In Vitro and In Vivo Evaluation of the Antidiabetic, Antioxidant, and Antibacterial Activities of the Hydroethanolic Extract of Dysphania ambrosioides L.

ABSTRACT

Dysphania ambrosioides L. (Amaranthaceae) is a medicinal plant widely used in traditional Moroccan medicine for the management of diabetes‐related disorders. This study investigated the antidiabetic, antioxidant, and antibacterial activities of its hydroethanolic extract. Antidiabetic potential was evaluated through pancreatic α‐amylase inhibition (in vitro and in vivo) and intestinal glucose absorption (in situ). Antioxidant and antibacterial activities were assessed using standard chemical assays and selected bacterial strains. The extract showed significant inhibition of pancreatic α‐amylase in vitro (IC₅₀ = 1.22 ± 0.18 mg/mL). In vivo, it markedly reduced starch‐induced hyperglycemia in both normal and alloxan‐induced diabetic rats (p < 0.001) and decreased intestinal glucose absorption in situ (p < 0.05). It also exhibited broad‐spectrum antibacterial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, with MIC values ranging from 0.07 to 0.625 mg/mL and MBC values from 0.312 to 1.25 mg/mL. Moderate antioxidant activity was observed using the ABTS assay (IC₅₀ = 293.98 ± 3.11 mg/mL), along with a high total antioxidant capacity. These results demonstrate that Dysphania ambrosioides exerts antidiabetic effects through inhibition of carbohydrate digestion and glucose absorption, alongside antioxidant and antibacterial activities. This study provides scientific evidence supporting its traditional use and highlights its potential as a natural source of bioactive compounds.

Hydroethanolic extract of Dysphania ambrosioides exhibits notable antidiabetic, antioxidant, and antibacterial activities. It inhibits pancreatic α‐amylase in vitro and reduces postprandial hyperglycemia in vivo, while also decreasing intestinal glucose absorption. The extract shows broad‐spectrum antibacterial effects and moderate antioxidant capacity, supporting its traditional Moroccan use and highlighting its potential as a source of bioactive compounds.

1. Introduction

Dysphania ambrosioides (L.) is a member of the Amaranthaceae family and is referred to as “M'Khinza” in Morocco. Ethnobotanical research suggests that Dysphania ambrosioides has been traditionally employed by local Moroccans for treating a variety of ailments, such as rheumatism, fever, female infertility, and erectile dysfunction [1]. This plant exhibits antibacterial effects [2], antioxidant properties [3, 4], anti‐inflammatory properties [5, 6, 7], myorelaxant and antispasmodic properties [8], according to pharmacological research. Additionally, this herb is employed as a vermifuge to treat gastrointestinal conditions and to benefit expectant mothers.

Dysphania ambrosioides has attracted increasing pharmacological interest due to its richness in biologically active secondary metabolites. Previous studies have shown that this species contains a wide diversity of phytochemical compounds, including saponins, alkaloids, tannins, flavonoids, and phenolic acids, which may explain the numerous biological activities attributed to it [4, 9]. Furthermore, several recent studies have investigated the polyphenolic composition of Dysphania ambrosioides, highlighting the presence of phenolic acids and flavonoids such as gallic acid, caffeic acid, ferulic acid, quercetin, rutin, and kaempferol [10, 11]. These compounds are widely recognized for their ability to neutralize reactive oxygen species, reduce oxidative stress, and protect biomolecules against oxidative damage. In addition, the polyphenols may contribute to promising antidiabetic effects through several mechanisms, including the inhibition of digestive enzymes α‐amylase and α‐glucosidase, improvement of insulin sensitivity, as well as reduction of oxidative stress associated with hyperglycemia [12, 13].

