# In Vitro andIn Vivo Anti-Helicobacter Activities ofEryngium foetidum (Apiaceae),Bidens pilosa (Asteraceae), andGalinsoga ciliata (Asteraceae) againstHelicobacter pylori

Authors: Laure brigitte Kouitcheu Mabeku; Bertrand Eyoum Bille; Eveline Nguepi
Journal: BioMed Research International
 (2016)

---

## Abstract

This study was performed to evaluate the antimicrobial activities of extracts ofBidens pilosa, Galinsoga ciliata, andEryngium foetidum against 6 clinical strains ofHelicobacter pylori in vitro andin vivo. Broth microdilution method was usedin vitro.In vivo, Swiss mice were inoculated withH.pylori and divided into 5 groups; the control group received the vehicle and the four others received 125, 250, and 500 mg/kg of methanol extract ofEryngium foetidum and ciprofloxacin (500 mg/kg) for 7 days, respectively.Helicobacter pylori colonization and number of colonies in gastric biopsies culture were assessed on days 1 and 7 after treatment. The lowest MIC value (64 μg/mL) and the best spectrum of bactericidal effect (MBC/MIC = 1) were obtained with the methanol extract ofEryngium foetidum. The number ofH.pylori infected animals was 17% (plant-extract) and 0% (ciprofloxacin) compared to 100% for the infected untreated group. Plant-extract (381.9
±
239.5 CFU) and ciprofloxacin (248
±
153.2 CFU) significantly reduced bacterial load in gastric mucosa compared to untreated, inoculated mice (14350
±
690 CFU).Conclusion. The present data provided evidence that methanol extract ofEryngium foetidum could be a rich source of metabolites with antimicrobial activity to fightHelicobacter pylori infections.


---

## Body

## 1. Introduction

Helicobacter pylori is a Gram negative microaerophilic helical bacillus that affects the gastric mucosa and can be found attached to epithelial cells of the human stomach [1]. It is an etiologic agent of peptic ulcer disease, primary gastritis, gastric mucosa-associated lymphoid-tissue lymphoma, and gastric adenocarcinoma [2]. Approximately 20% of persons infected withH.pylori develop related gastroduodenal disorders during their lifetime [3]. Approximately 50% of persons areHelicobacter pylori positive around the world, with the developing countries having a prevalence of 80%–90% and industrialized countries like the United States having ≈35%–40% [3]. As the prevalence, the annual incidence ofH.pylori infection is high in developing countries (≈4%–15%) compared with industrialized countries (0.5%) [4]. Low socioeconomic status, overcrowding, poor sanitation or hygiene, and living in a developing country are the risk factors of infection [2]. The recurrence of gastroduodenal disorders is substantially reduced by the eradication therapy ofH.pylori infection. This eradication therapy entails two of the following antimicrobial agents; metronidazole, amoxicillin, tetracycline, or clarithromycin in association with a proton pump inhibitor or bismuth [5]. Quadruple regimens are used as a salvage therapy when triple therapy regimens failed [5]. The emergence of drugs resistanceH.pylori strain is the most common causes of treatment failure [6]. The failure of 38% and 55% of cases, respectively, to therapy regimens containing metronidazole and clarithromycin has been documented, especially when these antimicrobial agents are used to treat infection with an organism resistant to one of the latter [7]. Consequently, there is the necessity for the development of new antimicrobial agents from natural sources for better chemotherapeutic applications [8]. Literature clearly shows thein vitro assessment of anti-Helicobacter properties of medicinal plant products [9–14]. However, because of short stomach transit time and poor acid stability, many compounds within vitro anti-Helicobacter properties become ineffectivein vivo [15–17]. Therefore, thein vitro therapeutic activity of plant products must be confirmed by anin vivo test. Even though no animal model of theH.pylori infection exactly matches the human disease, experimental models on some animals such as primates, domestic cats and dogs, gnotobiotic piglets, ferrets, Mongolian gerbils, and mice provide important insights into thein vivo evaluation of new antimicrobials.Eryngium foetidum is an annual small plant (approximately 30 cm high) in the family Apiaceae, used as a medicinal herb and spice. It is used as health foods in view of its significant amount of calcium, iron, carotene, riboflavin, proteins, and vitamins A, B, and C content [18]. In traditional medicine, this plant is used in the treatment of various ailments such as fevers, chills, vomiting, burns, hypertension, headache, earache, stomachache, asthma, arthritis, snake bites, scorpion stings, diarrhea, malaria, and epilepsy [19, 20]. In addition, the aerial part of this plant is a rich source of essential oils which can be exploited in the cosmetic industry [20]. Eryngial (E-2-dodecenal) is the main constituent of volatile compounds of the plant [21]. Studies demonstrating the anthelmintic, anti-inflammatory, analgesic, anticonvulsant, anticlastogenic, anticarcinogenic, antidiabetic, and antibacterial activity of this plant have been reported [22–25].Despite its popular uses to calm stomach pain, its activity againstH.pylori which is a major cause of gastric ulcer has not been experimentally confirmed [26]. This study was therefore carried out to evaluate thein vitro andin vivo antibacterial activity ofEryngium foetidum on clinical strains ofH.pylori in order to identify potential sources of materials for the synthesis of new drugs to fightHelicobacter pylori infections.


