Thabrew MI; Arawwawala LDAM

Review Article

Austin Biol. 2016; 1(2): 1007.

An Overview of In Vivo and In Vitro Models that can be used for Evaluating Anti-Gastric Ulcer Potential of Medicinal Plants

Thabrew MI¹* and Arawwawala LDAM²

¹Institute of Biochemistry, University of Colombo, Sri Lanka

²Industrial Technology Institute, Bauddhaloka Mawatha, Sri Lanka

*Corresponding author: Thabrew MI, Professor, Institute of Biochemistry, Molecular Biology & Biotechnology, University of Colombo, No. 90, Cumaratunga Munidasa Mawatha, Colombo 03, Sri Lanka

Received: April 01, 2016; Accepted: June 14, 2016; Published: June 16, 2016

Abstract

Many people in the world suffer from peptic ulcers. Of the two main types of peptic ulcers (gastric and duodenal) that develop in humans, gastric ulcers are the most commonly found. Although many medications are currently available for the management of gastric ulcers, prolonged use of these drugs may lead to series of adverse effects such as thrombocytopenia, nephrotoxicity, hepatotoxicity, gynecomastia and impotence. With the increasing tendency for use of herbal drugs for the alleviation of various disease conditions, associated with the belief that natural compounds produce less toxic side effects, much research is now being carried out worldwide, to investigate the potential of plants and plant based medicines to protect against the development of gastric ulcers or alleviate symptoms associated with this condition. In this review, the authors hope to present an overview of the in vivo and in vitro experimental models that have been used in different laboratories of the world during the past few decades, to carry out such investigations, along with the underlying mechanisms of ulcer induction in each method. The aim is to sensitize other researchers about the different experimental models available for carrying out investigations to discover the gastroprotective potential of plants or herbal remedies used in traditional systems of medicine, for the management of gastric ulcers and develop novel plant based drugs that could be used for their prevention and cure.

Keywords: Gastric ulcer models; Medicinal plants; Gastroprotection; Ulcer index

Introduction

Ulcers are lesions of the skin or mucous membrane characterized by the superficial inflamed dead tissue [1]. Among different types of ulcers that can develop, peptic ulcers are the most common. Peptic ulcers can develop on the inside lining of the stomach (gastric ulcer) or the small intestine (duodenal ulcer) [2]. Gastric ulcer, one of the widest spread, is believed to be due to an imbalance between aggressive and protective factors [3]. Studies have shown that gastric ulcer occurs in at least 10% of the world’s population [4]. The major protective factors include adequate blood flow, secretion of prostaglandins, mucin, nitric oxide, bicarbonate and growth factors. Aggressive agents include increased secretion of hydrochloric acid and pepsin, inadequate dietary habits, free oxygen radicals, the consumption of non-steroidal anti-inflammatory drugs and alcohol, stressful conditions and infection with Helicobacter pylori [5,6].

Several drugs such as anticholinergic drugs, histamine H2- receptor antagonists, antacids and irreversible proton pump inhibitors have been used for the treatment of gastric and duodenal ulcers [7]. However, prolonged use of these drugs may lead to series of adverse effects such as thrombocytopenia, nephrotoxicity, hepatotoxicity, gynecomastia and impotence [7,8]. Due to such unpleasant side effects produced by conventional drugs, there is an urgent need of more effective and safer treatments with fewer side effects, for the treatment of gastro-duodenal ulcers. Therefore, during the past few years, there has been an increasing interest in the development of plant based gastroprotective agents that are believed to produce less toxic side effects [9]. In this context it is important to first screen plants or herbal remedies with traditional ethno-medicinal uses in gastric ulcer management, for validation of their reputed antiulcer activity. To carry out such investigations it is essential to have credible experimental models.

Several in vivo and in vitro models are available to evaluate the antiulcer activity of medicines/plants. However, selection of a suitable model has proven to be difficult as each model has significant advantages as well as disadvantages. Further information about these various in vivo and in vitro models are scattered in the literature, and difficult to find. The main aim of this review is to present to the various researchers interested in carrying out studies on the gastroprotective potential of plants or herbal remedies, a comprehensive overview of available in vivo and in vitro models that could be used for this purpose, along with the underlying mechanisms of ulcer induction in each method. Thus, it gives a broad view of the issue that will help to select the most appropriate model for the validation of existing traditional therapies for gastric ulcers and development of novel plant based drugs that could be used for their prevention and cure.

