ABSTRACT
Background:
Gastroesophageal reflux disease (GERD) is a chronic, relapsing disorder. In this era of modern and fast-track lifestyle and food habits, the incidence of GERD is rapidly increasing. Currently, proton pump inhibitors (PPIs) are the primary choice of treatment. However, the associated side effects and a high relapse rate give rise to the need to explore alternative therapies.
Objective:
The study aimed to evaluate HAGE-101912, an herbal combination, in different experimental models of GERD.
Methods:
Antacid activity was assessed based on H+/K+ATPase inhibitory activity of parietal cells using artificial gastric juice. Tonic contraction of the lower esophageal sphincter (LES) was evaluated using an AD Instrument. A GERD model of the pylorus and fundus ligation (preventive and curative models) in rats was selected to assess the efficacy of HAGE-101912 at a dose of 250 mg/kg body weight, and various parameters such as the gastric pH, gastric volume, total acidity, gross esophageal ulcer index, and histopathological changes were evaluated. The prokinetic activity was assessed using the phenol red method.
Results:
HAGE-101912 increased the acid-neutralizing capacity (P < 0.001), decreased H+/K+ATPase activity (P < 0.01), and increased the contraction of the LES. In the preventive model, HAGE-101912 significantly reduced the gastric acid volume (P < 0.01), total acidity (P < 0.001), and gross esophageal ulcer index (P < 0.01); increased the gastric acid pH (P < 0.01); and protected the esophageal epithelium. In addition, HAGE-101912 increased gastric emptying and gastrointestinal transit through its prokinetic activity (P < 0.05).
Conclusion:
HAGE-101912 has a beneficial effect in GERD as it effectively inhibits the H+/K+ATPase, increases the gastric pH, restores the LES function, protects the esophageal epithelium, and increases gastric emptying and transit.
KEYWORDS: Acid neutralizing, GERD, herbal combination, H+/K+ATPase, prokinetic
INTRODUCTION
GERD is a chronic, relapsing disorder that is caused by the reverse flow of gastric acid into the esophagus from the stomach. It leads to the erosion of the inner esophageal mucus epithelium. Untreated GERD leads to Barrett’s esophagus (B.E) and increases the risk of esophageal adenocarcinoma (E.A). About 40% of adults in the world suffer from GERD.
The weekly prevalence of GERD is estimated to be 10–20% in western countries and around 5% in Asian countries.[1] It has been reported that both men and women are equally prone to GERD. Recent studies confirm that in addition to gastric juice, reflux contents have a mixture of gastric acid, duodenal fluid, bile acid, and pancreatic juice.[2] The common symptoms of GERD are heartburn, belching, hoarseness, difficulty swallowing food, tooth erosion, asthma symptoms, and sleep disturbance. A weak lower esophageal sphincter (LES), reduced LES pressure, weak esophageal mucosal defense, poor diet (acidic, spicy foods, etc.), alcohol consumption, smoking, hiatal hernia, and delayed gastric emptying are the causative factors of GERD.[3]
Administering proton pump inhibitors (PPIs) is the first line of treatment for GERD. Other medications such as antacids and H2 blockers are also used in the preliminary phase. Long-term use of PPIs has drawbacks such as refractory complications (prevalence of 10% to 40%) and other complications such as calcium deficiency and weak bones. Recent studies found that long-term use of PPIs results in an increased risk of gastric cancer and other CNS complications such as dementia.[4,5]
The current study was conducted to evaluate the efficacy of the herbal combination HAGE-101912 in various experimental models of GERD. HAGE-101912 contains Centella asciatica aerial part extract, Benincasa hispida spray-dried fruit juice, Cuminum cyminum seed aqueous extract, the aerial part of the plant of Tinospora cordifolia extracts, and deglycyrrhizinated Glycyrrhiza glabra root extract.
