Pharmacokinetic and phytochemical screening of Allium subhirsutum leaves for antimicrobial and anti-inflammatory properties

Riadh Badraoui; Yasser Al-Hazmi; Faten Brahmi; Hmed Ben-Nasr; Emira Noumi; Nouha Bouali; Mohd Adnan; Arif J Siddiqui; Vincenzo De Feo; Mejdi Snoussi

doi:10.1038/s41598-025-18825-x

. 2025 Oct 6;15:34806. doi: 10.1038/s41598-025-18825-x

Pharmacokinetic and phytochemical screening of Allium subhirsutum leaves for antimicrobial and anti-inflammatory properties

Riadh Badraoui ^1,^2,^3,^✉, Yasser Al-Hazmi ^1,², Faten Brahmi ⁴, Hmed Ben-Nasr ⁵, Emira Noumi ^1,², Nouha Bouali ^1,², Mohd Adnan ¹, Arif J Siddiqui ¹, Vincenzo De Feo ⁶, Mejdi Snoussi ^1,²

PMCID: PMC12501031 PMID: 41053188

Abstract

This study aimed to screen the phytochemical composition of the leaves of Allium subhirsutum L. methanolic extract (ASE) by Gas Chromatography-Mass Spectrometry (GC-MS). Furthermore, the antioxidant, antibacterial and anti-inflammatory effects were examined using combined approaches: in silico, in vitro and in vivo on carrageenan-induced acute inflammation in rats. Inflammatory biomarkers (C-reactive protein and fibrinogen) oxidative injury parameters (thiobarbituric acid reactive substances, advanced oxidation of protein products, catalas, superoxide dismutase, and glutathione peroxidase) levels were assessed in the inflamed paws and compared to controls. The identified compounds in ASE possessed acceptable pharmacokinetic and ADMET (for absorption, distribution, metabolism, excretion and toxicity) properties. They bound the targeted receptors (Tyrosyl-tRNA synthetase and Gyrase of S. aureus, Human peroxiredoxin 5 and Cyclooxygenase-2) with acceptable affinities and established strong molecular interactions. ASE exhibited bacteriostatic and fungistatic action against different tested microbial strains. Moreover, histological examinations of paw edema revealed that ASE (given by gavage at 100 mg/kg BW for 10 days) ameliorate inflammation and oxidative stress status as outlined by anti-edematous, antioxidant and inflammatory biomarkers. Our investigation provided evidence that ASE could be useful in the management of oxidative stress, bacterial infections and acute inflammation. The results of the in silico assay supported these effects.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-18825-x.

Keywords: Allium subhirsutum, Pharmacokinetics, Acute inflammation, Oxidative injury, Antimicrobial activity, Molecular docking, GC-MS

Subject terms: Computational biology and bioinformatics, Drug discovery, Immunology, Microbiology, Systems biology

Introduction

All over the world, several population still relies on traditional use of medicinal plants and phytotherapy for health promotion. Garlic is commonly used by traditional practitioners for preparation of herbals medicine, and also considered as food spicy. In this context, Allium subhirsitum L. is a perennial plant, which belongs to the garlic family. A. subhirsutum extract (ASE) have been reported to possess tremendous health benefits and have been used in medical purposes^1,2. Recent studies reported the ethno-pharmacological use of the bulbs for therapeutic claims. Several biological activities including wound-healing², inhibition of tumor angiogenesis in a rat model of induced skeletal metastases by Walker 256/B cells^1,3 thanks to the medicinally active phytochemicals that are responsible for its bioactivities^2,4. Garlic plants, such as A. subhirsutum, showed considerable health benefits including antioxidant^3,4, hypolipidemic and anticancer potential⁴. Nevertheless, both anti- antimicrobial and inflammatory effects of A. subhirsutum are not well documented and might benefit from experiments in murine models of inflammation.

Carrageenan (CAR)-induced paw edema is a producible model of acute inflammation, which mimic human inflammatory cases^5–7. The key features of this experimentally-induced inflammation include increase in vascular permeability and leucocytes infiltration, which lead to paw edema within the inflamed foci^8–10. Both acute and chronic inflammations associated disruption of the oxidative/antioxidative balance as a result of high levels of reactive oxygen species (ROS)^11–13.

Excessive ROS may contribute to oxidative injury through alteration of cellular components; proteins, lipids and nucleic acids^14,15. ROS can, already, initiate, accelerate and enhance the inflammatory process through direct effect on the release of several cytokine and other inflammatory factors.

Regular antiinflammatory drugs, specifically Nonsteroidal Anti-inflammatory Drugs (NSAIDs) and Corticosteroids, are still playing key roles in pain and inflammation controls, but they have discouraging profile of several drawbacks^16,17. Thus, the increased need for safe medication and health promotion based on the use active compounds such as phenols and flavonoids that are found in most plants and nutraceutical compounds^18,19. The laters possessed promising potentials to reduce oxidative injury and inflammatory burden and several pathological disorders. Similarly to the garlic bulbs, ASE might possess antimicrobial and antiinflammatory activity but this is not well documented and require further investigations.

Considering these potential effects, the current study investigated the beneficial effects of ASE using combined (i) in silico approach by ADMET properties, affinities and molecular interactions of ASE phytochemicals with several receptors: tyrosyl-tRNA synthetase and Gyrase of S. aureus, Human peroxiredoxin 5 and Cyclooxygenase-2 (1JIJ, 2CXT, 1HD2 and 1CX2, respectively); (ii) in vitro antimicrobial assay on some selected microorganisms; and (iii) in vivo assay in a rat model of acute inflammation.

Materials and methods

Plant material and extraction procedure

A. subhirsutum samples were purchased from Hail region and the bulbs were used for plant growing in pots at laboratory conditions from May to July 2022. The A. subhirsutum leaves have been collected in this period as they are well developed. The Voucher specimen number is 7427, which is deposited in EGE University’s Herbarium as previously reported⁴. A. subhirsutum leaves were freshly collected from young seedling plants and washed with sterile distilled to eliminate debris. Twenty grams of the obtained plant material (Leaves) were mixed with 400 mL of pure methanol in 1 L-glass-bottle. After incubation for 72 h at room temperature, the filtrate was dried at 45 °C in the incubator chamber.

phytochemical composition

A Gas Chromatograph system (Shimadzu Nexis GC-2030) equipped with a QP2020 NX Mass Spectrometer was used to identify the compounds in methanolic extract of A. subhirsutum L. leaves. The rate of the carrier gas (helium) was 1mL/min associated a column with a 1.8 μm film thickness, 2.1 mm of internal diameter, 50 mm of length and 70 °C of temperature. The later was kept at this level for 2 min before steadily raising it by 5 °C/min until reaching 280 °C. The carrier gas was flowing at a rate of 1mL/min through an injection port of 250 °C and ionization voltage of 70 eV. The A. subhirsutum compounds were detected using a quadrupole mass spectrometer once expelled from the column associated MS data libraries WILEY8.LIB and NIST08 as previously reported²⁰.

Biological properties of A. subhirsutum methanolic extract

Antimicrobial activities

The diameter of growth inhibition zone was assessed on agar medium using the disc diffusion assay on several bacterial and candida strains (Table 1) as previously described²¹. The studied strains included the major common human infectious bacteria that are known with antibacterial resistance. The disc diffusion assay was run in triplicate. MICs (for Minimal inhibitory concentrations) and MBC/MFCs values (for Minimal bactericidal/fungicidal concentrations) have been studied as previously described²². To interpret the character of the tested extract we used the method reported by Gatsing et al. (2009)²³. Ampicillin and amphotericin B have been used as standard drugs for the antibacterial and antifungal activities, respectively.

Table 1.

List of bacterial and fungal strains used for antimicrobial activities.

