Comparison Effect of an aqueous extract of Alpinia and Fenugreek seeds on Male Rats with Gastric Ulcer

This study aimed to investigate the effects of aqueous extracts of Fenugreek seeds (FSAE) and Alpinia (AAE) on ethanol-induced stomach ulcers in rats. For five weeks, thirty male rats weighing 150 -170 gram. Then, divided into two major groups. The first one included five rats were fed a basal diet as part of a normal control group (negative group). To induce stomach lesions, twenty-five rats in the second group were fasted overnight and subsequently administered a single oral dose of 5 ml per kg of ethanol. These groups were split into 5 subgroups, as follow: (G 2:positive control group, which was not given any treatment and was given the basal diet, G 3: Rats given the basal diet, besides 250 ml/kg/daily of FSAE orally, G 4: Rats given the basal diet, in addition to 500 ml/kg/daily of FSAE orally, G 5: Rats given the basal diet, in addition to 250 ml/kg/daily of AAE orally: and G 6: Rats given the basal diet, in addition to 500 ml/kg/daily of AAE orally. Histological examinations, chemical assays, and biochemical assessments were conducted on both serum and gastrointestinal mucosa samples. The results revealed that the overall antioxidant capacity of FSAE was notably greater than that of AAE. However, there were no observable differences in the overall levels of flavonoids and phenols between FSAE and AAE.When compared to the positive control group, serum levels of AST, ALT, creatinine, urea, and uric acid were significantly lower in all groups that received support from FSAE (250 and 500 ml/kg) and AAE (250 and 500 ml/kg). For G6 and G4, respectively, the best findings were obtained. The study found that groups with varying percentages of FSAE and AAE had higher serum levels of GSH, GPX, SOD, and PH, but lower levels of MDA and MPO.The G6 achieved the best outcomes. Various FSAE and AAE treatments significantly increased stomach mucosa GSH, GPX, SOD, and PH levels, while MDA and MPO levels decreased in all groups compared to the untreated group.Compared to the positive control group, all groups receiving diets supplemented with different proportions of FSAE and AAE exhibited significant enhancements in stomach histopathology. Among these, G6 demonstrated the most favorable outcomes. Consequently, this study recommends the utilization of Alpinia aqueous extract as a potent agent for gastric protection. It also suggests its therapeutic potential in managing gastrointestinal disorders linked with oxidative stress and inflammation, especially in the prevention and treatment of ethanol-induced gastric ulcers.

The application of Trigonella foenum seed produced an antiulcer effect.Omeprazole, a drug that blocks the proton pump and is used to treat digestive disorders like gastritis, gastric ulcers, duodenal ulceration, and gastroesophageal reflux disease, has effects similar to those of fenugreek seeds.In a rat model where ethanol was used to induce stomach ulcers, the gel portion and aqueous extract from fenugreek seeds have effects on mucosal glycoproteins, and ant secretory activity has a protective role against ulcers (Pandian et al., 2002).According to (Negm and Aboraya, 2023), the composition of A. officinarum powder (per 100 grams dried) consists of 2.23 grams of fat, 5.56 grams of protein, 3.24 grams of ash, 59.94 grams of carbohydrates, 11.76 grams of moisture, and 17.27 grams of fiber.Fenugreek seeds contain approximately 25% fiber, 23-26% proteins, 0.9% lipids, and 58% carbohydrates (Wani and Kumar, 2018).

Aim of the study
Therefore, the aim of this study was to assess the impact of an aqueous extract derived from Trigonella Fenugreek seeds and Alpinia officinarum Hance on male rats afflicted with gastric ulcers.

Materials:
-Dried Fenugreek seeds (Trigonella) and Alpinia (Alpinia officinarum) were obtained from the National Research Centre in Dokki, Cairo, Egypt.
-Starch and oil were bought from a neighbourhood market in Cairo, Egypt.
-Thirty albino male rats (Sprague Dawley Strain), weighing between 150 and 170 grams, were obtained from the Food Technology Research Institute in Giza.