## 2. Materials and Methods

### 2.1. Bacterial Strains

The clinical microorganisms used here were obtained from the Gastroenterology Department of Laquintinie Hospital, Douala, Cameroon. These microorganisms were collected from gastric biopsies of patients complaining of upper gastrointestinal pain, hematemesis, or/and dyspepsia requiring gastroendoscopic examination. Six strains ofH.pylori were freshly isolated and suspended in Brain Heart Infusion (BHI) broth supplemented with 5% horse serum and 20% glycerol and stored at −80°C until further use.


### 2.2. Animals

Swiss albino mice of 18–22 g weight obtained from Animal House, University of Dschang, Cameroon, were selected as experimental animals. These animals were fed with standard pellet diet and tap water.


### 2.3. Statement on Ethics Approval

Throughout the experiments, all animals received human care according to the criteria outlined in the internationally accepted principle guidelines of the European Union on Animal Care (CEE Council 86/609). Animal experiments were designated and approved by the Cameroon National Ethics Committee (Reg. number. FWA-IRB00001954).


### 2.4. Plant Material

The leaves ofBidens pilosa (Asteraceae) andGalinsoga ciliata (Asteraceae) were collected in Dschang (west region of Cameroon) and leaves ofEryngium foetidum (Apiaceaes) in Kumba (southwest region of Cameroon). Identification of the plant was done at the National Herbarium Yaoundé (voucher specimen numbers 42254/HNC, 57409/HNC, and 42131 HNC, resp., in the listed order. HNC: Cameroon National Herbarium). The plant materials were then air-dried at room temperature and ground into a fine powder.


### 2.5. Chemicals and Culture Media

Culture media (Columbia Agar, Brain Heart Infusion (BHI), lacked horse blood, Horse Serum, Vitox Supplement) and CampyGen gas pack were obtained from Oxoid, Basingstoke, England. Doxycycline (doxycycline 200 mg, Combitic Global Caplet, India), metronidazole (metronidazole Tablets BP 500 mg, Strides Arcolab, India), and ciprofloxacin (zoflox, ciprofloxacin 750 mg, Odypharm) were used as reference antibiotics for the determination of the MIC and MBC and for thein vivo test were obtained from local pharmacy. P-Iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was used to indicate microbial growth [27].


### 2.6. Extraction

Dried powdered plant material was weighed (250 g) and soaked in 3.5 L of ethyl acetate (EA) or methanol (MeOH) for 72 hours. The plant mixture was then filtered and concentrated under reduced pressure using a rotary evaporator. The extract was further concentrated by allowing it to stand overnight in an oven at 30°C.


### 2.7.In Vitro Study

#### 2.7.1. Antimicrobial Assay: Determination of MIC and MBC

The clinical strains (H.pyloriα1,H. pyloriα2, H.pyloriα3, H.pyloriα4, H.pyloriα5, andH.pyloriα6), identified using Gram staining and enzymatic properties (catalase, oxidase, and urease), were employed to evaluate thein vitro anti-Helicobacter activity of the plant-extracts. Methanol and ethyl acetate extracts ofBidens pilosa,Galinsoga ciliata, andEryngium foetidum were used for the determination of MICs by the INT broth microdilution method [27] using 96-well plates. Twofold dilutions of each extract were prepared in the test wells in BHI broth supplemented with 5% horse serum (BHI-serum). The final extract concentrations ranged from 0.002 to 1.024 mg/mL. One hundred microliters of inoculums prepared from 48 h colonies on supplemented Columbia Agar (Columbia Agar + 5% (v/v) lacked horse blood and 1% (v/v) Vitox) at McFarland turbidity standard 3 was added to 100 μL of the extract-containing culture medium. Control wells were prepared with culture medium and bacterial suspension and broth, only respectively. Ciprofloxacin, doxycycline, and metronidazole at concentration ranges of 0.002 to 0.128 mg/mL were used as positive control. The plates were covered with a sterile plate sealer; the contents of the wells were mixed with a shaker and incubated for 3 days at 37°C under microaerophilic conditions. After incubation, 40 μL of 0.2 mg/mL INT was added per well and incubated at 37°C for 30 min. Living bacteria reduced the yellow dye to pink. The sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth is known as the MIC. The MBC was determined by adding 50 μL aliquots of the preparations which prevented the color change of the medium after incubation during MIC assays, to 150 μL of BHI-serum. These preparations were incubated at 37°C for 72 h under microaerophilic conditions. The lowest concentration of extract, which did not produce a color change after addition of INT, was considered as the MBC. Each MIC and each MBC were determined in triplicate and the mean values are recorded.