Code	Bacterial strains tested
M₁₁	Staphylococcus hominis
M₁₆	Pseudomonas aeruginosa
M₁₄	Klebsiella pneumoniae
M₆	Escherichia coli
M₁₇	Acinetobacter baumannii
M₈	Enterobacter cloacae
M₉	Enterobacter faecium
M₁₉	Pseudomonas aeruginosa
M₁₂	Staphylococcus aureus
M₁₃	Staphylococcus epidermidis
M₂₂	Staphylococcus aureus (MRSA)
M₇	Enterobacter faecalis

Code	*Candida* spp. strains tested
A₁	Candida utilis ATCC 9255
A₈	Candida tropicalis ATCC 1362
A₄	Candida guillermondii ATCC 6260
A₁₅	Candida albicans ATCC 20402

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Computational study

Pharmacokinetic properties

The phytochemicals found on A. subhirsutum extract, have been used to predict the bioavailability and ADMET (for absorption, distribution, metabolism, excretion and toxicity) parameters based on the physicochemical properties as previously described^9,15.

Molecular Docking and interaction assessment

The tridimensional (3D) structures of tyrosyl-tRNA synthetase (TyrRS) of S. aureus (1JIJ), Gyrase of S. aureus (2XCT), Human peroxiredoxin 5 (1HD2), and Cyclooxygenase-2 (1CX2) have been retrieved from RCSB databases. Both TyrRS and Gyrase are commonly targeted by antibiotics and drug-design and development as the bacterium is largely responsible for hospital-acquired infections. Widely expressed in tissues and mainly localized in mitochondria, peroxisomes and cytosol, 1HD2 belongs to the emerging family of peroxidases, which are able to reduce H₂O₂ and alkyl hydroperoxides, and involved in the oxidative/antioxidative status. COX-2 is mainly induced by cytokines, endotoxins and mitogens in the immune cells and is of key responsibility of the increased levels of prostaglandins during inflammatory responses. The 3D chemical structures of the A. subhirsutum identified compounds were collected from PubChem databases or drawn using ChemDraw software packages. Both ligands and macromolecules have been prepared, specifically by removal of water molecules, addition of polar hydrogens and Kollman charges^3,9. The docking procedure was based on the CHARMm force field was applied for each phytochemical compound with the different targeted receptors, using vina software packages. The major reasons behind the selection of these macromolecules 1JIJ, 2XCT, 1HD2, and 1CX2 are their key role in antibacterial, antioxidant and anti-inflammatory pathways, and they are also commonly targeted in similar studies^21,23.

Animals and in vivo experimental procedure

Acute inflammation was induced in Wistar rats of 8–9 weeks. Rats were purchased from SIPHAT Company (central pharmacy of Tunisia). The rats were acclimatized to the animal housing conditions (12 h/12 h cycle of light/dark and at 23 ± 2 °C of temperature) at least for one week prior to the beginning of the experimental procedure. Twenty four rats have been used in this study. Drinkable water was given ad libitum together with rats’ standard laboratory diet. The rats were randomly divided into 4 groups:

i)
The control group composed of 6 non-inflamed rats (CTRL), which received injections of physiological saline (0.9%).
ii)
The inflamed group composed of 6 rats (CAR group), which received sub injection of carrageenan (10 mg/kg of BW by gavage).
iii)
The inflamed and treated group composed of 6 rats (CAR + ASE), which received daily injections of ASE (100 mg/kg BW by gavage) for 10 days then carrageenan injections in the day number 11.
iv)
The reference group composed of 6 rats (CAR + INDO), which received daily injections of indomethacin, as a reference compound, for 10 days then carrageenan.

Carrageenan-induced paw edema, which manifested by variation in paw thickness before and after the CAR injections. A digital caliper was used to assess the inhibitory effect of ASE as compared to INDO for the first five hours following the injection of CAR.

The percentage of inhibition was calculated as follow:

% of inhibition = (Vc – Vt / Vc) × 100.

Where:

Vc = Volume of paw edema in CTRL rats.

Vt = Volume of paw edema in ASE/INDO rats.

Five hours after the injections, the rats were anesthetized and sacrificed. The rats were anesthetized by intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) followed by decapitation. Both blood samples and paws were removed for hematological, biochemical and histological studies. The study experiments were performed in accordance with relevant guidelines and regulations and approved by the local Ethical Committee Guidelines for the care and use of laboratory animals of Faculty of Medicine of Sfax–University of Sfax (12/ES/15_2022). Furthermore, the study was carried in accordance with ARRIVE guidelines.

Sample preparation

After sacrifice, the blood samples have been collected for each rat and their plasma were aliquoted and stored at -80 °C until used. Some other blood samples were sed for hematological numeration. The injected paw’s tissues, in close vicinity to the inflamed area, were mixed with 10% (w/v) phosphate buffer saline, pH = 7.4 then centrifuged (20 min at 9000 rpm). The obtained supernatant were used to assess the oxidative stress status.

Inflammatory biomarkers

An automatic analyzer (COBAS INTEGRA 400” C-Reactive protein) was used to measure the CRP levels (in mg/L) by turbidimetric method. The level of fibrinogen was also assessed as described in the Clauss’s method²⁴. The study was based on the assessment of fibrinogen conversion into fibrin in the presence of thrombin excess and recording the time required for clotting time, which is negatively correlated to the fibrinogen level in the sample. Fibrinogen levels were expressed in g/L.

Hematological parameters

Some hematological parameters were assessed and recorded. This includes numeration of red blood corpuscules, white blood cells count, and platelets (RBC, WBC and PLT, respectively) using a hematological automate analyzer (KX21 hemogram). Hematological parameters have been assessed.

Oxidative injury assessment

Protein quantification was assessed as described by Lowry et al.²⁵ based on the use of bovine serum albumin as standard.

TBARS and AOPP pro-oxidant assays

Thiobarbituric acid reactive substances (TBARS) levels have been assessed as described by Draper and Hadley²⁶ based on the spectrophotometrically concentration measurement of malondialdehyde (MDA) and MDA like compounds at 532 nm. The TBARS levels were expressed as nmol of MDA/mg of protein.

Advanced oxidation of protein products (AOPP) levels have been assessed as described by Kayali²⁷ based on the spectrophotometric records at 340 nm. AOPP levels were expressed as was nmoles of AOPP/mg of protein using the extinction coefficient of 261 cm/mM.

SOD, CAT and GPx antioxidant assays

Enzymatic activity of superoxide dismutase (SOD) was assessed as described by Beauchamp and Fridovich²⁸ based on the spectrophotometric records at 560 nm. The SOD activity was expressed as Units/mg of protein. Enzymatic activity of catalase (CAT) was assessed as described by Aebi²⁹ based on spectrophotometrically analyzes at 240 nm. The CAT activity was expressed as µmoles H₂O₂ consumed/min/mg of protein. Enzymatic activity of glutathione peroxidase (GPx) was assessed as described by Flohé and Gunzler³⁰. The GPx activity was expressed as µmoles of GSH oxidized/min/mg of protein.

Histological examination

The inflamed foci of the different paw of rats have been explored for histopathological features. The tissues have processed for standard histological examination purposes following fixation in 10% of formalin solution and embedding in paraffin. Section of 5 μm-thick were cut then stained with hematoxylin-eosin before qualitative histopathological examination underneath microscope (Olympus Inc., Tokyo, Japan).

Statistical analyses

All data represent the mean and standard error of the mean (mean ± SEM). Statistical analyses included the one-way analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test using Graphpad (SPSS, CA) software. Statistical differences were considered significant when p values where less than 5%.

Results

Screening of the chemical composition

The fragmentation patterns of the main identified compounds are listed in below (Fig. 1). The yield of extraction was 15.67 ± 1.15%. As shown in Table 1, fourty-one phytochemicals are reported in the methanolic extract of A. subhirsutum leaves. This extract was dominated by trehalose (17.2%); methyl methanethiolsulfonate (11.43%); 5-oxo-DL-Proline (6.21%); 1-methylcyclopropanemethanol (6.09%); hexadecanoic acid (5.61%); (Z, Z)-9,12-octadecadienoic acid (4.6%); 2-(hydroxymethyl)-2-nitro-1,3-propanediol (3.61%); phytol (3.59%); cyclamic acid (3.54%); propanethial S-oxide (3.44%); diallyl trisulfide (2.58%); allyl methyl trisulfide (2.46%); but-3-enyl (E)-2-methylbut-2-enoate (2.25%); neophytadiene (2.22%); disulfide, methyl 2-propenyl (2.13%); and 1-butanol, 3-methyl-, acetate (2.06%). The list of the identified molecules and their structures are shown in Table 1.

