INTRODUCTION:

Investigation and use of natural products for treatment are more efficient, safer, and economical compared to chemical drugs, according to the World Health Organization (WHO)¹. Among several countries, Indonesia is widely recognized for its traditional medicine, known as jamu, which has been used to treat various diseases and maintain health. A major ingredient in jamu is obtained from Curcuma genus², such as mango ginger (C. amada Roxb.) and black turmeric (C. aeruginosa Roxb).

Curcuma amada and C. aeruginosa contain various secondary metabolites, contributing to their broad pharmacological potential.

Important phenolic compounds reported from the rhizomes of C. amada and C. aeruginosa are curcuminoids, phenolics, and flavonoids. Curcumin compounds are well-known as antioxidant and anti-inflammatory agents, effectively treating various inflammation-related diseases^3,4.

Various pharmacological activities have been reported from C. amada, including antioxidant⁵, anti-hyperpigmentation⁶, anti-diabetic⁷, rheumatoid arthritis treatment⁸, and skin diseases⁹. Meanwhile, C. aeruginosa is a traditional remedy for diarrhea, rheumatism, fever, cough, and asthma¹⁰. Pharmacological research reports that C. aeruginosa has antioxidant^11,12, anti-inflammatory¹³, antiviral^14,15, and anticancer¹⁶ activities. Despite these numerous investigations, there have been no reports on the activities of C. amada and C. aeruginosa as anti-ulcer drugs. Curcuma genus reported for its anti-ulcer activity includes Curcuma longa Linn. and Curcuma purpurascens BI rhizomes^17,18.

Gastric ulcers are lesions that affect the mucous membrane of the stomach or duodenum. Peptic ulcers are generally caused by increased gastric acid levels that cause gastric mucosa disorders. Several causes of this disease have been reported, including smoking habits, stress, infection, and alcohol consumption. In addition, the administration of non steroids anti inflammatory drugs (NSAIDs) contributes to the cause of peptic ulcer disease. Many anti-ulcer agents are used, showing severe side effects on the human body. Therefore, research on gastric ulcer drugs with fewer side effects has increased significantly in recent years. This research aimed to evaluate the potential of C. amada and C. aeruginosa as herbal medicine for treating gastric ulcer.

MATERIALS AND METHODS:

Chemicals and Plant Material:

Chemicals such as absolute ethanol, NaOH, diethyl ether, and HCl purchased from Merck^®, Omeprazole sourced from Dexa Medica^®. Sucrose and alcian blue puchased from Sigma Aldrich^®, while C. amada and C. aeruginosa were obtained in Palembang, South Sumatra. Subsequently, plant vouchers of C. aeruginosa and C. amada were identified and stored in the Herbarium of Andalas University with voucher numbers 171/K-ID/ANDA/III/2023 and 172/K-ID/ANDA/III/2023, respectively.

Preparation of C. amada and C. aeruginosa Ethanol Extract:

The ethanol extract of C. amada and C. aeruginosa rhizomes were prepared using the maceration method. Initially, 500 g of C. amada and C. aeruginosa powder was macerated with 95% ethanol at a ratio of 1:10, three times. All macerates were evaporated using a rotary evaporator at 60°C until thick extract of C. amada (ERTM) and C. aeruginosa (ERTI) were obtained.

Determination of Total Flavonoid Content:

Total flavonoid content was determined using the AlCl₃ colorimetric method. Initially, 10mg of extract of ERTM and ERTI rhizome were dissolved in methanol to a volume of 10 mL. Subsequently, 3 mL of extract solution was added with 0.2 mL of 10% AlCl₃, 0.2 mL of 1 M Na-acetate, and filled with distilled water to 10 mL, followed by incubation for 30 minutes. Absorbance was measured using Uv-Vis spectrophotometry (Biobase^®) at λ 430 nm. The total flavonoid content was calculated from the quercetin standard curve and expressed as (mg QE/g) quercetin equivalent. The total flavonoid content was calculated from the quercetin standard curve and expressed as (mg QE/g) quercetin equivalent¹.