According to a research study, this species is utilized in flok medicine for the management of diabetes [14, 15, 16]. Diabetes mellitus is a complex metabolic ailment characterized by chronic hyperglycemia arising from inadequate insulin secretion, insulin resistance, or both, leading to aberrant glucose, protein, and lipid metabolism [13]. Indeed, this disorder hastens the degradation of the vascular system, culminating in extremely severe effects such as micro‐ and macro‐angiopathy. Diabetes encompasses a multitude of deleterious sequelae that can culminate in serious morbidity and mortality. These include but are not limited to angina pectoris, acute myocardial infarction, cerebrovascular accident, cardiac complications (coronary artery disease), arterial plaque buildup (atherosclerosis), and neuropathic manifestations [15]. To treat high blood glucose levels, a variety of synthetic oral hypoglycemic medications, such as sulfonylureas, biguanides, and alpha‐glucosidase inhibitors, are employed. Nonetheless, prolonged use has been associated with side effects such as hypoglycemia, headache, dizziness, nausea, and weight gain [17]. Therefore, the search for more effective and safer antidiabetic therapeutic alternatives for diabetes management has become a major goal in recent research [17, 18]. Between 2012 and 2030, the number of diabetics is predicted to increase by about 180 million, a stunning 48% increase over 2012, resulting in 2.7% annual growth, more than double the world adult population's annual growth rate [19]. According to recent national and international health reports, including those of the International Diabetes Federation (2021) and the Ministry of Health of Morocco (2022), diabetes affects approximately 1.5 million people in Morocco [14]. In Eastern Morocco, the prevalence of this disease has been estimated at 10.2%, which may be associated with unhealthy lifestyle habits, including high sugar consumption, poor dietary patterns, and insufficient physical activity [20]. Moreover, this botanical species has the capacity to exert influence in the combat against pathogenic microorganisms and reactive oxygen species, stemming from both endogenous and exogenous sources, such as ultraviolet radiation and tobacco consumption [14]. The propagation of these radicals poses a harmful threat to biological systems due to their capacity to extract electrons from diverse cellular locations, such as the plasma membrane and even DNA itself [21].

Although Dysphania ambrosioides has been traditionally used in Moroccan folk medicine for the management of several ailments, particularly those related to diabetes, its underlying mechanisms of action remain insufficiently explored, especially regarding the combined evaluation of in vitro and in vivo antidiabetic effects alongside its antioxidant and antibacterial properties. The main objective of this study is therefore to evaluate the antidiabetic potential of the hydroethanolic extract of Dysphania ambrosioides by investigating its effects on pancreatic α‐amylase inhibition (in vitro and in vivo) and intestinal glucose absorption. Furthermore, the study aims to characterize its antioxidant and antibacterial activities.

2. Materials and Methods

2.1. Plant Identification and Extraction

The plant material (Figure 1) was collected in «Guercif» (Eastern Morocco) in March 2022 (34°14' N, 3°22' W). The taxonomic classification of the plant was ascertained by Pr. Mostafa Elachouri, a faculty member of the Mohammed First University's Faculty of Sciences in Oujda, Morocco. A voucher specimen has been deposited in the Herbarium of Mohammed I University, Oujda, Morocco, under the identifier HUMPOM44. After dehydrating the botanical sample at a temperature of 25°C, a total of 450 g of floral material underwent maceration using 70% ethanol concentration. This process was conducted with continuous agitation for 48 h, under ambient conditions. Subsequently, the hydroethanolic extract of Dysphania ambrosioides (DAHE) was subjected to drying at 40°C, yielding a dehydrated extract.

FIGURE 1.

Photograph of Dysphania ambrosioides (May 2022).

2.2. Antihyperglycemic Effect of DAHE Extract

2.2.1. Experimental Animals

The experiments were conducted using 18 Wistar rats (200–300 g) and 24 mice (20–32 g). All animals were housed under standard laboratory conditions in air‐conditioned rooms with controlled lighting cycles (12 h light/12 h dark), with free access to food and water throughout the experimental period. The food was removed 24 h before the experiment. The animals received adequate care in accordance with the Declaration of Helsinki guidelines 1975. The animals underwent a 24‐h fasting period prior to the commencement of the experiment. Comprehensive steps were taken to ensure the animals' welfare, adhering to internationally recognized guidelines for the care and utilization of laboratory animals, as published by the United States National Institutes of Health 2012 (NIH Publication No. 85‐23, revised 1985), National Research Council (NRC, 2011). In addition, the experimental protocol was approved by the ethics committee of Sidi Mohamed Ben Abdellah University in Fez. This research, identified by the code USMBA‐SNAMOPEQ 2024‐10. where the study adhered to the minimum statistically significant number of rats required, and multiple tests are conducted on the same animal.