Antimicrobial biological activities of ASE

The growth inhibition zone (mGIZ) recorded on agar Petri dishes grew in a concentration-dependent way (Table 2). The mGIZ varied from 8.33 ± 0.58 mm (P. aeruginosa, M₁₆) to 17.00 ± 1.00 mm (S. epidermidis, M₁₃) at 3 mg/disc. For Candida species, the mGZ (mm) was about 13.33 ± 0.58 mm for all tested species. Using the assay proposed by Gatsing and colleagues (2009)²³ based on the calculated MBC/MIC ratio, A. subhirsutum exhibited bacteriostatic action against the different tested bacterial strains, except S. hominis (Bactericidal action).

Table 2.

Tentative identification of phytoconstituents from A. subhirsutum methanolic extract by using the GC-MS technique.

Peak	Area (%)	RT (min)	Molecular weight	Chemical formula	CAS number	Name
1	11.43	1.464	126	C₂H₆O₂S₂	2949-92-0	Methyl methanethiolsulfonate
2	6.09	1.659	86	C₅H₁₀O	2746-14-7	1-Methylcyclopropanemethanol
3	1.87	1.763	74	C3H₆S	870-23-5	Allyl mercaptan
4	3.54	2.037	179	C₆H₁₃NO₃S	100-88-9	Cyclamic acid
5	0.79	2.426	118	C₆H₁₄O₂	3453-99-4	2,2-Dimethoxybutane
6	0.54	2.675	113	C₇H₁₅N	7003-32-9	2-Methylcyclohexylamine
7	1.14	2.844	113	C₇H₁₅N	55683-33-5	3-methyl-1-(1-methylethyl)- Azetidine
8	3.44	3.024	90	C₃H₆OS	32157-29-2	Propanethial S-oxide
9	2.13	4.229	120	C₄H₈S₂	2179-58-0	Disulfide, methyl 2-propenyl
10	0.92	5.052	136	C₁₀H₁₆	2633-80-9	2-Allylbicyclo [2.2.1] heptane
11	2.25	5.366	154	C₉H₁₄O₂	-	But-3-enyl (E)- 2-methylbut-2-enoate
12	0.28	5.696	114	C₅H₆O₃	4100-80-5	dihydro-3-methyl-2.5-Furandione
13	0.52	6.360	126	C₂H₆O₂S₂	2949-92-0	S-Methyl methanethiosulphonate
14	0.97	6.528	126	C₆H₆O₃	118-71-8	Maltol
15	1.91	6.730	146	C₆H₁₀S₂	2179-57-9	Diallyl disulphide
16	2.06	6.873	130	C₇H₁₄O₂	123-92-2	1-Butanol, 3-methyl-, acetate
17	1.33	7.533	101	C₅H₁₁NO	4801-58-5	Piperidin-1-ol
18	2.46	7.684	152	C₄H₈S₃	34135-85-8	Allyl methyl trisulfide
19	0.38	8.139	143	C₆H₉NO₃	4931-66-2	L-Proline-5-oxo-methyl ester
20	1.01	8.534	114	C₆H₁₀O₂	38653-49-5	Allyl-1,3-Dioxolane
21	0.29	8.730	128	C₇H₁₂O₂	2549-59-9	7-methyl-2-Oxepanone
22	1.05	9.032	113	C₇H₁₅N	3230-23-7	4-Ethylpiperidine
23	0.96	9.438	134	C₄H₆O₅	6915-15-7	Malic Acid
24	0.40	9.630	143	C₈H₁₇NO	7037-49-2	4-Piperidinepropanol
25	2.58	10.094	178	C₆H₁₀S₃	2050-87-5	Diallyl trisulfide
26	0.45	10.169	150	C₉H₁₀O₂	7786-61-0	2-Methoxy-4-vinylphenol
27	0.71	10.427	150	C₉H₁₀O₂	876-02-8	4-hydroxy-2-methylacetophenone
28	0.66	10.978	144	C₇H₁₂O₃	102539-71-9	Oxan-3-ylacetic acid
29	3.61	11.788	151	C₄H₉NO₅	126-11-4	2-(hydroxymethyl)-2-nitro-1,3-Propanediol
30	6.21	12.686	129	C₅H₇NO₃	149-87-1	5-oxo- DL-Proline
31	0.25	13.422	198	C₁₀H₁₄O₄	-	Fumeric acid, ethyl 2-methylallyl ester
32	0.72	14.258	194	C₇H₁₄O₆	3396-99-4	Alpha-D-Galactopyranoside, methyl
33	0.39	15.579	228	C₁₄H₂₈O₂	544-63-8	Tetradecanoic acid
34	2.22	16.460	278	C₂₀H₃₈	504-96-1	Neophytadiene
35	5.61	17.665	256	C₁₆H₃₂O₂	57-10-3	Hexadecanoic acid
36	0.67	17.460	216	C₁₂H₂₄O₃	1883-13-2	3-hydroxydodecanoic acid
37	17.20	17.920	342	C₁₂H₂₂O₁₁	99-20-7	Trehalose
38	0.25	18.505	228	C₁₄H₁₂O₃	131-57-7	Oxybenzone
39	3.59	19.135	296	C₂₀H₄₀O	150-86-7	Phytol
40	4.60	19.360	280	C₁₈H₃₂O₂	60-33-3	(Z, Z)-9,12-Octadecadienoic acid
41	1.03	19.573	284	C₁₈H₃₆O₂	57-11-4	Octadecanoic acid

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%: Percentage; RT: Retention Time; Molecular weight: g/mol; CAS number: Chemical Abstracts Service.

Figure 2 in below represents an example of the diameter of growth inhibition zone recorded on Mueller Hinton agar using 3 mg/disc as A. subhirsutum methanolic extract concentration.

Fig. 2 — Antibacterial activity against *A. baumannii* recorded on Mueller Hinton agar tested at 3 mg/disc using disc diffusion method.

Using the microdilution assay, the MICs values for bacterial strains varied from 4.687 mg/mL to 9.375 mg/mL the different tested bacteria. While MBCs values ranged between 37.5 mg/mL and 150 mg/mL for the same bacterial strains.

In addition, a concentration about 9.375 mg/mL of A. subhirsutum extract was sufficient to inhibit the growth of the different tested yeast strains. While high concentrations (75–150 mg/mL) are needed to completely kill fungal growth (Table 3).

Table 3.

Results of the antimicrobial activities of Allium subhirsutum L. (Leaves) methanolic extract obtained from disc diffusion and microdilution assays as compared to standard drugs (Ampicillin and amphotericin B).

