Topical Emulsion Containing Lavandula stoechas Essential Oil as a Therapeutic Agent for Cutaneous Wound Healing

: Background and objectives: The present research was designed to evaluate the chemical composition of Lavandula stoechas essential oil (EOLS) as well as the in vivo wound-healing property. The chemical composition of EOLS was identiﬁed by gas chromatography mass spectrometry. Nine-teen compounds of EOLS were reported. Linalool was identiﬁed as the major chemical compound (24.87%), followed by linalyl acetate (19.10%). EOLS showed a high content of oxygenated compounds (63.54%). In vivo wound healing activity of the topical cream prepared from EOLS (0.5% w / w ) was assessed using a circular excision wound model. The wound area (mm 2 ) in all animal groups was estimated and measured on day 0, 4, 8, 11, and 16. Results: The EOLS formulation cream (0.5% v / w ) showed the highest effect on wound models when compared to reference Madecassol ® (Asiaticoside). On days 4, 11, and 16, wound contractions were 26.4%, 78%, and 96.3% for the EOLS-treated group, and 8.5%, 64.1%, and 86.1% for the vehicle cream-treated group. Animals treated with EOLS cream showed a signiﬁcant decrease in the epithelization period, wound area, and scar thickness, whereas the rate of wound contraction signiﬁcantly increased. This is the ﬁrst such report to be published. Histological analyses were also consistent with the results of the excision experimental method. Treatment with EOLS cream formulation resulted in decreased inﬂammation and an increased rate of tissue perfusion and proliferation as well as remodeling, along with re-epithelization. Conclusions: Our results support the use of EOLS in the development of pharmaceuticals for the management of wounds, and/or inﬂammatory-related diseases. Additional studies are needed to elucidate and explain the exact mechanism of its pharmacological activity.


Introduction
Wound diseases and inflammatory-related illnesses have been more common in recent decades. Cure complexity, an increase in multidrug-resistant bacteria, side effects of medical therapies, and medication costs are both reasons for the need for the identification and development of new, efficient treatments with low toxicity and low cost [1]. Aromatic herbs and medicinal plants as well as volatile oils derived from them have long been used for therapeutic and medicinal uses [2]. Many papers and publications in recent years have identified the immense ability of these essential oils (EOs) and their chemical compounds, with several articles highlighting their antinociceptive, fungicidal, antioxidant, and antitumor properties [2][3][4][5][6].

Determination of Chemical Composition of Essential Oil
Analysis and identification of the volatile compounds were performed using a Shimadzu GC-17A gas chromatograph coupled with a Shimadzu QP-5050A MS detector (Shimadzu Corporation, Kyoto, Japan). The GC-MS system was equipped with a TRACSIL Meta.X5 (95% dimethylpolysiloxane and 5% diphenylpolysiloxane) column (60 m × 0.25 mm, 0.25 µm film thickness; Teknokroma S. Coop. C. Ltd., Barcelona, Spain). Analyses were carried out using He as a carrier gas at a column flow rate of 0.3 mL/min and a total flow of 3.9 mL/min in a split ratio of 1:50 and the following program: (a) 50 • C for 0 min; (b) increase of 3 • C/min from 80 • C to 240 • C and hold for 1 min; (c) increase of 25 • C/min from 240 • C to 300 • C and hold for 3 min. The temperatures of the injector and detector were 230 • C and 300 • C, respectively. All compounds were identified by comparison of their mass spectra with NIST05 and Wiley spectral library collections.

