Enhanced antimicrobial activity of silver nanoparticles‐Lonicera Japonica Thunb combo

Abstract

Silver metals have long been known to possess antimicrobial properties. Recently, even the nanoparticle version of silver (AgNPs) has also been established as antimicrobials. In this study AgNPs were combined with extracts of the medicinal plant Chinese honeysuckle, Lonicera japonica Thunb. The antimicrobial activity of the AgNPs‐herb was tested against pathogenic Escherichia coli CMCC44113. Using different AgNPs or herb (honeysuckle water extract or HWE) ratios in the presence of a fixed concentration of E. coli CMCC44113, potencies were found to be proportional with concentrations. The antimicrobial activities of AgNPs‐HWE combo were significant enhanced, when compared with solely AgNPs or HWE. Thus, atomic force microscopic and propidium monoazide‐PCR were used to probe the damages caused by AgNPs‐HWE combo on the cell morphology and cell membrane integrity of E. coli. The mechanism of AgNPs‐HWE combo against E. coli may attribute to AgNPs leads to cell wall lysis and damages cell membrane integrity, and thus increases the penetration of HWE into the bacterium, which results in more serious damage to bacterial cells. These findings indicated that AgNPs‐herb was more potent than the AgNPs alone and holds promise for the development of nanoparticle enhanced herbal pharmaceuticals.

1 Introduction

Antibiotic resistance of pathogenic microorganisms has been increasing rapidly in recent years due to the massive use and abuse of conventional antibiotics [1]. Self‐protection mechanisms in microorganisms such as genetic modification, production of drug degrading enzymes and/or modifications in antibiotics‐specific binding sites were induced because of administration of antibiotics [1, 2]. Numerous studies have been focused on reduction of the use of conventional antibiotics as well as research for new antimicrobial components that are less potent in inducing antibiotic resistance. These researches on new antimicrobials converged into metals (e.g. silver, zinc, copper, etc.) and botanicals (e.g. herbal medicine) as possible alternatives.

Several chemical forms of silver including silver metals have long been known to exhibit antimicrobial activities. Ag⁺ ion in the form of a silver nitrate solution has been the typical antimicrobial form. The biocidal effect of Ag⁺, with its broad spectrum activity including bacterial, fungal, and viral agents, has been achieved at submicromolar concentrations [3]. In addition, oxidised bulk silver has also been reported to exhibit antimicrobial properties that had been ascribed to its surface oxide layer and/or release of Ag (I) species [4, 5]. Recent advances in technology and increased consumer demand for beneficial health materials are giving rise to many new products that use silver because of its highly effective antibacterial property [6]. Silver has been used for medical applications and as a household good because of its non‐toxic ability to sterilise and deodorise [7, 8, 9].

Recently, the nanoparticle version of silver (AgNPs) has also been shown to exhibit antimicrobial properties [10, 11, 12, 13]. These AgNPs had been prepared through a variety of synthetic methods [13, 14, 15, 16, 17]. For example, wound dressings with sputtered AgNPs have been used clinically to suppress microbial infection in burn wounds [18]. AgNPs possess effective broad‐spectrum activity against bacteria with less probability to cause microbial resistance than conventional antibiotics [19, 20]. In this paper, the antimicrobial properties of AgNPs were evaluated.

Traditionally, herbal medicines have been evaluated for antimicrobial and therapeutic effects in China. Most of these traditional Chinese herbal medicines serve as important sources of phytochemicals that are generally accepted to bring health benefits, including antimicrobial, antitumorigenic, antimutagenic, anti‐inflammatory, anti‐allergenic, and protection against ischemic heart diseases [21, 22, 23, 24, 25]. Among these Chinese herbal medicines, Lonicera japonica Thumb, or honeysuckle (using the flower‐bud or full‐blown flower), is one of the major multi‐functional medicinal herbs. Consuming this plant has long been acknowledged in traditional Chinese medicine for its antimicrobial, anti‐inflammatory, anti‐aging, cancer prevention, and many other medical applications. Our interest lies on the antimicrobial potency of honeysuckle and the possible enhancement with emerging AgNPs.