Code	Bacterial Strains Tested	A. subhirsutum L. methanolic extract				Ampicillin
Code	Bacterial Strains Tested	GIZ ± SD	MIC	MBC	MBC/MIC ratio	GIZ ± SD	MIC	MBC
M₁₁	S. hominis	13.67 ± 0.58	9.375	18.75	2; Bactericidal	10.33 ± 0.57	0.625	1.25
M₁₆	P. aeruginosa	8.33 ± 0.58	9.375	75	> 4; Bacteriostatic	6.00 ± 0.00	2.5	5
M₁₄	K. pneumonia	15.33 ± 0.58	9.375	150	> 4; Bacteriostatic	6.00 ± 0.00	0.625	5
M₆	E. coli	13.67 ± 0.58	9.375	150	> 4; Bacteriostatic	6.00 ± 0.00	1.25	5
M₁₇	A. baumannii	14.67 ± 0.58	9.375	150	> 4; Bacteriostatic	10.33 ± 0.57	0.625	2.5
M₈	E. cloacae	14.67 ± 0.58	9.375	75	> 4; Bacteriostatic	6.00 ± 0.00	0.625	1.25
M₉	E. faecium	13.33 ± 0.58	4.687	150	> 4; Bacteriostatic	6.00 ± 0.00	0.625	5
M₁₉	P. aeruginosa	10.67 ± 0.58	4.687	37.5	> 4; Bacteriostatic	6.00 ± 0.00	0.625	1.25
M₁₂	S. aureus	12.67 ± 0.58	4.687	150	> 4; Bacteriostatic	6.00 ± 0.00	0.625	1.25
M₁₃	S. epidermidis	17.00 ± 1.00	4.687	37.5	> 4; Bacteriostatic	24.00 ± 1.00	0.312	0.625
M₂₂	S. aureus (MRSA)	14.33 ± 0.58	4.687	150	> 4; Bacteriostatic	6.00 ± 0.00	0.625	1.25
M₇	E. faecalis	16.33 ± 0.58	4.687	37.5	> 4; Bacteriostatic	6.00 ± 0.00	0.312	2.5

Code	Fungal Stains Tested	*A. subhirsutum* L. methanolic extract				Amphotericin B
Code	Fungal Stains Tested	GIZ ± SD	MIC	MFC	MFC/MIC ratio	Amphotericin B
A₁	C. utilis ATCC 9255	13.33 ± 0.58	9.375	150	> 4; Fungistatic	11.67 ± 0.57	0.78	1.56
A₈	C. tropicalis ATCC 1362	13.33 ± 0.58	9.375	150	> 4; Fungistatic	14.33 ± 0.57	0.195	0.78
A₄	C. guillermondii ATCC 6260	13.33 ± 0.58	9.375	75	> 4; Fungistatic	12.67 ± 0.57	0.195	0.39
A₁₅	C. albicans ATCC 20402	13.33 ± 0.58	9.375	75	> 4; Fungistatic	12.67 ± 0.57	0.195	0.39

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Computational study

ADME profiling

The ADME characteristics of all identified compounds in A. subhirsutum extract revealed that A. subhirsutum compounds can considered as bioactive compounds with acceptable absorption and permeation (Table S1) associated low toxicological profiles (Table 4).

In fact, the predicted physicochemical, lipophilicity, and druggability showed that:

Table 4.

Toxicity profiles of the identified compounds in A. subhirsutum methanolic extract. Number and name of the compounds are same listed in Table 1.

Entry		Identified compounds in A. subhirsutum methanolic extract
Entry		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
T ₁	AMES toxicity	Yes	No	No	No	No	No	No	No	No	No	No	No	Yes	No	No	No	No	No	No	No
T ₂	Maximum tolerated dose (human)	1.058	1.062	1.164	1.062	0.987	0.903	0.794	1.063	0.834	0.129	0.846	1.197	1.058	0.466	0.674	0.938	1.088	0.727	1.149	0.905
T ₃	hERG I inhibitor	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No
T ₄	hERG II inhibitor	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No
T ₅	Oral Rat Acute Toxicity (LD₅₀)	2.632	1.839	2.213	1.814	1.925	2.376	2.267	2.073	2.512	1.521	1.872	2.02	2.632	2.162	2.375	1.766	2.45	2.845	1.999	1.864
T ₆	Oral Rat Chronic Toxicity (LOAEL)	1.803	1.656	1.622	1.19	2.169	1.658	1.692	1.368	1.726	2.28	2.401	2.521	1.803	1.839	1.847	2.411	1.514	1.728	2.39	2.206
T ₇	Hepatotoxicity	No	No	No	Yes	No	Yes	Yes	No	No	No	No	No	No	No	No	No	No	No	No	No
T ₈	Skin Sensitisation	No	No	No	No	Yes	Yes	Yes	No	No	No	Yes	Yes	No	No	Yes	Yes	Yes	Yes	No	Yes
T ₉	T. pyriformis toxicity	-0.48	-1.325	-0.63	0.262	-0.275	-0.575	0.151	-0.428	0.72	0.911	0.38	-1.312	-0.48	-0.294	1.371	-0.188	-0.85	1.416	-0.597	-0.772
T ₁₀	Minnow toxicity	2.242	2.277	2.101	2.308	2.058	2.296	1.743	2.124	1.386	0.807	1.336	2.299	2.242	2.644	0.79	1.361	2.685	1.051	3.089	1.871

Entry		Identified compounds in *A. subhirsutum* methanolic extract
Entry		21	22	23	24	25	26	27	28	29	30	31	32	33	34	35	36	37	38	39	40	41
T ₁	AMES toxicity	No	No	No	No	No	Yes	No	No	Yes	No	No	No	No	No	No	No	No	No	No	No	No
T ₂	Maximum tolerated dose (human)	0.924	0.764	1.212	0.759	0.582	1.067	0.476	1.279	1.371	1.464	0.922	1.898	-0.559	0.272	-0.708	0.878	1.505	0.511	0.05	-0.827	-0.791
T ₃	hERG I inhibitor	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No
T ₄	hERG II inhibitor	No	No	No	No	No	No	No	No	No	No	No	No	No	Yes	No	No	No	No	Yes	No	No
T ₅	Oral Rat Acute Toxicity (LD₅₀)	2.127	2.286	1.818	2.051	2.711	2.076	2.08	1.695	1.609	1.6	2.098	1.281	1.477	1.473	1.44	1.414	1.776	1.913	1.607	1.429	1.406
T ₆	Oral Rat Chroni Toxicity (LOAEL)	2.17	1.6	3.104	1.271	1.857	2.019	2.268	2.602	1.941	2.619	2.371	3.321	3.034	1.158	3.181	2.546	4.072	1.864	1.043	3.187	3.33
T ₇	Hepatotoxicity	No	Yes	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	Yes	No
T ₈	Skin Sensitisation	Yes	Yes	No	Yes	Yes	Yes	Yes	No	No	No	Yes	No	Yes	Yes	Yes	Yes	No	No	Yes	Yes	Yes
T ₉	T. pyriformis toxicity	-0.826	-0.116	0.285	-0.726	2.008	0.071	0.285	-0.11	-0.518	0.241	0.096	0.285	0.978	1.65	0.84	0.287	0.285	1.316	1.884	0.701	0.65
T ₁₀	Minnow toxicity	2.107	2.147	3.348	2.249	0.516	1.957	1.765	2.38	3.345	3.378	1.903	4.603	-0.601	-2.039	-1.083	0.523	9.618	0.266	-1.504	-1.31	-1.565

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T₁: Categorical (Yes/No); T₂: Numeric (log mg/kg/day); T₃: Categorical (Yes/No); T₄: Categorical (Yes/No); T₅: Numeric (mol/kg); T₆: Numeric (log mg/kg_bw/day); T₇: Categorical (Yes/No); T₈: Categorical (Yes/No); T₉: Numeric (log ug/L); T₁₀: Numeric (log mM).

The molecular weight of the tested molecules ranged from 74.14 g/mol (Compound 3 = Allyl mercaptan) to 342.30 g/mol (Compound 37 = Trehalose).
The number of heavy atoms varied from 0 to 23 (Trehalose).
The highest number of aromatic heavy atoms was 12 for the compounds Oxybenzone.
The number of rotatable bonds varied from 1 to 16 and the number of H-bond acceptors from 0 to 11, and the number of H-bond donors varied from 0 to 8.

A typical medicinal chemistry metric for maximizing a drug’s capacity to enter cells is called PSA. Molecules that are difficult to penetrate cell membranes are those having polar surfaces larger than 140 angstroms squared (Å²). A PSA of less than 90 Å² is usually required for chemicals to cross the blood-brain barrier (BBB) and function on receptors in the central nervous system (CNS).

Our predicted results revealed that 4 out of 41 molecules tested have a TPSA value (Å²) higher than 90 angstroms squared interpreted to be less able to cross the BBB. None of the identified molecules displayed TPSA value (Å²) higher than 140 angstroms which can be interpreted as molecules unable to easily penetrate cell membrane. Regarding the predicted consensus Log Po/w, all compounds were found to be highly lipophilic (Consensus values ranging from 0.19 to 7.07). The bioavailability score for almost all identified compounds were about 0.17 to 0.58 highlighting their good oral bioavailability.

