Medicinal plants have been used as a primary source of drugs to treat various ailments for ages due to their broad biological and pharmacological activities. They also play a critical role in identifying potential therapeutic agents [1]. In addition, there is a popular notion that since plants are natural, they are safe and have no side effects, making them a safer alternative to chemical drugs [2]. However, some medicinal herbs are poisonous and cause serious side effects or death [3]. Therefore, an adequate scientific evaluation emphasizing the toxicological qualities is critical while assessing the potential and efficacy of medicinal plants [4]. Despite the widespread use of herbal medications in health care in developing countries, little evidence-based information is available about their toxicity and adverse effects [5]. Several studies have shown that particular plants may have various detrimental effects on human health. Thus, rigorous research is imperative to ensure the safety of plants for human consumption, forecast toxicological consequences, and offer adequate evidence for dosage selection during disease treatment in humans [6]. Moreover, medicinal plants are recommended as promising sources for innovating new antimicrobials [7] and antioxidants [8].

Arbutus unedo, belonging to the family Ericaceae, is a widespread folkloric perennial medicinal plant found across North Africa and the Mediterranean. It is extensively used in Moroccan traditional medicine to treat various gastrointestinal, cutaneous, and urinary tract disorders [9]. A. unedo leaves have been used as a depurative, antioxidant, antidiarrheal, diuretic, urinary antiseptic, and astringent [10, 11], as well as in treating a variety of dermatologic, cardiovascular, urologic, and renal illnesses, besides hypertension and diabetes [12, 13]. The industry of Moroccan aromatic and medicinal plants (AMP) is characterized by their variety and richness. Due to the biological diversity and climatic changes in this country , over 800 of the 4,200 plant species are aromatic and/or have medicinal properties. Therefore, traditional medicine is prevalent in Morocco [12]. The current study aimed to evaluate the phytochemical, anti-diabetic, antioxidant, antibacterial, and acute and subchronic toxicity of Moroccan A. unedo leaves due to their significant features in Moroccan traditional medicine and the lack of sufficient scientific studies regarding its bioactivity.

1. Plant material and extraction procedure

The A. unedo leaves were collected from the wild in Morocco’s Taza region (34°13’N, 4°1’W, 510 m altitude, 900 mm annual rainfall average) and authenticated at the Rabat scientific institute. The voucher specimen was stored at the herbarium of the Botanical Department of the Scientific Institute of Rabat, Morocco, with the code (RAB 101549). The powdered leaves were extracted twice separately with 90% methanol and hot distilled water using an orbital shaker for 24 hours under normal conditions , followed by filtration and evaporation under reduced pressure to yield aqueous and methanol extracts.

2. Phytochemical screening

Qualitative analyses of A. unedo leaves were carried out using conventional phyto-analytical methods described in previous studies [14] to screen the following phytochemical groups: tannins, anthraquinones, terpenoids, flavonoids, free quinones, saponins, and alkaloids.

3. Mineral composition

The mineral composition (P, Na, K, Zn, Fe, Mn, Mg, and Ca) of A. unedo leaves was determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES, JobinYvonUltima 2) [15]. The powdered AU leaves (150 mg) were rinsed with 2 mL of 70% HNO3 solution before burning at 110℃ in a Teflon beaker. After adding 0.5 mL of hydrofluoric acid (HF), the covered beaker was placed in a sand bath. The sample combinations were heated until they formed a clear solution. After removing the top, the fluid was evaporated until completely dried. Finally, 2.0 mL of HCl was added, followed by 25 mL of 2.0 M HCl extraction.

4. Phenolic content

The total phenolic contents were determined using the Folin-Ciocalteu reagent (FCR) as described in our previous study [13].

5. Flavonoid and condensed tannin content

The total flavonoids and condensed tannins were estimated through colorimetric assays as described in our previous studies [9, 13].

6. Antioxidant activity

The in vitro antioxidant effect of leaf extracts was estimated using the ferric reducing and DPPH (2,2’-Diphenyl-1-picrylhydrazyl hydrate) antioxidant power assays according to our previous studies [13, 15].

