Chemical Composition and In Vitro Anti-Helicobacter pylori Activity of Campomanesia lineatifolia Ruiz & Pavón (Myrtaceae) Essential Oil

Helicobacter pylori is the most common cause of gastritis and peptic ulcers, and the number of resistant strains to multiple conventional antimicrobial agents has been increasing in different parts of the world. Several studies have shown that some essential oils (EO) have bioactive compounds, which can be attributed to antimicrobial activity. Therefore, EOs have been proposed as a natural alternative to antibiotics, or for use in combination with conventional treatment for H. pylori infection. Campomanesia lineatifolia is an edible species found in the Brazilian forests, and their leaves are traditionally used for the treatment of gastrointestinal disorders. Anti-inflammatory, gastroprotective, and antioxidant properties are attributed to C. lineatifolia leaf extracts; however, studies related to the chemical constituents of the essential oil and anti-H. pylori activity is not described. This work aims to identify the chemical composition of the EO from C. lineatifolia leaves and evaluate the anti-H. pylori activity. The EO was obtained by hydrodistillation from C. lineatifolia leaves and characterized by gas chromatography–mass spectrometry analyses. To assess the in vitro anti-H. pylori activity of the C. lineatifolia leaf’s EO (6 μL/mL–25 μL/mL), we performed broth microdilution assays by using type cultures (ATCC 49503, NCTC 11638, both clarithromycin-sensitive) and clinical isolate strains (SSR359, clarithromycin-sensitive, and SSR366, clarithromycin-resistant). A total of eight new compounds were identified from the EO (3-hexen-1-ol (46.15%), α-cadinol (20.35%), 1,1-diethoxyethane (13.08%), 2,3-dicyano-7,7-dimethyl-5,6-benzonorbornadiene (10.78%), aromadendrene 2 (3.0%), [3-S-(3α, 3aα, 6α, 8aα)]-4,5,6,7,8,8a-hexahydro-3,7,7-trimethyl-8-methylene-3H-3a,6-methanoazulene (2.99%), α-bisabolol (0.94%), and β-curcumene (0.8%)), corresponding to 98.09% of the total oil composition. The EO inhibited the growth of all H. pylori strains tested (MIC 6 μL/mL). To our knowledge, the current study investigates the relation between the chemical composition and the anti-H. pylori activity of the C. lineatifolia EO for the first time. Our findings show the potential use of the C. lineatifolia leaf EO against sensitive and resistant clarithromycin H. pylori strains and suggest that this antimicrobial activity could be related to its ethnopharmacological use.