Determination of Total Phenolic Content:

Total phenolic content was determined using the Folin-Ciocalteu reagent based on the previous report. A total of 0.1 mL of extract solution was added to 7.9 mL of distilled water and 1.5 mL of Folin-Ciocalteu reagent. The test solution was vortexed for one minute, followed by the addition of 20% Na₂CO₃ was the volume reached 10 mL. The solution was incubated for one hour and the absorbance was measured using a UV-Vis spectrometer at λ 760 nm. The total phenolic content was calculated using a gallic acid calibration curve and expressed in milligrams of gallic acid equivalent per gram of extract (mg GAE/g)^1,19.

Determination of Total Curcumin Content:

The quantification of curcumin in the extract was measured by spectrometry using the curcumin standard. The calibration curve for the curcumin standard was obtained from the absorbance of curcumin solutions in the concentration range of 5, 10, 20, 30, 40, 50, and 60 ppm using a UV-Vis spectrometer at λ 424 nm. The results were expressed as milligrams of curcumin equivalent per gram of extract (mg CE/g)²⁰.

Acute Toxicity Test:

The acute toxicity test followed the OECD 423 protocol²¹.

Antiulcer Activity Test of Extract:

The testing procedure received approval from the Ethics Committee of Ahmad Dahlan University with No. 0222211083. The anti-ulcer effect test of ERTM and ERTI was carried out following the procedure reported previously²². A total of 45 male Wistar rats (BW 180-200 g) were acclimatized in the laboratory environment for one week. Furthermore, all rats were randomly divided into nine groups, consisting of control, ERTM, and ERTI tests. The normal control and positive control groups were given 0.5% NaCMC and 20 mg/kg BW omeprazole, respectively. The negative control group consisted of animals that did not receive any pre-treatment but were induced with absolute ethanol. Meanwhile, the test groups were given pre-treatment with ERTM and ERTI at doses of 100, 200, and 400 mg/kg body weight, respectively. After pre-treatment for 14 days, rats were fasted for 24 hours. The next day, rats were induced to develop ulcer by administering 1 mL/200 mg of absolute ethanol orally, except for the normal group. Subsequently, rats were left undisturbed for ±2hours to maximize ethanol's effect on the stomach. This was followed by anesthetization with diethyl ether and dissection to collect the stomach organs for observations of the protective effects of extract. These observations included measuring the area of the mucosal lesions, and gastric biochemical parameters such as pH, volume, total gastric acidity, and mucin levels, with histopathological examination of the stomach.

Measurement of Gastric Mucosal Lesion:

The gastric ulcer was photographed with a digital camera and the lesion area was measured using Image J software. The severity of the lesion was assessed based on the redness of the lesion area in gastric mucosa²³. The Ulcer index (UI) and the percentage of ulcer inhibition for each animal were determined following the reported methods^24–26. Ulcer Index (UI) = [Average number of ulcer + Average ulcer score + Percentage of animals with ulcer]:10.

Ulcer area (ethanol) – ulcer area (test)

% Ulcer Inhibition = ----------------------------------- × 100

Ulcer area (ethanol)

Measurement of Gastric Biochemical Parameters:

The physicochemical properties of gastric fluid, including volume, pH, and acidity, were easured using previously reported procedures^22,27. Gastric wall mucus content, namely mucin, was measured using spectrometry, based on the formation of gastric mucus complex with Alcian Blue Stain (AB). Following the literature, mucin was complexed with 10 mL of 0.1% AB. Subsequently, the mucin-AB complex was extracted using 0.5 M MgCl₂ and emulsified with diethyl ether. The resulting emulsion was centrifuged at 3600 rpm for 10 minutes and the absorbance mucin-AB complex in the aquaeus layer was read at a wavelength of 580 nm. Mucin content was expressed as µg AB/g tissue.

