Chemical Composition of the Essential Oil of Mastic Gum and their Antibacterial Activity Against Drug-Resistant Helicobacter pylori

Graphical Abstract Mastic gum is derived from the tree named Pistacia lentiscus that is grown only in Island Hios of Greek. Since Mastic was first reported to kill Helicobacter pylori (H. pylori) in 1998, there has been no further study to elucidate which component of mastic specifically shows the antimicrobial activity against H. pylori. In this study, we examined which component of mastic gum was responsible for anti-H. pylori activity. We prepared the essential oil of mastic gum and identified 20 constituents by GC–MS analysis. Ten standard components were assayed for anti-H. pylori activity, and it clarified that α-terpineol and (E)-methyl isoeugenol showed the anti-H. pylori activity against four different H. pylori strains that were established from patients with gastritis, gastric ulcer and gastric cancer. These components could be useful to overcome the drug-resistance H. pylori growth in stomach.


Introduction
Helicobacter pylori (H. pylori), gram-negative bacterium, induces chronic gastric infection of one-half of the world population. Infection with H. pylori is often associated with a viable proportion of duodenal ulcer, gastric ulcer, and gastric carcinoma [1,2]. During chronic infection process of H. pylori for decades, the bacterium infection initially induces chronic gastritis, and progresses to atrophic gastritis and metaplasia and then to cancer [3]. Intestinaltype gastric cancer shows a multi-step carcinogenic process, from atrophic gastritis to intestinal metaplasia to dysplasia. In Japan, Fukase et al. [4] reported that gastric cancer was inhibited by H. pylori eradication in postendoscopic gastric mucosal resection of gastric cancer in a multi-center randomized control trial. As an approach for prevention of gastric cancer, H. pylori infection has been eradicated by combined treatment with several antimiciobial agents plus proton pump inhibitors. However, this approach does not always provide satisfactory benefits for whole population because of the high cost of therapeutic agents and the emergence of antibiotic resistance [5]. A new approach for anti-H. pylori vaccines has seen recently developed, and some of these vaccines have had some success in the eradicative animal models, and efforts to prove these vaccines efficaciens are underway in human vaccine trials [6].
Further development of potent antimicrobial drugs is expected to improve the therapeutic and preventive effects against pathogenesis by H. pylori. Zaidi et al. [7] have isolated bactericidal constituents from the plant named Mallotus philippinenesis that are effective against clarithromycin-and metronidazole-resistant strains of Japanese and Pakistani H. pylori. On the other hand, mastic gum, which is a resin secreted from the stem of Pistacia lentiscus, was found to be effective against H. pylori [8]. In our present study, we examined which component of the essential oil prepared from mastic gum could inhibit the growth of H. pylori. The bactericidal activity of the mastic components will be discussed in association with their drug resistance reversal effects.

Chemical Composition of the Essential Oil of Mastic Gum
GC-MS analysis of the essential oil of mastic gum led to the identification of the components, which are listed in Table 1. A typical GC-MS chromatogram of the essential oil of mastic gum is illustrated in Fig. 1. The identification of the components was based on comparison of their mass spectra with those of NIST and Wiley libraries, as well as on comparison of their retention times [9,10] and of the standard components analyzed. The major constituent of the essential oil was a-pinene (Peak 1; 82.26 %), and totally 20 components were identified from the essential oil of mastic gum.

