- Email: [email protected]
Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway
European Journal of Medicinal Chemistry 96 (2015) 436e444
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway Xiu-Mei Li a, 1, Xue-Gang Luo a, *, 1, Chuan-Ling Si c, d, Nan Wang a, Hao Zhou a, Jun-Fang He a, Tong-Cun Zhang a, b, * a Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China b Institute of Biology and Medicine, Wuhan University of Science and Technology, Wuhan, 430000, PR China c Tianjin Key Laboratory of Pulp & Paper, Tianjin University of Science & Technology, Tianjin, 300457, PR China d State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, 150040, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Accepted 15 April 2015 Available online 16 April 2015
Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-b-(200 -acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-b-(200 -acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-b-(200 -acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Hypericum ascyron L. Antimicrobial activity Preparative high performance liquid chromatography Structure identification Structureeactivity relationship Apoptosis
1. Introduction Antimicrobial resistances against human pathogenic microorganisms have developed to be more and more serious due to the indiscriminate use of antimicrobial drugs in the treatment of infectious diseases. This situation, the undesirable side effects of certain antibiotics and the emergence of previously uncommon
* Corresponding authors. Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China. E-mail addresses: [email protected] (X.-G. Luo), [email protected] (T.-C. Zhang). 1 The first two authors contributed equally to the project. http://dx.doi.org/10.1016/j.ejmech.2015.04.035 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
infections [1e3] forced scientists into looking for new antimicrobial substances from various sources. Lots of evidence showed that plants, especially traditional herbs, represent potential source of new anti-infective agents [1]. They could markedly mitigate infectious diseases, but lack adverse side effects which are often associated with traditional antimicrobial agents, including hypersensitivity, allergic reaction, and immunosuppression [4e6]. In particular, Hypericum is one important genus of traditional Chinese herb medicine, which could produce secondary metabolites with antimicrobial properties [7]. Among 484 Hypericum L. (Guttiferae/Hypericaceae) species which are widespread in warm temperate areas throughout the world, only Hypericum perforatum is widely used in official medicine [8]. Hypericum ascyron L. is a perennial herbaceous plant genus of the Guttiferae family. It is commonly found in Japan and the
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
northeast, the basin of Yellow River and Yangtze River of China. It common names include Hong-Han-Lian, Huang-Hai-Tang, and Hunan-Lian-Qiao. The aerial parts of the plant have been used as a traditional Chinese medicine for the treatment of wounds, swelling, headache, nausea, abscesses, abnormal menstruation and promoting lactation [9]. Recent studies have revealed many mechanisms underlying the pharmacological activities of H. ascyron L., including of histamine-release inhibitory [10], anti-inflammatory and analgesic effects [11], anti-oxidant activity [12] glucosidase inhibitory, anti-diabetic activity [13], and anticancer [14]. However, the antibacterial activity of H. ascyron L. was not reported. Plants are widely accepted as good sources of novel antimicrobial agents. Screening of antimicrobial activities to find which types of bacteria are susceptible to plant extracts is useful, however the investigation of underlying mechanism is also crucial for drug development. To explore the possible antibacterial activity compounds and antibacterial mechanism of H. ascyron L.. Firstly, the crude extract of H. ascyron L. was prepared and its function on the proliferation of bacterial cells was analyzed. Secondly, to identify the compounds which are responsible for the antibacterial activity, a bioassay-guided fractionation was employed using preparative HPLC, and the structural characterization of the antibacterial compounds was analyzed using NMR and electrospray ionization tandem mass spectrometry (ESI/MS). Thirdly, biochemical methods were utilized to explain the bacterial cell death mechanism. 2. Materials and methods 2.1. General experimental procedures Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm; Agela Technologies, Inc; China) and Kromasil C18 column (250 4.6 mm i.d.; 5 mm; AkzoNoble, Sweden) were used for column chromatography. For preparative HPLC, a preparative-HPLC system (Agilent Technologies, Inc; USA) consisting of an Agilent 1200 series pump, manual injector, binary pump, and a 1200 series variablewavelength UV-VIS detector was used for all analyses. For a analytical HPLC system (Agilent Technologies, Inc; USA) consisting of an Agilent 1100 series pump, manual injector, a binary pump and a 1100 series variable-wavelength UV-VIS detector was used for all analyses. NMR spectra (1H, 13C, COSY, HMBC, HSQC) were recorded in DMSO-d6 spectrometer with a Bruker Avance III 400 MHz equipped with a cryoprobe and using TMS as the internal reference. Mass spectrometry (MS) was carried out using a micromass ESI instrument (Agilent Technologies, Inc; USA) using ESI-ionization in positive mode. 2.2. Plant material All the samples of H. ascyron L. were collected from Hailaer (China). All specimens, which were authenticated by Ya-hong Sun (Genhe city of Inner Mongolia agriculture and animal husbandry bureau, Genhe, PR China), were dried in the shade until the weight remained constant. 2.3. Microbial strains The antimicrobial activity of H. ascyron L. extract and its fractions were tested against the following microorganisms: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC 10031), Staphylococcus aureus (ATCC 25923) and Micrococcus luteus (ATCC 10240). All strains were provided by China General Microbiological Culture Collection Center (CGMCC) and maintained in Mueller-Hinton agar (Hopebio, China) and stored at 20 C.
437
2.4. Antibacterial activity evaluation The antimicrobial efficacy of all the samples was evaluated using cup plate method. Mueller-Hinton agar plates were prepared by pouring 10e15 mL of the medium into each sterile Petridish and were allowed to set at room temperature. The bacterial cell suspension was standardised to the optical density of 0.1 at 600 nm using a spectrophotometer. Thus final concentration of microorganisms in the inoculum was adjusted to 105 CFU/mL and was inoculated over the surface of agar medium using a sterile cotton swab. The Oxford cups were placed in each plate. Each Oxford cup was added 200 mL of the designed concentration of H. ascyron L. extract and fractions. The petri plates were then incubated at 37 C for 18 h. 2.5. Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetraazolium bromide (MTT) assay was used for measuring the proliferation of bacterial cells. Briefly, bacterial cells (105 CFU/mL) were inoculated into Mueller-Hinton broth at 200 mL/well in 96-well microtiter plates. Two fold serial dilutions of H. ascyron L. extract were added to wells containing bacterial cells. After 24 h of incubation at 37 C, each concentration was assayed in triplicate (n ¼ 3). Twenty-four hours later, 10 mL of the MTT (5 mg/mL) reagent was added to each well and the plates were incubated for 4 h at 37 C. Then, DMSO (100 mL) was added to terminate the reaction, and the plate was shaken slightly to redissolve the crystals formed. The absorbance of each well was measured using Synergy 4 microplate reader (BioTek Instruments, Winooski, VT, USA). All the results were expressed as the inhibition ratio of cell proliferation calculated as [(A-B)/A] 100%, where A and B were the average numbers of viable cells of the control and samples, respectively. MBC, defined as the minimum concentration required to killing 99.9% of a bacteria inoculums, was determined by reinoculating 20 mL of each culture medium from the microtiter plate wells onto Mueller-Hinton agar plates. After 18 h of incubation at 37 C, MBC value was determined by visually inspecting the agar plates for bacterial growth. MIC and MBC measurements were performed at least in triplicate (n ¼ 3). 2.6. Bioassay-guided isolation The aerial parts of the dried plants were harvested and then selected through a 60 mesh sieve. Each sample powder (1.00 g) was accurately weighed and extracted using 30 mL (60%, v/v) ethanolwater solution by ultrasonic extraction for 30 min at 30 C [14]. The extract obtained was centrifuged for 10 min at 5000 rpm with a Hermle Z206A centrifuge (Denville Scientific Inc., USA).The extract was collected and filtered through a membrane filter with a pore size of 0.22 mm (Agela Technologies, Inc; China). The extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The gradient of elution program was as follows: from 0 to 5 min, acetonitrile followed a linear change from 5% to 10%; from 5 to 10 min, acetonitrile linearly changed from 10% to 15%; from 10 to 15 min, solvent A linearly changed from 15% to 17%; from 15 to 30 min, acetonitrile was isocratic at 17%; from 30 to 60 min, acetonitrile linearly changed from 17% to 25%; from 60 to 90 min, acetonitrile linearly changed from 25% to 45%. The flow rate was at 15 mL/min with a 10 mL injection volume, The absorbance was monitored at 275 nm [14] to give 10 fractions (as shown in Fig. 1(A)) based on pre-HPLC detection peaks at different retention time. Each fraction was tested for antibacterial activity and the
438
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 1. Bioguided screening of antibacterial activity compounds from H. ascyron L. (A) Fractionation of the extract from H. ascyron L., the extract was fractionation into 10 fractions based on pre-HPLC detection peaks at different retention time. (B) and (C) Antibacterial activity of 10 fractions. Each fraction was tested for antibacterial activity and the bioactive fractions were combined to obtain fraction 8. This was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) eluting and affording compound 1 and compound 2. (D) Structure of compound 1 and compound 2, with rings named and positions numbered.
bioactive fractions were combined to obtain fraction 8. This was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) eluting and affording compound 1 and compound 2. The compound 1 and compound 2 were identified by MS and NMR. 2.7. HPLC analysis of fraction 8 Fraction 8 was separated on a Kromasil C18 column (250 mm 4.6 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The gradient of elution program was as follows: from 0 to 10 min, acetonitrile followed a linear change from 20% to 23%; from 10 to 30 min, acetonitrile linearly changed from 23% to 25%; from 30 min to analysis end, acetonitrile was isocratic at 25%. The flow rate was at 1.0 mL/min with a 10 mL injection volume, the absorbance was monitored at 275 nm. 2.8. Effect of fraction 8 on the growth of M. luteus cells M. luteus cells (105 CFU/mL) were inoculated into MuellerHinton broth at 200 mL/well in microtiter plates. Fraction 8 (0, 2.5, 5 mg/mL) was added to wells containing bacterial cells and incubated at 37 C. For each treatment, the growth of M. luteus was determined by automated absorbance measurements at 600 nm (Bioscreen C, Labsystems, Helsinki, Finland) at every hour.
