apigenin 6-C-α-arabinofuranosyl 8-C-α-arabinopyranoside (1) and a new bibenzyl, bulbotetusine (2). The absolute configuration of 2 was determined by the comparison of experimental and calculated eCD. Compounds 1 and 2 showed no obvious cytotoxic activity against any five human tumour cell lines.

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

Funding
This work was financially supported with a grant from the national natural Science Foundation of China [grant number 81160393].