In vivo models that can be used for study of gastroprotection

Peptic ulcers can be induced by physiological, pharmacological or surgical manipulation in several animal systems. However, rodents are the most commonly used as in vivo experimental models. The principles of those that are most frequently used by researchers investigating the gastroprotective effects of plants or herbal remedies, along with their underlying mechanisms of action, are described below.

Absolute ethanol-induced gastric lesions: Gastric mucosal injury may occur when defense mechanisms are impaired by noxious substances such as gastric acid and HCl secretion into the gastric lumen [10]. Administration of absolute ethanol by gavage has long been used as a reproducible method to induce gastric injury in experimental animals [11]. Ethanol can promote the development of gastric lesions by exposing the mucosa to the hydrolytic and proteolytic actions of hydrochloric acid and pepsin [12]. Moreover, ethanol can stimulate gastric acid secretion, resulting in microvascular injuries that facilitate vascular permeability, through reflex release of gastrin and histamine from sensitive nerve terminals present in the gastric mucosa [13]. It is known that intra-gastric administration of ethanol results in gastric mucosal injury characterized by disturbances in microcirculation, mast cell secretary products, inhibition of prostaglandin synthesis, reduction in mucus production and reactive species [14]. Ethanol is also known to increase cellular oxidative stress [15] and produce alterations in gastric cell calcium levels [16] that may lead to the pathogenesis of gastric mucosal injury.

The variety of damaging effects mediated by ethanol has been exploited in developing the ethanol induced gastric ulcer model for testing the gastroprotective potential of various plants/ natural compounds. However, because this model is independent of gastric acid secretion, it is not suitable to evaluate protection against ulceration dependent on acid secretion. Because ethanol can directly enhance the levels of free radicals that can mediate alterations in cell structure and function or contribute to other mechanisms that support oxidative damage [14] and can also mediate direct toxic effects on the gastric mucosa resulting in reduced secretion of bicarbonates and gastric mucous production [17], it is more appropriate to use this model for evaluating the gastroprotective potential of test materials that have cytoprotective and/or antioxidant activities (Table 1).

Table 1: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the absolute ethanol induced gastric lesion model.




  
    Plants
    Ref:
      No.
    Type of extract and    doses
    Type of animal    species used in the experiment
    Dose of ethanol used    to induce ulceration
    Reference drug
  
  
    Acacia ferruginea DC.
    [5]
    Bark
      Ethyl acetate fraction of    acetone extract
      (100 mg/kg)
    Male Wistar rat
      (180-220 g)
    99.5% absolute ethanol (5mL/kg,    p.o)
    Omeprazole
      (20 mg/kg, p.o)
  
  
    Alpinia calcarata Roscoe
    [18]
    Rhizomes
      Hot water extract
      (500, 750, 1000 mg/kg)
    Cross-bred male albino rats
      (200-250 g)
    Absolute ethanol
      (1 mL/kg, p.o)
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Cenostigma macrophyllum Tul.
    [19]
    Leaves
      Hydroalcoholic fraction of    ethanol extract
      (50, 100, 200 mg/kg)
    Male Swiss mice
      (20-30 g)
    Absolute ethanol
      (0.5 mL, p.o)
    Carbenoxolone
      (100 mg/kg, p.o)
  
  
    Conyza blinii H. Lev.
    [20]
    Whole plant
      Total saponins
      (5, 10, 20 mg/kg)
    Male & Female Sprague -    Dawley rats
      (180-200 g)
    Absolute ethanol
      (1 mL, p.o)
    Colloidal bismuth subcitrate
      (14.4 mg/kg, p.o)
  
  
    Curcuma xanthorrhiza Roxb.
    [21]
    Leaves
      Cold ethanolic extract
      (250, 500 mg/kg)
    Male� Sprague - Dawley rats
      (200-250 g)
    Absolute ethanol (5mL/kg, p.o)
    Omeprazole
      (20 mg/kg, p.o)
  
  
    Margaritaria    discoidea Baill.
    [22]
    Stem bark
      Ethanolic extract
      (50, 100, 150 mg/kg)
       
      Hexane, ethyl acetate,    dichloromethane, butanol and aqueous fractions
      (150 mg/kg from each fraction)
    Male Wistar albino rats
      (100-150 g)
    Absolute ethanol
      (1mL, p.o)
    Misoprostol
      (0.1 mg/kg, p.o)
  
