Anti-Helicobacter pylori Effects of Thymus caramanicus Jalas Essential Oils: A New Antimicrobial Approach

Background Helicobacter pylori are the principal causative factor in the etiological factors of chronic, active, or type B gastritis; peptic and duodenal ulcers; stomach carcinoma; and epithelial tissue lymphoid malignancies. It infects more than half of the population worldwide. To reduce H. pylori production, pharmacological therapy of H. pylori diseases typically involves using threefold treatment methods. However, as a result of such therapy, antimicrobial resistance is commonly developed. Alternative therapeutics for H. pylori diseases are thus of particular interest. Methods Thyme essential oils (EOs) obtained from T. caramanicus Jalas plants in Iran were tested for antibacterial activity against H. pylori obtained from 320 poultry specimens in this investigation. Antibacterial activity was measured using inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs). The impact of T. caramanicus Jalas essential oils on H. pylori isolate cagA, vacA, and babA2 gene expression was evaluated using a quantitative real-time PCR method (p < 0.05). Results The chemical content of these EOs varied significantly according to chromatographic examination. Thymol, carvacrol, and terpinene-4-ol are the most abundant components in these EOs. H. pylori was recognized as a Helicobacter species with a 175-bp PCR product of 16S rRNA in 20/20 (100%). According to PCR results, all 20 (100%) isolates belonged to H. pylori. The EOs inhibited H. pylori in a dose-dependent manner, with T. caramanicus Jalas being the most effective, followed by pterygium EOs in decreasing order. At 8 mg/mL of T. caramanicus Jalas EOs, IZs against H. pylori were 27.4 ± 0.42 mm, and at 8 mg/mL of pterygium, IZs against H. pylori were 1 ± 0.02. T. caramanicus Jalas essential oils were used to treat all bacteria, and the findings showed that T. caramanicus Jalas had a substantial inhibitory impact on the expression of cagA, vacA, and babA2 virulence-related genes (p < 0.05). Conclusions In a dose-dependent manner, the EOs of T. caramanicus Jalas EO demonstrated a high degree of antimicrobial property against H. pylori bacteria. The most efficient EOs were those from T. caramanicus Jalas with relative concentrations of thymol and carvacrol, followed by the coumarin-dominated pterygium EO with reduced antibacterial activity.


