One new lignan derivative from the Combretum alfredii Hance

Abstract One new lignan derivative 2,3-dimethyl-4-(4-methoxyphenyl)-6,7-dihydroxynaphthalene (1), together with five known compounds (2–6), were isolated from ethanol extract of the branches and leaves of Combretum alfredii Hance collected in Hainan Province, China. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. Compounds 1, 2, 5 and 6 were isolated from the genus of Combretum for the first time. All compounds were evaluated for their antibacterial activities. Compounds 1 and 2 showed significant antibacterial activities against six pathogenic bacteria.


Introduction
The Combretum comprise about 250 species and distribute throughout the tropics and subtropics, of which only 12 species are distributed in China (Hutching et al. 1996;Eloff et al. 2008;Dawe et al. 2013). The Combretum species are extensively used in traditional medicine against inflammation, infections, diabetes, malaria, bleeding, diarrhoea, digestive disorders and so on (Hoareau et al. 1999;Edeoga et al. 2005;Lima et al. 2012). And also Combretum extracts or isolates have shown in vitro bioactivities such as antibacterial, antifungal, anti-inflammatory and cytotoxic cativities (Dawe et al. 2013). There were a series of interesting compounds isolated from the plants of Combretum, such as terpenoids, flavonoids, phenanthrenes and stilbenoids (Hutching et al. 1996;Eloff et al. 2008). In the course of screening for biologically and chemically novel natural compounds from the plants of Combretum, C. alfredii Hance was chosen for further phytochemical investigation, which is one of the species growing on Hainan Province, P. R. China (Hutching et al. 1996;Eloff et al. 2008;Dawe et al. 2013).
In our previous work on this genus, 10 compounds were isolated from C. alfredii Hance such as ellagic acids and triterpenoids, and some of them showed significant antibacterial activities and artemia salina activities (Wang et al. 2015). In our ongoing research for new bioactive compounds from C. alfredii Hance, bioassay guided fraction of the bioactive extract led to the isolation of one new lignan derivative 2,3-dimethyl-4-(4-methoxyphenyl)-6,7dihydroxynaphthalene (1), together with five known compounds (2−6) ( Figure 1). This paper mainly deals with the isolation, structural characterisation and antibacterial evaluation of the isolated compound against six pathogenic bacteria.
The biological activity of all isolated compounds was evaluated with six pathogenic bacteria, including Staphylococcus aureus (ATCC 27154), Bacillus subtilis (ACCC 11060), Escherichia coli (ATCC 25922), Micrococcus luteus (ATCC 10240), Micrococcus tetragenus (ATCC 13623) and Staphylococcus albus (ATCC 8799). Compound 1 showed strong antibacterial activity against E. coli and S. albus with the same MIC values of 1.25 μg/mL, and showed significant antibacterial activity against B. subtilis and M. tetragenus with the same MIC values of 2.5 μg/mL. Compound 2 showed significant antibacterial activity against M. luteus and S. albus with the same MIC values of 2.5 μg/mL. The other compounds showed weak or no antibacterial activity with the MIC values of more than 10 μg/mL.

Plant materials
The branches and leaves of C. alfredii Hance were collected in Sanya, Hainan Province, China, in June 2013 the fruiting season and authenticated by associate Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University (No. 20130608CO). They were not separated. The averaged diameter of the collected branches was about 2 cm. A voucher specimen was deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University.

Antibacterial activity assays
The antibacterial activities against six bacterial strains, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Micrococcus luteus, Micrococcus tetragenus, and Staphylococcus albus were determined by a serial dilution technique using 96-well microtiter plates. The compounds were dissolved in DMSO to give a stock solution. Bacterial species were cultured overnight at 37 °C in LB broth and diluted to 10 6 cfu/mL when used. LB broth was used as a blank control, and DMSO was used as a negative control, while ciprofloxacin was used as a positive control. The experiments were repeated three times (Pierce et al. 2008).

Conclusions
One new lignan derivative 2,3-dimethyl-4-(4-methoxyphenyl)-6,7-dihydroxynaphthalene (1), together with five known compounds (2-6), were isolated from ethanol extract of C. alfredii Hance collected in Hainan Province, China. The results showed that compounds 1, 2, 5 and 6 were isolated from the genus of the Combretum for the first time. Compound 1 providing a meaningful chemotaxonomic proof for the genus Combretum, and showed strong antibacterial activity against E. coli and S. albus with the same MIC values of 1.25 μg/mL. Compound 2 showed significant antibacterial activity against M. luteus and S. albus with the same MIC values of 2.5 μg/mL.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures.

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