  
    Piper tuberculatum� Jacq.
    [23]
    Fruits
      Dichloromethane fraction of    ethanol extract
      (10, 30, 100 mg/kg)
    Female Wistar rats (180-220 g)
    Absolute ethanol (0.5mL/200g,    b.wt, p.o)
    Carbenoxolone
      (100 mg/kg, p.o)
  
  
    Polygonum chinense Linn. 
    [24]
    Leaves
      Hot water extract
      (62.5, 125, 250, 500 mg/kg)
    Male & Female Sprague -    Dawley rats
      (200-250 g)
    Absolute ethanol (5mL/kg, p.o)
    Omeprazole
      (20 mg/kg, p.o)
  
  
    Trichosanthes cucumerina Linn.
    [25]
    Aerial parts
      Hot water extract
      (375, 500, 750,1000 mg/kg)
    Male & Female Wistar rats
      (200-225 g)
    Absolute ethanol (5mL/kg, p.o)
    Cimetidine
      (100 mg/kg, p.o)
       
      Sucralfate
      (400 mg/kg, p.o)
  
  
    Vochysia tucanorum 
      Mart.
    [26]
    Leaves
      Methanol extract
      (250, 500, 1000 mg/kg)
      Butanol fraction of methanolic    extract (37.5, 75, 150 mg/kg)
    Male Wistar rats
      (150-250 g)
    99.5% absolute ethanol (1 mL,    p.o)
    Carbenoxolone
      (100 mg/kg, p.o)
  
  
    Zanthoxylum rhoifolium Lam.
    [27]
    Stem bark
      Ethanol extract
      (62.5, 125, 250, 500 mg/kg)
    Male & female Swiss albino    mice
      (20-30 g)
    Absolute ethanol
      (1mL, p.o)
    N- acetylcysteine (750 mg/kg, i.p)



Table 1:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the absolute ethanol induced gastric lesion model.

To induce ulcers with ethanol, rats that have been fasted for 24-36 h are pretreated with vehicle or extracts or reference drug orally. After 1h, ulcers are induced by administration of absolute ethanol orally and kept for another hour after which rats are sacrificed, stomachs excised and severity of ulceration measured.

HCl/ethanol induced gastric lesions: This model may be considered to be an advanced model of the absolute ethanol induced ulcer model discussed before. Instead of ethanol only, a mixture of HCl and ethanol are used to induce ulceration. Gastric lesions induced by HCl/ethanol are due to direct necrotizing action on the gastric mucosa. Combination of ethanol with HCl is considered to accelerate the progress of ulcerogenesis and enhance gastric injury [28].

In this model, 18-24 h fasted mice are pretreated with vehicle or extracts or reference drug orally. After 1 h, gastric lesions are induced by administrating the ethanol/HCl mixture. The animals are euthanized after 30 min or 1 h, stomachs excised and severity of ulceration measured (Table 2).

Table 2: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the HCl/ethanol.




  
    Plants
    Ref:
      No.
    Type of extract and    doses
    Type of animal    species used in the experiment
    Dose of HCl/ethanol    used to induce ulceration
    Reference drug
  
  
    Celtis iguaneae Jacq.
    [29]
    Leaves
      Hexane extract
      (100 mg/kg)
    Male albino Swiss mice
      (25-35 g)
    0.45M HCl/60 % ethanol solution
      (10 mL/kg, p.o)
    Carbenoxolone
      (200 mg/kg, p.o)
  
  
    Cenostigma macrophyllum Tul.
    [19]
    Leaves
      Hydroalcoholic    fraction of ethanol extract
      (50, 100, 200    mg/kg)
    Male Swiss mice
      (20-30 g)
    0.15M HCl/40 %    ethanol solution
      (1 mL, p.o)
    Carbenoxolone
      (100 mg/kg, p.o) 
  
  
    Centaurea solstitialis Linn.
    [30]
    Flowers
      Sesquiterpene lactones:
      Chlorojanerin
      (59.2 mg/kg)
      13- acetyl solstitialin
      (179 mg/kg)
    Male & Female Balb-C mice
      (20-40 g)
    0.3M HCl/50 % ethanol solution
      (0.2 mL, p.o)
    Famotidine
      (20 mg/kg, p.o)
  