Introduction
Helicobacter pylori (H.pylori) is an underlying cause of the world's most common and chronic pathogenic bacteria, afecting about half of the country's population.Peptic ulcer disease, gastritis, stomach carcinoma, and functional dyspepsia are all believed to be caused by H. pylori [1][2][3].Extradigestive diseases (idiopathic thrombocytopenic purpura, vitamin B12 insufciency, and unexplained iron defciency anemia) were also added to the criteria for H. pylori elimination [4,5].Te prevention of H. pylori has proven difcult for clinicians recently due to the pathogen's developing antibiotic resistance.H. pylori diseases are generally treated with a proton pump inhibitor and two antimicrobials: amoxicillin, clarithromycin, or metronidazole (triple therapy) [6].Levofoxacin can be used instead of clarithromycin during the frst treatment, with greater effcacy [7].Furthermore, an alternate experimental approach is required when regional clarithromycin tolerance exceeds 20% [8].When the combination therapy fails, a bismuthbased quadruple medication (bismuth salts, tetracycline, and metronidazole with PPI) or a non-bismuth-based quadruple treatment (levofoxacin, nitazoxanide, and doxycycline plus PPI) should be advised [9].Only symptomatic individuals require treatment.As a result, asymptomatic patients serve as a reservoir for H. pylori isolates in the community, particularly antibacterial drug strains that spread rapidly [10].A dietary line focused on maintaining a low level of H. pylori concentration in the stomach mucosa would help people with microbial infections, limiting the establishment of acute gastroenteritis and an increased risk of gastric ulcers [11].Urease is a crucial molecule that allows H. pylori to survive and colonize by starting the hydrolysis reaction, which produces ammonia, which is used to neutralize gastric acid and generate a favorable pH condition.As a result, urease is regarded as a crucial objective in developing and applying antimicrobial agents [12].
Vaccination, probiotics, photodynamic inactivation, phage treatment, and phytomedicine have all been researched as medicinal natural antimicrobial agents in past years [13].Te World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population.Te medicinal potential of plant species is because of the occurrence of secondary phytoconstituents, which have numerous functions, such as antioxidant, antimicrobial, cytotoxic, anticancer, and antiviral [14,15].
Numerous researches have been conducted in the quest for anti-H.pylori activities and gastroprotective efect in plants and plant herbal blends [16,17].Furthermore, natural-source components have been studied extensively as possible efective natural compounds for treating H. pylori infection.Antimicrobial, antifungal, antiviral, antiparasitic, and antioxidant activities have been discovered in herbal extracts [16].Only a few studies have examined the impact of particular herbal extracts on H. pylori survival and proliferation [17].Te in vitro function of herbal extracts is poorly understood.Te frequency of H. pylori in the gastrointestinal of mice treated with lemongrass was dramatically decreased compared to control animals, according to research by Ohno et al. [17].Hartmani et al. evaluated the anti-H.pylori efcacy of a 2 : 1 blend of Satureja hortensis and Origanum vulgare subsp.hirtum aromatic plants.Te combination efciently eliminated this infection in 70% of the mice in vivo [18].Plant-derived products have an imperative biological role against certain pathogenic organisms and were considered to be a major source of modern drugs [18].
Tymus (Lamiaceae) plants are well known as fragrant plants from the Eastern Mediterranean.In the Persian environment, 14 of the 215 species farmed globally are found [19].T. caramanicus Jalas (Avishan) is an indigenous plant to Iran, and the leaves have historically been used for rheumatism, skin problems, and antimicrobial purposes [20].In various regions of Iran, Tymus taxa are used as culinary herbs and teas.Te antioxidant properties of pterygium and the composition and antioxidant capacity of the extracts of T. caespititius, T. camphoratus, and T. mastichina from Portugal have been documented [20][21][22].Plant oils from various Tymus species, particularly T. revolutus, T. pubescens, T. serpyllum, T. kotschyanus, have been shown to have antibacterial action [23].Te plant oils from T. caramanicus were high in carvacrol at various developmental stages.Te compounds also showed antimicrobial efects against Gram-positive and Gram-negative ATCC microbial species.Tymol has also been shown to have antimicrobial properties [24].Tymol has been proposed as the physiologically active substance of thyme in the treatment of H. pylori [24,25].
Te objective of this study was to fnd EOs having potent antibacterial properties against H. pylori proliferation.T. caramanicus EOs were tested in vitro for anti-H.pylori activities.We investigated thyme EO's phytochemical profle and urease inhibitory capabilities with varied anti-Helicobacter activity.

Plant Identifcation and Collecting. Between July and
September 2021, aerial portions of T. caramanicus Jalas were gathered.Tese wild plants were discovered in the research sites' common grounds.Furthermore, these taxa are not classifed as threatened or protected.As a result, no special authorization was needed to collect and study the plants indicated.Voucher specimens had been kept in the Herbarium of I.A.U.Shahrekord Branch (no.231).

Plant Material Preparation and Essential Oil Extraction.
For the extraction of the essential oil in this study, the methodology of Chimnoi et al. [26] was used.Freshly harvested T. caramanicus Jalas plants (1.0 kg) were introduced to 2 L ethanol in a 4 L round-bottom glass beaker (with vegetal material/extraction solvent rate � 1/10 (w/v)) and exposed to water distillation for 3 hours using product obtained type apparatus.Te essential oil was lighter than water and could be separated using a separating funnel.Te oil was dried over anhydrous sodium sulfate.Te amount of each oil was then measured in milliliters (mL), dried over anhydrous sodium sulfate, and kept in the dark at 4 °C until use.