Gastric Histopathological Observations:

The preparation of test specimens was carried out using a standard method. Initially, the gastric organs were treated with 10% formalin, dehydrated with a series of alcohol concentrations, and processed using routine procedures. The specimens were stained with hematoxylin and eosin and observed using a digital microscope. Subsequently, further analysis was performed on photomicrographs of the tissue²⁸.

Data Analysis:

In this research, the data obtained were processed using SPSS version 25. Analysis started with testing for normality using the Shapiro-Wilk test, followed by a homogeneity test with Levene's statistic. Subsequently, a one-way ANOVA was conducted with a 95% confidence level (p<0.05). Differences between groups were analyzed using the post-hoc Tukey test with a significance level of p<0.05.

RESULTS:

Flavonoid, Curcumin, and Phenolic Contents of Total Extract:

The results of the measurements of flavonoid, curcumin, and total phenolic contents in the extract of ERTM and ERTI rhizomes are presented in (Table 1).

Table 1. Flavonoid, curcumin and phenolic contents of ERTM and ERTI

Extract	Flavonoid (mgQE/g)	Phenolic (mgQE/g)	Curcumin (mgCE/g)
ERTM	66.62	95.59	20.15
ERTI	43.10	62.30	8.82

Acute Toxicity of ERTM and ERTI:

Preliminary tests at graded doses of 300, 2000, and 5000 mg/kg BW showed no toxic symptoms in rats, thereby the main test was conducted at a dose of 5000mg/kg BW. The results showed no toxic symptoms, weight loss, or death in rats given ERTM and ERTI at a dose of 5000mg/kg BW, therefore the LD₅₀ was more than 5000mg/kg BW. The results of the acute toxicity test are shown in (Table 2).

Table 2. The Results of Acute Toxicity Test of C. amada (ERTM) and C. aeruginosa (ERTI) Extracts

Groups	Mortality	Toxic Symtomps	Body Weight of Rats (g)
Groups	Mortality	Toxic Symtomps	Day-0	Day-14
Normal Control	0	None	212.33 ± 1.01	215.66 ± 1.57
ERTM 5000 mg/kg	0	None	217.74 ± 13.69	220.29 ± 12.21
ERTI 5000 mg/kg	0	None	197.15 ± 3.30	200.43 ± 12.81

Antiulcer Effect of Extract:

The severity of ulcer was observed macroscopically, as shown in (Figure 1). This observation was followed by quantification using UI, while the prevention effectiveness was quantified by the percentage of inhibition, as presented in (Table 3).

Figure 1. The effect of Curcuma amada (ERTM) and C. aeruginosa (ERTI) ethanol extract on rats with gastric ulcers caused by absolute ethanol.

Table 3. The Effect of ERTM and ERTI on ulcus index and percentage of inhibition (n=5)

Groups	Ulcus Index ± SD	Percent Inhibition (%)
Normal	0.00 ± 0.00^bc	100
Ethanol	11.58 ± 0.06^ab	0
Omeprazole	6.98 ± 0.09^ac	39.74
ERTM 100	11.30 ± 0.11^ab	2.38
ERTM 200	10.69 ± 0.08^abc	7.65
ERTM 400	7.01 ± 0.11^ac	39.40
ERTI 100	11.26 ± 0.07^ab	2.49
ERTI 200	10.60 ± 0.01^abc	8.21
ERTI 400	6.99 ± 0.09^ac	39.41

Description: ^a,b,ceach state significantly different compared to the normal, omeprazole and ethanol groups (p<0.05).

The group induced with ethanol showed the most severe ulceration, characterized by redness of gastric, ulcer spots, and hemorrhagic stress. Ulceration decreased progressively in the groups receiving extract pre-treatment. As shown in Table 3, increasing doses improved gastric protection in the treated groups. ERTM and ERTI at a dose of 100mg/kg BW did not provide a significant protective effect compared to the negative control group (p>0.05). Meanwhile, at a dose of 400 mg/kg BW, there was a protective effect similar to the omeprazole group (p>0.05). These results confirmed that rats not receiving extract treatment lacked defense against ethanol-induced ulceration.

