In Vitro and In Vivo Study of Combined Effect of Some Algerian Medicinal Plants and Probiotics against Helicobacter pylori

Helicobacter pylori causes gastritis, peptic ulcers and gastric cancer and affects more than half of the world’s population. Although this infection can have serious consequences, no novel cure or remedy have been discovered, the present therapy still relies on a variety of known antibiotics and anti-secretory agents. In the present study, the potential effect of combinations between methanolic extracts of four Algerian medicinal plants, garlic (Allium sativum), red onion (Allium cepa), cumin (Cuminum cyminum L.) and fenugreek (T. foenum-graecum L.), with different strains of lactic acid bacteria against H. pylori, was investigated. Similarly, the in vivo antibacterial effect of fenugreek extract combined with Bifidobacterium breve on H. pylori colonization potential to confirm the enhanced effect of the mixture was explored. Helicobacter pylori was inhibited by all combined mixtures of extracts and probiotics with varying results. The highest anti-H. pylori activities were found in fenugreek/B. breve, cumin/B. breve, garlic/B. breve and onion/B. breve combinations with inhibition diameters of 29, 26, 23 and 25 mm, respectively. Preliminary studies on the effect of probiotics against H. pylori revealed that the inhibition was due to lactic acid and bacteriocins and it was also related to the presence of phenolic compounds in the studied plants, such as gallic acid, caffeic acid, quercetin and vanillic acid. Fenugreek extract was found to inhibit the growth of H. pylori in a concentration-dependent manner. When H. pylori-infected rats were administered B. breve, the infection rate of H. pylori was significantly reduced, while the combination of B. breve and fenugreek extract effectively inhibited H. pylori. In addition, the B. breve and fenugreek extract mixture significantly reduced gastritis in H. pylori-infected rats. These results suggest that this complex mixture may be an alternative to treating diseases caused by H. pylori infection.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped bacterium, which causes gastric infection, leading to diverse outcomes ranging from asymptomatic gastritis to invasive adenocarcinoma [1,2]. Numerous factors have been identified that may contribute to the development of adverse H. pylori infection outcomes; these can be grouped into a triad of risk factors, including host, pathogen and environmental characteristics that interact to promote disease progression [2]. Over half of the people on the planet are its hosts, making this bacterium one of the most common infectious factors in humans [3]. About 70% of the patients showing H. pylori infection of the stomach display no symptoms, only around 10% to 20% will likely develop peptic ulcers and only 1-2 percent will be diagnosed with gastric cancer or mucosa-associated lymphoid tissue lymphoma [4]. In a previous study in Algeria, the prevalence of H. pylori infection was 66.1%, more prevalent The possibility of combined therapy using phenolic phytochemical-linked medicinal plants and lactic acid bacteria with probiotic potential on H. pylori inhibition may represent a novel and affordable approach to treating H. pylori-associated peptic ulcers and active chronic gastritis and impacting their long-term effects [31].
Based on the rationale above, the objective of this research was to evaluate the antimicrobial potential of combined methanolic extracts of some medicinal plants from Algeria, garlic, onion, cumin and fenugreek, with different probiotic strains, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lacticaseibacillus rhamnosus GG, Lacticaseibacillus rhamnosus LA80, Lactobacillus helviticus, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Lacticaseibacillus casei, Lactobaccilus acidophilus, Lactobacillus lactis and Streptococcus thermophilus, against H. pylori both in vitro and in vivo. To our best knowledge, this study is the first report on the effect of combined Algerian medicinal plants with probiotics against H. pylori responsible for gastro-duodenal diseases.

Plant Material
Samples of garlic (Allium sativum) bulb, onion (Allium cepa) bulb, fenugreek (Trigonella foenum-graecum L.) seeds and cumin (Cuminum cyminum L.) seeds were sorted, washed and dried at room temperature, then ground into a fine powder and sieved through a 200 µm sieve. Resulting powders were used to prepare methanolic extracts using a maceration method; all the extraction procedures were performed and conditions were set as described by Bouhenni [32,33]. Briefly, the methods applied to investigate the chemical composition of the tested plant extracts were:

Determination of Total Phenolic Content
An amount of 0.5 mL of varying concentrations of each used extract and 2.5 mL of Folin-Ciocalteu (1/10 dilution in water) were mixed with 1 mL of sodium carbonate (20%). This mixture was incubated in the dark at room temperature for 30 min. The absorbance of the solution was measured at 765 nm using a UV Vis spectrophotometer A calibration curve was established using gallic acid as standard. The results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g of Dry Matter.

Determination of Total Flavonoids Content
The total flavonoid content of plant extracts was determined using the aluminium chloride method. 1.5 mL of various concentrations was mixed with 75 µL of aluminium chloride solution and 0.5 mL of sodium acetate solution; the mixture was completed with distilled water until a volume of 2.5 mL. After an incubation period of 30 min at room temperature in the dark, the absorbance of the solution was measured at 415 nm using a UV-Vis spectrophotometer. The results were expressed as milligrams of quercitin equivalent (QE) per 100 g of Dry Matter.

