INTRODUCTION:

‘Vidanga’ (Embelia ribes) is a potent Ayurvedic plant belonging to the Myrsinaceae family, encompassing around a thousand species of shrubs and trees divided among thirty-three genera. Notably, genera such as Myrsine, Maesa, Rapanea, and Embelia are extensively utilized within traditional and ethnobotanical practices for their diverse therapeutic applications including anthelmintic properties, anti-tuberculosis effects, blood purification, bronchitis treatment, tumor management, appetite stimulation, alleviation of indolent conditions, jaundice remedy, mental disorder treatment, mouth ulcer relief, pleurisy alleviation, relief from stimulating sore throat, and management of skin diseases, usually administered in the form of powder^1,2. The Ayurvedic Pharmacopoeia of India (API) recognizes Embelia ribes as associated with 'Vidanga'³. However, it is crucial to note that Embelia ribes face endangerment as a medicinal plant due to its high fruit-bearing capacity and long-standing demand in the pharmaceutical and Ayurvedic industries⁴.

In recent years, the challenge of drug productivity has led to increased interest in finding new therapeutic indications for known drugs. This strategy involves exploring known drugs for novel applications⁵. This approach has yielded promising results, such as in the case of Korean medicine prescriptions, where Samjunghwan has been indicated as an anti-aging therapy and has demonstrated significant efficacy in managing hyperlipidemia^6,7. Furthermore, the historical application of Morus nigra L. juice in managing tonsillitis and sore throat, combined with its ability to reduce pro-inflammatory cytokines, underscores the potential of utilizing traditional remedies⁸.

The particular interest is the fruit extracts of Embelia ribes (Er) and Embelia tsjeriam-cottam (Et), which have demonstrated enhanced free radical scavenging potential⁹. Additionally, the active compound Embelin is a common feature in both Er and Et, showcasing their shared characteristics^10,11,12. Quantitative analysis conducted by Subramaniam et al. in 2005¹³ revealed that Embelin content in Er (4.33%) and Et (3.96%) exhibited non-significant variations. In Ayurveda, 'Vidanga' holds a prominent position as one of the most popular herbs for fortifying the body's natural gastrointestinal defenses¹⁴. Its versatility extends to aiding digestion, preventing constipation, and possessing specific pharmacological properties, including the provision of centrally acting non-narcotic, orally effective analgesic activity, carminative, anxiolytic activity, anti-urolithiatic activity, anti-implantation activity etc.^{15,16,17,18,19,20}. Moreover, 'Vidanga' is a common component in several polyherbal formulations designed to address gastrointestinal issues, which are readily available in the market, including products like Abana, Gasex, Geriforte, Sanjivani Vati, Koflet, Krumiva-Kar Kwath, Livomyn, Liv-52, Livosin, Mahamanjisthadi Kwatha, Vidangirishta, Vidanga lauha, and Vidangidi lauha^21,22,23.

However, the surging demand for the endangered species Embelia ribes (Er), exceeding its annual availability by over 500 metric tons, underscores the need for sustainable solutions²⁴. In this context, the present study aims to comparatively validate the anti-ulcerogenic properties of Er and Et fruit extracts. The primary objective is to assess the efficacy of these extracts in treating ethanol-induced stomach ulcers in an animal model, thereby contributing to the current understanding of safe and effective stomach ulcer management.

MATERIALS AND METHODS:

Plant materials and reagents:

Er and Et were collected from village Kemse in the Koyna area of Sahyadri Hills of Maharashtra with prior permission from the Maharashtra State Biodiversity Board (MSBB/Research/Desh-5/811/2021-22). Dr. Suresh Jagtap, Taxonomist, Associate Professor at BVDU-IRSHA, Pune, India identified and authenticated the plants. The voucher specimen was taken to the Herbaria of Medicinal Plants Conservation Centre in Pune (MPCC1526, MPCC2743). Tris-HCl buffer (60 mL, 0.9 units), N-succinyl phenyl-alanine-P-nitroanilide substrate, chymotrypsin, chymostatin, and omeprazole were supplied by Sigma Aldrich (St. Louis, MO, USA).