  
    Copaifera    langsdorffii Desf. 
    [6]
    Leaves
      70 % aqueous ethanolic extract
      (50,250,500 mg/kg)
       
      Isolated compounds: a humulene, � caryophyllene oxide, kaurenoic acid, quercitrin and afzelin
      (30 mg/kg/compound)
    Swiss mice
      (25-30 g) 
    0.3M HCl/50 % ethanol solution
      (0.2 mL, p.o)
    Omeprazole
      (30 mg/kg, p.o)
  
  
    Helicteres sacarolha A. St.-Hil.
    [31]
    Leaves
      Hydroethanolic extract
      (20, 50, 250 mg/kg)
    Albino mice-Swiss- Webster    strain
      (25-30 g) 
    0.3M HCl/60 % ethanol solution
      (0.3 mL, p.o)
    Carbenoxolone
      (100 mg/kg, p.o)
  
  
    Vochysia tucanorum
      Mart.
    [26]
       
    Leaves
      Methanol extract
      (250, 500, 1000 mg/kg)
    Male Swiss mice
      (25-40 g)
    0.3M HCl/60 % ethanol solution
      (0.2 mL, p.o)
    Lansoprazole
      (30 mg/kg, p.o)
  
  
    Zanthoxylum rhoifolium Lam.
    [27]
       
    Stem bark
      Ethanol extract
      (62.5, 125, 250, 500 mg/kg)
    Male & female Swiss albino    mice
      (20-30 g)
    0.15M HCl/40 %    ethanol solution
      (1 mL, p.o)
    N- acetylcysteine (750 mg/kg,    i.p)



Table 2:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the HCl/ethanol.

Water-immersion stress or cold-resistant stress induced gastric ulcers: Gastric ulcers induced by water –immersion stress or cold resistant stress [32] in rats or mice are known to resemble human peptic ulcers, both grossly and histopathologically [33]. The restraint technique developed by Brodie and Hanson [34] when coupled with the cold water or ordinary water immersion method developed by Levine [35] is reported to induce stress lesions in a synergistic manner.

Stress induced ulcers are mediated mainly by the release of histamine that results in an increased acid secretion, decreased mucus production, pancreatic juice reflux and poor flow of gastric blood. Generation of reactive oxygen species and inhibition of prostaglandin synthesis also promote stress induced ulcer formation [36,37]. This model has been widely used for assessing the gastroprotective effects of various test agents (Table 3), especially those with mucus enhancing and cytoprotective properties.

Table 3: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the stress models.




  
    Water-immersion    stress model
  
  
    Plants
    Ref:
      No.
    Type of extract and doses
    Type of animal species used in    the experiment
    Temperature
    Reference drug
  
  
    Centaurea solstitialis Linn.
    [30]
    Flowers
      Sesquiterpene lactones:
      Chlorojanerin
      (59.2 mg/kg)
      13- acetyl solstitialin
      (179 mg/kg)
    Male & Female    Sprague-Dawley arts
      (120-200 g)
    19-22 ^°C
    Famotidine
      (20 mg/kg, p.o)
  
  
    Copaifera malmei Harms.
    [38]
    Leaves
      Infusion
      (25, 100, 400 mg/kg)
    Male & Female Wistar albino    arts
      (120-200 g)
    19 ± 1 ^°C
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Helicteres sacarolha A. St.-Hil.
    [31]
    Leaves
      Hydroethanolic extract
      (20, 50, 250 mg/kg)
    Wistar strain rats
      (180-220 g)
    22 ^°C
    Ranitidine
      (50 mg/kg, p.o)
  
  
    Sambucus ebulus Linn.
    [39]
    Leaves
      n-buatnol fraction (540, 816    mg/kg) and remaining water sub extract (843 mg/kg) of methanolic extract
    Male Wistar strain rats
      (120-150 g)
    17-22 ^°C
    Famotidine
      (2 mg/kg, p.o)
  
  
    Cold-resistant stress    model 
  
  
    Celtis iguaneae Jacq.
    [29]
    Leaves
      Hexane extract
      (100 mg/kg)
    Male albino Swiss mice
      (25-35 g)
    4 ^oC for 2 h
    Ranitidine
      (100 mg/kg, p.o)
  
  
    Cenostigma macrophyllum Tul.
    [19]
    Leaves
      Hydroalcoholic fraction of    ethanol extract
      (50, 100, 200 mg/kg)
    Male Swiss mice
      (20-30 g)
    4 � 1 ^oC for 3 h
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Zanthoxylum rhoifolium Lam.
    [27]
    Stem bark
      Ethanol extract
      (125, 250, 500 mg/kg)
    Male & female Wistar rats
      (180-240 g)
    3 � 1 ^oC for 4 h
    Cimetidine
      (100 mg/kg, p.o)



Table 3:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the stress models.