In this paper, we evaluated the antimicrobial properties of AgNPs in combination with the Chinese traditional medicinal herb, Lonicera japonica Thumb in the form of a water extracts (honeysuckle water extract or HWE). The combination of nanoparticle and traditional medicinal herb, AgNPs‐HWE was used to develop the hybrid pharmaceutical material, AgNPs‐herbal antimicrobial herb (AgNPs‐herb). Using different AgNPs or herb ratios in the presence of a fixed concentration of E. coli CMCC44113, and the antibacterial activities of AgNPs or herb in different concentrations were recorded. The synergetic antimicrobial effects were examined when combined the AgNPs and HWE. Furthermore, we preliminarily probe the mechanism of action of AgNPs‐HWE combo against pathogens by using atomic force microscopic (AFM) and propidium monoazide (PMA)‐PCR techniques.

2 Materials and methods

2.1 AgNPs synthesis and characterisation

The AgNPs were synthesised using a modified published method [26]. Briefly, silver tetradecanoate and Ag(n‐C₁₃ H₂₇ COO) was placed in flask, and triethyl amine was added, the subsequent reaction solution was carried out at 80°C for 2 h. Bis(amine)silver(I) carboxylate was formed as an intermediate that is a mild reducing agent to produce the nanoparticles. The insoluble precursor was disappeared and formed a homogeneous solution of silver nanoparticles. The nanoparticles were precipitated by using the acetone, collected by filtration, and dried under vacuum. The core size of the AgNPs was established using transmission electron microscopy (TEM). The characterised AgNPs were coated with amphiphilic polymers to create the colloidal form in water and at the same time incorporate reactive carboxyl groups on the particle surface [27]. The carboxyl groups on the surface of AgNPs were useful for bioconjugation. The hydrodynamic size of the AgNPs was measured using a light scattering instrument (Zetatrac Ultra 151, MicroTrac, Inc, USA).

2.2 Herbal plant material

Honeysuckle was purchased from the Traditional Chinese herbal drug store located in Nanchang, China. The water extract of the honeysuckle, which was used in all the studies in this paper, was prepared as follows: honeysuckle (45 g) (grown in North China, 2012 using the flower or buds) were added to 1 L distilled water (95°C) on a battery of percolators for 2 h. The extract obtained by above procedure was defined as 1 × HWE, and 5×, 4×, 3× and 2×HWE were concentrated from 1 × HWE respectively. The resulting HWE was purified through a 0.22 μm sterile filter. The HWE was frozen and stored at −20°C until it was used for the experiments.

2.3 Bacterial strain

The bacteria indicator strain, E. coli CMCC44113 (provided by the National Center for Medical Culture Collection (CMCC) in Beijing, China) was grown in Tryptic Soy Agar (TSA). The harvested cells were stored by freezing in Tryptic Soy Broth (TSB)/20% glycerol solution (vol/vol) and kept at −20°C. A fresh culture of E. coli CMCC44113 was prepared by incubating aerobically at 37°C for 24 h. The cells were extracted with 85% saline solution at a concentration of 1 on the McFarland scale which is equivalent to 3 × 10⁸ cells/mL. A 10 μl (3 × 10⁶ cells) aliquot of this solution was diluted to 10 ml with TSB and mixed thoroughly to prepare 3 × 10⁵ cells/mL E. coli solution. The final bacteria solution that was used in the antimicrobial studies involved 10 µL (3 × 10³ cells) of this less concentrated solution and was added to 1 ml of TSB for a final concentration around 3000 microorganisms unless specified.

2.4 Antimicrobial activity of AgNPs at different concentrations

The nanoparticles were dispersed in distilled water and the AgNPs concentration was adjusted to 10 mg Ag/ml. The final concentration of the AgNPs was established using absorbance at λabs = 500 nm. To carry out Ag concentration measurement, the AgNPs colloids were diluted 100 times with deionised water. The absorbance (A ₅₀₀) was measured using a HP UV‐vis absorption spectrophotometer. The A ₅₀₀ was compared with a standard and the concentration was calculated using the following equation

$$C_{\mathrm{Ag}}=A_{500}\times \mathrm{Dilution}\mathrm{factor}\times \mathrm{Calculation}\mathrm{factor}\mathrm{mg}/\mathrm{ml}$$

An aliquot of the aqueous AgNPs was added to 1 ml of the bacteria TSB suspension containing about 3000 E. coli cells (each test sample was counted prior to use) to give a AgNPs concentration of 0, 0.1, 0.2, 0.5, and 1 mg/ml. The E. coli/ nanoparticle mixture was incubated aerobically at 37°C for 2 h. The live bacteria numbers were determined using standard microdilution method. The samples were diluted with TSB to provide decreasing concentrations (geometric series, with a coefficient of 10) from a concentration C down to C/10n. After incubation for 24–48 h at 37°C the colony forming units (CFU, which were the remaining live bacterial cells that was previously exposed to the nanoparticles for 2 h) were counted.