#### 2.7.2. Preliminary Phytochemical Analysis

The qualitative phytochemical analysis ofEryngium foetidum methanol extract was performed following the methods of Parekh and Chanda [28] to determine the presence of alkaloids (Mayer, Wagner, and Dragendorff test), flavonoids (alkaline reagent, Shinoda), phenolics (lead acetate, alkaline reagent test), triterpenes and steroids (Liebermann-Burchard Test), saponins (foam test), tannins (gelatine), anthraquinones (ether-chloroform 1 : 1 v/v, NaOH 10% w/v), and anthocyanins (1% HCl, heating). The results were qualitatively expressed in term of positive (+) and negative (−) signs.


### 2.8.In Vivo Anti-Helicobacter pylori Assessment

#### 2.8.1. Inoculation of Experimental Animals

Methanol extract ofEryngium foetidum, the most active plant-extract according to the MIC value obtained, was chosen for furtherin vivo anti-Helicobacter assessment. For this purpose, seventy-two mice were allowed to acclimatize for one week before initiation of experiment. After the acclimatization period, the animals were fasted for 12 h and sixty of them were infected with 0.2 mL of 108 CFU/mL ofH.pyloriα2 suspension, four times in one week with a 24 h interval between each inoculation. The inoculation day was considered as day 0 and subsequent days as day 1, day 2, and day 3 up to day 21. A group of uninfected mice serving as a normal group received sterile solution of the vehicle (200 μL of 0.25% Tween 80).


#### 2.8.2. Distribution of Animals

Inoculated mice were distributed into five groups of 12 animals and allowed to rest for 24 h after the last inoculation. Group I, a negative control group, received sterile solution of the vehicle; group II was treated with ciprofloxacin (500 mg/kg) and served as positive control; test groups III to V were treated, respectively, with 125, 250 and 500 mg/kg ofEryngium foetidum extract, once daily for seven consecutive days. The mice were allowed to fast for 12 h after the last day of treatment and six from each group were sacrificed with ethyl ether. The rest of animal were sacrificed after a posttreatment period of seven days. The stomach of each animal was removed and opened through the longer curvature with sterile surgical instruments. The stomach and duodena, covering all subtypes of mucosa, were used for culturingH.pylori.


#### 2.8.3. Culture and Bacterial Load

Stomach biopsies were scraped off and homogenized with 0.5 mL of PBS in a 1.5 mL Eppendorf tube. The homogenized sample was serially diluted 10-fold. Each 0.1 mL of homogenate was plated on supplemented Columbia Agar and incubated at 37°C for 2 to 3 days under microaerophilic conditions [29].H.pylori was identified through macroscopic characteristics (small translucent colonies), microscopic characteristics and Gram staining (Gram negative curved rods), and enzymatic properties (urease, catalase, and oxidase positive). The bacterial load was estimated by colony count and expressed as log10 CFU per milliliter of homogenate.


### 2.9. Statistical Analysis

The results were expressed as means ± standard deviation. Before analysis, bacterial densities were expressed in log10. The analysis of variance followed by the paired Student’s t-test was used for the statistical evaluation of data. The differences between groups were considered significant at p
<
0.05.


## 2.1. Bacterial Strains

The clinical microorganisms used here were obtained from the Gastroenterology Department of Laquintinie Hospital, Douala, Cameroon. These microorganisms were collected from gastric biopsies of patients complaining of upper gastrointestinal pain, hematemesis, or/and dyspepsia requiring gastroendoscopic examination. Six strains ofH.pylori were freshly isolated and suspended in Brain Heart Infusion (BHI) broth supplemented with 5% horse serum and 20% glycerol and stored at −80°C until further use.


## 2.2. Animals

Swiss albino mice of 18–22 g weight obtained from Animal House, University of Dschang, Cameroon, were selected as experimental animals. These animals were fed with standard pellet diet and tap water.


## 2.3. Statement on Ethics Approval

Throughout the experiments, all animals received human care according to the criteria outlined in the internationally accepted principle guidelines of the European Union on Animal Care (CEE Council 86/609). Animal experiments were designated and approved by the Cameroon National Ethics Committee (Reg. number. FWA-IRB00001954).


## 2.4. Plant Material

The leaves ofBidens pilosa (Asteraceae) andGalinsoga ciliata (Asteraceae) were collected in Dschang (west region of Cameroon) and leaves ofEryngium foetidum (Apiaceaes) in Kumba (southwest region of Cameroon). Identification of the plant was done at the National Herbarium Yaoundé (voucher specimen numbers 42254/HNC, 57409/HNC, and 42131 HNC, resp., in the listed order. HNC: Cameroon National Herbarium). The plant materials were then air-dried at room temperature and ground into a fine powder.


