Constituents of Psoralea corylifolia Fruits and Their Effects on Methicillin-Resistant Staphylococcus aureus

Two new flavonoids, bakuisoflavone (1) and bakuflavanone (2), together with 15 known compounds, were isolated from the fruits of Psoralea corylifolia, and their structures were characterized by spectroscopic data. The effects of the isolated compounds on methicillin-resistant Staphylococcus aureus were also examined. We found that two compounds, isobavachalcone (10) and bakuchiol (12), showed noticeable antibacterial effects on the MRSA strains examined. Quantitation of the major constituents, including anti-MRSA constituents, was then performed. The results showed individual contents of 1.26%–16.49% (w/w) among the examined compounds in the ethyl acetate extract from P. corylifolia fruits.


Introduction
Psoralea corylifolia L. (Fabaceae) is a plant species indigenous to China. Fruits of this plant have been used traditionally for various diseases, including gynecological bleeding, vitiligo and psoriasis.

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A number of chemical constituents, including flavonoids and coumarins, have been isolated from this plant. Some of these compounds exhibit antioxidant [1], antiplatelet [2], estrogenic [3], immunomodulatory, and antitumor properties [4]. Antibacterial effects of several constituents on Staphylococcus aureus and S. epidermidis [5] have been reported. A previous report showed that bakuchiol, the main constituent, was also effective on methicillin-resistant S. aureus (MRSA) [6].
Staphylococcus aureus causes a variety of human diseases, ranging from minor skin infections to severe sepsis, and MRSA has become one of the most frequently encountered antibiotic-resistant bacteria [7]. Several prenylated phenolics obtained from licorice have shown noticeable antibacterial effects on MRSA [8]. Since a number of prenylflavonoids and related compounds were isolated from P. corylifolia [5,9,10], this plant species is expected to be a resource of lead compounds for new anti-MRSA drugs.
Our preliminary research showed that P. corylifolia fruit extract exhibited remarkable antibacterial effects on MRSA. Therefore, we scrutinized the fruit constituents and isolated 17 compounds, including two new ones, and found that several compounds showed potent antibacterial activity on MRSA. This paper describes the structures of the new compounds, as well as the antibacterial effects of the phenolic constituents on MRSA.
The 13 C-NMR spectrum ( Figures S6 and S7) revealed 20 carbon signals (15 for the flavanone skeleton and five for the 2-hydroxyl-3-methyl-3-butenyl moiety), although duplication of several signals was also observed ( Table 1). In the HMBC spectrum (Figures 2 and S8), correlations between H-1′′ of the C5 (2-hydroxy-3-methyl-3-butenyl moiety) unit to the C-5, C-6, and C-7 signals of the flavanone skeleton indicated that this C5 unit is located at C-6. Bakuflavanone (2) was thus characterized to be 4′,7-dihydroxy-6-(2-hydroxy-3-methyl-3-butenyl)-flavanone. The duplication of NMR signals was attributed to racemization at one of the asymmetric centers. The circular dichroism (CD) spectrum of this compound showed a prominent negative Cotton effect at ~306 nm, indicating the S configuration at C-2. Therefore, this compound was determined to form a mixture at the C-2′′asymmetric center.

Antibacterial Effects of Isolated Compounds
The antibacterial effects of the compounds isolated from P. corylifolia on MRSA were examined by the liquid dilution method [23]. Minimum inhibitory concentrations (MIC) of the tested compounds are shown in Table 2.

Structure-Activity Relationships
Previous studies have shown that bakuchiol (12) and isobavachalcone (10) exhibit antibacterial effects on MRSA [24,25], and the present study also showed antibacterial effects of these two on MRSA OM481 and OM584 strains. On the other hand, compounds 13-15, each of which is oxidized at the terminal prenyl group, showed weaker antibacterial effects than that observed for bakuchiol (12), which is unoxidized at the terminal prenyl group. Similarly, bakuisoflavone (1) and bakuflavanone (2), in which the prenyl group is oxidized to 2-hydroxy-3-methyl-3-butenyl group in each structure, showed weaker antibacterial effects than those of the corresponding unoxidized compounds, bavachin (5) and neobavaisoflavone (6). Therefore, we assumed that hydroxylation of the prenyl group weakened the antibacterial effects. Bavachinin (4), in which the hydroxy group at C-7 in bavachin (5) is substituted with a methoxyl group, showed a weaker antibacterial effect relative to that of compound 5. This comparison suggests the importance of the presence of the phenolic hydroxyl group in the flavonoid skeleton.
Based on these observations, we concluded that the presence of both phenolic hydroxy groups and the lipophilicity due to the benzene rings and the prenyl group are required for the potent antibacterial effects on MRSA, and these may contribute to the interaction of the compounds at bacterial cell membranes. Analogous observations have also been reported on the effects of licorice phenolics on MRSA [8] and on vancomycin-resistant Enterococci (VRE) [26][27][28].