Antibacterial Activity of Mastic Components
We next examined which component inhibits the growth of H. pylori. Commercially available 10 compounds were tested for antibacterial activities against H. pylori clinical strains. The results were summarized in Table 2. Surprisingly, some of them showed antibacterial activity againt clarithromycin (CAM)-and/or metronidazole (MNZ)resistant strains. Figure 2 shows most potent anti-pyloritic activity of (E)-methyl isoeugenol and a-terpineol not only against drug sensitive strains (#09-292) but also against drug resistant strains (#09-87, #09-224 and #09-243). These 10 compounds also showed antibacterial activity against three different strains (E. coli, S. aureus, B. subtilis) [9]. The antibacterial activity of the mastic gum was first reported about two decades ago [11]. In 2007, Paraschos et al. reported that the acidic fraction of mastic gum showed the potent bactericidal activity against H. pylori clinical strains and the most active compound was isomasticadienolic acid [12]. In our screening analysis of the essential oil of mastic gum, we identified 20 chemical compositions, and an (E)-methyl isoeugenol was found to show the potent antibacterial activity against four H. pylori strains including CAM-and MNZ-resistant strain. Another compound, a-terpineol also showed antibacterial activity, but its effect was less than that of (E)-methyl isoeugenol. In the previous report [9], verbenone, a-terpineol, and linalool showed higher antibacterial activity than all other components, and (E)-methyl isoeugenol showed moderate activity against E. coli, S. aureus, B. subtillis. (E)-methyl isoeugenol and a-terpineol are the trace components of the essential oil. However, in our present study, these components first showed much higher antibacterial activity against H. pylori when compared with the same dose of the essential oil itself.
H. pylori infection is known to be involved in gastric and duodenum ulcer, gastritis and metaplasia, gastric cancer and MALT lymphoma [13][14][15][16][17]. H. pylori infection can be eradicated by combined treatment with several antimicrobial agents plus proton pump inhibitors. Fukase et al. have reported that the eradication of H. pylori reduces the incidence of gastric cancer [4]. In Japan, combined treatment with proton pump inhibitor (PPI), amoxicillin (AMPC) and CAM have been applied as first line eradication therapy, and MNZ is replaced CAM as second line eradication therapy [18]. However, the subsequent increase in bacterial resistance to CAM in Japan  [19]. Our present study demonstrated antibacterial effects of (E)-methyl isoeugenol and a-terpineol against CAMand MNZ-resistant H. pylori strains ( Table 2). These compounds derived from the mastic gum could be further useful for eradication of H. pylori including drug-resistant H. pyroli.

Preparation of the Essential Oil of Mastic Gum
The essential oil was prepared according to the manufactural protocol in the Japanese Pharmacopoeia. Mastic gums (30 g) in a 300 mL round bottom flask was set to a prescribed distillation apparatus and refluxing with 300 mL of distilled water for 5 h to yield 0.9 mL of essential oil (731.2 mg, 2.4 % yield).

Chemical Composition of the Essential Oil of Mastic Gum
The GC-MS analysis of the essential oil was undertaken using a Shimadzu QP-5050A GC-MS system (Kyoto, Japan), operating in electron ionization (EI) mode with an ionization energy of 70 eV. The instrument was equipped with an INERTCAP-5MS/SIL capillary column (30 m, i.d. 0.25 mm, GL Sciences, Tokyo Japan) with helium as carrier gas at 1.4 mL/min flow rate. Column temperature was initially kept for 2 min. at 60°C, gradually increased to 180°C at a rate of 3.5°C/min then increased to 280°C at a rate of 10°C/min and kept for 5 min. The injector and interface were set to 300 and 280°C, respectively, The gas chromatograph operated in the split mode with a split ratio of 94:1. The mass spectrum was monitored starting at m/z 40 and ending at m/z 500, with a scan The chemical composition of the essential oil was analyzed using NIST and Wiley registry of mass spectral data (Shimadzu Corporation).

H. pylori Clinical Strains
In this study, we used four clinical isolates of H. pylori which were obtained from patients who underwent endoscopic examination at Oita University Hospital, Oita, Japan. Based on the Epsilometer test (E test) for drug susceptibility, these strains were assessed as sensitive or resistant to clarithromycin (CAM) and metronidazole (MNZ) at minimum inhibitory concentration (MIC) of 1 lg/mL (CAM) and 16 lg/mL or higher (MNZ), respectively. The clinical background and drug sensitivity of the 4 strains were as followed. CAM-sensitive, MNZ-resistant strain #09-87 derived from atrophic gastritis; CAM-resistant, MNZ-sensitive strain #09-224 from gastric ulcer; CAM-resistant, MNZ-resistant strain #09-243 from atrophic gastritis; CAM-sensitive, MNZ-sensitive strain #09-292 from gastric cancer.

Antibacterial Activity Test
H. pylori culture suspension was used to inoculate the plates, and discs containing 1, 10, 100 lg of compounds were applied onto culture plates. The plates were incubated under microaerophilic conditions for 3 days at 37°C on Mueller Hinton II Agar (Becton Dickinson, Franklin Lakes, NJ, USA) plate supplemented with 7 % horse blood without antibiotics. The antibacterial activities were evaluated by measuring a diameter of the inhibition ring. Fig. 2 Antibacterial activity of (E)-methyl isoeugenol and aterpineol against #09-292 H. pylori clinical strain. a control, b 100 lg/disk of (E)-methyl isoeugenol