Effect of Extract on Gastric Fluid Biochemistry:

The effect of gastric protection on gastric biochemical parameters is shown in (Figure 2). Ethanol induction increased gastric fluid volume in the negative control group compared to the normal group (p<0.05) (Figure 2). The groups receiving pre-treatment with ERTM and ERTI showed a significant decrease in gastric fluid production, particularly at 200 and 400mg/kg BW doses, which did not differ from the omeprazole group (p>0.05). The pH of gastric fluid in the negative control group was the lowest compared to the others, with values different from the normal and positive control groups (p<0.05). However, the pH of gastric fluid in the 400mg/kg BW dose group was approximately the same as the normal and positive control groups (p<0.05). The decrease in total acidity values in the treatment groups was consistent with changes in gastric fluid volume, with low total acidity showing effective inhibition of gastric acid secretion. The negative control group showed the highest total acidity, significantly different from both the normal and omeprazole groups (p<0.05). Meanwhile, the test groups showed a linear decrease in total acidity values with increasing doses. The 400 mg/kg BW dose group had total acidity values identical to the normal and omeprazole groups (p>0.05).

Figure 2. Effect of ERTM and ERTI pre-treatment on changes in gastric biochemical parameters. Data are shown as mean±SD (n=5), where ^a,b,cindicate that the differences between the normal, omeprazole and ethanol groups are significant (p<0.05).

Mucin production is one of the main mechanisms of gastric defense. In this research, the untreated rats group showed the lowest mucin levels, significantly different from the normal group (p<0.05).

The omeprazole-treated group showed the highest mucin levels, even compared to the normal group (p<0.05). The ERTM treatment groups at doses of 200 and 400 mg/kg BW showed mucin levels comparable to both the normal and omeprazole control groups.

Gastric Histopathology:

Microscopic analysis showed that the cells in the mucosal layer of the normal group appeared intact, with regular glandular structures and closely arranged epithelial cells. However, the negative control group showed severe structural disintegration of the surface epithelium with necrotic lesions penetrating the submucosal layer. The positive control group, showed protection characterized by reduced mucosal damage, fewer necrotic lesions, and mild disturbances on the mucosal epithelial surface. The 100 mg/kg BW dose group showed significant mucosal damage and numerous necrotic lesions, with a damage reduction observed at the 200 mg/kg BW dose. The 400 mg/kg BW dose group showed the best protection, indicated by a histological profile similar to the omeprazole control, with minimal necrotic lesion and mucosal damage, as presented in (Figure 3).

Figure 3. Microscopic photo of stomach tissue in experimental rats. (H & E staining, Magnification 400x).

DISCUSSION:

Phenolic and flavonoid are secondary metabolites found in many types of vegetables and medicinal plants. Moreover, curcuminoids are the main compounds found in the Curcuma genus. In addition to providing colour and flavour to plants, phenolic, flavonoid, and curcumin play an important role as natural antioxidant; therefore, plants containing these compounds have the potential to be used as medicinal plant²⁹. The quantitative analysis showed that ERTM and ERTI contained flavonoid, phenolic, and curcumin metabolites (Table 1); the phenolic content of C. amanda found was relatively the same as the literature³⁰. The results of this study are consistent with other reports, showing that the phenolic content of C. aeruginosa is higher than the flavonoid content³¹. Phenolic and flavonoid compounds have been well-known to have antioxidant and anti-inflammatory activities, which are the basis for evaluating the anti-ulcer effects of ERTM and ERTI. Furthermore, acute toxicity tests were conducted to determine the exposure range where death is expected to occur³². The results of the acute toxicity test showed LD₅₀ > 5000 mg/kg BW (Table 2), so the extracts were included in the practically non-toxic category²¹.