Introduction
Helicobacter pylori, discovered by Marshall and Warren [1], is a Gram-negative spiral flagellated bacterium found in the stomachs of patients with chronic gastritis and peptic ulcers, and infects the stomachs of approximately half the global population [2]. The current treatment of H. pylori infection involves a combination of an antisecretory drug, such as proton pump inhibitors (PPI) or H 2 -blockers, together with two or three antimicrobials [3][4][5][6][7]. As expected for a multidrug therapeutic regimen, several side effects may be associated. In these cases, the patient may abandon treatment, contributing to the emergence of drugresistant strains of H. pylori [8]. Bi et al. [9] demonstrated that herbal medicines are effective in treating gastric ulcers and have fewer side effects and lower recurrence rates. Moreover, the combination of herbal medicines and conventional anti-gastric ulcer drugs displays a synergistic effect against gastric ulcers [10,11] In 2017, the World Health Organization listed clarithromycin-resistant H. pylori in the high priority category, in which prescribed treatment requires attention. The main point of this problem is that drug resistance has been markedly increasing and, consequently, the success of therapeutic regimens that include clarithromycin for eradicating H. pylori infection has been declining [12].
The genus Campomanesia is a member of the Myrtaceae family that comprises more than 100 genera and 4000 species around the world [13]. In Brazil, 41 species are known, of which 32 are endemic. These plants are represented by shrubs and trees found in the Brazilian Amazon rainforest, Atlantic forest, Cerrado and Caatinga, occurring in several regions of the country [14]. The Campomanesia species are popularly known as "guavira", "guabirobeira", or "gabiroba", characterized by fruits with a citrus aroma and flavor that are used to prepare liqueurs, juices and sweets [15,16]. In addition, several species of Campomanesia are traditionally used for medicinal purposes, such as dysentery, stomach problems, diarrhea [17], cystitis and urethritis [18], as well as hepatic disorders [19]. Due to empirical knowledge of the health benefits associated with these species, the biological and pharmaceutical effects associated with Campomanesia have been the object of studies by different research groups [20][21][22][23].
When considering the antimicrobial activity, the essential oil obtained from C. guazumifolia showed strong inhibition for Staphylococcus aureus, Escherichia coli and Candida albicans, and demonstrated significant antioxidant activity [21]. In a study on the antimicrobial potential of essential oils from Cerrado plants against multidrug-resistant foodborne microorganisms, the essential oil from C. sessiliflora showed strong inhibition in clinical Staphylococcus strains, which cause bovine mastitis and are related to milk-borne diseases [22]. Furthermore, the antimicrobial and antibiofilm activities of the EO of the C. aurea showed sufficient capability against Listeria monocytogenes [23]. These results reveal the antimicrobial potential of the essential oils obtained from the Campomanesia species.
Although several studies showed that the plants and plant extracts/constituents exhibit anti-H. pylori activity and gastroprotective action, in recent years, few studies have described the effects of specific essential oils on H. pylori growth and viability [24][25][26].
Previous studies conducted by our research group demonstrated that C. lineatifolia Ruiz & Pavón leaf extracts protect the gastric mucosa against experimental ethanoland indomethacin-induced gastric lesions [27]. Furthermore, the gastroprotective effects appear to be related to their antioxidant properties and polyphenolic contents. In a study of Brazilian medicinal plants in lipopolysaccharide (LPS)-stimulated THP-1 cells, Henriques et al. [28] demonstrated the in vitro tumor necrosis factor α (TNF-α) inhibitory activity of C. lineatifolia.
By considering a causal relationship between inflammation, gastric ulcers and H. pylori infection, we proposed a study to determine whether C. lineatifolia essential oil (EO) has anti-H. pylori activity. In the present study, we also aimed to identify the chemical composition of the EO that could be related to anti-H. pylori activity so that it can be used as an alternative or adjunctive agent to the current therapy for H. pylori infection.
As far as we know, our study investigates, for the first time, the chemical composition and the anti-H. pylori activity from the essential oil of C. lineatifolia.

