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INTRODUCTION
Gastric ulcers are lesions in the mucosal lining of the digestive (gastrointestinal) system. The disease is characterized typically based on the anatomical location which is immersed in acid and pepsin [1]. Based on site of attack, gastric ulcer is recognized as the most prevalent chronic diseases of the peptic ulcer. A gastric ulcer occurs as a result of extended starvation, disruption in the barrier of gastric mucosa due to stress, nutrient deficiency, decreases in mucus secretion associated with prolonged or overdose ingestion of non-steroidal anti-inflammatory drugs (NSAID), smoking habit and infection with Helicobacter pylori [2]. Though gastric ulcer is a non-fatal disease, the symptom manifestation elicits discomfort, disturbs the daily routines and triggers mental distress in patient.
In the past decades, gastric ulcer affected a concerning number of people globally. Studies have revealed that the risk for developing a gastric ulcer is expected to be 10% approximately and the incidence peaks more than 80% by the age of 50 and above [3]. Though overall improvisation in hygiene and healthcare quality has influenced the declining rate in peptic ulcer especially in gastric ulcers, the amplified usage of NSAIDs has caused an increase in NSAIDs induced gastric ulcers. The complications of gastric ulcer impose significant morbidity and economic load on the health system and the society. According to literature, the total costs of gastric ulcer management have been expected to reach at least seven billion annually [4,5].
Therapeutic management of gastric ulcers continues to be developed, with the aim of pain relieving, ulcer restoration and halting reoccurrence. Hence, the current pharmacological treatment is based on the inhibition of gastric acid secretion or proton-pump inhibitors. Such derivatives are typically used to manage gastric ulcer clinically but in the long-term, they can’t be tolerated due to their safety profile. Despite their successful healing rate, the incidence of reoccurrence within a year was frequently reported. This occurrence is mainly associated with accumulation of neutrophil and production of reactive oxygen species which lead to an incomplete healing process. The treatment regime is also associated with various side effects such as gynecomastia, infertility, osteoporosis, vitamin (iron, magnesium, and B12) deficiencies and hypergastrinemia. These symptoms can last for a long period, during and after discontinuation of the treatment. Therefore, there is an urgent need for development of novel drugs that can be administered for long-term with reduced side effects [6,7].
Plant derived compounds have been used in traditional medicine since the ancient times for various ailments and diseases treatment. Now, medicinal herbs have become clinically viable for the management/treatment of gastric ulcer over synthetic drugs due to their potentially improved safety and efficacy [8]. Ocimum sanctums is used in Ayurveda and Siddha systems for asthma, cold, colic pain, cough, digestive disorders, diarrhea, headache and fever. Ocimum sanctum fresh leaves and stem extract yields an important bioactive compound, called cirsilineol. Cirsilineol is a rich phenolic antioxidant compound with various pharmacological importance such as anti-bacterial activity, anti-cancer activity, anti-diabetic activity, anti-fertility activity and anti-lipidemic activity [9-12]. Despite the extensive use of cirsilineol both traditionally and clinically, there was no data on gastroprotective mechanisms. Thus, this study was conducted to provide a scientific base for the use of cirsilineol for its anti-ulcer action against gastric ulcer.
METHODS
Chemicals
Cirsilineol of an analytical standard were purchased from Sigma-Aldrich, St. Louis, MO, USA for the present investigation. The following chemicals were acquired from Sigma-Aldrich: hydrochloric acid (HCl)/ethanol, sodium hydroxide (NaOH), topfer reagent, cyanide/ferricyanide reagent, trichloroacetic acid, thiobarbituric acid, tetrahydrobenzofluorene, aprotinin, pro-inflammatory ELISA kit and haematoxylin and eosin (H&E) staining solution. Other chemicals and solvents incorporated in this experiment were also from analytical grade.
Experimental protocol
Animal model
A total of 24 male Sprague–Dawley rats (6–8 weeks, 150–180 g) were caged under controlled laboratory environment and allowed to tap water and rodent pellets ad libitum. The acclimatization to experimental condition was performed for seven days in animal house prior to commencement of the experimentation. All further protocols were performed to minimize suffering. The experimental protocol was design according to international guidelines was approved by the Institutional Animal Ethics Committee. The ethical approval was provided by animal ethics committee of Xichang People’s Hospital (approval no.: XCRMYY0910).
Study design
The rats were separately categorized into four groups and each had six animals (n = 6). The dosage selection for cirsilineol (dissolved in 0.1% Tween 80) was done according to results of preliminary studies. The administration of cirsilineol was made via oral route 30 min before exposure to HCl/ethanol.