Methods: Chemical analysis Quantifying the aggregate quantities of flavonoids, phenols, and antioxidants
The total flavonoids and phenols in the aqueous extracts of alpinia and fenugreek seeds were quantified following the methodologies described by (Ivanov et al., 2015) & (Liu et al., 2009).Preparation of Alpinia &Fenugreek seeds aqueous extracts 100 grams of dried alpinia and fenugreek seeds were boiled in distilled water for 15 minutes, then chilled and filtered in a sterile (vial.Nwabufo and Olusanya, 2017).

Biological Experiment
Feed cups were utilized for diet provision to reduce food wastage, while a glass tube that was placed through the cage's wire provided the rats with water.following the method outlined by Reeves et al., 1993.The components for the basal diet, totaling one hundred grams, were prepared.Animal investigations were conducted with the consent of the UResearch Animal Facility-Institutional Animal Care and Use Committee, Cairo University, under approval number URAF-E-4-23.
The components of the basal diet were formulated according to the (AIN technique, 1993), comprising choline chloride (0.20%), cellulose (5%), a vitamin mixture (1%), maize oil (10%), casein (12%), mineral mixture (4%), and starch (remaining quantity).After period of adaptation , the animals were divided into two main groups.The first one, termed the normal control , consisted of five rats fed the standard diet for five weeks.The second group of 25 rats received a single oral dose of 5 milliliters per kilogram of ethanol after an overnight fast to induce stomach lesions, as per the protocol outlined by (Tolulope et al., 2019).Subsequently, this group was further divided into five subgroups, each comprising five rats.Group 2: Positive control group, receiving only the basal diet without any additional treatment.Group 3: Rats fed the basal diet, supplemented with an oral dose of 250 ml/kg of Fenugreek seeds aqueous extract.Group 4: Rats fed the basal diet, supplemented with an oral dose of 500 ml/kg of Fenugreek seeds aqueous extract.Group 5: Rats fed the basal diet, supplemented with an oral dose of 250 ml/kg of Alpinia aqueous extract.Group 6: Rats fed the basal diet, supplemented with an oral dose of 500 ml/kg of Alpinia aqueous extract.Every week for the five weeks of the experiment, food consumption was noted and each rat's weight was recorded.Using the following formula, the body weight gain and food efficiency ratio (FER) were calculated in accordance with (Chapman et al., 1959): Rats were fasted for an entire night at the conclusion of the experiment before being slaughtered.After blood collection, the blood was centrifuged.For biochemical analysis of inflammatory measures and oxidative damage markers, serum was separated and kept at -20°c.For instance, ELISA kits (catalog NO.MBS2636626), (catalog NO.MBS744364), (catalog No.CSB-E12144r), (catalog NO.CSB-E08555r), (catalog NO.CSM-E08722r) were used to analyze malondialdehyde (MDA), glutathione peroxidase (GPX), glutathione (GSH), superoxide dismutase (SOD), myeloperoxidase (MPO) and PH, respectively.(Reitman & Frankel's, 1957) analysis of aspartate amino transferase (AST) and alanine amino transferase (ALT) was followed.Serum uric acid was measured using the methods described by (Fossati et al., 1980), creatinine by (Bartels & Bohmer, 1971) and urea by (Marsh et al., 1965).

Histopathological Examination
Each rat's stomach was removed, photographed (Table 1), and then subjected to a histopathological examination in accordance with (Bancroft et al., 2012).

Statistical analysis
The findings are presented as mean ± SD.According to (McClave & Benson, 1991), oneway analysis of variance, or "ANOVA," was used to statistically analyze the data.The aqueous extract from fenugreek seeds exhibits notably higher total antioxidant activity compared to that from alpinia officinarum, as demonstrated in Table 2 illustrates.Conversely, phenols and flavonoids in total were not significantly present between fenugreek seeds or alpinia aqueous extract.According to (Negm and Aboraya, 2023), the composition of alpinia officinarum aqueous extract includes 46.51± 0.40 GAE/g total phenols, 30.97± 1.85 QE/g total flavonoids, and 3.05± 0.26 DPPH% antioxidant activity.Conversely, fenugreek seed extract exhibits substantial antioxidant potential with total phenolic contents of 454.93 ± 3.57 mg GAE/g, total flavonoid contents (TFC) of 135.04 ± 2.12 µg/CE, and total antioxidant capacity (TAC) of 162.51 ± 3.81 per gram of dry plant extract, as highlighted by (Fatima et al., 2022).

