The Evaluation of Proton Pump Inhibitor Bioactive Fraction DLBS2411 from Cinnamomum burmannii (Nees & T. Nees) in Animal Model of Gastric Ulceration Healing

Corresponding Author: Raymond Rubianto Tjandrawinata Dexa Laboratories of Biomolecular Sciences, Industri Selatan V Block PP no.7, Jababeka Industrial Estate II, Cikarang, West Java 17550, Indonesia Ph: +62-21-89841901 Fax: +62-21-89841905 Email: raymond@dexa-medica.com Abstract: DLBS2411 is a bioactive fraction from Cinnamomum burmannii that is believed to possess anti-ulceration activity. In this study acetic acid was administered to induce gastric ulcer. Rats were divided into six groups: Healthy animals, untreated animals (acetic acid-induced), treatment group of BAF DLBS2411 25 mg kg −1 Body Weight (BW), BAF DLBS2411 50 mg kg −1 BW and positive control group of omeprazole 2 mg kg −1 and sucralfate 100 mg kg −1 BW. The efficacy of DLBS2411 was measured through histopathology examination and ulceration observation. Hematological parameters were also evaluated before and after treatments to indicate the safety profile of DLBS2411. The untreated animals developed more serious injuries (i.e., bigger and more severe ulcers) than the positive control groups and DLBS2411 treatment groups. DLBS2411 treatment with doses of 25 and 50 mg kg −1 and sucralfate 100 mg kg −1


Introduction
Peptic ulcers are injuries in the mucosal areas immersed in gastric acid and pepsin, wherein normally the areas is covered with mucin secreted from mucus cells. Ulceration results from imbalance between gastroprotective activity and aggressive factors such as acidpepsin secretion, mucosal barrier, mucus secretion, blood flow, cellular regeneration, prostaglandins and epidermal growth factors (Bae et al., 2011;De Barros et al., 2008). It is one of the most common gastrointestinal disorders in clinical practice which is generally treated with modern medicines such as sucralfate and omeprazole. Considering potential side effects arising from these medicines, such as constipation, decreased bone mineral density and hematopoietic events sue to higher aluminium concentration, novel medicines should be sought for as an alternative in treatment of internal diseases (Dallwig, 2010;Papich, 2016;Odeh and Oliven, 2001;Yanagihara et al., 2015).
In recent years, we have conducted many studies on the potential use of herbal extracts for treatment of different kinds of diseases, especially on Cinnamomum burmannii extract for diabetes treatment. Combined with Lagerstroemia speciosa, C. burmannii has been found to induce insulin sensitivity in vitro as well as in patients in clinical studies Sitepu et al., 2016;Manaf et al., 2016;Kartolo et al., 2016;Tjandrawinata et al., 2013;Tandrasasmita et al., 2011;Tandrasasmita et al., 2014;Tjandrawinata et al., 2011). DLBS2411, a bioactive fraction containing C. burmannii, has previously been proven to affect hydrogen potassium adenosine triphosphate (H + /K + ATPase) activity as well as to down-regulate the gene expression. DLBS2411 was also reported to possess antioxidative activity (Tjandrawinata et al., 2013). Such pharmacological property would be beneficial for treatment and prevention of peptic ulcer. This paper further delved into the potential of DLBS2411 as an ulcer healing agent fractionated from C. burmannii. In this regard, DLBS2411 was studied in rat model of gastric ulceration.

Preparation of DLBS2411
The dried C. burmannii bark was milled and macerated in water for 2 h and concentrated to approximately 1/10 of its initial volume and fractionated with methylene chloride to allow the aqueous phase to separate from the organic phase. The aqueous phase was then concentrated to eliminate any residual methylene chloride. The resulting concentrate was dried in an oven and the dried product was milled using Cone Mill (Comil underdrium 197,Quadro,Canada) and stored in a well-sealed container.

Animals
Thirty five young adult male Wistar rats (Rattus norvegicus) weighing around 250-300 grams were used in this study and housed individually in polysulfone cage and maintained under standard conditions (12 h light-12 h dark cycle, 25±2°C; 30-70% humidity).
All procedures in this experiment has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Dexa Laboratories of Biomolecular Sciences (DLBS), with protocol number DIS-DLBS-PROC-006 and carried out in accordance with the Association for Assessment and Accreditation Laboratory Animal Care (AAALAC) International.

Experimental Procedures
The experiment was carried out according to the method by Okabe et al. (1971) with minor modifications.

Pilot Study (Ulcer Producing Study)
Twelve hours prior to blood sampling and surgery, rats were fasted and placed in metabolic cages (no food, water ad libitum), in order to avoid any interferences of gastric acidity degree that could cause disturbance in ulcer induction. After fasting, blood was collected and surgery was performed to induce ulcer (Day-0). Three days after surgery, five rats were euthanized with pentobarbital injection.
Twelve hours before blood sampling and surgery, rats were fasted and placed in the metabolic cage (no food, water ad libitum). After fasting, blood samples were collected and surgery was performed to induce ulceration with acetic acid. Three days after surgery (day 1 of treatment), the animals were treated with placebo (for healthy animals and untreated animals), DLBS2411 at dose of 25 and 50 mg kg −1 BW (for treatment groups), omeprazole 2 mg kg −1 BW and sucralfate 100 mg kg −1 BW (for positive control groups) for seven days. At the end of the experiment, rats were euthanized using pentobarbital injection. Pathology condition of gaster, ulcer area and hematological data were observed in this study.
The schematic description of the experimental treatment is provided in Fig. 1.