MATERIALS AND METHODS
Chemicals
Pepsin and omeprazole were procured from Tokyo chemical industry Chemicals Pvt. Ltd., Tamil Nadu, India. A H+/K+ATPase Colorimetric Assay Kit was from Elabscience, United States. Thiopental was obtained from Neon Laboratories Ltd., Thane, Maharashtra, India. All additional chemicals and reagents were from Sigma Aldrich Chemicals Private Limited, Bengaluru, India, and HiMedia Laboratories Private Limited, India, and were of analytical grade.
Experimental animals
Female Wistar rats were procured from a central animal house facility of Himalaya Wellness Company in India. The animals were placed under the controlled conditions of temperature, relative humidity, and light cycle before and during the study. All were fed with standard rat pellet feed and Reverse osmosis (RO) purified, water ad libitum. The experiment was carried out under protocol no. 198/19, which was approved by the Institutional Animal Ethics Committee (IAEC) of Himalaya Wellness Company. The animals were treated according to the guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and The Ministry of Environment and Forests, Government of India.
Procedure
Antacid activity using modified artificial gastric juice (in vitro)
The method of Sandhya s et al. was followed. Briefly, the test solution of HAGE-101912 was prepared at a concentration of 1 mg/ml with demineralized (DM) water. The same concentration of sodium bicarbonate (NaHCO3) was also prepared and used as a reference standard. Water was taken as a control. The pH of all the solutions was checked using a pH meter (Servewell Instruments Pvt. Ltd., Bengaluru, India).
To prepare artificial gastric juice (AGJ), 2 g of NaCl and 3.2 mg of pepsin enzymes were dissolved in 500 ml of DM water. Then, 7 ml of concentrated hydrochloric acid was added, the volume was made up to 1000 ml with DM water, and the pH was adjusted to 1.2.
To determine the neutralizing capacity on AGJ, 90 ml of the prepared test solutions was added to 100 ml AGJ. The change in pH was noted to examine the neutralizing effect.
The neutralizing capacity of the test solutions was also evaluated by Fordtran’s method of titration.[6]
H+/K+ATPase activity (ex vivo)
The H+/K+ ATPase enzyme was prepared by the method described by Reyes Chilpa et al.[7]
The prepared enzyme source was incubated with HAGE-101912 (250 μg) for 1 hour, and then the H+/K+ATPase activity was evaluated as per the procedure mentioned on the colorimetry kit. Omeprazole (250 μg) was used as a reference standard. This method was performed in triplicate.
H+/K+ATPase activity (in vivo)
Female Wistar rats (200–250 g) were used for the in vivo H+/K+ATPase activity. The rats were segregated into three groups of six each. Group 1 served as the control and received DM water (10 mL/kg). Group 2 served as the reference standard and received omeprazole (10 mg/kg). Group 3 received HAGE-101912 (250 mg/kg) for a period of 7 days. The dose was selected based on the LD50 studies. On the seventh day, the gastric mucosa was collected and dispersed in ice-cold saline (1:10) and H+/K+ATPase activity was evaluated.[8]
Tonic contraction of lower esophageal sphincter (ex vivo)
The strips of LES was prepared by the method described by Tsai CC et al.[9] After a 30 min equilibration period, pre-response of the muscle strips was confirmed by adding 0.001 M acetyl choline and then washed off. Further HAGE-101912 was added in a cumulative dose manner (3, 6, 12, 24, 48, 96 μg/mL), and the contraction was recorded.
Gastro-esophageal reflux disease model in rats (in vivo preventive design model)
Female Wistar rats (200–250 g) were divided into four groups of six each. Groups 1 and 2 served as the sham control and positive control (disease control), respectively, and received DM water at a dose of 10 mL/kg. Groups 3 and 4 received omeprazole (10 mg/kg) and HAGE-101912 (250 mg/kg), respectively. Rats were administered with respective treatment orally using an oral catheter for 2 weeks. On the 14th day, overnight fasted rats were anesthetized with Isoflurane and mid-line laparotomy was performed as per Kwon OJ et al. to induce GERD. Gastric acid was collected, and the entire stomach with esophagus was collected immediately and subjected for the estimation of total acidity and gross pathology.[10]
The collected esophagi were evaluated for the gross morphology scoring and ulcer index.[11] After gross evaluations, the tissues were stored in 10% neutral formalin buffer (NBF) for histopathological evaluation.