Analysis of Essential
Oils.N-hexane was used to dilute 2 µL of T. caramanicus Jalas essential oils to 1 mL.A Shimadzu GC-2010 Plus gas chromatography system, connected to a Shimadzu QP2010 Ultra mass spectrometer, was used to evaluate 1 µL of essential oil liquids utilizing GC-MS (Shim-Pol, Izabelin, Poland).
A silicon dioxide capillary column ZB-5MS (30 m, 0.25 mm i.d.) with a layer thickness of 0.25 m was employed to isolate substances (Phenomenex, Torrance, CA, USA).Te following oven temperature program was launched at 50 degrees Celsius, held for 3 minutes, then raised at 8 degrees Celsius each minute to 250 degrees Celsius and held for 2 minutes.Te spectrum analyzers were set to electron-impact operation, with a wavelength of 40-500 amu, a 70-eV electron afnity, and a scan rate of 0.20 seconds per scanning.Te pump, connector, and ion generator were all held at temperatures of 250, 250, and 220 degrees Celsius, respectively.Split infusion (1 μL) was performed using a split frequency of 1 : 20 with helium as the gas phase at a fow rate of 1.0 mL/min.Te stability indices were calculated using the same operation 2 Evidence-Based Complementary and Alternative Medicine circumstances as a similar set of n-alkanes (C8-C20).A computer-assisted spectrum library (NIST 2011 Gaithersburg, MD, USA; MassFinder 2.1 and 4.0, Hamburg, Germany) used mass spectroscopy of reference compounds and MS information from the literature to identify substances.

Isolation and Identifcation of the Helicobacter Genus.
Tree hundred and twenty poultry samples were randomly gathered from farms in the Shahrekord region, Iran.Te Gram-negative, S-, or C-shaped bacteria were seen by transferring the colonies on slants and staining them with gram.It was discovered that rod and coccoid forms exist.Biochemical tests were performed on the purifed cultures to confrm their identity.As a control strain, the H. pylori ATCC 700392 strain was used.Te Helicobacter genus was identifed using 16S rRNA (Table 1).Lactofeed Biotech Group approved oligonucleotide sequences (Iran).Wilkins-Chalgren Anaerobe Broth-enhanced colonies were subcultured.Using a DNA extraction kit, genomic DNA was isolated from bacteria (Cinna-colon, Iran).Te process was carried out according to the manufacturer's instructions.

Statistical Analysis.
For each group, the data were reported as the mean + SEM.Te statistical analysis was conducted using computer software (SPSS version 20).Oneway analysis of variance (ANOVA) was used to compare the groups, followed by LSD post hoc multiple comparisons.p values of less than 0.05 were deemed statistically signifcant.Evidence-Based Complementary and Alternative Medicine Te monoterpene hydrocarbons are the primary distinguishing characteristics of the two EOs.T. caramanicus Jalas were used to make these EOs.However, the essential characteristics of T. caramanicus Jalas EO are the bicyclic monoterpenoids (3) and pinene (6).In contrast, the essential oil of pterygium is characterized by a monocyclic molecule, 2,2-bis(4-hydroxydiphenyl) propane.Monoterpene alcohols are the signifcant components in the two T. caramanicus Jalas chemically investigated EOs.Tis category includes essential oils hydrodistilled from T. caramanicus Jalas.Tymol, carvacrol, and terpinene-4-ol are the most abundant components in these EOs.Aside from alcohols, the existence of monoterpene hydrocarbons is a distinguishing feature of the EOs listed.p-Cymene is a critical component of thyme and oregano EOs, whereas additional terpinenes, such as c, α, and ε-terpinene (=terpinolene), as well as -terpineol, were found in tea tree EO.

