Antimicrobial Mechanism of Saponin from Sapindus mukorossi against Escherichia c

Faling LEI1, Lei XIAO1, Chunyan YAO2*, Zengli WANG

1. Infinitus (China) Company Ltd., Xinhui 529156, China; 2. Zhejiang Academy of Medical Sciences, Hangzhou 310013, China; 3. Healthy Star Bio-Tech R&D Ltd., Shanghai 200335, China

Abstract [Objectives] To investigate the antimicrobial activity and mechanism for saponin from Sapindus mukorossi against Escherichia coli. [Methods] E. coli was used as the test bacteria, and the antimicrobial effect of Sapindus saponin was studied by the minimum inhibitory concentration method. The antimicrobial mechanism of Sapindus saponin was studied in terms of growth curves, membrane potential, activity of cells, cell surface morphology and cell internal structure. [Results] The results indicate that Sapindus saponin can inhibit the growth of E. coli, the minimum inhibitory concentration is 5 mg/mL and has a significant inhibitory effect on the growth of E. coli. After treated with Sapindus saponin, the membrane potential of E. coli increased by 16.7, 17.3 and 19.1 times after exposition to 0.5, 1, 2 MIC Sapindus saponin respectively. PI staining results show that cell viability decreased and permeability of cell membranes increased. The results of SEM and TEM further corroborate the membrane damage, the cell was damaged after exposition to Sapindus saponin. [Conclusions] The antimicrobial mechanism of Sapindus saponin were to changed membrane potential of cell, and damage the cell membrane structures.

Key words Sapindus mukorossi, Saponin, Escherichia coli, Antimicrobial mechanism

1 Introduction

SapindusmukorossiGaertn, also known as soapnut and soapberry, is a deciduous tall tree generally growing in forests below 1 000 m altitude in China, as well as in the lowland area of Taiwan of China, India, and Japan[1]. The fruits ofS.mukorossicalled "wu-huan-zi" in China, means non-illness fruit[2], have been used as expectorant and natural surfactants in ancient China[3]. Pharmacological properties ofS.mukorossiare explored to show that the plants with effects of antimicrobial, anti-inflammatory, antitumor, antihypertensive, antihyperglycemic, insecticidal and reduce the impact of heavy metals in the soil[4-10]. Furthermore, the fruit pericarp is widely used as a natural detergent and its extract is commercially utilized as a foam stabilizing and emulsifying agent in cleansers, shampoos and cosmetics, because the pericarp is abundant in saponin of high surface activity[11].

As a natural antimicrobial ingredient, Sapindus saponin has been reported to have good antimicrobial activity. Antimicrobial activities of leaf and fruit extracts of this plant were also evaluated on some bacteria and fungus[12]. As a potential antimicrobial substance, Sapindus saponin possess a series of advantages including being found in a wide range of sources, easy to obtain, and relatively low cost. At present, the study on antimicrobial effect on Sapindus saponin mainly stays at the stage of bacteriostatic effect, and how it is to achieve antimicrobial function is still unclear, it is very necessary to study its antimicrobial mechanism.

2 Materials and methods

2.1 Materials and instruments

2.1.1Materials: Dry fruit pericarps ofS.mukorossiwere collected on the Zhangpu Danshan Seedling field in Fujian Province, China.EscherichiacoliATCC8739, was preserved in the microbiological lab, Zhejiang Academy of Medical Sciences (Hangzhou, China). Mueller-Hinton broth was purchased from Qingdao Hope Bio-Tcehnology Co., Ltd. Propidium iodide (PI), DiBAC4(3) were obtained from Beyotime Biotechnology. PBS, glutaraldehyde, uranyl acetate and lead citrate were purchased from Sinopharm Chemical Reagent Co., Ltd.

