Evaluation And Mechanism Of Active Factors In Mastic Gum Against Helicobacter Pylori

Backgrounds:Helicobacter pylori (H. pylori, Hp) are closely associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. It is classified as I grade carcinogen by world health organization (WHO) in 1994. Antimicrobial therapy for this infection is a complex issue, and the following drugs are currently used in combination: proton-pump inhibitors and/or bismuth, metronidazole, clarithromycin, and amoxicillin. These methods have some obvious disadvantages, such as the bad obedience of patients, relapse and reinfection, high ratio of toxicity and drug resistance and high cost of the drugs. Among them, the high ratio of drug resistance is the most serious problem for the failure of treatment. People are looking for new ways and new drugs which can overcome these barriers.Mastic gum (MG) is a natural resinous exudate obtained from the stem and main leaves of Pistacia lentiscus var. chia, a small evergreen tree that is cultivated almost exclusively on the island of Chios. MG was used as a flavouring substance in many countries. On condition of intended use as flavoring agents, MG was considered as innocuity and has no side effect. It is reported that MG had therapy effect for gastric ulcer caused by H. pylori, and had bactericidal activity for H. pylori in vitro. The chemical composition of MG has recently been studied, and some components of MG were identified as antibacterial substances, but as yet no correlation between the antibacterial activity against Hp and the composition of MG has been reported.Objectives:This study examined the antibacterial activity against H. pylori of some chemical compositions of MG both in vitro and in vivo. The purpose was to find out the active compositions in MG, evaluate their pharmacological characters and investigate their antibacterial mechanism, thus set a ground for further research. Methods:1. 10 compositions of MG,α-terpineol, verbenone, linalool, limonene,β-caryophyllene, methyl isoeugenol,γ-terpinene, trans-anethole, myrcene and farnesol were selected. The disk diffusion susceptibility method and broth dilution susceptibility method were used in order to examine their drug sensitivities against H. pylori. MICs and MBCs were determined, and the MIC50,MIC90,MBC50,MBC90 was determined for the most effective composition.2. Hp infection model of BALB/c mice was applied, the infection rate was evaluated by PCR method, culture method and pathological method. After infection, mice were separately treated with the three components with the highest drug sensitivity. Salad oil was used as solvent control, metronidazole, which was one of the most used antibiotics in clinical, was used as positive group. 20 mice were used in every group, and drugs were given intragastricly 1 time every day for 14 days. During the treatment period, the statuses of mice were recorded. The colonization of H. pylori was assessed on the 8th day of treatment, the 1st day after completion of each treatment and 1 month after the completion of each treatment in order to determine the eradication rate. Especially, the composition with the highest eradication rate was further estimated for the curative dose through the same way.3. Simple pharmaceutical and toxicological characters of the composition with the highest eradication rate were evaluated. H. pylori survival in 1/2MIC was exposed to 1/2MIC of test compounds in test tubes and incubated at 37°C for 24 h. This procedure was repeated for up to 14 cycles. The MICs were determined in the 7th day and 14th day, and morphological changes of the bacteria during the course of continued exposure were recorded. The induced resistance bacteria were cultured in cultures with no drugs for 14 generations, and the MICs were also monitored. At the same time, the MICs of the component with the highest eradication rate against the induced resistance bacteria were also determined. This component was also placed at 4℃, room temperature(25℃) and 37℃for 18 monthes, and the MICs were monitored during this period. Tube square matrix method was used to determine the interaction among the three most sensitive components.The safeties of components were evaluated by maximum tolerance dose method and LD50 method. During the periods, blood indexes, biochemical indexes, weight of rats, weight of organs, and pathology changes were detected.4. The bactericidal mechanism of the component with the highest eradication rate was evaluated through two ways. After the adding of this component, the morphological changes of H. pylori were detected by light microscope and electron microscope. Gastro epithelial cell GES-1 was cultured, normal H. pylori and H. pylori treated by this component were added in and cultured together when the cells growed up to 80%. Adhesive attractions were recorded in immersion objective.Results:1. All the selected compounds except myrcene and farnesol have bactericidal activity against Hp. Particularly,β-caryophyllene, linalool,γ-terpinene,α-terpineol and trans-anethole have higher activity, their MIC against H. pylori NCTC11637 are as follows: 625μg/ml, 156μg/ml, 156μg/ml, 625μg/ml, 39μg/ml. The three most effective components were linalool,γ-terpinene and trans-anethole. The MIC50, MIC90, MBC50 and MBC90 for trans-anethole were as follows: 39μg/ml, 78μg/ml, 78μg/ml and 156μg/ml.2. The H. pylori infection rate of BALB/c is 100%(8/8). On the 8th day of treatment,γ-terpinene and linalool have no significant difference compared with solvent control group (P>0.05). Trans-anethole and positive control group have significant difference compared with solvent control group (P<0.05). On the1st day after completion of treatment and 1 month after the completion of each treatment, all the groups have significant difference compared with solvent control group. The eradication rate of all the groups are lower than 50 %( 3/6) in the 8th day of treatment, but are higher than 66.7 %( 3/6) on the 1st day after completion of treatment. Especially, the eradication rate of trans-anethole approaches 83.3 %( 5/6), which is identical to the positive control group. 1 month after the completion of each treatment, the eradication rates are almost the same as 1st day after completion of each treatment. Further investigation on the curative dose of trans-anethole suggests that it is comparablely effective at the dose of 250mg/kg.3. The pharmaceutical characters of trans-anethole. When induced, the MIC of trans-anethole for H. pylori strain 11637 didn't change. At the same time, the MIC of metronidazole changed a lot, H. pylori could still grow when the concentration of metronidazole was 512 times of the original MIC, but the same strain of H. pylori couldn't survive in the MIC concentration of trans-anethole. Three compounds,γ-terpinene, linalool and trans-anethole have the highest activity. The FIC of them are 1.5, 2.0, 1.5, thus they are independent in the bactericidal activity against H. pylori. After storing in 4℃, room temperature(25℃) and 37℃for 18 monthes, the MIC of trans-anethole against H. pylori stays the same. The LD50 of trans-anethole is 3086 mg/kg, TI﹥20.6. When the used dose approaches 5000 mg/kg, it induced the increase of glutamic pyruvic transaminase (GPT) in blood and intestinal tympanites.4. The morphous and structure change a lot when treated with trans-anethole. H. pylori cells became swelled and necrosis. The cell membrane breaks up or even disappears, which results in the efflux of content, and this change becomes more and more obvious as time goes by. Inhibition test of H. pylori adhesion indicates that the adhesive ability of H. pylori decreased after treating by trans-anethole.Conclusions:1.γ-terpinene, linalool and trans-anethole are bactericidal molecules against Helicobacter pylori in mastic gum. They have bactericidal activity against H. pylori both in vitro and in vivo. Trans-anethole can be further researched as a candidate for treatment of H. pylori infection.2. Trans-anethole have a lower chance of inducing resistance, and it does not have cross-resistance to metronidazole. It is stable when stored in storing in 4℃, room temperature(25℃) and 37℃, and may have a broad antimicrobial spectrum.3. Trans-anethole is safe in BALB/c mice when used as a candidate for treatment of H. pylori infection. It induced damage of liver when used in high dose.4. Trans-anethole kills H. pylori through multiple mechanisms, including changing the membrane permeability of H. pylori and blocking its adhesion to Gastric epithelium.