Separation and Identification of Four New Compounds with Antibacterial Activity from Portulaca oleracea L.

The Portulaca oleracea L. (P. oleracea) has been used to treat bacillary dysentery for thousands of years in China. Pharmacology studies on P. oleracea have also showed its significant antibacterial effects on the enteropathogenic bacteria, which might reveal the treatment of P. oleracea in cases of bacillary dysentery to some extent. To date, however, the therapeutic basis of P. oleracea treating on bacillary dysentery remains unknown. We determined the antibacterial effective fraction of P. oleracea in a previous study. The current study, which is based on our previous study, was first designed to isolate, identify and screen antibacterial active constituents from P. oleracea. As a result, four new compounds (1–4), portulacerebroside B (1), portulacerebroside C (2), portulacerebroside D (3) and portulaceramide A (4) along with five known compounds (5–9) were isolated, and structures were established by their physico-chemical constants and spectroscopic analysis. The antibacterial activities against common enteropathogenic bacteria were evaluated for all compounds and the new compounds 1–4 showed significant antibacterial effect on enteropathogenic bacteria in vitro, which might contribute to revealing the treatment of P. oleracea in cases of bacillary dysentery.


Introduction
The genus of Portulaca is an annual herb which taxonomically belongs to the family of Portulacaceae. Although it originates from India, Portulaca has been widely distributed in other temperate and tropical areas of the world [1,2]. In China, there are six species of Portulaca, among which Portulaca oleracea L. (P. oleracea) has been used as a traditional Chinese medicine (TCM) for thousands of years. P. oleracea, cold in nature and acid in flavor, possesses the efficacies of clearing away the heat evil and detoxifying and cooling blood to stop diarrhea. In clinical situations, P. oleracea has been used to treat acute appendicitis, scrofula ulcer, pediatric pertussis, burns and scalds, psoriasis [3], hemorrhinia, uterine bleeding, urinary tract infections, lung abscess, mumps, and especially more effective in bacillary dysentery which manifested feeling cold or fever, bellyache, diarrhea, tenesmus, and mucus pus blood stool [2]. Chemical studies on P. oleracea showed its main constituents of fatty acids, terpenes, alkaloids, coumarins, flavonoids, and volatile oil [2]. In addition, the only cerebroside of portulacerebroside A was reported in 2008 [4]. Pharmacology studies on P. oleracea showed its activities of antibacterial [5,6], hepatoprotective [7,8], anti-inflammatory, analgesia [2,9], muscle relaxant [10], neuroprotective [11], anti-oxidant [1], and anti-aging [12], but studies on the corresponding therapeutic basis were far from sufficient. Obviously, the treatment of P. oleracea on bacillary dysentery results from its antibacterial effect on the enteropathogenic bacteria; however, the corresponding active components in P. oleracea were unknown. Consequently, we screened and obtained the antibacterial fraction (EtOAc extract) from P. oleracea. The effective fraction could inhibit and kill the common enteropathogenic bacteria in vitro effectively, based on which a bioassay-guided isolation and phytochemical study of P. oleracea was performed and four new along with five known compounds were obtained from the effective fraction. The structures of known compounds 5-9 were determined by detailed 1D-and 2D-NMR analyses, ESI-MS and comparison of their spectral data with literature values was undertaken. In this paper, the isolation and structural elucidation of the new compounds 1-4 was described. We also investigated the antibacterial effects of compounds 1-9 against common enteropathogenic bacteria in vitro.  (Tables 1 and 2) showed characteristics of a cerebroside with a 2-hydroxy fatty acid fraction as the aglycone of 1. Methanolysis of 1 obtained a fatty acid methyl ester (FAME) and a long-chain base (LCB). The FAM was determined as 2-hydroxypentadecanoic acid methyl ester by Gas Chromatography-Mass Spectrometer (GC-MS) analysis. The 1D-TOCSY spectrum of 1 showed a correlation between δH 4.22 (1H, m, H-3) and 5.46 (2H, m, H- 8,9), which suggested the olefinic bond was located in the LCB. To determine the location of the olefinic bond in LCB, the dimethyl disulfide (DMDS) derivatives of the LCB was analyzed by ESIMS and characteristic fragment ion of m/z 187 [M + H] + was obtained. Therefore, the olefinic bond was located at C-8 and C-9. The LCB was further determined as 2-aminooctadec-8-ene-1,3-diol by GC-MS analysis ( Figure 1). The specific rotation [α] 22 D = −5.8° (c = 0.02, CHCl3) of the FAM confirmed that the absolute configuration of C-2′R [13]. The 2S, 3R stereochemistry was determined by comparing of the 13 C-NMR data of C-2 and C-3 with those in references [14][15][16]. The trans-configuration (E) of the olefinic bond in 1 was determined by signals at δC 33.2/32.1 of the two carbons next to the olefinic bond in 13 C-NMR spectrum [9]. The 1 Hand 13 C-NMR data (Tables 1 and 2) were further assigned by the spectra of DEPT, HSQC, 1 H-1 H COSY, and HMBC. Consequently     ) of the FAM confirmed that the absolute configuration of C-2′R, which is the same with 1. The 2S, 3R stereochemistry was determined by comparing of the 13 C-NMR data of C-2 and C-3 with those of in references [17,18]. The trans-configuration (E) of the olefinic bond in 2 was determined by C-6 signal at δC 34.2 in 13 C-NMR spectrum [16]. The 1 H-and 13 C-NMR data (Tables 1  and 2 ) of the FAM confirmed that the absolute configuration of C-2′R [13]. The 2S, 3S, and 4R stereochemistry was determined by comparing of the 13 C-NMR data of C-2, C-3, and C-4 with those of in reference [19]. The trans-configuration (E) of the olefinic bond in 4 was determined by signals at δC 33.3/33.0 of the two carbons next to the olefinic bond in 13 C-NMR spectrum [16]. The 1 H-and 13 C-NMR data (Tables 1 and 2 The known compounds were identified as friedelin (6) [20], 3-acetylaleuritolic acid (7) [21], 4α-methyl-3β-hydroxylfriedelan (8) [22], cycloartenol (9) [23], and lupeol (10) [24] by comparing their NMR spectroscopic and physical data with the literature values ( Figure 3). The antibacterial activities of compounds 1-9 against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Shigella flexneri (S. flexneri), and Salmonella typhi (S. typhi) were investigated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined. Compounds 1-4 showed significant antibacterial activity on common enteropathogenic bacteria in vitro (Table 3), while other compounds did not show any antibacterial effects on the enteropathogenic bacteria at the tested concentration (data not shown). The structures of compounds 1-4 involve a sphingoid base and an amide-linked fatty acyl chain. The amphipathic molecules exhibit diverse biological activity, including broad antibacterial activities against both Gram positive and negative bacteria in this study.

