Plectranthus amboinicus extract for use in cosmetic scarring

Abstract

A method for promoting scar maturation or reducing the risk of scar formation in a subject, the method comprising administering to a skin site of the subject in need thereof an effective amount of a topical formulation comprising an extract of Plectranthus amboinicus (PA), optionally in combination with an extract of Centella asiatica (CA), and one or more excipients.

Description

[Background technology]

[0001] The aesthetics of transverse scars in perceptible areas can not only affect appearance but also induce functional impairment or pathological conditions such as hypertrophic scars, resulting in patient suffering and financial loss. Therefore, postoperative scar cosmesis is a frequent challenge for surgeons. Currently available scar management methods include compression therapy, oral medications, surgical excision, local injections, laser therapy, radiation therapy, cryotherapy, and topical medications. These treatments focus on reducing postoperative scar formation.

[0002] Topical preparations are generally non-invasive, painless, and easy to administer, resulting in high patient compliance. Currently available topical anti-scarring agents typically include traditional silicone-based products, corticosteroids, imiquimod, mitomycin C, and onion extract (Kim et al., Update on Scar Management: Guidelines for Treating Asian Patients. 2013;132(6):1580-1589). However, the efficacy of these topical preparations is unsatisfactory, and surgical scar cosmesis remains a challenging task. Summary of the Invention

[0003] The present disclosure is based, at least in part, on the development of methods for promoting scar (e.g., post-surgical scar) maturation using topical formulations, and the use of quantitative digital photography assays to assess the progression of scar maturation. Topical formulations for use in the methods disclosed herein include an extract of Plectranthus amboinicus (PA), and optionally an extract of Centella asiatica (CA), as well as excipients such as viscosity-increasing agents, emulsifiers, ointment bases, antibacterial agents, and solvents.

[0004] Thus, in some aspects, the present disclosure describes a method for promoting scar maturation or reducing the risk of scar formation in a subject, the method comprising administering an effective amount of a topical formulation to a skin site in need thereof, the topical formulation comprising a Plectranthus amboinicus (PA) extract, optionally in combination with a Centella asiatica (CA) extract and one or more excipients. In some embodiments, the scar is a post-surgical scar. In some embodiments, the scar is caused by skin damage (e.g., traumatic scarring). In some embodiments, the subject (e.g., a human patient) has undergone a surgical procedure at the skin site (e.g., has a scar formed at the surgical site). In other embodiments, the subject (e.g., a human patient) is scheduled for a surgical procedure at the skin site.

[0005] In some embodiments, the PA extract comprises salvigenin, cirsimaritin, rosmarinic acid, carvacrol, or a combination thereof.

[0006] In some embodiments, the PA extract may be prepared by a process including: (i) mixing a portion of PA (e.g., the aerial part) with an extraction solvent to produce a first PA extract; (ii) filtering and concentrating the first PA extract to produce a concentrated PA extract; (iii) contacting the concentrated PA extract with a hydrophobic interaction chromatography resin; and (iv) eluting the column with an eluent to produce a PA extract. In some cases, the extraction solvent is acetone, butyl methyl ether, ethanol, ethyl acetate, isopropyl alcohol, methanol, or a mixture thereof. Alternatively, or in addition, the eluent comprises a solvent having a polarity index of about 2.1 to 5.4. In some cases, the eluent comprises a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, and hexane. In some specific examples, the PA extract may be prepared by the process described in Example 1 below.

[0007] In some embodiments, the topical formulations disclosed herein may further comprise a CA extract, which may comprise asiaticoside.

[0008] In some embodiments, the topical formulation comprises a PA extract and a CA extract in a weight ratio of 1:1 to 1:4. In one example, the weight ratio of the PA extract to the CA extract is 1:4. For example, the topical formulation may comprise about 0.25% (w / w) PA extract and / or about 1% (w / w) CA extract.