In Vitro Anti-Helicobacter pylori Activity Evaluation
The antimicrobial activity of the C. lineatifolia EO at 6 µL/mL-25 µL/mL concentrations was evaluated against ATCC 49503; NCTC116328 (both clarithromycin-sensitive strains), and SSR359; SSR366, clarithromycin-sensitive clinical isolate, clarithromycin-resistant clinical isolate, respectively. Clarithromycin was used as a positive control with inhibitory activity for the H. pylori growth in a concentration range of 0.03 µg/mL-256 µg/mL. It was shown that the EO was able to inhibit the growth of all the H. pylori strains at the lowest concentration tested, with a calculated MIC of 6 µL/mL. Clarithromycin inhibited the H. pylori growth at 0.03 µg/mL in the sensitive strains (ATCC 49503, NCTC11638, and SSR359) and at 8 µg/mL in the clarithromycin-resistant clinical isolate (SSR366).
The phytochemical analysis of the Psidium guajava leaves, Myrtaceae family, showed the presence of the ether 1,1-diethoxyethane (1) as one of the constituents that confer fruit aroma [38].
Compound 6, β-curcumene, was identified as one of the major constituents in the essential oil from the rhizomes of Curcuma amada and C. longa (Zingiberaceae) [48,49]. Curcuma sp. is traditionally used to treat various intestinal diseases, fever and jaundice [50] and its anti-H. pylori activity has been described [51,52]. In the Myrtaceae family, γ-curcumene is found in the essential oil of Eucalyptus microtheca leaves [32] and β-curcumene in Myrcia sylvatica [53].
The treatment for H. pylori infection commonly involves first-line triple therapy with clarithromycin (a combination of second-generation PPI, amoxicillin and clarithromycin for 14 days) [7]. In areas of high clarithromycin resistance (> 15%), a quadruple bismuth therapy (PPI, bismuth salt, tetracycline, nitroimidazole) or a concomitant therapy (PPI, clarithromycin, metronidazole, amoxicillin) is recommended [3][4][5]. However, these regimens may increase the risk of side effects and the cost of treatment, which brings difficulties for their implementation in clinical practice and often causes treatment abandonment by the patient. In addition, the use of multiple antimicrobial agents for H. pylori infection may increase the risk of future microbial resistance [56]. In this scenario, new regimens and approaches that allow the minimal use of antimicrobials for a shorter treatment period are needed.
Plant-based medicines are effective in treating gastric ulcers and have fewer side effects and lower recurrence rates, and when used in monotherapy or in combination with conventional drugs, they can become an alternative to treat certain gastric ulcers and prevent their recurrence [9]. Clinical studies have already demonstrated the efficacy of some natural products for the treatment of gastrointestinal diseases. For example, capsaicin (derived from the peppers of the Capsicum genus) protected the gastric mucosa in healthy volunteers who took acetylsalicylic acid [57] and decreased the intensity of dyspeptic symptoms in patients with functional dyspepsia [58]. Curcumin, from Curcuma longa (turmeric), improved dyspeptic symptoms, improved quality of life, and provided a satisfactory equivalent to omeprazole in a double-blind, placebo-controlled trial in patients diagnosed with functional dyspepsia [59]. Patients treated with a Maytenus ilicifolia (espinheira-santa) extract for 28 days showed a significant improvement compared to the placebo group, with regard to the overall dyspeptic symptoms, and especially for symptoms of heartburn and gastralgia [60].
Regarding the essential oils, Korona-Glowniak et al. [24] evaluated the in vitro activity of the five essential oils, silver fir, pine needle, tea tree, lemongrass and cedarwood oils, against H. pylori. The most active against the clinical strains of H. pylori were cedarwood oil and oregano oil. Moreover, the cedarwood oil inhibited the urease activity at sub-inhibitory concentrations, suggesting that this essential oil can be a useful component of the plant preparations supporting the eradication of H. pylori therapy. The Pelargonium graveolens oil exhibited good activity against H. pylori at a MIC of 15.63 µg/mL, and once combined with clarithromycin, a significant synergistic effect appeared at a fractional inhibitory concentration index of 0.38 µg/mL [25]. Thus, the combination of herbal medicines with anti-H. pylori activity could improve the outcome for gastric ulcer patients.
The alcohol, 3-hexen-1-ol, is described as one of the majority constituents of the EO of Fagopyrum esculentum (Polygonaceae) flowers and is related to the antimicrobial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and Gram-negative bacteria, Escherichia coli and Pseudomonas lachrymans [61]. Pino et al. [62] also described the presence of 3-hexen-1-ol as the majority constituent of the EO from Galinsoga parviflora (Asteraceae) leaves and demonstrated that this EO exhibited antimicrobial activity against Gram-positive bacteria S. aureus and Bacillus cereus. Similarly, this alcohol corresponded with the majority constituent in the C. lineatifolia EO, and together with the data described in the literature, one can suggest its potential anti-H. pylori activity, observed in this study.
It has been shown that β-curcumene and α-bisabolol, the majority constituents of the EOs of Curcuma sp. (Zingiberaceae) and Plinia cerrocampanensis (Myrtaceae), respectively, have anti-H. pylori activities [46,47,49]. In addition, Brehm-Stecher & Johnson [66] described that bisabolol is able to increase bacterial wall permeability and, consequently, susceptibility to antibiotics of clinical importance, suggesting a possible mechanism for its activity.
Studies indicate that plant essential oils have bactericidal effects against H. pylori, and this activity could be related mainly to the terpenoid content, followed by other compounds, such as phenols, alcohols, aldehydes and ketones [24,26,36,67]. Additionally, the EOs have equivalent bactericidal effects against both the antibiotic-susceptible strains and other H. pylori strains that resist antibiotics, suggesting that EOs may be promising candidates to treat H. pylori infection [68].
All these findings related to the compounds identified in the EO of C. lineatifolia may suggest that the anti-H. pylori activity of the EO may originate from one of its main constituents or even synergism between them. However, it is important to note that the antibacterial assay was performed as an initial screen to determine whether the C. lineatifolia EO demonstrated anti-H. pylori activity. In the current study, the microbroth dilution assay data presented demonstrates the bacteriostatic effect of the EO at a MIC of 6 µL/mL, as H. pylori growth was inhibited at all concentrations of EOs tested (6 µL/mL-25 µL/mL). Furthermore, lower concentrations of EO (i.e., < 6 µL/mL) could possibly demonstrate anti-H. pylori activity, and will be the subject of our future MIC and minimum bactericidal concentration (MBC) studies after purifying additional EO from fresh C. lineatifolia leaves to determine whether the EO also possesses bactericidal activity.
These results showed the potential of the C. lineatifolia EO against clarithromycinsensitive and resistant H. pylori-type cultures and clinical isolate strains, making the species promising in the treatment of gastric ulcers associated with H. pylori infection.