Operating Procedure
After 12 h of fasting, rats were anesthetized with ketamine mixed with xylazine at dose of 40-80 and 5-10 mg kg −1 BW, respectively, via intra-peritoneal route. Acetic acid solution (80% v/v, 500 µL) was placed on serosal surface of the gaster in its corpus region and allowed to remain in contact for 30 sec. The area that were in contact with acid were rinsed gently with saline and the abdomen were closed with suture. For healthy animals, acetic acid solution was replaced with aquadest for injection. Neomycin powder was applied at the site of operation in order to avoid post surgical infection. Day 3 post surgery was considered as day 1 of treatment.

Histopathology Examination
At the end of the treatment, the animals were euthanized. Gastric samples were collected and fixed into neutral buffer formalin. These samples were processed using tissue processor with different concentrations of ethanol and stained with hematoxylin eosin and examined under 10× magnification.
Severity of gastric ulcer was assessed according to the method by Karakoyun et al. (2009) where the result of the examination was divided into four parameters which are desquamation of surface epithelium (0-3), hemorrhage, focal necrosis and mucosal congestion (0-3), glandular cells degeneration (0-3) and inflammatory cell infiltration (0-3) with a maximum total score of 12 for the most severe condition.

Statistical Analysis
Data of each group should be normally distributed and the variance should be homogenous in order to be analyzed with one-way analysis of variance (ANOVA). The continuous data were expressed as mean ± S.E.M. (standard error of mean) followed by post hoc test for multiple comparisons (Tukey's HSD or Games-Howell test), using SPSS ® 20 Statistics software. Throughout the analysis, parametric variables were log transformed in order to meet the underlying distributional assumptions of the statistical models otherwise, the corresponding nonparametric test results were used. All statistical tests were done at 5% level of significance considered as statistically significant.

Pilot Study (Ulcer Producing Study)
A pilot study was performed prior to the full study of DLBS2411 on acetic acid-induced ulceration. The purpose of this pilot study was to determine whether acetic acid (80% v/v, 500 µL) could induce gastric ulcer on the mucous layer. Pathology and histopathology examination showed that the treatment successfully induced gastric ulcer as shown in Fig. 2.
From the histopathology examination, it was apparent that edema and hemorrhagia with a lot of neutrophils infiltration were found in all groups of the ulcer producing study (Fig. 2B). Neutrophils were found in the serosa area and spreading to the mucosa of gastric. According to these results, contact with acetic acid (80% v/v, 500 µL) was able to induce the ulcer formation successfully. Therefore, this method was valid for further study, i.e., induction of ulcerations.

Full Study
Following the pilot study, a full study investigating the effect of DLBS2411 on gastric ulcers were performed. Figure 3 shows that the biggest ulceration was seen in untreated animals, as compared to treatment and positive control groups.  Histopathology examination showed the presence of edema and hemorrhage in the untreated animals. Macrophages mixed with neutrophils were also found in the cytoplasm from the serosa to submucosa area in dark brown color (hemosiderin). Details of mucus formation and the severity of gastric ulcer were determined through histopathology examination as shown in Fig. 4, while ulcer area of each group is shown in Fig. 5. Based on the histopathology results, there was no abnormal condition observed in the healthy animals (Fig. 4A). Untreated animals developed severe infiltration of lymphocytes, plasma cell, macrophages mixed with a fewer numbers of neutrophils, eosinophils and mast cells in the serous and mucous layer of gastric. A number of brown-pigmented macrophages were also found in the cytoplasm (Fig. 4B). Treatment groups of BAF DLBS2411 at dose of 25 and 50 mg kg −1 BW showed segmented erosion on the mucosal surface ( Fig. 4C and D). Overall mucous muscular and serous areas were found to be thickened by granular tissues. Omeprazole-treated group showed a segmented edema and capillary dilatation on the submucous and muscular mucous areas which led to the development of edema thickening, mononuclear cell infiltration and granular tissues in the serous area (Fig. 4E). Mild to moderate macrophage infiltration mixed with eosinophils were also found. Fibrous tissues mixed with severe infiltration were found on the gastric serosa and mucous of the sucralfate-treated group (Fig. 4F). Vasculitis was also found on the submucous area of this group (Fig. 4F).
Treatment with BAF DLBS2411 at dose of 25 mg kg −1 , 50 mg kg −1 and sucralfate reduced the gastric ulcer area up to 54.8, 36.53 and 14.85%, respectively, compared to that of the untreated animals (Fig. 5). While treatment with omeprazole for 7 days did not cause any marked reduction of the gastric ulcer size from that of the untreated animals (Table 1).       Histopathology data showed that under normal condition (i.e., the healthy animals), rats did not develop any ulcer. The most severe gastric ulcers were found in the untreated animals. Treatment groups of BAF DLBS2411 at dose of 25 mg kg −1 , omeprazole and sucralfate developed only mild to moderate gastric ulcers. Small ulcers were found in BAF DLBS2411 at dose of 50 mg kg −1 group. A semi-quantitative severity scoring data according to the method developed by Karakoyun et al. (2009) is shown in Fig. 6.
Wound healing process was scored based on granulation in tissues developed in gaster following treatments. Based on the observation, treatment with DLBS2411 at doses 25 and 50 mg kg −1 were found to produce only mild to thick granulation, which were similar to that of omeprazole and sucralfate (Fig. 7). Treatment with BAF DLBS2411 at dose of 25 mg kg −1 , 50 mg kg −1 and positive control group treated with omeprazole and sucralfate reduced the severity of gastric ulcer as much as 45, 56.67, 36.67 and 20%, respectively, compared to that of the untreated animals (Table 2).     Hematology examination before treatment showed that prior to the study the animals were in excellent condition. The result of hematology examination after treatment were found to be significantly different on the platelet, MCH and PCT value of ulcer producing group due to time differences between pilot study and full study which was performed on day-3 and day-10 after induction of gastric ulcer. There was no significant difference in the hematological value among groups on full study (Table 3 and 4). This result indicates that all treatment agents did not affect the hematological value.