2.5 Antimicrobial activity of HWE with and without AgNPs

The HWE was evaluated for antimicrobial activity with and without the AgNPs. For this study, 250 μl of HWE was added to 750 μl of the bacteria TSB suspension which contained approximately 2250 E. coli cells. To this, 250 μl of distilled water was added without any AgNPs and this was used for control.

To evaluate the antimicrobial effect of both the AgNPs and the HWE, the same volume of 3 × HWE was added to AgNPs for a final nanoparticle concentration of 0.1 mg/ml. The mixture was incubated at 37°C for 1 h with gentle shaking. It is anticipated that this process allowed the herbal effective components to be loaded onto the nanoparticles through surface charge interaction. After incubation, the AgNPs‐HWE mixture was added to 750 μl of the bacteria TSB suspension which contained approximately 2250 E. coli cells. For the AgNPs control, 250 μl of distilled water was added to the AgNPs at 0.1 mg/ml final concentration.

The above mixtures were incubated aerobically at 37°C for 2 h to allow the formulation to react with the bacteria. After incubation, each of the mixtures were plated on a TSB agar plate and incubated aerobically at 37°C for 12 h, and then the CFU was counted.

2.6 AFM analysis of AgNPs‐HWE combo against E. coli

AFM observation was used to evaluate the antimicrobial effect of the AgNPs‐HWE combination on the E. coli cells. In this study, E. coli (10⁶ cfu/mL) alone or treated with AgNPs‐HWE combo were washed with PBS and transferred on mica piece sample holders and air dried. E. coli nanostructures were imaged with AJ‐III AFM (Shanghai AJ Nano‐Science Development Co., Ltd, Shanghai, China) in a tapping mode (cantilever length 100 μm) with Nanosytems ACT Si probes at a scan rate of 1.6 Hz at a scan size was 6.7 μm × 6.7 μm. The height and amplitude images were collected and further analysed using iNano SPM‐ELeChem software (Shanghai AJ Nano‐Science Development Co., Ltd, Shanghai, China) to display the cells surface.

2.7 PMA treatment and PMA‐PCR analysis

E. coli (10⁶ cfu/ml) treated with AgNPs, AgNPs‐HWE combo or alone were washed with PBS. PMA treatment, DNA extraction, and PMA‐PCR were performed as described before [28]. Briefly, the cells were treated with 5 μg/ml of PMA (Biotium, USA) in the dark for 5 min and subsequently exposed to a 500‐W halogen light for 5 min and extracted the genomic DNA of E. coli O157:H7 using the boil method as described in our previous work [28]. The primers for the unique gene of E. coli O157:H7 flic was used in PCR [29].

2.8 Statistical analyses

All the tests were performed at least in triplicate analysis. The data gathered were analysed using the statistical program SigmaPlot 16 (SPSS, Inc., Chicago, USA).

3 Results and discussion

3.1 Characterisations of AgNPs

The AgNPs were initially synthesised in organic solvent before these were converted to dispersions of nanoparticles in water. Characterisation with TEM showed that the size of the hydrophobic AgNPs was approximately 3 nm in diameter (as shown by the images on Fig. 1 on the middle). After conversion to the colloidal form through encapsulation with the polymer, the hydrodynamic size was recorded at approximately 15–17 nm (light scattering datum on Fig. 1 on the left‐hand side). The zeta potential of the AgNPs was measured at −28 mV. These results are in agreement with what is expected for AgNPs. The colour of the aqueous AgNPs solution was very yellowish black (Fig. 1 right‐hand side) and the light scattering was as expected. There was no indication of aggregation of the AgNPs after conversion into the colloid solution.

Fig. 1.

