Background
Helicobacter pylori (
H. pylori) is known to be a major pathogen in the development of gastritis, peptic ulcer disease, gastric adenocarcinoma, and mucosa associated lymphoid tissue (MALT) lymphoma [
1,
2]. Therefore,
H. pylori eradication is important for the management of these diseases. However, eradication rate for
H. pylori has decreased due to antibiotic resistance of
H. pylori. Its resistance rates to clarithromycin and metronidazole in East Asia and Europe have been reported to be 17–34 and 28–65%, respectively [
3‐
7]. Therefore, new alternatives or adjuvant approaches are needed for
H. pylori eradication, especially in the area where there is high antibiotic resistance rate of
H. pylori. Previous study has reported that foods or components of foods have anti-
H. pylori activities by facilitating penetration of antibiotics to
H. pylori by damaging cell membrane, inhibiting urease activity of
H. pylori, inhibiting
H. pylori adhesion to gastric mucosa, and interfering with cell division process of
H. pylori [
8]. Among these mechanisms, inhibiting urease activity of
H. pylori can help eradicate
H. pylori by altering optimal pH and inhibiting colonization of
H. pylori [
9‐
11].
A recent meta-analysis has shown that the intake of citrus fruits can reduce the incidence of gastric cancer in the area with high prevalence of
H. pylori [
12]. Another report has shown that phytochemical constituents of citrus peels possess biological activities, including anticancer, immunostimulation, and antigenotoxic effects [
13]. Oranges, lemons, limes, grapefruit, and tangerines are well-known examples of citrus fruits.
Citrus aurantifolia (
C. aurantifolia), also known as key lime, is one of widely consumed citrus fruits in many cultural cuisines and juice production. It has antibacterial activities against
Mycobacterium tuberculosis, Staphylococcus aureus, and others. Among various constituents of
C. aurantifolia, citral, 4-Hexen-3 one, oleic acid, and palmitic acid have been found to possess antibacterial activities [
14‐
19]. However, it is currently unclear whether
C. aurantifolia and its constituents have anti-
H. pylori activities. Therefore, the objective of this study was to evaluate anti-
H. pylori activities of
C. aurantifolia and its constituents and their possible inhibitory effects on urease activity of
H. pylori.
Methods
Key lime (C. aurantifolia) extraction
Slices of C. aurantifolia were dried in a constant drying oven (VS-4150ND, VISION SCIENTIFIC, Daejeon, Korea) at temperature of 50 °C. Dried C. aurantifolia slices were mixed with liquid nitrogen and ground into fine powders using a mortar and pestle. Powders of C. aurantifolia (1 g) were then dissolved in 30 ml of sterile distilled water and incubated at room temperature for 24 h. Dissolved C. aurantifolia was filtered using a 0.45 µm pore syringe filter (Corning, NY 14831-001, USA). Twofold serial dilutions of C. aurantifolia extract (original concentration, 34 mg/ml) were made with distilled water (1:1 to 1:1024).
We used 4-hexen-3 one, oleic acid, and palmitic acid as constituents of
C. aurantifolia to determine their antimicrobial activities and inhibitory effects on urease activity of
H. pylori [
14,
19]. For each constituent [citral (Sigma-Aldrich #W230316, USA), 4-hexen-3 one (Sigma-Aldrich #H13001, USA), oleic acid (Sigma-Aldrich #O1008, USA), and palmitic acid (Sigma-Aldrich #P0500, USA)], we prepared the following concentrations: 1, 2, 5, 10, 50, 100, 200, 400, 500, and 1000 µg/ml.
Helicobacter pylori strain ATCC 43526 and triple drug resistant (TDR) H. pylori strains
We used standard
H. pylori strain (ATCC
® CRL-43526™, USA) and TDR
H. pylori strains isolated from gastric antrum and body from 18 patients with gastric epithelial neoplasm. Methods of isolation and culture for
H. pylori were the same as those described in our previous study [
20].