Extraction:

Dried fruits of Er and Et were crushed (1.0 kg) and extracted with ethanol using a soxhlet apparatus (Rotamantal, Remi) for 16-24 hours at 60-80ºC each⁸. In a rotary evaporator (IKA RV 10) at 45 °C, solvents were evaporated under decreased pressure, leaving a limited quantity of plant extract.

Animal care:

Animal concern and laboratory animal usage were conducted by the Committee for Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The Ethical Committee of PAVO Research Solutions, 78, Brushellz Industrial Park, Behind Toyota Showroom, Opposite GSFC, off NH 48 (old NH 8), Village, Gujarat, India gave their clearance (IAEC Approval Protocol Number: PAVO/IAEC/2021/02/008) dated on 17th April 2021. The rats obtained were housed in plastic cages and acclimatized for one week. They were housed at room temperature and given free access to clean water while being fed a conventional animal diet. Measurement of BWG, food, and water consumption were noted on a daily basis.

In vitro anti-ulcer assay:

The extracts were subjected to an in-vitro anti-ulcer assay utilizing chymostatin as the reference medication, as described by Saleem et al., 2016²⁴. All extracts and standard medicines were determined for their α-chymotrypsin inhibitory activity.

Extract preparation for in vivo anti-ulcer assay:

The dried-out extracts of ethanol were regenerated at a dose of 50 mg/kg body weight. Omeprazole was chosen as the study's anti-ulcer medicine of choice and the medicine was made available at Bharati Vidyapeeth University's medical store in Pune. The medication was prepared and given through the mouth at a dose of 10 mg per kilogram of body weight.

Study Plan:

After completion of acclimatization, all animals were randomized based on body weight. For the study, 42 adult Wistar rats with 141 and 222 g weights were randomly allocated into seven groups (G1-G7), each with five animals. Distilled water was granted to Group G1 (healthy control). Group G2 (ulcer control) was administered with 90% ethanol (5 mL kg_1). Group G3 (Omeprazole) was given Omeprazole (10 mg per kg body weight every day). Group G4 and G5 were given Er fruit extract 100 and 200 mg per kg body weight every day. Group G6 and G7 were given Et fruit extract 100 and 200 mg per kg body weight every day^25,26,27.

Ulcer induction:

After a 24-hour the test groups refrained from eating and ethanol was given orally. After 1 hour of ethanol (90%, 5 ml/kg) ingestion, gastric ulcers developed. Treatment of rats in the test groups was initiated after 24 hours and continued for 14 days²⁸. The methodology for the study plan, the sacrifice of animals, and the Total ulcer and mucosa area were explained in the Supplementary material.

Sacrifice of animals:

Isoflurane anesthesia was given to all the rats 24 h after the last administration. Stomachs were excised by midline incisions and saline washed. A high-resolution camera was used to obtain gross images of stomachs at a set distance of 10 cm from the tissue. The photos were then loaded into Image J software to be measured and ulcer parameters were determined.

Ulcer score:

A grid line was generated on the imported image to determine the ulcer score. The mean ulcer score for each group was then calculated. The user interface was calculated using the formula:

Ulcer index = [Ulcerated area] / [Total mucosa surface area] x100

The curative ratio was calculated using the formula:

Curative ratio = [UI control] x [UI treated] / [UI control]

Biochemical Analysis:

A portion of the stomach was thawed and used for antioxidant testing. Thawed tissues were homogenized in a 0.32M sucrose solution with 2% Triton X-100. For MDA, GSH, GPx, and CAT tests, the remaining stomach parts were homogenized in 50M potassium phosphate (pH 7.5) and 1M EDTA. Glucose, TG, total cholesterol, HDL cholesterol, and LDL cholesterol were all measured in serum. At 4°C, homogenized tissues were treated to sanitation for 30 seconds at a time. Thereafter, at 4°C, homogenized tissues were centrifuged for 10 minutes at 4000 rpm/min. Methods of the analysis were explained.

