In Vivo Study of Healing Effects of Sida acuta Leaf Extracts on Helicobacter pylori Induced Ulceration in Mice

Helicobacter pylori is a human pathogen that is widely distributed and known to cause peptic ulcer, gastritis and gastric cancer. Treatment is usually by combination of acid inhibitory therapy and antibiotics because of drug resistance problems. Studies now focus on the use of medicinal plants for treatment of H. pylori induced ulcers because they are inexpensive and have limited side effects. This study was undertaken to determine the effects of cold water and ethanol extracts of Sida acuta leaves on H. pylori induced ulcer in mice. Oral administration with the aid of a feeding tube of 0.5 ml of 10 CFU/ml of H. pylori (Accession number LT799736) in phosphate buffer saline (pH 6.8) was used to induce ulcer in mice. Effects of various concentrations of cold water and ethanol extracts of S. acuta leaves on the ulcer parameters and histology examinations of stomach tissues of mice were investigated after 2 weeks of administration of extracts into the mice. Mice treated with clarithromycin served as positive control while mice administered with H. pylori but not treated served as negative control. One-way analysis of variance (ANOVA) was used for data analysis and results were considered significant if p < 0.05. Bacterial load of stomach tissue showed significant reduction from 15.4 × 10 CFU/ml to 3.5 × 10 CFU/ml and 2.6 × 10 CFU/ml for cold water and ethanol extracts respectively. Reduction in ulcer severity (2.00 ± 0.10 to 0.080 ± 0.05), ulcer index (8.50 ± 1.10 to 4.26 ± 0.03) and gastric volume (1.32 ± 0.12 to 0.54 ± 0.13) were observed. Histology of stomach tissues of mice treated with extracts revealed clear granulation indicating tissue repair and wound healing. S. acuta extracts were observed to enhance ulcer healing in a mice model. How to cite this paper: Ekwealor, C.C., Okorie, C.C., Ukoha, C.C. and Mba, A.N. (2020) In Vivo Study of Healing Effects of Sida acuta Leaf Extracts on Helicobacter pylori Induced Ulceration in Mice. Journal of Biosciences and Medicines, 8, 1-14. https://doi.org/10.4236/jbm.2020.89001 Received: July 12, 2020 Accepted: September 4, 2020 Published: September 7, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access C. C. Ekwealor et al. DOI: 10.4236/jbm.2020.89001 2 Journal of Biosciences and Medicines

treatment of H. pylori induced ulcers because they are inexpensive and have limited side effects. This study was undertaken to determine the effects of cold water and ethanol extracts of Sida acuta leaves on H. pylori induced ulcer in mice. Oral administration with the aid of a feeding tube of 0.5 ml of 10 7 CFU/ml of H. pylori (Accession number LT799736) in phosphate buffer saline (pH 6.8) was used to induce ulcer in mice. Effects of various concentrations of cold water and ethanol extracts of S. acuta leaves on the ulcer parameters and histology examinations of stomach tissues of mice were investigated after 2 weeks of administration of extracts into the mice. Mice treated with clarithromycin served as positive control while mice administered with H. pylori but not treated served as negative control. One-way analysis of variance (ANOVA) was used for data analysis and results were considered significant if p < 0.05. Bacterial load of stomach tissue showed significant reduction from 15.4 × 10 6 CFU/ml to 3.5 × 10 6 CFU/ml and 2.6 × 10 6 CFU/ml for cold water and ethanol extracts respectively. Reduction in ulcer severity (2.00 ± 0.10 to 0.080 ± 0.05), ulcer index (8.50 ± 1.10 to 4.26 ± 0.03) and gastric volume (1.32 ± 0.12 to 0.54 ± 0.13) were observed. Histology of stomach tissues of mice treated with extracts revealed clear granulation indicating tissue repair and wound healing. S. acuta extracts were observed to enhance ulcer healing in a mice model. How to cite this paper: Ekwealor, C.C., Okorie, C.C., Ukoha, C.C. and Mba, A.N.