In Vivo Wound Healing Activity Preparation of Test Samples for Bioassay
The tissue repair property was determined using an excision wound model (two rats per group). EOLS was formulated into a topical cream emulsion (0.5%, w/w) for the in vivo wound models ( Table 1). The abovementioned cream was made by precisely measuring the hydrophilic and lipophilic phase components, placing them in different beakers, and heating them. The lipophilic process was created by melting the waxes and emulsifiers (stearic acid, cetylic alcohol, stearyl alcohol and ceteareth-20) and constantly combining the substances. The water-soluble ingredients (octyldodecanol, xanthan gum and glycerin) were dissolved in deionized water to create the aqueous form. The two phases were heated to 65 • C before all of the components were dissolved. When the oil and water phases were both at the same temperature (65 • C), the aqueous phase was gradually combined with the lipophilic phase while stirring until the cream melted and cooled. To create a semisolid cream base (Figure 1), the topical emulsion When the oil and water phases were both at the same temperature (65 °C), the aqueous phase was gradually combined with the lipophilic phase while stirring until the cream melted and cooled. To create a semisolid cream base (Figure 1), the topical emulsion was cooled. Following the creation of a wound with a surgical instrument, a quantity of each test ointment was added topically to the wounded area. The animals in the vehicle group received only the cream base, while the rats in the positive control group received Madecassol ® 1% (Asiaticoside) cream.

Circular Excision Wound Model
A spherical excision method has been used to track tissue repair and closure time. Each animal group was sedated with 0.01 mL of Thiopental Rotexmedica ® (Sodium thiopental). Shaving was used to remove the animals' back hairs. Every animal had a superficial wound shaped on the dorsal inter-scapular area by excising the skin with a 2 cm biopsy punch; the wounds were left wide open [17].
The EOLS cream, the standard drug (Madecassol, 1%), and the vehicle ointments base were applied topically once daily until the wound recovered properly (day 16). Every other day, translucent tracing paper was used to track the development of the wound region. Furthermore, the wound region was assessed using an AutoCAD method. Using the formula given, wound contraction was determined as a percent of the contraction in the wounded area: % wound contraction initial wound area specific day wound area initial wound area 100 Each group of animals had a tissue sample obtained from the repaired skin for histology examination.

Histology Examination
On day 17, the end of the study, skin samples from each group were isolated. Specimens were put in 10% buffered formalin, handled, and paraffin-blocked before being sectioned into 5 micrometer sections and stained with (hematoxylin and eosin) H&E stains. A light microscope (Olympus CX41) was used to examine skin tissues, which were rated as medium (+), intermediate (++), or extreme (+++) for epithelial or cutaneous remodeling. To score epithelial tissue or subcutaneous remodeling, re-epithelization or ulcers in the skin, fibroblast formation, polymorphonuclear cells, neo-vascularization, and collagen depositions in the layers of the skin were investigated. Ultimately, all tissue repair treatments were integrated and designed for wound healing processes such as infection, regeneration, and remodeling in all groups [17]. Histopathologic findings were deemed non-parametric, and no statistical study was carried out.

Circular Excision Wound Model
A spherical excision method has been used to track tissue repair and closure time. Each animal group was sedated with 0.01 mL of Thiopental Rotexmedica ® (Sodium thiopental). Shaving was used to remove the animals' back hairs. Every animal had a superficial wound shaped on the dorsal inter-scapular area by excising the skin with a 2 cm biopsy punch; the wounds were left wide open [17].
The EOLS cream, the standard drug (Madecassol, 1%), and the vehicle ointments base were applied topically once daily until the wound recovered properly (day 16). Every other day, translucent tracing paper was used to track the development of the wound region. Furthermore, the wound region was assessed using an AutoCAD method. Using the formula given, wound contraction was determined as a percent of the contraction in the wounded area: % wound contraction = initial wound area − specific day wound area initial wound area × 100 Each group of animals had a tissue sample obtained from the repaired skin for histology examination.

Histology Examination
On day 17, the end of the study, skin samples from each group were isolated. Specimens were put in 10% buffered formalin, handled, and paraffin-blocked before being sectioned into 5 micrometer sections and stained with (hematoxylin and eosin) H&E stains. A light microscope (Olympus CX41) was used to examine skin tissues, which were rated as medium (+), intermediate (++), or extreme (+++) for epithelial or cutaneous remodeling. To score epithelial tissue or subcutaneous remodeling, re-epithelization or ulcers in the skin, fibroblast formation, polymorphonuclear cells, neo-vascularization, and collagen depositions in the layers of the skin were investigated. Ultimately, all tissue repair treatments were integrated and designed for wound healing processes such as infection, regeneration, and remodeling in all groups [17]. Histopathologic findings were deemed non-parametric, and no statistical study was carried out.