2.2.2. Diabetes Induction

Healthy mice were fasted for 14 h and were rendered diabetic by intraperitoneally. (i.p) injection of a diabetogenic substance which is Alloxan (Allx) at a concentration of (100 mg/kg) and was solubilized in phosphate buffer (pH = 4.5; Na₂HPO₄, 0.1 M; C₆H₈O₇, 0.1 M). Subsequent to administering Allx injection, animals were provided unrestricted access to feed until the seventh day, when the levels of fasting blood glucose in mice were assessed. Mice exhibiting blood glucose levels equal to or exceeding 1.26 g/L were deemed to have diabetes and were included in the study

2.2.3. In Vitro α‐Amylase Inhibitory Activity

The activity of amylase inhibition of DAHE was determined by spectrophotometric assay employing 3,5‐dinitrosalicylic acid (DNSA) [22]. The extract was tested at various concentrations of 0.45, 0.91, 1.82, 3.64, and 7.27. 200 µL of the extract was mixed with 200 µL α‐amylase and incubated at 37°C for 10 min. Afterward, we added 200 µL of the 1% starch solution to the reaction mixture, followed by re‐incubating the mixture for 15 min at 37°C. The reaction was terminated by adding 600 µL of DNSA (containing 30 g of KNaC₄H₄O₆·4H₂O in 20 mL of 2 M NaOH and 40 mL of 96 mM of 3,5‐Dinitrosalicylic acid). The resulting solution was then heated for 8 min in a boiling water bath and brought to room temperature. All measurements were performed in triplicate. The absorbance was measured at 540 nm. The percent inhibition of the activity of amylase inhibition was computed utilizing the subsequent formula:

$$\%\text{Inhibition}=\left[(\text{OD Control}-\text{OD treatment}/\text{OD control}\right)]\times 100$$

2.2.4. In Vivo α‐Amylase Inhibitory Activity

An experimental approach was adopted in order to confirm the inhibitory impact of DAHE on α‐amylase in vivo, as described by [23]. Fasted healthy and Allx‐diabetic the mice were categorized into four groups, (n = 6; male/female = 1). Distilled water (10 mL/kg) was given to Group 1. DAHE was administered to groups 2 and 3 at doses of 125 and 250 mg/kg, respectively. Acarbose was given to Group 4 at a dose of 10 mg/kg. The oral starch tolerance test was executed in the following manner: the test substance (distilled water, DAHE, or acarbose) was given at t = 0 min. Glycemia was evaluated after 30 min, and the rats were given 3 g/kg of starch. Following that, blood glucose variations were recorded at 30, 60, and 120 min.

2.2.5. In Situ, Intestinal Glucose Absorption

In 36‐h‐fasted normal rats that had been given an intramuscular dose of pentobarbital (50 mg/kg) to put them to sleep, the effect of DAHE on intestinal absorption of glucose was examined using an intestinal perfusion technique [24].DAHE (125 mg/kg) suspended in perfusion solution and with glucose (1 g/L) was perfused with a peristaltic pump at 0.53 mL/min for 60 min. Three groups of rats (n = 6; male/female = 1) were used: The control group was perfused with perfusion solution supplemented with only glucose, the positive control group received phlorizin (0.1 mM) solution, and the group received perfusion solution with added 125 mg/kg of DAHE. After 1 h, the perfusates were gathered to estimate glucose concentrations in final solutions using a commercial kit of glucose oxidase‐peroxidase, and the intestinal glucose absorption was assessed in mg/10 cm/1 h.

