Antimicrobial Characterization of Inula britannica against Helicobacter pylori on Gastric Condition

Abstract

The antimicrobial effects of methanol and ethanol extracts of Inula britannica against several Helicobacter pylori strains (26695, J99, and SS1) were evaluated in vitro, to determine their applicability as functional foods. In the paper disc diffusion method, the antimicrobial effects of the I. britannica extracts against the H. pylori strains were apparent. Viable cell counting also showed that the extracts at 100 μg/ml concentration dramatically decreased the viability of the H. pylori strains. In particular, the methanol and ethanol extracts at a concentration of 100 μg/ml reduced the H. pylori SS1 cell number to 2.46 log CFU/ml and 1.08 log CFU/ml, respectively. In the presence of 100 μg/ml extracts, the urease production of H. pylori SS1 was decreased to more than 30%, whereas that of H. pylori J99 and H. pylori 26695 was decreased to about 20%, relative to the controls. The extracts inhibited the attachment of the H. pylori strains to human gastric AGS cells as well as caused the detachment of already attached H. pylori cells. In addition, the H. pylori morphology was changed to a coccoidal shape in the presence of the extracts. In conclusion, the I. britannica extracts were effective against H. pylori strains in vitro, irrespective of genotype status, and could therefore be used as novel functional foods.

Introduction

Helicobacter pylori infection is associated with the pathogenesis of chronic gastritis, peptic ulcers, mucosa-associated lymphoid tissue type lymphoma of the stomach, and gastric carcinoma [7]. It is as a worldwide health problem, with an infection rate of approximately 50% [1]. H. pylori has been identified as a Group 1 carcinogen by the International Agency for Research on Cancer [5]. The bacteria may cause damage to gastric epithelial cells via protein encoded by its cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) [2]. H. pylori cagA-positive strains are associated with gastric ulcer, duodenal ulcer, and gastric cancer [20].

The pathogenicity of H. pylori is initiated at its germination stage in gastric cells [23]. H. pylori secretes urease into the strong acidic environment of the stomach and changes urea into ammonia, thus neutralizing its surroundings for survival. The inhibition of urease activity therefore becomes a strong bacteriostatic means by which to hinder H. pylori’s survival inside the stomach. Moreover, methods to suppress the attachment of H. pylori to the gastric mucosa, which is the initial step of infection, and to detach already colonized bacterial cells can be as effective a management strategy as killing H. pylori directly [19].

To reduce the side effects of antimicrobials such as antimicrobial resistance, constipation, and gastrointestinal disorder, the use of natural products as anti-H. pylori substances has been reported, including probiotics, plant extracts, curcumin, and catechin [1,4]. Cranberry extract was demonstrated to suppress the H. pylori-stimulated secretion of interleukin-8 from stomach cells [19]. In addition, synthetic antimicrobial peptides and flavonoids have demonstrated anti-H. pylori effects both in vivo and in vitro [21,27].

Inula britannica is used in traditional Chinese and Kampo medicines as a herbal remedy for treating digestive disorders, bronchitis, and inflammation. I. britannica extract has demonstrated biological efficacy, such as anticancer, antioxidant, anti-inflammatory, antibacterial, and hepatoprotective activities [10,13]. The flower of I. britannica and Inula helenium L. has demonstrated antimicrobial effects against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Bacillus cereus, and their antimicrobial substances are reported as essential oils, flavonoids, etc. [6,13]. Therefore, I. britannica has sufficient potential as a herbal medicine with digestion-enhancing properties. The function of I. britannica depends mainly on the composition of its terpenoids and flavonoids. However, whether or not I. britannica has antimicrobial activity against H. pylori infection of gastric cells has not yet been reported.

In this study, the efficacy of I. britannica extracts as an herbal remedy against H. pylori was evaluated by testing the following: (i) their antimicrobial effect against H. pylori, using the paper disc method, urease production assay, and viable cell counting method; (ii) their induction of morphological changes of H. pylori; and (iii) their abilities to inhibit the adherence of H. pylori to gastric cells and to detach H. pylori from gastric cells.

 

Materials and Methods

Bacterial Strains, Media, and Culture Condition

Helicobacter pylori strains 26695 (cagA+), J99 (cagA+ and vacA+), and SS1 (cagA+) were obtained from the H. pylori Korean Type Culture Collection (HpKTCC, Korea). The culture technique for the growth of the strains followed the method of Joo et al. [9]. The H. pylori strains were activated on Brucella agar (Difco Laboratories, USA) supplemented with 5% horse serum, at 37℃ in 10% CO2 and 100% humidity. Liquid cultures were combined with 3 ml of Brucella broth containing 10% horse serum in 90 mm petri dishes, where the inoculated cell density had an OD600 value of 0.01 and was subjected to subculturing at every 24 h.