## 2.5. Chemicals and Culture Media

Culture media (Columbia Agar, Brain Heart Infusion (BHI), lacked horse blood, Horse Serum, Vitox Supplement) and CampyGen gas pack were obtained from Oxoid, Basingstoke, England. Doxycycline (doxycycline 200 mg, Combitic Global Caplet, India), metronidazole (metronidazole Tablets BP 500 mg, Strides Arcolab, India), and ciprofloxacin (zoflox, ciprofloxacin 750 mg, Odypharm) were used as reference antibiotics for the determination of the MIC and MBC and for thein vivo test were obtained from local pharmacy. P-Iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was used to indicate microbial growth [27].


## 2.6. Extraction

Dried powdered plant material was weighed (250 g) and soaked in 3.5 L of ethyl acetate (EA) or methanol (MeOH) for 72 hours. The plant mixture was then filtered and concentrated under reduced pressure using a rotary evaporator. The extract was further concentrated by allowing it to stand overnight in an oven at 30°C.


## 2.7.In Vitro Study

### 2.7.1. Antimicrobial Assay: Determination of MIC and MBC

The clinical strains (H.pyloriα1,H. pyloriα2, H.pyloriα3, H.pyloriα4, H.pyloriα5, andH.pyloriα6), identified using Gram staining and enzymatic properties (catalase, oxidase, and urease), were employed to evaluate thein vitro anti-Helicobacter activity of the plant-extracts. Methanol and ethyl acetate extracts ofBidens pilosa,Galinsoga ciliata, andEryngium foetidum were used for the determination of MICs by the INT broth microdilution method [27] using 96-well plates. Twofold dilutions of each extract were prepared in the test wells in BHI broth supplemented with 5% horse serum (BHI-serum). The final extract concentrations ranged from 0.002 to 1.024 mg/mL. One hundred microliters of inoculums prepared from 48 h colonies on supplemented Columbia Agar (Columbia Agar + 5% (v/v) lacked horse blood and 1% (v/v) Vitox) at McFarland turbidity standard 3 was added to 100 μL of the extract-containing culture medium. Control wells were prepared with culture medium and bacterial suspension and broth, only respectively. Ciprofloxacin, doxycycline, and metronidazole at concentration ranges of 0.002 to 0.128 mg/mL were used as positive control. The plates were covered with a sterile plate sealer; the contents of the wells were mixed with a shaker and incubated for 3 days at 37°C under microaerophilic conditions. After incubation, 40 μL of 0.2 mg/mL INT was added per well and incubated at 37°C for 30 min. Living bacteria reduced the yellow dye to pink. The sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth is known as the MIC. The MBC was determined by adding 50 μL aliquots of the preparations which prevented the color change of the medium after incubation during MIC assays, to 150 μL of BHI-serum. These preparations were incubated at 37°C for 72 h under microaerophilic conditions. The lowest concentration of extract, which did not produce a color change after addition of INT, was considered as the MBC. Each MIC and each MBC were determined in triplicate and the mean values are recorded.


### 2.7.2. Preliminary Phytochemical Analysis

The qualitative phytochemical analysis ofEryngium foetidum methanol extract was performed following the methods of Parekh and Chanda [28] to determine the presence of alkaloids (Mayer, Wagner, and Dragendorff test), flavonoids (alkaline reagent, Shinoda), phenolics (lead acetate, alkaline reagent test), triterpenes and steroids (Liebermann-Burchard Test), saponins (foam test), tannins (gelatine), anthraquinones (ether-chloroform 1 : 1 v/v, NaOH 10% w/v), and anthocyanins (1% HCl, heating). The results were qualitatively expressed in term of positive (+) and negative (−) signs.


## 2.7.1. Antimicrobial Assay: Determination of MIC and MBC

The clinical strains (H.pyloriα1,H. pyloriα2, H.pyloriα3, H.pyloriα4, H.pyloriα5, andH.pyloriα6), identified using Gram staining and enzymatic properties (catalase, oxidase, and urease), were employed to evaluate thein vitro anti-Helicobacter activity of the plant-extracts. Methanol and ethyl acetate extracts ofBidens pilosa,Galinsoga ciliata, andEryngium foetidum were used for the determination of MICs by the INT broth microdilution method [27] using 96-well plates. Twofold dilutions of each extract were prepared in the test wells in BHI broth supplemented with 5% horse serum (BHI-serum). The final extract concentrations ranged from 0.002 to 1.024 mg/mL. One hundred microliters of inoculums prepared from 48 h colonies on supplemented Columbia Agar (Columbia Agar + 5% (v/v) lacked horse blood and 1% (v/v) Vitox) at McFarland turbidity standard 3 was added to 100 μL of the extract-containing culture medium. Control wells were prepared with culture medium and bacterial suspension and broth, only respectively. Ciprofloxacin, doxycycline, and metronidazole at concentration ranges of 0.002 to 0.128 mg/mL were used as positive control. The plates were covered with a sterile plate sealer; the contents of the wells were mixed with a shaker and incubated for 3 days at 37°C under microaerophilic conditions. After incubation, 40 μL of 0.2 mg/mL INT was added per well and incubated at 37°C for 30 min. Living bacteria reduced the yellow dye to pink. The sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth is known as the MIC. The MBC was determined by adding 50 μL aliquots of the preparations which prevented the color change of the medium after incubation during MIC assays, to 150 μL of BHI-serum. These preparations were incubated at 37°C for 72 h under microaerophilic conditions. The lowest concentration of extract, which did not produce a color change after addition of INT, was considered as the MBC. Each MIC and each MBC were determined in triplicate and the mean values are recorded.