2.3. Antibacterial Assays

2.3.1. Bacteria Strains

In order to evaluate the antibacterial efficacy of DAHE extract, our study has targeted the bacterial strains Escherichia coli 021 and Bacillus cereus (isolated from a clinical specimen) sourced from a provincial hospital located in Taza, Morocco.; Pseudomonas aeruginosa ATTC 27853 and Staphylococcus aureus ATTC 29213 (American Type Culture Collection: ATCC) were isolated from food products in the LMBBM, Sciences and Technogies Faculty, Fez Morocco. Bacteria were stocked at inclined Luria‐Bertani (LB) agar medium at 4°C. Before test, bacteria were revived by subculturing in a suitable culture medium: LB at 35°C for 20‐24 h. The final bacterial concentrations about 10⁶ CFU/mL bacteria have been applied for the antibacterial screening according to the National Committee for Clinical Laboratory guidelines [25].

2.3.2. Agar‐Disc Diffusion Assay

The investigation of the antibacterial properties of the DAHE (Dichrostachys cinerea) extract was carried out utilizing the agar disc‐diffusion assay, with minor modifications to a previously reported protocol [26]. Briefly, sterile disks (6 mm diameter) containing 8 µL of DAHE extract was deposited in the surface of Muller‐Hinton agar medium, previously inoculated with bacteria (1 × 10⁸ to 2 × 10⁸ CFU/mL). Chloramphenicol (10 µg/disc) Ciprofloxacin (10 µg/disc) were as standard antibiotics. After incubation at appropriate temperature for 18‐24 h at 37°C, the antibacterial activity was reported as a inhibition zone diameter in millimeters. Test was performed in three independent experiments.

2.3.3. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

Minimum Inhibitory Concentration (MICs) were performed in sterile 96 well‐microplate using micro‐dilution technique as reported [2]. A 0.15% (w/v) concentration of bacteriological agar was employed to emulsify the examined extracts within the culture medium. First, 50 µL of Muller Hinton broth was allocated to all experimental wells, with the exception of the initial well where 100 µL of DAHE extract (100 mg/mL). Extract was successively diluted by the transfer of 500 µL from the first to the 10th well (0.194 to 100 mg/mL). The 11th well was considered as growth control and the 12th was used as sterility control (negative control). Then, 50 µl of inoculum preparation (about 10⁶ CFU/mL) Was introduced into every well. (First to 11th). After incubation at 37°C for 18‐24 h, 20 µl of p‐iodonitrotetrazoliumchloride (TTC) was added to each well as an indicator of bacterial growth. The microplate was subjected to another incubation of 1 h at 25°C. Subsequently, then bacterial growth was indicated by coloration transformation from black to red. Finally, Minimum Bactericidal Concentration (MBCs) were ascertained by inoculating 5 µL from the blanc wells (No color) on (LB) plates and followed by incubation at 37°C for 18‐24 h.

2.4. Antioxidant Activity

2.4.1. Total Antioxidant Capacity (TAC) Assay

Phosphomolybdenum methods was applied for the evaluation of the TAC of DAHE extract [27]. In short, 0.1 mL of DAHE was mixed with mixture solution of 0.6 M of sulfuric acid (H₂SO₄), 28 mM sodium phosphate (Na₃PO₄), and 4 mM ammonium molybdate ((NH₄)6MO₇O₂₄). Then, the obtained mixture was incubated in a boiling water at 94°C for 2 h. The absorbance was read at 695 nm, with a blank containing only reagent solution. The experiments were performed in three time and data presented as ascorbic acid equivalent per gram of extract (mg AAE/g E).

2.4.2. ABTS Radical Scavenging Assay

Discoloration test of ABTS^∙+ was employed to investigate the antiradical ability of DAHE extracts as described by [28]. In short, the cationic radical (ABTS^∙+) was obtained by reacting 10 mL of ABTS (2 mM) with 0.1 mL of potassium persulfate (K₂S₂O₈) (70 mM). After the incubated at obscurity for 15 h, the obtained ABTS^∙+ solution was diluted with methanol until attaining an optical density of 0.7 at 734 nm. Then, 0.2 mL of DAHE was added to 2 mL of the diluted ABTS^∙+ solution and allowed to react for 2 min. After, the absorbances was measured at 734 nm. The antiradical capacities are established as the concentration of DAHE that showed 50% inhibition (IC₅₀). This value was measured by plotting the inhibition degrees against the tested concentrations. Ascorbic acid and BHT were used as standards. The experiments were performed in three time and IC₅₀ values were presented as means ± SD.