2.9. Apoptosis analysis 2.9.1. Annexin V-FITC/PI staining M. luteus cells apoptosis was measured by Annexin V-FITC and PI staining method following the manufacturer’s instructions (Tianjin Sungene Biotech Co., Ltd., China). After exposure to fraction 8 (0, 2.5 mg/mL) at 37 C for 0, 2, 4 and 8 h, cells were then harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min and washed once with cold PBS. Then M. luteus cells were suspended in1 mL 1 Binding Buffer, centrifuged 8000 rpm for 10 min, and then remove the Binding Buffer. Resuspend cells in 1 mL 1 Binding Buffer, adjust cell concentration to 105 (CFU/mL). 100 mL of cells (105 CFU/mL) was added to each 1.5 mL microtubes. Then 5 mL of Annexin V-FITC was added to the 1.5 mL microtubes, respectively. Gently vortex each microtube and incubate for 10 min in room temperature, protected from light. 5 mL of PI solution was added to the 1.5 mL microtubes and incubated for 5 min in room temperature, protected from light. Finally, 500 mL of PBS was added to the 1.5 mL microtubes and vortex gently, respectively. The analysis of the cells was done by flow cytometry (FACScan; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were sorted into living, necrotic, early apoptotic, and late apoptotic cells. The relative ratio of early and late apoptotic cells were counted for further comparison. This assay was repeated more than three times.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
2.9.2. TUNEL assay for apoptotic DNA fragmentation The 30 -OH of the DNA fragments in apoptotic cells were labeled and stained by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling method using an apoptosis in situ detection kit (Promega, Germany), according to the manufacturer’s instructions. Briefly, after treatment with the fraction 8(0, 2.5 mg/mL) at 37 C for 0, 2, 4 and 8 h, apoptotic M. luteus cells (105 CFU/mL) were harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min and washed once with cold PBS, fixed with 4% paraformaldehyde for 25 min. Fixed cells were washed once with PBS and centrifuged at 8000 rpm for 10 min. Cells were permeabilized with 70% ethanol for at least 24 h at 20 C. Cells were centrifuged at 8000 rpm for 10 min and rewashed once with PBS. Excess liquid was removed by centrifuging at 8000 rpm for 10 min. Cells were covered with 200 mL of Equilibration Buffer for 5e10 min at room temperature, then centrifuged at 8000 rpm for 10 min. Cells were added 100 mL of rTdT incubation buffer and incubate at 37 C for 60 min with aluminum foil to protect from direct light. The reactions were terminated with 2X SSC for 15 min at room temperature. Cells were washed with fresh PBS for three times to remove unincorporated fluorescein-12-dUTP. Cells were stained with 40 ,6diamidino-2-phenylindole solution (1 mg/mL) for 15 min at room temperature in the dark. Cells were washed with PBS for three times. Fluorescent microscopy (Olympus, FV1000, Japan) was used to capture the image of the fluorescein-labeled TUNEL-positive cells. The cell nucleus was labeled in blue by DAPI (Invitrogen, Molecular Probes, Eugene, OR) and the nick-ends were labeled in green. A merge between the nucleus (blue) and nick-end (green) labeling showed up as purple.
2.9.3. DNA fragmentation assay DNA fragmentation is an important index of apoptosis. Usually DNA ladder was observed could determine the cell apoptosis. M. luteus cells were incubated with fraction 8 (0, 2.5 mg/mL) at 37 C after 0, 2, 4 and 8 h, 105 (CFU/mL) cells were then harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min at 4 C and washed once with PBS. DNA was extracted using the Apoptosis, DNA Ladder Extraction Kit (Beyotime, China) as follows: 1 mL of the sample cracking liquid was added 5 mL of proteinase K (20 mg/mL) and blended. Then each of harvested samples was added 500 mL of cracking liquid with proteinase K. Cells were Vortex blended. Then all samples were digested at 50 C water bath for the night. Each sample was added 500 mL of Tris balanced phenol. Cells were intensely Vortex blended, and then centrifuged at 12,000g for 5 min at 4 C. Phenol and the intermediate phase were slowly sucked out. The rest of the water phase was extracted with the same volume Tris balance phenol again and centrifuged at 12,000g for 5 min at 4 C. Phenol and the intermediate phase were slowly sucked out again. The rest of the water phase was extracted with the same volume chloroform once again. About 300 mL of supernatant was slowly sucked out, then 60 mL of 10 mM ammonium acetate and 600 mL of anhydrous ethanol were added to each of sample, and mixed upside down several times. The samples were stored at 20 C for 1 h, and then centrifuged at 12,000g for 10 min at 4 C. The supernatant was abandoned. Each of samples was added 600 mL 70% ethanol, and mixed upside down about twice times. Samples were centrifuged at 12,000g for 10 at 4 C. The supernatant of all samples were carefully abandoned. Each of samples was immediately dissolved with 50e100 mL TE. The DNA was then electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide (EB) staining. The gel was photographed under ultraviolet light.
439
2.10. Detection of membrane potential (Djm) M. luteus cells were incubated with fraction 8 (0, 2.5 mg/mL) at 37 C after 0, 2, 4 and 8 h, 105 (CFU/mL) cells were then harvested by centrifuging (8000 rpm, 10 min) and washed twice with cold PBS. Each harvested sample was re-suspended in 500 mL of Rhodamine 123 (1 mM) for 30 min at 37 C, and washed twice with PBS. Then M. luteus cells were analyzed by FACScanto (BD Biosciences). 2.11. Statistical analysis All the data were expressed as mean ± SD (standard deviation, SD) and all the statistical analysis was performed using SPSS statistical software package (SPSS Inc., Chicago, IL, USA). Furthermore, p<0.05 was considered statistically significant. 3. Results 3.1. Bioassay-guided screening of the antibacterial ingredients of H. ascyron L. The antibacterial effects of H. ascyron L. extract were tested on several micro-organisms (E. coli, E. cloacae, K. pneumonia, S. aureus and M. luteus), and the results showed that its antimicrobial activity were mainly against Gram-positive bacteria, especially M. luteus (data not shown). The results are in agreement with previous studies of E. coli, E. cloacae and K. pneumoniae were more resistant to most plant extracts than Gram-positive bacteria [8]. Therefore, M. luteus was used as the indicator bacteria for the screening of the antibacterial activity compounds from H. ascyron L.. The extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) to afford 10 fractions based on preparative HPLC detection peaks at different retention time (as shown in Fig. 1A). Each fraction was then tested for the antibacterial activity. As demonstrated in Fig. 1B and C, except the mixture (the extract from H. ascyron L.), fraction 1, fraction 2, and fraction 10, all the other fractions exhibited varying levels of antibacterial activity against M. luteus at 10 mg/mL. Among 10 fractions, the fraction 8 showed the highest antibacterial ability, and the diameter of inhibition zone on the tested M. luteus was 14.53 ± 0.19 mm. Fraction 8 was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm), and then afforded two main pure compounds (Compound 1 and Compound 2), as shown in Fig. 1D. Compound 1 was isolated as a yellow amorphous powder, with molecular formula C23H22O12 deduced from the [Mþ1]þ peak at m/ z 490 in the MS and supported by 1H, 13C and 13C DEPT spectra. The spectral data with those reported kaempferol 3-O-b-(200 -acetyl) galactopyranoside spectrum data are basically identical in literature [15]. Compound 1 was identified as kaempferol 3-O-b-(200 acetyl) galactopyranoside. Compound 2 was isolated as a yellow amorphous powder, with molecular formula C15H10O7 deduced from the [M1] þ peak at m/z 302 in the MS and supported by 1H, 13C and 13C DEPT spectra. The spectral data with those reported quercetin spectrum data are basically identical in literature [16]. Therefore, compound 2 was identified as quercetin. Based on the HPLC analysis of fraction 8 (Fig. 2), fraction 8 mainly included kaempferol 3-O-b-(200 -acetyl) galactopyranoside, quercetin and two impurity peaks (1 and 2), the content of kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin in the fraction 8 was above 90% (See supplementary material). Therefore, we considered kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin were the crucial elements of fraction 8 possessing antibacterial activity.
440
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 2. HPLC analysis of fraction 8. Fraction 8 was separated on a Kromasil C18 column (250 mm 4.6 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The flow rate was at 1.0 mL/min with a 10 mL injection volume, the absorbance was monitored at 275 nm.