## 2.7.2. Preliminary Phytochemical Analysis

The qualitative phytochemical analysis ofEryngium foetidum methanol extract was performed following the methods of Parekh and Chanda [28] to determine the presence of alkaloids (Mayer, Wagner, and Dragendorff test), flavonoids (alkaline reagent, Shinoda), phenolics (lead acetate, alkaline reagent test), triterpenes and steroids (Liebermann-Burchard Test), saponins (foam test), tannins (gelatine), anthraquinones (ether-chloroform 1 : 1 v/v, NaOH 10% w/v), and anthocyanins (1% HCl, heating). The results were qualitatively expressed in term of positive (+) and negative (−) signs.


## 2.8.In Vivo Anti-Helicobacter pylori Assessment

### 2.8.1. Inoculation of Experimental Animals

Methanol extract ofEryngium foetidum, the most active plant-extract according to the MIC value obtained, was chosen for furtherin vivo anti-Helicobacter assessment. For this purpose, seventy-two mice were allowed to acclimatize for one week before initiation of experiment. After the acclimatization period, the animals were fasted for 12 h and sixty of them were infected with 0.2 mL of 108 CFU/mL ofH.pyloriα2 suspension, four times in one week with a 24 h interval between each inoculation. The inoculation day was considered as day 0 and subsequent days as day 1, day 2, and day 3 up to day 21. A group of uninfected mice serving as a normal group received sterile solution of the vehicle (200 μL of 0.25% Tween 80).


### 2.8.2. Distribution of Animals

Inoculated mice were distributed into five groups of 12 animals and allowed to rest for 24 h after the last inoculation. Group I, a negative control group, received sterile solution of the vehicle; group II was treated with ciprofloxacin (500 mg/kg) and served as positive control; test groups III to V were treated, respectively, with 125, 250 and 500 mg/kg ofEryngium foetidum extract, once daily for seven consecutive days. The mice were allowed to fast for 12 h after the last day of treatment and six from each group were sacrificed with ethyl ether. The rest of animal were sacrificed after a posttreatment period of seven days. The stomach of each animal was removed and opened through the longer curvature with sterile surgical instruments. The stomach and duodena, covering all subtypes of mucosa, were used for culturingH.pylori.


### 2.8.3. Culture and Bacterial Load

Stomach biopsies were scraped off and homogenized with 0.5 mL of PBS in a 1.5 mL Eppendorf tube. The homogenized sample was serially diluted 10-fold. Each 0.1 mL of homogenate was plated on supplemented Columbia Agar and incubated at 37°C for 2 to 3 days under microaerophilic conditions [29].H.pylori was identified through macroscopic characteristics (small translucent colonies), microscopic characteristics and Gram staining (Gram negative curved rods), and enzymatic properties (urease, catalase, and oxidase positive). The bacterial load was estimated by colony count and expressed as log10 CFU per milliliter of homogenate.


## 2.8.1. Inoculation of Experimental Animals

Methanol extract ofEryngium foetidum, the most active plant-extract according to the MIC value obtained, was chosen for furtherin vivo anti-Helicobacter assessment. For this purpose, seventy-two mice were allowed to acclimatize for one week before initiation of experiment. After the acclimatization period, the animals were fasted for 12 h and sixty of them were infected with 0.2 mL of 108 CFU/mL ofH.pyloriα2 suspension, four times in one week with a 24 h interval between each inoculation. The inoculation day was considered as day 0 and subsequent days as day 1, day 2, and day 3 up to day 21. A group of uninfected mice serving as a normal group received sterile solution of the vehicle (200 μL of 0.25% Tween 80).


