Potential effect of Raw Honey on Gastric Mucosal Healing in
Aspirin-induced Rats

 

Devyani Diah Wulansari¹*, Aguslina Kirtishanti¹, Reine Risa Risthanti²,
Devyana Dyah Wulandari³, Lili Soetjipto⁴, Dwi Winarni⁵

¹Department of Clinical and Community Pharmacy, Faculty of Pharmacy,

University of Surabaya, Surabaya, Indonesia.

²Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Surabaya, Surabaya, Indonesia.

³Medical Laboratory Technologyst Study Program, Faculty of Health,

Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia.

⁴Faculty of Medicine, University of Surabaya, Surabaya, Indonesia.

⁵Faculty of Science and Technology, University of Airlangga, Surabaya, Indonesia.

 

ABSTRACT:

Background: Raw honey is a natural ingredient which has a variety of nutrients that can be used for alternative treatments for peptic ulcer disease. This study was carried out to examine the antiulcer effects of Raw Honey against Aspirin induced gastritis in rats. Methodology: Wistar rats were separated into 6 groups. Aspirin suspension 200mg/kgBW was given orally to groups 2-6 for 2 days. Then, group 1 and 2 received carboxymethylcellulose (CMC), groups 3-5 were orally forced-fed with 3.5, 7, and 14mL/kgBW of raw honey, and group 6 received 100mg/kgBW Cimetidine. The tested animals were killed after receiving therapy for 15 days and the gastric mucosa was observed macroscopically of the ulcer index and microscopically through histopathological preparations. The antioxidant effect of raw honey was identified from the lipid peroxidation marker (MDA). Conclusion : Treatment with 7 and 14 ml/kgBW of raw honey promotes gastric mucosal repair based on the macroscopic and microscopic observations. Significant decreases in the levels of the lipid peroxidation marker (MDA) was observed. Significance was defined as p<0.05 compared to the ulcer control group (Group 6).

 

 

 

INTRODUCTION: 

Peptic ulcer disease (PUD) is one of the most common gastroenterological diseases that refers to damage to the mucosal wall of the stomach (gastric ulcer) and duodenum (duodenal ulcer), which penetrates about 0.5 cm through the muscularis mucosa to the deeper layers of the stomach submucosa^1,2 and is characterized by the appearance of round to oval reddish lesions with a diameter of 0.3 cm to 0.6 cm and the edges of the ulcer are well defined³.

 

The World Health Organization (WHO) states that there are about 8 million people suffering from peptic ulcer disease worldwide every year (2000-2016). The prevalence of peptic ulcer disease is influenced by age, ethnicity, gender, geographical conditions, and socio-economic status^4,5.

 

PUD is associated with the presence of H. Pylori infection and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as Aspirin^6,7, smoking, excessive alcohol use, emotional stress and psychosocial factors, etc⁸. NSAIDs can cause damage to the gastric mucosa by two mechanisms, namely directly irritating the gastric epithelium or inhibiting endogenous mucosal prostaglandin synthesis⁹. The local (direct) irritating effect on the gastric mucosa is associated with the acidic nature of these NSAIDs. Whereas systemic inhibition of PG synthesis is the primary means by which NSAIDs cause gastric ulcers. Nonselective NSAIDs, including ASA, inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) and reduce platelet aggregation, which can increase the risk of bleeding. Blocking of the COX-2 enzyme pathway results in beneficial analgesic and anti-inflammatory effects. However, the blocking of the COX-1 enzyme, which produces PG results in inhibition of PG production, which provides protection to the stomach^10,11. Therefore, NSAIDs have been widely used as referral inducers to evaluate the gastroprotective activity of several therapeutic agents¹². Several causes and risk factors affect the secretion of acid in the gastric mucosa. The high level of acidity in the gastric mucosa results in injury to the mucosa which then causes discomfort in the stomach, excruciating pain in the upper abdomen, heartburn, a feeling of fullness in the stomach, and stomach cramps¹³. In practice, histamine-2 receptor antagonist is a class of drugs more widely used by the public¹⁴, and also has a different effectiveness is not significant with PPI drug class in overcoming the condition of peptic ulcer¹⁵.