In water-immersion stress model, animals are fasted for a period of 24-36 h prior to the experiment and treated with vehicle or test drug or reference drug. After 30 min. animals are placed individually in each compartment of a stress cage and immersed vertically up to the xyphoid level in a water bath and kept for 7 h to induce stress ulcer. After 7 h animals are sacrificed and ulceration quantified.

In cold resistant stress model animals are fasted for a period of 18 h prior to the experiment treated with vehicle or test drug or reference drug. After 1 h, rats are individually restrained in plastic cages in a refrigerator for 2-4 h and sacrificed. Finally, stomachs are taken out and severity of ulceration measured.

Pylorus-ligated-induced peptic ulcer: The ligation of the pylorus end of the stomach causes accumulation of gastric acid in the stomach which in turn produces ulcers. Ulcers result from a breakdown of the gastric mucosal barrier resulting from auto digestion of the gastric mucosa. This model is useful for evaluating the (a) cytoprotective effects of drugs that increase secretion of mucus and (b) anti-secretory drugs that reduce secretion of gastric aggressive factors such as acid and pepsin.

Pylorus ligation is performed according to the method described by Shay et al. [40] with slight modifications. In brief, animals fasted for 48 h, are pretreated with vehicle or extracts or reference drug orally, and after 1 h pyloric end of the stomach is ligated under ether anesthesia (Table 4). Stomach is then placed carefully in the abdomen and the wound sutured by interrupted sutures. Animals are sacrificed after 4 h and their stomachs are removed and longitudinally excised along the greater curvature. The inner surface is then examined for ulcerative lesions. The gastric volume and acidity are then assessed according to method described by Shay et al. [40].

Table 4: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the pylorus-ligated-induced peptic ulcer model.




  
    Plants
    Ref:
      No.
    Type of    extract and doses
    Type of    animal species used in the experiment
    Reference    drug
  
  
    Margaritaria    discoidea Baill.
    [22]
    Stem    bark
      Ethanolic extract
      (50, 100, 150    mg/kg)
    Male Wistar albino    rats
      (100-150 g)
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Muntingia calabura 
      Linn.
    [41]
    Leaves
      Methanolic extract
      (100, 200, 500    mg/kg)
    Male Sprague Dawley    rats
      (180-200 g)
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Piper tuberculatum 
      Jacq.
    [23]
    Fruits
      Dichloromethane    fraction of ethanol extract
      (10, 30, 100 mg/kg)
    Female Wistar rats
      (180-220 g)
    Piplartine
      (15 mg/kg, p.o)
  
  
    Sambucus ebulus Linn.
    [39]
    Leaves
      n-buatnol fraction (540,    816 mg/kg) and remaining water sub extract (843 mg/kg) of methanolic extract
    Male Wistar strain    rats
      (120-150 g)
    Famotidine
      (2 mg/kg, p.o)
  
  
    Trichosanthes cucumerina Linn.
    [42]
    Fruits
      50% ethanol extract
      (500 mg/kg)
    Male & female    Wistar rats
      (200-250 g)
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Zanthoxylum rhoifolium Lam
    [27]
    Stem    bark
      Ethanol extract
      (125, 250, 500    mg/kg)
    Male & female    Wistar rats
      (180-240 g)
    Cimetidine
      (100 mg/kg, p.o)



Table 4:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of the pylorus-ligated-induced peptic ulcer model.