Determination of Condensed Tannins Content
An amount of 1 mL of each extract was mixed with 2.5 mL of 4% methanol vanillin solution and 2.5 mL of H 2 SO 4 . After 15 min, the absorbance was measured at 500 nm. Condensed tannin contents were expressed as milligrams of Catechin equivalent (CE) per 100 g of Dry Matter.

Determination of Hydrolysable Tannins Content
An amount of 500 µL of the extract was added to 3.5 mL of the ferric chloride solution. The contents were then quickly mixed and the absorbance read at 660 nm, 15 secs after the addition of the extract solution. Hydrolysable tannin content was expressed as milligrams of Tannic acid equivalent (TAE) per 100 g of Dry Matter.
Phenolic compounds were identified qualitatively by the HPLC method (Supplementary Materials) and antioxidant activity was measured using a DPPH assay [32,33]. The extracts were carefully stored and later used for assessing their anti-H. pylori effects.

Probiotic Strains
Twelve probiotic strains were used in this study ( Table 1): six of them (6) were obtained from the culture collection of the Laboratory of Sciences and Technics of Animal Production, Faculty of Agriculture-Abdelhamid Ibn Badis University, Mostaganem-Algeria; one (1) strain was obtained from the Laboratory of Natural and Local Bioresources, University of Hassiba Benbouali, Chlef-Algeria and the remaining strains (5)  Fresh gastric biopsy specimens obtained from patients who underwent upper gastrointestinal endoscopy or from symptomatic patients with a positive rapid urease assay at the Youcef Damardji Tiaret Hospital, Algeria were transferred to sterile physiological saline, transported to the laboratory and processed within 4 h after sampling. Biopsy specimens were minced and homogenized in physiological sterile saline (0.5 mL) and then plated immediately on plates containing Colombia agar supplemented with 5% horse blood. The plates were incubated at 37 • C under microaerophilic conditions (10% CO 2 ) using BD GasPak™ (kingtec, Taiwan) for a maximum of 10 days. Suspected isolates were identified as H. pylori by conventional methods using Gram stain, urease, catalase and oxidase tests, API Campy (Bio Merieux. Cedex. France) and polymerase chain reaction (PCR).

Animals
Sixty healthy male Wistar rats (200 ± 3.81 g) obtained from the Pasteur Institute of Algiers (Algeria) were used to evaluate the combined effect of plant extract and probiotic bacteria against H. pylori. The animals were kept in individual cages under appropriate environmental conditions (temperature 22 ± 1 • C, 12/12 h light-dark cycle and relative humidity of 60 ± 10%) for two weeks adaptation period at the Veterinary Sciences Institute, The disc diffusion test was used for primary screening of the susceptibility of H. pylori to the plant extracts. Bacterial suspensions adjusted to McFarland turbidity standard 0.5 (1.5 × 10 −8 CFU/mL) were inoculated on plates containing Muller-Hinton agar with 5% of horse blood. Filter paper discs (6 mm diameter) impregnated with 60 µL of different concentrations of each plant extract (10,20,30,40,50,60,70,80,90, 100, 150, 250, 500 and 1000 µg/mL) were placed on the inoculated agar surfaces. Methanol at a concentration of 80% (60 µL) was used as a negative control. The plates were observed for 2 to 3 days at 37 • C under microaerophilic conditions. The antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the plant extracts [36].

Evaluation of the Minimum Inhibitory Concentration (MIC) of Plant Extracts with the Agar Dilution Method
The extracts presenting an inhibition zone ≥9 mm in diameter were chosen to assay the Minimum Inhibitory Concentration (MIC) with the agar dilution method using Mueller Hinton agar with 5% of horse blood. Methanol (80%) was used in the assay as a negative control. Concentrations of each extract were prepared in methanol 80% and 1 mL of each solution was incorporated in 20 mL of appropriate melted agar and poured into a Petri dish. The final concentrations of the extracts in the medium ranged from 90 to 1000 µg/mL. Agar plates were inoculated with 1 mL of bacteria suspension (1.5 × 10 8 CFU/mL). The plates were incubated for 5-7 days at 37 • C under microaerophilic conditions. The MIC was defined as the lowest concentration of plant extract inhibiting visible growth [37].

Evaluation of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Plant Extracts with the Broth Dilution Method
Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC) were measured using tube dilution methods. Fresh bacterial suspensions were prepared in Mueller Hinton broth and adjusted to McFarland turbidity standard 0.5. Serial concentrations were prepared from crude plant extracts within the range of 90 to 1000 µg/mL; then, 1 mL of each extract was added to tubes containing 8 mL of Mueller Hinton broth. Finally, 1 mL of a 1:1000 dilution of bacteria adjusted was added to obtain a 10 mL final volume. Controls of bacteria without extract were also prepared under similar conditions. The tubes were incubated for 2-3 days at 37 • C before recording the MIC. A volume of 0.1 mL of each suspension was spread onto Columbia agar plates containing 5% horse blood. After incubation in microaerophilic conditions at 37 • C for 72 h, the colonies formed were subsequently computed. The MBC was defined as the lowest concentrations of the plant extract, inducing complete inhibition of colony formation of the test bacteria at the latter cultivation [38].