Ethanol is a classic inducer used for gastric ulceration due to its ability to penetrate gastric mucosa, triggering oxidative stress, and inflammatory signaling pathways that lead to gastric epithelial damage^13,33. Ethanol damages the mucosa, beginning with microvascular injury and progressing to increased vascular permeability, edema, and epithelial lifting³⁴. Some studies used NSAIDs such as indomethacin as peptic ulcer inducers^1,24. However, in this study, we used absolute ethanol with several considerations: Ethanol-induced injuries are acute, causing erosion, bleeding, and perforation in a short period³⁵. In addition, ethanol takes less time to cause mucosal damage than indometacin^1,24. Ethanol causes gastric ulcers by increasing pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8³⁶. This research showed that ethanol significantly induced ulceration, characterized by redness of the gastric with clear hemorrhagic lesion and visible ulcer spots, as shown in (Figure 1). The ethanol group has the highest UI and the lowest percentage of inhibition, as presented in (Table 3) . The severity of ulceration was supported by increased volume and total acidity of gastric fluid, along with a decrease in pH and gastric mucus content. However, rats pre-treated with ERTM and ERTI showed increased gastric mucosal defense factors, as evidenced by a reduction in UI and an increase in the percentage of inhibition. The 400 mg/kg BW dose group showed protective effects comparable to the normal and omeprazole groups (p>0.05) (Figure 2).

The antiulcer activity of the Curcuma genus has been previously reported, namely C. longa and C. pupurascens. The antiulcer activity shown by ERTM and ERTI was lower when compared to the activity of C. purpurascens ethanol extract¹⁸ but better than C. longa¹⁷. Functional and morphological changes in the gastric caused by ethanol were partly due to increased gastric acid secretion. Excessive gastric acid secretion can cause erosion of the mucosa which is the protective layer of the stomach, resulting in hemorrhagic and necrotic lesions in the stomach. Therefore, reducing gastric acid secretion has been reported as an effective method to protect the stomach from alcohol-induced injury³⁷. This research showed that ERTM and ERTI could suppress gastric acid secretion in line with increasing doses. At a dose of 400 mg/kg BW, the pH and total acidity of gastric fluid in the test rats were comparable to the group given omeprazole (Figure 2).

The pathophysiology of ethanol-induced gastric ulcer included various factors, such as disruption of gastric mucin formation³⁸. The hemorrhagic and necrotic lesion caused by ethanol were associated with decreased in gastric wall mucus (mucin) secretion³⁹. Mucin is the main barrier to gastric defense against ethanol-induced erosion. Therefore, quantifying mucin levels is important for evaluating the mechanisms of defense effects produced by the test compounds⁴⁰. This study showed that ERTM and ERTI could increase mucin levels comparable to omeprazole (Figure 2). The increase in mucin levels led to a good defense effect against ulceration, as evidenced by gastric profile of the 400 mg/kg BW dose group (Figure 1); showing that ERTM and ERTI provided gastroprotective effects by increasing mucin level.

Gastric mucosal damage caused by ethanol is associated with oxidative stress. Generally, ethanol metabolism induces the formation of reactive oxygen species (ROS), which cause endothelial damage to gastric blood vessels, microcirculation disorders, and ischemia²⁸. The antioxidant activity that allows effective ROS scavenging³⁷, plays a crucial role in producing gastroprotective effects. The previous reports have shown that ERTM can inhibit DPPH and NO radicals⁴⁰, increase the activity of endogenous antioxidants such as catalase and superoxide dismutase (SOD)⁴¹, and reduce MDA levels in rats serum^10,42. Similarly, ERTI has been reported to scavenge DPPH and ABTS radicals⁷. The antioxidant activity of ERTM and ERTI comes from curcumin, which can scavenge free radicals and enhance the cytoprotective action of endothelial cells experiencing oxidative stress⁴³. Furthermore, C. aeruginosa has been reported to contain the flavonoid naringenin, which is capable of increasing the activity of antioxidants such as SOD, catalase, and glutathione (GSH)^2,11. Naringenin has been shown to suppress the activity of nuclear factor-κB (NF-κB), pro-inflammatory cytokine such as TNF-α, IL-6, and IL-8, and reduce the levels of nitric oxide (NO), malondialdehyde (MDA) in ethanol-induced animal models. In addition, naringenin can inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins through suppression of NF-κB and mitogen-activated protein kinase (MAPK) signals in ethanol-stimulated gastric epithelial cells³⁶.