2.4.3. Statistical Analysis

The data were represented in terms of mean value along with the standard deviation (SD). Furthermore, to determine the variations between groups, a one‐way analysis of variance (ANOVA) was conducted utilizing GraphPad Prism for Windows (Ver. 5, San Diego, CA), which was followed by a Tukeys post hoc test. A statistically significant difference was acknowledged when the p‐value was less than 0.05.

3. Results and Discussion

3.1. Experimental Validation

3.1.1. In Vitro α‐Amylase Inhibitory Activity

As demonstrated in Figure 2, in vitro experiments indicate that the presence of DAHE resulted in a significant reduction in the activity of α‐amylase, as compared to the control group. The IC₅₀ values for this inhibition were calculated to be 1.22 ±0.18 mg/mL, demonstrating a notable effect. Furthermore, at higher concentrations of DAHE (7.27 mg/mL), it was observed that the inhibitory impact of DAHE surpassed that of acarbose, with inhibition percentages of 82% and 85.21%, respectively. The strong inhibitory activity observed for DAHE at higher concentrations may be explained by its rich phytochemical composition, particularly phenolic acids and flavonoids. These compounds are well documented for their ability to inhibit carbohydrate‐hydrolyzing enzymes such as α‐amylase through different mechanisms, including interaction with catalytic amino acid residues and alteration of enzyme conformation, leading to reduced substrate accessibility [29]. Moreover, several recent studies have reported that plant extracts rich in polyphenols exhibit dose‐dependent inhibitory effects comparable to or even higher than acarbose at elevated concentrations [30, 31]. Therefore, the marked inhibitory activity of DAHE at 7.27 mg/mL is likely attributed to the synergistic action of its bioactive constituents, supporting its potential as a natural source of antidiabetic agents.

FIGURE 2.

Inhibitory activity of DAHE and acarbose against α‐amylase at various concentrations. Data are presented as means ± standard deviation (n = 3). * p<0.05; ** p<0.01; *** p<0.001 in comparison to the control. DAHE: Dysphania ambrosioides hydroethanolic extract.

3.1.2. In Vivo α‐Amylase Inhibitory Activity

The glycaemic response of a control group increased significantly following starch administration, with levels rising from 1.06 g/L to 1.78 g/L as depicted (Figure 3A). Conversely, the presence of DAHE at t = 30 min led to a notable reduction in postprandial glycaemia when contrasted to control (p<0.001 and p<0.01, respectively), with a similar reduction observed at t = 60 min (p<0.001) when administered at doses of 125 and 250 mg/kg. However, there was no significant difference in glycaemia at t = 120 min when compared to control. Acarbose demonstrated comparable activity to DAHE at a dose of 10 mg/kg.

FIGURE 3.

Effect of DAHE on blood sugar levels following excessive starch intake in healthy (A) and diabetic mice (B) (n = 6). * p<0.05; ** p<0.01; *** p<0.001 in comparison to the control group. DAHE: Dysphania ambrosioides hydroethanolic extract, Acar: acarbose.

The effect of DAHE on blood glucose levels was evaluated in alloxan‐induced diabetic mice following an oral starch overload. The results showed a significant reduction in postprandial blood glucose levels at 30, 60, and 120 min after administration of two doses of DAHE (p < 0.001). Furthermore, at 60 min post‐overload, glycemic levels in mice treated with 250 mg/kg of DAHE were comparable to those observed in the acarbose‐treated group (Figure 3B).