Evaluation of Growth Kinetics of H. pylori in the Presence of Plant Extracts
The ability of several plant extracts to suppress H. pylori growth was tested by adding 1 mL of each plant extract on the 6th hour of development, while the optical density was subsequently measured every 2 h with UV-Vis spectrophotometer at 600 nm to follow the development of H. pylori in the presence of the extract [39]. Cell-free culture supernatants of probiotics were prepared as described by Kim et al. [40]. The culture supernatant was collected from a 24 hr culture by centrifugation at 4.000× g rpm/10 min. The resulting supernatants were filter-sterilized (pore size 0.22 mm). H. pylori cultures were plated on fresh Mueller Hinton agar plates containing 5% horse blood (1.5 × 10 8 CFU/mL) and wells were drilled into the agar by using sterile Pasteur pipettes; 60 µL of fresh probiotic strains cell-free culture supernatants were introduced in the agar wells. Plates were incubated for 48 to 72 h under microaerophilic conditions at 37 • C and the diameters of inhibition zones around the wells were measured. The antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by each probiotic.

Evaluation of Growth Kinetics of H. pylori in the Presence of Probiotics
A pre-culture tube of the H. pylori strain was inoculated in Mueller Hinton broth and incubated at 37 • C for 18 h, from which three standardized (0.5 MacFarland) culture tubes were prepared and the measurement of the bacterial growth of the pathogen strain was carried out by measuring the optical density every 2 h with UV-Vis spectrophotometer at 600 nm. After 6 h, 1 mL of each supernatant (normal/organic acid-free/H 2 O 2 free prepared as described below) was added to the culture tubes; the optical density was then measured every 2 hr up to 24 hr and the OD = f (t) curve was drawn [39].

Influence of Organic Acids (Lactic and Acetic Acids)
Lactic acid bacteria can produce inhibitor molecules of H. pylori, such as organic acids, bacteriocins and hydrogen peroxide. To ensure the presence of these molecules, we took an 18 h culture of different strains of probiotics which were transferred to 50 mL of modified MRS broth and incubated at 37 • C for 18 h. After incubation, the tubes were centrifuged at 4.000× g rpm/10 min in order to recover the supernatant [39].

Influence of Bacteriocins
In order to study the effect of bacteriocins on the growth of H. pylori, the effect of organic acids, in particular lactic and acetic acids, was eliminated and the supernatant was neutralized (pH = 7) by adding a 0.1 N NaOH solution [39].

Influence of Hydrogen Peroxide
The effect of H 2 O 2 was inhibited by incubating the supernatant fluid with catalase enzyme solutions prepared to a final concentration of 1 mg mL −1 in phosphate-buffered saline (pH 7.0).
The inhibitory effect of the agent was tested by eliminating the possible effect of organic acids by adjustment of the cells-free supernatants' pH to 7 and that of hydrogen peroxide in the presence of catalase. Fresh Mueller Hinton agar plates containing 5% horse blood were inoculated with 1.5 × 10 8 CFU/mL of H. pylori; filter paper discs (6 mm diameter) impregnated with 30 µL of fresh supernatants of probiotics +30 µL of plant extracts were placed on the inoculated agar surfaces (each probiotic has been combined with all four extracts). Plates were incubated for 48 to 72 h under microaerophilic conditions at 37 • C and the diameters of inhibition zones around discs were measured.

Evaluation of Growth Kinetics of H. pylori in the Presence of Combined Solutions (Plant Extracts with Probiotics)
Different mixtures of probiotics with plant extracts were examined for inhibition of H. pylori growth. The measurement of H. pylori growth was carried out by measuring the optical density with UV-Vis spectrophotometer at 600 nm every 2 h after adding 500 µL of probiotic supernatant +500 µL of plant extract on the 6th hour of growth (each probiotic has been combined with all four extracts).
At the end of the experiment, the rats were euthanized with diethyl ether and a full necropsy was performed.

Histopathologic Analysis of Gastric Tissue Samples
After removal, the stomach of each animal was opened through the longer curvature with sterile surgical instruments. Urease activity was determined in one half and the other half was fixed in 10% buffered formalin, trimmed to include all areas of the stomach, then processed by standard methods [41] and embedded in paraffin. From each block, two 5 µm sections were made, one being stained by hematoxylin and eosin (H&E) and the other by a Giemsa stain for H. pylori detection. All cases were examined in a blind manner by a veterinary pathologist, according to criteria established by Lee et al. [42]. Histopathological examination completed for stomach specimens were ranked according to the intensity of H. pylori colonization as follows: severe infection (3), moderate infection (2), mild infection (1) and free from infection (0) [43]. Gastritis was defined by the presence of lymphocytic or neutrophilic infiltration.
In Giemsa-stained sections, examining the different anatomical regions of the H. pyloriinfected and treated rats, H. pylori bacteria colonizing the glands were graded on a scale of 0 to 4 as follows: 0, absence of bacteria; 1, bacteria isolated and randomly distributed; 2, reduced number of bacteria; 3, large number of bacteria and 4, very high number of bacteria.