## 2.8.2. Distribution of Animals

Inoculated mice were distributed into five groups of 12 animals and allowed to rest for 24 h after the last inoculation. Group I, a negative control group, received sterile solution of the vehicle; group II was treated with ciprofloxacin (500 mg/kg) and served as positive control; test groups III to V were treated, respectively, with 125, 250 and 500 mg/kg ofEryngium foetidum extract, once daily for seven consecutive days. The mice were allowed to fast for 12 h after the last day of treatment and six from each group were sacrificed with ethyl ether. The rest of animal were sacrificed after a posttreatment period of seven days. The stomach of each animal was removed and opened through the longer curvature with sterile surgical instruments. The stomach and duodena, covering all subtypes of mucosa, were used for culturingH.pylori.


## 2.8.3. Culture and Bacterial Load

Stomach biopsies were scraped off and homogenized with 0.5 mL of PBS in a 1.5 mL Eppendorf tube. The homogenized sample was serially diluted 10-fold. Each 0.1 mL of homogenate was plated on supplemented Columbia Agar and incubated at 37°C for 2 to 3 days under microaerophilic conditions [29].H.pylori was identified through macroscopic characteristics (small translucent colonies), microscopic characteristics and Gram staining (Gram negative curved rods), and enzymatic properties (urease, catalase, and oxidase positive). The bacterial load was estimated by colony count and expressed as log10 CFU per milliliter of homogenate.


## 2.9. Statistical Analysis

The results were expressed as means ± standard deviation. Before analysis, bacterial densities were expressed in log10. The analysis of variance followed by the paired Student’s t-test was used for the statistical evaluation of data. The differences between groups were considered significant at p
<
0.05.


## 3. Results

### 3.1. MIC and MBC Determination of Plant-Preparations and Antibiotics

Plant species showed different anti-Helicobacter activity with each other with MIC values ranging from 64 to >1024 μg/mL. The methanol extract ofE.foetidum shows the best activity for MIC values between 64 and 512 μg/mL against 3/6 (50%) evaluated strains. Methanol extract fromE.foetidum also displayed the best spectrum of bactericidal effect with a ratio of MBC/MIC 1 obtained on two testedH.pylori strains. MIC values from 128 to 512 μg/mL were also recorded withE.foetidum (AE),Bidens pilosa (MeOH),Bidens pilosa (AE),Galinsoga ciliate (MeOH), andGalinsoga ciliata (AE) extracts, respectively, against 2/6 (33.33%), 4/6 (66.66%), 1/6 (16.66%), 4/6 (66.66%), and 4/6 (66.66%) of the evaluated bacteria. The best MIC value (64 μg/mL) was obtained with the methanol extract fromE.foetidum againstH.pyloriα1 andH.pyloriα2.


### 3.2. Phytochemical Screening

According to Table2, alkaloids, phenols, flavonoids, anthraquinones, and sterol are the chemical compounds present in the methanol extract ofE.foetidum.


### 3.3. Culture

After the cessation of treatment, the number ofH.pylori positive animals in treated groups (plant-extract and ciprofloxacin) was lower than the number recorded in the untreated infected group (control group) (Table 3). None of the animals treated with the highest dose of plant-extract (500 mg/kg) or ciprofloxacin wasH.pylori positive. However, only 83.33% of animals treated with 125 and 250 mg/kg of the plant-extract wereH.pylori negative. There were no differences among the groups treated with three doses of plant-extract and ciprofloxacin at the second posttreatment period of time. Almost all the infected treated animals wereH.pylori negative at the second posttreatment period, indicating the lasting action of the plant-extract or ciprofloxacin.


### 3.4. Bacterial Load

Infected mice showed stableH.pylori colonization of gastric mucosa as shown in the number of bacterial counts (Figure 1). The bacterial load of gastric mucosa in infected mice was significantly reduced following the administration of 500 mg/kg ofE.foetidum (381.9
±
239.5 CFU) and ciprofloxacin (248
±
153.2 CFU) compared to that recorded with untreated infected mice (14350
±
690 CFU).Figure 1
Effects of different treatment on the recovery ofH.pylori from infected mice. Each circle represents the bacterial count for one animal. Inf: infected + vehicle; Inf + Ef 125: infected +E.foetidum pinnatum 125 mg/kg; Inf + Ef 250: infected +E.foetidum250 mg/kg; Inf + Ef 500: infected +E.foetidum500 mg/kg; Inf + Cp 500: infected + ciprofloxacin 500 mg/kg; Uninf: normal group. Data column of the different treatment with superscript ∗ are significantly different compared with infected control group (p
∗
<
0.05).


## 3.1. MIC and MBC Determination of Plant-Preparations and Antibiotics

Plant species showed different anti-Helicobacter activity with each other with MIC values ranging from 64 to >1024 μg/mL. The methanol extract ofE.foetidum shows the best activity for MIC values between 64 and 512 μg/mL against 3/6 (50%) evaluated strains. Methanol extract fromE.foetidum also displayed the best spectrum of bactericidal effect with a ratio of MBC/MIC 1 obtained on two testedH.pylori strains. MIC values from 128 to 512 μg/mL were also recorded withE.foetidum (AE),Bidens pilosa (MeOH),Bidens pilosa (AE),Galinsoga ciliate (MeOH), andGalinsoga ciliata (AE) extracts, respectively, against 2/6 (33.33%), 4/6 (66.66%), 1/6 (16.66%), 4/6 (66.66%), and 4/6 (66.66%) of the evaluated bacteria. The best MIC value (64 μg/mL) was obtained with the methanol extract fromE.foetidum againstH.pyloriα1 andH.pyloriα2.


