For thousands of years, mankind has used various plants found in its environment to treat all kinds of diseases[1].

The therapeutic virtues of plants are showing renewed interest thanks to the improvement of extraction techniques and the progress of structural analysis methods for the discovery of new active ingredients. It is estimated that two thirds of today's drugs are of natural origin, obtained by hemisynthesis or modification of a natural product [2] .

The family Arecaceae contains 200 genera and 3000 species. Chamaerops humilis L. is a medicinal plant of the Arecaceae family. It is widespread in North Africa, especially in the western Mediterranean region [3].

humilis L. (Mediterranean dwarf palm) is an important floristic element of the western Mediterranean region, being the only palm species naturally distributed in both Europe and Africa [4] .

Several studies have shown the beneficial effects of C. humilis against chronic diseases such as cancer, ulcers, and kidney stones [5].Therefore, the present study was initiated to evaluate for the first time the anti-inflammatory activity of the methanolic extract of the fruits of C. humilis in albino Wistar rats and the antimicrobial activity of the essential oil and methanolic extract of C. humilis extracts.

The fruits of C. humilis were collected during the fall season in the mountains of Tlemcen (western Algeria). The plant was botanically identified by the Algerian Forestry Department.

The fruits were thoroughly washed to remove dirt, then cut into small pieces and dried in the shade at room temperature for 15 days. The dried plant material was then pulverized and used for extraction.

Essential Oil Extraction

Essential oil of air-dried fruit. (250 g) was obtained by hydrodistillation for 5 h using a Clevenger type apparatus according to [6]. The oil was separated from the hydrosol and dried over anhydrous sodium sulfate and stored in sealed vials at 4°C.

Preparation of methanolic extract

50 g of the fruit were first degreased with 250 ml of light petroleum. After 24 hours of continuous stirring, the solution is filtered through Whatman paper. The pomace obtained is delipidated twice with the same solvent before extraction [7].

Determination of total polyphenols.

The Folin-Ciocalteu method was applied. The Folin-Ciocalteu reagent oxidizes phenols to phenolate ions in an alkaline medium, resulting in the formation of a blue molybdotungstic complex. This coloration is measured spectrophotometrically at a wavelength of 765 nm. The absorbance is proportional to the amount of phenols present in our extracts. The results are expressed in mg of phenolics per g of dry residue from a gallic acid standard curve [8].

Determination of flavonoids

Quantitative determination was performed according to the colorimetric method described by [9].

In hemolysis tubes, 1500µl of distilled water is added to 500ul of mother methanol solution (2mg/ml). At zero time, a volume of 150µl of 5% NaNO2 is added. After 5 minutes of reaction, 150µl of 10% AlCl3 (dissolved in methanol) is added. After 11 minutes, 500µl of NaOH (1M) is added. The absorbance is read immediately on a spectrophotometer at 510 nm. A standard series is prepared from a series of catechin solutions with the following concentrations: (20; 50; 100; 200; 400; 600; 800; 1000 µg/ml). The flavonoid content is expressed from the calibration curve in milligrams of catechin equivalents per area of dry methanolic residue (mg EC/g dry residue).

Antimicrobial Activity Microbial strains

The essential oil and methanolic extract of C. humilis fruits were evaluated against five bacterial reference strains, namely Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, Staphylococcus aureus ATCC 43300, Bacillus subtilis ATCC 11778 and a yeast Candida albicans ATCC 10231.

Preparation of Inocula

Inocula of the bacterial and yeast strains were prepared from overnight broth cultures. Suspensions were adjusted to 0.5 McFarland or an optical density of 0.08 to 0.13 at a wave length of 625 nm, equivalent to 108 cfu/mL [10] (CLSI, 2006).

Disk Diffusion Assay

Essetial Oil and methanolic extract of C.humilis were tested for antimicrobial activity using the disc diffusion technique on solid media [11]. Discs of sterile Whatman paper (6 mm) were impregnated with 4 μL of EO and then placed on plates of Mueller-Hinton agar (Pronadisa TM, Spain) inoculated with an inoculum of 10⁸ CFU/mL. The plates were then incubated for 24 hours at 37°C for bacteria and 30°C for yeast. Results were recorded by measuring the growth inhibition zones around the discs. All assays were performed in triplicate.

Determination of minimum inhibitory concentration (MIC)

The MIC was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute [10] .

DMSO was used to dissolve the EO. A serial twofold dilution of the oil was prepared in a 96- well microliter plate in the range of 40.00-0.08 mg/mL inoculated in Mueller-Hinton broth. The MIC was defined as the lowest concentration of essential oil and methanolic extract that inhibited visible growth. All assays were performed in triplicate.

Acute Toxicity Study:

Principle:

It consists of determining the lethal dose (LD50). The LD50 is the dose of a substance that can cause the death of 50% of an animal population under certain experimental conditions. This dose makes it possible to determine the symptoms of intoxication and to compare substances with respect to their toxic potential.

Toxicity Procedure:

The acute toxicity of the methanolic extract of C. humilis was evaluated in Swiss albino mice fasted for 5 days. The animals were randomly assigned to 10 male mice. The control group received the same amount of distilled water in the same manner.

After administration, the animals were observed for 5 days to study their general behavior in comparison with the control group (death, restlessness, respiration, asthenia).

Study of anti-inflammatory activity:

In order to highlight the anti-inflammatory activity of the methanolic extract of C.humilis for the first time, an experimental model of acute inflammation of the mouse leg induced by carrageenin at 1% was evaluated according to the method of [12] .