This method was established using the ABTS radical cation decolorization technique, as described by Tuberoso et al. [16]. The samples were expressed as TEAC doses (mg TE/g edw) while the absorbance was measured at 734 nm.

7. Antibacterial activity

Methanolic and aqueous extracts of A. unedo leaves were screened for antibacterial activity using the agar disc-diffusion method, as reported previously [14], with minor modifications. The test was conducted in a sterile Petri dish with a diameter of 90 mm containing Nutrient agar material (Oxoid, France). Briefly, a suspension of the test microorganism in Nutrient broth (0.1 mL at 10⁷ CFU/mL) was distributed over Nutrient agar. The extracts were absorbed on a sterile paper disc (5 µL per 6 mm diameter disc) and deposited on the surface of previously streaked agar medium (1 µg each Petri dish). Petri dishes were incubated at 37℃ for 24 hours, and the inhibitory zone diameter was measured in millimeters. Each test was conducted in triplicate. The measurement scale was as follows: zones of inhibition > 15 mm were considered significantly inhibitory; zones of inhibition 10-15 mm were considered moderately inhibitory; and zones of inhibition 10 mm were regarded non-inhibitory.

The minimum inhibitory concentration (MIC) of each plant extract was evaluated using microbroth dilution in 96-well microplates according to the procedure described previously [14]. In each microplate row, decreasing amounts of each extract were formulated in DMSO using the serial two-fold dilution technique. After adding 20 µL of 0.5 McFarland bacterial suspension and 160 µL of Mueller-Hinton broth (MHB, Biokar, Beauvais, France), the microplates were incubated at 37℃ for 24 hours. Further, 40 µL of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Schaffhausen, Switzerland) was added at a concentration of 0.2 g/mL and incubated for 30 minutes at 37℃. The TTC stains bacterial cells in red color, indicating the presence of bacteria in the wells. The MIC was defined as the lowest concentration that did not exhibit detectable bacterial growth. However, the minimal bactericide concentration (MBC) was obtained by sub-culturing 50 µL from a microplate well that lacked bacterial growth on Mueller-Hinton agar (Biokar, Beauvais, France). The plates were then incubated at 37℃ for 24 hours. The concentration at which no media growth occurred was designated MBC. As a reference test, chloramphenicol (30 µg/disc) (Sigma-Aldrich, Schaffhausen, Switzerland) was used, and the experiment was performed thrice to get a statistically significant result [17]. MBC/MIC values were also computed to determine if the compound has bactericidal or bacteriostatic properties.

8. In vitro antidiabetic activity

The antidiabetic effect of A. unedo leaves was assessed using the procedures described in our previous study. Briefly, the extracts were tested for their ability to block the enzymatic actions of α-amylase and α-glucosidase [18].

9. Toxicological study

Healthy male Wistar rats weighing between 230 and 270 grams and Swiss Albino mice weighing between 25 and 30 grams were obtained from the animal unit of the Faculty of Medicine and Pharmacy at Mohamed V University in Rabat, Morocco. All rats and mice used in this study were housed in communal cages at a temperature of 25℃ and were exposed to a 12-hour cycle of light and 12-hour darkness with constant access to food and drink.

The current investigation was performed according to the ethics of “Guide for the Care and Use of Laboratory Animals,” 8th edition, prepared by the National Academy of Sciences [19]. From Mohammed V University in Rabat, ethical approval was obtained (protocol code # UA-2021-02).

Organization for Economic Testing of Chemicals (no 423) specified that the acute oral toxicity of extracts should be determined using Swice female mice [20]. Mice were separated into three groups of six animals based on weight and size. All animals had unrestricted access to tap water and the same kind of chow throughout the research, except for a short fast before the oral administration of the single doses of A. unedo leaf extract. Oral administration of 2,000 mg/kg and 5,000 mg/kg was chosen for the treatment mice group, whereas the untreated control group was administered with distilled water, and the vehicle was dosed. The overall behavior of mice was observed continuously for the first hour after injection and regularly for the first 24 hours (with specific emphasis on the first 4 hours). Further, mice were observed daily for the following two weeks. We tracked deaths, changes in body weight, and symptomatological disorders during the observation period.

