Prinsepia utilis Royle seeds obtained from Yunan province, China were identified and authenticated as Prinsepia utilis Royle seeds by the Kunming Institute of Botany, Chinese Academy of Sciences.

The dried seeds of Prinsepia utilis Royle was crushed and extracted with distilled water and then the aqueous extract was precipitated in ethanol. The supernatant was collected and condensed, subsequently extracted by petroleum ether, ethyl acetate and n-butanol respectively. The antibacterial activity in vitro of these three extracts was tested by disc diffusion method in vitro. As a result, the ethyl acetate extracts showed satisfactory antibacterial activity.

The ethyl acetate extract was purified by Silica gel column chromatography. The open glass column (45 mm by 600 mm) was packed with Silica gel G (100 - 200 mesh, Merck, Qingdao). The column, loaded with the ethyl acetate extract, was eluted with chloroform-methanol-acetone (8:7:0.5 [vol/vol/vol]) solvent system. Each fraction (50 mL) of the eluate was tracked by thin-layer chromatography (TLC) (silica gel GF₂₅₄ [0.2 mm thick]; Merck). Seven fractions were collected, concentrated in the rotary evaporator and dried under vacuum, then used for assay of their antibacterial activity by disc diffusion method. The active fraction, which showed a high antibacterial activity, was further purified by repeatedly re-crystallized with acetone.

The bacterial strains used in this study were Staphylococcus aureus ATCC25923, Escherichia coli ATCC44102 and Salmonella sp. CMCC(B)50041 were supplied from the Department of Food Engineering Sciences, Sichuan Agricultural University, Ya’an, China.

The disc diffusion method was used to evaluate the antibacterial activity [11] [12] . An inoculum of the test microbes were prepared for 24 h in Mueller Hinton Broth (MHB) cultures and inoculums were further diluted 1:10 in sterile MHB to obtain a final inoculum of 1 × 10⁶ CFU/mL. 200 μl of the bacterial inoculum were spread evenly onto the surface of Mueller Hinton agar plates by a sterile L stick, before setting aside the seeded agar plate (9 cm in diameter) to solidify for 15 minutes. Sterile filterpaper disks with 6 mm diameter were used to screen the antimicrobial activity. Each sterile disk was impregnated with 10 μl of the fraction at the concentration of 20, 10, and 5 mg/ml respectively; Oregano oil (used as positive control) and Dimethylsulfoxide (used as negative control) were placed on the surface of the seeded plates. The plates were covered and incubated at 37˚C for 24 h, then diameter of inhibition zone was measured. Each test was performed in triplicate.

The MICs and MBCs of active fractions were determined by the broth microdilution method, recommended by the Clinical and Laboratory Standards Institute/NCCLS [13] . The concentrations of active fractions ranging from 20 to 0.313 mg/mL and an inoculum of 1 × 10⁶ CFU/mL were used. The MIC was taken as the lowest con- centration of active fractions in the wells of the microtiter plate that showed no turbidity after 24 hours of incubation at 37˚C. The lowest concentration showing no visible growth on sterile agar plates by subculture was taken as MBC. Oregano oil was used as positive control and DMSO as negative control. All the experiments were performed in triplicate and the results were averaged.

The results were expressed as the mean ± S.D. (Standard Deviation). The differences between the fractions were using one-way analysis of variance (ANOVA) by SPSS for Windows Programmer (SPSS 13.0; SPSS Inc., Chicago, IL, USA). A p value < 0.05 was considered significant statistically.

Antibacterial activity each fraction obtained from the ethyl acetate extract of Prinsepia utilis Royle by silica gel column chromatography was shown in Table 1. Oregano oil (positive control) had been well known for its bacteriostatic properties [14] . We found that anterior fractions were active against all bacteria tested at concentration of 20 mg/mL, while the Fr.6 and Fr.7 showed no effect on all bacteria and produced no zone of inhibition.

The white snowflake-like compound A was isolated from the Fr.1 by re-crystallization with acetone. TLC indicated that Compound A has only one spot by petroleum ether, chloroform and ethanol (Figure 1). Fr.1, Fr.4 and compound A had a strong inhibitory effect, the inhibition zone diameter against standard S. aureus ATCC- 25923 (14.16 mm, 15.26 mm, 14.03 mm) were greater than that of oregano oil (13.76 mm) in the same concentration, they had the same inhibition effect as oregano oil against standard E. coli ATCC44102 and Salmonella CMCC(B)50041.

The MICs and MBCs of active fractions were shown in Table 2. Out of the three bacterial species tested, the range of MIC values of active fractions separated from the ethyl acetate extract of Prinsepia utilis Royle against

Note: “—”means no zone ofinhibition.

S. aureus ATCC25923 (0.625 mg/mL to 10 mg/mL) were lower compared with Gram-negative bacteria E. coli ATCC44102 and Salmonella CMCC(B)50041 (2.5 mg/mL to 20 mg/mL). The MICs of Fr.1 and Fr.4 against S. aureusATCC25923 were the lowest, 0.625 mg/mL. The MIC of Oregano oil had better antibacterial effects on E. coli ATCC44102 and Salmonella CMCC(B)50041 (1.25 mg/mL, 2.5 mg/mL) than S. aureus ATCC25923 (5 mg/mL).

Based on the results obtained from the MICs, the MBCs were higher than the MIC values of active fractions against the bacteria. The MBC values of Fr.1 and Fr.4 against S. aureus ATCC25923 (2.5 mg/mL, 1.25 mg/mL) were 4 times and 2 times greater than MIC values (0.625 mg/mL, 0.625 mg/mL), respectively. The MICs and MBCs of the compound A were the same against S. aureus ATCC25923 (1.25 mg/mL), while the MBC value was 2 times greater than its MIC values for E. coli ATCC44102, greater than 20 mg/mL for Salmonella CM- CC(B)50041.