3.1.3. In Situ, Intestinal Glucose Absorption

Based on the findings demonstrated in Figure 4, the uptake of glucose in the jejunum was quantified as 17 mg/10 cm/h in the control group without the presence of DAHE. Conversely, in the presence of DAHE at a dose of 125 mg/kg, the intestinal glucose absorption rate was significantly diminished compared to the control group, with a measured quantity of 11.71 mg/10 cm/h (p<0.05). Moreover, the intestinal absorption rate of phlorizin was also considerably reduced by 4.71 mg/10 cm/h (p<0.001).

FIGURE 4.

Effect of DAHE on intestinal glucose absorption in rats (n = 6). * p<0.05; *** p<0.001 as compared to the control. DAHE: Dysphania ambrosioides hydroethanolic extract.

This investigation aimed to assese the inhibitory effect of DAHE on intestinal glucose absorption and α‐amylase activity as a potential strategy to manage postprandial hyperglycemia and diabetes [32]. The study employed a comprehensive approach, including in vitro, in situ, and in vivo tests, as well as an in vitro assay, to directly assess the impact of DAHE on α‐amylase activity. However, since in vitro analyses are limited, further examination was necessary. To corroborate these findings in vivo, we examined the effects of DAHE on the intestinal glucose absorption pathway in normal and alloxan‐diabetic mice, where DAHE significantly reduced intestinal glucose absorption (p < 0.05). The investigation revealed that DAHE strongly inhibits α‐amylase activity both in vitro and in vivo, thereby reducing intestinal glucose uptake. Moreover, DAHE was found to be more inhibitory activity than acarbose in inhibiting α‐amylase assay. Therefore, DAHE (125 mg/kg) had a rapid effect on alloxan‐diabetic mice, significantly (p<0.001) lowering blood sugar levels after starch loading. The hydrolysis of starch is facilitated by a double‐displacement mechanism that utilizes specific carboxylic acids as active sites to break down a covalent β‐glycosyl enzyme intermediate [24]. The effect of DAHE on the activity of α‐amylase enzyme was evaluated using selected concentrations (0.45, 0.91, 1.82, 3.64, 7.27 mg/mL), which were chosen based on a comprehensive range of concentrations tested initially. The concentration‐response curve, comprising of potency, slope, and maximum efficiency, was established from the concentration series to determine the IC₅₀ value (1.22 ± 0.18 mg/mL). Notably, certain extracts, such as Ammodaucus leucotrichus aqueous extract, were also examined during the experiment [13], Olea europaea extracts [33], Zizyphus lotus aqueous extract [17], P. jaubertii methanolic extract were shown to have an anti‐α‐amylase effect. With IC₅₀ values of 20.40 and 31.91 g/mL, respectively, aqueous extract of Z. lotus leaves and fruits has demonstrated a high enzymatic inhibitory activity compared to hydroethanolic extract of Dysphania ambrosioides [34]. Additionally, it has been reported that these extracts contain polyphenolic compounds that play a key role in the inhibition of enzymes associated with type 2 diabetes [1, 35]. The phytochemical profiling demonstrated that Dysphania ambrosioides contains a wide diversity of bioactive constituents, particularly flavonoids, phenolic acids, and other polyphenolic compounds, in agreement with previous phytochemical investigations of this species [36]. The observed inhibitory effects of the hydroethanolic extract on digestive enzymes can be plausibly linked to these identified phenolic constituents. In particular, flavonoids such as quercetin, luteolin, catechin, epicatechin, myricetin, and kaempferol have been widely reported to exert significant α‐amylase inhibitory activity [9, 37], In addition, phenolic acids, including rosmarinic acid, and ferulic acid, are also recognized for their contribution to enzyme inhibition and antidiabetic potential [38, 39, 40]. However, it should be emphasized that further in‐depth studies are required to confirm the underlying mechanisms and to validate these effects in vivo. Indeed, this study has certain limitations due to its primarily focus on hyperglycemia, without complementary in vivo evaluations such as body weight monitoring, blood biochemical parameters, behavioral assessment, or histopathological examination.