Statistical Analysis
Mean values of treated groups (EOLS and Madecassol creams) were compared with those of a vehicle group and analyzed using statistical tests. Comparison between different groups was carried out using the one-way analysis of variance (ANOVA). Differences with p < 0.05 between experimental groups were considered statistically significant. Statistical data analysis was carried out using XLStat 2014 software (Addinsoft, Paris, France).
As previously mentioned, variations in the chemical composition of EOs presented in several reports and publications are most likely due to differences in distillation operational conditions as well as the chemical components of lavender, which are affected by plant types and varieties, phenological transformations, storage, extraction methods, climatic and growth conditions, and harvesting time [11,16,42].
Granger et al. [43] studied eight samples of EOLS from different areas (Southern France, Corsica, Spain, and Turkey). All EOs were characterized by the importance of camphor and/or fenchone, which represented 74−98% of the EO, with one or the other ketone predominant.
The comparison of EO chemical composition among the analyzed Lavandula species indicated that numerous components such as carvacrol, thymol, limonene, farnesene and thymol methyl ether, were restricted to Lavandula multifidi and L. coronopifolia. Additionally, all major L. stoechas constituents (fenchone, comphor and lavandulyl acetate) and a number of minor ones (eucalyptol, borneol, bornyl acetate, myrtenyl acetate, and cadalene) were absent in L. coronopifolia and L. multifida. This chemical difference across species implies that the concentration of organic compounds may be useful in assessing chemotaxonomy. The chemical compositions of lavender essential oils vary greatly due to environmental factors such as latitude, atmospheric pressure, relative humidity, and precipitation, which influence the relative chemical substances in the oil based on how, where, and when the plant was grown and collected [44,45]. The percentage of linalool, the oil's main chemical component, has been observed to be altered significantly in response to fluctuations in various environmental conditions. For illustration, scientists found one population of lavender cultivated in North Greece with linalool percentages fluctuating from 48.71% to 35.1% over a few weeks due to temperature changes and the duration since the most recent precipitation.

Effect of Lavender Essential Oil on Percent Wound Contraction and Area
The use of EOs in skincare is rapidly expanding around the globe. Due to the extreme renewed interest in phytochemicals such as EOs, it is important to understand their potential in wound healing for application areas in human wellbeing and health. On days 0, 4, 8, 11, and 16, the wound area (mm 2 ) was determined and analyzed in all animal types ( Figure 3). When compared to the control group, animals treated with EOLS cream formulation (0.5%, w/w) had a smaller wound area.

Effect of Lavender Essential Oil on Percent Wound Contraction and Area
The use of EOs in skincare is rapidly expanding around the globe. Due to the extreme renewed interest in phytochemicals such as EOs, it is important to understand their potential in wound healing for application areas in human wellbeing and health. On days 0, 4, 8, 11, and 16, the wound area (mm 2 ) was determined and analyzed in all animal types (Figure 3). When compared to the control group, animals treated with EOLS cream formulation (0.5 %, w/w) had a smaller wound area.  Figure 4 illustrates the wound-healing development as a result of wound contraction. When EOLS topical emulsion was applied to rats, the percent wound contraction rate improved when compared to control group animals ( Figure 5). In the excision wound model, the EOLS formulation cream was shown to have therapeutic potential, while the vehicle group had no substantial wound healing activity. On days 4, 11, and 16, wound contractions were 26.4%, 78%, and 96.3% for the EOLS-treated group, and 8.5%, 64.1%, and 86.1% for the vehicle cream-treated group.  Figure 4 illustrates the wound-healing development as a result of wound contraction. When EOLS topical emulsion was applied to rats, the percent wound contraction rate improved when compared to control group animals ( Figure 5). In the excision wound model, the EOLS formulation cream was shown to have therapeutic potential, while the vehicle group had no substantial wound healing activity. On days 4, 11, and 16, wound contractions were 26.4%, 78%, and 96.3% for the EOLS-treated group, and 8.5%, 64.1%, and 86.1% for the vehicle cream-treated group.