In addition, the predicted results showed that almost all identified compounds in the tested A. subhirsutum methanolic extract exhibited important pharmacokinetic properties. In fact, the BBB that controls how many chemicals can enter the CNS from the blood.

Out of the 41 substances, 35 displayed strong BBB permeability, indicating good BBB dispersion. In addition, 17 compounds were expected to not be absorbed by the digestive system and may consequently be ejected.

When using skin penetration coefficients (log Kp) as a measure of the transport of chemicals through mammalian epidermis, the majority of phytoconstituents showed good skin penetration.

The bioavailability radars showed the pink area as the ideal range for each attribute (lipophilicity, size, polarity, solubility, saturation, and flexibility), which can be used to determine druglikeness. The bioavailability findings showed that not all tested compounds fell into the pink zone for at least five parameters and are therefore thought to be drug-like molecules.

The Boiled egg model (Fig. 3) of all compounds was generated. The fact that some of them, including compounds 29, 23, 30, 19, 12, 24, 17, 22, 2, and 36 appear in the white ellipse indicates that the gastrointestinal tract can passively absorb them. While the others with red points were discovered to not be P-gp substrates, compounds 34 and 39 with a blue point suggests that it may be.

Fig. 3 — Boiled Egg model of all the ASE identified compounds, reported in Table 1.

Toxicity prediction

The predicted toxicity of the compounds identified in the A. subhirsutum methanolic extract are shown in Table 4. Four of the identified compounds have mutagenic potential (AMES negative) namely compounds 1 (Methyl methanethiolsulfonate), 13 (S-Methyl methanethiosulphonate), 26 (2-Methoxy-4-vinylphenol), and 29 (2-(hydroxymethyl)-2-nitro-1,3-). All tested compounds have log_LC50>-0.3 (Minnow toxicity); They are regarded as molecules without high acute toxicity. Furthermore, the rat lethal dose (LD₅₀) expressed in (mol/kg) ranged between 1.281 mol/kg (Alpha-D-Galactopyranoside, methyl) and 2.711 mol/kg (Diallyl trisulfide). The predicted LOAEL, in rats, expressed as a numeric value equal to (log mg/kg_bw/day) ranged from 1.19 (Cyclamic acid) to 4.072 (Trehalose). None of the tested molecules was able to inhibit the human ether-related gene (hERG) cardiac potassium channel (hERG I). The compounds 34 (Neophytadiene) and 39 (Phytol) were able to inhibit the hERG II. For the hepatotoxicity prediction, five identified molecules (Cyclamic acid, 2-Methylcyclohexylamine, 3-methyl-1-(1-methylethyl)- Azetidine, 4-Ethylpiperidine, and Z, Z)-9,12-Octadecadienoic acid) were predicted to induce liver injury in silico. Finally, it was predicted that the maximum tolerated dose for humans is about 1.898 (log mg/kg/day) for the compound 32 (Alpha-D-Galactopyranoside, methyl).

Molecular Docking findings

The Tables 5 and 6 exhibit the bindings affinities and the interacting residues. They exhibit also the deep embedding for each of the targeted receptors. Both ligands and macromolecules have been prepared, specifically by removal of water molecules, addition of polar hydrogens and Kollman charges^9,31,32. Molecular docking, which was based on the CHARMm force field, was applied for each phytochemical compound with the different targeted receptors^33,34, using vina software packages. The major reasons behind the selection of these macromolecules 1JIJ, 2XCT, 1HD2, and 1CX2 are their key role in antibacterial, antioxidant and anti-inflammatory pathways, and they are also commonly targeted in similar studies^31,34.

Table 5.

Binding affinity (kcal/mol^− 1) of the Allium subhirsutum identified compounds with the four studied receptors: 1JIJ, 2XCT, 1HD2, and 1CX2.

Compound No.	Targeted receptors
Compound No.	1JIJ (antibac)	2XCT (antibac)	1HD2 (antiox)	Cyclooxygenase-2 (1CX2)
1	−4.1	−3.0	−3.4	−3.7
2	−4.0	−3.5	−3.7	−4.4
3	−2.9	−2.3	−2.4	−3.3
4	−5.9	−4.9	−5.1	−6.5
5	−4.3	−3.9	−3.8	−4.5
6	−5.6	−4.4	−4.8	−5.5
7	−4.5	−3.8	−4.2	−5.1
8	−3.4	−2.7	−3.1	−3.8
9	−3.4	−2.6	−2.9	−3.6
10	−5.5	−4.7	−5.1	−6.3
11	−5.0	−4.1	−4.3	−6.9
12	−5.6	−4.6	−4.8	−5.5
13	−4.1	−3.1	−3.4	−3.7
14	−6.0	−4.7	−5.1	−5.6
15	−3.6	−3.1	−3.2	−4.6
16	−3.7	−3.0	−3.1	−4.6
17	−5.1	−3.9	−4.5	−5.0
18	−3.4	−2.7	−3.1	−3.8
19	−5.6	−4.0	−4.9	−6.1
20	−4.6	−3.7	−4.0	−4.9
21	−5.8	−4.7	−5.1	−6.2
22	−5.2	−4.2	−4.3	−5.4
23	−5.7	−4.3	−4.9	−5.2
24	−5.8	−4.4	−5.1	−5.9
25	−3.9	−3.2	−3.2	−4.5
26	−6.2	−4.8	−4.9	−6.4
27	−6.6	−5.4	−5.1	−6.8
28	−5.8	−4.5	−4.8	−5.8
29	−5.2	−3.8	−4.3	−4.4
30	−5.8	−4.2	−5.0	−6.0
31	−5.5	−4.4	−4.4	−5.0
32	−6.8	−5.5	−5.2	−6.2
33	−5.0	−4.3	−4.3	−7.5
34	−5.4	−4.9	−4.3	−7.2
35	−5.2	−4.6	−3.8	−6.4
36	−3.4	−2.7	−3.1	−3.8
37	−7.5	−6.9	−6.2	−6.3
38	−7.7	−7.0	−5.6	−8.3
39	−5.3	−5.0	−4.1	−7.4
40	−5.0	−4.5	−4.3	−7.2
41	−3.4	−2.7	−3.1	−3.8

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Table 6.

Binding affinity, conventional H-bonds and the closest embedding of the best A. subhirsutum compounds with 1JIJ, 2XCT, 1HD2, and 1CX2.

No.	Affinity (Kcal/Mol)	No. Conventional H-Bonds	Closest Interacting Residues
No.	Affinity (Kcal/Mol)	No. Conventional H-Bonds	Interaction type & Residues	Closest Embedding (Distance, Å)
38	1JIJ	4	Conventional H-Bond: Asp40 (1.99), Tyr170 (2.67), Gln174 (2.25), Asp40 (2.72) Pi-Sigma: His50 (3.78) Alkyl: Ala39 (3.82) Pi-Alkyl: Ala39 (4.73), Leu70 (5.07)	Asp40 (1.999)
38	1CX2	3	Conventional H-Bond: Thr206 (1.98), His386 (2.75), Ala202 (2.29) Pi-Cation: His207 (4.57) Pi-Donor Hydrogen Bond: Trp387 (3.17) Pi-Sigma: His207 (3.64) Pi-Pi T-shaped: His207 (4.78) Amide-Pi Stacked: Trp387/His388 (4.89) Pi-Alkyl: His207 (4.43), His386 (4.29), Leu391 (4.79)	Thr206 (1.984)
37	1HD2	8	Conventional H-Bond: Asn76 (2.12), Asp77 (2.64), Arg124 (2.72), Arg124 (2.20), Arg124 (2.53), Ala42 (2.09), Pro100 (2.69), Val75 (2.52)	Ala42 (2.09)
38	2XCT	2	Conventional H-Bond: Lys1043 (1.93), His1079 (1.94) Pi-Sigma: Val1045 (3.80) Alkyl: Lys1043 (5.07) Pi-Alkyl: His1046 (4.52), Ala1034 (5.44), Ala1089 (5.48)	Lys1043 (1.932)

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Compounds no. 37 and 38 have been predicted to process best binding score, the richest molecular interactions, and were also tightly embedded to the studied receptors. For instance, compound no. 37 established eight conventional H-bonds with the receptor 1HD2 (Figs. 4 and 5). The involved amino acid residues include Asn76, Asp77, Ala42, Pro100, Val75, and four times Arg124. 2XCT and compound no. 38 have good binding affinity of -8.3 kcal/mol, showed 2 conventional H-bonds only but associating a rich network of bonding: Pi-Sigma (Val1045), Alkyl (Lys1043) and Pi-Alkyl (His1046, Ala1034, and Ala1089).