3.2. MIC and MBC of fraction 8 To further detect the antibacterial ability of fraction 8, the MIC was tested on M. luteus cells in designed concentration (0.16 mg/ mL, 0.31 mg/mL, 0.63 mg/mL, 1.25 mg/mL, 2.5 mg/mL, 5 mg/mL) and showed a significant decrease in viable cells number was detected after treatment for 18 h. Besides, the anti-proliferative effect exhibited a dose-dependent manner, and the inhibitory rate increased to 100% when the dose of fraction 8 was over 5 mg/ mL, indicating that the MIC value was 5 mg/mL (Fig. 3A). Furthermore, the MBC was determined by re-inoculating 20 mL of each culture medium from the 5 mg/mL microtiter plate wells onto Mueller-Hinton agar plates. After being incubating at 37 C for 18 h, there was no bacteria growth on 5 mg/mL tested Mueller-Hinton agar plates, suggested that 5 mg/mL was also the MBC, implying that fraction 8 could be regarded as a bactericidal medicine. In addition, to further indicate the effect of antibacterial activity of fraction 8 based on the growth cycle of M. luteus, the growth cycle of M. luteus was also performed. As shown in Fig. 3B, the growth of M. luteus was significantly inhibited by fraction 8, especially from the exponential phase to the stationary growth phase. In addition, consistent with the MIC evaluated in the MTT assay, M. luteus cell was inhibited fully at 5 mg/mL. These results further confirmed the validity of MIC value. 3.3. Effects of fraction 8 on the apoptosis of M. luteus Previous studies have showed that apoptosis markers, including of phosphatidylserine exposure, chromosome condensation and DNA fragmentation, could be observed in bactericidal antibiotic treatment [17]. Therefore, we sought to determine whether apoptosis was involved in the antibacterial effect of fraction 8. To determine the apoptosis-stimulating effects of fraction 8 on M. luteus, the flow cytometry analysis using Annexin V-FITC/PI staining was performed. As shown in Fig. 4A, a small percentage of apoptotic cells was found in the negative group (1.7 ± 0.7%) after
Fig. 3. MIC and the growth cycle of M. luteus. (A) MIC value of fraction 8 on M. luteus. M. luteus cells were treated with fraction 8 from 0.16 mg/mL to 5.0 mg/mL for 18 h at 37 C. (B) Effect of fraction 8 on the growth cycle of M. luteus. M. luteus cells were treated with fraction 8 (0 mg/mL, 2.5 mg/mL, 5 mg/mL) for 8 h at 37 C. For each treatment, the growth of M. luteus was determined by automated absorbance measurements at 600 nm, detected absorption value every hour.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
441
Fig. 4. Apoptosis analysis. (A) Results of annexin V/PI double stain assay. The apoptosis rate of M. luteus cells treated with fraction 8 (0 mg/mL, 2.5 mg/mL) for 0, 2, 4 and 8 h was detected by the flow cytometry using fluorescently-labeled annexin V/PI double stain. (B) Results of TUNEL assays. The apoptotic status of M. luteus cells treated by fraction 8 (0 mg/ mL, 2.5 mg/mL) for 0, 2, 4 and 8 h was determined by TUNEL assay (green channel). DAPI (blue channel) is used to locate the nuclei of the cells. (C) The gel electrophoresis image obtained after DNA fragmentation assay for apoptosis detection. M. luteus cells were cultured at 37 C for 0, 2, 4 and 8 h in the presence or absence of the indicated concentration of fraction 8. DNA was isolated and visualized on a 1.0% agarose gel stained with ethidium bromide. From left lanes are: Lane M: Marker; Lane 1: 0 h (CON); Lane 2: 2 h (CON); Lane 3: 4 h (CON); Lane 4:8 h (CON); Lane 5: 0 h (2.5 mg/mL); Lane 6: 2 h (2.5 mg/mL); Lane 7: 4 h (2.5 mg/mL); Lane 8:8 h (2.5 mg/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubating 8 h. However, the percentage of apoptotic cells significantly increased from 0.0 ± 0.2% to 21.3 ± 0.8% after being treated with 2.5 mg/mL of fraction 8 from o hour to 8 h. The results implied that fraction 8 could time-dependently induce M. luteus cells death through apoptosis pathway. To further define the mechanism underlying fraction 8mediated cell death, M. luteus cells were treated with fraction 8 (0, 2.5 mg/mL) for 0, 2, 4 and 8 h and DNA fragmentation was measured using the TUNEL assay. Fraction 8 treatment activated apoptosis in M. luteus cells, as evidenced by increased fluorescence
intensity, which was detected by labeling DNA single-strand breaks with dUTP-fluorescein at the 30 eOH ends of the nuclei. As demonstration in Fig. 4B, the green fluorescence intensity was almost undetectable in the control cells (100%), whereas the fluorescence intensity increased significantly in a time-dependent manner in the fraction 8-treated M. luteus cells. The results demonstrate that fraction 8 effectively induced apoptosis in M. luteus cells. Furthermore, when apoptosis occurs, DNA is cleaved into oligonucleosomal fragments of 180e200 bp [18]. In the present study,
442
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 5. Effects of fraction 8 on membrane potential. Membrane potential (Djm) was analyzed using Rho123. The M. luteus cells were treated with the designed concentration of fraction 8 (0 mg/mL, 2.5 mg/mL), at 37 C for 0, 2, 4 and 8 h. M. luteus cells were incubated with Rho123 (1 mM) for 30 min and then harvested for analysis by flow cytometry, M1 reflects the reduction of Djm.
as indicated by the agarose gel electrophoresis, exposure of M. luteus cells to the fraction 8 at concentrations of 2.5 mg/mL for 2, 4 and 8 h could result in marked DNA fragmentation, whereas the control (untreated with fraction 8) did not exhibit any DNA ladder, as shown in Fig. 4C. 3.4. Effects of fraction 8 on membrane-mediated apoptosis pathways To test whether fraction 8 could induce apoptosis of M. luteus through destroying the cell membrane, we measured the disruption of cell membrane potential (Djm) using Rho123 stain. As shown in Fig. 5, when M. luteus cells were treated with 2.5 mg/mL of fraction 8 for indicated periods, indicating the collapse of Djm.
Fig. 6. Potential antibacterial mechanism of fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) attached to the membrane surface from electrostatic attraction, and resulted in the disturbance of the target membrane, as well as structural change and fracture of the membrane. Fraction 8 (kaempferol 3-O-b(200 -acetyl) galactopyranoside couple with quercetin) entered the cytoplasm through membrane pores, and subsequently, two parallel reactions took place, namely, increase of the level of ROS and interaction with genome DNA, which caused cell death.
The observations indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. 4. Discussion Among 484 Hypericum L. (Guttiferae/Hypericaceae) species, widespread in warm temperate areas throughout the world, only H. perforatum is widely used in official medicine. H. sampsoni, H. ascyron L., H. foliosum, H. geminiflorum and H. scabrum containing considerable amounts of other acylphloroglucinol derivatives have the potential to demonstrate antibacterial and cytotoxic activity [8]. In our present investigation, the results showed that H. ascyron L. extract possessed antibacterial effect and mainly inhibited the proliferation of Gram-positive bacteria, especially M. luteus. A possible explanation for these observations may be associated with the significant structural differences between Gram-positive and Gram-negative bacterial cells walls, as the latter possess an outer membrane and a unique periplasmic space [19]. The resistance of Gram-negative towards antibacterial substances is due to its outer hydrophilic membrane surface that is rich in lipopolysaccharide molecules and thus creates a barrier to numerous antibiotic molecules. This composition makes the cell wall impermeable to lipophilic solutes, and the porins in the cell wall do not allow the penetration of high molecular mass hydrophilic solutes, with an exclusion limit of about 600 Da [20]. Further, enzymes in the periplasmic space are capable of breaking down molecules introduced from the outside. Gram-positive bacteria (M. luteus) does not have such an outer membrane and cell wall structure. Antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane resulting in leakage and coagulation of the cytoplasm [21]. In addition, the lack of an outer lipopolysaccharide membrane in the Gram-positive bacteria could allow increased permeability of Hypericum metabolites into cells [22,23]. Therefore, the extract of H. ascyron L. displayed obvious antibacterial activity to Gram-positive bacteria, especially M. luteus.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
The discovery of active compounds from natural products is a time-consuming process. An effective strategy developed recently is the use of pre-fractionated natural product libraries, which not only reduces the background that is a problem with crude libraries, but also speeds the purification of active compounds [24]. The method has two steps: the preparation of fractions from crude extracts and the isolation of individual compounds from the active fractions. In order to cut down the isolated and purified time, in our present investigation, the extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) to afford 10 fractions based on preparative HPLC detection peaks at different retention time (Fig. 1A). Each fraction was then tested on M. luteus (Fig. 1B and C), except the mixture (the extract from H. ascyron L.), fraction 1, fraction 2, and fraction 10, all the other fractions exhibited varying levels of antibacterial activity against M. luteus at 10 mg/mL. Among 10 fractions, the fraction 8 showed the highest antibacterial ability. We obtained the active fraction in a short time. The MIC and MBC values of fraction 8 on M. luteus were 5 mg/mL. In addition, we determined that the effect of antibacterial activity of fraction 8 (2.5, 5 mg/mL) based on the growth cycle of M. luteus, found the growth of M. luteus was significantly inhibited, especially from the exponential phase to the stationary growth phase. These results implied that fraction 8 could be regarded as a bactericidal medicine. We isolated and obtained two key antibacterial activity compounds by identified as kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin. The content of the two compounds in fraction 8 was above 90%. We considered that kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin of fraction 8 played a fatal role in antibacterial action. Through the literature retrieval, we found that kaempferol 3-O-b-(200 -acetyl) galactopyranoside was described for the first time from Clusiaceae and first reported in antibacterial activity. Previous studies showed that kaempferol 3-O-b-(200 acetyl) galactopyranoside was confirmed in aconitum paniculatum [15] and possessed certain antioxidation [25]. Quercetin is one of a group of over 4000 naturally available plant phenolics whose isolation and biological identification were first described by SzentGyorgyi in 1936 [26]. In addition, quercetin was identified in H. ascyron in 1980 [16]. Various studies have shown that the pharmacological effects of quercetin include protective effects against cancer, cardiovascular diseases, ischemic injuries, and antiinflammatory, immunemodulatory, gastroprotective activities, antioxidant, antibacterial, as well as enhancement of stress resistance and extension of the life span of various organisms from bacteria to vertebrates [27-30]. Flavonoids of structure factors play an important role in biological activity. Flavonoids molecules of structureeactivity relationship studies have shown that the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5 and 7 in the A ring are the first group of antibacterial activity, followed by the hydroxyl at position 3 in the C ring for antibacterial activity of such compounds. The hydroxyl at position 30 and 40 in the B ring also have certain antibacterial activity [31,32]. As shown in Fig. 1D, quercetin includes the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7, 3, 30 and 40 . Kaempferol 3-O-b-(200 -acetyl) galactopyranoside possesses the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7 and 40 . These results are in agreement with previous studies of flavonoid molecules possess the characteristic of antibacterial activity. Apoptosis is commonly known as physiological programmed cell death and is different from necrosis [33]. Cell apoptosis involves the biological regulation of the numbers and vital activities of cells, and is an important metabolic process. Apoptosis occurs through the activation of cellintrinsic suicide machinery [34]. One key mechanism involved in the function of antibacterial drugs is the activation of the apoptotic pathway. The results of Annexin V/PI
443
double staining demonstrated the apoptosis rate of M. luteus cells was increased with the extension of the exposure time to fraction 8, in a time dependent manner (Fig. 4A). The apoptosis effects of fraction 8 on M. luteus cells were further confirmed via TUNEL assay (Fig. 4B). In addition, an important feature of apoptosis is the fragmentation of genomic DNA into integer multiples of 180e200 bp, which results in a characteristic ladder upon agarose gel electrophoresis. As shown in Fig. 4C, the experiment results showed that the DNA of M. luteus cells presented typical ladder-like pattern after treatment with fraction 8 at 2.5 mg/mL, in a time dependent manner, whereas the control indicated no evident DNA ladder. These results demonstrated that fraction 8 could induce the death of M. luteus cells by apoptosis pathway. Recent research has revealed evidence of apoptotic death in bacteria. Apoptosis-like death in E. coli was described by Dwyer et al.. In addition to membrane depolarization and DNA fragmentation, they found that this apoptosis like death is also characterized by phosphatidylserine exposure to the outer leaflet of the plasma membrane and chromosome condensation [17]. Apoptosis-like death is induced not only by DNA-damaging agents but also by various antibiotics, including spectinomycin, ampicillin, and gentamicin. The results may differ from ours because we firstly found the compounds from the nature product could induce M. luteus cells apoptosis death. Reactive oxygen species (ROS) include free radicals such as superoxide (O-2), hydroxyl radical (OH-), and non-radical derivatives of oxygen such as H2O2, which is mainly derived from the respiratory chain in mitochondria [35]. ROS generation and oxygen stress have been reported as common anti-bactericidal episodes caused by many antibacterial compounds [36]. ROS generation and disruption of the membrance potential (DJm) contributed to drug induced apoptosis [37]. In our present work, we found that the membrane potential of M. luteus cells treated with fraction 8 was remarkably lost (Fig. 5), demonstrating that fraction 8 induced irreversible apoptosis phenomenon by membrane-mediated apoptosis. In addition, Mirzoeva and colleagues showed that quercetin could cause an increase in permeability of the inner bacterial membrane and a dissipation of the membrane potential [38]. Therefore, we speculate the antibacterial mechanism of fraction 8 as shown in Fig. 6. Potential antibacterial mechanism of fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) attached to the membrane surface from electrostatic attraction, and resulted in the disturbance of the target membrane, as well as structural change and fracture of the membrane. Fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) entered the cytoplasm through membrane pores, and subsequently, two parallel reactions took place, namely, increase of the level of ROS and DNA fragmentation, which caused cell death. 5. Conclusion In summary, through screening we identified kaempferol 3-O-b(200 -acetyl) galactopyranoside and quercetin, as potential antibacterial compounds in H. ascyron L. Kaempferol 3-O-b-(200 -acetyl) galactopyranoside is described for the first time from Clusiaceae and first reported in antibacterial activity. Using TUNEL labeling, Annexin V-FITC/PI and DNA gel electrophoresis, we found that bacteria cell death with features of apoptosis after exposure to the antibacterial activity from H. ascyron L. at moderate concentration. We report for the first time an assessment and possible mechanism of kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin could induce cells death by apoptosis pathway. In addition, we found kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin could disrupt cells membrane. Therefore, we speculated the antibacterial activity compounds induced irreversible apoptosis phenomenon by membrane-mediated apoptosis. Our data might
444
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs. H. ascyron L. is a promising antibacterial species against antibiotic multi-resistant bacteria because of its natural occurrence, wide distribution in China and Japan.