Antimicrobial susceptibility testing
We stored H. pylori strains at − 80 °C. After thawing and culture of standard H. pylori strain and 18 TDR H. pylori strains, we measured minimum inhibitory concentrations (MICs) by agar dilution method for antibiotics and by modified media dilution method for C. aurantifolia extract and each constituent of C. aurantifolia. We made agar plates using Muller Hinton agar containing 5% sheep blood (Hanilcomed, Korea), 1% IsoVitalex (BD Biosciences), and one of the following drug concentrations for MIC assay: 2–32 µg/ml of metronidazole, 0.25–4 µg/ml of clarithromycin, 0.125–2 µg/ml of amoxicillin and levofloxacin, and 1–16 µg/ml of tetracycline. All antibiotics used in this investigation were purchased from Sigma (St. Louis, MO, USA) except clarithromycin which was obtained from Abbott Laboratories (Abbott Park, IL, USA). We added 10 ml of agar solution into 100 π plate and then cooled down. H. pylori strain ATCC 43526 (Manassas, VA USA,) was used as a quality control organism. Antibiotic concentrations used in this study were based on cutoff levels related to Laboratory Standards Institute (CLSI) clinical breakpoints for resistance. All MICs were interpreted using CLSI breakpoints. Antibiotic resistance was defined as follows: amoxicillin, MIC ≥ 0.5 µg/ml; clarithromycin, MIC > 1.0 µg/ml; metronidazole, MIC > 8 µg/ml; tetracycline, MIC > 4 µg/ml; and levofloxacin, MIC > 1 µg/ml.
We tested MIC for C. aurantifolia and four constituents of C. aurantifolia. We mixed 6 × 108 CFU/ml H. pylori in twofold serial dilutions of C. aurantifolia extract (34 mg/ml–33.2 µg/ml, 1:1 to 1:1024) or in serial concentrations of its four constituents (1–1000 µg/ml), respectively. These mixtures of H. pylori with C. aurantifolia extract or its four constituents (5 µl each) were dropped immediately onto agar plates. We determined MIC levels of C. aurantifolia extract and each constituent based on invisible H. pylori colony on the agar plate after 7 days of incubation.
Urease activity inhibition test
We harvested
H. pylori in 0.9% saline and then prepared mixtures of 6 × 10
8 colony forming units (CFU)/mL of
H. pylori with two-fold serially diluted solution of
C. aurantifolia extract (1:1 to 1:1024). We prepared 6 × 10
8 CFU/ml of
H. pylori with citral, 4-hexen-3-one, oleic acid, or palmitic acid in the following concentrations: 10, 50, 100, 200, 400, 500, and 1000 µg/ml
. H. pylori strains with each constituent were incubated at room temperature for 10 min. We used 0.9% saline as a control. We added each
H. pylori strain (6 × 10
8 CFU/ml) in 5 into 200 µl of the following mixture: 1.5% urea (Bioshop, Canada Inc.) and 0.1% EDTA with 0.02% cresol red solution (Bioshop, Canada Inc.). The mixture ratio was 2:1. The reaction was incubated at room temperature for 20 min. After that, we measured urease activity at absorbance of 590 nm using a VersaMax™ ELISA reader (MOLECULAR DEVICES, Silicon Valley, CA, USA) [
21,
22]. Urease inhibition test for each
H. pylori strain was repeated three times.
Statistical analysis
Analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences in urease activity depending on the concentration of C. aurantifolia extract and its constituents. Urease activities were shown as mean ± standard deviation (SD). All reported P values were two-sided and P < 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS software, version 23 (IBM Corp, Armonk, NY, USA).
Discussion
Our present study showed that C. aurantifolia extracts could inhibit urease activity of antibiotic-susceptible H. pylori strain and TDR H. pylori strains in vitro in a dose-dependent manner. Among constituents of C. aurantifolia, citral and 4-hexen-3-one showed dose-dependent inhibition of urease activities of antibiotic-susceptible H. pylori strain and TDR H. pylori strains. Furthermore, citral and 4-hexen-3-one showed inhibitory effects on the growth of antibiotic-susceptible H. pylori strain and TDR H. pylori strains.