Group I: 0.1% Tween 80 (normal saline)
Group II: HCl/ethanol
Group III: cirsilineol (20 mg/kg) + HCl/ethanol
Group IV: cirsilineol (40 mg/kg) + HCl/ethanol
Prior to 24 h fasting, HCl/ethanol (0.1 ml/20 g) mixture (0.15 M HCl in 98 % ethanol) was used to induce the formation of gastric lesions in the rats through oral administration, following the method of Jin et al. [13]. Normal rats were substituted with the equal volume of 0.1% Tween 80. Subsequently, all the rats were sacrificed after 1 h of sample administration. The blood sample was collected and stomach was excised with the gastric contents were aspirated before washing with normal saline for the gastro-protective assessment. All biochemical test protocols were performed following El-Maraghy et al. [14] and Yang et al. [15].
Measurement of gastric ulcerative lesions
The cleansed stomach was cut open along the great curvature, washed again with iced cold phosphate buffer (PBS) solution for assessment total ulcer area using inverted microscope with digital camera.
Measurement of ulceration index
Following, the stomach sample were subjected for the determination of gastric ulcer area (mm2) using the image J software. The length and width of the ulcer was measured using a ruler to assess the total ulcer area. The following formula was used to obtain the percentage of ulcer inhibition (%) by the treatment and the obtained ulcer index was expressed as mean score value for each rat:
Percentage inhibition (%) = [Ulcerated area (model) – Ulcerated area (treated)] / Ulcerated area (model) × 100%
Measurement of pH and total acidity
The pH of gastric juice content was obtained using digital pH meter by dipping the electrode into the beaker containing gastric juice. Thereafter, one milliliter of centrifuged gastric juice titrated against 0.1 N NaOH using the Topfer reagent for determination of free acidity and 1% phenolphthalein in alcoholic solution for total acidity, indicated by changes in color (red to colorless to pink). The sum of both titrations was for total acidity.
Measurement of hemoglobin value
A clean and dry test tube was used to pipette out 5 ml of buffered cyanide/ferricyanide reagent. Approximately, 0.02 ml of blood was mixed into the tube and incubated at 30°C. The resulting reaction mixture was subjected to absorbance reading at 546 nm. The concentration of hemoglobin was estimated using the equation; C (g/ 100 ml) = 36.77 × Abs.
Measurement of pro-inflammatory cytokines
A portion of the stomach tissue was homogenized in PBS (pH 7.4) and centrifuged at a high speed (10,000 ×g) for 20 min at 4°C using centrifuge machine. The supernatant’s protein concentration was determined in the homogenate of stomach tissue samples. The tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) concentrations in were measured using commercially available ELISA kit. The homogenized supernatant yielded to a roughly of 10% w/v of homogenate. Then, the supernatant was further centrifuged at 1,500 rpm for 20 min, and the recovered supernatant was used for the determination of inflammatory mediators by ELISA kits. The results obtained were expressed as cytokines concentration in lung tissue homogenates (µg/mg protein). The cytokines levels were determined by comparing to the standard graph.
Measurement of lipid peroxidation activity
The measurement of lipid peroxidation, the end product of malondialdehyde (MDA) was determined using TBARS method. Briefly, the stomach’s homogenate supernatant was heated at a low pH (HCl) with TBA at 90°C for 30 min to produce a pink chromogen. The reaction mixture was cooled and centrifuged before subjecting to spectrophotometric absorbance at 535 nm. An extinction coefficient of 1.56 × 105 M−1 cm−1 was used to calculate the TBARS value for the sample. The results are expressed as lipid peroxidation (LPO) µmol/mg protein.
Measurement of superoxide dismutase enzymes (SOD) activity
The SOD value in the supernatant of stomach homogenate, was determined following Marklund and Marklund [16] method. Principally, the SOD concentration was determined on its capability to increase the rate of auto-oxidation of tetrahydrobenzofluorene in the tested sample. One unit of SOD was assumed as enzyme amount of halted pyrogallol oxidation by half. The SOD activity was recorded spectrophotometrically at 525 nm and articulated as U/mg protein.
Measurement of catalase (CAT) activity
CAT activity was done according to the method prescribed by Aebi [17]. The stomach homogenate was diluted with distilled water and the resulting absorbance was recorded at 240 nm. One unit of CAT was demarcated as the enzyme amount essential to decompose 1 µmol of hydrogen peroxide (H2O2)/min. The CAT enzyme activity was expressed as U/mg protein.