 

Honey is one of the natural products produced by bees and has a distinctive sweet taste, contains various compounds that are beneficial to health such as minerals, carbohydrates, proteins, enzymes, amino acids, phenolic compounds, vitamins, and organic acids¹⁶. The mineral content contained in honey includes K, Ca, Mg, Na, Mn, and Fe whose alkaline nature serves to neutralize excessive stomach acid, so that the level of gastric acidity can be maintained under normal conditions¹⁷. Honey also has antibacterial activity because it contains glucose oxidase  enzyme which can convert glucose in honey into gluconic acid and hydrogen peroxide, where the latter is the main antibacterial agent that can destroy the outer membrane of bacterial cells, causing damage to bacterial cells¹⁸. In addition to peroxide compounds, honey also has antibacterial action due to non-peroxide compounds, namely methylglyoxal (MGO) compounds that can inhibit the formation of biofilms on bacteria¹⁹. Honey also contains a large number of compounds such as flavonoids and other polyphenols that have antioxidant activity. In addition, the high viscosity of honey can also help protect the gastric mucosa²⁰.

 

Currently, research on the effectiveness of processed honey as gastrotherapy has been widely known, however research on the effectiveness of raw honey or honey that has not been processed and heated as gastrotherapy is still very limited. Raw honey has a more complex nutritional content compared to processed honey, because the processing/heating process can affect the biochemical composition contained in honey²¹. Based on the description above, it is necessary to carry out further exploration of the effectiveness of raw honey on peptic ulcers.

 

MATERIALS AND METHODS:

Materials and Reagents:

This study used cimetidine as a standard antiulcer drug obtained from PT. Kimia Farma, Jakarta - Indonesia. The drug is administered orally using an oral probe once a day for 15 days at a dose of 100mg/kgBW^22,23 suspended with 0.5% CMC Na. The test material used in this study was raw honey produced from Apis dorsata bees with monoflora nectar extraflora Acacia mangium nectar from South Sumatra, Indonesia. Honey that has not undergone processing or heating, was given to the test group orally using an oral probe 1x a day for 15 days at a dose of 3.5ml/kgBW, 7ml/kgBW, and 14 ml/kgBW^24,25 suspended with 0.5% CMC Na.

 

Animal Stock:

The animals used in this study were 30 male white rats of the Wistar strain (Rattus norvegicus) for treatment, 2-3 months old, weighing 200-300grams with healthy physical conditions²⁶. Animals were obtained from the Surabaya Pharmacy Veterinary Center and developed at the Pharmacology-Toxicology Laboratory of the University of Surabaya (Ethics No. 111:113:114:116/KE/XII/2019). During the rearing period, animals were randomly divided into 6 groups and received standard Hi-Pro-Vite CP511B feed, placed in separate cages, and maintained cage sanitation to avoid coprophagy^27,28.

 

 

Figure I. The scheme of the experimental design

 

 

Experimental Design:

After 15 days of acclimatization, rats were randomly divided into six groups. Rats in group 1 functioned as normal controls, while rats in group 2-6 functioned as rats with Aspirin-induced gastritis at a dose of 200 mg/kgBW for 2 days. Gastritis animals in groups 3, 4 and 5 were given raw honey orally at 3.5, 7, and 14 mL/kgBW, respectively, and gastritis animals in group 6 were given oral treatment with standard drug cimetidine at a dose of 100mg/kgBW for 15 consecutive days. A simplified treatment scheme is given in Figure 1.

 

Phytochemical Screening:

To determine the presence of several phytoconstituents such as tannin, saponin, and flavonoids and terpenoids, the extracts prepared using the sequential solvent extraction technique were submitted to many qualitative tests using following method²⁹:

 

a) Tannin Test:

Raw honey samples was added 0.1% FeCl3. If the colour changes to yellow/greenish brown after being homogenized, it was indicated that it contains a positive tannin compounds.

b) Saponin Test:

The raw honey sample was added with distilled water and then shaken until foamy, then was added with olive oil and shaken, if the foam is still visible and does not disappear when shaken, it was indicated that it contains a positive (+) saponin compounds.

c) Flavonoid Test:

Raw honey was added with dilute ammonia and then homogenized. If there is a change in color to yellowish, it was indicated that it contains a positive (+) for flavonoid compounds.

d) Terpenoid Test:

Raw honey was added with acetic acid until submerged, then allowed to stand for 15 minutes. After that, 5 drops were transferred into a test tube and concentrated sulfuric acid was added. The presence of triterpenoids is indicated by a color change to red orange or purple.