General
The NMR spectra were measured on Bruker AVANCE 400 MHz NMR instrument (Bruker SpectroSpin, Karlsruhe, Germany), and chemical shifts are given as δ (ppm) while the coupling constants are given in Hz. Xero Q Tof MS spectrometer (Waters, Milford, MA, USA) was used to measure and analysis the HRESIMS data. Volatile derivatives from compounds were analyzed on a GC-MS (Angilent, California, CA, USA) instrument. Waters 2535 instrument coupled with a Waters Sunfire prep C18 OBD (19 × 250 mm i.d.) column, a UV-2998 (Waters, MA, USA), and RI-2414 detector as a Preparative HPLC (Waters, MA, USA) was used to prepare compounds. FTIR-8400S (Shimadzu, Kyoto, Japan) was used to record the IR Spectra data; Column chromatographies including Macroporous resin (AB-8 Crosslinked Polystyrene, Nankai Chemical Plant, Tianjin, China), silica gel (200-300 mesh, Haiyang Chemical Group Co. Ltd., Qingdao, China), and ODS-A (120A, 50 mm; YMC, Kyoto, Japan) were also employed. A microplate reader (BMG FLUOStar OPTIMA, Ortenberg, Germany) was used to monitor the growth of the bacterial strains.

Bacterial Strains and the Preparation of Inoculums
The bacterial strains of E. coli (ATCC25922), S. aureus (ATCC25923), S. flexneri (ATCC12022), and S. typhi (ATCC14028) from American Type Culture Collection were provided by Department of Microbiology and Immunology, Heilongjiang university of Chinese medicine. Strains from refrigerated stock cultures were inoculated into common agar plate and incubated at 37 °C for 18 h. The bacteria were activated in nutrient broth and incubated at 37 °C for another 18 h. The concentrations of strains for antibacterial test in vitro were 5 × 10 5 CFU·mL −1 .

Plant Materials
We collected the aerial part of P. oleracea from the Dongfanghong Forestry Agency (Jixi, China) and identified by Lianjie Su of Heilongjiang University of Chinese Medicine. The voucher specimen (No. 20130814) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine, China.

Dimethyl Disulfide Derivative of LCBs from 1 and 4
According to the reference [18], LCBs from 1 and 4 (0.5 mg) were dissolved in dimethyl disulfide (DMDS, 0.2 mL), respectively, and then iodine (1 mg) was added into the solutions. The mixtures were stored in a small-volume sealed vial at 60 °C for 40 h. The reaction was ended with aqueous Na2S2O3 (5%), and then we extracted the mixtures with n-hexane (0.3 mL). The extracts were concentrated respectively to give the DMDS derivatives of LCBs from 1 and 4.

Compounds 1-9 Serial Dilution
A microdilution method was used to determine the MICs of the compounds on 96-well cultivated plates according to the previous report [27]. The compounds 1-9 were dissolved in nutrient broth with 10% DMSO and 32.0 mg·mL −1 solutions (pH 7.2) were obtained, respectively. There were 12 wells in each row of a microplate, to each of the first ones we added 100 μL compound solution, and to the remaining 11 wells we added 100 μL broth culture. For serial dilution, 100 μL each compound solution was added into the second well and then 100 μL was sequentially transferred to the following wells until the 10th well. The last two wells served as growth control and sterility check. After that, 100 μL of inoculum was added into each well except the last well in which 100 μL broth was added instead. Amoxicillin was used as a positive drug.

Determination of MICs and MBCs
The growth of the bacterial strains in the microplates was monitored at 37 °C for 20 h using a microplate reader. Standard antibacterial agent amoxicillin was also screened under identical conditions for comparison. Considering the role of DMSO, the same experiment was carried out with 10% DMSO and showed no activity against any bacterial strains. MIC was expressed as the mean concentration between the well showing growth and that showing no growth.
After MIC testing, the microplates set up for the MICs determination were used to determine the MBC as described previously [28]. For each well showing no bacterial growth, the entire volume was spread onto nutrient agar plates and subcultured. The MBC was defined as the lowest concentration of the compounds showing no bacterial growth after incubating for 20 h.

Conclusions
We investigated the chemical constituents of P. oleracea based on its antibacterial activity and nine compounds were obtained, including three new cerebrosides and a new ceramide.  (4), respectively. Antibacterial tests in vitro showed that new compounds could significantly inhibit or kill the common enteropathogenic bacteria, which might contribute to revealing the usefullness of P. oleracea as a treatment for bacillary dysentery.