Sub-chronic toxicity was evaluated for 90 days in accordance with OECD Test Guidelines 408 with some modifications [21]. The animals were kept in three groups of six each, in the same manner, described earlier. After grouping the animals , the treatment group received daily gastric gavage at two dosages (250, 500 mg/kg) of the aqueous extract evaluated during the 90-day period, whereas the control group received a physiological solution (vehicle). The dosages were delivered orally to the animals through an esophageal probe. Feeding sessions were held on a consistent schedule throughout the day. Weighing of all animals started shortly before the treatment, continued weekly throughout the exposure period, and culminated on the day of sacrifice. Changes in weekly weight and behavior were calculated and documented.

10. Statistical analysis

The findings were expressed using the mean of three replicates and standard deviation. Antibacterial and antioxidant activities were performed by analysis of variance (one-way ANOVA). The student’s t-test was performed for toxicological evaluation to establish statistical comparisons between the control and treatment groups. Statistical significance was defined as p-values less than 0.05.

1. Phytochemical screening

Qualitative analysis of A. unedo leaves identified the following compounds: tannins, flavonoids, terpenoids, and anthraquinones. However, alkaloids and saponins were not detected in A. unedo leaves (Table 1). Our preliminary screening revealed that the phytochemical groups identified in A. unedo leaves might have a variety of therapeutic actions against pathological illnesses and disorders.

(-) absent; (+) present; (++) present with moderate concentration; (+++) present with high concentration..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

Phytochemical screening of Arbutus unedo leaves.

Phytochemical class	Leaves of Arbutus unedo
Tannins	+++
Anthraquinones	++
Terpenoids	+
Flavonoids	+
Free quinones	+
Saponins	-
Alkaloids	-

(-) absent; (+) present; (++) present with moderate concentration; (+++) present with high concentration..

2. Mineral composition

The mineral composition of A. unedo leaves (mg/kg) is shown in Table 2. We measured eight mineral elements viz., zinc (Zn), iron (Fe), phosphorus (P), sodium (Na), magnesium (Mg), potassium (K), and calcium (Ca) in A. unedo leaves.

Table 2

The levels of mineral contents in the leaves of Arbutus unedo.

Mineral	Content mg/kg dw
Ca	4,150.04 ± 170.56
Fe	179.60 ± 8.58
Mg	1,391.48 ± 51.34
P	1,119.46 ± 40.86
Na	82.30 ± 1.82
K	8,254.74 ± 119.69
Zn	8.13 ± 0.40

3. Phenolic contents

Table 3 shows the total tannins, flavonoids, and phenolic contents in A. unedo leaves. The phenolic content of A. unedo methanolic leaf extract was significantly higher than the aqueous leaf extract (31.83 ± 0.29 mg GAE/g edw and 20.99 ± 0.17 mg GAE/g edw, respectively). Similarly, the methanolic extract contained more flavonoids (16.66 ± 1.47 mg RE/g edw) than the aqueous extract (12.06 ± 2.04 mg RE/g edw). On the other hand, the condensed tannins in the methanolic extract did not vary significantly from the aqueous extract (18.22 ± 4.12 mg CE/g edw and 17.47 ± 0.60 mg CE/g edw, respectively).

*The results are expressed as (1)mg of gallic acid equivalent, (2)mg of rutin equivalent, (3)mg of catechin equivalent. Data are reported to mean (n = 3) ± standard error. The same letter (a, b) was assigned to the values of the same column that does not have a significant difference (p < 0.05)..

&md=tbl&idx=3' data-target="#file-modal"">Table 3

Total phenolic, flavonoid, and tannin contents of Arbutus unedo leaves*.

Extracts	Phenolic content(1)	Flavonoid content(2)	Tanin content(3)
Methanolic extract	31.83 ± 0.29a	16.66 ± 1.47a	18.22 ± 4.12a
Aqueous extract	20.99 ± 0.17b	12.06 ± 2.04b	17.47 ± 0.60a

*The results are expressed as (1)mg of gallic acid equivalent, (2)mg of rutin equivalent, (3)mg of catechin equivalent. Data are reported to mean (n = 3) ± standard error. The same letter (a, b) was assigned to the values of the same column that does not have a significant difference (p < 0.05)..