2.1.2Instruments: SW-CJ-1G purification workbench (Suzhou Purification Equipment Co., Ltd.), THZ-300 shaking incubator (Shanghai Yiheng Scientific Instruments Co., Ltd.), YXQ-75G vertical autoclave (Shanghai Boxun Medical Biological Instrument Corp), UV-3802 UV/Vis Spectro-photometer (Unico (Shanghai) Instrument Co., Ltd.), Multiskan FC microplate readers (Thermo Fisher Scientific), E80I fluorescence microscopy (Nikon), Regulus 8100 scanning electron microscopes (Hitachi, Ltd.), H-7650 transmission electron microscopy (Hitachi, Ltd.).

2.2 Methods

2.2.1Extraction and isolation of theS.mukorossiextract.S.mukorossiextract (SE) was prepared in accordance with the following method: the pericarp powder ofS.mukorossi(1.0 kg) were extracted with nine times their weight of 70% ethanol at 60 ℃, and first extraction for 3 h, second extraction for 3 h, third extraction for 2 h. The extracted solution was filtered and combined together, then solvent was removed under reduced pressure to generate a crude extract. The extract was subjected to D101 macroporous resin and eluted successively with aqueous ethanol gradient system (0%, 30%, 70%). The 70% ethanol fractions were combined and removed ethanol under reduced pressure, the content of total saponins of SE determined by UV was 160 mg/mL.

2.2.2Determination of the minimum inhibitory concentration. The antimicrobial activity againstE.coliof SE was determined by minimum inhibition concentration (MIC) values, The MIC was determined according to methods previously reported with certain modifications[13]. Tests were performed after reactivating the bacteria in Mueller-Hinton broth for subsequent culture for 24 h at 37 ℃. Mixed the bacterial suspension adjusted to 1×106CFU/mL, and diluted the SE with sterile liquid medium. Took 96-well microtiter plate format, added 50 μL SE with different density and 50 μL of each antibiotic dilution into the respective well, pipetted 100 μL of broth in the sterility control well and 50 μL in the growth control well. Inoculated each well containing the antibiotic solution and the growth control well with 50 μL of the bacterial suspension, obtained the final desired inoculum of 5×105CFU/mL, incubated microtiter plate at 37 ℃ for 16-20 h. The MIC is defined as the lowest concentration of the antimicrobial agent that inhibits visible growth of the tested isolate as observed with the unaided eye.

2.2.3Growth curves. A freshly grown axenic culture of (1×105CFU/mL)E.coliwas inoculated into flasks containing the liquid nutrient medium, SE was added to three of the cultures to obtain final concentration of 0.5, 1 and 2 MIC. The control culture include liquid nutrient medium and without SE. Bacteria were further cultured at 37 ℃ at 150 rpm in an orbital shaking incubator under aerobic conditions, and cell growth was monitored by reading theOD600nm value at 6 h interval[14].

2.2.4Fluorometric assay for membrane potential.E.colicells were grown in Mueller-Hinton broth at 37 ℃ to an optical density at 600 nm of 0.5 (approximately 1×107CFU/mL). Bacteria were mixed with final concentration 0, 0.5, 1, 2 MIC SE respectively, and incubated at 37 ℃ for 30 min. Then, 1 μmol/L membrane potential-sensitive fluorescent probe DiBAC4(3) was added followed by the addition of extract. After 5 min, fluorescence was measured using the spectrofluorometer at the excitation and emission wavelength of 492 and 515 nm, respectively[15].

2.2.5Cell viability assay. Cell viability was determined by PI with reference to the method of Yan[16]. The dye exhibits significantly enhanced fluorescence on binding to intracellular nucleic acids. Logarithmic phase bacteria were harvested by centrifugation at 3 500 rpm for 10 min at 4 ℃, washed three times with PBS (pH 7.4) and adjusted the bacteria concentration of 1×107CFU/mL. Bacteria were mixed with final concentration 0.5, 1, 2 MIC SE respectively, the control culture include liquid nutrient medium and without SE, and incubated at 37 ℃ for 6 h. PI was added into each well to a final concentration of 2.0 μg/mL, and kept at room temperature for 30 min in the dark. Take 10 μL stained bacterial suspensions were deposited onto glass slides and covered with coverslips. Bacteria were marked and photographed in fluorescence microscopy, excitation and emission wavelengths were set at 535 and 615 nm, respectively.