NSAID’s induced gastric ulcers: Gastric ulcers are known to be induced by excessive use of many Non-Steroidal Anti-Inflammatory Drugs (NSAID’s) such as indomethacin, aspirin and ibuprofen [43]. This property has been exploited for the development of NSAID’s induced gatric ulcer models in rats. This is one of the most commonly used models for investigating antiulcer properties of test agents. NSAID’s induce gastric ulcers by inhibiting prostaglandin synthesis via the cyclooxygenase pathway [43,44]. In the stomach, prostaglandins protect against mucosal injury by stimulating bicarbonate and mucus secretion, maintaining mucosal blood flow and regulating mucosal cell turnover and repair [45,46]. NSAID’s can also induce mucosal damage by enhancement of reactive oxygen free radical production and neutrophil infiltration [46]. Furthermore, NSAID’s (especially those of acidic nature) can exert direct cytotoxic effects on epithelial cells and disrupt surface active phospholipids on the mucosal surface, independent of effects on synthesis of prostaglandins, thus making the mucosa more susceptible to damage by luminal acid [47]. This model is therefore useful for evaluating the ability of antisecretory and cytoprotective agents to protect against gastric ulcer development (Table 5).

Table 5: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of NSAID’s.




  
    Plants
    Ref:
      No.
    Type of    extract and doses
    Type of    animal species used in the experiment
    Dose of    NSAID to induce ulceration
    Reference    drug
  
  
    Cenostigma macrophyllum Tul. 
    [19]
    Leaves
      Hydroalcoholic    fraction of ethanol extract
      (100 mg/kg)
    Wistar rats
      (200-250 g)
    Indomethacine
      (30 mg/kg, s.c.)
    Cimetidine
      (100 mg/kg, p.o)
  
  
    Copaifera    malmei Harms.
    [38]
    Leaves
      Infusion extract
      (25,100, 400 mg/kg)
    Male & Female    Wistar rats
      (180-220 g)
    Piroxicam
      (200 mg/kg, p.o)
    Ranitidine
      (100 mg/kg, p.o)
  
  
    Trichosanthes cucumerina Linn.
    [25]
       
       
      [42]
    Aerial    parts
      Hot water extract
      (750 mg/kg)
       
      Fruits
      50% ethanolic    extract
      (500 mg/kg)
    Male & Female    Wistar rats
      (200 -225 g)
       
      Wistar rats
      (200-250 g)
    Indomethacine    (25mg/kg, p.o)
       
      Aspirin
      (500 mg/kg, p.o)
    Cimetidine
      (100 mg/kg, p.o)
       
      Cimetidine
      (100 mg/kg, p.o)
  
  
    Margaritaria    discoidea Baill.
    [22]
    Stem    bark
      Ethanolic extract
      (50, 100, 150    mg/kg)
    Male Wistar albino    rats
      (100-150 g)
    Indomethacine
      (80 mg/kg, p.o)
    Omeprazole
      (100 mg/kg, p.o)
  
  
    Glycyrrhiza    glabra 
      Linn.
    [48]
    Roots    & Rhizomes
      70 % aqueous    ethanolic extract (50,100,150 & 200�    mg/kg)
    Male & Female    Swiss mice
      (25-30 g)
    Indomethacine
      (60 mg/kg, p.o)
    Cimetidine
      (100 mg/kg, p.o)



Table 5:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of NSAID’s.

To evaluate gatroprotective potential of test agents, rats fasted for 24-36 h are orally administered the selected NSAID dissolved in an appropriate vehicle (e.g. water, 1% carboxymethyl cellulose) and after 1 h treated with the different doses of the test agent. After 4 h post treatments with the test agent, rats are sacrificed, stomachs excised and severity of ulceration measured.

Histamine-induced gastric ulcers: Histamine is known to be one of the important factors mediating formation of gastric ulcers, and this is the basis of the histamine induced gastric ulcer model [49]. Histamine released from mast cells binds with receptors present on the surface of parietal cells and causes activation of adenylate cyclase which converts ATP into c-AMP. This conversion is responsible for enhanced secretion of HCl from parietal cells [50]. Histamine also has vasodilating ability and reduces mucus production. These pharmacological effects of histamine have been exploited in producing the histamine-induced ulcer model. This model is useful for evaluating the antisecretory effects of drugs and other agents that function as H2-receptor antagonists.

To induce ulcers with histamine, animals fasted for 18- 24 h are subcutaneously administered histamine phosphate. The animals are sacrificed after 2 h or 4 h following histamine administration and the stomachs dissected out after pylorus and cardiac ligation [51]. The gastric content is collected into a centrifuge tube for determination of pH and total acidity.