Isolation and Identifcation of the Helicobacter.
In 320 cases of poultry fesh, the presence of H. pylori was evaluated.Te 20 positive Helicobacter spp.were detected by urease, oxidase, and catalase assays after 4 hours of incubation, respectively, by a purple hue, a blue/purple color, and the generation of oxygen bubbles.Helicobacter spp. was found in 20 of 320 (6.25%) poultry meat specimens.Te 16SrRNA gene PCR amplifcation was used to confrm all of the strains.Te electrophoretically displayed PCR results from 20 Helicobacter spp.were identifed from 320 poultry fesh specimens.H. pylori was recognized as a Helicobacter species with a 175-bp PCR product of 16S rRNA in 20/20 (100%).According to PCR results, all 20 (100%) isolates belonged to H. pylori.

Antibacterial Tests of T. caramanicus Jalas EOs against H. pylori.
Te EOs inhibited H. pylori in a dose-dependent manner, with T. caramanicus Jalas being the most efective, followed by T. caramanicus EOs in decreasing order (Figure 2(a)).Te 8 mg/mL dosage of these T. caramanicus Jalas EO produced around 27.4 ± 0.42 mm inhibitory zones against H. pylori.Te 8 mg/mL dosages, except pterygium EOs, resulted in less than 5 mm inhibitory zones (Table 3).

Results of Minimum Inhibitory Concentration (MIC) of
Cefxime and EOs of T. caramanicus Jalas.In this test, the minimum inhibitory concentration of cefxime was compared to the performance of two extracts of T. caramanicus Jalas EOs.It is noted that T. caramanicus Jalas EOs have a high inhibitory concentration on H. pylori strains ranging from 16% to 72%.
Te lowest number in each row of samples determined the best MIC performance.Tis is because the bacteria stopped growing at the same low dose of the drug.Te more efectively bacterial growth is inhibited at a low concentration, the better candidate the essential oil is for treatment (Table 4).

Antibacterial Virulence Gene Expression Analysis of EOs.
Te virulence-related genes cagA, vacA, and babA2 in MDR bacterial strains were assessed using a quantitative real-time PCR approach.T. caramanicus Jalas essential oils were used to treat all bacteria, and the fndings showed that T. caramanicus Jalas had a substantial inhibitory impact on    Evidence-Based Complementary and Alternative Medicine the expression of cagA, vacA, and babA2 virulence-related genes (p < 0.05) (Figure 2(b)).Te T. caramanicus Jalas signifcantly reduced the expression of cagA, vacA, and babA2 genes in all MDR strains, which could be due to the interaction of thymol with transcription factors, which causes these factors to be inactivated, virulence genes to be not transcribed, and virulence gene expression to be reduced (Figure 2(c)).