Preparation of I. britannica Extract

I. britannica flowers were obtained from traditional herbal markets (Seoul, Korea) and used in the dry state. The voucher specimen (No. 20131011001) has been deposited in the Biotechnology Laboratory of Konkuk University, Seoul, Korea. Ground plant materials (30 g) were soaked in 300 ml of solvent at room temperature for 24 h, using a rotating shaker [12]. The solvents used were methanol and ethanol. The extracts were evaporated on a rotary evaporator at 40℃ and then filtered through filter paper. The concentrates were then freeze-dried. Samples for experimental use were prepared by diluting the freeze-dried concentrates with dimethyl sulfoxide (DMSO) to concentrations of 2.5, 5, and 10 mg/ml, followed by filtration through a 0.45 μm syringe filter.

Paper Disc Diffusion Method

The paper disc diffusion method was carried out primarily to confirm the antibacterial activity of the I. britannica extracts against H. pylori [14]. The three activated H. pylori strains were inoculated at 107 CFU/ml on Brucella agar containing 5% horse serum. Each sample was absorbed onto paper discs (8 mm in diameter) that were placed on the Brucella agar. The discs were incubated in a CO2 incubator set to 10% CO2 and 100% humidity, at 37℃ for 3 days. The size of the clear zone around the discs was measured to confirm the antibacterial activity.

Viable H. pylori Count Method

H. pylori strains were inoculated in 2,970 μl of Brucella broth containing 10% horse serum, in 90 mm petri dishes. Then 30 μl of I. britannica extract, at a final concentration of 0, 25, 50, 75, or 100 μg/ml, was added to the broth cultures. The plates were incubated at 37℃ in 10% CO2 and 100% humidity. For the control, the extracts were substituted by 30 μl of distilled water and DMSO. After 24 h incubation, a sample of each treatment was spread on Brucella agar containing 10% horse serum, and then incubated at 37℃ in 10% CO2 and 100% humidity. The viable cell number was counted and presented as log CFU/ml.

Inhibitory Effect of I. britannica on Urease Activity in H. pylori

The inhibitory effect of the I. britannica extracts on urease activity was measured using a modification of the method described by Malekzadeh et al. [18]. In brief, 2,850 μl of urea broth, 50 μl of I. britannica extract, and 100 μl of H. pylori culture medium were added to a 60 mm petri dish. The initial H. pylori number in the urea broth was about 1 × 107 CFU/ml, and the final concentrations of the I. britannica extracts were 25, 50, 75, and 100 μg/ml. DMSO (50 μl) was added instead of I. britannica extract, as the control. The mixture was incubated in a CO2 incubator set to 10% CO2 and 100% humidity, at 37℃ for 3 h, and subsequently the absorbance of the culture at 560 nm was measured using a spectrophotometer. The inhibitory effect on urease activity was calculated using Eq. (1):

Adhesion Inhibition and Detachment of H. pylori on AGS Cells

AGS (a human gastric cancer cell line) was obtained from the Korean Cell Line Bank (KCLB, Korea). The cells were cultured in RPMI 1640 (Gibco, USA), containing 10% fetal bovine serum (Gibco) and 1% streptomycin/penicillin (Gibco), at 37℃ in an atmosphere of 5% CO2 and 95% air.

For the adhesion assays, AGS cells were seeded in 96-well tissue culture plates at a concentration of 1 × 105 cells/well to obtain confluence and incubated for 24 h before use. To initiate the assay, 1 ml (1 × 107 CFU/well) of H. pylori and 100 μg/ml of I. britannica extract in RPMI medium containing 0.5% yeast extract were added to the monolayer of AGS cells. The plate was then incubated for 2 h at 37℃ in 5% CO2 and 95% air. Non-adherent H. pylori cells were removed by washing the plates three times with P BS. Each w ell was t hen repl enished with 2 00 μl of urea broth for 3 h, following which the absorbance at 560 nm was measured using a microplate reader (Molecular Devices, USA). The inhibitory effect on urease activity was calculated using Eq. (1).