The sections were analyzed for erosion of the mucosal layer/ulcer formation, hyperkeratosis, sloughing of keratin, and submucosal edema.
Gastro-esophageal reflux disease model in rats (in vivo curative design model)
Eighteen female Wistar rats (200–250 g) were divided into three groups of six each based on their mean body weight. Group 1 served as the control and received DM water at a dose of 10 mL/kg. Groups 2 and 3 received omeprazole and HAGE-101912 at a dose of 10 mg/kg and 250 mg/kg, respectively. Under isoflurane anesthesia, a mid-line laparotomy was performed, and the fundus and pyloric sphincter were ligated using a 3-0 silk thread. After 6 hours, the rats were again anesthetized with isoflurane, and the pylorus was unligated. Treatment was continued for 7 days. On the 7th day, the esophagus was collected along with the stomach, and gross ulcer scoring was performed based on severity score (1, mild; 2, moderate; and 3, severe) and stored in 10% NBF for histopathological evaluation. The tissues were analyzed for epithelium thickness.[12-14]
Prokinetic activity by phenol red method (in vivo)
Twenty-four female Wistar rats (200–250 g) were segregated into four groups of six each based on their mean body weight. Group 1 served as the normal control or 0 min control and received 10 mL/kg DM water. Group 2 served as the 15 min control and received 10 mL/kg DM water. Groups 3 and 4 received domperidone and HAGE-101912 at a dose of 10 mg/kg and 250 mg/kg, respectively. After 7 days of treatment, all rats received 2 mL of semi-solid test meal of 1.5% methyl cellulose solution containing phenol red (50 mg/100 mL). the prokinetic activity was performed as described by Jang Y et al.
The gastro-intestinal transit was measured by evaluating the distance travelled by phenol red and the total length of the small intestine.[15,16]
Statistical analysis
All the values were expressed as the mean ± standard error of the mean (SEM). The results were statistically analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test. The Prism GraphPad 6.07 software (GraphPad Software Inc, San Diego, CA, USA) was used for statistical analysis. A P value <0.05 was considered as statistically significant.
RESULTS
Evaluation of in vitro antacid activity by modified AGJ
The pH of HAGE-101912 was found to be 4.99 ± 0.32, whereas the pH of DM water and sodium bicarbonate was found to be 6.68 ± 0.13 and 9.13 ± 0.27, respectively. In both the methods of acid-neutralizing capacity test, it was observed that HAGE-101912 and sodium bicarbonate significantly increased the pH [Figure 1a] and H+ ion consumption [Figure 1b] compared with that of the control (P < 0.001).
Figure 1.
Effect of HAGE-101912 on acid neutralizing capacity. (a) Change in pH; (b) H+ ions consumed. (***p<0.001 compared to control)
Evaluation of H+/K+ATPase activity
H+/K+ATPase, the proton pump found in parietal cells, is responsible for the acidification of stomach contents. The greater the reduction in H+/K+ATPase activity better is the proton pump inhibitor activity. In the present study, HAGE-101912 was evaluated for H+/K+ATPase activity in both ex vivo and in vivo models, and omeprazole was used as a reference standard.
Figure 2a illustrates the ex vivo H+/K+ATPase activity. A significant decrease in H+/K+ATPase activity was observed in HAGE-101912 (P < 0.01) and omeprazole (P < 0.001) at 250 μg compared to the control.
Figure 2.
Effect of HAGE-101912 on H+/K+ATPase activity. (a) H+/K+ATPase activity (ex vivo); (b) H+/K+ATPase activity (in vivo)
Figure 2b illustrates the in vivo H+/K+ATPase activity. Administration of HAGE-101912 (250 mg/kg) and omeprazole (10 mg/kg) for 7 days significantly decreased in H+/K+ATPase activity in both HAGE-101912 (P < 0.01) and omeprazole (P < 0.001) treated animals when compared with the control.