Discussion
Te plant's naturally extracted oils have been utilized in food storage, medicine, complementary therapy, and ecological cures for hundreds of years [27].According to the previous research [28][29][30], natural compounds have antimicrobial efects on various bacteria.Moreover, aromatic plants difered in their antibacterial action.By measuring antimicrobial and  Evidence-Based Complementary and Alternative Medicine antivirulence ability, we evaluated anti-Helicobacter characteristics of T. caramanicus Jalas essential oils commercially available.T. caramanicus Jalas (thyme) oil showed the highest activity against H. pylori strains.Tese EOs have signifcant biocompatibility against H. pylori, based on the biosynthetic pathway parameters of O'Donnell et al. [30].Te remaining EOs had good biocompatibility, with MICs ranging from 2 to 4%.Of the T. caramanicus Jalas EOs tested, the T. caramanicus Jalas essential oil had the lowest minimum inhibitory concentration (MIC).Essential oils have previously been shown to have anti-H.pylori activity [31], and of the 13 plant extracts evaluated for their inhibitory activity on H. pylori in the study [32], the essential oils of Cymbopogon citratus (lemongrass) and Lippia citriodora (lemon verbena) were found to have the most potent antimicrobial activities for H. pylori strain ATCC 49503.Bergonzelli et al. (2003) studied the efects of 60 plant extracts on the development of H. pylori in vitro and found that 30 essential plant oils had a potent efect followed by 15 essential oils with a moderate efect.Carvacrol, isoeugenol, nerol, citral (�neral + geranium), and sabinene were found to have the strongest anti-H.pylori actions among the chemicals found in these oils [33].Our research found monoterpenoids to be the most prevalent elements of the T. caramanicus Jalas EOs.Tis category of terpenoids accounts for over 90% of the total content of these herbal extracts.Sesquiterpenoids make up a minor portion of their overall makeup.Terpenoids are water-insoluble to relatively water-insoluble, although they dissolve in the phospholipid bilayer.Terpenoids are thought to have antibacterial properties because of their capacity to break or penetrate lipid structures, resulting in a loss of membrane stability, dissipation of the proton gradient, and disruption of internal pH equilibrium [34,35].Te varying MIC values of the EOs may be related to their diferent phytochemical compositions.Monoterpene hydrocarbons were the most abundant components in pterygium, the least efective EO against H. pylori of the two EOs studied, implying marginal biocompatibility against H. pylori.Despite this minor variation, T. caramanicus Jalas had lower MIC values for the EOs than the pterygium group, probably due to the presence of monoterpene alcohols.Te MIC values obtained increased as the concentration of EO increased.Tymol is a potent antibacterial agent, because of hydroxyl group and delocalized electrons essential for disrupting bacterial membranes [33], binding to transmembrane and intracellular molecules, and altering membrane fuidity, potassium ion permeability, and ATP contamination in microorganisms.Carvacrol and coumarin have a substantial inhibitory impact against H. pylori by disrupting and depolarizing the cell membranes by attacking membrane proteins and internal drug targets.However, the antimicrobial efect of carvacrol was diminished in the presence of thymol [36].Tymol or carvacrol identifed in T. caramanicus Jalas EO was discovered to be the primary components in our investigation, with the other two being identifed in trace levels.Previous substances found in T. caramanicus Jalas EOs, such as -terpinene, _-longipinene, geranyl acetate, longifolene, (E)-α-carryophyllene, α-humulene, caryophyllene oxide, cedrol, and α-eudesmol, have been demonstrated to have no antimicrobial efect against Gram-negative bacteria in other research studies [32,37,38].Moreover, certain substances with no antimicrobial activities have been observed to have a synergistic impact when combined with antimicrobial drugs; for instance, the existence of p-Cymene in combination with carvacrol may boost the oil's antibacterial efect [39].Te anti-H.pylori efcacy of the T. caramanicus Jalas EO is derived from monoterpene alcohols and aldehydes in pterygium EOs.Geranial and neral in T. caramanicus Jalas EOs have also been identifed as compounds with increased anti-H.pylori antibacterial activities when compared to other materials in various research types [19].
A very diferent scenario existed in the instance of T. caramanicus Jalas EOs.Tere were no monoterpenoids found in this oil.Coumarin is the most prevalent in this EO, with cedrane being the most distinctive component.To date, no studies have been conducted on the biological action of pterygium EOs and their metabolites against H. pylori.Coumarin, which belongs to the pterygium family, has been shown to have anti-H.pylori action, as have the major components found in pterygium essential oils.Its antimicrobial property against Streptococcus mutants and antifungal characteristics has been described as dependent on DNA polymerase suppression [40].Pterygium oil was not one of our research's most efective essential oils.
Te generation of reactive oxygen and nitrogen molecules is linked to H. pylori infection preceding gastric mucosa colonization.As a result, antioxidants can be considered a supplemental medication for treating H. pylori [41].Using nutritious nutritional supplements containing compounds with potent antioxidant activity can boost the body's defenses and prevent H. pylori from multiplying [42].Furthermore, most research has shown that infectious disease reduces the number of antioxidants in stomach juice [43].In vivo and in vitro research has revealed that antioxidants like vitamin C and astaxanthin scavenge not only oxidative stress but also have antibacterial action against H. pylori [44].Bibi et al. (2017) earlier hypothesized a relationship between the existence of the H. pylori babA2/cagA+/vacAs1 genotype and the prevalence of gastroenteritis, stomach carcinoma, and ulcerative colitis [45].In H. pylori isolates recovered from clinical specimens of human and animal populations, a high incidence of vacA, cagA, iceA1, oipA, and babA2 genotypes has also been described [45][46][47].
Moreover, H. pylori isolates recovered from varying dietary specimens have shown a signifcant frequency of these genes [48,49].Previous studies have linked the H. pylori genotypes vacA, cagA, iceA, oipA, and babA2 to interleukin-8 and cytotoxin exudation, attachment to gastric epithelial cells, increase in the frequency of infammatory impact, vacuolization, apoptosis process in gastric epithelial cells, stomach ulcers ulceration, and increased intense neutrophilic incursion [47][48][49][50][51].In this study, the T. caramanicus Jalas signifcantly reduced the expression of cagA, vacA, and babA2 genes in all MDR strains, which could be due to the interaction of thymol with transcription factors, which causes these factors to be inactivated, Evidence-Based Complementary and Alternative Medicine virulence genes to be not transcribed, and virulence gene expression to be reduced.Te use of essential oils of T. caramanicus Jalas EO may aid in efectively managing H. pylori, which is widely disseminated worldwide.Furthermore, as the multiresistant profle grows, as does the public's desire to consume "green goods," the usage of these EO will aid in managing H. pylori infection, perhaps reducing the pathogen's spread from asymptomatic carriers.