Principle

The principle is to test the inhibitory effect of the methanolic extract of the fruit of C. humilis on the acute inflammation induced by the injection of 0.1 ml of carrageenin at 1% under the plantar fascia of the left hind leg of mice [13,14] .

Procedures:

Mice were divided into 3 groups of 10 animals each and fasted for 5 days prior to the experiment. The groups were divided as follows

G.01: C. humilismethanolic extract was administered orally at a dose of 250 mg/kg body weight (treatment group);

G.02: mice in the reference group were given sodium diclofenac (25 mg/kg) at a dose of (10 mg/kg) bw (reference group);

G.03: controls received distilled water (control group).

30 min after the last gavage, 0.1 ml of 1% carragenin in isotonic saline (NaCl 0.9%) was injected under the plantar fascia of the left hind leg of the mice.After 5 h, the mice were sacrificed and the hind legs were cut at the joint and weighed on an analytical balance.

Statistical Analyses :

All in vivo data are presented as mean standard deviation. Data were analyzed by analysis of variance followed by Tukey's multiple comparison test. Analyses were performed with XLSTAT software. P < 0.05 was used as the criterion for statistical significance.

In this study, the extraction of C. humilis fruits yielded 0.2% essential oil and 46.14% methanolic extract. The total polyphenol content in the methanolic extract was determined to be 0.3724±0.1523mg EAC/mg dry weight0.3724±0.1523mg EAC/mg dry weight, and the flavonoid content was found to be 9.2516±0.5433mg quercitrin equivalent/mg dry residue9.2516±0.5433mg quercitrin equivalent/mg dry residue.

The antimicrobial activity of both essential oil and methanolic extract was evaluated against different strains of bacteria and yeast. The essential oil showed significant antimicrobial. activity against Candida albicans (14.666 ± 0.577 mm inhibition zone, MIC 2.5 mg/mL), Staphylococcus aureus (11.333 ± 1.154 mm inhibition zone, MIC 10 mg/mL) and Escherichia coli (11.333 ± 1.000 mm inhibition zone, MIC 5 mg/mL). However, the methanolic extract showed no significant activity against any of the microbial strains tested. (Figure 1 And Table 1)

Figure 1: Antimicrobial activity of essential oil and methanolic extract of C. humilis fruits.

Table 1 Minimum inhibitory concentration (MIC)

Toxicological tests showed no signs of toxicity, behavioral changes, or mortality in mice treated at doses up to 5000 mg/kg for 5 days, suggesting that the extract is safe for use at these concentrations. In the anti-inflammatory study, the methanolic extract administered at a dose of 250 mg/kg showed a 46.41% inhibition of carrageenan-induced paw edema in mice. In comparison, the reference drug diclofenac (25 mg/kg) produced a 68.99% inhibition. .( Table 2 ) .

Table 2: Anti-inflammatory activity of C. Humilis

The results of this study highlight the therapeutic potential of C. humilis fruits. The methanolic extract showed moderate but significant anti-inflammatory activity, demonstrating 46.41% inhibition of carrageenan-induced paw edema in mice at a dose of 250 mg/kg. Although the efficacy was lower than that of diclofenac (68.99% inhibition), the activity of the extract supports its traditional use in the treatment of inflammatory conditions. [11]

The high content of polyphenols and flavonoids in the extract is likely responsible for its anti-inflammatory effects, as these compounds are known to inhibit key enzymes involved in the inflammatory process and reduce the production of inflammatory mediators such as prostaglandins and leukotrienes [15].

The antimicrobial activity of Chamaerops humilis essential oil was more pronounced against Gram-positive bacteria (Staphylococcus aureus) and the yeast Candida albicans, with significant inhibition zones and low minimum inhibitory concentrations (MIC). In contrast, Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pneumoniae were resistant to both the essential oil and the methanolic extract, probably due to their complex cell wall structure, which acts as a barrier to hydrophobic substances [16] . The higher efficacy of the essential oil against Gram-positive bacteria is consistent with previous studies, suggesting that its lipophilic components interact more effectively with the simpler cell walls of Gram- positive organisms [17].

Toxicological evaluation showed no signs of acute toxicity in mice treated with the methanolic extract at doses up to 5000 mg/kg, confirming its safety for oral administration. This finding supports its potential for therapeutic use, but also underscores the need for further studies on chronic toxicity and long-term effects [18].

Overall, these results validate the traditional medicinal use of Chamaerops humilis and open avenues for its application in the development of anti-inflammatory and antimicrobial agents. The relatively lower anti-inflammatory activity compared to standard drugs and the limited antimicrobial spectrum of the methanolic extract suggest that further optimization of extraction methods or combination with essential oils may enhance its efficacy. Furthermore, the variability in antimicrobial results compared to other studies highlights the need to standardize methodologies and evaluate a wider range of microbial strains to fully characterize its bioactivity [11,19].

The study confirms the therapeutic potential of C. humilis fruits, particularly through its methanolic extract and essential oil. The methanolic extract demonstrated significant anti- inflammatory activity in vivo, supporting its traditional use in managing inflammatory conditions. The essential oil exhibited noteworthy antimicrobial activity against select pathogens, especially Staphylococcus aureus, Escherichia coli, and Candida albicans.

The findings suggest that the bioactive compounds, including polyphenols and flavonoids, contribute to these biological effects. Furthermore, the absence of acute toxicity underscores the safety of the methanolic extract for oral use.

This work provides a strong foundation for the development of Chamaerops humilis-based therapeutic agents, bridging traditional medicine and modern pharmacological research.

Acknowledgement

We are grateful to the Algerian forest conservation services for identifying the plant. 

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