For measurement of the detachment ability, AGS cells were seeded at a concentration of 1 × 105 cells/well in 96-well tissue culture plates and incubated for 24 h to form a monolayer. Then, 1 ml (1 × 107 CFU/well) of H. pylori in RPMI medium containing 0.5% yeast extract was applied to the washed monolayer of AGS cells and incubated for 2 h. This was followed by addition of 100 μg/ml of I. britannica extract and further incubation for 2 h. Non-adherent H. pylori cells were removed by washing the plates three times with PBS. Each well was then treated with 200 μl of urea broth for 3 h, after which the absorbance at 560 nm was measured using a microplate reader (Molecular Devices). The inhibitory effect on urease activity was calculated using Eq. (1).

Analysis of Morphological Change of H. pylori Strains Using Scanning Electron Microscopy (SEM)

The H. pylori strains were inoculated in 3 ml of Brucella broth containing 10% horse serum and 100 μg/ml of I. britannica extract and incubated at 37℃ for 24 h. Aliquots of the cultures were subjected to SEM analysis [13]. To do this, the bacterial suspension was centrifuged and the cells were washed gently with PBS. Fixation of the cells was performed in a 2.5% glutaraldehyde solution, with overnight incubation at 4℃. After dehydration using a sequential ethanol series (from 60% to 100%), the specimens were dried and then coated with gold. Morphological changes of the bacterial cells were observed on a scanning electron microscope.

Statistical Analysis

Each treatment of experiments was performed at least three times, and the results are presented as the mean ± standard deviation. Analysis of variance and Duncan’s multiple range tests of the experimental data were performed using SPSS 18 software (IL, USA).

 

Results and Discussion

Antimicrobial Effect of I. britannica Extracts as Determined by Paper Disc Diffusion Method

I. britannica flowers consist mainly of terpenoids and flavonoids (quercetin, naringenin, keampferol, hesperetin, etc.), materials that are known to have antioxidant, anticancer, and antimicrobial effects, among others [13,17]. The antimicrobial effect of the I. britannica extracts was evident from the paper disc diffusion results (Table 1 and Fig. 1). There was no difference in antimicrobial effect between the methanol and ethanol extracts, and the inhibition zone increased with increasing concentration of the extract. The strength of the antimicrobial effect on the H. pylori strains was in the order of 26695 < J99 < SS1. Regardless of the type of extract used, moderate inhibition was observed in H. pylori SS1 and H. pylori J99, and slight inhibition was observed for H. pylori 26695 in the presence of 10 mg/ml of the extract. H. pylori strains are reported to be susceptible to metronidazole, an affordable anti-H. pylori therapy worldwide [8]. Similar to the results of our study, H. pylori SS1 was more susceptible to metronidazole than H. pylori 26695 and H. pylori J99.

Table 1.The results are presented as the mean ± standard deviation. a–jMean values with different superscripts within each row are significantly different (p < 0.05).

Fig. 1.Inhibition effect of I. britannica extracts using the disc diffusion method. (A) H. pylori 26695 with I. britannica methanol extract; (B) H. pylori 26695 with I. britannica ethanol extract; (C) H. pylori J99 with I. britannica methanol extract; (D) H. pylori J99 with I. britannica ethanol extract; (E) H. pylori SS1 with I. britannica methanol extract; (F) H. pylori SS1 with I. britannica ethanol extract.

Antimicrobial Effect of I. britannica Extracts Determined by Viable H. pylori Count Method

The antimicrobial effect of the I. britannica extracts was also shown using the viable H. pylori count method (Fig. 2). Regardless of the H. pylori strain and type of extract tested, the I. britannica extracts showed noticeable antimicrobial effects at 75 and 100 μg/ml. At 100 μg/ml, the methanol and ethanol extracts reduced the H. pylori SS1 cell number to 2.46 log CFU/ml (a 5.8 log CFU/ml reduction) and 1.08 log CFU/ml (a 7.3 log CFU/ml reduction), respectively. The ethanol extract had a stronger antimicrobial effect than the methanol extract. A reason for this is that the main component of I. britannica extracts is quercetin [13], the solubility of which is higher in ethanol than in methanol [24]. The I. britannica extracts had stronger antimicrobial effects against H. pylori SS1 than 200 μM hesperetin, the highest concentration of flavonoids published that gave a 2.5 log CFU/ml reduction at 122 μg/ml [21].

Fig. 2.Inhibition effect of I. britannica extracts against (A) H. pylori 26695, (B) H. pylori J99, and (C) H. pylori SS1, using the viable H. pylori count method. ■, Methanol extract; □, ethanol extract. a-gMean values with different superscripts within each figure are significantly different (p < 0.05).