Histological Examination
Histopathology was performed on skin samples. Histological analyses supported the findings of the excision experimental study. Representative photomicrographs (Figure 6) stained with H&E were also used to demonstrate the wound healing phase. Among the experimental classes, different stages of wound healing processes were studied. Among the experimental groups, wound healing stages (inflammation, proliferation, and remodeling) were observed and documented (Table 4). Wound healing processes were delayed in the negative control group, while quicker remodeling was found in the test groups in different degrees.

Histological Examination
Histopathology was performed on skin samples. Histological analyses supported the findings of the excision experimental study. Representative photomicrographs ( Figure 6) stained with H&E were also used to demonstrate the wound healing phase. Among the experimental classes, different stages of wound healing processes were studied. Among the experimental groups, wound healing stages (inflammation, proliferation, and remodeling) were observed and documented (Table 4). Wound healing processes were delayed in the negative control group, while quicker remodeling was found in the test groups in different degrees. Various steps in wound healing processes were observed during the experimental period, including inflammation, regeneration, and remodeling. In the negative control group, there were delays in wound repair processes, as well as inflammation, monocyte cells, and cellular necrosis ( Figure 6B). Histological analysis of this group revealed macrophage aggregation with poor collagenation. The dermis had a high number of fibroblasts, while the control had less new blood vessel formations. Accumulation of collagen, connective tissues, and blood vessels with epidermal covering at the wound margin was observed in the EOLS topical cream formulation-treated group ( Figure 6D,E). Treatment with this EOLS cream formulation resulted in reduced inflammation, enhanced tissue perfusion and regeneration, remodeling, and re-epithelization. In the EOLS cream formulation-treated animals, there were less macrophage and more collagen fibers, with less scar formation. Ben Djemaa et al. [13] demonstrated that the topical application of Lavandula aspic volatile oil raises the amount of epithelial cells and has a great influence on the closure of the wound.