Light scattering (LS) properties (left‐hand side), TEM image (middle) and colloidal solution (right‐hand side) of AgNPs. The LS pattern I indicates that the AgNPs have an average size of 15–17 nm

3.2 Antimicrobial activity of AgNPs at various concentrations

Using an indicator strain, pathogenic E. coli CMCC44113, the antimicrobial property of different concentrations of AgNPs on its own was evaluated. The results shown in Fig. 2 a indicated that the AgNPs exhibited very significant concentration dependent bacterial inhibitory activity. The live bacteria number decreased significantly with an increase in the concentration of AgNPs. At 0.1 mg/ml, only 29% of the population was able to form colonies. This decreased to 16% at 0.2 mg/ml and 8% at 0.5 mg/ml. At 1 mg/ml AgNPs, the number of bacteria colonies was down to 4% in comparison with the control which indicated that they were hardly able to survive. This is in agreement with previous studies on the antimicrobial properties of AgNPs [4, 5, 13, 14, 15, 16, 17, 18]. Hence, we were able to show the strong antimicrobial activity of AgNPs at a significantly low concentration.

Fig. 2.

Antimicrobial activity of AgNPs (a) and HWE (b) at various concentrations against a starting concentration of 2250 CFU/mL E. coli, respectively (N = 3)

3.3 Antimicrobial activity of honeysuckles and AgNPs‐HWE combo

Just like other herbal fruits and vegetables that are known for the prevention of a number of chronic diseases, honeysuckle has been described for its chemopreventive, antimicrobial, anti‐adherence, and antioxidant properties [21, 24]. The antimicrobial activity of honeysuckle may be attributed to the prevention of adhesion of the microorganisms to tissue cells and/or implant surfaces which is a prerequisite for colonisation and infection of a number of pathogens in the urinary tract, mouth, and in open wounds [30, 31].

Before preparing the AgNPs‐HWE combination, the antimicrobial property of the HWE alone was evaluated using pathogenic E. coli. The results in Fig. 2 b indicated that the HWE on its own had significant antimicrobial activity. Even at a low concentration of 1 × HWE, the E. coli population decreased by 50%. The E. coli population was completely wiped out when 5 × HWE was used.

The combination of AgNPs with HWE showed significant enhancement of the antibacterial effect (p < 0.05). As shown in Fig. 3 a, the survival rates of E. coli incubation with 3 × HWE or 0.1 mg/ml AgNPs were approximately 42.57% or 22.75%. However, the mixture of 3 × HWE and 0.1 mg/ml AgNPs exhibited a 4.24% survival rate, which is significant lower than 9.68% (42.57% × 22.75% = 9.68%), indicating the synergistic antibacterial effect was occurred between AgNPs and HWE. In addition, with 3 × HWE and 0.1 mg/ml AgNPs (Fig. 3 b) the bacterial growth inhibition was more than fivefold compared with the AgNPs alone resulting in no growth in the medium. This indicated that in the presence of 3 × HWE only one tenth of the AgNPs concentration was required to eradicate the bacteria.

Fig. 3.

Enhanced antimicrobial activity of AgNPs‐HWE combo compared with solely HWE and AgNPs (a). (b) shows plates containing the mixture of E. coli, the HWE, AgNPs‐HWE combo. The AgNPs and HWE used for this test were 0.1 mg/ml and 3 × HWE, respectively. (N = 3)

3.4 Physical evidence of AgNPs‐HWE combo antimicrobial

Having observed the potency of the AgNP‐HWE combination, we evaluated the antimicrobial mechanism of action. The antimicrobial mechanism of AgNPs‐HWE combo was analysed for physical evidences of microbial damage by observing with AFM. As indicated in Fig. 4, untreated E. coli cells (C group) showed a smooth surface, and few evidences of surface break points can be found on the profile map. However, when E. coli was treated with AgNPs‐HWE combo (T group), the profile map showed a rough surface and some cellular debris which indicated fractures on the cell surface. Degree of roughness (Ra) as analysed by the AFM software showed 16.309 nm for C group and 36.894 nm for T group. This indicated that the degree of roughness of the E. coli surface when treated with AgNPs‐HWE combo was 1.26 times as compared with that of the control. Such difference in the degree of roughness that was performed in triplicate analysis is taken as evidence of fractures on the E. coli cell surface. After the treatment with AgNPs‐HWE combo, the E. coli cell morphology showed changes in size, shape and surface roughness (on the cell wall and membrane).

Fig. 4.