[14]
[15] [16]
Declaration of interest [17]
The authors report no declarations of interest. [18]
Acknowledgments
[19]
This work was financially supported by National Natural Science Foundation of China (31171303, 31170541, 31171297, 31270837), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1166), State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (K2013101), Natural Science Foundation of Tianjin City (13JCZDJC29400).
[21]
Appendix A. Supplementary data
[23]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.04.035.
[24]
[20]
[22]
[25]
References [1] C. Iris, A. Zampini Marta, I. Vattuone Maria, Antibacterial activity of Zuccagnia punctata Cav. ethanolic extracts, J. Ethnopharmacol. 102 (2005) 450e456. [2] A. Marchese, G.C. Shito, Resistance patterns of lower respiratory tract pathogens in Europe, Int. J. Antimicrob. Ag. 16 (2001) 25e29. [3] K. Poole, Overcoming antimicrobial resistance by targeting resistance mechanisms, J. Pharmand. Pharmacol. 53 (2001) 283e284. [4] R. Dall'agnol, A. Ferraz, A.P. Bernardi, D. Albring, C. Nor, E.E.S. Schapoval, Bioassayguided isolation of antimicrobial benzopyrans and phloroglucinol derivatives from Hypericum species, Phytother. Res. 19 (2005) 291e293. [5] S. Gibbons, Anti-staphylococcal plant natural products, Nat. Prod. Rep. 21 (2004) 263e277. [6] P.K. Mukherjee, G.S. Saritha, B. Suresh, Antimicrobial potential of two different Hypericum species available in India, Phytother. Res. 16 (2002) 692e695. [7] S.A. Sarkisiana, M.J. Janssenb, H. Mattab, G.E. Henryb, K.L. LaPlantea, D.C. Rowleya, Inhibition of bacterial growth and biofilm production by constituents from Hypericum spp, Phytother. Res. 26 (2012) 1012e1016. [8] G. Stojanovi, A. Dorevic, A. Smelcerovic, Do other Hypericum species have medical potential as St. John’s Wort (Hypericum perforatum)? Curr. Med. Chem. 20 (2013) 2273e2295. [9] JiangSu New Medical College, Dictionary of Chinese Crude Drugs, Scientific Technological Publishers, 1977, p. 1002. [10] J. Wang, N.L. Wang, X.S. Yao, Inhibitory activity of Chinese herbal medicines toward histaminerelease from mast cells and nitric oxide production by macrophage-like cell line RAW264.7, J. Nat. Med. 60 (2006) 73e77. [11] J.M. Lv, W. Jia, C.Y. Li, The anti-inflammatory and analgesic effects of guttiferae hypericum ascyron linn, Pract. Clin. J. Integr. Tradit. Chin. West. Med. 8 (2008) 87e89. [12] U.K. Sang, S.U. Chon, B.G. Heo, Y.S. Park, S. Gorinstein, Total phenolics level, antioxidant activities and cytotoxicity of young sprouts of some traditional Korean salad plants, Plant. Food. Hum. Nutr. 64 (2009) 25e31. [13] W.Y. Kang, Y.L. Song, L. Zhang, a-Glucosidase inhibitory and antioxidant
[26] [27] [28]
[29]
[30] [31] [32] [33]
[34] [35]
[36] [37]
[38]
properties and antidiabetic activity of Hypericum ascyron L, Med. Chem. Res. 20 (2011) 809e816. X.M. Li, X.G. Luo, Li K. M N, W. Li, D.Y. Ma, et al., Quality and antitumour activity evaluation of extract of Hypericum ascyron, Biomed. Chromatogr. (2014), http://dx.doi.org/10.1002/bmc.3169. G. Fico, A. Braca, A.R. Bilia, F. Tome, I. Morelli, Flavonol glycosides from the flowers of Aconitum paniculatum, J. Nat. Prod. 63 (2000) 1563e1565. Z.Q. Wang, X.R. Wang, Studies on the active principles of Hong Han Lian (Hypericum ascyron L.), Acta. Pharm. Sin. 6 (1980) 365e367. D.J. Dwyer, D.M. Camacho, M.A. Kohanski, J.M. Callura, J.J. Collins, Antibioticinduced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis, Mol. Cell. 46 (2012) 561e572. A. Saraste, K. Pulkki, Morphologic and biochemical hallmarks of apoptosis, Cardiovasc. Res. 99 (2000) 528e537. C.F. Duffy, R.F. Power, Antioxidant and antimicrobial properties of some Chinese plant extracts, Int. J. Antimicrob. Ag. 17 (2001) 527e529. -Pie boji, N. Eze, A.N. Djintchui, B. Ngameni, N. Tsabang, J. Gangoue D.E. Pegnyemb, et al., The in-vitro antimicrobial activity of some traditionally used medicinal plants against beta-lactam-resistant bacteria, J. Infect. Dev. Ctries. 3 (2009) 671e680. Y. Gao, M.J. Van belkum, M.E. Stiles, The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C, Appl. Environ. Microbiol. 65 (1999) 4329e4333. F. Tian, B. Li, B. Ji, J. Yang, G. Zhang, Y. Chen, et al., Antioxidant and antimicrobial activities of consecutive extracts from Galla chinensis: the polarity affects the bioactivities, Food. Chem. 113 (2009) 173e179. S.A. Sarkisiana, M.J. Janssenb, H. Mattab, G.E. Henryb, K.L. LaPlantea, D. Rowleya, Inhibition of bacterial growth and biofilm production by Constituents from Hypericum spp, Phytother. Res. 26 (2012) 1012e1016. F.E. Koehn, New strategies and methods in the discovery of natural product anti-infective agents: the mannopeptimycins, J. Med. Chem. 51 (2008) 2613e2617. , N.D. Tommasi, Antioxidant A. Braca, G. Fico, I. Morelli, F.D. Simone, F. Tome and free radical scavenging activity of flavonol glycosides from different aconitum species, J. Ethnopharmacol. 86 (2003) 63e67. J.B. Harborne, Nature, distribution and function of plant flavonoids, Prog. Clin. Biol. Res. 213 (1986) 15e24. M.Y. Li, T.Y. Zhu, Quercetin in a Lotus leaves extract may be responsible for antibacterial activity, Arch. Pharm. Res. 31 (2008) 640e644. T.B. Ng, F. Liu, Y. Lu, Antioxidant activity of compounds from the medicinal herb aster tataricus, Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 136 (2003) 109e115. M. Bathori, I. Zupko, A. Hunyadi, Monitoring the antioxidant activity of extracts originated from various Serratula species and isolation of flavonoids from Serratula coronata, Fitoterapia 75 (2004) 162e167. C. Kanadaswami, L.T. Lee, P.P. Lee, The antitumor activities of flavonoids, In. Vivo 19 (2005) 895e909. J.T. Zhang, Y.D. Song, CNDO/2 Study on flavonoids 13, J. Shanxi. Agr. Univ., 1993, pp. 134e140. Y.H. Jiang, Study on Antimicrobial Components in Lotus Leaves, 2007. Y.F. Sun, C.K. Song, H. Vernstein, F. Unger, Z.S. Liang, Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway, Food. Chem. 138 (2013) 1998e2007. D.R. Green, G.I. Evan, A matter of life and death, Cancer. Cell. 1 (2002) 19e30. A. Erental, Z. Kalderon, A. Saada, Y. Smith, H. Engelberg-Kulkaa, Apoptosis-like death, an Extreme SOS Response in Escherichia coli, Mbio 5 (2014) 1426e1440. J. Nordberg, E.S.J. Arner, Reactive oxygen species, antioxidants, and the mammalian thioredoxin system, Free. Radic. Bio. Med. 31 (2001) 1287e1312. R.S. Ye, H.Y. Xu, C.X. Wan, S.S. Peng, L.J. Wang, H. Xu, et al., Antibacterial activity and mechanism of action of 3-poly-L-lysine, Biochem. Biophys. Res. Co. 439 (2013) 148e153. O.K. Mirzoeva, R.N. Grishanin, P.C. Calder, Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria, Microbiol. Res. 152 (1997) 239e246.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway Xiu-Mei Li a, 1, Xue-Gang Luo a, *, 1, Chuan-Ling Si c, d, Nan Wang a, Hao Zhou a, Jun-Fang He a, Tong-Cun Zhang a, b, * a Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China b Institute of Biology and Medicine, Wuhan University of Science and Technology, Wuhan, 430000, PR China c Tianjin Key Laboratory of Pulp & Paper, Tianjin University of Science & Technology, Tianjin, 300457, PR China d State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, 150040, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2014 Accepted 15 April 2015 Available online 16 April 2015
Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-b-(200 -acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-b-(200 -acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-b-(200 -acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Hypericum ascyron L. Antimicrobial activity Preparative high performance liquid chromatography Structure identification Structureeactivity relationship Apoptosis
1. Introduction Antimicrobial resistances against human pathogenic microorganisms have developed to be more and more serious due to the indiscriminate use of antimicrobial drugs in the treatment of infectious diseases. This situation, the undesirable side effects of certain antibiotics and the emergence of previously uncommon
* Corresponding authors. Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China. E-mail addresses: [email protected] (X.-G. Luo), [email protected] (T.-C. Zhang). 1 The first two authors contributed equally to the project. http://dx.doi.org/10.1016/j.ejmech.2015.04.035 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
infections [1e3] forced scientists into looking for new antimicrobial substances from various sources. Lots of evidence showed that plants, especially traditional herbs, represent potential source of new anti-infective agents [1]. They could markedly mitigate infectious diseases, but lack adverse side effects which are often associated with traditional antimicrobial agents, including hypersensitivity, allergic reaction, and immunosuppression [4e6]. In particular, Hypericum is one important genus of traditional Chinese herb medicine, which could produce secondary metabolites with antimicrobial properties [7]. Among 484 Hypericum L. (Guttiferae/Hypericaceae) species which are widespread in warm temperate areas throughout the world, only Hypericum perforatum is widely used in official medicine [8]. Hypericum ascyron L. is a perennial herbaceous plant genus of the Guttiferae family. It is commonly found in Japan and the
