ReseaOurchers' findings for ArJ essential oil antimicrobial activity are consistent with previously published data ^{[17][24][69][70][71][72]}[17,24,82,83,84,85]. Benmansour et al. demonstrated that ArJ essential oil had an excellent inhibitory effect against tested MRSA (methicillin-resistant Staphylococcus aureus), S. aureus, and B. subtilis [17], which is consistent with ouresearchers' results. Benderradji et al. showed that petroleum ether and ethyl acetate extracts of A. sahariensis leaves had the highest inhibitory activity against most tested strains. The most reported significant inhibition zone was obtained with chloroform extract of the plant against Pseudomonas ^[69][82]. These findings partially corroborate ouresearchers' results, since ArJ essential oil could not kill Pseudomonas, which might be a consequence of the essential oil and extract’s differing phytochemical contents, as well as species variations. Elazzouzia et al. demonstrated that the essential oil of A. ifranensis had highly potent antibacterial activity against the tested S. aureus ^[73][55], which is, again, consistent with ouresearchers' results. Kazemi et al. demonstrated that the essential oil of the aerial parts of A. kermanensis had highly potent antibacterial activity against B. subtilis, P. aeruginosa, and S. aureus, which is partially consistent with researcheours' results ^[72][85]. Al-Wahaibi et al. demonstrated that essential oils derived from A. judaica and A. herba-alba had potent antimicrobial potential against the tested organisms, including Aspergillus fumigatus, Syncephalastrum racemosum, Geotricum candidum Candida albicans, Streptococcus pneumoniae, Bacillus subtilis, and Escherichia coli, except Pseudomonas aeruginosa; these results are consistent with ouresearchers' results [24]. The results of ouresearchers' experiment study indicated that ArJ essential oil has highly potent antimicrobial activity, demonstrating that ArJ essential oil could be a promising antimicrobial drug candidate and can cure various human infections, e.g., wound infections, boils, acne, etc., caused by various life-threatening pathogens, including bacteria and fungi. These results encouraged us to conduct wound healing testing on an animal model to verify the antimicrobial properties of ArJ essential oil in-vivo.

5. In Vivo Skin Burn Wound Healing

In thise entrcurrent study, the second-degree burn was induced on female rats based on the recent publication that observed significant wound healing in second and third-degree wounds ^[74][57]. The choice of three-month-old female rats was due to their quicker wound healing and greater wound contraction ability as compared to males ^[75][86].

5.1. Morphological Appearance and Histological Analysis of the Wounds

The observations of wounds over three weeks of treatment revealed the significant progression in the healing process among the treated groups. At the time of burn induction, the skin burns produced were whitish in color and round in shape. After one week of the treatment, a crust developed on the wound along with the disappearance of edema in the treated groups (ArJ and SS groups). The wound area started decreasing by the second week of the treatment; however, edema formation was still prominent in the untreated burn area. At the end of 3 weeks, the treatment wound area for both the ArJ and SS groups demonstrated recovery, while the untreated zone did not recover completely (Figure 3).

Figure 3. Morphological appearances of Artemisia judaica (G1)- and silver sulfadiazine (SS) (G2)-treated wounds at various time points.

Wound areas were measured using freely available Image J software. No significant differences were observed among the groups after the induction of skin burn. Two weeks after the treatment, the ArJ group’s wound area decreased significantly (p  = 0.04), while the SS group’s wound size remained insignificant compared to the negative group (Supplementary file, Figure S2).

Wound healing is a complex restorative process of injured tissue to its original state ^[76][87], and it involves hemostasis, inflammation, proliferation, and remodeling ^[77][88] to prevent complex metabolic alteration affecting body organ systems. During the process of hemostasis, blood coagulation occurs, while the inflammation process ensures safety from invasive pathogens ^[77][88], thereby facilitating the proliferation step ^[78][89] towards remodeling the tissue maturation process ^[78][79][89,90]. During skin burn, cells and tissues are damaged substantially, thereby involving a complicated healing network compared to wound incision ^[76][87]. Based on the deepness of the burn wounds, they are categorized as first-, second-, and third-degree burns. The first-degree burn is generally red or gray without any blisters and normal capillary network, while in a second-degree burn, blisters and partial-thickness damage to the dermis are observed. Second- and third-degree burns are treated similarly ^[80][91]. In thise entrcurrent study, second-degree skin burn wounds were induced, which healed over 3 weeks for the ArJ and SS groups.

H&E staining was performed for all the animal groups (Figure 4). The H&E staining demonstrated epidermis integrity and the degree of neutrophilic infiltration in the dermis and capillaries of the ArJ and SS groups compared to the untreated wound zone. Wound healing visual appearance for ArJ was not enlarged, most probably due to the balm effect of paraffin (Figure 4B). Tissue sections were analyzed qualitatively for the amount of fibroblast, collagen, inflammation, and neovascularization in SS and ArJ groups. The data demonstrated increased collagen, fibroblast, and neovascularization, with decreased inflammation in the SS and ArJ groups compared with the negative control group (Supplementary file, Figure S3).