Evaluation of tonic contraction of LES
HAGE-101912 showed contraction in isolated LES in a dose-response manner. HAGE-101912 induces the tonic LES contraction at 3 μg/ml and showed an increase in contraction up to 96 μg/ml. [Figure 3].
Figure 3.
Effect of HAGE-101912 on tonic contraction of LES muscle (ex vivo)
Gastro-esophageal reflux disease model (preventive design model)
The increased gastric juice volume was significantly decreased in the groups treated with HAGE-101912 (P < 0.01) and omeprazole (P < 0.001), compared with that in the positive control group. There was a significant increase in pH of gastric juice in HAGE-101912-treated group (P < 0.01) and omeprazole-treated group (P < 0.001) compared with that in the positive control group. A significant decrease (P < 0.001) in total acidity of gastric juice was observed in both omeprazole-treated and HAGE-101912-treated group compared with that in the positive control group. The gross esophageal ulcer index was significantly (P < 0.01) decreased in HAGE-101912-treated and omeprazole-treated groups compared with that in the positive control group [Table 1].
Table 1.
Effect of HAGE-101912 on gastric parameters in GERD model
| Parameters | Sham control | Positive control | Omeprazole (10 mg/kg) | HAGE-101912 (250 mg/kg) |
|---|---|---|---|---|
| Gastric juice volume (ml) | 0.14±0.07 | 2.60±0.17a | 1.13±0.13b | 1.74±0.22c |
| Gastric juice pH | 2.92±0.11 | 2.21±0.01a | 3.93±0.03b | 3.81±0.01c |
| Gastric juice acidity (mmol/L) | - | 102.20±3.64 | 33.33±2.11b | 41.45±2.61b |
| Gross esophageal ulcer index | 0.00±0.00 | 0.27±0.01a | 0.03±0.02c | 0.04±0.02c |
All values are expressed as the mean±standard error of the mean (SEM); aP<0.001 compared to Sham control; bP<0.001 and cP<0.01 compared to Positive control
In histopathological evaluation, the scoring pattern was performed based on the extent of severity of damage of the esophagus: score 0 for no erosion/abnormality, score 1 for mild erosion, score 2 for moderate erosion, and score 3 for severe erosion and ulcer formation.
GERD by surgical induction showed erosion of the epithelium layer of the esophagus in the positive control animals; it showed a significant increase in the erosion or ulcer formation (P < 0.01), hyperkeratosis (P < 0.001), and sloughing of keratin (P < 0.001) in the positive control group compared with that in the sham control animals. HAGE-101912 significantly reduced erosion or ulcer formation (P < 0.05), hyperkeratosis (P < 0.01), and sloughing of keratin (P < 0.05) compared to a positive control [Table 2 and Figure 4].
Table 2.
Histopathological evaluation of esophagus of control and HAGE-101912 treated animals
| Parameters | Sham control | Positive control | Omeprazole (10 mg/kg) | HAGE-101912 (250 mg/kg) |
|---|---|---|---|---|
| Erosion/ulcer formation | 0.00±0.00 | 3.33±0.21b | 2.25±0.25 | 1.00±0.43d |
| Hyperkeratosis | 0.00±0.00 | 2.64±0.15a | 2.50±0.22 | 1.29±0.18c |
| Sloughing of keratin | 0.00±0.00 | 2.73±0.19a | 2.67±0.21 | 1.42±0.20d |
| Sub-mucosal edema | 0.00±0.00 | 2.55±0.21a | 2.67±0.21 | 2.29±0.28 |
All values are expressed as the mean±standard error of the mean (SEM); aP<0.001 and bP<0.01 compared to Sham control; cP<0.01 and dP<0.05 compared to Positive control
Figure 4.