This model has been used to evaluate gastroprotection by fixed oil (3 mL/kg) [52] and cold ethanolic leaf extract of Ocimum sanctum Linn. (50, 100 mg/kg) [53]. Gastric ulceration was induced by intraperitoneal administration of histamine acid phosphate using male and female guinea pigs (300-350 g). Omeprazole (10 mg/kg p.o) was used as reference drug [53].

Serotonin-induced gastric ulcers: Serotonin is a vasoconstrictor and ulceration induced by serotonin is believed to arise from a disturbance of gastric mucosal microcirculation [52]. In this model, rats fasted for 24 h are pretreated with vehicle or extracts or reference drug. After 30 min serotonin creatinine sulphate (20-50 mg/kg) is administered subcutaneously and animals sacrificed (Table 6). Their stomachs longitudinally excised after 6-18 h along the greater curvature. The inner surface is then examined for ulcerative lesions [54,55].

Table 6: Examples of medicinal plants whose gastroprotective activity has been evaluated by use of serotonin.




  
    Plants
    Ref:
      No.
    Type of extract and    doses
    Type of animal    species used in the experiment
    Dose of serotonin    to induce ulceration
    Reference drug
  
  
    Centaurea solstitialis Linn.
    [30]
    Flowers
      Sesquiterpene lactones:
      Chlorojanerin
      (59.2 mg/kg)
      13-acetyl solstitialin
      (179 mg/kg)
    Male & Female Balb-C mice
      (20-40 g)
    50 mg/kg
      (0.5 mL, s.c)
    Famotidine
      (20 mg/kg, p.o)
  
  
    Ocimum sanctum 
      Linn.
    [52]
       
       
       
    Seeds
      Fixed oil
      (1, 2 mL/kg)
    Wistar strain albino rats
      (160-200 g)
    20 mg/kg
      (0.5 mL, s.c)
    Not stated
  
  
    Sambucus ebulus
      Linn.
    [39]
    Leaves
      n-buatnol fraction
      (495 mg/kg)
    Male Wistar strain rats
      (120-150 g)
    50 mg/kg
      (0.5 mL, s.c)
    Famotidine
      (2 mg/kg, p.o)



Table 6:  Examples of medicinal plants whose gastroprotective activity has been evaluated by use of serotonin.

Acetic acid-induced gastric ulcers: Takagi et al. [56] developed a model for inducing chronic gastric ulcers in rats by sub-mucosal injection of acetic acid. These ulcers resemble chronic ulcers in humans both grossly and histologically. To overcome certain problems, the original method has undergone several modifications and the one that is currently most favoured is that developed by Okabe and Pfeiffer [57]. This method involves application of acetic acid solution intraluminally. The acetic acid induced ulcer model is most suitable for evaluating the effects of various test agents on the healing process of chronic peptic ulcers and also for screening their antisecretory and cytoprotective effects (Table 7).

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Citation: Thabrew MI and Arawwawala LDAM. An Overview of In Vivo and In Vitro Models that can be used for Evaluating Anti-Gastric Ulcer Potential of Medicinal Plants. Austin Biol. 2016; 1(2): 1007.

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Plants	Ref: No.	Type of extract and doses	Type of animal species used in the experiment	Dose of acetic acid to induce ulceration	Reference drug
*Celtis iguaneae* Jacq.	[29]	Leaves Hexane extract (100 mg/kg)	Male albino Swiss mice (25-35 g)	50 �L of 20% acetic acid (sub mucosal injection)	Ranitidine (50 mg/kg, p.o)
*Copaifera malmei* Harms.	[38]	Leaves Infusion (12.5, 50, 200 mg/kg)	Male & Female Swiss albino mice (25-30 g)	50 �L of 30% acetic acid (sub mucosal injection)	Cimetidine (50 mg/kg, p.o)
*Ocimum sanctum* Linn.	[53]	Leaves Cold ethanolic extract (50, 100 mg/kg)	Male & Female Sprague Dawley rats (180-200 g)	60 �L of 40% acetic acid (local application to serosal surface of the stomach)	Omeprazole (10 mg/kg, p.o)

An Overview of In Vivo and In Vitro Models that can be used for Evaluating Anti-Gastric Ulcer Potential of Medicinal Plants

Abstract

Introduction

In vivo models that can be used for study of gastroprotection

In vitro methods for evaluation of gastroprotection

Assessment of anti- Helicobacter pylori activity

Conclusion

References