A) Measurement of Gastric Mucosal Reduced GSH:

GSH was measured using Bio diagnostic kits that were based on the Ellman method Siegers et al. 1988²⁹. The GSH concentration in the stomach mucosa was measured in the U/g of tissue.

B) Measurement of Gastric Mucosal Lipid Peroxide Measured as MDA:

Stomach mucosal MDA was quantified using the commercial diagnostic kit (Crest Biosystems, Goa, India). At an optical density of 535 nm, the color formed was measured. The MDA concentration in the stomach mucosa was measured in nmol/g of tissue.

C) Measurement of Gastric Mucosal GPx:

The activity of gastric mucosal GPx was evaluated by a commercial diagnostic kit (Crest Biosystems, Goa, India). The rate of NADPH oxidation at 340 nm was measured using H2O2 as the substrate to evaluate GPx activity. GPx activity was measured in U/g of tissue.

D) Measurement of Gastric Mucosal SOD:

SOD activity in the gastric mucosa was assessed using the commercial diagnostic kit (Crest Biosystems, Goa, India). This assay relies on SOD's capacity to prevent the nitro blue tetrazolium dye from being reduced by phenazine methosulphate. SOD activity was measured in U/mg tissue.

E) Measurement of Gastric Mucosal CAT:

The CAT activity was monitored using the commercial diagnostic kit (Crest Biosystems, Goa, India). The test involves reacting H2O2 with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to produce a colored chromophore with a wavelength of 510 nm. CAT activity was measured in U/g of tissue.

Histopathological analysis:

The histopathological investigation was performed on a section of the stomach tissue that had been fixed in 10% formol saline. For the histoarchitectural outline, slides were stained with H&E.

Statistical analysis:

Graph Pad Prism was used for statistical analysis. Body Weight, feed consumption, hematology, clinical biochemistry, etc. data were subjected to parametric ANOVA. Dunnett's test was performed to compare the test item treatment group to the control group if ANOVA revealed statistical significance (P<0.05).

RESULT:

In-vitro anti-ulcer assay (α-chymotrypsin assay)

The results outcomes revealed that non-significant change was observed in Er (87.2%) and Et (86%) fruit extracts at 200 mg/kg body weight and exhibited maximum in-vitro antiulcer activity, where chymostatin was comparable to standard. (figure 1). Et followed by Er extracts at 100 mg/kg body weight showed α-chymotrypsin inhibitory activity was also moderate to good.

Figure 1. Values are given as mean ± SEM for % inhibition of a-Chymotrypsin activity in each group.

(P ≤ 0.001, Turkey’s post hoc comparisons).

Effect of ‘Vidanga’ Extracts on Body Weight Gain (BWG) and Food and Water Consumption

The non-significant change was observed in percentage weight gain (p = 0.772), day-to-day food ingestion (p =0.123), and water intake (p =0.058) (Table 1) in rats when treated with Er and Et fruit extracts in comparison with the healthy group.

Table 1: Effect of Er and Et fruit extracts on % BWG and food and water consumption.

Treatment	% BWG	Food consumption (gm/rat/day)	Amount of water consumption (ml/day)
HC	19.16 ± 2.9	3.96 ± 2.1	10.84 ± 2.6
Er 100mg/kg	22.57 ± 4.56	3.98 ± 1.98	10.03±3.24
Er 200mg/kg	22.52 ± 5.02	3.98 ± 2.15	10.01 ± 4.2
Et 100mg/kg	22.36 ± 3.98	5.99 ± 3.3	11.07 ± 4.32
Et 200mg/kg	22.65 ± 4.32	7.09 ± 4.43	11.14 ± 3.04

HC: Healthy Control, Er 100: Embelia ribes 100 mg/kg, Er 200: Embelia ribes; 200 mg/kg, Et 100:

Embelia tsjerium-cottam;100 mg/kg, Et 200: Embelia tsjerium-cottam; 200 mg/ kg; Data are mean ± SEM (n=6)

Effect of ‘Vidanga’ extracts on Serum Glucose, TG, Total Cholesterol, HDL-Cholesterol, and LDL-Cholesterol:

As compared with the healthy control rats, treatment with Er and Et fruit extracts (200 mg/kg) for 14 days resulted in non-significant changes in serum glucose (P = 0.721), TG (P = 0.543), total cholesterol (P = 0.460), HDL-cholesterol (P = 0.387), and LDL-cholesterol (P = 0.411). (Table 2).