Quantitative Analysis of the Major Constituents in P. corylifolia Fruit Extract
In the present study, several anti-MRSA constituents were found in P. corylifolia fruits, indicating that this plant may be a valuable resource for lead compound development of anti-MRSA drugs. To evaluate the usefulness of this plant, quantitative analysis of the antibacterial constituents was performed. Ethyl acetate extract from P. corylifolia was used for HPLC analysis. Quantitation of the major constituents, including anti-MRSA constituents, was based on UV absorption at 280 nm.
The results are summarized in Table 3. Among the peaks caused by constituents (Figure 3), isobavachalcone (10) (peak 10) and bakuchiol (12) (peak 12) showed especially potent anti-MRSA effects. Among the estimated compounds, bakuchiol (12) has the highest ethyl acetate extract content. This observation is in accord with the fact that this compound, a typical meroterpene composed of a phenolic structure and a monoterpene structure, was isolated as the major constituent from P. corylifolia [29]. The bavachinin (4) content, which is biogenetically derived from bavachin (5), was much higher than its mother compound, 5. The chalcones isobavachalcone (10) and corylifol B (11) also showed high contents in the extract. These results shown in the present study suggested that the HPLC method can be utilized for quality control with respect to the antibacterial resource. Table 3. Contents of major constituents in P. corylifolia EtOAc extract.

Antibacterial Assay
Two strains of MRSA, OM481 and OM584, clinical isolates from Okayama University Hospital that were stored in a Department of Microbiology laboratory, were used in this study. MICs were estimated by a liquid dilution method. Briefly, tested sample solutions of compounds 1-17 were diluted two-fold serially, and pre-cultured bacterial solutions were mixed on 96-well plates and incubated at 37 °C for 24 h. The lowest concentration among the tested samples at which the visible growth was completely inhibited was regarded as the MIC.

Quantitative Analysis of the Major Constituents in P. corylifolia Fruit Extract
Quantitative analysis of the constituents of the P. corylifolia fruit extract was carried out on a Hitachi HPLC-DAD D-2000 HSM system (Hitachi, Tokyo, Japan), involving an L-2455 DAD detector, equipped with a YMC-Pack Pro C18 (6.0 mm i.d. × 150 mm) column in an oven at 40 °C. The photodiode array detector was set for obtaining UV absorption from 200 to 400 nm, and the chromatograms at 280 nm were used for the quantitative analyses. The mobile phase was programmed using solvents A [water-acetonitrile-formic acid (60:40:1)] and B [water-acetonitrile-formic acid (20:80:1)] with the gradient mode as follows: 0%-25% B at 0-15 min, 25%-50% B at 15-35 min, 50%-75% B at 35-45 min, 75%-100% B at 45-60 min. The flow rate was set at 1.0 mL/min. Solutions of nine compounds (4, 5, 6, 8, 10, 11, 12, 16 and 17), used as the external standards, were prepared in series (from 0.01 to 1.00 mg/mL) and 2 µL each of the solutions were applied to HPLC, to produce nine individual regression lines to give equations shown in Table 4. Under these HPLC conditions, the relative standard deviation (RSD) 3.7% of the peak area was observed for eight injections of the solution of isopsoralen (17). The MeOH solution of the EtOAc extract of P. corylifolia fruit (1 mg/mL, 2 µL) was applied to the HPLC analysis, and three independent experiments were conducted for the quantitation of the nine constituents in the extract. The characters x and y represent the amount of the compound injected and the relative peak area shown by the data processor of the HPLC system, respectively. b Square of correlation coefficient for x and y.