## 3.2. Phytochemical Screening

According to Table2, alkaloids, phenols, flavonoids, anthraquinones, and sterol are the chemical compounds present in the methanol extract ofE.foetidum.


## 3.3. Culture

After the cessation of treatment, the number ofH.pylori positive animals in treated groups (plant-extract and ciprofloxacin) was lower than the number recorded in the untreated infected group (control group) (Table 3). None of the animals treated with the highest dose of plant-extract (500 mg/kg) or ciprofloxacin wasH.pylori positive. However, only 83.33% of animals treated with 125 and 250 mg/kg of the plant-extract wereH.pylori negative. There were no differences among the groups treated with three doses of plant-extract and ciprofloxacin at the second posttreatment period of time. Almost all the infected treated animals wereH.pylori negative at the second posttreatment period, indicating the lasting action of the plant-extract or ciprofloxacin.


## 3.4. Bacterial Load

Infected mice showed stableH.pylori colonization of gastric mucosa as shown in the number of bacterial counts (Figure 1). The bacterial load of gastric mucosa in infected mice was significantly reduced following the administration of 500 mg/kg ofE.foetidum (381.9
±
239.5 CFU) and ciprofloxacin (248
±
153.2 CFU) compared to that recorded with untreated infected mice (14350
±
690 CFU).Figure 1
Effects of different treatment on the recovery ofH.pylori from infected mice. Each circle represents the bacterial count for one animal. Inf: infected + vehicle; Inf + Ef 125: infected +E.foetidum pinnatum 125 mg/kg; Inf + Ef 250: infected +E.foetidum250 mg/kg; Inf + Ef 500: infected +E.foetidum500 mg/kg; Inf + Cp 500: infected + ciprofloxacin 500 mg/kg; Uninf: normal group. Data column of the different treatment with superscript ∗ are significantly different compared with infected control group (p
∗
<
0.05).