Effect of ‘Vidanga’ Extracts on the Severity of Gastric Lesion UI:

In rats treated with ethanol (5 ml/Kg bwt); a UI increase was observed in comparison to the control (𝑃 = 0.001) (Table 3). There was a decrease in UI with the treatment of omeprazole (10 mg/kg) or Er and Et fruit extracts in ethanol-injected rats viz. 90.04 %, 62.58 %, and 79.47 %, respectively, as compared to the ethanol-injected rats (𝑃 = 0.00) (Table 3). However, a non-significant curative ratio was observed after the treatment using lower concentrations of both extracts (100 mg/kg).

Table 2: Effect of Er and Et fruit extracts on serum glucose, TG, total cholesterol, HDL- cholesterol, and LDL cholesterol

Treatment	Serum Glucose	Triglycerides (TG)	Total Cholesterol	HDL-Cholesterol	LDL Cholesterol
HC	3.53 ± 1.11	0.35 ± 0.21	1.34 ± 0.31	1.37 ± 0.07	0.27±0.0
Ulcer 90% Ethanol (5 ml/Kg bwt	3.60 ± 0.12^#	0.35 ± 0.09^#	1.25 ± 0.10^#	1.33 ± 0.08^#	0.22±0.1^#
Omaprezole (10 mg/kg)	3.61 ± 0.09^*	0.33 ± 0.10^*	1.26 ± 0.07^**	1.30 ± 0.06^*	0.25±0.0^*
Er 100 mg/kg b.wt	3.65 ± 0.13^*	0.36 ± 0.06^*	1.29 ± 0.10^*	1.31 ± 0.09^*	0.26±0.01^*
Er 200 mg/kg b.wt	3.67 ± 0.05^**	0.33 ± 0.03^*	1.19 ± 0.11^*	1.30 ± 0.04^*	0.28±0.02^*
Et 100 mg/kg b.wt	3.66 ±0.13^*	0.34 ± 0.09^*	1.28 ± 0.10^*	1.31 ± 0.09^*	0.22±0.05^*
Et 200 mg/kg b.wt	3.70 ± 0.06^**	0.33 ± 0.04^*	1.18 ± 0.08^**	1.21 ± 0.13^*	0.24±0.02^*

The results are expressed as mean ± SD (n=6). *P < 0.05, **P < 0.01 compared to ulcer group and #P < 0.05 compared to healthy control

Table 3: Effect of Er and Et fruit extracts on the gastric lesion (UI)

Treatment regimen	Ulcerated Area (mm²)	Total Stomach Area (mm²⁾	Ulcer Index (mm²)	Curative ratio (%)
HC	0	0	0	0
Ulcer 90% Ethanol (5 ml/Kg bwt)	11±1.96^#	35±19.35^#	31.43±12.35^#	0.00
Omeprazole (10 mg/kg)	1±0.01^**	32±2.62^*	3.13±1.89^*	90.04
Er 100 mg/kg b.wt	4±0.99^*	33±1.79^*	12.12±2.21^*	61.43
Er 200 mg/kg b.wt	2±0.11^**	31±3.43^*	6.45±1.74^*	79.47
Et 100 mg/kg b.wt	7±1.07^*	35±10.02^**	20.00±1.01^*	36.36
Et 200 mg/kg b.wt	4±0.09^*	34±3.32^*	11.76±0.99^*	62.58

Each value is the mean ± SEM (𝑛 = 6), *P < 0.05, **P < 0.01 compared to the ulcer group, and #P < 0.05 compared to the healthy control.