Introduction
Helicobacter pylori is one of the most important human pathogens, affecting more than half of the world's population [1]. The prevalence of H. pylori infection varies widely according to geographical area, patient age and socioeconomic status [2]. In general, people in developing nations, residents of developed countries with low socioeconomic status and poor level of hygienic social environment have a higher prevalence of infection [3] [4].
The range of isolation is between 70% -90% in developing countries and 25% -50% in developed countries [5]. In various regions of sub-Saharan Africa, for example, 61% -100% of the population may harbor the pathogen [6]. Infection with H. pylori is usually acquired in early childhood and persists for life [7].
While over 80% of infected individuals are asymptomatic [8], the infection can lead to peptic ulcer, gastritis, and gastric cancer [1] [9], thus, it has been recognized as the principal agent leading to gastric cancer and as a class I carcinogen by WHO. H. pylori uniquely colonizes the stomach where it induces inflammation and affects gastric physiology [9]. Motility has been shown to be essential for successful in vivo colonization by H. pylori and is provided by its sheathed flagella [10].
H. pylori eradication is very necessary and the management strategy depends on whether the patient is a first-time or a chronic non-steroidal anti-inflammatory drugs (NSAID) user [11]. Drugs that have demonstrated efficacy include amoxicillin, clarithromycin, metronidazole, tetracycline and bismuth [4]. In general, monotherapy is not recommended for the treatment of H. pylori infection due to poor clearance rates and problems of drug resistance [12]. Eradication is usually achieved with a combination of acid-inhibiting therapy and antibiotics.
The addition of anti-secretory agents to antibiotics accelerates the ulcer healing process [13]; however, such combination therapy does not always successfully eradicate H. pylori and is expensive [14]. Many studies now focus on the use of medicinal plants for the treatment of H. pylori, because of the limited side effects on tissues and it's inexpensive nature [15] [16] [17]. They have also been used in traditional medicine to treat a wide range of diseases including digestive disorders such as ulcers [18].
Sida acuta otherwise known as broom weed is a shrub belonging to Malvaceae family. The plant is widely distributed in the subtropical regions and has many traditional usages that varied from one region to another [19]. All the plant parts exert various pharmacological properties which include antiplasmodial, antimicrobial, antioxidant and cytotoxic activities [20]. In Nigeria, S. acuta known as Udo by the Igbos [21] and Iseketu by the Yorubas [22] has been reported to be

Microorganism Used
Helicobacter pylori (Accession number LT79936) was used for ulcer induction in mice.

Preparation of Plant Materials
The leaves were washed under running tap water, dried at 25˚C and ground into fine powder using an electric blender (Moulinex

Experimental Design, Induction of Ulcer Using H. pylori and Treatment
A total of fifty four healthy swiss mice weighing 17 -20 grams were housed in polypropylene cages under controlled conditions of temperature (25˚C) with 12 h light dark cycles [24]. They were allowed to acclimatize for 7 days and given free access to feeds and water ad libitum.
Mice were divided into six groups (A-F), with 6 mice in groups A-C and 12 mice in groups D-F. Mice with the exception of those in group A were orally administered with 0.5 ml of 10 7 CFU/ml of H. pylori in phosphate buffer saline (pH 6.8) with the aid of a feeding tube to induce ulcer. The mice received a total of 3 doses given at 3 days interval to enable proper pathogen incubation and disease establishment [25]. Group A mice were administered with 0.5 ml of phosphate buffered saline only (healthy control) while in Group B H. pylori was administered without treatment (negative control). In Group C, H. pylori was administered and treated with 0.5 ml of 50 mg/ml clarithromycin (positive control), Group D has 12 of the mice administered with H. pylori but 6 of them were treated with 0.5 ml of 100 mg/ml cold water extract and the other 6 treated with 0.5 ml of 100 mg/ml ethanol extract. Group E was similarly administered with H. pylori but 0.5 ml of 200 mg cold water and ethanol extracts were used for treatment. In Group F as earlier stated, H. pylori was administered and treated with 0.5 ml of 400 mg/ml of cold water and ethanol extracts. The treatment with clarithromycin and leaf extracts were carried out twice daily for 14 days.

Animal Sacrifice
The mice were anaesthetized by chloroform inhalation for 1min before sacrifice.
Mice stomach was opened through the greater curvature and the contents used for estimation of bacterial count, evaluation of ulcer parameters and histology studies.

Bacterial Count
Stomach biopsies were scraped off and homogenized with 0.5 ml of phosphate buffered saline (PBS) and the homogenate serially diluted 10-fold. 0.1 ml of 10 −6 dilution of the homogenate was inoculated on Columbia Agar (Oxoid) and incubated at 37˚C for 5 days under microaerophilic conditions. The bacterial load was estimated by colony count and expressed as log 10 CFU per milliliter of homogenate [17].

Evaluation of Ulcer Parameters
Determination of Ulcer Index: The gastric contents were removed from the mice and blood cloths washed off with water. Mice were fixed on a board and ulcer formation examined using a 10× magnifier lens. The following scores/ratings were used to evaluate the ulcer index as well as the severity of gastric lesions [26]. Determination of Gastric Volume: Gastric content was collected and centrifuged at 300 rpm for 10 min to remove any solid mass and the volume of gastric juice determined using a graduated measuring cylinder [27].
Determination of pH: The pH of the gastric contents of both the control and the treated groups were determined using the method of [27]. An aliquot of 0.5 ml of gastric juice was diluted with 0.5 ml distilled water and the pH of the solution measured using digital pH meter.
Determination of Total Acidity: An aliquot of 0.5 ml of gastric juice was diluted with 0.5 ml of distilled water and was taken into a 50 ml conical flask. One drop of phenolphthalein indicator was added and titrated with 0.1NNaOH until a permanent pink color was observed [27]. The volume of 0.01NNaOH consumed was noted and total acidity calculated as shown below.