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
northeast, the basin of Yellow River and Yangtze River of China. It common names include Hong-Han-Lian, Huang-Hai-Tang, and Hunan-Lian-Qiao. The aerial parts of the plant have been used as a traditional Chinese medicine for the treatment of wounds, swelling, headache, nausea, abscesses, abnormal menstruation and promoting lactation [9]. Recent studies have revealed many mechanisms underlying the pharmacological activities of H. ascyron L., including of histamine-release inhibitory [10], anti-inflammatory and analgesic effects [11], anti-oxidant activity [12] glucosidase inhibitory, anti-diabetic activity [13], and anticancer [14]. However, the antibacterial activity of H. ascyron L. was not reported. Plants are widely accepted as good sources of novel antimicrobial agents. Screening of antimicrobial activities to find which types of bacteria are susceptible to plant extracts is useful, however the investigation of underlying mechanism is also crucial for drug development. To explore the possible antibacterial activity compounds and antibacterial mechanism of H. ascyron L.. Firstly, the crude extract of H. ascyron L. was prepared and its function on the proliferation of bacterial cells was analyzed. Secondly, to identify the compounds which are responsible for the antibacterial activity, a bioassay-guided fractionation was employed using preparative HPLC, and the structural characterization of the antibacterial compounds was analyzed using NMR and electrospray ionization tandem mass spectrometry (ESI/MS). Thirdly, biochemical methods were utilized to explain the bacterial cell death mechanism. 2. Materials and methods 2.1. General experimental procedures Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm; Agela Technologies, Inc; China) and Kromasil C18 column (250 4.6 mm i.d.; 5 mm; AkzoNoble, Sweden) were used for column chromatography. For preparative HPLC, a preparative-HPLC system (Agilent Technologies, Inc; USA) consisting of an Agilent 1200 series pump, manual injector, binary pump, and a 1200 series variablewavelength UV-VIS detector was used for all analyses. For a analytical HPLC system (Agilent Technologies, Inc; USA) consisting of an Agilent 1100 series pump, manual injector, a binary pump and a 1100 series variable-wavelength UV-VIS detector was used for all analyses. NMR spectra (1H, 13C, COSY, HMBC, HSQC) were recorded in DMSO-d6 spectrometer with a Bruker Avance III 400 MHz equipped with a cryoprobe and using TMS as the internal reference. Mass spectrometry (MS) was carried out using a micromass ESI instrument (Agilent Technologies, Inc; USA) using ESI-ionization in positive mode. 2.2. Plant material All the samples of H. ascyron L. were collected from Hailaer (China). All specimens, which were authenticated by Ya-hong Sun (Genhe city of Inner Mongolia agriculture and animal husbandry bureau, Genhe, PR China), were dried in the shade until the weight remained constant. 2.3. Microbial strains The antimicrobial activity of H. ascyron L. extract and its fractions were tested against the following microorganisms: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC 10031), Staphylococcus aureus (ATCC 25923) and Micrococcus luteus (ATCC 10240). All strains were provided by China General Microbiological Culture Collection Center (CGMCC) and maintained in Mueller-Hinton agar (Hopebio, China) and stored at 20 C.
437
2.4. Antibacterial activity evaluation The antimicrobial efficacy of all the samples was evaluated using cup plate method. Mueller-Hinton agar plates were prepared by pouring 10e15 mL of the medium into each sterile Petridish and were allowed to set at room temperature. The bacterial cell suspension was standardised to the optical density of 0.1 at 600 nm using a spectrophotometer. Thus final concentration of microorganisms in the inoculum was adjusted to 105 CFU/mL and was inoculated over the surface of agar medium using a sterile cotton swab. The Oxford cups were placed in each plate. Each Oxford cup was added 200 mL of the designed concentration of H. ascyron L. extract and fractions. The petri plates were then incubated at 37 C for 18 h. 2.5. Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetraazolium bromide (MTT) assay was used for measuring the proliferation of bacterial cells. Briefly, bacterial cells (105 CFU/mL) were inoculated into Mueller-Hinton broth at 200 mL/well in 96-well microtiter plates. Two fold serial dilutions of H. ascyron L. extract were added to wells containing bacterial cells. After 24 h of incubation at 37 C, each concentration was assayed in triplicate (n ¼ 3). Twenty-four hours later, 10 mL of the MTT (5 mg/mL) reagent was added to each well and the plates were incubated for 4 h at 37 C. Then, DMSO (100 mL) was added to terminate the reaction, and the plate was shaken slightly to redissolve the crystals formed. The absorbance of each well was measured using Synergy 4 microplate reader (BioTek Instruments, Winooski, VT, USA). All the results were expressed as the inhibition ratio of cell proliferation calculated as [(A-B)/A] 100%, where A and B were the average numbers of viable cells of the control and samples, respectively. MBC, defined as the minimum concentration required to killing 99.9% of a bacteria inoculums, was determined by reinoculating 20 mL of each culture medium from the microtiter plate wells onto Mueller-Hinton agar plates. After 18 h of incubation at 37 C, MBC value was determined by visually inspecting the agar plates for bacterial growth. MIC and MBC measurements were performed at least in triplicate (n ¼ 3). 2.6. Bioassay-guided isolation The aerial parts of the dried plants were harvested and then selected through a 60 mesh sieve. Each sample powder (1.00 g) was accurately weighed and extracted using 30 mL (60%, v/v) ethanolwater solution by ultrasonic extraction for 30 min at 30 C [14]. The extract obtained was centrifuged for 10 min at 5000 rpm with a Hermle Z206A centrifuge (Denville Scientific Inc., USA).The extract was collected and filtered through a membrane filter with a pore size of 0.22 mm (Agela Technologies, Inc; China). The extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The gradient of elution program was as follows: from 0 to 5 min, acetonitrile followed a linear change from 5% to 10%; from 5 to 10 min, acetonitrile linearly changed from 10% to 15%; from 10 to 15 min, solvent A linearly changed from 15% to 17%; from 15 to 30 min, acetonitrile was isocratic at 17%; from 30 to 60 min, acetonitrile linearly changed from 17% to 25%; from 60 to 90 min, acetonitrile linearly changed from 25% to 45%. The flow rate was at 15 mL/min with a 10 mL injection volume, The absorbance was monitored at 275 nm [14] to give 10 fractions (as shown in Fig. 1(A)) based on pre-HPLC detection peaks at different retention time. Each fraction was tested for antibacterial activity and the
438
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 1. Bioguided screening of antibacterial activity compounds from H. ascyron L. (A) Fractionation of the extract from H. ascyron L., the extract was fractionation into 10 fractions based on pre-HPLC detection peaks at different retention time. (B) and (C) Antibacterial activity of 10 fractions. Each fraction was tested for antibacterial activity and the bioactive fractions were combined to obtain fraction 8. This was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) eluting and affording compound 1 and compound 2. (D) Structure of compound 1 and compound 2, with rings named and positions numbered.
bioactive fractions were combined to obtain fraction 8. This was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) eluting and affording compound 1 and compound 2. The compound 1 and compound 2 were identified by MS and NMR. 2.7. HPLC analysis of fraction 8 Fraction 8 was separated on a Kromasil C18 column (250 mm 4.6 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The gradient of elution program was as follows: from 0 to 10 min, acetonitrile followed a linear change from 20% to 23%; from 10 to 30 min, acetonitrile linearly changed from 23% to 25%; from 30 min to analysis end, acetonitrile was isocratic at 25%. The flow rate was at 1.0 mL/min with a 10 mL injection volume, the absorbance was monitored at 275 nm. 2.8. Effect of fraction 8 on the growth of M. luteus cells M. luteus cells (105 CFU/mL) were inoculated into MuellerHinton broth at 200 mL/well in microtiter plates. Fraction 8 (0, 2.5, 5 mg/mL) was added to wells containing bacterial cells and incubated at 37 C. For each treatment, the growth of M. luteus was determined by automated absorbance measurements at 600 nm (Bioscreen C, Labsystems, Helsinki, Finland) at every hour.