Plant Material
Campomanesia lineatifolia leaves were collected in February 2020 from Minas Gerais, Brazil (19 • 52 9.87 S; 43 • 58 12.04 W). The species was identified by Dr Marcos Sobral from the Botany Department of Biological Sciences, the Institute at the Federal University of Minas Gerais (UFMG), Belo Horizonte. A voucher specimen (BHCB 150.606) was deposited at the UFMG Herbarium. The registration in the National System of Genetic Heritage and Associated Traditional Knowledge Management (SisGen) was carried out and given the code A216C7C.

Isolation of the Essential Oil
The EO was extracted from the fresh leaves of C. lineatifolia by hydrodistillation using a Clevenger-type apparatus. The extraction was carried out for 3 h by mixing 143 g of plants in 1000 mL of distilled water. The EO were dried with sodium sulphate anhydrous (Dinâmica Química Contemporânea Ltda), and concentrated under reduced pressure by a rotatory evaporator to evaporate water. The pure oil was stored at −25 • C until further analysed. The essential oils' yield was 0.5 mL.

Analysis of the Essential Oil
The essential oil chemical composition assessments and the identification of the main constituents were conducted by GC-MS analyses. The GC-MS analysis was performed in triplicate using a Hewlett-Packard 6890N gas chromatograph, coupled with a 5975B mass selective detector (MSD; Agilent Technologies, Inc., Santa Clara, CA, USA) operating at 70 eV over a mass range of 35-500 amu and a scanning speed of 0.34 and equipped with a DB-5MS fused-silica capillary column (5% phenylmethylsiloxane; length-30 m, internal diameter-0.25 mm, film thickness-0.25 µm). The oven temperature was raised from 70 • C to 290 • C at a heating rate of 5 • C/min and held isothermal for 10 min; injector temperature, 250 • C; interface temperature, 300 • C; carrier gas, helium at 1.0 mL/min. The sample oil was dissolved in Et 2 O at the ratio of 1:1000 and injected into a pulsed split mode. The flow rate was 1.5 mL/min for the first thirty seconds (0.50 min), then modified to 1.0mL/min for the remainder of the run; the split ratio was 40:1. The identification of the constituents was based on the comparison of their linear retention indices relative to the series of n-hydrocarbons (C6-C40), and on computer matching against commercial (NIST or Wiley library) and home-made library mass spectra that were built up from pure substances and components of known oils, and MS literature data.