Statistical Analysis
The data were collected and statistically analyzed by use of MS Excel 2007 and presented as mean ± SD of three replicates.

Results of Phytochemical Analysis and Screening
Phytochemical analysis showed that phenols are the largest group of metabolites in the four studied plants. Results of phytochemical screening of our plants using HPLC revealed the presence of caffeic acid, isoquercetin, vanillic acid, myricetin 3-0-rutinoside, syringaresinol, citrusine, rosmarinic acid and p-coumaric acid in cumin seeds, gallic acid, sinapic acid, caffeic acid, asterogenic acid, pyrogallol, hyperoside and ferulic acid in fenugreek seeds, gallic acid, quercitin, rutin, hyperoside and karempferol in red onion and one molecule for garlic extract which is gallic acid [32,33].

Results of Biochemical Identification
After incubation for 5 days at 37 • C in a microaerophilic atmosphere, the results of the reading were reflected by the appearance of small colonies 1 to 2 mm in diameter. The colonies were grayish or transparent in color, shiny, round and had a regular outline. Microscopic observation in the fresh state showed that H. pylori is a small curved, mobile bacillus.
The Gram stain performed on smears from the individual colonies indicated the presence of Gram-negative bacteria. The biochemical tests showed that the strain was positive for urease, catalase and oxidase and therefore had a significant enzymatic activity.

Results of Evaluation of DIZ of Plant Extracts Using the Disc Diffusion Method
The inhibitory effect of the methanolic extracts on the growth of H. pylori is shown in Table 2. The results indicated a concentration-dependent increase in the DIZ against H. pylori for all plant extracts. The DIZ started from 6 mm for the different plant extracts at 10 µg initial concentration. High DIZ were recorded for the cumin and fenugreek extracts at 90 µg concentrations and above, with slight primacy of the fenugreek extract (16.00 ± 0.00) at 1000 µg.

Concentrations (µg) DIZ (mm) Garlic Extract Onion Extract Cumin Extract
Fenugreek Extract 10 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 20 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 30 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 The results of plant extracts induced MIC and MBC against H. pylori are presented in Table 3. The strongest MIC and the MBC against H. pylori were obtained with the fenugreek extract at 100 and 150 µg/mL, respectively. The other extracts rendered as follows: cumin extract 150 µg/mL (MIC) and 250 µg/mL (MBC), while for the onion and garlic extracts, the concentrations needed to be much higher to attain the same MIC and MIBs. This suggested that fenugreek extract provided the best antibacterial effect at the lowest concentrations among all extracts evaluated in the present study. The inhibition kinetics evaluation showed that plants extract had antibacterial activity against H. pylori (Figure 2). Almost similar growth kinetics were recorded for all plant extracts with relatively overlapping curves up to 6 h when adding plant extracts, followed by a marked decrease in H. pylori growth recorded with the fenugreek extract compared to other extracts.

tracts
The inhibition kinetics evaluation showed that plants extract had antibacterial activity against H. pylori (Figure 2). Almost similar growth kinetics were recorded for all plant extracts with relatively overlapping curves up to 6 h when adding plant extracts, followed by a marked decrease in H. pylori growth recorded with the fenugreek extract compared to other extracts. According to the results, fenugreek extract caused the highest growth inhibition of H. pylori, followed by cumin, garlic and onion, respectively.

Evaluation of DIZ of Probiotics against H. pylori Using Well Diffusion Assay
The antibacterial activity of 12 probiotic strains was evaluated in vitro by the well diffusion method against H. pylori. Table 4  According to the results, fenugreek extract caused the highest growth inhibition of H. pylori, followed by cumin, garlic and onion, respectively.

Results of Evaluation of Growth Kinetics of H. pylori in the Presence of Probiotics
The results of evaluation of the growth kinetics of H. pylori showed a slight decrease in the presence of probiotics (Figures 3-5). An important inhibition effect was recorded when the cell-free supernatant obtained from a culture of probiotics was used, indicating that a diffusible molecule (organic acids) should be present in this supernatant. However, a slightly lower inhibitory effect was obtained using bacteriocin-free and H 2 O 2-free (neutralized) supernatants.
Influence of organic acids (lactic and acetic acids) The results of evaluation of the growth kinetics of H. pylori showed a slight decrease in the presence of probiotics (Figures 3-5). An important inhibition effect was recorded when the cell-free supernatant obtained from a culture of probiotics was used, indicating that a diffusible molecule (organic acids) should be present in this supernatant. However, a slightly lower inhibitory effect was obtained using bacteriocin-free and H2O2-free (neutralized) supernatants.  Influence of organic acids (lactic and acetic acids)  Among the tested probiotics, B. breve was responsible for an important growth decrease in H. pylori that could be explained by the high production of organic acids.  Looking at the inhibition by organic acids, a highly reduced growth rate of H. pylori was induced by B. breve, suggesting its ability to synthesize more bacteriocins than the other tested probiotics.  Looking at the inhibition by organic acids, a highly reduced growth rate of H. pylori was induced by B. breve, suggesting its ability to synthesize more bacteriocins than the other tested probiotics.  Looking at the inhibition by organic acids, a highly reduced growth rate of H. pylori was induced by B. breve, suggesting its ability to synthesize more bacteriocins than the other tested probiotics.  Influence of hydrogen peroxide (H2O2) No significant changes were observed with the suppression of H2O2 activity for all tested bacteria suggesting that it has no effect on H. pylori growth.