Measurement of serum biochemical parameters
The blood samples were collected in heparinized/non-heparinized syringes and placed into polystyrene micro-tubes. After clotting process, the blood was centrifugation at 4,000 ×g for 7 min, to obtain the serum. The serum was separated using EDTA-treated pasteur pipettes. The remaining blood cells were washed with isotonic saline and the buffy coat was discarded and processed further for the preparation of serum biochemical analysis before storing in −80°C. The serum samples were analyzed using auto-analyzer to evaluate possible changes in serum biochemical parameters (liver function test, renal function test and lipid profile).
Measurement of histopathological analysis
The section of stomach sample was fixed flat on filter papers using 10% formaldehyde for 48 h. The tissues were then processed sequentially in graded alcohol for dehydration. Further, the tissues were impregnated with paraffin wax for half an at least hour at 56°C and formed into a block. The blocks were sliced into 5 µm by rotary microtome, dewaxed, rehydrated and stained with H&E stain. The slides were viewed by light microscope for histological significance.
Statistical evaluation
The analytical method was validated as mean ± SE of at least triplicate experiments and calculated using SPSS 19.0 software (IBM Co., Armonk, NY, USA). Assessments between experimental groups were scrutinized with the Student-Newman-Keuls multiple comparison test. p < 0.05 were deliberated as statistically significant.
RESULTS
Effect of cirsilineol on the gastric ulcer markers in HCl/ethanol induced gastric ulcer rats
In Fig. 1, the effect of orally administered cirsilineol on HCl/ethanol-induced gastric lesion in rats was shown as ulcer index and ulcer markers (total acidity, pH and hemoglobin). The ulcer scores seen in the rats treated with cirsilineol (Group III) were significantly lower (p < 0.05) than those in the ulcer group (II) alone. The sizes of the ulcer areas were also reduced in the rats treated with higher dose of cirsilineol (Group IV) (p < 0.01). Similarly, the impact of cirsilineol (Group III & IV) on gastric acidity was ameliorated compared to the ulcer group (II). The effect was normalized to the control group (I). As for the pH of the gastric contents, a slight increase was observed in the rats treated with cirsilineol (Group III) than the ulcer group (II). Rats treated with higher dose of cirsilineol (Group IV) implied the same increase in pH level. A similar pattern was observed in hemoglobin level when cirsilineol was treated in rats, induced with HCl/ethanol.
Effect of cirsilineol on the production of inflammatory mediators in HCl/ethanol induced gastric ulcer rats
In Fig. 2, the gastroprotective action of cirsilineol showed the involvement of inflammatory signals against HCl/ethanol-induced gastric lesion in rats. In ulcer group (II), the TNF-α expression was up regulated markedly (p < 0.05) as well as IL-1β, IL-6 levels than in control group (I). Treatment with cirsilineol diminished TNF-α, IL-1β, and IL-6 levels significantly (p < 0.01) upon induction by HCl/ethanol. In other way, oral administration of cirsilineol produced a significant alteration which was normalized to the control group (I).
Effect of cirsilineol on the production of anti-oxidative expression in HCl/ethanol induced gastric ulcer rats
In Fig. 2, the antioxidant defense property of cirsilineol was shown by evaluating the SOD, CAT, and LPO activity against HCl/ethanol-induced gastric lesion in rats. The SOD and CAT activities decreased in the ulcer group (II), respectively, compared with those in the control group (I). However, in cirsilineol treated groups (III & IV), a significant (p < 0.01) increase in SOD and CAT activities were observed. On the other hand, the LPO content was significantly (p < 0.05) higher in the gastric model group (II), compared with that in the control group (I). In cirsilineol treated groups (III & IV), the LPO contents were lower (p < 0.01), compared with those in the ulcer group (II).
Effect of cirsilineol on the production of liver function enzymes in HCl/ethanol induced gastric ulcer rats
In Fig. 3, the oral administration of cirsilineol on HCl/ethanol-induced gastric lesion in rats led to significant alteration in liver function parameters (alkaline phosphatases [ALP], alanine aminotransferase [ALT], and aspartate aminotransferase [AST]). The ingestion of HCl/ethanol in group II causes a significant (p < 0.05) elevation of ALP, ALT, and AST levels in serum compared with that of the control group (I). The administration of cirsilineol moderately prevented the elevation of serum level of ALP, ALT, and AST (p < 0.05).
Effect of cirsilineol on the production of metabolic acidosis in HCl/ethanol induced gastric ulcer rats
In Fig. 4, the activity of cirsilineol on the electrochemical balance in HCl/ethanol-induced gastric lesion in rats were illustrated. The ulcer group (II) presented a decline in the total carbon dioxide level resulting in high anion gap (p < 0.05) in comparison to control group (I). Following oral administration with cirsilineol, a significant (p < 0.05) elevation in the carbon dioxide level, thus lowering the anion gap and elevation in which was almost comparable to control group (I).