 

Gastric Ulcer Induction:

The material used as an inducer for gastric ulcers is aspirin³⁰ (Cardio-Aspirin Tablet 100mg) from PT Kimia Farma, Jakarta - Indonesia, suspended with 0.5% CMC Na. Before being induced, the test animals were put on fasting condition for 1 x 24 hours. Then the drug was given once a day for 2 consecutive days orally using an oral probe at a dose of 200mg/kgBW³¹ to experimental animals in groups 2-6.

 

Sampling:

After 15 days the experimental animals were treated, then subjected to fasting condition for 24 hours and anesthetized using Thiopental Sodium 30mg/kgBW intraperitoneally³². Then, the rat's stomach was dissected by making small incision in the abdominal midline of the rat. Rats were sacrificed with decapitation 4 hours after pyloric ligation³³. All gastric juices were separated to measure the total acidity, then gastric tissue was isolated to measure the ulcer degree macroscopically and microscopically. Furthermore, blood samples were taken intracardiac³⁴ and used to measure MDA levels.

 

Measurement of Total Acidity of Gastric Juice:

The gastric fluid obtained was centrifuged for 10 minutes at 3000rpm. The filtrate formed was then titrated with the acidimetric method against 0.01 N NaOH to pH 7³⁵.

 

Total acidity (mEq/4 hours) =

V gastric fluid (ml) x Vol. NaOH (ml) x Normality 0.01 NaOH.

 

Measurement of Lipid Peroxidation Markers (MDA):

The blood samples obtained were then centrifuged to collect the serum. Then the serum obtained was measured for MDA levels by the Placer method. The animal serum that has been formed is pipetted 0.5ml, added 9ml of PBS solution, after that 4ml of supernatant is taken, added 1ml of 15% TCA, and 1ml of TBA reagent, then heated with a water bath of 80ºC for 15 minutes, then cooled at a temperature of chamber for 60 minutes, after being cooled it was centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was measured using a spectrophotometer at a wavelength of 532nm which was then calculated for levels (MDA) in the sample using a regression equation from the results of standard MDA measurements³⁶.

 

Observation of the degree of gastric ulcer macroscopically:

Macroscopic observations were carried out visually by stretching the isolated gastric organs on a board and observing the number and diameter of the ulcers formed. The antiulcer effect was evaluated based on the state of the stomach (ulcer) which was assessed using a score based on the number and severity of ulcers. Indicators of the number and severity of ulcers can be seen based on the following criteria^38,40:

 

Tabel 1. Score System Based On the number and severity of ulcers

 

Furthermore, the degree of ulcer formed was assessed using the Ulcer Index (UI) with the following equation^{38, 41}:

U             =  U N + U S + (U P ) 10 -1

U             =  Ulcer Index

U N         =  Score of ulcers per animal

U S          =  Ulcer severity score per animal

U P          = Percentage of animals with ulcers from a group

 

The percentage protection was calculated by using the formula³⁸

% Protection = ((UI control – UI test)/UI Control) x 100

 

Microscopic Observation of Gastric Ulcer:

Gastric tissue samples that have been opened on the major curvature with a cross section were then fixed with 10% buffered neutral formalin (BNF) solution for at least 24 hours, then dehydrated in graded ethanol solution of 70%, 80%, and 90%. after that clearing was performed using xylol 3 times. Then the tissue was infiltrated and embedded using paraffin and cooled at room temperature. The paraffin blocks formed were cut with a thickness of 3µm-4µm using a microtome³⁹. Staining with Hematoxylin was carried out for 8 minutes, then rinsed with running water, followed by washing with Lithium carbonate for 15-30 seconds and rinsing with running water. Ultimately the samples were stained with Eosin for 2-3 minutes. And washed with running water, prior to drying.