Cell morphology and cell membrane integrity of E. coli were examined by AFM and PMA‐PCR, respectively. Height images (C1 and T1) and amplitude images (C2 and T2) of E. coli by AFM (in tapping mode, scanning size 6.7 μm × 6.7 μm). C: no treatment, T: treated with AgNPs‐HWE combo at 3 × HWE with 0.1 mg/ml AgNPs). The scale bar = 1 μm. (A): PMA‐PCR results, the lanes 1–4 are 0.1 mg/ml AgNPs, 0.1 mg/ml AgNPs‐ 3 × HWE combo, positive control and negative control, respectively. Lane M is DL2000 marker. (N = 3)

3.5 PMA‐PCR assessment of E. coli cell membrane integrity

We employed PMA‐PCR to analyse the cell membrane integrity of E. coli after exposure to AgNPs, AgNPs‐HWE combo, respectively. PMA can selectively penetrate compromised membranes of dead cells, adhere to intracellular DNA irreversibly, and subsequently eliminate the positive signals from dead bacteria. The reduction of PCR products that is dependent on the amount of damaged cell has been used to indicate membrane disruption of cells [32]. As shown in Fig. 4 a, slight PCR products were observed when treated with 0.1 mg/ml AgNPs, indicated that PMA entre into cells and bind to DNA. However, When the E. coli treatment with AgNPs‐HWE combo, PCR products were barely seen, suggesting cell membrane of E. coli were subjected more serious damage than solely AgNPs treatment. These observations may be taken as evidences of weakening of the cell integrity that may eventually lead to cell wall fracture that was caused by the AgNPs treatment. These resulted in leaky bacteria that allowed for the easy penetration of HWE into the cells. This eventually resulted in enhanced cell death.

3.6 Possible mechanism of action of AgNPs‐HWE combo

The complex components of the water extracts of HWE were mainly including volatiles, flavonoids, organic acids, triterpenes and inorganic elements. Chlorogenic acid, an important member of organic acids in HWE, is the principal active ingredient of HWE while it has been considered as a promising candidate for resisting HIV virus [33]. The exact mechanism of action of HWE on microbes is still unclear, but the possible mechanism of action of chlorogenic acid has been suggested according to the non‐competitive inhibition of intracellular arylamine N‐acetyltransferase of bacterium [34, 35, 36]. When the cells of E. coli were exposed to AgNPs, many pits and gaps were observed in bacterial cells by AFM, and the cell membrane was fragmentary. Accordingly, we present a possible explanation for the enhancement of the synergistic antibacterial effect with the combination of HWE and AgNPs. AgNPs act on the cell wall, which lead to cell wall lysis and increase cell membrane permeability and thus increase the penetration of chlorogenic acid into the bacterium. More chlorogenic acid reacts with arylamine N‐acetyltransferase, which results in more serious damage to bacterial cells. In addition, when AgNPs were mixed with HWE, the active components of HWE such as chlorogenic acid to be loaded onto the nanoparticles through surface charge interaction due to the AgNPs coated with reactive carboxyl groups. More AgNPs and effective components of HWE could simultaneously react to the bacteria and then resulted to more bacterial death. Thus, our future focus will be on identifying the intrinsic active component of the HWE. We will use the active component in the development of natural antimicrobial agents that can be used in the food industry as well as for medical and disinfection applications. We believe that novel composite materials such as AgNPs are good candidates for the enhancement of the medicinal effects of honeysuckle and other Chinese herbal medicine.

4 Conclusion

Silver nanoparticle was combined with honeysuckle towards the development of a promising antimicrobial pharmaceutical material. The antimicrobial activity of water extracts from honeysuckle was established against Escherichia coli CMCC44113. Various concentrations of the AgNPs were mixed with the HWE to establish nanoparticles enhancement of antimicrobial potency. The combination of 3 × HWE with 0.1 mg/ml AgNPs led to bacterial growth inhibition that was 200% more effective compared with 1 mg/ml AgNPs and 5 × HWE alone, respectively. Observations under AFM indicated that the AgNPs‐HWE treated bacteria showed cell wall fractures and cell membrane lesion. Moreover, PMA‐PCR results demonstrated the cell membrane permeability was increased by AgNPs‐HWE, which may have resulted in leaky cell wall that eventually resulted in cell death. These findings indicated that AgNPs‐HWE combo was more potent than the AgNPs alone and holds promise for the development of nanoparticle enhanced herbal pharmaceuticals.