Table (3): The impact of administering aqueous extracts of Alpinia officinarum Hance and Trigonella foenum graecum on nutritional parameters in rats with gastric ulcer
Mean values in the same row with distinct superscript letters indicate a significant difference P ≤ 0.05.
When examining the body weight gain, feed efficiency ratio, and feed intake of the control group (G) in comparison to normal rats, the data presented in Table (3) indicated a non-significant differences.Moreover, when comparing both control rats ("G"1 and 2) to the treatment groups (3, 4, 5, and 6), there was no significant difference observed in body weight gain, feed efficiency ratio, or feed intake.The results were validated by (Teixidor-Toneu et al., 2016).They found A. officinarum is combined with other herbs in a msahan mixture, which is used as traditional medicine in Morocco.To cure conditions like musculoskeletal diseases, gynecological conditions, and general health, the plant materials are pulverized, dried, and combined with meals.Similar to this, the Marrakech region of Morocco uses the roots to heal colds and promote weight gain.(Javaid et al., 2021) found that chronic administration of Alpinia led to reduction in weight gain and Lee index of rats compared to the obesogenic diet-fed rats.

Table (4): The impact of administering the aqueous extract of Alpinia officinarum Hance and Trigonella foenum graecum on liver and kidney function in rats with gastric ulcers
Mean values in the same row with distinct superscript letters indicate a significant difference P ≤ 0.05.Data presented in Tab. 4 revealed a notable elevation in serum levels of AST, ALT, creatinine (Cr), urea (UN), and uric acid (UA) among positive rats in comparison to normal control rats.Conversely, rats administered aqueous extracts containing 250 and 500 ml of fenugreek seeds, as well as 250 and 500 ml of Alpinia per kg, exhibited significantly reduced serum levels of AST, ALT, UN, UA, and Cr when contrasted with the positive control group.Our results agreed with (Kaviarasan and Anuradha, 2007) stated that It was discovered that fenugreek seed possesses hepatoprotective qualities.Fenugreek seed polyphenolic extract functions as a shield against ethanol-induced hepatic abnormalities.Additionally, (Kaviarasan and Anuradha, 2007) reported that fenugreek powder's ethanolic extract inhibited ethanol-induced toxicity in Chang liver cells, and that its protective activity is equivalent to that of the common hepatoprotective drug sylimarin.It was discovered that fenugreek polyphenolic extract prevented ethanol-induced liver damage by regulating hepatic injury indicators (ALT, AST, ALP, LDH, and GGT), boosting hepatocyte viability, and lowering apoptotic nuclei.Creatinine levels significantly decreased when fenugreek was taken orally.prior studies demonstrating enhanced kidney functioning following fenugreek supplementation and has a favorable impact on the elderly's hepatic enzyme regulation (Marine et al., 2020).In another study, confirmed that Alpinia officinarum improving liver function parameters, and suppressing oxidative stress, as well as modifying antioxidant defense mechanisms (Niazvand et al., 2023).Additionally, (Rajesh et al., 2013) revealed that, decrease AST, ALT levels when compared to model control group indicating that Alpinia Officinarum is effective in reducing abnormal AST, ALT levels.(Lin et al., 2018) found Rhizoma Alpiniae Officinarum extract, which has high contents of total phenolics and flavonoids, showed significant hypouricemic and renal protective effects on hyperuricemic rats, and reducing urea nitrogen and creatinine levels.(Palanirajan et al., 2022) indicated a reduction in urea, uric acid, and creatinine levels upon administration of Alpinia purpurata extract.Additionally, it was observed that serum marker enzymes including Protein, AST, ALT, ALP, and LDH were significantly elevated (p<0.05) in the prostate cancer-induced group.However, oral administration of Alpinia purpurata for 16 weeks to both induced and simultaneously treated groups markedly normalized these values.