2.2.6Scanning Electron Microscopy. The bacteria strains were cultured overnight in an orbital shaker set at 37 ℃ and 200 rpm, then bacterial cultures were adjusted to 5×105CFU/mL. Bacteria were mixed with final concentration 0.5, 1, 2 MIC SE respectively, the control culture include liquid nutrient medium and without SE, all samples incubated at 37 ℃ for 6 h. After treatment, the indicators were collected by centrifugation and washed by PBS (pH 7.2), bacteria cultures were incubated with 2.5% glutaraldehyde overnight at 4 ℃. Following three washes with PBS, the samples were hydrated with ascending concentration of ethanol (25% for 5 min, 50% for 10 min, 75% for 10 min and three changes of 100% ethanol for 10 min each). Subsequently, the cells were dried by CO2for 4 h and coated with gold. The samples were examined with a SU8100 scanning electron microscopy[17-18].

2.2.7Transmission Electron Microscopy. The bacteria strains were cultured overnight in an orbital shaker set at 37 ℃ and 200 rpm, then bacterial cultures were adjusted to 5×105CFU/mL. Bacteria were mixed with final concentration, 0.5, 1, 2 MIC SE respectively, the control culture include liquid nutrient medium and without SE, incubated at 37 ℃ for 6 h. After treatment, the indicators were collected by centrifugation and washed by PBS (pH 7.2), cells were fixed with 2.5% glutaraldehyde overnight at 4 ℃. Then cells were post-fixed in 1% osmium tetroxide at 4 ℃ for 2 h. After washed again, the cells were dehydrated in a series of ethanol solutions and permeated in white resin. Embedding was done at 60 ℃ for 48 h. Approximate 70 nm thin sections were prepared on copper grids and stained with 2% uranyl acetate and lead citrate. Ultrastructure observation was performed on a transmission electron microscope[18].

3 Results and analysis

3.1 Minimum inhibitory concentrationBroth microdilution was used for minimum inhibitory concentration test, the concentration of the test solution is in the range of 80-0.001 mg/mL, SE exhibits inhibitory activity againstE.coli, the MIC is 5 mg/mL.

3.2 Effects of SE onE.coligrowth curvesThe growth curve ofE.coliis shown in Fig.1. Compare with the control group, the growth ofE.coliwas significantly slowed down after the action of 0.5 MIC SE, and with an increase in its concentration, there was an increased inhibition in the growth ofE.coli. When growth for 0-12 h,E.coligrew rapidly in the control group, and only slightly increased inE.coliunder MIC and 2 MIC conditions. When growth for 24-30 h, the culture ofE.colicontinued to grow, but the bacteria in the SE group had enter the decline period. Thus, SE has a significant inhibitory effect on the growth ofE.coli.

3.3 Fluorometric assay for membrane potentialDiBAC4(3) is a fluorescent dye used for monitoring change in the membrane potential. The fluorescence of the dye is enhanced when the dye enters the cell membrane as a result of membrane depolarization. The fluorescent intensity of 0.5, 1 and 2 MIC increased by 16.7, 17.3 and 19.1 times (Fig.2), respectively, when compared with untreated cells. Results indicate that SE treatment caused rapid depolarization.

Fig.1 Effects of SE on the growth curve ofEscherichiacoli

Fig.2 Membrane potentials ofEscherichiacolitreated by SE

3.4 Cell viability assayPropidium iodide is a fluorescent intercalating agent that can be used to stain cells. PI is not membrane-permeable, making it useful to confirm cell viability based on membrane integrity. The results of PI staining ofE.coliunder different treatments are shown in Fig 3. Bacterial cells treated without the addition of SE (Fig.3A) showed scarce or even no fluorescence. Fluorescence began to appear after treatment with 0.5 MIC of SE for 6 h, and the fluorescence intensity increased obviously with the increase of the concentration of SE (Fig.3C and Fig.3D), indicating that SE caused damage toE.colicell membrane and eventually resulted in bacteria died.