4. Antioxidant activity in A. unedo

The antioxidant properties of the A. unedo leaves were determined using the FRAP, ABTS radical scavenging capacity, and DPPH approaches (Table 4). The DPPH findings indicated a dose-dependent impact, confirmed by the computation of IC50 dosages. The methanol and aqueous extracts of A. unedo leaves showed significant antioxidant activity, with IC50 values of 0.01 ± 0.22 and 0.01 ± 0.98 mg/mL, respectively. The antioxidant activity of these extracts remained lower than that of ascorbic acid (IC50 = 0.003 ± 0.667 mg/mL), the positive control. Similarly, the methanolic extract demonstrated significantly higher antioxidant activity than the aqueous extract (51.58 ± 0.56 mg TE/g extract) in the ABTS test (34.00 ± 0.56 mg TE/g extract). In addition, the FRAP approach revealed that the methanol extract had maximum reducing power (920.66 ± 122.65 mg AAE/g extract), followed by the aqueous extract (403.33 ± 15.27 mg AAE/g extract).

Data are reported to mean (n = 3) ± standard error..

&md=tbl&idx=4' data-target="#file-modal"">Table 4

Antioxidant content of Arbutus unedo.

Tested compound	DPPH (IC50 mg/mL)	ABTS (mg TE/g extract)	FRAP (mgAAE/g extract)
Methanolic extract	0.01 ± 0.22	51.58 ± 0.56	920.66 ± 122.65
Aqueous extract	0.01 ± 0.98	34.00 ± 0.56	403.33 ± 15.27
Ascorbic acid	0.003 ± 0.67	-	-

Data are reported to mean (n = 3) ± standard error..

5. In vitro antidiabetic activities

In vitro antidiabetic activity analysis of A. unedo leaves in both extracts is shown in Table 5. The methanol extract exhibited the highest anti-glucosidase and anti-amylase activity, with IC50 values of 0.099 ± 1.21 and 1.350 ± 0.32 mg/mL, respectively. Interestingly, the methanolic extract inhibited α-glucosidase more effectively than the acarbose medication (p < 0.05), with IC50 values of 199.53 ± 1.12 and 396.42 ± 5.16 g/mL, respectively. In contrast, the aqueous extract was less efficient than the methanolic extract against α-glucosidase and α-amylase, with IC50 values of 0.196 ± 4.21 and 1.320 ± 0.26 mg/mL, respectively.

Data are reported to mean (n = 3) ± standard error..

&md=tbl&idx=5' data-target="#file-modal"">Table 5

Digestive enzymes inhibition activity (α-glucosidase and α-amylase) of Arbutus unedo extracts.

Products	α-amylaseIC50, μg/mL	α-glucosidaseIC50, μg/mL
Methanolic extract	1.350 ± 0.32	0.099 ± 1.21
Aqueous extract	1.320 ± 0.26	0.196 ± 4.21
Acarbose	0.396 ± 5.16	0.199 ± 1.12

Data are reported to mean (n = 3) ± standard error..

6. In vitro antibacterial potential

The antibacterial activity of A. unedo leaves (methanolic and aqueous extracts) is expressed in inhibition diameters (Table 6). Table 7 depicts the minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and MBC/MIC values. Both extracts had different levels of antibacterial activity against all bacteria except for Pseudomonas aeruginosa. The methanolic extract was the most effective against the three bacteria that were most likely to be affected.

*The width of the inhibition zone (mm) around the disks (6 mm) impregnated with 20 μL (60 mg/mL) of the extracts; positive control: chloramphenicol (30 µg/disc), negative control: methanol 20 μL (90%), (–): no activity (6.0 mm disc diameter). At p < 0.05, values in the same column that do not have a common letter (a to c) differ significantly..

&md=tbl&idx=6' data-target="#file-modal"">Table 6

Antibacterial activity of Arbutus unedo leaves (methanolic and aqueous extracts) against four bacterial strains*.