Effect of cirsilineol on the production of kidney function markers in HCl/ethanol induced gastric ulcer rats
In Fig. 5, the oral administration of cirsilineol on HCl/ethanol-induced gastric lesion in rats resulted in a significant alteration of kidney function parameters (urea, creatinine, total protein, albumin and globulin). The oral administration of cirsilineol in HCl/ethanol (groups III & IV) causes a significant (p < 0.05) ameliorative effect of kidney markers compared with that of the ulcer group (II). The administration of cirsilineol were normalized to normal control group (I).
Effect of cirsilineol on the production of lipid markers in HCl/ethanol induced gastric ulcer rats
In Fig. 5, the triglyceride, high-density lipoprotein (HDL) cholesterol and total cholesterol activity of oral administration of cirsilineol against HCl/ethanol-induced gastric lesion in rats are presented. The figures reveal that the administration of cirsilineol (groups III & IV) greatly reduces (p < 0.05) the concentration of triglyceride when compared with the ulcer group (II). However, the administration of cirsilineol (groups III & IV) increases the concentration levels of total and HDL of cholesterol level with a significant difference when compared with the ulcer group (II) (p < 0.05).
Effect of cirsilineol on the production of histological evidence in HCl/ethanol induced gastric ulcer rats
In Fig. 6, the histological alteration in orally administrated cirsilineol against HCl/ethanol-induced gastric lesion in rats are illustrated. The figures reveal that the induction of HCl/ethanol induced gastric lesion, which was evidenced by congestion of inflammatory cell, loss of mucous membrane and necrosis. The administration of cirsilineol (group III) greatly reduces the loss of mucous membrane and inflammatory cells when compared with the ulcer group (II). The effect of cirsilineol was almost normalized to the control group (I). In addition, the histological analysis of gastric mucosa in stomach specimen treated with higher dose of cirsilineol (group IV) showed comparable patterns as cirsilineol treated group III, suggesting the treatment with cirsilineol at lower dose is sufficient to prevented HCl/ethanol induced gastric injury in rats.
DISCUSSION
The ideal way to resolve stomach complications are possibly to find ways to restore gastric homeostasis with certain medication which resembles dietary intakes to address their symptoms. Herein, this investigation evaluates the gastro-protective effect of the cirsilineol against HCl/ethanol-induced gastric injury in rats. HCl/ethanol-induced gastric ulcer were utilized in this study as an experimental model to investigate the protective action of cirsilineol extracted from Ocimum sanctum. This model was preferred as it is closely features the development of ulcers in humans [18]. In this model, HCl induce damage in the lining of stomach mucosa, while ethanol causes severe lesion by suppressing the production of mucus and enzymes. Subsequently, these creates formation of oxidative stress which changes in the permeability of gastric mucosa to produce hemorrhagic lesions, mucosal friability, extensive sub mucosal edema, inflammatory cells infiltration, and epithelial cell loss in the stomach, which are distinctive features of injury [19].
In the marker accessing assay, oral administration of HCl/ethanol induced gastric ulcer in rats promoted gastric ulcer area compared to the control group. Combination of HCl/ethanol are considered as a ‘gastro-toxic’ agent that causes gastric mucus diminution that considerably injures the cell membrane of stomach [20]. Following oral administration with cirsilineol, the gastric lesions were significantly reduced. Possibly cirsilineol are effective against detrimental effect of HCl/ethanol, which could hold a gastric mucosal membrane protective activity. For these reasons, a further validation was carried out to examine the effect of cirsilineol in the role of gastro-protection. The observations showed protective indexes which supported by the percentage of lesion inhibition caused by cirsilineol in HCl/ethanol induced gastric ulcer rats. These results are in parallel with previous work which evaluated the gastro-protection of various synthetic compounds and authenticate the gastro-protective role of gallic acid against ethanol-induced gastric ulcer in rats [21].
In the present study, an increase in total acidity and hemoglobin concentration while decrease in pH were observed in HCl/ethanol induced gastric ulcer rats. The degree of gastric ulceration has been associated with increase in the acidity and hemoglobin activity. In addition, gastritis can root to mal-absorption of iron because gastric acid is required for the iron absorption which often results in hemoglobin deficiency disorder such as anemia [22]. Treatment with cirsilineol was found to improve the pH level, which shows the degree of acidity reduction. Hemoglobin level was also found to be enhanced in cirsilineol animals. These modulating activity are highly anticipated for gastro-protective effect of an anti-ulcer agent. This anti-ulcerogenic effect were in agreement with a previous study conducted by Mahendran et al. [23] using Garcinia cambogia extract which amend the hemoglobin, volume and total acidity level of gastric juice in ulcerated rats.