3.2. Antibacterial Activity

In the last two decades, emergence of antibiotic‐resistant starins is considered as a major threat to the world's health, food security, and progress. Although it's a natural occurrence, the excessive use of synthetic antimicrobials in both humans and animals is hastening the process. Therefore, the quest for antimicrobial substances of natural origin would provide a suitable substitute. Currently, the use of medicinal plants in therapy is developing considerably each year thanks to the progressive discovery of their properties and their medicinal efficacy, in particular antibacterial, anti‐inflammatory, antioxidant, antiseptic, antiviral, and antifungal.

The present study supports the hypothesis that plant extracts exhibit significant antimicrobial activity and may serve as effective natural antimicrobial agents. It was found that using the disc‐diffusion method, the DAHE has remarkable bactercidal ability against both Gram+ and Gram‐ starins with a high zone of inhibitions, those microorganisms were B. cereus (17.24±0.05 mm); S. aureus (24.81±0.13 mm), P. aeruginosa (12.56±0.4 mm) and E.coli (14.28±1.66 mm). With significant activity when compared to the standard antibiotic Chloramphenicol and Ciprofloxacin (Table 1 and Figure 5).

TABLE 1.

Determination of inhibition zones of Dysphania ambrosioides hydroethanolic extract (DAHE).

Bacteria	Zones inhibition (IZ) (mm)
	DAHE	Chloramphenicol	Ciprofloxacin
B. cereus	17.24±0.05a	15 ± 1.15b	13 ± 0.42c
S. aureus	24.81±0.13a	14 ± 0.1b	18 ± 1.12c
P. aeruginosa	12.56±0.4a	10 ± 1.1b	10 ± 0.42b
E. coli	14.28±1.66a	22 ± 0.21b	15 ± 0.20a

The values are expressed as mean ± standard deviation (n = 3). Significant differences were analyzed using ANOVA followed by Tukey's test (p < 0.05). Different letters indicate statistically significant differences between groups.

FIGURE 5.

Antibacterial activity of DAHE extract using disc diffusion method. Chloramphenicol Ciprofloxacin were used as control. Data are presented as means ± standard deviation (n = 3). Final bacterial concentration of 10⁶CFU/mL.

Indeed, numerous scientific studies on medicinal plants have shown that Gram+ bacteria are the most sensitive to tested substances than Gram‐ bacteria, primarily due to differences in the cell wall structure [41, 42, 43]. Previous investigations have reported the antibacterial effects of Dysphania ambrosioides extracts. The results are in agreement with those obtained by [44] who indicated that extracts of Dysphania ambrosioides exhibit good antibacterial activity on Gram+ bacteria (S. aureus, B. subtilis); While a weak antibacterial effect was noticed for Gram‐ bacteria such as E. coli, P. aeruginosa.

On the other side, as shown in Table 2. DAHE extract has shown noticeable antibacterial activities with MIC and MBC value rangining between 0.07 and 0.625, and between 0.312 and 1.25 mg/mL, respectively.

TABLE 2.

Determination of MIC, MBC, and MBC/MIC values of DAHE extract in mg/mL.

Microorganisms	DAHE			Chloramphenicol
	MIC	MBC	MBC/MIC	MIC	MBC	MBC/MIC
B. cereus	0.07	0.625	8.92	8	8	1
S. aureus	0.312	0.625	2	32	32	1
P. aeruginosa	0.312	0.312	1	128	128	1
E. coli	0.625	1.25	2	8	16	2

These findings confirmed the previous published data and indicated that these plant extracts can be employed as antimicrobial agents. The MIC and MBC of the Dysphania ambrosioides extracts were reported to be as low as 0.42 mg/mL (MIC) for Staphylococcus aureus and Enterococcus faecalis, 4.29 mg/mL (MIC) for Enterococcus faecalis [10]. Moreover, Ouadja and his colleagues indicated that Dysphania ambrosioides extracts show antibacterial activity with some variability according to the resistance of the bacterial strains used in the test, the experimental methods used, as well as the plant origin, and the parts used to prepare the extracts can affect the ability of the plant [3, 45].