Compound	Mean zone of inhibition of bacteria (mm)
	Escherichia coli	Salmonella typhimurium	Pseudomonas aeruginosa	Staphylococcus aureus
Methanolic extract	14.5 ± 0.5c	12.2 ± 0.4c	–	18.2 ± 0.4c
Aqueous extract	9.2 ± 0.3b	8.0 ± 0.1b	–	12.4 ± 0.3b
Chloramphenicol	28.6 ± 0.3a	21.8 ± 0.3a	17.7 ± 0.2c	30.8 ± 0.7a
90% methanol	–	–	–	–

*The width of the inhibition zone (mm) around the disks (6 mm) impregnated with 20 μL (60 mg/mL) of the extracts; positive control: chloramphenicol (30 µg/disc), negative control: methanol 20 μL (90%), (–): no activity (6.0 mm disc diameter). At p < 0.05, values in the same column that do not have a common letter (a to c) differ significantly..

*(–): no activity at the maximal concentration used, MIC and MBC values are expressed as mg/mL..

&md=tbl&idx=7' data-target="#file-modal"">Table 7

MIC, MBC and MBC/MIC values of tested extracts*.

Microorganism	Methanolic extract (mg/mL)				Aqueous extract (mg/mL)
	MIC	MBC	MBC/MIC		MIC	MBC	MBC/MIC
Escherichia coli	25	50	2		50	50	1
Salmonella Typhimurium	50	50	1		50	100	2
Staphylococcus aureus	12.5	25	2		25	50	2
Pseudomonas aeruginosa	–	–			–	–

*(–): no activity at the maximal concentration used, MIC and MBC values are expressed as mg/mL..

7. Toxicological investigation

The acute oral toxicity investigation revealed that the A. unedo aqueous leaves extract had no impact on the change in body weight of mice during the observation period compared to the witness (Fig. 1).

Figure 1. Changes in the body weights of mice treated with Arbutus unedo leaf extract. Each point represents the mean ± SD (n = 6). No significant difference.

The chronic oral toxicity of A. unedo leaves aqueous extract was studied in rats using the oral route daily at dosages of 250 and 500 mg/kg for three months.

Table 8 shows the body weight changes in control and aqueous A. unedo leaf extract-treated rats. Rats gained weight over time, and no significant difference in mean weight growth was observed between treated and control rats.

Table 8

Body weight changes in rats treated with A. unedo extracts.

Days	Control	250 mg/kg	500 mg/kg
D0	229.97 ± 3	215.46 ± 2	218.16 ± 09.84
D30	241.93 ± 3	221.37 ± 1	239.71 ± 02.54
D60	256.62 ± 2	238.17 ± 2	261.23 ± 18.03
D90	271.21 ± 2	244.58 ± 2	284.12 ± 39.24

Table 9 summarizes the impact of A. unedo leaves aqueous extract on many hematological parameters. When the aqueous extract was administered at doses of 250 mg/kg and 500 mg/kg, there was no significant difference between the mean levels of different hematological parameters tested in the animals in the treatment groups and their values in the control groups.

Table 9

Hematological parameters of males’ rats after 90 days of treatment with Arbutus unedo extract.

Tests	Control	250 mg/kg	500 mg/kg
Red blood cells (106 μL−1)	8.1 ± 0.2	8.3 ± 0.3	8.4 ± 0.1
White blood cells (103 μL−1)	12.3 ± 0.3	12.6 ± 0.4	12.8 ± 0.4
Hemoglobin (g/dL)	11.7 ± 0.6	11.9 ± 0.7	12.1 ± 0.6
Hematocrit (vol %)	41.1 ± 0.7	44.4 ± 1.3	46.7 ± 1.7
Platelets (×104 L−1)	81.7 ± 1.2	83.5 ± 2.0	84.8 ± 0.8
Lymphocytes (%)	77.41 ± 0.6	78.55 ± 0.8	78.88 ± 0.2
Neutrophils (%)	15.4 ± 1.4	15.7 ± 1.6	15.8 ± 1.2

After 90 days of the treatment, the aqueous extract-treated mice showed a substantial drop in blood pressure. Compared to the control, no change was detected in the other values (creatinine, urea, ALT, and ASAT) (Table 10). Overall, Fig. 2 presents the results of the current study.