Inflammation is a primary mechanism underlying gastric lesions³. It occurs through the upregulation of TNF-α and other pro-inflammatory factors, exacerbating ulcer⁴⁰. Ethanol consumption has been reported to upregulate TNF-α and IL-1β while downregulating the anti inflammatory cytokine IL-10. On the other hand, curcumin, a major component in ERTM and ERTI, can inhibit the production of pro-inflammatory cytokines both in vitro and in vivo⁴⁴, making it is an effective and safe natural anti inflamatory agent³⁸. In addition, curcumin can reduce inflammation by regulating downstream nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), and Activator Protein-1 (AP-1)^2,8. The ethanol extract of C. Aeruginosa, with its curcumin content, shows anti-inflammatory activity by inhibiting the production of IL-6 and TNF-α, and phagocytic activity⁴⁵ as well as C. amada⁴⁶. This study demonstrates that ERTM and ERTI are capable of preventing inflammation in ethanol-induced gastric rats due to the phenolic, flavonoid, and curcumin content of the extracts. The mechanism of anti-ulcer activity is by reducing gastric acid secretion and increasing mucosal production, thereby preventing gastric injury caused by absolute ethanol.

Photomicroscopic of the ethanol group show ulceration characterized by the damaged mucosal layer forming necrotic wounds extending to a relatively wide muscular mucosae layer (Figure 3). Absolute ethanol causes an increase in neutrophil infiltration in the early process of ulcer formation. Generally, ethanol is capable of releasing vasoactive molecules in the gastric, reducing blood flow, and increasing gastric acid secretion, leading to gastric mucosal damage and injury⁴⁷. Ethanol’s capacity to increase the regulation of pro-inflammatory cytokines, such as TNF-α and IL-1β and reduce the regulation of anti-inflammatory cytokine IL-10, worsens tissue damage⁴⁸. This study shows that ERTM and ERTI enhance gastric protection and reduce neutrophil infiltration into gastric tissue, thereby increasing gastric defence from erosion caused by ethanol. The mechanism of gastric protection by ERTM and ERTI occurs through reducing oxidative stress and inflammation. Based on observations of biochemical and histopathological parameters, the protective ability of ERTM is slightly superior to ERTI. This capability is related to the higher levels of flavonoid, phenolic, and curcumin content in the C. amada extract compared to C. aeruginosa.

CONCLUSION:

The finding of this study indicated that ethanol extract of C. amada and C. aeruginosa enhanced gastric defense against ethanol-induced ulceration. The protective effect against ulcer occurred by reducing acid secretion and increasing mucosal levels, as well as improving the structure of gastric epithelial layer. The extract of C. amada and C. aeruginosa were non-toxic in acute toxicity study and could be used as herbal medicine for gastric ulcer.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

ACKNOWLEDGEMENT:

Thank you to the head of the Pharmacology Laboratory of the Department of Pharmacy, Sriwijaya University, and the head of the Barokah Palembang Clinical Laboratory.

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Received on 11.03.2025 Revised on 13.08.2025

Accepted on 10.11.2025 Published on 16.03.2026

Available online from March 18, 2026

Research J. Pharmacy and Technology. 2026;19(3):1130-1136.

DOI: 10.52711/0974-360X.2026.00160

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