Effect of Er and Et fruit extracts on Gastric Mucosal GPx, SOD, and CAT activities:

Table 4 shows the results of the enzymatic antioxidant analyses. In the varied treatment regimens, there was a nonsignificant change in CAT activity. When rats were given ethanol (ulcer), both stomach mucosal GPx (148.83 U/g tissue) and SOD (0.33 U/g tissue) activity were significantly lower than in healthy control rats (P = 0.000 and 0.015). Treatment with omeprazole, Er and Et fruit extracts significantly increased gastric mucosal GPx for both the concentrations (𝑃 = 0.035 and 0.005) and SOD enzyme activity for the concentrations (𝑃 = 0.010 and 0.012, resp.) (Table 4). On the other hand, highly increased activity among all the groups was observed in Omeprazole and Er (200 mg/kg).

DISCUSSION:

The most common method for testing anti-ulcerogenic medicines in rats is to use an ethanol-induced gastric ulcer model^30,31. Apart from this, ethanol ingestion increases the production of malondialdehyde and reduces glutathione production which induces oxidative stress by reducing gastric blood flow. However, this rat model parallels the etiology of ulcers in humans³², whereas ethanol-induced stomach ulcers are associated with oxidative stress and ROS³³. The current observations demonstrated the anti-ulcerative effect of fruit extracts of Er and Et preventing gastric ulcers caused by ethanol. To treat the mucosal injury, the effect of these extracts on oxidative stress indicators and antioxidant enzymatic activity was considerable and dose-dependent. Furthermore, the rat stomach mucosa's macroscopic appearances showed anti-ulcerogenic potential.

Chymotrypsin is a digestive proteolytic enzyme produced by the pancreas that is used in the small intestine and helps to digest proteins. The α-chymotrypsin enzyme speeds up the hydrolysis reaction that breaks down peptide bonds in the absence of a catalyst³⁴. However, plants are good sources of PIs and are now treated as vital signaling molecules in many biological activities such as apoptosis, inflammation, blood clotting, etc.³⁵. In the present study, Er (87.2%) and Et (86%) fruit extracts at 200 mg/kg body weight exhibit maximum in-vitro antiulcer activity as compared to the standard chymostatin. In the gastrointestinal tract, pro-inflammatory responses are induced through the proteases that are commonly found in ulcer manifestation and engage protease-activated receptors. Er, and Et have shown a non-significant variation to inhibit α-chymotrypsin activity. Similarly, Casearia sylvestris extract exhibits the anti-ulcerogenic effect of protease inhibition³⁶.

Our observation suggests that fourteen days of feeding of Er and Et fruit extracts did not alter % BWG (p = 0.772), daily food consumption (p =0.123) and water intake (p =0.058), serum glucose (𝑃 = 0.721), TG (𝑃 = 0.543), total cholesterol (𝑃 = 0.460), HDL-cholesterol (𝑃 = 0.387), and LDL-cholesterol (𝑃 = 0.411) compared with healthy control rat. Previously, several studies reported similar assessments for medicinal plants with anti-ulcerogenic activity^37,38.

Macroscopic and histopathological findings revealed that Er and Et fruit extracts have a strong anti-ulcerative effect against ethanol-induced stomach ulcers. The results from these groups suggest Er and Et fruit extracts at 200mg/kg concentration provide better remediation against ethanol-induced gastric ulcers. In those treated with Er and Et fruit extracts, total ulcer area, total ulcer score, and ulcer index were comparable to those treated with regular Omeprazole. However, Er and Et fruit extracts (200 mg/kg) exhibit the highest curative ratio (79.57% and 62.58% respectively), demonstrating comparable anti-ulcerogenic potential as Omeprazole (90.04%). The hydrochloric acid in ethanol weakens the stomach's protecting mucosal membrane³⁹. Gastric mucous protects the mucous membranes against internal discredit by chemicals and acids⁴⁰. Our results show that fruit extracts of Er and Et (200 mg/kg) caused intact gastric mucous as compared to the ethanol-treated rat. Ethanol treatment in rats resulted in ulcer formation with severe lesions and extensive apparent hemorrhagic necrosis of the gastric mucosa. While, when the gastric ulcer was treated with Er and Et fruit extracts at 200 mg/kg promoted cytoprotecting against mucosal injury.