Helicobacter pylori eradication rates have decreased while their resistance rates to antibiotics have increased. To improve eradication rates of
H. pylori, alternative treatments such as antibiotics combined with plant extracts, probiotics, curcumin, honey, and antioxidants have been suggested [
8]. Previous study has shown that lime juice concentrates have good inhibitory effects on both Gram-negative and Gram-positive bacterial strains, with MIC in the range of 12.5–50 μg/ml [
23]. Another study has demonstrated that hexane extract of fruit peels of
C. aurantifolia exhibits inhibitory effect against antimicrobial resistant
M. tuberculosis strains, with MIC in the range of 25–50 μg/ml [
24]. Among constituents from
C. aurantifolia, palmitic acid, linoleic acid, oleic acid, 4-hexen-3-one, and citral are active against
M. tuberculosis strains [
14,
25]. We selected four available constituents (palmitic acid, oleic acid, 4-hexen-3-one, and citral) from
C. aurantifolia that showed antimycobacterial activity. In our study,
C. aurantifolia extract decreased the number of
H. pylori ATCC 43526 colonies and TDR
H. pylori colonies. Constituents of
C. aurantifolia also showed inhibitory effects against
H. pylori strain ATCC 43526, with MIC of citral at 5–10 μg/ml and MIC of 4-hexen-3-one at 20–50 μg/ml. Furthermore, citral showed inhibitory effects against 18
H. pylori strains with triple drug resistance. Its MIC ranged from 2 to 100 μg/ml. In addition, 4-hexen-3-one showed inhibitory effects against 18
H. pylori strains with triple drug resistance. Its MIC was in the range of 20–200 μg/ml.
Biglar et al. have shown that
C. aurantifolia can inhibit the activity of Jack-bean urease (IC
50 = 28 μg/ml) [
26]. Our study also showed that
C. aurantifolia extract could inhibit urease activity of
H. pylori at dilution of 1:64 to 1:1. Among constituents from
C. aurantifolia, citral and 4-hexen-3-one showed dose-dependent inhibitory effects on urease activity of
H. pylori. It is known that
H. pylori can neutralize acid in its environment by producing urease which breaks down urea in the stomach to carbon dioxide and ammonia. These chemicals then react with strong acids in the gastric environment to produce a neutralized area around
H. pylori [
27]. Previous animal study has shown that
H. pylori is unable to colonize at gastric mucosa with normal physiological pH in urease-negative mutant piglet [
28]. Recently, another study has demonstrated that bacterial load is decreased within 5–7 days in a urease knockout infection mouse model [
29]. Urease expression is required for establishing initial colonization and maintaining chronic infection [
2,
29]. In the present study,
C. aurantifolia extract and its constituents showed inhibitory effects on urease activity of
H. pylori, suggesting that they might have potential as adjuvants to enhance
H. pylori eradication.
In this study, we did not show the association between antibacterial effect and inhibition of urease activity. Bactericidal effect of C. aurantifolia may affect the growth of H. pylori colonies, leading to inhibition of urease activity and vice versa. Although low dose of C. aurantifolia extract showed no obvious effect on the growth of H. pylori, it showed inhibitory effect on urease activity of H. pylori. Further studies are needed to evaluate the mechanism involved in the antibacterial effect of C. aurantifolia and the causal association between its inhibition of urease activity and bactericidal effects.
In conclusion, C. aurantifolia and its constituents attenuated urease activities of H. pylori strains. Citral and 4-hexen-3-one had antimicrobial effects on H. pylori strains with triple drug resistance, suggesting that C. aurantifolia might have potential as a therapeutic agent to control H. pylori strains that cause eradication failure with other antibiotics. Future studies are needed to evaluate the efficacy and toxicity of C. aurantifolia in vivo.
Authors’ contributions
CHP: study concept and design; analysis and interpretation of data; drafting and finalizing the manuscript; study supervision. SML: Carrying out the experiment; analysis and interpretation of data; drafting the manuscript. SYP: Analysis of electronic medical records; analysis and interpretation of data; drafting the manuscript. MJK: Carrying out the experiment. EAC, CHJ, HSK, SKC and JSR: Patient recruitment and care. All authors read and approved the final manuscript.