 

Statistic Analysis:

Data analysis to compare between groups was conducted using ANOVA test with SPSS 16 software . The results showed a descriptive table to determine the largest, smallest average and median values. In addition, Test of Homogenety of Variances table was obtained determine the homogeneity of a data and conditions can be used to perform a one-way ANOVA test. The p value > 0.05 indicates homogeneous data so it can be concluded that there is a significant difference, while the p value < 0.05, it can be concluded that there is no significant difference. The Tukey HSD test was performed to compare the significance values between groups.

 

RESULT:

Phytochemical Screening:

The results of the phytochemical qualitative analysis test showed that raw honey which was tested for tannins showed a color change to brown and gave a stable froth on the saponin test. Likewise, the flavonoid test and the terpenoid test showed a color change. Based on the test results, it was indicated that raw honey contains tannins, saponins, flavonoids, and terpenoids.

 

Tabel 2. Phytoconstituents of Raw Honey

 

Effect of Raw Honey on Stomach Acidity:

Animal treated with raw honey at a dose of 3.5 ml/kgBW and 7ml/kgBW for 15 days did not give a significant difference in pH value compared to the control group with p values of 0.971 and 0.997, respectively. Likewise, in the total gastric acidity value, the group that was given raw honey at a dose of 3.5 ml/kgBW and 7ml/kgBW did not give a significant difference with p values 0.173 and 0.056, respectively. However, the group of animals treated with raw honey at a dose of 14ml/kgBW showed significant pH values (p<0.01) and total gastric acidity (p<0.01) compared to the control group, as shown in table II.

 

Effect of Raw Honey on the Degree of Stomach Ulcers

The antiulcer activity of raw honey can be seen from the ulcer index and % protection shown in table II. Animals with ulcer which have been treated with raw honey at a dose of 3.5ml/kgBW, 7ml/kgBW and 14ml/kgBW showed significant differences compared to the control group with the lower case values of 0.002; 0.003; 0.000, respectively The decrease in ulcer grade was in line with the administration of cimetidine therapy at a dose of 100 mg/kgBW as an antiulcer  which provided significant protection in treating ulcers (61.09%) compared to the control group (p<0.001). Ulcer animals induced by Aspirin and treated with CMC-Na gave a visually clear picture of bleeding lesions as shown in Figure II. However, raw honey therapy provide  the recovery from and decrease the severity of bleeding that occurred with a decrease in the level of bleeding. So, it could be indicated that raw honey plays a role in suppressing the formation of ulcers. Figure II and table II show that the administration of raw honey at a dose of 14 ml/kgBW (e) provided a significant inhibition of ulcer formation and was comparable to the ulcer healing profile given by the cimetidine 100 mg/kgBW group (f).

 

Table 3. The pH values ​​of gastric juices, total acidity, ulcer index and % protection are mean±SD of 5 test animals in each group.

Statistical analysis using ANOVA followed by the Tukey HSD test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

 

(a)                                   (b)

 

(c)                                    (d)

 

(e)                                    (f)

Figure II. Effect of raw honey on macroscopic appearance of gastric mucosa of Aspirin-induced ulcer rats. (a) Normal group , (b) CMC Na control group, (c) Raw honey group at a dose of 3.5 ml/kgBB, (d) Raw honey group at a dose of 7 ml/kgBB, (e) Raw honey group at a dose of 14 ml/kgBB, (f) Cimetidine group 100 mg/kgB

 

Effect of Raw Honey on MDA Concentration:

Raw honey also influences lipid peroxidation activity in the systemic circulation. This can be see in the ulcer animal group that was treated with raw honey at a dose of 3.5 ml/kgBW, where it gave a significantly different profile for the decrease in MDA concentration compared to the control group, as shown in Figure III. Meanwhile, in the ulcer animal group, which was given raw honey at a dose of 7 ml/kgBW and 14 ml/kgBW showed a significantly higher decrease in MDA concentration than the 3.5 ml/kgBW dose when compared to the control group with both p values <0.001. However, when compared with the cimetidine group of 100 mg/kgBW, the ulcer animal group Treated with the highest dose did not provide a significant difference with p value = 0.997

 

Effect of Raw Honey on Histopathological Appearance of Gastric Mucosa with HE Staining:

Histological description of the normal group in number (a) Figure IV shows that there is no intrusion in the surface epithelial cells of the gastric tissue, gland cells to the external muscularis layer all of which appear intact. There was severe damage to the epithelial cells and necrotic lesions that reached the submucosal observed in the control group (figure IV.b). Meanwhile, in the group of ulcer animals treated with raw honey at a dose of 3.5 ml/kgBW as shown in Figure (IV.c), moderate epithelial cell damage and necrotic lesions occurred up to the muscularis layer of the mucosa. Meanwhile, the group that was given raw honey at a dose of 7 ml/kgBW (figure IV.d) showed mild erosion of the epithelial cells and erosion occurred up to the glandular cell layer. The last group with the highest dose of raw honey showed slight changes in the shape of the epithelial cells and gland cells, no erosion was seen as shown in figure (IV.e). Ultimately, in the group given cimetidine 100 mg/kgBW, the epithelial cell components were slightly changed but the gland cells appeared intact (Fig. IV.f). These results indicate that raw honey has a repair effect on gastric ulcers which increases in dose dependent manner.

 

 

Figure III. Effect of raw honey on MDA lipid peroxidase parameters in blood of Aspirin-induced ulcer rats. All values ​​were expressed as mean ± SD. P values ​​in all groups were significantly different from the control group with values ​​*p<0.05, **p<0.01, ***p<0.001. Data were analyzed by ANOVA using Statistical Package for the Social Sciences Software (SPSS 16)

 

(a)                                   (b)

 

(c)                                    (d)

 

(e)                                    (f)

Figure IV. Histopathological picture of gastric tissue. (a) Normal group. (b) Ulcer animal control group given CMC Na. (c) Ulcer animal group given raw honey therapy at a dose of 3.5 ml/kgBW. (d) Ulcer animal group given raw honey therapy at a dose of 7 ml/kgBW. (e) Ulcer animal group given raw honey therapy at a dose of 14 ml/kgBW. (f) Ulcer animal group given cimetidine therapy at a dose of 100 mg/kgBW.

 

DISCUSSION:

Uncontrolled gastric acid secretion can cause peptic ulcer which can be a serious problem for human health worldwide. Various factors are involved in the pathogenesis of peptic ulcer in humans such as chronic NSAID use, stress, H. pylori infection, alcohol consumption, smoking and improper diet. Aspirin is a non-steroidal anti-inflammatory drug that induces ulcers by inhibiting prostaglandin synthesis in the stomach by blocking the cyclooxygenase enzyme⁴². Nonsteroidal anti-inflammatory drugs also cause an inflammatory response that increases reactive oxygen species in the gastric mucosa⁴³. Therefore, in this study, the gastric ulcer-inducing compound used was Aspirin at a dose of 200mg/kgBW. Aspirin has been reported to cause mucosal damage due to various factors such as inhibition of prostaglandin synthesis, increasing gastric acid secretion, increasing H+ ion back diffusion, decreasing mucin secretion and breaking down the gastric mucosal barrier⁴⁴.

 

 

Although there are many drugs available to treat peptic ulcer disease such as H-2 receptor antagonists, proton pump inhibitors (PPI), antacids and anti-muscarinics, the administration of these therapies can cause some side effects in patients, and does not provide complete recovery⁴⁵. Honey has been widely discussed by all religious books, and accepted by all generations, traditions and civilizations, both ancient and modern. In an effort to Further explore the potency, this study was conducted to find a safe and curative agent for the treatment of gastric ulcers using natural and widely found ingredients. In this study, ulcer animals in the control group showed gastric pH with a value of 3.96. However, with the administration of honey, the pH of gastric juices increase gradually depending on the dose reaching a maximum of 5.2460 (Table II). To prevent gastric ulcers, gastric mucosal protective agents must be strengthened to overcome the triggering factors for ulcers^46,47. Therefore, one of the modalities in the prevention and treatment of gastric ulcers is to suppress the rate of gastric acid secretion or neutralize it in the gastric mucosa. Treatment with raw honey at a dose of 14ml/kgBW gave a significantly higher pH change than the control group and showed a comparable increase in pH compared to the ulcer animal group treated with Cimetidine 100 mg/kgBW). This indicates that raw honey has an inhibitory effect on gastric acid secretion and its inhibitory action is thought to be similar to that of cimetidine in influencing gastric acid secretion. Cimetidine is the standard drug used in this study that works by inhibiting histamine release as a result of H2 receptor blockade, inhibiting intracellular adenylate cyclase, Na-K TPase, and parietal cell proton pumps, thereby reducing gastric acid secretion⁴⁸.