Quantifying the DIZ Using the Disc Diffusion Method
Results of the determination of DIZ of medicinal plants combined with probiotics against H. pylori are shown in Table 5). The combined effect of most probiotics with plant  No significant changes were observed with the suppression of H 2 O 2 activity for all tested bacteria suggesting that it has no effect on H. pylori growth.

Quantifying the DIZ Using the Disc Diffusion Method
Results of the determination of DIZ of medicinal plants combined with probiotics against H. pylori are shown in Table 5). The combined effect of most probiotics with plant extracts is quite similar, except for B. longum, B. bifidum and B. breve showing highly distinct DIZ values. The highest DIZ values were obtained with all B. breve combinations, especially with fenugreek extract giving 28.67 ± 0.58 mm DIZ against the H. pylori strain.

Evaluation of Growth Kinetics of H. pylori in the Presence of Combined Extract Plants with Probiotics
The results of the evaluation of H. pylori growth kinetics showed a remarkable decrease in the presence of probiotics combined with plant extracts (Figures 6-9). All probiotics have induced an important decrease in the growth of H. pylori in combination with plant extracts. However, the most notable decrease in growth was recorded with the fenugreek extract when combined with B. breve (Figure 8).

Evaluation of Growth Kinetics of H. pylori in the Presence of Combined Extract Plants with Probiotics
The results of the evaluation of H. pylori growth kinetics showed a remarkable decrease in the presence of probiotics combined with plant extracts (Figures 6-9). All probiotics have induced an important decrease in the growth of H. pylori in combination with plant extracts. However, the most notable decrease in growth was recorded with the fenugreek extract when combined with B. breve (Figure 8).

Results of the In Vivo Study
During the acclimation and the experimental periods, most of the rats did not show any treatment-dependent clinical signs, except those in group 9 (HP + TFE + TBB), where severe weight loss that could be attributed to the number of gavage (three consecutive gavages per day) was observed. No gross lesions were observed during the necropsy at the end of the experimental period.
The histological results obtained by administering the combination between B. breve and fenugreek extract in order to evaluate their effect on H. pylori-induced stomach inflammation in vivo were shown in Figure 10.

Results of the In Vivo Study
During the acclimation and the experimental periods, most of the rats did not show any treatment-dependent clinical signs, except those in group 9 (HP + TFE + TBB), where severe weight loss that could be attributed to the number of gavage (three consecutive gavages per day) was observed. No gross lesions were observed during the necropsy at the end of the experimental period.
The histological results obtained by administering the combination between B. breve and fenugreek extract in order to evaluate their effect on H. pylori-induced stomach inflammation in vivo were shown in Figure 10.

Results of the In Vivo Study
During the acclimation and the experimental periods, most of the rats did not show any treatment-dependent clinical signs, except those in group 9 (HP + TFE + TBB), where severe weight loss that could be attributed to the number of gavage (three consecutive gavages per day) was observed. No gross lesions were observed during the necropsy at the end of the experimental period.
The histological results obtained by administering the combination between B. breve and fenugreek extract in order to evaluate their effect on H. pylori-induced stomach inflammation in vivo were shown in Figure 10.