Fig. 4 — 3D view of (A) compound No. 38-1JIJ, (B) Compound No. 38-2XCT, (C) Compound No. 37-1HD2 and (D) Compound No. 38-1CX2.

Fig. 5 — 2D view of diagram of interactions (A) 38-1JIJ, (B) 38-2XCT, (C) 37-1HD2 and (D) 38-1CX2.

In vivo assessment of ASE effects

Inflammation inhibitory effects activity

Changes of edema size were recorded following CAR injection in the paws of the rats. Following CAR injection, the rat paws of CAR, CAR + ASE and CAR + INDO groups increased in diameter, as a result of edema associated inflamed foci. The maximum increase in the size of edema was around the third hour after CAR injection. This increase was more prominent in CAR group, which received no treatment. In fact, both ASE and INDO significantly decreased the acute inflammation as induced by CAR, specifically during the first 3 h (Table S2). After the first 3 h, ASE and INDO inhibited the edema by 58% and 31%, respectively.

Five hours of CAR injection, the decreased paw edema reached about 80%, which represent the most significant anti-inflammatory.

Inflammatory biochemical and hematological markers

After sacrifice, CRP and fibrinogen, as inflammatory biochemical markers, have been assessed for each rat. Table 7 exhibited that both parameters increased significantly in CAR group (p < 0.05) once compared with CTRL. However, ASE and INDO lowered back these change to reach values slightly higher than the CTRL group, specifically for fibrinogen of CAR + ASE for which the level was comparable to the CTRL (1.84 vs. 1.87). Hence, similarly to INDO, the reference drug, ASE significantly prevented the increase of CRP and fibrinogen following the induced acute inflammation.

Table 7.

Biochemical inflammatory markers and hematological parameters in CTRL, CAR, CAR + ASE and CAR + INDO.

	CTRL	CAR	CAR + ASE	CAR + INDO
CRP (mg/L)	0.81 ± 0.01	1.13 ± 0.03 ^a	0.87 ± 0.02 ^b	0.88 ± 0.04 ^b
Fibrinogen (g/L)	1.84 ± 0.16	2.11 ± 0.22 ^a	1.88 ± 0.27 ^a	1.87 ± 0.31 ^b
WBC (10³/µL)	12.43 ± 0.53	18.74 ± 0.93 ^a	15.02 ± 0.66 ^b	16.76 ± 0.54 ^{a, b}
Platelets (10³/mm³)	463 ± 19.21	867 ± 13.62 ^a	548 ± 20.13 ^b	602 ± 18.98 ^{a, b}

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All data represent mean ± SEM. ^ap<0.05 vs. CTRL and ^bp<0.05 vs. CAR group.

While number of red blood corpuscles (RBC), did not varied (data not shown), numeration of both WBC and PLT showed significant increases as compared to CTRL. However, significant decrease in numerations were noticed in CAR + ASE and CAR + INDO groups as compared to CAR group. Nevertheless, the numerations were still not close to normalcy once compared with CTRL group of rats.

Oxidative stress assessment

Table 8 exhibits that CAR increased the skin levels of TBARS and AOPP once compared to the CTRL rats. ASE and INDO significantly reduced the increased levels. Furthermore, the alleviation by ASE was more prominent than INDO.

Table 8.

Pro-oxidant and antioxidant markers in the skin tissues of CTRL, CAR, CAR + ASE and CAR + INDO groups.

	CTRL	CAR	CAR + ASE	CAR + INDO
Pro-oxidants
TBARS	0.39 ± 0.03	1.12 ± 0.05 ^a	0.52 ± 0.05 ^b	0.63 ± 0.04 ^b
AOPP	0.74 ± 0.03	0.99 ± 0.05 ^a	0.82 ± 0.05 ^b	0.80 ± 0.05 ^b
Antioxidants
SOD	3.72 ± 0.31	1.89 ± 0.22 ^a	2.56 ± 0.29 ^b	2.87 ± 0.26 ^b
GPx	9.32 ± 0.62	4.37 ± 0.49 ^a	6.84 ± 0.61 ^b	7.21 ± 0.53 ^b
CAT	1.36 ± 0.08	0.76 ± 0.07 ^a	0.97 ± 0.04 ^b	1.02 ± 0.09 ^b

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All data represent mean ± SEM. ^ap<0.05 vs. CTRL and ^bp<0.05 vs. CAR group.

The activities of enzymatic anti-oxidants (SOD, CAT and GPx) were also assessed and the results are shown in Table 8, which reported significantly lower activities in CAR rats as compared to CTRL rats. However, the CAR injected rats that had been pre-treated with ASE or treated with INDO showed significantly restored activities that were close to the healthy CTRL rats.

Histological findings

The histopathological features in the paw tissues have been assessed using standard histology. The results are shown in Fig. 6. CTRL rats showed healthy histological aspect of the epidermal and its underlying tissues; dermis and skeletal muscle. However, CAR group exhibited vascular dilatation and acute edematasis associated infiltration of leucocytes, particularly the polynuclear neutrophils. The CAR + ASE and CAR + INDO rats exhibited similar results and less prominent inflammatory attributes. In fact, their histological slides exhibited less leakage of neutrophils and intermuscular and dermis edema.

Discussion

To the best of our knowledge, this study reports for the first time the phytochemical composition of the methanolic extract of A. subhirsutum leaves through Gas chromatography–mass spectrometry (GC–MS). Fourty-one compounds have been identified, which are dominated mainly by sulfur compounds and trehalose (17.2%); methyl methanethiolsulfonate (11.43%); 5-oxo-DL-Proline (6.21%); 1-methylcyclopropanemethanol (6.09%); hexadecanoic acid (5.61%); (Z, Z)-9,12-octadecadienoic acid (4.6%); 2-(hydroxymethyl)-2-nitro-1,3-propanediol (3.61%); phytol (3.59%); cyclamic acid (3.54%); propanethial S-oxide (3.44%); diallyl trisulfide (2.58%); allyl methyl trisulfide (2.46%); but-3-enyl (E)-2-methylbut-2-enoate (2.25%); neophytadiene (2.22%); disulfide, methyl 2-propenyl (2.13%); 1-butanol, 3-methyl-, acetate (2.06%) were the dominant identified constituents.

Previous works have studied the phytochemical composition of organic extracts from A. subhirsutum bulbs, leaves and flowers^22,33–38. In fact, Snoussi et al. (2022)²² reported the identification of 25 phytoconstituents from hairy garlic methanolic extract using the LC-MS method, with sebacic acid, 4-Oxomytiloxanthin, cepharanthine, methyl gamboginate, hexadecasphinganine, 11-alpha-acetoxykhivorin, and linolenoyl lysolecithin being the dominant components.

Ethanolic extract of leaves and bulbs of A. subhirsutum L. from Padova, Italy contained several bioactive chemicals³⁶. Alliin, allicin, gamma-glutamyl (S)-allylcysteine, luteolin, methoxy quercetin isomer, glucosyl gallate, and N-trans-feruloyl-tyramine dominated the ethanolic extract from the dried, ground-up bulbs. Additionally, using the LC-ESI-MS/MS approach, Emir and colleagues reported the discovery of thirty different polyphenols in the methanolic extract of A. subhirsutum L. aerial portions.