## 4. Discussion

In this study, the antimicrobial activities of the ethyl acetate and methanol extract of three medicinal Cameroonian plants were evaluated against sixH.pylori strains.In vitro, the tested plants displayed selective antibacterial activities and this activity was different within the same plant species from one extraction solvent to another. In fact, the type of solvent used largely affects the effectiveness of the extracts. The differences observed in the antibacterial activities of the plants species could be due to the differences in their chemical composition and in the mechanism of action of their bioactive constituents [30]. It is known that the antibacterial activity of a plant-extract is considered significant when MIC values are less than 100 μg/mL, moderate when being between 100 and 625 μg/mL, and weak when being greater than 625 μg/mL [31]. Thus, the MIC value of 64 μg/mL obtained withE.foetidummethanol extract againstH.pyloriα1 andH.pyloriα2 can be considered significant. The active principles of the extract of this plant should be assignable to different secondary metabolites belonging to a wide variety of compound classes. The preliminary phytochemical analysis of the tested plant-extract revealed the presence of alkaloids, phenols, flavonoids, anthraquinones, and steroid (Table 2). The result of the antimicrobial activity ofE.foetidumobtained herein correlates with that of Lingaraju et al. [22] showing that the ethyl acetate extracts of this plant have antimicrobial activity againstBacillus subtilis,Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa, and fungusCandida albicans using agar well diffusion method. The activity of the methanol extract ofE.foetidumagainstH.pyloriα1 andH.pyloriα2 was similar to that of doxycycline, a standard drug againstH.pyloriα1 (Table 1). This is quite remarkable particularly because the standard antibiotics are in the purified form whereas the extracts are mixtures of both pharmacological and nonpharmacological substances. Anti-Helicobacter activity of the methanol extract ofE.foetidumwas better than that of metronidazole, another antibiotic used in the treatment ofH.pylori infection. Metronidazole-containing regimens have recently been shown to have limited effectiveness because of the increasing prevalence of resistance to this drug [32]. Its prevalence varies from 10 to 90% in different countries [33]. Studies of Ndip et al. [34] and Tanih et al. [32] carried out, respectively, in Cameroon and South Africa, documented a very high resistance rate of 95.5% to metronidazole.Table 1
MIC/MBC of crude plant-extracts and antibiotics (µg/mL).
Crude extracts/antibiotics
H. pylori stains
H. pyloriα1
H. pyloriα2
H. pyloriα3
H. pyloriα4
H. pyloriα5
H. pyloriα6
E. foetidum(MeOH)
64/64
64/64
512/512
>1024
>1024
>1024
E. foetidum(AE)
256/256
512/512
>1024
>1024
>1024
>1024
Bidens pilosa (MeOH)
1024/1024
512/1024
>1024
256/1024
512/1024
256/512
Bidens pilosa (AE)
1024/1024
>1024
>1024
256/512
>1024
1024/1024
Galinsoga ciliata (MeOH)
512/1024
256/512
>1024
>1024
128/512
256/512
Galinsoga ciliata (AE)
256/512
>1024
256/512
256/512
>1024
128/512
Doxycycline
64/64
32/32
32/32
32/32
4/16
32/32
Ciprofloxacin
8/8
32/32
2/2
<2
16/16
8/8
Metronidazole
>128
>128
>128
>128
>128
>128
All the values given in the table are means of three determinations. MeOH: methanol; AE: ethyl acetate.Table 2
Phytochemical screening of methanol extract ofE. foetidum.
Compounds
Methanol extract ofE. foetidum
Alkaloids
+
Flavonoids
+
Phenolics
+
Saponins
−
Tannins
−
Triterpenes
−
Anthraquinones
+
Anthocyanins
−
Steroid
+
+: present; −: absent.Table 3
Effects of different doses ofE. foetidumand ciprofloxacin on H. pylori gastric colonization of infected and normal mice.
Treatment group (mg/kg)
Tests
1 day after cessation of treatment
7 days after cessation of treatment
Animals number
Animals number
1
2
3
4
5
6
Negative (%)
1
2
3
4
5
6
Negative (%)
Normal
Urease
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Catalase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Oxidase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Gram st
−
−
−
−
−
−
100
−
−
−
−
−
−
100
H. pylori + vehicle
Urease
+
+
+
+
+
+
0
+
+
+
+
+
+
0
Catalase
+
+
+
+
+
+
0
+
+
+
+
+
+
0
Oxidase
+
+
+
+
+
+
0
+
+
+
+
+
+
0
Gram st
+
+
+
+
+
+
0
+
+
+
+
+
+
0
H. pylori+ E. foetidum, 125
Urease
−
−
−
+
+
+
50
−
−
+
+
−
−
67
Catalase
−
−
−
+
−
−
83
−
−
−
−
−
−
100
Oxidase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Gram st
−
−
−
+
+
−
67
−
−
−
+
−
−
83
H. pylori+ E. foetidum, 250
Urease
−
+
+
−
−
−
67
−
−
−
−
−
+
83
Catalase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Oxidase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Gram st
−
+
−
−
−
−
83
−
−
−
−
−
−
100
H. pylori+ E. foetidum, 500
Urease
−
−
−
−
−
+
83
−
−
−
−
−
−
100
Catalase
−
−
−
−
−
+
83
−
−
−
−
−
−
100
Oxidase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Gram st
−
−
−
−
−
+
83
−
−
−
−
−
−
100
H. pylori+ ciprofloxacin, 500
Urease
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Catalase
−
−
−
−
+
−
83
−
−
−
−
−
−
100
Oxidase
−
−
−
−
−
−
100
−
−
−
−
−
−
100
Gram st
−
−
−
+
−
−
83
−
−
−
−
−
−
100
All the percentages given in the table are means of the six animals (n
=
6) that tested negative per group. (+): positive test; (−): negative test.Since thein vitro anti-Helicobacter properties of a compound can become ineffectivein vivo, the most active plant-extractin vitro was also evaluatedin vivo on the mouse model. As indicated by the confirmation tests for the presence ofH.pylori and bacterial load, both methanol extract ofE.foetidum(500 mg/kg) and ciprofloxacin significantly reducedH.pylori colonization of gastric mucosa in infected mice as compared to infected untreated mice. Thus, our data showed that the anti-Helicobacter activity of the methanol extract ofE.foetidumwas effectivein vitro andin vivo. The activity of the plant-extract demonstrated herein is very important when considering that the tested plant-extracts are from edible plant parts and also when considering the medical importance of the tested bacteria. In fact,H.pylori is a virulent pathogen as evidenced by its low infective dose and high prevalence in human populations and the principal cause of type B gastritis, peptic ulcer disease, gastric adenocarcinoma, and MALT-lymphoma [35]. It has also been classified as a Class I carcinogen by the World Health Organization [35]. Furthermore, the continuous emergence of drugs resistant toH.pylori drastically reduced the limited range of antibiotics that have efficacy in the treatment of this infection leading globally to an increase in the frequency of therapeutic failure [36].In conclusion, the present data provided evidence that methanol extract ofE.foetidum could be a rich source of metabolites with antimicrobial activity to fightHelicobacter pylori infections. Further investigations on purification and structure elucidation of the compounds are in progress.



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*Source: 2171032-2016-08-18.xml*