Data are reported to mean (n = 3) ± standard error. The same letter (a, b) was assigned to the values of the same column that does not have a significant difference (p < 0.05)..

&md=tbl&idx=10' data-target="#file-modal"">Table 10

Biochemical parameters of males’ rats after 90 days of treatment with A. unedo extract.

Tests	Control	250 mg/kg	500 mg/kg
AST (U/L)	142.22 ± 0.1	143.31 ± 0.3	143.5 ± 0.1
ALT (U/L)	54.2 ± 0.2	56.31 ± 0.25	56.5 ± 0.4
Creatinine (mg/L)	3.38 ± 0.2	3.7 ± 0.1	3.88 ± 0.2
Urea (g/L)	0.26 ± 0.02	0.28 ± 0.01	0.29 ± 0.02
Glucose (g/L)	1.10 ± 0.15b	0.92 ± 0.02a	0.90 ± 0.01a

Data are reported to mean (n = 3) ± standard error. The same letter (a, b) was assigned to the values of the same column that does not have a significant difference (p < 0.05)..

Figure 2. A summary of the biological characteristics of Moroccan Arbutus unedo leaves.

Historically, plants have been used as the primary source of medicinal compounds to cure various ailments. The discovery of novel compounds with biological activity is currently one of the most challenging tasks. This can only be accomplished by conducting a phytochemical screening of the plant extract to identify its phytochemical composition.

The phytochemical investigation of the A. unedo leaf aqueous extract revealed the presence of various phytochemical compounds, including tannins, anthraquinones, terpenoids, flavonoids, and free quinones. The phytochemical classes identified in the first screening have been implicated in several therapeutic actions against pathological illnesses and disorders. Our findings are consistent with Dib et al. [22].

The analysis of phenolic content revealed that the methanol extract of A. unedo leaves contained significantly higher polyphenols and flavonoids than the aqueous extract. These distinctions result from the polarity of solvents used to extract the phenolic compounds. Our findings differed from those of Oliveira and colleagues from Portugal [23], who stated that the aqueous and methanol A. unedo leaf extracts contained 172.21 ± 6.29 and 149.28 ± 5.33 mg GAE/g, respectively, of total phenolics. Tannin contents in A. unedo leaves were comparable in both extracts. Earlier, Tannins in A. unedo leaves have been identified in a few studies [24].

Numerous analytical approaches could be used to determine the antioxidant potential of natural compounds in vitro. Due to the wide variety of phytochemical compositions and oxidative processes, we used the combination of FRAP, ABTS, and DPPH assays to examine A. unedo leaf extracts. In this study, the methanol extract exhibited better results in all three tests than the water extract. The estimation of antibacterial activity indicated that both methanolic and aqueous A. unedo leaf extracts were effective against the tested bacteria (p < 0.05), with the methanol extract showing significantly better efficiency, except for P. aeruginosa, which was resistant to the extracts (Table 6). Bacteria that showed sensitivity to these extracts using agar-diffusion were subjected to MIC and MBC assays. The MIC, MBC, and MBC/MIC values are shown in Table 7. The MIC values of the methanolic extract were lower than those of the aqueous extract against the tested microorganisms. Lower MIC and MBC values suggest that less drug concentration is required to prevent the organism’s growth; hence, the methanolic extract is speculated as a more effective antibacterial agent. MBC/MIC values were between 1 and 2, suggesting that the antibacterial compounds included in these extracts are bactericidal against the three tested bacteria. A previous study carried out on A. unedo leaf extracts collected from another place in Morocco (Ouezzane Province) stated that the methanolic extract exhibited noticeable antibacterial activity against all tested bacteria (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa) [25].