2.9. Apoptosis analysis 2.9.1. Annexin V-FITC/PI staining M. luteus cells apoptosis was measured by Annexin V-FITC and PI staining method following the manufacturer’s instructions (Tianjin Sungene Biotech Co., Ltd., China). After exposure to fraction 8 (0, 2.5 mg/mL) at 37 C for 0, 2, 4 and 8 h, cells were then harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min and washed once with cold PBS. Then M. luteus cells were suspended in1 mL 1 Binding Buffer, centrifuged 8000 rpm for 10 min, and then remove the Binding Buffer. Resuspend cells in 1 mL 1 Binding Buffer, adjust cell concentration to 105 (CFU/mL). 100 mL of cells (105 CFU/mL) was added to each 1.5 mL microtubes. Then 5 mL of Annexin V-FITC was added to the 1.5 mL microtubes, respectively. Gently vortex each microtube and incubate for 10 min in room temperature, protected from light. 5 mL of PI solution was added to the 1.5 mL microtubes and incubated for 5 min in room temperature, protected from light. Finally, 500 mL of PBS was added to the 1.5 mL microtubes and vortex gently, respectively. The analysis of the cells was done by flow cytometry (FACScan; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were sorted into living, necrotic, early apoptotic, and late apoptotic cells. The relative ratio of early and late apoptotic cells were counted for further comparison. This assay was repeated more than three times.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
2.9.2. TUNEL assay for apoptotic DNA fragmentation The 30 -OH of the DNA fragments in apoptotic cells were labeled and stained by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling method using an apoptosis in situ detection kit (Promega, Germany), according to the manufacturer’s instructions. Briefly, after treatment with the fraction 8(0, 2.5 mg/mL) at 37 C for 0, 2, 4 and 8 h, apoptotic M. luteus cells (105 CFU/mL) were harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min and washed once with cold PBS, fixed with 4% paraformaldehyde for 25 min. Fixed cells were washed once with PBS and centrifuged at 8000 rpm for 10 min. Cells were permeabilized with 70% ethanol for at least 24 h at 20 C. Cells were centrifuged at 8000 rpm for 10 min and rewashed once with PBS. Excess liquid was removed by centrifuging at 8000 rpm for 10 min. Cells were covered with 200 mL of Equilibration Buffer for 5e10 min at room temperature, then centrifuged at 8000 rpm for 10 min. Cells were added 100 mL of rTdT incubation buffer and incubate at 37 C for 60 min with aluminum foil to protect from direct light. The reactions were terminated with 2X SSC for 15 min at room temperature. Cells were washed with fresh PBS for three times to remove unincorporated fluorescein-12-dUTP. Cells were stained with 40 ,6diamidino-2-phenylindole solution (1 mg/mL) for 15 min at room temperature in the dark. Cells were washed with PBS for three times. Fluorescent microscopy (Olympus, FV1000, Japan) was used to capture the image of the fluorescein-labeled TUNEL-positive cells. The cell nucleus was labeled in blue by DAPI (Invitrogen, Molecular Probes, Eugene, OR) and the nick-ends were labeled in green. A merge between the nucleus (blue) and nick-end (green) labeling showed up as purple.
2.9.3. DNA fragmentation assay DNA fragmentation is an important index of apoptosis. Usually DNA ladder was observed could determine the cell apoptosis. M. luteus cells were incubated with fraction 8 (0, 2.5 mg/mL) at 37 C after 0, 2, 4 and 8 h, 105 (CFU/mL) cells were then harvested to 1.5 mL microtubes. Cells were centrifuged at 8000 rpm for 10 min at 4 C and washed once with PBS. DNA was extracted using the Apoptosis, DNA Ladder Extraction Kit (Beyotime, China) as follows: 1 mL of the sample cracking liquid was added 5 mL of proteinase K (20 mg/mL) and blended. Then each of harvested samples was added 500 mL of cracking liquid with proteinase K. Cells were Vortex blended. Then all samples were digested at 50 C water bath for the night. Each sample was added 500 mL of Tris balanced phenol. Cells were intensely Vortex blended, and then centrifuged at 12,000g for 5 min at 4 C. Phenol and the intermediate phase were slowly sucked out. The rest of the water phase was extracted with the same volume Tris balance phenol again and centrifuged at 12,000g for 5 min at 4 C. Phenol and the intermediate phase were slowly sucked out again. The rest of the water phase was extracted with the same volume chloroform once again. About 300 mL of supernatant was slowly sucked out, then 60 mL of 10 mM ammonium acetate and 600 mL of anhydrous ethanol were added to each of sample, and mixed upside down several times. The samples were stored at 20 C for 1 h, and then centrifuged at 12,000g for 10 min at 4 C. The supernatant was abandoned. Each of samples was added 600 mL 70% ethanol, and mixed upside down about twice times. Samples were centrifuged at 12,000g for 10 at 4 C. The supernatant of all samples were carefully abandoned. Each of samples was immediately dissolved with 50e100 mL TE. The DNA was then electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide (EB) staining. The gel was photographed under ultraviolet light.
439
2.10. Detection of membrane potential (Djm) M. luteus cells were incubated with fraction 8 (0, 2.5 mg/mL) at 37 C after 0, 2, 4 and 8 h, 105 (CFU/mL) cells were then harvested by centrifuging (8000 rpm, 10 min) and washed twice with cold PBS. Each harvested sample was re-suspended in 500 mL of Rhodamine 123 (1 mM) for 30 min at 37 C, and washed twice with PBS. Then M. luteus cells were analyzed by FACScanto (BD Biosciences). 2.11. Statistical analysis All the data were expressed as mean ± SD (standard deviation, SD) and all the statistical analysis was performed using SPSS statistical software package (SPSS Inc., Chicago, IL, USA). Furthermore, p<0.05 was considered statistically significant. 3. Results 3.1. Bioassay-guided screening of the antibacterial ingredients of H. ascyron L. The antibacterial effects of H. ascyron L. extract were tested on several micro-organisms (E. coli, E. cloacae, K. pneumonia, S. aureus and M. luteus), and the results showed that its antimicrobial activity were mainly against Gram-positive bacteria, especially M. luteus (data not shown). The results are in agreement with previous studies of E. coli, E. cloacae and K. pneumoniae were more resistant to most plant extracts than Gram-positive bacteria [8]. Therefore, M. luteus was used as the indicator bacteria for the screening of the antibacterial activity compounds from H. ascyron L.. The extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) to afford 10 fractions based on preparative HPLC detection peaks at different retention time (as shown in Fig. 1A). Each fraction was then tested for the antibacterial activity. As demonstrated in Fig. 1B and C, except the mixture (the extract from H. ascyron L.), fraction 1, fraction 2, and fraction 10, all the other fractions exhibited varying levels of antibacterial activity against M. luteus at 10 mg/mL. Among 10 fractions, the fraction 8 showed the highest antibacterial ability, and the diameter of inhibition zone on the tested M. luteus was 14.53 ± 0.19 mm. Fraction 8 was subjected to an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm), and then afforded two main pure compounds (Compound 1 and Compound 2), as shown in Fig. 1D. Compound 1 was isolated as a yellow amorphous powder, with molecular formula C23H22O12 deduced from the [Mþ1]þ peak at m/ z 490 in the MS and supported by 1H, 13C and 13C DEPT spectra. The spectral data with those reported kaempferol 3-O-b-(200 -acetyl) galactopyranoside spectrum data are basically identical in literature [15]. Compound 1 was identified as kaempferol 3-O-b-(200 acetyl) galactopyranoside. Compound 2 was isolated as a yellow amorphous powder, with molecular formula C15H10O7 deduced from the [M1] þ peak at m/z 302 in the MS and supported by 1H, 13C and 13C DEPT spectra. The spectral data with those reported quercetin spectrum data are basically identical in literature [16]. Therefore, compound 2 was identified as quercetin. Based on the HPLC analysis of fraction 8 (Fig. 2), fraction 8 mainly included kaempferol 3-O-b-(200 -acetyl) galactopyranoside, quercetin and two impurity peaks (1 and 2), the content of kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin in the fraction 8 was above 90% (See supplementary material). Therefore, we considered kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin were the crucial elements of fraction 8 possessing antibacterial activity.
440
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 2. HPLC analysis of fraction 8. Fraction 8 was separated on a Kromasil C18 column (250 mm 4.6 mm i.d., 5 mm). Acetonitrile and water with 0.1% acetic acid were used as mobile phases. The flow rate was at 1.0 mL/min with a 10 mL injection volume, the absorbance was monitored at 275 nm.