Photomicrograph of esophagus showing the severity of lesions and erosion of epithelium layer in the preventive design model. (a) Normal histoarchitecture of the esophagus in Sham control; (b) Positive control animal esophagus showed significant erosion of epithelium layer; (c) The esophagus of omeprazole-10 mg/kg-treated animals showed mild to moderate erosion of epithelium layer; (d) The esophagus of HAGE-101912-250 mg/kg-treated animals showed mild to moderate erosion of epithelium layer. 5-point star (★) =Normal keratinized squamous mucosal epithelium, Diamond (♦)=Normal Tunica muscularis layer of the esophagus in Sham control. The red arrow marks indicate moderate to severe erosion in the positive control group. The blue and black arrows indicate mild to moderate erosion of the epithelial layer in omeprazole- and HAGE-101912-treated groups, respectively
In the curative design model, HAGE-101912-treated group showed a reduction in gross esophageal ulcer index compared to the control group and was at par with the omeprazole-treated group [Figure 5a]. Esophageal epithelium thickness is the major parameter in identifying the severity of damage in the curative design model (chronic reflux model). A decrease in esophageal epithelium thickness was observed in HAGE-101912-treated group compared to the control group and was at par with the omeprazole group [Figures 5b and 6].
Figure 5.
Effect of HAGE-101912 in an experimental model of gastro-esophageal reflux disease (curative design model) (a) Gross esophageal ulcer index; (b) Esophageal epithelium thickness
Figure 6.
Photomicrograph of esophagus showing esophagus epithelium thickness in the curative design model (a) Control animal esophagus thickness; (b and c) Omeprazole (10mg/kg) and HAGE 101912 (250mg/kg)-treated animals showed a decrease in esophageal epithelium thickness compared to control animals, respectively
Evaluation of prokinetic activity by phenol red method
An increase in the percentage gastric emptying was observed in HAGE-101912-treated group, whereas a significant (P < 0.05) increase in the percentage gastric emptying was observed in domperidone-treated group compared with that in the control group [Figure 7a]. A significant (P < 0.05) increase in gastro-intestinal transit was observed in both HAGE-101912-treated and domperidone-treated groups compared with that in the control group [Figure 7b].
Figure 7.
Effect of HAGE-101912 on prokinetic activity. (a) Percentage gastric emptying; (b) Percentage gastro-intestinal transit
DISCUSSION AND CONCLUSION
Herbal combination HAGE-101912 was prepared based on the available literature in the traditional books of Ayurveda and on the scientific evidence available in the modern literature on the efficacy of individual herbs on different targets of GERD. HAGE-101912 was evaluated for anti-GERD activity using various in vitro, ex vivo, and in vivo experimental models.
The gastric acid pH plays an important role in acidity and reflux. Gastric acid reflux with low pH results in more damage to the esophagus. Antacids are the drugs known to neutralize the acid by increasing the pH of gastric acid so that erosion can be minimized. In this context, antacid activity of HAGE-101912 was estimated and it was found that it significantly neutralizes the artificial gastric juice compared to the control. This indicates the antacid property of HAGE-101912, which may be because of the presence of Benincasa hispida herb, which is known to decrease acid volume secretion, acid output, and pepsin activity; thereby, it exhibits antacid activity.[17]
The proton pumps are responsible for regulating the gastric acid secretion. It exchanges potassium from the intestinal lumen with cytoplasmic hydronium. It is the primary enzyme responsible for the acidification of the stomach contents and the activation of the digestive enzyme pepsin. PPIs are the drugs that inhibit gastric acid secretion by blocking the H+/K+ATPase enzyme (proton pump). Reduction in gastric secretion results in minimizing gastritis and esophageal erosion in GERD conditions.[7,8] HAGE-101912 showed proton pump inhibitory activity in both ex vivo and in vivo models, which may be because it contains a substantial concentration of polyphenols and flavonoids, which are known to exhibit significant proton pump inhibitory activity.[7,18]
