Antibacterial Activity of the Flavonoids from Dalbergia odorifera on Ralstonia solanacearum

Phytohemical investigation on the heartwood of Dalbergia odorifera resulted in the isolation of nine flavonoids. Their structures were elucidated as sativanone (1), (3R)-vestitone (2), (3R)-2',3',7-trihydroxy-4'-methoxyisoflavanone (3), (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone (4), carthamidin (5), liquiritigenin (6), isoliquiritigenin (7), (3R)-vestitol (8), and sulfuretin (9) based on their spectral data. All compounds were evaluated for their inhibitory activity against Ralstonia solanacearum. This is the first report about anti-R. solanacearum activity of the compounds from D. odorifera.


Introduction
Ralstonia solanacearum, the pathogen that is the causal agent of bacterial wilt, is one of the best-known bacterial diseases, and is found in tropical, subtropical, and some temperate regions of the World. This soilborne pathogen attacks more than 200 plant species, including many agriculturally important crops [1]. This bacterium can also be free-living as a saprophyte in water or in the soil in the absence of host plants [2]. Streptomycin is widely used in agriculture, but the overuse of it can lead to bacterial resistance [3]. Thus, it is very necessary to search for more potent anti-R. solanacearum compounds.
The heartwood of Dalbergia odorifera T. Chen, named "Jiangxiang" in Chinese traditional medicine, was used in China and Korea for the treatment of blood stagnation syndrome, ischemia, swelling, necrosis and rheumatic pain [4,5]. Previous chemical investigations on this plant have led to the isolation of flavonoids and phenolic compounds [6][7][8]. Some flavonoids have been reported to possess various pharmacological effects such as anti-inflammatory, antibacterial, antiplasmodial, antinephritic, neuroprotective and antioxidant activities [9][10][11][12][13][14]. During the course of our screening for anti-R. solanacearum agents from tropical medicinal plants, the crude ethanol extract of the heartwood of D. odorifera showed anti-R. solanacearum activity. In this paper, we described the isolation, identification and anti-R. solanacearum activity of compounds 19.
Compounds 14 belong to the isoflavanone class. Compound 1 showed lower activity than the other compounds, and this may be due to the absence of the 2'-OH group, suggesting that this 2'-OH is a favorable group for activity. Compounds 24 had a B-ring OH group (2' position), and 3 had a B-ring OH group (3' position), while 4 had a C-ring OH group (3 position). Lower activity of 3 compared to that of 2 seemed to be because the 3'-OH and 2'-OH formed a stable five-membered ring, which reduced the inhibition of the 2'-OH group. Compound 4 had slightly reduced inhibition compared with 2, which leads us to speculate that the 3-OH and 2'-OH formed an unstable six-membered ring. Compound 8 belong to the isoflavane class which lack the C(4)=O in the C-ring compared with 2, and its activity was higher than that of 2. The result suggests that the presence of C(4)=O will reduced the inhibitory effect.

General
The NMR spectra were recorded on a Bruker AV-400 spectrometer, using TMS as an internal standard. Column chromatography was performed with silica gel (Marine Chemical Industry Factory, Qingdao, China) and Sephadex LH-20 (Merck). TLC was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, China) plates.

Plant Materials
The dried heartwood of D. odorifera was purchased from the Haikou Free Market of Agricultural Products, Hainan Province, China, in October, 2010. The specimen was identified by Professor Zheng-fu Dai of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (No. 20101009) has been deposited.

Extraction, Fractionation and Identification of the Flavonoids
The dried and crushed heartwood of D. odorifera (8.4 kg) was extracted three times with 95% ethanol (50 L) at room temperature for three weeks totally. The ethanol extract was then filtered through absorbent gauze, and the filtrate was concentrated on a rotary evaporator under reduced pressure at 50 °C to remove ethanol, resulting in a crude ethanolic extract. This was partitioned with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate phase (477.0 g) was submitted to column chromatography (CC) over silica gel eluted with a mixture of chloroform and methanol . These data were consistent with those reported in [19].  C-4′). These data were in accordance with those reported previously [19].

Bacterial Strains
The R. solanacearum strain was obtained from Professor Ming-he Mo of the Key Laboratory of Protection and Utilization of Biological Resources, Yunnan University, and maintained on a nutrient agar (NA) slant at 4 °C.

Antibacterial Activity
These compounds were individually tested for in vitro antibacterial activity against R. solanacearum strain by the filter paper disc agar diffusion method [22]. The NA medium was mixed with suspension (2 mL) containing 10 7 CFU/mL of R. solanacearum, and then poured into Petri-plates to a uniform depth of 5 mm and was allowed to solidify. The isolated compounds dissolved in dimethyl sulfoxide (DMSO) (1.6 µL, 50 mg/mL) were impregnated on sterile filter paper discs (6 mm diameter) and then applied aseptically to the surface of the agar plates. Streptomycin sulfate (1.6 µL, 50 mg/mL) was used as positive control. The plates were incubated at 37 °C for 24 h. Then the diameters of the inhibition zones including the 6 mm disc diameter were measured. Experiments were done in triplicate, and the results were mean values.

Conclusions
In conclusion, a total of nine compounds including four isoflavanones 14, two flavanones 5 and 6, one chalcone 7, one isoflavane 8 and one aurone 9 were isolated from D. odorifera and identified by comparison of their NMR data with data reported in the literature. In addition, all compounds were evaluated for their inhibitory activity against R. solanacearum. Among the nine flavonoids, compound 8 exhibited the strongest antibacterial activity and compounds 2, 6, and 7 showed strong antibacterial activity. This is the first report of the anti-R. solanacearum activity of the compounds from D. odorifera.