Analysis of Inhibition Effect on Urease Activity in H. pylori

The most characteristic biochemical feature of H. pylori is its strong ability to produce urease [21,25]. Urease is an enzyme that decomposes urea into ammonia and CO2. The ammonia thus produced neutralizes gastric acid, thereby self-protecting H. pylori from the strong acidic conditions of the stomach. Therefore, the inhibition of urease activity by I. britannica could be used as an indirect method to confirm its bactericidal activity against H. pylori. The urease inhibitory activity of the I. britannica extracts appeared to be the strongest against H. pylori SS1, whereas it showed a similar inhibitory activity against H. pylori 26695 and H. pylori J99 (Table 2). With 100 μg/ml extract treatment, the urease activity of H. pylori SS1 was decreased by more than 30%, whereas that of the other strains was decreased by about 20%. The ethanol extract was slightly more effective than the methanol extract, similar to the results of the viable cell count method. Likewise, the I. britannica extract had a stronger antimicrobial effect against H. pylori SS1 than did 200 μM hesperetin (a 6.32% decrease at 122 μg/ml) [21].

Table 2.The results are presented as the mean ± standard deviation. a-lMean values with different superscripts in table 2 are significantly different (p < 0.05).

Adhesion Inhibition and Detachment of H. pylori on AGS Cells

H. pylori is well adapted to its host because of its attachment ability, which facilitates colonization, resistance to mucus turnover and gastric peristalsis, evasion from the human immune system, and efficient delivery of proteins into the gastric cell, such as the product of cagA [23]. Therefore, whether the I. britannica extracts could inhibit the adhesion of H. pylori and cause its detachment were investigated by testing the microbe’s urease production in the gastric cell model (Table 3). The strength of the adhesion-inhibiting ability of the I. britannica extracts was in the order of J99 > SS1 > 26695. The urease activity of the three H. pylori strains was decreased by 10.35–32.39% and 21.37–47.17%, respectively, with 100 μg/ml of the methanol and ethanol extracts. On the other hand, the strength of the detachment ability of the I. britannica extracts was in the order of 26695 > SS1 > J99, where the urease activity was decreased by 2.68–17.97% and 10.73–23.90%, respectively, in the presence of 100 μg/ml of the methanol and ethanol extracts. In the case of H. pylori 26695, the extracts were better at detaching the attached bacterial cells than inhibiting their attachment to the gastric cells. Conversely, the extracts were better at inhibiting the attachment ability of H. pylori J99 and H. pylori SS1. Lee and Chang [15] reported that Lactobacillus plantarum NO1 could bring about the detachment of H. pylori KCCM 41756 from AGS cells, showing a 33–44% inhibitory effect on urease activity. A high-molecular-mass constituent of cranberry juice inhibited the adhesion of H. pylori strains BZMC-25, EHL-65, and 17874 to sialic acid glycol-conjugates of human gastric mucus [3]. Lactobacillus johnsonii La1, Lactobacillus salivarius, Lactobacillus acidophilus, and Weissella confusa have all been shown to inhibit the attachment ability of H. pylori [16].

Table 3.The results are presented as the mean ± standard deviation. a-gMean values with different superscripts within attachment or detachment are significantly different (p < 0.05).

Morphological Changes of H. pylori in the Presence of I. britannica Extracts

The SEM images of H. pylori strains 26695, J99, and SS1 all showed morphological changes in the presence of I. britannica extracts (Fig. 3). Untreated cells had a bacilli shape, whereas the extract-treated cells were coccoidal shaped. The ethanol extract resulted in more coccidal-shaped cells than the methanol extract. Coccodial forms of H. pylori strains have been reported to be the degenerative form of cells in their death phase, where these morphology changes were shown to be related to loss of culturability and reduction in the total amounts and integrity of RNA and DNA [11,26]. These results were similar to that seen after exposures to bismuth and antibiotics [22]. Therefore, the I. britannica extracts could have led to the H. pylori strains entering their death phase. The greater morphological changes brought about by the ethanol extract were similar to the results of the viable cell counting method and urease production assay, which showed the superiority of the ethanol extract over the methanol extract in anti-H. pylori activities.

Fig. 3.Morphological changes of H. pylori strains in the presence of I. britannica extracts. (A) H. pylori 26695 without I. britannica extract; (B) H. pylori 26695 with I. britannica methanol extract; (C) H. pylori 26695 with I. britannica ethanol extract; (D) H. pylori J99 without I. britannica extract; (E) H. pylori J99 with I. britannica methanol extract; (F) H. pylori J99 with I. britannica ethanol extract; (G) H. pylori SS1 without I. britannica extract; (H) H. pylori SS1 with I. britannica methanol extract; (I) H. pylori SS1 with I. britannica ethanol extract.