LES is a specialized circular smooth muscle at the junction of the esophagus and stomach which allows the passage of bolus into the stomach and meanwhile prevents the backflow of gastric contents from the stomach to the esophagus.[19] Oblique sling and circular muscles are the two separate muscle components of LES. The upper sling muscles contract and open the sphincter to allow the food to move into the stomach. The lower circular muscle generates basal tone by contracting, which helps to prevent reflux of gastric contents into the esophagus. A dysfunctional LES allows the reflux of large amounts of gastric juice. A relaxed LES results in the opening of circular muscle which will end up in the backflow of gastric contents. HAGE-101912 showed a concentration-dependent tonic contraction of LES, indicating that it restricts the reflux by contracting the lower circular muscle of LES. Deglycyrrhizinated Glycyrrhiza glabra is one of the ingredients of HAGE-01912 which is known to improve impaired LES muscle contraction. The perceived effect may be because of the presence of deglycyrrhizinated Glycyrrhiza glabra.[20]
The GERD was mimicked in rats by tying the pylorus and fundus part of the stomach, which results in an increase in pressure and ends up in reflux; it is a well-established in vivo model to screen the drugs for GERD. In this model, the secreted gastric acid could remain in the stomach by ligating the pylorus end and will further spread into the esophagus because of the fundus ligation, and a typical GERD condition can be achieved. The HAGE-101912 was tested in both preventive and curative GERD models. In the preventive model, the animals were treated for 2 weeks before the induction of the disease. However, in the curative model, the disease was induced first and the treatment was followed for 1 week.[21] HAGE-101912 was found to be effective in reversing the condition of GERD by decreasing the gastric acid secretion, increasing the gastric juice pH, and decreasing the total acidity. It also decreased the esophageal erosion/ulcers, hyperkeratosis, and epithelial thickness, which are the typical pathological markers of GERD. HAGE-101912 showed protection in both preventive and curative models. However, it showed better results in the preventive model compared to curative. The ingredients of HAGE-101912, like Cuminum cyminum, Centella asciatica, and Tinospora cordifolia have the capacity to increase the mucin secretion, by which erosion of epithelium is minimized. They are also reported for increasing the gastric pH and decreasing the total acidity of gastric juice.[22-24]
One of the most common problems with GERD patients is delayed gastric emptying. It results in an increased volume and pressure in the stomach, which causes the reflux of gastric contents. Prokinetic drugs increase gastric emptying and gastro-intestinal transit, which help in reducing the symptoms of GERD.[25] In this study, HAGE-101912 increased the percentage of gastric emptying and significantly increased the percentage of gastro-intestinal transit compared to the control group. This showed that HAGE-101912 along with other activities possesses prokinetic activity which will be useful in reducing the symptoms of GERD. This activity may be because of Benincasa hispida, which has been reported for prokinetic activity.
HAGE-101912 acts holistically to ameliorate the condition of GERD. It exerts its effects by decreasing the gastric secretion, increasing the gastric pH, inhibiting the H+/K+ATPase enzyme, and accelerating the gastric emptying and gastro-intestinal transit. It decreased the esophageal erosion/ulcers, hyperkeratosis, and epithelial thickness of esophageal tissue. Thus, it can be recommended as such or as an adjuvant in the treatment of GERD. Further experiments and clinical investigation are in progress to confirm the same.
Institutional animal ethical committee approval for conducting animal studies
Inbred Wistar rats were procured from the central animal house facility (R&D, Himalaya Wellness Company, Bengaluru, Karnataka, India. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Himalaya Wellness Company (Protocol no. 198/19), and the animals received care as per the guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), The Ministry of Environment and Forests, Government of India.
Financial support and sponsorship
The research was supported by M/S Himalaya Wellness Company, Bengaluru, India.
Conflicts of interest
The authors are employees of Himalaya Wellness Company (Bengaluru, Karnataka, India) during the research work. The authors declare no other conflict of interest.
Acknowledgements
The authors acknowledge Phytochemistry, Natural Product Innovation, Preclinical Pharmacology and Toxicology Department, R&D Center and Scientific Publication team, Himalaya Wellness Company, Bengaluru for providing support during the study.
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