Conclusion
EO of T. caramanicus Jalas demonstrated a high degree of antimicrobial property in a dose-dependent manner against H. pylori strains.Its efectiveness may be due to its composition in bioactive molecules those with relative concentrations of thymol and carvacrol.However, T. caramanicus Jalas EOs showed a reduced antibacterial activity.In the end, the essential oil of T. caramanicus Jalas represents a better candidate for the formulation of an effective treatment against gastric ulcer caused by H. pylori strains.

Figure 1 :
Figure 1: Te structures of the key constituents found in the essential oils under investigation.Table 2's compound numbers are the same.
Te temperature cycle program also included initial denaturation at 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 20 seconds and 60 °C for 40 seconds.Te primer sequences of the target genes, including cagA, vacA, babA2, and 16S rRNA (internal control), are given in Table tration (MIC) was determined for all H. pylori isolates, with the procedure modifed by adding resazurin after culture to see H. pylori development.On each microplate, there was a reasonable DMSO control (at a fnal concentration of 10%), a positive control (containing inoculum without the tested essential oils), and negative control (containing the tested essential oils without inoculum).Subculturing 5 μL of2.6.Antibacterial Virulence Gene Expression Analysis of EOs.A quantitative real-time PCR was used to evaluate the efect of the T. caramanicus Jalas essential oils on the cagA, vacA, and babA2 gene expression of H. pylori isolate.Total RNA was extracted using a whole RNX-Plus kit (Cin-naGen Co., Iran) from isolates after treatment with sub-MIC values of the T. caramanicus Jalas essential oils.Te cDNA was synthesized according to the YTA Kit Protocol (Yekta Tajhiz, Iran).A real-time PCR test was performed with YTA SYBR Green master mix (Yekta Tajhiz, Iran), and the 16SrRNA gene was used as an internal control.15 μL of reaction volume consisted of 0.5 μL cDNA, 0.5 μL forward primer, 0.5 μL rivers primer, 10 μL master mix, and 3.5 μL of double sterile distillation water was used for real-time PCR.

Table 2
lists the volatile components in T. caramanicus Jalas EOs in the elution frequency from a ZB-5MS phase.Te chemical content of these EOs varied signifcantly according to chromatographic examination.Figure1depicts the architecture of the most prominent terpenoids discovered in the studied EOs.

Table 1 :
List of primers used in this study.

Table 2 :
Chemical composition of the analyzed essential oils.

Table 3 :
Antibacterial activity of the T. caramanicus Jalas EOs using agar disk difusion method.