A1 A2
(A) Skin microscopic image of normal rats: A1 (X10), A2 (X40)  Our data indicate that EOLS topical cream formulation could aid in the rapid healing of acute and chronic wounds by preserving the injury site from infections, inhibiting inflammatory cells, and forming connective tissue in the healed tissue (Table 5). This research will add scientific proof to the folkloric use of the Lavendula genus in tissue regeneration. Plant-derived EOs, such as Lavendula, are being used in the treatment of inflammation and burns [10]. Lavender oil, in particular, has a long history of use in wound repair. It has been stated that lavender EOs have a wide range of pharmacological properties that could be beneficial in the wound healing process [46][47][48]. Antimicrobial agents play a vital part in tissue regeneration. They function as a shield against microbial attacks and protect the injured area from a variety of infections. The wound healing activity of the LSEO could be also attributed to their antimicrobial effects. Iran Lavandula Angustifolia -Assessment of the effect of nanoemulsion cream containing lavender volatile oil and licorice extract on the healing of deep skin -A nanoemulsion comprising lavender EO and licorice extract promotes wound healing at many phases, including wound contraction, tissue regeneration, and molecular processes such as increased expression of TGF-1, type I, and type III collagen genes.  Various steps in wound healing processes were observed during the experimental period, including inflammation, regeneration, and remodeling. In the negative control group, there were delays in wound repair processes, as well as inflammation, monocyte cells, and cellular necrosis ( Figure 6B). Histological analysis of this group revealed macrophage aggregation with poor collagenation. The dermis had a high number of fibroblasts, while the control had less new blood vessel formations. Accumulation of collagen, connective tissues, and blood vessels with epidermal covering at the wound margin was observed in the EOLS topical cream formulation-treated group ( Figure 6D,E). Treatment with this EOLS cream formulation resulted in reduced inflammation, enhanced tissue perfusion and regeneration, remodeling, and re-epithelization. In the EOLS cream formulation-treated animals, there were less macrophage and more collagen fibers, with less scar formation. Ben Djemaa et al. [13] demonstrated that the topical application of Lavandula aspic volatile oil raises the amount of epithelial cells and has a great influence on the closure of the wound.
Our data indicate that EOLS topical cream formulation could aid in the rapid healing of acute and chronic wounds by preserving the injury site from infections, inhibiting inflammatory cells, and forming connective tissue in the healed tissue (Table 5). This research will add scientific proof to the folkloric use of the Lavendula genus in tissue regeneration. Plant-derived EOs, such as Lavendula, are being used in the treatment of inflammation and burns [10]. Lavender oil, in particular, has a long history of use in wound repair. It has been stated that lavender EOs have a wide range of pharmacological properties that could be beneficial in the wound healing process [46][47][48]. Antimicrobial agents play a vital part in tissue regeneration. They function as a shield against microbial attacks and protect the injured area from a variety of infections. The wound healing activity of the LSEO could be also attributed to their antimicrobial effects. The wound healing effect of ointments containing 5% and 10% of pennyroyal and lavender was tested in vivo (Wistar albino rats).
-Both 5% and 10% Lavandula and Mentha ointments enhanced the wound healing progression in comparison with a control. -No statistically significant difference was noted between lavender oil at 5% and 10% groups and the reference drug (Cicatryl-Bio cream) over 9 to 12 days. - The percent of wound contraction with lavender cream (10%) group was reported to be statistically higher (p < 0.001) in comparison with the positive group (Cicatryl-Bio) at the 18th day. -Rats treated with lavender and Mentha ointments have a good re-epithelization, with a great amount of granulation tissue formation and higher collagen quantity.
Kazemi et al. [49] Iran Lavandula Angustifolia -Assessment of the effect of nanoemulsion cream containing lavender volatile oil and licorice extract on the healing of deep skin wound in an animal model.
-A nanoemulsion comprising lavender EO and licorice extract promotes wound healing at many phases, including wound contraction, tissue regeneration, and molecular processes such as increased expression of TGF-1, type I, and type III collagen genes. - The increased antioxidant activities of superoxide dismutase and glutathione peroxidase resulted in lower MDA levels, a byproduct of lipid peroxidation. -Nanoemulsion of lavender EO significantly decreased the wound area more than other groups.
Carbone et al. [50] Italy Lavandula intermedia -Production of nanostructured lipid carriers for the combined delivery of lavender EO and ferulic acid. -Determination of its impact in the wound healing properties.
-The mutual delivery of lavender EO and ferulic acid significantly stimulated cell migration with greater efficacy in comparison with the free drug solution and the carrier without the lavender oil. - The potential combined activity of the lavender oil and the antioxidant ferulic acid co-delivered in nanostructured lipid carriers in helping cell growth and tissue regeneration, demonstrating a potent approach in the wound healing treatment.
-The addition of Lavandula EO and Ag NPs to the nanofiber dressings increased their hydrophilicity and assured the proliferation of chicken embryo fibroblasts cultivated in vitro on these fiber dressings. - The antimicrobial efficacy of the nanofiber was tested against E. coli and S. aureus, indicating that the dressings had outstanding bactericidal effects. - The composite nanofiber dressings have a high potential for application as multipurpose wound dressings, providing protection against microbes while also stimulating tissue regeneration.   Iran