 

ROS is one of the main destructive mechanisms of aspirin in gastric cells. On the other hand, a significant decrease in MDA concentration in animals treated with raw honey, may indicate a reduction in oxidative injury to the stomach⁴⁹. MDA is a useful compound as a biomarker of lipid peroxidation because it can be measured in body fluids⁵⁰. Lipid peroxidation leads to loss of membrane fluidity, disruption of ion transport and membrane integrity and ultimately loss of cellular function⁵¹. By lowering the concentration of MDA, it can be beneficial for tissue protection, thus make raw honey is potent as as a cytoprotective agent. This study demonstrated a dose-dependent decrease in MDA in raw honey (Figure III). 

 

Raw honey was used as a potential anti-ulcer agent in this study because it did not undergo a heating process that could change its components and biological activity. Meanwhile, the processed honey through a heating process is intended to reduce the water content to prevent honey fermentation, dissolving the core sugar to slow down granulation and homogenizing the honey for consumer preferences. The content of HMF and the enzymatic activity of honey which is an indicator of honey freshness will also change during the heating process. In addition, the heating process on honey can also damage the quality and biological activity as well as for its originality⁵². Based on the results of phytochemical screening on raw honey in table 1, it shows the presence of flavonoids, saponins, alkaloids, and tannins. The health benefits of flavonoids are related to their antioxidant activity with several studies demonstrating the ability of these compounds to bind to reactive oxygen species. Flavonoids also have the ability to stabilize membranes and several flavonoids have been reported to increase the gastric mucosal prostaglandin concentration. In addition to its ability to bind free radicals, flavonoids with their antioxidant properties are able to chelate transition metal ions, inhibit enzymes that play a role in the oxidation process, reduce acid secretion and inhibit pepsinogen production. Gastroprotective effects of saponins have been reported in various references^53,54. Tannins are known to have styptic properties, due to their ability to shrink surface pores (astrigen) and are hemostatic⁵⁵.

 

Peptic ulcers in this study were induced by aspirin. It can inhibit the COX-1 and COX-2 cyclooxygenase enzymes, which cause the accumulation of intracellular arachidonic acid which inhibits prostaglandin synthesis⁵⁶. Changes in prostaglandin levels can increase acid secretion in the gastric mucosa which disrupts gastric balance and increases ROS. Based on this explanation, prostaglandins play an important role in preventing peptic ulcers and provide a protective function by increasing the production of bicarbonate and mucus, therefore in the context of peptic ulcer healing it is important to prevent prostaglandin suppression⁵⁷. Raw honey at a dose of 14 ml/kgBW showed a significant decrease in ulcer index (p < 0.001) compared to the control group and gave a similar ulcer healing rate in both the control group and the normal group. These results suggest the possible involvement of prostaglandins and mucus in the antiulcer activity of raw honey. Therefore, it is suggested that the administration of honey and cimetidine helps control acid secretion in the gastric lumen and total acidity which suggests that honey might modulate prostaglandin secretion and ROS production. The findings of this study recommend that raw honey and cimetidine have significant anti-ulcer activity.

 

CONCLUSION:

Our results showed that raw honey exerted a significant dose-dependent cytoprotective effect on Aspirin-induced ulcers (p < 0.05). In conclusion, honey has an effective ingredient for healing ulcers, which is suggested for treating gastric ulcers. Honey that has been tested on rats is recommended for ulcer patients in hospital. In future research, it is recommended to quantitatively analyze the phytoconstituents of honey which have a dominant role in treating ulcers and explored the effect of honey on cyclooxygenase 2 (COX-2) inhibition with immunohistochemical techniques.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank to the University of Surabaya for providing the facilities in carrying out this research

 

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Accepted on 19.07.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1153-1160.