Discussion
H. pylori isolated from biopsy samples of gastric ulcer patients were identified by different tests as described above. The detection of the ureC gene of the H. pylori strain was further confirmed by PCR amplification of the ureC gene specific to the strain of H. pylori. Our results of PCR were similar to the previous study by Lage et al., (1995) that showed the amplification of curves for the targeted gene (ureC) [35].
The evaluation of the antibacterial effect of methanolic extracts from tested plant materials against H. pylori clinical isolates using a disc diffusion test and by evaluating the MIC on solid media and MBC on broth media indicated an inhibitory activity in high percentages. The results presented in this study emphasize the significant antibacterial effect of methanol extracts, especially for fenugreek extract, which was higher than that of cumin, onion and garlic extracts, respectively; all tested plants showed substantial but broadly different anti-H. pylori effects with an MBC ranging from 150 to 1000 µg/mL. Similar results were obtained by other investigators for anti-H. pylori activities of the studied plants. Thus, O'Gara et al. [17] and Cellini [44] showed that garlic exhibited a potential anti-H. pylori effect with MIC ranging from 250 to 500 µg/mL and a MBC ranging from 250 to 500 µg/mL [17,44]. The in vitro anti-H.pylori activity of extracts and compounds obtained from garlic has been extensively documented [44,45]. An aqueous garlic extract had a MIC of 40 µg/mL against H. pylori, and for other garlic compounds (allicin, ajoenes, vinyldithiins, thiosulphinates), the MIC values were approximately 10 to 25 µg/mL [45,46]. Similarly, the other plants studied in this research represented subject to investigations of other groups. Onion extracts presented a good anti-H. pylori activity, according to Yordanov et al. [18], while cumin showed a MIC of 691 µg/mL against H. pylori [19] and, finally, fenugreek was observed to moderately (68%) inhibit the growth of H. pylori according to Manjegowda and Dharmesh [20]. The bactericidal activity of plant preparations against H. pylori significantly depends on the type of the extract and its components, concentration and exposure time and the density of the tested bacterial strains [47]. In previous studies, phytochemical screening of four plants was carried out and the results revealed the presence of eight phytochemical compounds in cumin extract, seven molecules in fenugreek extract, five compounds found in red onion extracts and one compound in garlic [32,33]. From these phytochemical studies, phenols are the largest group of metabolites in the studied plants and many compounds are believed to have remarkable anti-H. pylori effect in vitro and, to some extent, in vivo. Studies that have attempted to elucidate the mechanism of action of plant extracts or compounds on H. pylori are available. Some of these studies, for example, investigated the effect of various compounds on bacterial virulence factors such as urease, adhesion, vacuolization, motility or on specific metabolic enzymes [48]. The antimicrobial activity of the secondary metabolites of plants is related to the -OH group bound to the phenolic ring [49].
The anti-H. pylori mechanism of action of some of the phytochemicals listed above is described. Some of the phenolic acids demonstrated antimicrobial activity similar to antibiotics [50]. For instance, quercetin showed activity against several bacteria causing inhibition of FabZ enzyme [51] and, in the same way, to kaempferol by inhibition of peptidoglycan synthesis, activity of β-lactamase, decrease in fatty acids and increase in cytoplasmic membrane permeability [52]. Caffeic acid is able to rupture cell membranes, interfere with aerobic metabolism [53] and inhibit efflux pump [54]. In a separate study, several phenolic acids, including vanillic acid, syringic acid, p-coumaric acid and ferulic acid, from Polish wild mushrooms demonstrated intermediate antibacterial activities against a range of Gram-negative and Gram-positive bacteria [55]. Hyperoside, ferulic acid, rutin and ellagic acid found in the extracts of three species of Potentilla displayed a good anti-bacterial activity against both Gram-positive and Gram-negative bacteria [56].
The anti-H. pylori activity in vitro of garlic extracts and compounds has been extensively reported; for example, the gallic acid potentially enhanced the activity of antibiotics against several P. aeruginosa isolates by altering the cell wall integrity [57]. In their study, Borges et al., found that ferulic acid and gallic acid had antimicrobial activity against four pathogenic bacteria by producing local damage and leakage (pore formation) of cellular components [58].
Out of the tested probiotic strains, Bifidobacterium breve showed the highest antibacterial activity against H. pylori. Moreover, the antibacterial activity of the other Bifidobacterium strains, B. bifidum and B. longum, was also higher than that of the reference strains (L. rhamnosus GG, L. rhamnosus LA80 and L. helviticus) and of isolated probiotic strains (L. fermentum, L. plantarum, L. casei, L. acidophilus, L. lactis and S. thermophilus). Further studies on the inhibition kinetics showed that B. breve presents the highest antibacterial activity against H. pylori when compared to other probiotic strains. Regarding the results of growth kinetics of H. pylori in the presence of probiotics, when investigating the influence of different variables (organic acids, bacteriocins and hydrogen peroxide), it was clear that the inhibition effect was due to the presence of organic acids, such as lactic and acetic acids and bacteriocins. Thus, in accordance with the bacterial death criteria of Pearson et al. [59], the effect of L. fermentum UCO-979C strain at 24 h can be considered lethal for H. pylori. Further, after repeating this assay at shorter times and using the same Pearson criteria for interpretation of the results, the death of H. pylori after 6 h of treatment with B. breve was noticed. Nevertheless, other researchers found different results when evaluating the anti-H. pylori effect of some probiotics. Bae et al., investigated the inhibitory effects of different Bifidobacterium spp. on the growth of H. pylori and indicated that significant suppression of H. pylori growth occurred, especially in the presence of B. breve [60]. García et al., establish that L. fermentum, L. casei and L. rhamnosus induced an inhibition ranging from 2 to 5 mm [30]. Boyanova et al. [61], when looking at the anti-H. pylori effect of some Lactobacillus strains, indicated that three strains suppressed the growth of >86% of H. pylori strains at low pH values and two strains suppressed the growth of >53% of the test strains at neutral pH values [61]. Further studies by Boyanova et al. [62] revealed that the DIZ of some Lactobacillus strains were higher, from 13 to 16 mm, while Chen et al. [63] found that L. rhamnosus and L. acidophilus exhibited an inhibitory effect against H. pylori reference strain 26695 demonstrated by a DIZs of 12.3 and 11.3 mm, respectively [62,63]. However, Paucar-Carrión et al. [61], when analyzing the anti-H. pylori activity of L. fermentum, concluded that the activity was mild with DIZ between 1 mm and 2 mm [64].
Among probiotics, Bifidobacterium is one of the favorites, is generally used for the prevention of gastrointestinal infection and it is commonly incorporated in fermented dairy products or food supplements. Bifidobacterium exerts an in vitro anti-H. pylori effect and inhibits adhesion to the mucosa by competitive exclusion [65]. Several studies have demonstrated a direct relationship between the addition of potential probiotic strains and the in vitro inhibition of H. pylori growth. Such strains were Lactobacillus acidophilus [66] and Lactobacillus casei Shirota strain [26], which, among others, have an antagonistic effect on H. pylori. Since most organic acids are produced by the Bifidobacterium spp. inhibited the growth of H. pylori, while the H. pylori growth was not inhibited by organic acids produced in some other Bifidobacteria-cultured media, it was suggested that those Bifidobacterium strains might produce antibiotic-like compounds (bacteriocins) [60]. The results of the present study confirm the findings of Zacharof and Lovitt [67], which imply the effects of organic acids and other bioactive substances of the Bifidobacterium and Lactobacillus strains and a higher activity of bacteriocins at lower pH values [67]. The activity of the neutralized and catalase-treated supernatants was approximately similar with regard to the H. pylori growth inhibition effect. Bacteriocin activity depended on numerous factors of the test strains, such as the bacterial cell envelope/membrane composition, reduction in bacteriocin binding/insertion, bacteriocin sequestering and degradation or efflux pumping [68].