3.2. MIC and MBC of fraction 8 To further detect the antibacterial ability of fraction 8, the MIC was tested on M. luteus cells in designed concentration (0.16 mg/ mL, 0.31 mg/mL, 0.63 mg/mL, 1.25 mg/mL, 2.5 mg/mL, 5 mg/mL) and showed a significant decrease in viable cells number was detected after treatment for 18 h. Besides, the anti-proliferative effect exhibited a dose-dependent manner, and the inhibitory rate increased to 100% when the dose of fraction 8 was over 5 mg/ mL, indicating that the MIC value was 5 mg/mL (Fig. 3A). Furthermore, the MBC was determined by re-inoculating 20 mL of each culture medium from the 5 mg/mL microtiter plate wells onto Mueller-Hinton agar plates. After being incubating at 37 C for 18 h, there was no bacteria growth on 5 mg/mL tested Mueller-Hinton agar plates, suggested that 5 mg/mL was also the MBC, implying that fraction 8 could be regarded as a bactericidal medicine. In addition, to further indicate the effect of antibacterial activity of fraction 8 based on the growth cycle of M. luteus, the growth cycle of M. luteus was also performed. As shown in Fig. 3B, the growth of M. luteus was significantly inhibited by fraction 8, especially from the exponential phase to the stationary growth phase. In addition, consistent with the MIC evaluated in the MTT assay, M. luteus cell was inhibited fully at 5 mg/mL. These results further confirmed the validity of MIC value. 3.3. Effects of fraction 8 on the apoptosis of M. luteus Previous studies have showed that apoptosis markers, including of phosphatidylserine exposure, chromosome condensation and DNA fragmentation, could be observed in bactericidal antibiotic treatment [17]. Therefore, we sought to determine whether apoptosis was involved in the antibacterial effect of fraction 8. To determine the apoptosis-stimulating effects of fraction 8 on M. luteus, the flow cytometry analysis using Annexin V-FITC/PI staining was performed. As shown in Fig. 4A, a small percentage of apoptotic cells was found in the negative group (1.7 ± 0.7%) after
Fig. 3. MIC and the growth cycle of M. luteus. (A) MIC value of fraction 8 on M. luteus. M. luteus cells were treated with fraction 8 from 0.16 mg/mL to 5.0 mg/mL for 18 h at 37 C. (B) Effect of fraction 8 on the growth cycle of M. luteus. M. luteus cells were treated with fraction 8 (0 mg/mL, 2.5 mg/mL, 5 mg/mL) for 8 h at 37 C. For each treatment, the growth of M. luteus was determined by automated absorbance measurements at 600 nm, detected absorption value every hour.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
441
Fig. 4. Apoptosis analysis. (A) Results of annexin V/PI double stain assay. The apoptosis rate of M. luteus cells treated with fraction 8 (0 mg/mL, 2.5 mg/mL) for 0, 2, 4 and 8 h was detected by the flow cytometry using fluorescently-labeled annexin V/PI double stain. (B) Results of TUNEL assays. The apoptotic status of M. luteus cells treated by fraction 8 (0 mg/ mL, 2.5 mg/mL) for 0, 2, 4 and 8 h was determined by TUNEL assay (green channel). DAPI (blue channel) is used to locate the nuclei of the cells. (C) The gel electrophoresis image obtained after DNA fragmentation assay for apoptosis detection. M. luteus cells were cultured at 37 C for 0, 2, 4 and 8 h in the presence or absence of the indicated concentration of fraction 8. DNA was isolated and visualized on a 1.0% agarose gel stained with ethidium bromide. From left lanes are: Lane M: Marker; Lane 1: 0 h (CON); Lane 2: 2 h (CON); Lane 3: 4 h (CON); Lane 4:8 h (CON); Lane 5: 0 h (2.5 mg/mL); Lane 6: 2 h (2.5 mg/mL); Lane 7: 4 h (2.5 mg/mL); Lane 8:8 h (2.5 mg/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubating 8 h. However, the percentage of apoptotic cells significantly increased from 0.0 ± 0.2% to 21.3 ± 0.8% after being treated with 2.5 mg/mL of fraction 8 from o hour to 8 h. The results implied that fraction 8 could time-dependently induce M. luteus cells death through apoptosis pathway. To further define the mechanism underlying fraction 8mediated cell death, M. luteus cells were treated with fraction 8 (0, 2.5 mg/mL) for 0, 2, 4 and 8 h and DNA fragmentation was measured using the TUNEL assay. Fraction 8 treatment activated apoptosis in M. luteus cells, as evidenced by increased fluorescence
intensity, which was detected by labeling DNA single-strand breaks with dUTP-fluorescein at the 30 eOH ends of the nuclei. As demonstration in Fig. 4B, the green fluorescence intensity was almost undetectable in the control cells (100%), whereas the fluorescence intensity increased significantly in a time-dependent manner in the fraction 8-treated M. luteus cells. The results demonstrate that fraction 8 effectively induced apoptosis in M. luteus cells. Furthermore, when apoptosis occurs, DNA is cleaved into oligonucleosomal fragments of 180e200 bp [18]. In the present study,
442
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
Fig. 5. Effects of fraction 8 on membrane potential. Membrane potential (Djm) was analyzed using Rho123. The M. luteus cells were treated with the designed concentration of fraction 8 (0 mg/mL, 2.5 mg/mL), at 37 C for 0, 2, 4 and 8 h. M. luteus cells were incubated with Rho123 (1 mM) for 30 min and then harvested for analysis by flow cytometry, M1 reflects the reduction of Djm.
as indicated by the agarose gel electrophoresis, exposure of M. luteus cells to the fraction 8 at concentrations of 2.5 mg/mL for 2, 4 and 8 h could result in marked DNA fragmentation, whereas the control (untreated with fraction 8) did not exhibit any DNA ladder, as shown in Fig. 4C. 3.4. Effects of fraction 8 on membrane-mediated apoptosis pathways To test whether fraction 8 could induce apoptosis of M. luteus through destroying the cell membrane, we measured the disruption of cell membrane potential (Djm) using Rho123 stain. As shown in Fig. 5, when M. luteus cells were treated with 2.5 mg/mL of fraction 8 for indicated periods, indicating the collapse of Djm.
Fig. 6. Potential antibacterial mechanism of fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) attached to the membrane surface from electrostatic attraction, and resulted in the disturbance of the target membrane, as well as structural change and fracture of the membrane. Fraction 8 (kaempferol 3-O-b(200 -acetyl) galactopyranoside couple with quercetin) entered the cytoplasm through membrane pores, and subsequently, two parallel reactions took place, namely, increase of the level of ROS and interaction with genome DNA, which caused cell death.
The observations indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. 4. Discussion Among 484 Hypericum L. (Guttiferae/Hypericaceae) species, widespread in warm temperate areas throughout the world, only H. perforatum is widely used in official medicine. H. sampsoni, H. ascyron L., H. foliosum, H. geminiflorum and H. scabrum containing considerable amounts of other acylphloroglucinol derivatives have the potential to demonstrate antibacterial and cytotoxic activity [8]. In our present investigation, the results showed that H. ascyron L. extract possessed antibacterial effect and mainly inhibited the proliferation of Gram-positive bacteria, especially M. luteus. A possible explanation for these observations may be associated with the significant structural differences between Gram-positive and Gram-negative bacterial cells walls, as the latter possess an outer membrane and a unique periplasmic space [19]. The resistance of Gram-negative towards antibacterial substances is due to its outer hydrophilic membrane surface that is rich in lipopolysaccharide molecules and thus creates a barrier to numerous antibiotic molecules. This composition makes the cell wall impermeable to lipophilic solutes, and the porins in the cell wall do not allow the penetration of high molecular mass hydrophilic solutes, with an exclusion limit of about 600 Da [20]. Further, enzymes in the periplasmic space are capable of breaking down molecules introduced from the outside. Gram-positive bacteria (M. luteus) does not have such an outer membrane and cell wall structure. Antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane resulting in leakage and coagulation of the cytoplasm [21]. In addition, the lack of an outer lipopolysaccharide membrane in the Gram-positive bacteria could allow increased permeability of Hypericum metabolites into cells [22,23]. Therefore, the extract of H. ascyron L. displayed obvious antibacterial activity to Gram-positive bacteria, especially M. luteus.
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
The discovery of active compounds from natural products is a time-consuming process. An effective strategy developed recently is the use of pre-fractionated natural product libraries, which not only reduces the background that is a problem with crude libraries, but also speeds the purification of active compounds [24]. The method has two steps: the preparation of fractions from crude extracts and the isolation of individual compounds from the active fractions. In order to cut down the isolated and purified time, in our present investigation, the extract was separated on an Agela Akasil C18 column (250 mm 30 mm i.d., 5 mm) to afford 10 fractions based on preparative HPLC detection peaks at different retention time (Fig. 1A). Each fraction was then tested on M. luteus (Fig. 1B and C), except the mixture (the extract from H. ascyron L.), fraction 1, fraction 2, and fraction 10, all the other fractions exhibited varying levels of antibacterial activity against M. luteus at 10 mg/mL. Among 10 fractions, the fraction 8 showed the highest antibacterial ability. We obtained the active fraction in a short time. The MIC and MBC values of fraction 8 on M. luteus were 5 mg/mL. In addition, we determined that the effect of antibacterial activity of fraction 8 (2.5, 5 mg/mL) based on the growth cycle of M. luteus, found the growth of M. luteus was significantly inhibited, especially from the exponential phase to the stationary growth phase. These results implied that fraction 8 could be regarded as a bactericidal medicine. We isolated and obtained two key antibacterial activity compounds by identified as kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin. The content of the two compounds in fraction 8 was above 90%. We considered that kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin of fraction 8 played a fatal role in antibacterial action. Through the literature retrieval, we found that kaempferol 3-O-b-(200 -acetyl) galactopyranoside was described for the first time from Clusiaceae and first reported in antibacterial activity. Previous studies showed that kaempferol 3-O-b-(200 acetyl) galactopyranoside was confirmed in aconitum paniculatum [15] and possessed certain antioxidation [25]. Quercetin is one of a group of over 4000 naturally available plant phenolics whose isolation and biological identification were first described by SzentGyorgyi in 1936 [26]. In addition, quercetin was identified in H. ascyron in 1980 [16]. Various studies have shown that the pharmacological effects of quercetin include protective effects against cancer, cardiovascular diseases, ischemic injuries, and antiinflammatory, immunemodulatory, gastroprotective activities, antioxidant, antibacterial, as well as enhancement of stress resistance and extension of the life span of various organisms from bacteria to vertebrates [27-30]. Flavonoids of structure factors play an important role in biological activity. Flavonoids molecules of structureeactivity relationship studies have shown that the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5 and 7 in the A ring are the first group of antibacterial activity, followed by the hydroxyl at position 3 in the C ring for antibacterial activity of such compounds. The hydroxyl at position 30 and 40 in the B ring also have certain antibacterial activity [31,32]. As shown in Fig. 1D, quercetin includes the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7, 3, 30 and 40 . Kaempferol 3-O-b-(200 -acetyl) galactopyranoside possesses the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7 and 40 . These results are in agreement with previous studies of flavonoid molecules possess the characteristic of antibacterial activity. Apoptosis is commonly known as physiological programmed cell death and is different from necrosis [33]. Cell apoptosis involves the biological regulation of the numbers and vital activities of cells, and is an important metabolic process. Apoptosis occurs through the activation of cellintrinsic suicide machinery [34]. One key mechanism involved in the function of antibacterial drugs is the activation of the apoptotic pathway. The results of Annexin V/PI
443
double staining demonstrated the apoptosis rate of M. luteus cells was increased with the extension of the exposure time to fraction 8, in a time dependent manner (Fig. 4A). The apoptosis effects of fraction 8 on M. luteus cells were further confirmed via TUNEL assay (Fig. 4B). In addition, an important feature of apoptosis is the fragmentation of genomic DNA into integer multiples of 180e200 bp, which results in a characteristic ladder upon agarose gel electrophoresis. As shown in Fig. 4C, the experiment results showed that the DNA of M. luteus cells presented typical ladder-like pattern after treatment with fraction 8 at 2.5 mg/mL, in a time dependent manner, whereas the control indicated no evident DNA ladder. These results demonstrated that fraction 8 could induce the death of M. luteus cells by apoptosis pathway. Recent research has revealed evidence of apoptotic death in bacteria. Apoptosis-like death in E. coli was described by Dwyer et al.. In addition to membrane depolarization and DNA fragmentation, they found that this apoptosis like death is also characterized by phosphatidylserine exposure to the outer leaflet of the plasma membrane and chromosome condensation [17]. Apoptosis-like death is induced not only by DNA-damaging agents but also by various antibiotics, including spectinomycin, ampicillin, and gentamicin. The results may differ from ours because we firstly found the compounds from the nature product could induce M. luteus cells apoptosis death. Reactive oxygen species (ROS) include free radicals such as superoxide (O-2), hydroxyl radical (OH-), and non-radical derivatives of oxygen such as H2O2, which is mainly derived from the respiratory chain in mitochondria [35]. ROS generation and oxygen stress have been reported as common anti-bactericidal episodes caused by many antibacterial compounds [36]. ROS generation and disruption of the membrance potential (DJm) contributed to drug induced apoptosis [37]. In our present work, we found that the membrane potential of M. luteus cells treated with fraction 8 was remarkably lost (Fig. 5), demonstrating that fraction 8 induced irreversible apoptosis phenomenon by membrane-mediated apoptosis. In addition, Mirzoeva and colleagues showed that quercetin could cause an increase in permeability of the inner bacterial membrane and a dissipation of the membrane potential [38]. Therefore, we speculate the antibacterial mechanism of fraction 8 as shown in Fig. 6. Potential antibacterial mechanism of fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) attached to the membrane surface from electrostatic attraction, and resulted in the disturbance of the target membrane, as well as structural change and fracture of the membrane. Fraction 8 (kaempferol 3-O-b-(200 -acetyl) galactopyranoside couple with quercetin) entered the cytoplasm through membrane pores, and subsequently, two parallel reactions took place, namely, increase of the level of ROS and DNA fragmentation, which caused cell death. 5. Conclusion In summary, through screening we identified kaempferol 3-O-b(200 -acetyl) galactopyranoside and quercetin, as potential antibacterial compounds in H. ascyron L. Kaempferol 3-O-b-(200 -acetyl) galactopyranoside is described for the first time from Clusiaceae and first reported in antibacterial activity. Using TUNEL labeling, Annexin V-FITC/PI and DNA gel electrophoresis, we found that bacteria cell death with features of apoptosis after exposure to the antibacterial activity from H. ascyron L. at moderate concentration. We report for the first time an assessment and possible mechanism of kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin could induce cells death by apoptosis pathway. In addition, we found kaempferol 3-O-b-(200 -acetyl) galactopyranoside and quercetin could disrupt cells membrane. Therefore, we speculated the antibacterial activity compounds induced irreversible apoptosis phenomenon by membrane-mediated apoptosis. Our data might
444
X.-M. Li et al. / European Journal of Medicinal Chemistry 96 (2015) 436e444
provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs. H. ascyron L. is a promising antibacterial species against antibiotic multi-resistant bacteria because of its natural occurrence, wide distribution in China and Japan.