The MBC/MIC ratios for the DAHE extract against the different strains tested are listed in Table 2. The extract is considered bacteriostatic when MBC/MIC ratios are greater than four, while those with an MBC/MIC ratio of four or less are considered bactericidal [46]. Accordingly, the DAHE extract had effective bactericidal activity against all tested strains except for the B. cereus strain which indicated a bacteriostatic action. Previous studies highlighted the presence of phenolic components in hydroethanolic extracts of Dysphania ambrosioides [10, 47]. Flavonoids are a class of heterocyclic organic molecules that belong to the group of polyphenols [47]. Several investigation have demonstated the antimicrobial ability of flavonoids. The antibacterial effect of phenolic molecules such as quercetin, rutin, and chrysin has been elucidated by numerous studies and is explained by their effect on cell permeability, and disrupt the bacterial membrane [10, 48]. Phenolic acids, hydroxyl groups, and carboxylic acid groups of phenolic compounds can disrupt the proton motive force, ion flow, coagulation, and leakage of protoplasmic contents, leading to bacterial death [3, 49].

3.3. Antioxidant Activity of DAHE Extracts

3.3.1. ABTS Radical Scavenging Assay

DAHE extracts displayed promising antioxidant effects. As shown in Figure 6, DAHE extracts exhibited significant capacity to scavenge ABTS+ radical (IC₅₀ = 293.98±3.11 mg/mL) but less effective compared to the synthetic antioxidants ascorbic acid (IC₅₀ = 165.91±0.7 mg/mL) and BHT (IC₅₀ = 201.12±3.48 mg/mL).

FIGURE 6.

ABTS free radical scavenging activity of DAHE extracts, and BHT and Ascorbic acid were used as positive control. Data are presented as means ± standard deviation (n = 3). p> 0,05 compared to control.

3.3.2. Total Antioxidant Capacity (TAC) Assay

The total antioxidant capacity (TAC) test showed that DAHE extracts has significant antioxidant activity, corresponding to 301.52 ± 1.17 mg AAE/g E at minimal dilutions (1/20) and to 67.11 ± 0.1 mg AAE/g E at the maximum dilution (1/320) as presented in table 3.

TABLE 3.

Total antioxidant capacity of DAHE.

DAHE extracts (Dilution)	1/320	1/160	1/80	1/40	1/20
TAC(mg AAE/g E)	67.11 ± 0.1	92.43 ± 1.22	148.76 ± 0.3	183.02 ± 0.5	301.52 ± 1.17

The experiment was performed in three independent repetitions, and the data are expressed as mean ± SD.

The antiradical potential of DAHE extracts was strongly correlated by its ability to exchange an electron or a hydrogen atom [27, 50]. Several studies have confirmed the ABTS+ scavenging activity of Dysphania ambrosioides extracts, reporting greater IC₅₀ values than we obtained. Indeed, Ouadja and his colleagues [45] showed that DAHE extracts had potent effect to reduce ABTS radical with an IC₅₀ value of 45.33±2.47 µg/mL [45]. Another study carried out on DA methanolic extracts using ABTS radical indicated an IC₅₀ value equal to 224 ± 0.042 µg AA/g E [51]. This divergence in results could be due to several factors, such as the variation in the chemical composition, geographical origin of plants, part used, storage and extraction conditions [52, 53].

4. Conclusions

In this study, we investigated the antidiabetic potential of the hydroethanolic extract of Dysphania ambrosioides through the inhibition of α‐amylase activity and the reduction of intestinal glucose absorption. The results demonstrated a concentration‐dependent inhibitory effect compared with the reference standard, suggesting a measurable modulatory activity on carbohydrate‐digesting enzymes. In addition, the extract exhibited notable antioxidant and antibacterial activities under the tested experimental conditions. Overall, these findings indicate that the biological activities of Dysphania ambrosioides may be associated with its phytochemical composition; however, further in‐depth studies are required to confirm the underlying mechanisms and validate these effects in vivo. This work provides experimental evidence supporting the traditional use of Dysphania ambrosioides, while avoiding overstatement regarding its therapeutic applications and highlighting its potential as a source of bioactive compounds for future pharmacological investigations.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.