Antiseptic Activity and Phenolic Constituents of the Aerial Parts of Vitex negundo var. cannabifolia

Four phenolics, salviaplebeiaside (1), γ-tocopherol (2), chrysosplenol-D (4), and isovitexin (5), along with α-tocoquinone (3) and β-sitosterol (6) were isolated from the aerial parts of Vitex negundo var. cannabifolia. The isolation was performed using bio-assay tracking experiments. The structures of compounds 1-5 were established by spectroscopic means. The antibacterial activities of the compounds were assessed against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus and Pseudomonas fluorescens. Chrysosplenol-D (4) exhibited activities against all the four spoilage microorganisms.


Introduction
Vitex negundo var. cannabifolia (Verbenaceae) is a shrub growing mainly in Yangzi River basin of China [1]. The plant was used as herbal medicine for the treatment of many diseases, such as colds, malaria, inflammation, sores and beriberi [2]. Some iridoids, lignans and other components in the OPEN ACCESS species were reported in previous phytochemical studies [3][4][5]. The juice extracted from aerial parts of the plant has been used as a folk antiseptic in Anhui province of China for preventing meat from rotting. In this study, four phenolics (compounds 1, 2, 4 and 5), along with two other compounds (3 and 6), were isolated and identified. Their antibacterial activities were examined and compound 4 was found to exhibit inhibitory activity against the four tested bacterial spp..

Antibacterial Activities of the Crude Extracts
The EtOH extract of the powdered dry aerial parts of V. negundo var. cannabifolia was successively fractionated with petroleum ether (PE), CHCl 3 and n-BuOH. The n-BuOH fraction was further fractionated by Diaion HP-20 column chromatography (CC) using H 2 O, 50% EtOH and 95% EtOH in a sequential elution process to yield three fractions A-C.
The antibacterial activities of the EtOH extract, as well as the PE, CHCl 3 , B, C fractions against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus, and Pseudomonas fluorescens were evaluated by the hole plate diffusion method (see Experimental section 3.10 for more details) [6]. The EtOH extract and the four other fractions were individually dissolved and diluted with DMSO to obtain serial concentrations of 100, 50 and 25 mg·mL -1 . The inhibition activities were evaluated by diameters of the inhibition zones ( Figure 1). Figures 1A-D show the significant inhibitory activities of the crude extracts at all three tested concentrations. The activity against E. coli was enhanced as the polarity of the fractions decreased ( Figure 1A), and the PE fraction showed significantly higher activity against E. coli at the concentrations of 50 and 25 mg·mL -1 versus the other fractions. However, there was no significant difference in the inhibition activity against B. subtilis among the four fractions ( Figure 1B). The activity of fraction C against M. tetragenus was significantly higher than that of any other fraction ( Figure 1C). Furthermore, at concentrations 50 and 25 mg·mL -1 , both fractions C and B showed significantly higher activities compared to the PE and CHCl 3 fractions. In Figure 1D, the inhibitory activities of the fractions against P. fluorescens were enhanced with the increase of the fractions' polarity. Both fractions C and B displayed higher activities than the PE or CHCl 3 fraction at all three concentrations. Overall fraction B showed a stronger inhibition against P. fluorescens. These results suggest that the bio-active components against E. coli were present in the lower-polarity (PE) fraction, while those against M. tetragenus and P. fluorescens were located in higher-polarity fractions (C and B).
By comparison their spectroscopic data (see the Experimental section) with those reported in the references, compounds 2-5 were identified as γ-tocopherol, α-tocoquinone, chrysosplenol-D and isovitexin, respectively [9][10][11][12]. Compound 6 were identified as β-sitosterol by comparing its Rf value with that of an authentic sample in a TLC experiment.

Antibacterial Activities of Compounds 1-5
The antibacterial activities of 1-5 against E. coli, B. subtilis, M. tetragenus, and P. fluorescens were also evaluated by the hole plate diffusion method. Compound 4 exhibited weak activities against E. coli, B. subtilis and M. tetragenus, with minimal inhibitory concentration (MIC) values of 500, 500 and 250 μg·mL -1 , respectively. At the same concentrations, ampicillin sodium displayed much stronger inhibition against the corresponding microorganisms. Nevertheless, the inhibitory activity of 4 against P. fluorescens was comparable with that of ampicillin sodium (MIC, 500 μg·mL -1 , see Table 2).