Table (5): The impact of administering aqueous extracts of Alpinia officinarum Hance and Trigonella foenum graecum on gastric biochemical measures
Mean values in the same row with distinct superscript letters indicate a significant difference P ≤ 0.05.
The study assessed the impact of an aqueous extract from fenugreek seeds and an aqueous extract from alpinia on oxidative stress and gastric protection.Gastric biochemical markers, including GSH, GPX, SOD, MDA, MPO, and PH, were utilized to measure stomach acidity and identify oxidative stress.According to Table (5), Rats induced with ethanol displayed swift damage to their stomach mucosa and substantial changes in oxidative stress biochemical markers compared with control group.Damage to the stomach mucosa resulted in a notable decrease in GSH, GPX, SOD, and PH levels in the positive control group compared to the negative control group.However, rats administered aqueous extracts of fenugreek seeds and Alpinia exhibited a significant increase in PH, SOD, GPX, and GSH levels.Among these, the most promising outcomes were observed for G6 and G4, respectively.(Johnley et al., 2020) reported similar outcomes and noted that treatment with alpinia rhizomes extract showed a significant increase in SOD, CAT and GSH compared to the positive control group.Also (Singaravelu et al., 2018) found comparable outcomes and noted that, Compared to control positive group, fenugreek-treated group exhibited a substantial decrease in ulcer index, gastric juice volume, and acidity.An analysis of oxidative enzymes revealed a noteworthy rise in Catalase, SOD, and GSH levels in the treated group in contrast to the positive control group.Conversely, positive Control exhibited significantly higher MDA activity and MPO levels compared to negative Control.Conversely, rats administered Fenugreek seeds aqueous extract and Alpinia aqueous extract showed a notable reduction in MDA activity and MPO levels.The most notable outcomes were observed for G6.The findings of (Lin et al., 2021) provide support for these conclusions as they show that by enhancing SOD activity, an extract from the rhizomes of Alpinia officinarum can efficiently scavenge for oxygen free radicals created by ethanol.This, in turn, can increase hydrogen peroxide breakdown, inhibit lipid peroxidation to protect the stomach from oxidation, and avoid injury to the gastric mucosa.Also these conclusions are corroborated by the outcomes of (Elsayed and Hussein, 2019) who demonstrated that the fenugreek aqueous extract demonstrated a protective effect, which was demonstrated by a decrease in inflammation.After 0.54 6.94 b ± 0.27 6.03 c ± 0.29 6.85 b ± 0.16 5.94 c ± 0.17 4.52 d ± 0.52 7.58 a ± 0.28 PH Glutathione (GSH), Glutathione peroxidase (GPX), Superoxide dismutase (SOD), Malondialdehyde (MDA), Myeloperoxidase (MPO) & Potential hydrogen (PH) comparing the outcomes of several therapies, it was shown that fenugreek extract had the strongest inhibitory effect on MPO.The effects of Fenugreek seeds aqueous extract and Alpinia aqueous extract on inflammatory parameters and oxidative stress markers in serum were evaluated.Utilizing biochemical markers including GSH, GPX, SOD, MDA, MPO, and PH, we were able to measure blood acidity and identify oxidative stress.Table (6) displays these findings.In comparison to the negative control group, the ethanol treatment had lowerd the serum concentrations GSH, GPX, SOD& PH.However, rats fed aqueous extracts of fenugreek seeds and alpinia revealed a notable increase in GSH, GPX, SOD, and PH.The best results were for G6 and G4, respectively.Contrary, to the negative control group, the ethanol treatment raised MDA and MPO serum levels.Simultaneously, rats administered aqueous extracts of fenugreek seeds and Alpinia showed a considerable decrease in MDA and MPO when compared to G2, with G6 and G4 showing the best outcomes, respectively.These results are in agreement with (Darwish et al., 2020) that mention adding fenugreek seed powder to the diet causes rats' oxidative damage indicators to decrease.Also these results are in agreement with (Jin et al., 2019) who demonstrated that Alpinia conchigera significantly reduced ulcer index, total acidity and pH.furthermore, the extract improved of CAT and SOD relative to rats in respective control groups.These results may be due to Total flavonoids content of Alpinia Officinarum whereas (Lin et al., 2023) observed that Alpinia officinarum Hance's total flavonoid successfully reversed the ethanol-induced cell apoptosis (from 23± 1.3 to 8.11 ± 0.93%) and decreased the area of the stomach ulcer in rats (from 11.2 ± 1.89 to 2.19 ± 0.95).Additionally, it improved oxidative stress.