Figure 4. Histological analysis using hematoxylin and eosin staining of control (A), Artemisia judaica (B), and SS (C). Arrows (black) indicate neutrophil infiltration; magnification 40×.

5.2. Role of Antioxidants and Oxidative Stress Markers in Wound Healing

The ArJ ointment group demonstrated significantly increased antioxidant SOD (p = 0.03) and CAT (p < 0.01) enzymatic activities compared to the negative group. The SOD and CAT activities were comparable in the intact and negative control. The SS treated group demonstrated a significant difference in CAT activity compared to the negative group (p = 0.01) and was comparable to the ArJ group, while the differences were insignificant for SOD activity. The antioxidant activity contributing towards wound healing is in accordance with previous studies reporting enhanced wound healing due to potent antioxidant activities ^[81][92]. LP significantly increased in the negative control group (p < 0.0001) compared to the intact group, which accords with previously recorded data ^[82][93]. No significant differences in LP were observed in treatment groups with either ArJ or SS groups compared to the negative control, while the ArJ and SS treated groups exhibited significant increases (p < 0.0001) compared to the intact group (Table 5).

Table 5.

 Effect of 

Artemisia judaica

 ointment on antioxidant and oxidant levels in skin burn rat model.

Groups	CAT	SOD	LP
	ng/g
I. Intact control	1.35 ± 0.05 A,B	0.04 ± 0.00 A	836.9 ± 37.75 A
II. Negative control (skin burn without treatment)	1.11 ± 0.06 A	0.04 ± 0.01 A	1214 ± 51.46 B
III. Silver sulfadiazine	1.79 ± 0.204 B	0.19 ± 0.06 A,B	1197 ± 30.30 B
IV. Artemisia judaica	1.82 ± 0.17 B	0.37 ± 0.13 B	1291 ± 18.85 B

Values are denoted as means ± SEM (Supplementary file, Tables S1–S3). Statistical significance was performed using one-way ANOVA, followed by a post hoc test on GraphPad Prism 8.0.2. CAT = catalase, LP = lipid peroxide, SOD = Superoxide dismutase. The mean values that do not share a superscript letter (A,B) in the respective columns of superoxide dismutase (SOD), catalase (CAT), and lipid peroxide (LP) are significantly different (p < 0.05) using Tukey’s multi-group comparisons.

5.3. Role of Pro- and Anti-Inflammatory Markers in Wound Healing

The pro-inflammatory markers IL-1b and IL-6 values were comparable among all the studied groups, similar to the previously published articles ^[83][84][94,95]. At the same time, tumor necrosis factor α (TNF-α) significantly increased in the negative control group compared to the intact group (p < 0.0001). TNF-α values decreased significantly after treatment with SS (p < 0.02) and ArJ (p < 0.002) compared to the negative control group. TNF-α, the inflammatory cytokine activated during acute inflammation by macrophages/monocytes, plays a vital role in cell signaling, leading to necrosis or apoptosis. TNF-α participates in vasodilatation and edema formation and leukocyte adhesion to the epithelium through the expression of adhesion molecules. Furthermore, TNF-α regulates blood coagulation, contributes to oxidative stress at sites of inflammation, and indirectly induces fever. The data conforms with Gushiken and Periera, where TNF-α decreased after two weeks of treatment in skin wound tissue compared to the negative control ^[83][84][94,95].

The anti-inflammatory or the pro-angiogenic markers IL-10 and transforming growth factor beta 1 (TGF-b1) increased significantly in both ArJ (p < 0.0001 and p < 0.0001) and SS (p < 0.0001 and p < 0.0001) groups compared to the negative and intact control groups. However, differences in IL-10 and TGF-b1 levels in the negative control group were insignificant compared to the intact group (Table 6). The data confirm previous studies where IL-10 increased significantly in skin wound healing tissue after two weeks of treatment compared to the negative control group ^[83][84][94,95].

Table 6.

 Effect of 

Artemisia judaica

 ointment on inflammatory and pro-angiogenic markers in skin burn rat model.

Groups	IL-1b	IL-6	TNF-α	TGF-b1	IL-10
	ng/g
I. Intact control	20.77 ±1.95 A	806.1 ±10.20 A	9.57 ±0.55 A	4.19 ±0.24 A	3.54 ±0.19 A
II. Negative control (skin burn without treatment)	19.37 ± 2.33 A	776.2 ±32.77 A	15.54 ± 0.92 B	3.87 ± 0.09 A	2.99 ± 0.25 A
III. Sulfadiazine	23.55± 0.88 A	789.4 ±18.02 A	12.85± 0.26 C	19.28± 0.30 B	12.68± 0.15 B
IV. Artemisia judaica	19.55 ± 1.34 A	869.2 ±51.91 A	11.96 ± 0.34 C	19.18 ± 0.33 B	13.39 ± 0.35 B

Values are denoted as means ± SEM (Supplementary file, Tables S4–S8). Statistical significance was performed using one-way ANOVA, followed by a post hoc test on GraphPad Prism 8.0.2. The mean values that do not share a superscript letter (A–C) in the respective columns of interleukin-1 (IL-1b), IL-6, IL-10, transforming growth factor beta 1 (TGF-b1), and tumor necrosis factor α (TNF-α) are significantly different (p < 0.05) using Tukey’s multi-group comparisons.