In this study, we confirmed in in vitro experiments the inhibitory activity of B. breve complex mixture containing fenugreek extracts on H. pylori. The fenugreek extract inhibited the growth of H. pylori in a dose-dependent manner (100 µg/mL). In addition, the inhibitory effect on H. pylori of B. breve and fenugreek extract, when applied as a complex mixture rather than individual components, was confirmed to be superior.
H. pylori was inhibited by all combined mixtures of extracts and probiotics with varying results, while fenugreek/B. breve, cumin/B. breve, garlic/B. breve and onion/B. breve combinations exhibited relevant anti-H. pylori activities with DIZ of 26, 29, 23 and 25 mm, respectively. Preliminary studies on the effect of probiotics against H. pylori revealed that inhibition may be due to lactic acids and bacteriocins. However, the presence of phenolic compounds, such as gallic acid, caffeic acid, quercetin and vanillic acid, in plant extracts may also have an influence.
Few studies investigate the antimicrobial effects of combinations between probiotics and plant extracts. Lee et al. [69] demonstrated that the treatment with a complex mixture of L. paracasei HP7, including the extract of P. frutescens and G. glabra, could inhibit the growth of H. pylori and, thus, is a promising treatment for patients with gastric symptoms, such as gastritis caused by H. pylori infection [69]. Behrad et al. [70] recorded that a mixture of cinnamon extract, L. acidophilus LA-5 and NCFM, Bifidobacterium Bb-12, L.casei LC-10 and Streptococcus thermophilus Th-4 exhibited the strongest inhibitory effect on H. pylori growth in vitro (13.5 mm) [70]. Other studies contradict the synergic effect between plant extracts and probiotics. Kang et al., (2021) investigated the anti-H. pylori effects of Lactobacillus plantarum (pH3A), monolaurin, grapefruit seed extract and their synergies in vitro and in vivo [71]. Monolaurin and grapefruit seed extract suppressed H. pylori growth at a MIC of 62.5 ppm. Additionally, L. plantarum pH3A significantly inhibited H. pylori growth. In the in vivo study, H. pylori colonization of the mouse stomach was significantly reduced by L. plantarum treatment, but the addition of monolaurin or grapefruit seed extract did not contribute to these anti-H. pylori activities. Therefore, the L. plantarum strain can potentially be applied as an alternative anti-H. pylori therapy, but evidence of its synergy with monolaurin or grapefruit seed extract in vivo is still lacking. Sadeghi et al. [72] examined the synergistic effect of broccoli sprout extract with probiotics on H. pylori growth inhibition and the findings showed a synergistic effect of the mixture on bacterial inhibition and suggested the use of broccoli sprout extract and probiotic bacteria in a yogurt form that is effective in the therapy of H. pylori infection [72].
The histopathological examination of gastric mucosa showed a moderate inflammatory infiltration in the antrum and fundus of animals in the non-treated groups infected with H. pylori and severe colonization of antral mucosa with H. pylori bacteria by use of Giemsa stain. Gastric samples of H. pylori-infected animals treated with second-line therapy showed a reduced number of inflammatory cells in the lamina propria; however, no changes in colonizing bacteria were observed in the Giemsa-stained samples. Tissue samples of rats infected with H. pylori and treated with TFE2 and TBB showed a significant decrease in infiltrating inflammatory cells and colonizing bacteria scores (1-0 and 1-1) for inflammation and colonization, respectively. However, the group of rats receiving the lowest dose of fenugreek extract (TFE1, 150 mg/kg) showed a slight decrease in inflammatory infiltration and colonization of gastric mucosa (score 2-2) compared to the higher dose group animals.
All animals in preventive groups showed normal gastric histology with a significant reduction in bacterial colonization (score 1). However, the gastric mucosa of treated animals exhibited mild inflammation due to infiltration by lymphocytes from previous H. pylori infections. Animals in the H. pylori-infected group treated with fenugreek extract associated with B. breve showed normal gastric histology with a total absence of H. pylori characteristic forms of colonialization in Giemsa-stained samples.
Data from the current study suggest that administration of TFE, TBB or their associations to H. pylori-infected rats can moderately reduce the number of H. pylori colonizing the gastric mucosa with an important effect on associated gastritis. However, prophylactic administration of TFE, TBB or their associations could prevent gastric inflammation and considerably reduce or completely eliminate H. pylori colonization.
In vivo anti-H. pylori activity of the fenugreek extract and/or B. breve has been confirmed by histopathological and urease tests. These compounds have suggestively reduced the number of animals with a positive urease test and the number of detected microorgan-isms in histological sections stained by Giemsa, exceedingly even the standard medications, thus preventing the presence of these organisms in the gastric mucosa.
None of the animals in the control and treated groups exhibited atrophy or metaplasia of the gastric mucosa. Similarly, important differences in the antral and fundic mucosa with respect to colonization by H. pylori and the associated gastritis were observed between the treated and control groups, as also reported by Sgouras et al. [26]. It has been suggested in previous in vivo studies in rats that the fenugreek extract forms a protective barrier over the gastric epithelial lining due to the presence of galactomannans, H. pylori possibly interacting with epithelial cells through secretory molecules (vitexin-7-O-glucoside, vicenin-1, luteolin and orientin) or as a result of adherence [73].
The significant reduction in the severity of gastritis observed in animals treated with the extract indicated a possible role as an anti-inflammatory and anti-secretory agent [73,74]. However, this gastro-protective action was not attributed exclusively to the alteration of gastric pH or acidity since the response of the extract was consistent in both in vivo and in vitro studies [73]. Additionally, it has been suggested that the in vivo activity of the extract may not result solely from the topical action but may also take place from a systemic component [75].
Several probiotics showed beneficial effects in animal models of H. pylori infection. In mice infection models, a combination of probiotics-L. acidophilus R0052 + L. rhamnosus R0011 and L. casei Shirota strain + L. johnsonii strain La1-were found to reduce the effects of H. pylori infection by decreasing H. pylori colonization and alleviating H. pylori-induced inflammation of the gastric mucosa [26,27].
There are numerous possible mechanisms by which probiotic bacteria can prevent H. pylori adhesion [76]. Lactobacilli, such as L. johnsonii La1 [77] or L. acidophilus LB [78], may exert anti-adhesion activity by secreting antimicrobial substances and strains, such as L. reuteri, can inhibit H. pylori growth by competing with adhesion sites. Non-specific rather than specific receptor site blockage is the most likely mechanism [79].
In vivo studies demonstrated that previous colonization with probiotics prevents or reduces H. pylori infection in experimental animals [27]. Consequently, regardless of the mechanisms of inhibition of the H. pylori adherence to epithelial cells, probiotics could prevent H. pylori colonization by inhibiting bacterial adhesion to gastric epithelial cells [80].