Histology Examination
This was performed according to [28]. Olympus binocular microscope at ×10 and ×40 magnification lens. Photomicrographs of the tissue section were captured using Motic image plus 2.0 camera.

Statistical Analysis
Results of the study were expressed as mean ± standard deviation. Differences between mean on the effect of S. acuta leaf extracts on the gastric parameters were analyzed using one-way analysis of variance (ANOVA) using SPSS version 21 software. Values were considered significant if p < 0.05.

Bacterial Load Count
All the infected mice showed stable H. pylori colonization of gastric mucosa at week 0. There was a reduction in bacterial load of gastric mucosa following the administration of the various concentrations of cold water and ethanol extracts of S. acuta and clarithromycin after weeks 1 and 2. As shown in Figure 1

Evaluation of Ulcer Parameters
The results of effects of cold water extract and ethanol extract of S. acuta on the gastric parameters of ulcerated mice after 2 weeks of treatment are as shown in Table 1. The ethanol and cold water extracts of S. acuta leaves showed significant protection against H. pylori induced ulcer at doses 100 mg/ml, 200 mg/ml and 400 mg/ml.
Mice treated with S. acuta extracts and clarithromycin (Groups C-F) had their ulcer index and severity, gastric volume and total acidity considerably reduced when compared to those infected without treatment (Group B). The gastric volume was significantly (p < 0.05) increased in Group B (1.32 ± 0.12) when compared with those in Group A (0.40 ± 0.10).

Histological Examinations
Histopathological changes were observed in the stomach tissues of mice in

Discussion
Unsuccessful eradication of H. pylori after treatments, recurrent infections, and increasing resistance of H. pylori to therapeutic treatments (5% -20% of treatments have been reported to fail due to H. pylori resistance to treatments) all have led to continuous research on finding new ways of treatments [29].
In this study, the research was designed to induce ulcer in mice using H. pylori (Accession Number LT79936) and to investigate the efficacy of the various concentrations of cold water and ethanol extracts of S. acuta in the ulcerated mice.
The result obtained in this study showed that all the infected mice (Groups B-F) had stable colonization of gastric mucosa by H. pylori which significantly reduced after 2 weeks of administration of extracts. It was observed that effects of the extracts on the studied parameters varied according to the extraction solvent, the concentration of the extract used and the length of administration of the extracts. Ethanol extracts had a significantly higher activity against the H. pylori than the cold water extracts. This variation may probably be due to better solubility of the bioactive components of S. acuta in ethanol which agrees with the reports of many researchers [30] [31] [32] [33]. They noted that components of medicinal plants are better extracted with alcoholic solvents like ethanol and methanol. The extracts inhibitory activities against H. pylori was also confirmed by the H pylori count in gastric biopsy culture, where the decrease in bacterial population was observed from samples treated with both extracts (cold water and ethanol) and clarithromycin. Several studies have also reported that extracts from several other plants, such as Allium sativum [15], Centella asiatica leaf [16], Aframomum pruinosum seeds [17], Calophyllum brasiliense Camb. [34], turmeric [35] and garlic powder and oil [36] have activities against H. pylori.
From the result obtained, there was increase in gastric volume after H. pylori was orally administered into the mice (Group B). This could be as a result of report by [37]   The micrograph of mice stomach tissue of Group B mice revealed mucosal lipidosis and deep ulcer while that of those treated with clarithromycin (50 mg/ml), cold water and ethanol extracts (400 mg/ml each) of S. acuta, all exhibited clear granulation which represents tissue repair or wound healing characterized by fibrous scar tissues, aggregation of infiltrates, and hyperplasia of epithelial cells. In another study carried out by [39], H&E staining of the gastric mucosa of H. pylori infected mice showed extensive lesions, oedema, leucocytes infiltration and hemorrhage which showed a significant reduction after treatment with geraniol.

Conclusion
The findings in this study confirm positive efficacy of the ethanol and cold water extracts of S. acuta leaves against H. pylori and in controlling stomach ulcer whose efficacy is comparable to standard drug clarithromycin. Thus, S. acuta has the potential to be developed as a treatment for H. pylori associated peptic ulcer disease. Further studies to obtain bioactive compounds of S. acuta and their mechanism of action are highly recommended.