Plant Material
Aerial parts of V. negundo var. cannabifolia were collected in the rural area of Xuan-Cheng, Anhui Province, China, in summer of 2008, and identified by Prof. Sheng-Ni Tian, Anhui Agricultural University, and Prof. Yue-Hong Yan, Hunan University of Science and Technology. A voucher specimen was deposited at the Laboratory of Botany, School of Life Sciences, Anhui Agricultural University.

Extraction
Dry stems and leaves of V. negundo var. cannabifolia (3.0 kg) were ground to a fine powder, which were extracted with 95% EtOH three times (24, 48 and 48 h with 10 L each time) by percolation at room temperature. The EtOH percolate was concentrated in vacuo to a syrup (EtOH extract, 420 g). This syrup was suspended in H 2 O and the aqueous suspension (1000 mL) was successively extracted with PE (500 mL, 3 times), CHCl 3 (500 mL, 3 times), and n-BuOH (500 mL, 3 times) at room temperature. The PE and the CHCl 3 extracts, on concentration, yielded a dark green syrup (PE fraction, 60 g) and a brown syrup (CHCl 3 fraction, 40 g) respectively. The n-BuOH extraction, upon concentration under reduced pressure, afforded 90 g of a brown syrup. This syrup was further fractionated by Diaion HP-20 CC using H 2 O, 50% EtOH and 95% EtOH in a sequential elution process, yielding three fractions A-C.

Anti-bacterial Assays of the Crude Extracts
Anti-bacterial activities were evaluated by the hole plate diffusion method [6]. The test microorganisms were E. coli, B. subtilis, M. tetragenus, and P. fluorescens, which were obtained from the School of Basic Medical Sciences, Anhui Medical University, Hefei, P.R. China. The EtOH extract, PE and CHCl 3 fractions, as well as fractions B and C were individually dissolved and diluted with DMSO to obtain serial concentrations of 100, 50 and 25 mg·mL -1 . Six mm wide holes were bored with a sterilized steel borer into the Nutrient Agar Media (beef extract 3 g, peptone 10 g, agar 17 g, NaCl 5 g, H 2 O 1,000 mL, pH 7.2) in the Petri dish inoculated with the test microorganism. The solution of the compound (60 μL) at a specific concentration was added into each of the holes. DMSO was used as the negative control. The plates were then incubated at 37 °C for 24 hours. The diameters of the inhibition zones were measured and recorded. The assays were performed three times in order to guarantee reproducibility of results (see Figure 1).

Anti-bacterial Assays of the Compounds
Compounds 1-5 and ampicillin sodium (positive control) were individually dissolved and diluted with DMSO to obtain serial concentrations of 1000, 500, 250, 125 and 62.5 μg·mL -1 (for ampicillin sodium, the solutions were serially diluted from 1000 to 0.03 μg·mL -1 ). The anti-bacterial assays were also performed by the hole plate diffusion method as described above. The inhibition zones around the holes were measured and the MIC, which was defined as the lowest concentration being able to inhibit any visible bacterial growth, was recorded. The assays were performed three times for statistical analysis (see Table 2).

Statistical Analysis
The data in Figure 1 are presented as means ± SD. The values were evaluated by one-way analysis of variance (ANOVA), followed by Duncan's multiple range tests using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant at P < 0.05.

Conclusions
This is the first report on the isolation of salviaplebeiaside, γ-tocopherol and α-tocoquinone from the genus Vitex. Compounds 1-5 were reported from investigated species for the first time. The apoptosisinducing and antimalarial activities, as well as vascular relaxation effects of 4 had been reported [13][14][15][16]. With respect to antibacterial activities, the inhibition effects of 4 on Staphylococcus aureus, Cladosporium cucumerinum and Bacillus cereus were documented [11,17]. Our study indicated the inhibition activities of 4 on four spoilage microorganisms for the first time. It is known that P. fluorescens plays an important role in rotting of meat. The results of the present assays suggest chrysosplenol-D could be used as a potential antiseptic food additive. Furthermore, this compound might also be one of the key components to account for the medicinal usage of the plant.
In our previous work, the compositions of the essential oil from the aerial parts of V. negundo var. cannabifolia and their antiseptic activities were analyzed [18]. However, neither the essential oil, nor the compounds isolated from the PE fraction displayed any significant inhibitory activity against P. fluorescens.
A characteristic of phenolic-rich high-polar fraction in the fruits of the investigated species was revealed by previous phytochemical studies [5]. Our work also revealed that phenolics were the predominant components in the high-polar fraction of the aerial parts of the plant. As the high-polar fraction was confirmed as the significant antiseptic fraction by our bio-assays, the phenolics could be the key antiseptic constituents of V. negundo var. cannabifolia, even though more chemical examinations have to be done for this plant.