The methanolic extract of both air-dried and powdered bulbs and aerial portions, the primary phenolic acids found were benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, gallic acid, ferulic acid, p-coumaric acid, and genistein. The headspace volatiles from Turkish crashing bulbs of A. subhirsutum L. were first studied in 2018 by Küçük and colleagues by GC-MS method. Six substances, including allyl methyl disulfide, methyl (methylthiol) methyl disulfide, diallyl disulfide, methyl trans-propenyl disulfide, dimethyl sulfide, and allyl methyl trisulfide, were identified, according to their study.

Our findings were comparable to those of Saoudi and his colleagues (2021)² in terms of the phytochemical makeup. In comparison to the equivalent oil, they reported a high source of phenols, flavonoids and tannins. The solvent employed might be held responsible for the variation in the phytochemical makeup. In fact, our team reported varying levels of these types of chemicals while employing the same Allium species. The amount of phenolics and flavonoids in plant samples is also influenced by their place of origin.

The biological activities screening of A. subhirsutum methanolic extract from leaves newly revealed a potent antibacterial effect against several Gram-positive and Gram-negative microorganisms with high diameter of growth inhibition and low MICs and MBCs values. Similarly, the same extract showed acceptable effect against four Candida species (C. utilis ATCC 9255, C. guillermondii ATCC 6260, C. tropicalis ATCC 1362, and C. albicans ATCC 20402). Few studies have described the antimicrobial activities of A. subhirsutum organic extracts obtained using different organs. In fact, Snoussi and colleagues (2022)²² reported that the aqueous extract of A. subhirsutum bulbs was active against E. coli ATCC 35218, K. pneumoniae ATCC 27736, P. aeruginosa ATCC 27853, P. mirabilis ATCC 29245, P. mirabilis, S. sciuri, S. pyogens, P. aeruginosa, multi-drug resistant S. aureus, E. cloacae, Stenotrophomonas paucimobilis, A. baumannii, C. albicans ATCC 10231, C. vaginalis, C. albicans, and C. neoformans ATCC 14116 strain with a growth inhibition zone (mm ± SD) varying from 6.00 ± 0.01 to 15.66 ± 0.57.

The presence of several phytochemicals with promising activity can be credited for the stated biological characteristics of the studied extract^22,33,34.

In reality, substances such methyl methanethiolsulfonate that is generated in varying levels by members of the Alliaceae family, are an anti-oomycete agent with antibacterial and antimutagenic properties³⁹. The sulfur is also known to prevent several tumors including the colon tumors^34,40. Moreover, several identified sulfur compounds, are also well known to be effective compounds involved in the prevention and treatment of a number of human diseases, including those affecting the endocrine system, cardiovascular system, nervous system, infectious diseases, and cancers^41–44.

Similar to this, the hexadecenoic acid found in hairy garlic’s aqueous extract was reported to possess antiinflammatory, antibacterial, antioxidant, and antitumor bioactivities^45–47. The discovered fatty acids methyl ester have also been reported to have antioxidant and antimicrobial properties^48,49.

Neophytadiene (diterpene) has been shown to function as a plant metabolite, an algal metabolite, an anti-inflammatory and antibacterial agent. It is a diterpene and an alkene. In Tagetes lucida and Senecio doria, this natural substance was discovered^50–52. Several garlic plants, such as A. subhirsutum, were found to suppress the arachidonic acid as well as cyclooxygenase and lipooxygenase metabolites^1,7,53. Recent reports highlighted the inhibition of prostaglandins (PGs) by several phytochemicals^9,54,55.

C-reactive protein (CRP) is produced specifically in the liver and the inflammatory cells of the inflammatory foci^56,57. CRP is a key indicator of the inflammatory process. Its increase usually paralleled the inflammation severity^58,59. Increased levels of CRP in CAR treated groups paralleled previous reports who linked such increase specifically to IL-6^3,4,60. Increased concentrations of fibrinogen paralleled high risk of thrombotic diseases⁶¹, as thrombin and fibrin increased the production of interleukins (particularly IL-6 and IL-8) by both endothelial and mononuclear cells. Similarly, to INDO, ASE lowered back CRP and fibrinogen levels, which indicates the anti-inflammatory potential of our studied plant. The phytochemical profile of ASE might inhibit the release of inflammatory mediators such as PGs, histamines and serotonins.

The hematological analysis revealed increased count of WBCs and increased count of platelets. These findings supported previous results, which reported high release of inflammatory cytokines within the site of inflammation, which resulted in disruptions of WBCs and platelet in experimental inflammation in rats^62,63. Our results confirm the beneficial effects of ASE and its phytochemical composition.

In physiological conditions, an equilibrium exists between pro-oxidants and the enzymatic and non-enzymatic antioxidants. This equilibrium is commonly disrupted during several pathologies, including inflammatory and infectious diseases. In this context, high levels of ROS within the inflammatory foci and its neighboring areas were reported to be a key factor of cells and tissues damage^64–66. The inflammatory storm of cytokines is also an enhancer of ROS production. This could explain the significant increase in TBARS and AOPP following the injection of inflammatory mediators^9,16. Injection of CAR reduced SOD, CAT and GPx activities as well. Chemotaxis of additional inflammatory cells was also reported in disruption of pro-oxidant/antioxidant balance cases^9,67. Similarly to other garlic species, A. subhirsutum, possessed promising antioxidant proprieties, which have direct effects of ROS production and, therefore, on the inflammatory status^1,2,31.

Histological slides belonging to CAR exhibited local inflammatory features, particularly leucocytes infiltration and edema. These aspects have been commonly reported in either acute inflammatory animal models or human associated inflammatory diseases^9,15,68. ASE, as a rich mixture displayed promising anti-inflammatory, antioxidant and antimicrobial potentials^2,4,22. Recently, it was also reported that A. subhirsutum possessed anticancer effect, particularly against Walker 256/B-induced breast cancer skeletal metastases¹.

Molecular docking analysis was carried out between ASE identified compounds and some targeted receptors, which are related to the assessed bioactivities. It has been previously reported that such approach is useful to understand the relationship between the structure and the activities of the compounds, including those of A. subhirsutum. All the ASE identified compounds showed negative binding affinity that support their antimicrobial, antioxidant and anti-inflammatory activities as assessed by different experimental approaches.

Compounds no. 37 and 38 have been predicted to process best binding score, the richest molecular interactions, and were also tightly embedded to the several studied receptors. For instance, compound no. 37 established eight conventional H-bonds with the receptor 1HD2. The involved amino acid residues include Asn76, Asp77, Ala42, Pro100, Val75, and four times Arg124. 2XCT and compound no. 38 have good binding affinity of -8.3 kcal/mol, showed 2 conventional H-bonds only but associating a rich network of bonding: Pi-Sigma (Val1045), Alkyl (Lys1043) and Pi-Alkyl (His1046, Ala1034, and Ala1089). These molecular docking findings support the promising effects of A. subhirsutum compounds. These computational results are predictive findings that require experimental validations. Nevertheless, they paralleled the in vitro and in vivo results regarding the antimicrobial and anti-inflammatory results. Our results supported previous docking studies on TRL6 and N-acetylated Pam2Cys or A. subhirsutum aqueous extract to assess anti-inflammatory, immunotherapeutic potential as well as an antimicrobial effect^69–72. The limitations of the study included the pretreatment following the use of ASE, which indicate preventive effect only and not sure about the possible treatment and/or extrapolation on the inflammatory processes.

Conclusions

Overall, our study reports local injection of CAR resulted in a systemic response, which is characterized by disruptions in several parameters at different levels: biochemical, hematological and histological. ASE prevented inflammation and its potential oxidative injury and antimicrobial secondary infection. In fact, pre-treatment with ASE resulted in similar effect to the reference medication by INDO. The in silico results confirmed the in vivo and in vitro findings and indicated promising molecular interactions through 1JIJ, 1HD2, 2XCT, and 1CX2 pathways. The ADME and pharmacokinetic properties supported also these finding and encourage the eventual use of ASE to counteract inflammation, oxidative injury and microbial infections. Further research studies are required, particularly to decipher the potential effects on some key immune genes involved on the oxidative/antioxidative status and inflammatory pathways.