Lavandula stoechas
Investigation of the ability of herbal combination cream containing lavender and rose-scented geranium EOs and aloe vera gel in the improvement of symptoms in patients with superficial second-degree burns, in comparison with silver sulfadiazine (SSD) 1% cream.
-Trial indicated that the combination cream made of EOs and aloe vera gel is higher to SSD 1% cream in alleviation of pain. -This combination cream may be used as a natural and potent alternative for SSD cream in superficial second-degree burns.
Hartman and Coetzee [58] studied the impact of a combined effect of lavender and chamomile EOs (6% combined dose) on severe wound healing. The authors chose lavender EO for its documented skin-regenerative, antimicrobial, and anti-inflammatory characteristics, and they included chamomile EO for its observed anti-inflammatory and sedative attributes. The research included eight patients with chronic ulcers that had been present for three or more months, five of whom were allowed to treat with the EO blend and three of whom were treated with standard approaches such as Granulex or a boric acid and hydrogen peroxide regimen. Scientists concluded that the wounds treated with the EOs were more rapidly cured by the completion of the study (with a total healing time of 420 days off), with four of five wounds treated with essential oils.
An earlier randomized control experiment on 120 women found that lavender EO substantially decreased pain after episiotomy and erythema of incision sites when compared to a control [48]. Another randomized clinical experiment for episiotomy recently found comparable effects, with a substantial reduction in REEDA (redness, edema, ecchymosis, discharge, and approximation) levels and visual analogue scale score for pain when compared to control [59]. Both clinical investigations indicate that lavender EO has a therapeutic effect on wound healing. Moreover, in both an experimental animal model and a human investigation, topical therapy with lavender EO on aphthous ulceration resulted in a considerable ulcer size decrease as compared to the control [60]. Furthermore, there has been research [61] evaluating the mechanism of action of lavender EO on the wound healing process in an experimental animal model. This study found that topical application of lavender EO accelerated wound closure compared to a control group, which was followed by transcriptional activation of PDGF-A and EGF, which are growth factors that play key roles in the wound recovery process such as tissue repair and regeneration [61]. These clinical investigations and animal studies clearly demonstrate that lavender oil has wound healing properties.
Sheikhan et al. [62] conducted a clinical trial in an Iranian hospital, which supports these findings. At five days post-episiotomy, researchers discovered that the group of women treated with 0.96% lavender EO had significantly lower pain intensity and REEDA scores (p < 0.001 and p < 0.001, respectively) than those treated with betadine. Harpreet et al. [63] tried to compare the recovery of episiotomy wounds in postnatal mothers treated with lavender EO versus betadine. Using the REEDA scale as a comparison, scientists discovered that lavender EO was more efficient in wound healing for the first three days (p = 0.035), but by day five (p < 0.05), both treatments were equally effective.
Moreover, it has previously been suggested that Lavandula EO may enhance faster wound healing, related to its potential to influence extracellular matrix caused by plateletderived growth factors (PDGFs) and re-epithelialization produced by epidermal growth factors (EGFs) [45]. Our findings are also consistent with those reported by Ben Djemaa et al. [13], who revealed a significant decrease in wound area after using a cream carrying Lavandula EO; the authors hypothesize that this result could be linked to the EO's antibacterial, antifun-gal, and anti-inflammatory properties, which are attributed to the presence of oxygenated monoterpenes.
A previous research published in the journal Evidence-Based Complementary and Alternative Medicine examined the efficacy of various wound healing treatments. Transcutaneous electrical nerve stimulation (TENS), lavender EO, saline solution and povidoneiodine were all tested on an experimental animal model. The TENS and lavender EO groups repaired wounds quicker than the control groups. These data imply that lavender has wound-healing booster activity [64].
Han et al. [65] assessed the biological effects of EOs on human dermal fibroblast cells that had been exposed to simulated chronic inflammation. Inflammatory molecular stimuli such as Il-1b, TNF-α and IFNγ were used to induce inflammation. A commercial blend of lavender, frankincense, sandalwood, myrrh, Helichrysum, and rose EOs was tested. The researchers found that all of the EOs tested had a significant anti-proliferative effect on fibroblast cells (p < 0.01). Furthermore, lavender EO was one of the few oils that inhibited collagen III (a major component of granulation tissue), plasminogen activator inhibitor (PAI-1) (a protein that tends to cause reduced extracellular matrix degradation), and tissue remodeling-related proteins.

Conclusions
Taken together, our results provide evidence for EOLS's in vivo wound-healing capabilities, implying its use as a bioactive compound in the pharmaceutical and cosmetic industry sectors. Nevertheless, additional research and studies are needed to explore its health benefit on other dimensions of tissue regeneration, such as cytokine production or tissue remodeling at the cellular level, as well as to elucidate and describe the precise mechanism of its pharmacological effect.