Discussion
Previous study by Gunal et al. (2002) has documented that several evidence showing acetic acid-induced gastric ulcer becomes chronic within 2-3 days and heals completely within 2-3 weeks without perforation or penetration. The ulcer induced by acetic acid is assumed to be similar to that of chronic human ulcer with regards to its location, severity and chronicity as well as with regard to the healing process (Silva et al., 2013). Treatment with BAF DLBS2411 at doses of 25 mg kg −1 , 50 mg kg −1 (BW) and sucralfate reduced the gastric ulcer area up to 54.8, 36.53 and 14.85%, respectively, from that of the untreated animals. Treatment with omeprazole for 7 days, however, did not indicate any reduction of the ulcer area. The severity of the ulcer was improved in all treated groups: treatment group DLBS2411 25 mg kg −1 BW, DLBS2411 50 mg kg −1 BW, omeprazole and sucralfate, as compared to that of the untreated animals. The severity scoring for gastric ulcer was determined by histopathology examination.
Untreated animals had the most severe damage among the groups; while the treated groups such as treatment group DLBS2411 at dose of 25 mg kg −1 BW, omeprazole and sucralfate developed mild to moderate gastric ulcers. The mildest ulceration was developed in treatment group DLBS2411 at dose of 50 mg kg −1 BW. Treatment with DLBS2411 at dose of 25 and 50 mg kg −1 , omeprazole and sucralfate could reduce the severity of gastric ulcer as much as 45, 56.67, 36.67 and 20%, respectively, compared to the untreated animals. Severity of gastric injury was assessed semi-quantitatively using method from Karakoyun et al. (2009) where each measured parameter was categorized into 0-3, resulting a maximum total score of 12, as shown in Fig. 6.
The reduction of ulcer area and alleviation of ulcer severity based on the histopathology analyses indicated that DLBS2411 treatments, either at the dose of 25 or 50 mg kg −1 BW in rats, were comparable to those of omeprazole or sucralfate. This may have occurred due to the dual mechanisms of action of DLBS2411: A down-regulator of proton pump and a mucosal protective agent (Tjandrawinata et al., 2013). Extensive studies have been carried out to assess the pharmaceutical potential of C. burmannii. Several studies have shown its potential effect as antibacterial, anti-inflammatory and analgesic drug (Al-Dhubiab, 2012;Khatib et al., 2005;Wu and Chou, 2011). DLBS2411 treatment is known to elevate phosphorylation of IKK and activate nuclear factor-КB (NF-КB) which could stimulate mucus synthesis and mucosal blood flow. High level of NF-КB increased mucus synthesis by promoting cyclooxygenase-2 (COX-2) and PGE 2 expression, which increased the MUC5AC gene. Activation NF-КB also increased production of NO, which stimulated mucosal blood flow (Wulandari et al., 2016). These mechanisms of action are postulated to provide the basis of the healing process of gastric ulcer by C. burmannii. A number of clinical trials are currently in process to investigate the efficacy and safety of DLBS2411 as treatment for gastric ulcer patients (identified number NCT01573403 and NCT02262169 respectively).

Conclusion
From the result of the present study, DLBS2411 at dose of 25 and 50 mg kg −1 BW were effective for gastric ulcer treatment as measured by the reduction of ulcer size. It is also effective in alleviating ulcer severity as seen from the histopathology result. There was no significant difference in hematological value among groups in the full study, indicating that DLBS2411 did not affect hematology profile. This present study demonstrated that DLBS2411 at dose of 25 and 50 mg kg −1 BW (or equivalent to the human dose of 250 and 500 mg daily) demonstrated curative effect on acetic-induced gastric ulcer.