Supplementary Information

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Supplementary Material 1^{(1.1MB, doc)}

Acknowledgements

This research received no funds. We want to acknowledge the Laboratory of Histophysiology of Developmental and Induced Pathologies (LR19ES12), Faculty of Medicine of Sfax.

Author contributions

Conceptualization, R.B. and M.S.; Data curation, H.B.N. and A.J.S.; Formal analysis, Y.H., H.B.N., N.B. and M.A.; Funding acquisition, M.S.; Investigation, F. B and E.N.; Methodology, R.B., Y.H. and H.B.N.; Resources, M.A.; Software, R.B. and M.S.; Supervision, V.D.F.; Validation, F.B., N.B. and A.J.S.; Visualization, E.N.; Writing – original draft, R.B.; Writing – review & editing, R.B., V.D.F. and M.S.

Funding

This research received no funds.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Institutional review board statement

The animal study protocol was approved by the Ethics Committee of Faculty of Medicine of Sfax-University of Sfax (12/ES/15-2022).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(1.1MB, doc)}

Data Availability Statement

All data generated or analysed during this study are included in this published article.

[CR1] 1.Badraoui, R. et al. Allium subhirsutum L. as a potential source of antioxidant and anticancer bioactive molecules: HR-LCMS phytochemical profiling, in vitro and in vivo Pharmacological study. Antioxidants9, 1003. 10.3390/antiox9101003 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Saoudi, M. et al. The role of Allium subhirsutum L. in the Attenuation of dermal wounds by modulating oxidative stress and inflammation in Wistar albino rats. Molecules26, 4875. https://doi.org/10.3390/molecules26164875 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Badraoui, R. et al. Expression profiling of selected immune genes and trabecular microarchitecture in breast cancer skeletal metastases model: effect of α–tocopherol acetate supplementation. Calcif. Tissue Int.110, 475–488. https://doi.org/10.1007/s00223-021-00931-3 (2022). [DOI] [PubMed] [Google Scholar]

[CR4] 4.Zammel, N. et al. Antioxidant and anti-inflammatory effects of Zingiber officinale Roscoe and Allium subhirsutum: in silico, biochemical and histological study. Foods10 (6), 1383. 10.3390/foods10061383 (2021). [DOI] [PMC free article] [PubMed]

[CR5] 5.Capasso, F., Dunn, C. J., Yamamoto, S., Willoughby, D. A. & Giroud, J. P. Further studies on carrageenan-induced pleurisy in rats. J. Pathol.116, 117–124. 10.1002/path.1711160208 (1975). [DOI] [PubMed] [Google Scholar]

[CR6] 6.Radhakrishnan, R., Moore, S. A. & Sluka, K. A. Unilateral Carrageenan injection into muscle or joint induces chronic bilateral hyperalgesia in rats. Pain104, 567–577. 10.1016/s0304-3959(03)00114-3 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Posadas, I. et al. Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and displays differential nitric oxide cyclooxygenase-2 expression. Br. J. Pharmacol.142, 331–338. 10.1038/sj.bjp.0705650 (2004). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.Patil, K. R. et al. Animal models of inflammation for screening of Anti-inflammatory drugs: implications for the discovery and development of phytopharmaceuticals. Int. J. Mol. Sci.20 (18), 4367. 10.3390/ijms20184367 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.Lakhrem, M. et al. Antioxidant, and Anti-Inflammatory activities of sulfated polysaccharide derived from Codium tomentosum: Pharmacokinetic assay. Pharmaceuticals (Basel). 17 (6), 672. 10.3390/ph17060672 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Widyarini, S., Sugiyono, Akrom, A. M. & Paryuni, A. D. Carrageenan-Induced acute inflammation on Back-Skin of mice: histopathological features, number of inflammatory cells, and expression of COX-2, COX-1, and IL-6. World Vet. J.13 (4), 520–530. 10.54203/scil.2023.wvj55 (2023). [Google Scholar]

[CR11] 11.Hussain, T. et al. Oxidative stress and inflammation: what polyphenols can do for us?? Oxidative medicine and cellular longevity, 7432797. (2016). 10.1155/2016/7432797 [DOI] [PMC free article] [PubMed]

[CR12] 12.Mahmoud, A. M., Wilkinson, F. L., Sandhu, M. A. & Lightfoot, A. P. The interplay of oxidative stress and inflammation: mechanistic insights and therapeutic potential of antioxidants. Oxidative Med. Cell. Longev.1, 9851914. 10.1155/2021/9851914 (2021). [Google Scholar]

[CR13] 13.Bezerra, F. S. et al. Oxidative stress and inflammation in acute and chronic lung injuries. Antioxidants12 (3), 548. 10.3390/antiox12030548 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Jelic, M. D., Mandic, A. D., Maricic, S. M. & Srdjenovic, B. U. Oxidative stress and its role in cancer. J. Cancer Res. Ther.17 (1), 22–28. 10.4103/jcrt.JCRT_862_16 (2021). [DOI] [PubMed] [Google Scholar]

[CR15] 15.Jedli, O. et al. Attenuation of ovalbumin-induced inflammation and lung oxidative injury in asthmatic rats by Zingiber officinale extract: combined in Silico and in vivo study on antioxidant potential, STAT6 and TNF-α pathways. 3 Biotech.12 (9), 191. https://doi.org/10.1007/s13205-022-03249-5 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Harirforoosh, S., Asghar, W. & Jamali, F. Adverse effects of nonsteroidal antiinflammatory drugs: an update of gastrointestinal, cardiovascular and renal complications. J. Pharm. Pharm. Sci.16 (5), 821–847. 10.18433/j3vw2f (2013). [DOI] [PubMed] [Google Scholar]

[CR17] 17.James, M. W. & Hawkey, C. J. Assessment of non-steroidal anti-inflammatory drug (NSAID) damage in the human Gastrointestinal tract. Br. J. Clin. Pharmacol.56, 146–155. 10.1046/j.1365-2125.2003.01934.x (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR18] 18.Zhang, Y-J. et al. Antioxidant phytochemicals for the prevention and treatment of chronic diseases. Molecules20 (12), 21138–21156. 10.3390/molecules201219753 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Kumar, A. et al. Major phytochemicals: recent advances in health benefits and extraction method. Molecules28 (2), 887. 10.3390/molecules28020887 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Pharmacokinetic and phytochemical screening of Allium subhirsutum leaves for antimicrobial and anti-inflammatory properties

Riadh Badraoui

Yasser Al-Hazmi

Faten Brahmi

Hmed Ben-Nasr

Emira Noumi

Nouha Bouali

Mohd Adnan

Arif J Siddiqui

Vincenzo De Feo

Mejdi Snoussi

Abstract

Supplementary Information

Introduction

Materials and methods

Plant material and extraction procedure

phytochemical composition

Biological properties of A. subhirsutum methanolic extract

Antimicrobial activities

Table 1.

Computational study

Pharmacokinetic properties

Molecular Docking and interaction assessment

Animals and in vivo experimental procedure

Sample preparation

Inflammatory biomarkers

Hematological parameters

Oxidative injury assessment

TBARS and AOPP pro-oxidant assays

SOD, CAT and GPx antioxidant assays

Histological examination

Statistical analyses

Results

Screening of the chemical composition

Fig. 1.

Antimicrobial biological activities of ASE

Table 2.

Fig. 2.

Table 3.

Computational study

ADME profiling

Table 4.

Fig. 3.

Toxicity prediction

Molecular Docking findings

Table 5.

Table 6.

Fig. 4.

Fig. 5.

In vivo assessment of ASE effects

Inflammation inhibitory effects activity

Inflammatory biochemical and hematological markers

Table 7.

Oxidative stress assessment

Table 8.

Histological findings

Fig. 6.

Discussion

Conclusions

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Competing interests

Institutional review board statement

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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