Conclusions
The administration of a complex mixture of Bifidobacterium breve and T. foenum-graecum L. extract was more effective than that of B. breve alone or T. foenum-graecum L. extract alone, the combination induced a higher antibacterial effect against H. pylori and prevented the inflammation induced by H. pylori. The present study confirmed that the administration of this mixture reduced the damage of the gastric mucous membrane. The mechanism probably relied on the inhibitory effect of B. breve on the adhesion of H. pylori to the gastric mucosa combined with the increased secretion of gastric mucosal mucin induced by T. foenum-graecum L. extract. Thus, a complex mixture of B. breve, including the extract of T. foenum-graecum L., could be used to treat patients with gastric symptoms, including ulcers caused by H. pylori. These results demonstrated that the complex mixture of B. breve, including the extract of T. foenum-graecum L. is a promising therapeutic agent for patients with gastric symptoms, such as for gastroduodenal diseases caused by H. pylori infection.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11051242/s1, The molecular separation of garlic, red onion, fenugreek and cumin methanolic extracts was achieved by HPLC at three wavelengths: 254 nm, 326 nm and 360 nm. The findings obtained are visible in the peaks and retention time of chromatograms of each molecule. The results obtained are shown in the chromatograms with peaks and retention time of each molecule ( Figures S1-S4) [32,33]. HPLC results revealed the presence of five components in red onion extract ( Figure S1), one component in garlic extract (Figure S2), fifteen compounds in cumin extract ( Figure S3) and eight compounds in fenugreek ( Figure S4), The identification of molecules found in the samples is based on comparing their retention times (Rt) with that of pure standards under the same experimental conditions (Table S1)