Various diseases like peptic ulcers, gastric carcinoma, etc. are believed to be initiated owing to oxidative stress⁴¹. Disastrously, many times body’s immune defence neutralizing these free radicals is insufficient. Therefore, external sources of supply of natural products fulfill the requirements⁴². ‘Vidanga’ species have shown significant antioxidant activity⁸. As a by-product of regular cellular metabolic activities, living cells produce reactive oxygen species (ROS). While, excessive production of ROS, attacks proteins, leads to damage to DNA, and induces lipid peroxidation⁴³. Attenuation of gastric mucosal GSH promotes the accumulation of ROS which interns damage the cell membrane⁴⁴. In the present study, Er and Et fruit extracts caused a decrease in the malondialdehyde (MDA) level and preserved the stomach mucosal GSH.

Additionally, the antioxidant activity of the Er and Et fruit extracts may be attributed to their greater flavonoid contents⁸. Flavonoids have been shown to have antiulcerogenic effects in numerous investigations^45.46,47. SOD, CAT, and GPx, antioxidant enzymes found in stomach mucus, play an important role in the cellular defence mechanism. SOD catalyzes the disproportionation of O2- to H2O2 and provides cellular defence, which is then scavenged by CAT and GPx enzymes. The release of lipid hydroperoxides is associated with GPx activity as they play a vital role in cell protection⁴⁸. Ethanol treated group of rats decreases the SOD, CAT, and GPx activities in gastric mucosa, and elicits the accumulation of ROS which induces lipid peroxidation. Our study shows that fruit extracts of Er and Et maintain SOD, CAT, and GPx activities as compared to the healthy control. These observations confirm the findings of previous reports, and alterations in antioxidant enzyme activities in ethanol-exposed animals⁴⁹.

The findings suggest that Et demonstrates a comparable anti-ulcerogenic potential to Er, making it a viable alternative for the treatment of gastric ulcers. Given the endangered status of Er, utilizing Et as an accessible species with similar therapeutic potential could be a practical strategy for addressing various gastrointestinal illnesses.

CONCLUSION:

The current study demonstrated that fruit extracts from both Er and Et exhibit cytoprotective abilities comparable to those observed in the omeprazole-treated group of rats with ethanol-induced ulcers. This remedial effect can be attributed to the regulation of oxidative stress biomarkers and the activation of antioxidant enzymes, which scavenge reactive oxygen species (ROS) to protect mucosal cells. These findings underscore the ulcer-healing potential of Er and Et, indicating their closely similar efficacy. The study further investigated the anti-ulcerogenic properties of Er and Et, revealing non-significant variations in their activities. These results suggest that using Et alongside Er as a readily available and similar therapeutic agent for gastrointestinal disorders is a promising avenue for further exploration, potentially leading to the development of new natural therapeutics.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to the authorities of the Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University) for their overall support.

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Received on 22.11.2023 Modified on 20.03.2024

Research J. Pharm. and Tech 2024; 17(9):4543-4551.

DOI: 10.52711/0974-360X.2024.00702

Treatment regimen	Gastric Mucosal Glutathione Peroxidase Enzyme Activity (GPx) U/g tissue	Gastric Mucosal Superoxide Dismutase Enzyme Activity (SOD)U/g tissue	Gastric Mucosal Catalase Enzyme Activity (CAT) U/g tissue
HC	629.83±2.68	0.49±0.12	0.27±0.01
Ulcer 90% Ethanol (5 ml/Kg bwt	148.83±1.22^*	0.33±0.01^*	0.15±0.01*
Omaprezole (10 mg/kg)	1133.17±2.06^**	0.55±0.1^*	0.13±0.01**
Er 100 mg/kg b.wt	894.67±1.87^*	0.46±0.01^*	0.14±0.02^**
Er 200 mg/kg b.wt	1029.50±1.48^*	0.55±0.02^**	0.18±0.00^*
Et 100 mg/kg b.wt	887.17±2.23^*	0.47±0.01^*	0.15±0.01^**
Et 200 mg/kg b.wt	1025.17±2.65^*	0.56±0.01^*	0.18±0.01^*