A. unedo leaves collected from Turkey displayed antibacterial activity against S. aureus but did not inhibit E. coli or Salmonella enteritidis [26]. Another investigation from Croatia suggested that the aqueous extract of A. unedo leaves harbor potential antibacterial agents against urinary pathogenic bacteria, especially Enterococcus faecalis [27]. The potent antibacterial activity of A. unedo leaves is attributed to their richness in phenolic content [28]. Our findings were superior to the previous study [18]. In addition, several additional studies have confirmed that the leaves and fruits of A. unedo possess outstanding antioxidant qualities [11, 29, 30]. The antioxidant properties of A. unedo leaves may be connected, at least in part, to their total phenolic content. To our knowledge, this is the first study examining the in vitro inhibitory action of A. unedo leaf extracts against α-glucosidase and α-amylase. Indeed, carbohydrate hydrolase inhibitors may be beneficial in treating obesity and diabetes, as well as the associated health concerns. Therefore, screening for anti-enzymatic activity is beneficial in limiting sugar absorption. The results indicated that the methanolic extract inhibited the enzymes strongly and had a significantly higher inhibitory effect on α-glucosidase than the acarbose molecule. These observations are similar to our recent investigation on A. unedo roots from another location, which showed that α-glucosidase was efficiently suppressed at low quantities of root extracts [18].

Despite the vast utilization of therapeutic herbs in underdeveloped countries, little scientific information concerning their toxicity and adverse effects is available [5]. Indeed, several studies have revealed that various plants may have various detrimental effects on human health. Thus, comprehensive studies are essential to evaluate the safety of plant products for human consumption and to provide adequate information for determining their appropriate amounts in individuals [6].

To the best of our knowledge, this is the first report examining the safety and efficacy of A. unedo leaves aqueous leaf extract. Thus, the present study determined the toxicological profile of A. unedo leaves aqueous extract by evaluating sub-chronic oral toxicity in rats for 90 days and acute oral toxicity in mice for 14 days. The present investigation discovered that the aqueous extract of A. unedo leaves, used in traditional Moroccan medicine to treat various ailments, was reasonably non-toxic following treatment with 2,000 and 5,000 mg/kg of body weight of the extract. No fatalities or clinical manifestations of poisoning were recorded, indicating that the LD50 is more significant than 5,000 mg/kg. A. unedo leaf extract is classified as a Category 5 chemical under the Globally Harmonized System of Classification and Labeling of Chemicals. It is regarded to be an oral non-toxic substance [31].

During the treatment period, rats treated with (250 and 500 mg/kg; day) dosages exhibited no change in their overall physical appearance or body motions. There were no signs of tremors, cramping, drooling, diarrhea, or coma, as well as no unusual behaviors such as self-harm or walking backward. In comparison, rats gained weight over time (as predicted), with no statistically significant difference in mean weight increase between treated and control rats over the chronic dosage phase. It is widely established that using harmful chemicals leads to a decrease in body weight gain of at least a minimum amount [3].

The hematopoietic system is one of the most toxically susceptible organ systems in the body. It is a key indicator of the physiological and pathological state of a human or animal [4]. As a result, any alteration in hematological parameters indicates the presence of toxic substances [32, 33]. There was no difference in hematological parameters across several groups of rats in the present experiment. However, compared to the control group, rats treated with various dosages of A. unedo leaves aqueous extract over a 90-day period showed a substantial decrease in blood glucose levels at the end. Similarly, blood sugar levels were reduced in previous investigations, which demonstrated that A. unedo leaves aqueous extract has hypoglycemic properties [34, 35].

Moreover, the liver is a critical organ involved in the biotransformation of medications, and its proper function is monitored using a variety of blood biomarker enzymes [36]. Increased serum ALT levels are indicative of liver tissue enlargement and deterioration [18]. Apart from being a marker of liver impairment, the AST level is also used to detect muscle and cardiac problems [37]. A. unedo leaves extract had no significant effect on blood ALAT and AST levels (p > 0.05), indicating no rise in ALAT and AST levels in rats after treatment compared to the control. These findings imply that there are no apparent hepatotoxic consequences in rats.

Arbutus unedo leaves comprise potential natural molecules with valuable bioactive attributes. Our study indicates that A. unedo is safe for pharmaceutical, chemical, and food sectors use. Their efficacy can be investigated further to enhance their utilization in the pharmaceuticals and food industry.

Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R158) Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

The data used to support the findings of this study are included within the article.

The authors declare that they have no conflicts of interest.