Histopathological Examinations Examination of stomach Histopathologically:
Histopathological examination of stomach of rats from group 1 (normal control) exhibited the normal histoarchitecture of gastric layers (Figs. 1).In adverse, The rats' stomachs of group 2 (untreated group) revealed histopathological damage characterized by gastric mucosal focal necrosis accompanied with edema of submucosa (Figs. 2).On the other hand, stomach of rats from group 3 (250 ml/kg of Fenugreek seeds aqueous extract) showed slight submucosal edema and focal shortening of the stomach mucosa (Fig. 3).Otherwise, stomach of group 4 rats (500 ml/kg of Fenugreek seeds aqueous extract) showed slightly submucosal edema (Fig. 4).Likewise sections from group 5 (250 ml/kg of Alpinia aqueous extract) demonstrated submucosal edema (Fig. 5).Furthermore, marked improved picture was noticed in stomach of rats from group 6 (500 ml/kg of Alpinia aqueous extract), examined sections exhibited no histopathological lesions (Figs. 6).The results are in agreement with (Qu, et al., 2021) who reported that Alpinia officinarum has been shown in vivo studies to be able to prevent ethanol-induced damage to the gastric mucosa of mice by reducing inflammation and reducing pain.Additionally, (Brinal Figer et al., 2017) revealed that, a positive connection was found between the extract dosages and the protective response, meaning that an increase in dose was accompanied by an increase in protection.It was discovered that the highest concentration of extract-1000 mg/kg-completely prevented ulcer ulcers.(Helmy., 2011) who found that a popular spice and condiment in Indian homes is fenugreek the high concentration of flavonoids, phenol, saponins, and amino acids in the plant seeds has been shown to have substantial medicinal values.Animal models have been used to demonstrate the antiulcer efficacy of aqueous and gel.(Jelodar et al., 2005) reported that Fenugreek has two active alkaloids: lysine and L-tryptophan.It has galactomannans and mucilage, which help to preserve the stomach mucosa.The stomach's parietal cells' ability to secrete hydrochloric acid is inhibited by fenugreek seeds.It lessens lipid peroxidation, which is a primary factor in stomach mucosal inflammation.Additionally, (Khan et al., 2018) study reported that primary active component of Alpinia officinarum is the flavonoid galangin.According to earlier research, the antiulcer effects of polysaccharides were primarily attained through one or more of the following pathways: binding to the mucosa surface to provide a protective coating, lowering the secretions of gastric acid and pepsin, preserving the mucus barrier, or lowering oxidative stress or inflammatory response of the gastric mucosa.

CONCLUSION
Both fenugreek seeds aqueous extract and Alpinia aqueous extract demonstrated notable antiulcer activity against ethanol-induced gastric ulcers, likely attributed to their antioxidant properties.Additional investigations is needed precise mechanism implicit the antiulcer effects for these extracts.

Figure
Figure.1: A microscopy image of rat ' s stomach from G1 showing the normal histoarchitecture of gastric layers (Hematoxylin & Eosin stain, X 100, scale bar 100µm).

Figure. 2 :
Figure.2: A microscopy image of rat ' s stomach from G 2 showing focal necrosis of the stomach mucosa accompanied with edema of the submucosa (Hematoxylin & Eosin stain, X 100, scale bar 100µm).

Figure. 3 :
Figure.3: A microscopy image of rat ' s stomach from G 3 showing focal shortening of gastric mucosa and slight edema of the submucosa (Hematoxylin & Eosin stain, X 100, scale bar 100µm).

Figure
Figure.4: A microscopy image of rat ' s stomach from G 4 showing slight edema of the submucosa (Hematoxylin & Eosin stain, X 100, scale bar 100µm).

ml) Fenugreek seeds aqueous extract 89.54 a ± 0.18 0.64 a ± 0.14 0.28 a ± 0.08
Mean values in the same row with distinct superscript letters indicate a significant difference P ≤ 0.05.

(Zhang et al., 2020) reported
food intake bettween the groups was not significantly different .

Table ( 6): The impact of administering an aqueous extract comprising Alpinia officinarum Hance and Trigonella foenum graecum on inflammatory indicators and oxidative stress markers in serum
Mean values in the same row with distinct superscript letters indicate a significant difference P ≤ 0.05.