The following study proved that HCl/ethanol increased the gastric level of inflammatory cytokines, TNF-α, IL-1β, and IL-6, while cirsilineol, modulated the levels of TNF-α, IL-1β, and IL-6. In gastric ulcer, HCl/ethanol causes inflammation to initiates macrophages release and inflammatory cytokines (TNF-α, IL-1β, and IL-6) to promote neutrophil accumulation at the target site for instigation of mucosal barrier destruction [24]. This data was in a line with a study conducted on gallic acid against ethanol-induced gastric ulcer rats which shows its anti-inflammatory effect by inhibiting the levels of TNF-α and IL-6 elevation [25]. Pro-inflammatory cytokine TNF-α activates the NF-kβ pathway, triggering the transcription for macrophages and cytokines to mediate inflammation. Mechanism of cirsilineol against HCl/ethanol-induced gastric lesion was apparent from the results of reduced inflammatory cytokines, which automatically inhibits the activation of NF-kβ pathway. Oral administration of cirsilineol might have restored the healing mechanism by re-epithelialization of mucosal and submucosal of the stomach cell membrane in HCl/ethanol induced gastric ulcer rats. Restoration of mucosal layer is considered the first indication of healing process against gastric ulcer damage [26]. This mechanism was exhibited by cirsilineol that could also be a key factor in the prevention of gastric ulcer.
The ELISA assay corroborated that oral administration of HCl/ethanol causes severe oxidative stress in stomach tissue as witnessed in control group by significant increase in the level of lipid peroxidation (MDA) which concurrently inhibited of the activity of antioxidant enzymes level, CAT and SOD. Manifestation of oxidative stress prompts abnormal level of free radicals which depletes the concentration of antioxidant enzymes, which function as defense mechanism against damaging free radicals [27]. The administration of cirsilineol improves the cellular antioxidant defenses by elevating the CAT and SOD while decreasing the level of lipid peroxidation. More recently, a study by Kumari and Anbarusa [28] reported similar protective activity by C-phycocyanin against selenite cataract rat model, by its ability to scavenge the free radicals. Similarly, cirsilineol conserved the antioxidant activity (SOD and CAT), specifying its antioxidant potential, thus protecting the gastric mucosa against lipid peroxidation damage. This data further confirms that cirsilineol has gastro-protective properties against the development of HCl/ethanol ulcers in rats. The lipid profile (total cholesterol, HDL, triglyceride levels) is associated with oxidative stress since increase of triglyceride level in gastric model group can be related to the excessive formation of lipid peroxides. Cirsilineol was able to reduce triglyceride levels in a dose-dependent manner by increasing the antioxidant effect, suppressing the production of lipid peroxides. Moreover, the total cholesterol and HDL levels have slightly reversed towards normal upon administration of cirsilineol due to the reversal of oxidative stress condition.
Finally, the toxicity profile of cirsilineol was investigated by accessing the serum biochemical parameters of liver, kidney and lipid function markers. Upon exposure to toxic condition such as HCl/ethanol or drugs, liver is impaired where by the ALP, ALT, and AST are excessively secreted into bloodstream. Another indication of liver injury is the release of plasma proteins such as total protein, albumin, and globulin [29]. These are estimation of excretory and metabolic estimation of liver where the levels were suppressed is a signal of chronic liver damage. As for renal damage, biochemical indicators are serum urea and creatinine which are excreted out of the body. However, during renal failure, the excretion functions are impaired. In addition, the level of blood cholesterol is implicative of metabolic problem in the liver [30]. Here, ameliorative effect of cirsilineol was observed in several markers of liver and kidney against HCl/ethanol-induced gastric lesions in rats. This was evidently justified with histopathological analysis revealing reduced damage in the stomach ulcer suggesting the cirsilineol anti-ulcer activity against experimentally induced gastric ulcer. These results give a further insight into the effect of cirsilineol on the organs of the rats for the safety claim of the cirsilineol.
Overall, the obtained results suggest cirsilineol employed an appreciable anti-ulcerative effect against HCl/ethanol-induced gastric lesions in rats. This activity can be associated with the improvement of antioxidant defense system to attenuate the inflammatory response for the improvement of the lesion accountable for the development of gastric ulcers. In addition, improvement in the liver, kidney and lipid parameter displayed a non-toxic profile of cirsilineol, clinically. However, a further comprehensive study is required to determine the mechanisms responsible for the gastro-protective action exhibited by cirsilineol.
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