Note: A: Control, B: 0.5 MIC, C: 1 MIC, D: 2 MIC.

Fig.3 Fluorescence microscopy images ofEscherichiacolirespectively treated with different SE concentrations

3.5 Scanning Electron MicroscopyScanning electron microscopy (SEM) was used to observe the surface morphology change ofE.coli, bacteria were treated with SE at concentrations of 0.5, 1 and 2 MIC, respectively, untreated bacteria were set as control. The results were shown in Fig.5, the control cell (Fig.4A) exhibited distinctive features characterized by regular rod-shaped and intact surface, when treated with 0.5 MIC SE the cells were significantly changed, and became irregular and shriveled (Fig.4B). The cell walls of the tested bacteria treated at 1 and 2 MIC level showed more severe morphological destruction, cellular damage and cell contents leakage.

Note: A: Control, B: 0.5 MIC, C: 1 MIC, D: 2 MIC.

Fig.4 SEM images of the morphology ofEscherichiacolitreated by SE

3.6 Transmission Electron MicroscopyTransmission electron microscopy (TEM) images ofE.coliexhibited in Fig.5, the cell wall and membrane ofE.coliintact and cytoplasmic homogeneous without SE treatment (Fig.5A). After treatment with 0.5 MIC SE, the structures of cell wall and membrane changed，separation between them (Fig.5B). When the concentration of SE increases to 1 MIC, the cytoplasm is not uniform, and the cavity of the cells appears (Fig.5C). Exposure to 2 MIC led to complete disintegration of the cells, together with the disappearance of organelle organization, cellular lysis (Fig.5D).

4 Discussions

Plant materials have been regarded as ample sources of novel biomolecules with a broad spectrum of biological and pharmacological properties. Bioactive compounds from many plants with antimicrobial activity have been identified[19]. A variety of compounds including saponins, flavonoids, polyphenols are isolated from theS.mukorossi, and saponin has become a major research object because of its various biological activities. In our study, Sapindus saponin has antibacterial activity and its MIC is 0.5 mg/mL, it can completely inhibit the proliferation ofE.coliduring the growth cycle, therefore, we conducted further experiments to determine its mechanism of inhibition. Membrane potential is measured as the difference in electric potential between the interior and the exterior of a biological cell[20]. Depolarization can lead to abnormal cell metabolism and affect the production of ATP. In this experiment, the addition of SE causes the depolarization ofE.coli

Note: A: Control, B: 0.5 MIC, C: 1 MIC, D: 2 MIC.

Fig.5 TEM images ofEscherichiacolitreated by SE

cell membrane leads to abnormal cellular metabolic activity and bacterial death. Duan[21]reported that 1,3,8-trihydroxy-4-chloro-6-methyl-anthraquinone could efficiently induce the depolarization of bacterial cell membranes, which led to an increase in membrane permeability in bothS.aureusandB.cereuscells. Cell membrane is an important barrier structure to ensure that cells complete various physiological functions, and provides a relatively stable internal environment for cell growth and metabolism. If the fungal cell membrane is damaged and damaged, it will affect the normal growth and reproduction of the bacteria, and when the damage is serious, the bacteria will die. Propidium iodide is a membrane impermeable dye, which only penetrates cells through damaged membranes, the fluorescence intensity of cell increased obviously when treated with SE. Lee[22]reported that the fluorescence ofC.albicanscells increased by 76.20% when treated with curcumin at its MIC when compared with the negative control, indicating damage to the membrane structure. The results of SEM and TEM can also corroborate the membrane damage, we observed some important structural changes inE.colicells after exposition to SE, such as cell wall irregularities, cellular damage, disappearance of organelle organization and cell lysis.

In conclusion, the present study demonstrated SE exhibited potent antimicrobial effect. The antimicrobial activity results from its ability to changed pH and membrane potential of cell, and damage the cell membrane structures. The SE is a promising natural antibacterial agent.