Various plant and herbal products are economically cheap to procure and demonstrate modest therapeutic potency with minimum toxicity relative to synthetic drugs ^{[45][46][47][85]}[45,46,47,96]. Eupolin ointment, derived from an aqueous extract of C. odorata leaves, is the first Vietnam FDA-approved product ^[86][59]. The wound healing mechanism, even though it remains unclear, nevertheless enhanced blood flow, decreased inflammatory response, and reduced infection rates, all of which are contributing factors to angiogenesis. Rats are loose-skinned, in contrast to the tight human skin, with quicker constriction of the wound than the epithelization process; as such, rat wound healing, even though it is resemblant, is not entirely similar to wound healing in human skin ^[87][88][97,98]. However, rats are widely used animal models due to their genetic and behavioral similarity, ease of handling, and being economically viable. Thus, the rat skin burn model serves as a vital knowledge resource.

Mortality has been one of the major concerns in patients with deep burns due to infections, and researchers have attempted to minimize wound infection risk and accelerate the healing process ^[89][58]. Topical antimicrobial ointments are commonly employed for such purposes as SS 1% with low toxicity and have a potent antibacterial effect in burn wound therapy management ^[90][91][92][99,100,101].

Increased antioxidant levels have demonstrated wound healing potency ^[93][102] by protecting tissue from oxidative stress ^[94][95][103,104]. In thise entrcurrent study, ArJ demonstrated antioxidant (augmented SOD and CAT) enzymatic activities, validating the role of antioxidant enzymes, in addition to potentiating wound healing through increased anti-inflammatory levels. TGF-b1 is involved in wound healing, angiogenesis, immune regulation, and cancer. On the contrary, TGF-b1, along with inflammatory marker IL-6, helps in T helper 17 differentiation (Th17), aggravating inflammation ^[96][105]. In researcheours' study, the levels of TGF-b1 increased significantly while no statistical difference was observed for IL-1b and IL-6 in the ArJ group compared to the control group.

Moreover, IL-10 is a key regulator of the immune system by limiting the inflammatory response, which could otherwise cause tissue damage. In one study, IL-10 knockout mice were found prone to colitis ^[97][98][106,107], and blocking of IL-10 resulted in severe pathology. On the contrary, increased IL-10 levels cause chronic infection, and IL-10 blocking paved the way for pathogen clearance ^[99][108]. Mucosal secretion of IL-10 and TNF-α were augmented during wound healing, demonstrating the protective effect of IL-10 against inflammation. On the contrary, treatment with P. pinnata increased the serum IL-10 concentration while downregulating TNF-α and IL-6 [45]. ReseaOurchers' experiment study showed a significant increase in IL-10 concomitant with a significant decrease in TNF-α in ArJ compared to the negative control group, thus confirming the potential role of IL-10 in promoting wound healing.

The current findings for the in vivo and in vitro antioxidant activity and the anti-inflammatory effect of ArJ could be attributed to the presence of higher percentages of oxygenated monoterpenes (57.2%) in the plant essential oils ^[100][109]. Oxygenated monoterpenes have been found as major constituents in the plants used traditionally to accelerate wound healing, e.g., the plant essential oil of Helichrysum italicum ^[101][102][110,111] has exhibited a primary role in potential antimicrobial activity and anti-inflammatory effects ^[102][111]. Furthermore, some of the major ArJ essential oils reported as antioxidant and anti-inflammatory agents, e.g., thujone (both α and β-thujone, 9.55%), 1,8-cineole (2.56%), camphor (1.92%), and borneol (0.47%), have chiefly contributed to the rosemary anti-inflammatory effect ^[103][112]. Furthermore, 1,8-cineole antioxidant and anti-inflammatory effects have been reported, and the compound effect as an inhibitor for the inflammatory markers, TNF-α, IL-6, IL-8, LTB 4, PGE 2, and IL-1β, as well as down-regulation of 5-lipoxygenase (LOX) and cyclooxygenase (COX) pathways, are well documented ^[104][105][113,114]. Moreover, methyl cinnamate, a major constituent in ArJ essential oil (4.02%), has demonstrated potent anti-inflammatory activity ^[106][115]. All these compounds participated in the antioxidant and anti-inflammatory effects of the ArJ essential oil as well as in the wound healing activity. However, other identified constituents in this articlentry could also be playing a role in the demonstrated plant activities.