New Sesquiterpenoids and a Diterpenoid from Alpinia oxyphylla

The new compounds 2-methyl-6-isopropyl-7-hydroxymethyl naphthalene (1), oxyphyllenone H (2), epi-oxyphyllenone (6), (E)-labda-12,14-dien-15(16)-olide-17-oic acid (3), and two new natural products 4 and 5 were isolated from the ethyl acetate part of 95% ethanol extract of Alpinia oxyphylla, together with six known compounds 7–12. The inhibitory effects of compounds 1–12 on α-glucosidase were evaluated, and compounds 1, 3 and 6 showed moderate bioactive effect, with inhibitory rates of 10.3%, 10.0% and 11.5%, respectively, compared to the positive control acarbose (41.9%) at 20 µg/mL.


Introduction
Alpinia Oxyphylla Fructus is the dry ripe fruit of Alpinia oxyphylla Miq. (Zingiberaceae), which is widely distributed in South China. It has been used in Traditional Chinese Medicine (TCM) for the treatment of intestinal disorders, dieresis and dementia [1]. Previous phytochemical investigations of this medicinal plant have resulted in the isolation and identification of a series of sesquiterpenoids [2][3][4][5][6]. As

Results and Discussion
Compound 1 was isolated as a pale yellow oil, and the molecular formula was established by HR-EI-MS as C15H18O (m/z 214.1347 [M] + ), indicating seven degrees of unsaturation. The 1 H-, 13  The remaining connectivity was solved by HMBC correlations. In the HMBC experiment ( Figure 2), The correlations from H-1 to C-3, C-5 and C-10, from H-4 to C-5, C-6 and C-10, from H-8 to C-6, C-7 and C-10, from H-9 to C-5, C-7 and C-10 confirmed the existence a naphthalene ring; the long-range correlations from CH3-15 to C-1, C-2 and C-3 confirmed that the methyl group was anchored at C-2; the HMBC correlations from -CH2-14 to C-6, C-7 and C-8 determined the direct connection between the C-14 and C-7; the correlations of H-11 with C-5, C-6 and C-7, of CH3-12/13 with C-6 displayed that the isopropyl group was connected with C-6. Thus the structure of 1 was determined to be as shown in Figure 1, which was named as 2-methyl-6-isopropyl-7-hydroxymethyl naphthalene.  . These signals explained two degrees of unsaturation, and implied that there were two rings in the structure of 2. In the HMBC experiment (Figure 2), the long-range correlations from H-1 to C-2, C-3, C-5, C-9 and C-10, from H-4 to C-2, C-3, C-5, C-6, and C-10 revealed the presence of a cyclohex-2-enone ring system in 2; the correlations from CH3-14 to C-8,C-9 and C-10, from H-11 to C-5, C-6, C-7, and from H-7 to C-9, H-8 to C-6, implied that compound 2 contained one 1-isopropyl-4-methylcyclohexane ring fragment, which was fused with the cyclohex-2-enone ring at C-5 and C-10. Finally, the planar structure of 2 was determined. Analysis of the NMR data, especially the 2D-NMR, revealed that compound 6 also possessed the same planar structure as 2.
Though there existed distinct differences in the NMR spectra between 2 and 6, it was very difficult to analyze the relative configuration of compound 2/6 by NOESY correlation or by analyzing the coupling constant because of their significantly overlapped signals in the complex 1 H-NMR spectra. The absolute configurations of C-10 in compound 2/6 were determined by CD spectroscopy. The CD spectra of 2 displayed a positive Cotton effect at 320 nm and a negative one at 251 nm, which were completely identical with that of (−)-(1R,7S,10R)-1-hydroxy-11-norcadinan-5-en-4-one, whereas the CD spectra of 6 was nearly opposite to that 2, thus the stereochemistry of C-10 in 2/6 were determined to be the R/S configuration [15]. In addition, the stereoisomer 14 of 2/6 was always synthesized mixed with its isomer, and its structure was postulated from its 1 H-NMR and mass spectra [7]. This is the first report of the isolation from a natural source of pure stereoisomers of 14, which were unambiguously characterized by NMR and mass spectroscopy experiments.     (Figure 2), the long-range correlations from CH3-20 to C-1, C-5, C-9 and C-10, from CH3-18 and CH3-19 to C-3, C-4 and C-5 confirmed that C-1, C-5, C-9 and C-20 were connected with C-10, and C-3, C-5, C-18 and C-19 were connected with C-4, respectively. The correlations from H-7, H-8 and H-9 to C-17 determined the connectivity of carboxyl group (C-17) with C-8; The HMBC correlations of H-12 with C-13, C-14 and C-16, and the correlations of H-15 with C-16 implied that a furan-2(3H)-one ring was connected with C-12 by the olefinic C-13. Thus the planar structure of 3 was determined.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV spectra were obtained on a Shimadzu UV-2550 visibl spectrophotometer. IR spectra were recorded on a Shimadzu FTIR-8400S infrared spectrometer with KBr disks. HR-ESI-MS were obtained on a LTQ-Obitrap XL LC-MS spectrometer. NMR spectra were recorded on a Bruker Avance DRX-600 instrument at 600 MHz ( 1 H-) and 150 MHz ( 13 C-), with TMS as the internal standard. CD spectra were recorded on a JASCO J-815 spectropolarimeter, using CH3OH as solvent. Purification were performed by Semi-prep-HPLC (Waters 2535 Pump, 2998 Detector). Silica gel H for column chromatography (CC) and silica gel GF254 for preparative TLC were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Precoated plates of silica gel GF254 were used for TLC, and detected under UV light at 254/360 nm.

Plant Material
The

Inhibitory Activity on Glucosidase
The bioactive assay experiment of inhibitory activity on glucosidase was determined according to the method of reference [17].

Conclusions
This study reported four new compounds and two new natural products together with six known compounds isolated from the ethyl acetate part of 95% ethanol extract of Alpinia oxyphylla. Inhibitory effects of compounds on glucosidase were evaluated, and compounds 1, 3 and 6 showed moderate bioactivity compared to the positive control acarbose at 20 µg/mL.

Author Contributions
All the authors discussed and planned the paper. Lei Hou, Gang Ding, Baolin Guo, Wenhua Huang wrote the first draft version, and Xiaojian Zhang, Zhiyong Sun and Xiangfen Shi all made their comments and suggestions to the first draft.