[14]
[15] [16]
Declaration of interest [17]
The authors report no declarations of interest. [18]
Acknowledgments
[19]
This work was financially supported by National Natural Science Foundation of China (31171303, 31170541, 31171297, 31270837), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1166), State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (K2013101), Natural Science Foundation of Tianjin City (13JCZDJC29400).
[21]
Appendix A. Supplementary data
[23]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.04.035.
[24]
[20]
[22]
[25]
References [1] C. Iris, A. Zampini Marta, I. Vattuone Maria, Antibacterial activity of Zuccagnia punctata Cav. ethanolic extracts, J. Ethnopharmacol. 102 (2005) 450e456. [2] A. Marchese, G.C. Shito, Resistance patterns of lower respiratory tract pathogens in Europe, Int. J. Antimicrob. Ag. 16 (2001) 25e29. [3] K. Poole, Overcoming antimicrobial resistance by targeting resistance mechanisms, J. Pharmand. Pharmacol. 53 (2001) 283e284. [4] R. Dall'agnol, A. Ferraz, A.P. Bernardi, D. Albring, C. Nor, E.E.S. Schapoval, Bioassayguided isolation of antimicrobial benzopyrans and phloroglucinol derivatives from Hypericum species, Phytother. Res. 19 (2005) 291e293. [5] S. Gibbons, Anti-staphylococcal plant natural products, Nat. Prod. Rep. 21 (2004) 263e277. [6] P.K. Mukherjee, G.S. Saritha, B. Suresh, Antimicrobial potential of two different Hypericum species available in India, Phytother. Res. 16 (2002) 692e695. [7] S.A. Sarkisiana, M.J. Janssenb, H. Mattab, G.E. Henryb, K.L. LaPlantea, D.C. Rowleya, Inhibition of bacterial growth and biofilm production by constituents from Hypericum spp, Phytother. Res. 26 (2012) 1012e1016. [8] G. Stojanovi, A. Dorevic, A. Smelcerovic, Do other Hypericum species have medical potential as St. John’s Wort (Hypericum perforatum)? Curr. Med. Chem. 20 (2013) 2273e2295. [9] JiangSu New Medical College, Dictionary of Chinese Crude Drugs, Scientific Technological Publishers, 1977, p. 1002. [10] J. Wang, N.L. Wang, X.S. Yao, Inhibitory activity of Chinese herbal medicines toward histaminerelease from mast cells and nitric oxide production by macrophage-like cell line RAW264.7, J. Nat. Med. 60 (2006) 73e77. [11] J.M. Lv, W. Jia, C.Y. Li, The anti-inflammatory and analgesic effects of guttiferae hypericum ascyron linn, Pract. Clin. J. Integr. Tradit. Chin. West. Med. 8 (2008) 87e89. [12] U.K. Sang, S.U. Chon, B.G. Heo, Y.S. Park, S. Gorinstein, Total phenolics level, antioxidant activities and cytotoxicity of young sprouts of some traditional Korean salad plants, Plant. Food. Hum. Nutr. 64 (2009) 25e31. [13] W.Y. Kang, Y.L. Song, L. Zhang, a-Glucosidase inhibitory and antioxidant
[26] [27] [28]
[29]
[30] [31] [32] [33]
[34] [35]
[36] [37]
[38]
properties and antidiabetic activity of Hypericum ascyron L, Med. Chem. Res. 20 (2011) 809e816. X.M. Li, X.G. Luo, Li K. M N, W. Li, D.Y. Ma, et al., Quality and antitumour activity evaluation of extract of Hypericum ascyron, Biomed. Chromatogr. (2014), http://dx.doi.org/10.1002/bmc.3169. G. Fico, A. Braca, A.R. Bilia, F. Tome, I. Morelli, Flavonol glycosides from the flowers of Aconitum paniculatum, J. Nat. Prod. 63 (2000) 1563e1565. Z.Q. Wang, X.R. Wang, Studies on the active principles of Hong Han Lian (Hypericum ascyron L.), Acta. Pharm. Sin. 6 (1980) 365e367. D.J. Dwyer, D.M. Camacho, M.A. Kohanski, J.M. Callura, J.J. Collins, Antibioticinduced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis, Mol. Cell. 46 (2012) 561e572. A. Saraste, K. Pulkki, Morphologic and biochemical hallmarks of apoptosis, Cardiovasc. Res. 99 (2000) 528e537. C.F. Duffy, R.F. Power, Antioxidant and antimicrobial properties of some Chinese plant extracts, Int. J. Antimicrob. Ag. 17 (2001) 527e529. -Pie boji, N. Eze, A.N. Djintchui, B. Ngameni, N. Tsabang, J. Gangoue D.E. Pegnyemb, et al., The in-vitro antimicrobial activity of some traditionally used medicinal plants against beta-lactam-resistant bacteria, J. Infect. Dev. Ctries. 3 (2009) 671e680. Y. Gao, M.J. Van belkum, M.E. Stiles, The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C, Appl. Environ. Microbiol. 65 (1999) 4329e4333. F. Tian, B. Li, B. Ji, J. Yang, G. Zhang, Y. Chen, et al., Antioxidant and antimicrobial activities of consecutive extracts from Galla chinensis: the polarity affects the bioactivities, Food. Chem. 113 (2009) 173e179. S.A. Sarkisiana, M.J. Janssenb, H. Mattab, G.E. Henryb, K.L. LaPlantea, D. Rowleya, Inhibition of bacterial growth and biofilm production by Constituents from Hypericum spp, Phytother. Res. 26 (2012) 1012e1016. F.E. Koehn, New strategies and methods in the discovery of natural product anti-infective agents: the mannopeptimycins, J. Med. Chem. 51 (2008) 2613e2617. , N.D. Tommasi, Antioxidant A. Braca, G. Fico, I. Morelli, F.D. Simone, F. Tome and free radical scavenging activity of flavonol glycosides from different aconitum species, J. Ethnopharmacol. 86 (2003) 63e67. J.B. Harborne, Nature, distribution and function of plant flavonoids, Prog. Clin. Biol. Res. 213 (1986) 15e24. M.Y. Li, T.Y. Zhu, Quercetin in a Lotus leaves extract may be responsible for antibacterial activity, Arch. Pharm. Res. 31 (2008) 640e644. T.B. Ng, F. Liu, Y. Lu, Antioxidant activity of compounds from the medicinal herb aster tataricus, Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 136 (2003) 109e115. M. Bathori, I. Zupko, A. Hunyadi, Monitoring the antioxidant activity of extracts originated from various Serratula species and isolation of flavonoids from Serratula coronata, Fitoterapia 75 (2004) 162e167. C. Kanadaswami, L.T. Lee, P.P. Lee, The antitumor activities of flavonoids, In. Vivo 19 (2005) 895e909. J.T. Zhang, Y.D. Song, CNDO/2 Study on flavonoids 13, J. Shanxi. Agr. Univ., 1993, pp. 134e140. Y.H. Jiang, Study on Antimicrobial Components in Lotus Leaves, 2007. Y.F. Sun, C.K. Song, H. Vernstein, F. Unger, Z.S. Liang, Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway, Food. Chem. 138 (2013) 1998e2007. D.R. Green, G.I. Evan, A matter of life and death, Cancer. Cell. 1 (2002) 19e30. A. Erental, Z. Kalderon, A. Saada, Y. Smith, H. Engelberg-Kulkaa, Apoptosis-like death, an Extreme SOS Response in Escherichia coli, Mbio 5 (2014) 1426e1440. J. Nordberg, E.S.J. Arner, Reactive oxygen species, antioxidants, and the mammalian thioredoxin system, Free. Radic. Bio. Med. 31 (2001) 1287e1312. R.S. Ye, H.Y. Xu, C.X. Wan, S.S. Peng, L.J. Wang, H. Xu, et al., Antibacterial activity and mechanism of action of 3-poly-L-lysine, Biochem. Biophys. Res. Co. 439 (2013) 148e153. O.K. Mirzoeva, R.N. Grishanin, P.C. Calder, Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria, Microbiol. Res. 152 (1997) 239e246.