Bacterial Strains and Culture Conditions
The H. pylori strains were obtained from the American Type Culture Collection (ATCC 49503, clarithromycin-sensitive), National Collection of Type Cultures (NCTC11638, clarithromycin-sensitive), and from Tallaght University Hospital, Dublin 24, Ireland (SSR359, clarithromycin-sensitive clinical isolate, and SSR366, clarithromycin-resistant clinical isolate). All strains were maintained on Columbia blood agar (CBA) plates containing 5% sheep's blood (VWR) at 37 • C under microaerobic conditions (CampyGen, Oxoid). Bacteria were inoculated into a brain heart infusion (BHI) broth (Sigma) containing 10% foetal bovine serum (Gibco) and incubated under microaerobic conditions at 37 • C with shaking (120 rpm) for 24h to prepare the bacterial suspensions for the broth microdilution assays.

Broth Microdilution Assays
The determination of the minimum inhibitory concentration (MIC) of EO against H. pylori was evaluated using the broth microdilution assays in 96-well plates, according to the Standards for Antimicrobial Susceptibility Testing protocol [69], with adaptations [70]. The EO was diluted with the BHI broth supplemented with 10% foetal bovine serum and dimethylsulfoxide 2% (DMSO, Sigma) at 47 • C to prepare the concentrations at 6 µL/mL-25 µL/mL (0.6-2.5% v/v).
A total of 100 µL aliquots of overnight bacterial suspension (approximately 10 6 CFU/mL; 1:20 0.5 McFarland Standard) [70] was added to each well of a 96-well plate. The cultures were incubated with 100 µL aliquots of EO for 72 h at 37 • C under microaerobic conditions (CampyGen) with shaking (120 rpm). The absorbances at a wavelength of 600 nm were read using a Varioskan LUX Microplate Reader (Thermo Fisher Scientific).
The MIC values were determined as the lowest concentration at which no bacterial growth occurred. Clarithromycin was used as the positive control at 0.03 µg/mL-256 µg/mL (0.003-25.6% v/v).

Statistical Analysis
The experiments were performed in triplicate, and the results were expressed as mean ± SEM. A one-way ANOVA using GraphPad Prism 6.0, followed by a Bonferroni post-test, was performed, considering values of a p ≤ 0.05 as statistically significant. The MIC values were calculated considering the concentrations that did not differ statistically from the BHI broth control.

Conclusions
As far as we know, our study investigates, for the first time, the chemical composition of the essential oil from C. lineatifolia fresh leaves and its anti-H. pylori activity. Using the GC-MS technique, it was possible to identify eight new compounds for the species, representing 98.09% of the total oil composition, the major components of the alcohol 3-hexen-1-ol and the sesquiterpene α-cadinol.
Our findings demonstrate that the C. lineatifolia EO has anti-H. pylori activity at the lower concentration tested (MIC 6 µL/mL), suggesting a bacteriostatic property and a relation with its ethnopharmacological use as a medicinal plant for the treatment of gastric ulcers. Furthermore, the antibacterial activity against clarithromycin-resistant clinical isolates indicates that essential oils could be used in combination, as even antibiotics with high activity are not effective when used singly in therapeutic regimens involving patients with resistant H. pylori infection. Evidence for the use of medicinal plants in therapy shows that they have fewer adverse effects and lower rates of infection recurrence, as well as reducing time and improving the patient's compliance with treatment.
For the follow-on work to this study, additional EO from fresh C. lineatifolia leaves will be purified to determine whether the EO also possesses bactericidal activity by performing MBC assays. In addition, it will be necessary to examine each antibacterial component of essential oil separately and in combination to ascertain whether they act alone or synergistically. Furthermore, studies involving in vivo models of gastric ulcers are proposed to determine the mechanisms by which the chemical constituents described here exert their anti-H. pylori activity.  Data Availability Statement: All data generated or analyzed during this study are included in this article.