[0009] In some embodiments, the topical formulation comprises one or more excipients, which may include (a) a viscosity increasing agent, (b) an ointment base, (c) an antibacterial agent, and (d) an emulsifier. In some examples, the viscosity increasing agent in the topical formulation may be about 1.0-10% (w / w). In some examples, the ointment base in the topical formulation may be about 5-30% (w / w). In some examples, the antibacterial agent in the topical formulation may be about 0.01-0.2% (w / w). In some examples, the emulsifier in the topical formulation may be about 0.5-6% (w / w).

[0010] Exemplary viscosity-increasing agents include, but are not limited to, cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or combinations thereof. In one embodiment, the viscosity-increasing agent comprises cetostearyl alcohol. Exemplary ointment bases comprise one or more petrolatum compounds. In one embodiment, ointment bases used in topical formulations include liquid petrolatum and white petrolatum. Exemplary antibacterial agents comprise one or more paraben compounds. In one embodiment, antibacterial agents used in topical formulations include methylparaben and propylparaben. Exemplary emulsifiers include sorbitan and / or polysorbate. In one embodiment, emulsifiers used in topical formulations include sorbitan monostearate and polysorbate 60.

[0011] In some cases, one or more excipients in a topical formulation may further include one or more solvents, for example, a non-aqueous solvent such as propylene glycol, and water.

[0012] In specific examples, the topical formulations disclosed herein comprise (e.g., consist essentially of, or consist of) a total of about 0.5-5% (w / w) Plectranthus amboinicus extract and Centella asiatica extract, cetostearyl alcohol in an amount of about 1.0-10% (w / w), a combination of white petrolatum and liquid petrolatum in a total amount of about 5-30% (w / w), a combination of methylparaben and propylparaben in a total amount of about 0.01-0.2% (w / w), and a combination of sorbitan monostearate and polysorbate 60 in a total amount of about 0.5-6% (w / w).

[0013] In some embodiments, the topical formulation is in the form of a cream, gel, dressing, spray formulation, ointment, paste, patch, mask, or lotion. In some examples, the topical formulation is a topical cream formulation. In some embodiments, the topical formulation is administered to the skin site 1 to 4 times per day (e.g., twice daily).

[0014] Any of the methods disclosed herein may further comprise assessing the skin condition of the skin site to which the topical formulation is applied before, during, and / or after treatment. In some embodiments, the skin condition for assessment may include pigmentation, vascularity, suppleness, scar height, color characteristics, texture characteristics, or a combination thereof. In some embodiments, the assessing step comprises semi-quantitative scar assessment, quantitative digital photograph analysis, or a combination thereof.

[0015] In some embodiments, the evaluating step comprises quantitative digital photographic analysis comprising: (a) obtaining digital images of one or more scar regions and one or more normal skin regions at the skin site to which the topical formulation is applied; (b) processing the digital images to calculate color and texture characteristics of the scar regions and normal skin regions; and (c) comparing the color and texture characteristics of the scar regions with those of the normal skin regions to assess scar maturation.

[0016] Also within the scope of the present disclosure are topical formulations as disclosed herein for use in promoting post-surgical scar maturation or reducing the risk of post-surgical scar formation in a subject, as well as the use of the topical formulations for the manufacture of a medicament for use in promoting post-surgical scar maturation or reducing the risk of post-surgical scar formation in a subject.

[0017] Further, the present disclosure provides a method for assessing scar maturation during treatment, the method comprising: (a) obtaining digital images of one or more areas of scarring and one or more areas of normal skin at the site of skin to which the treatment is to be applied, before and during the course of the treatment; (b) processing the digital images to calculate color and texture features of the scar area and the normal skin area; and (c) comparing the color and texture characteristics of the scar area with those of normal skin areas to assess scar maturation; The method includes:

[0018] In some embodiments, the treatment comprises a topical preparation applied to the site of the scar.

[0019] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of some embodiments, as well as from the appended claims.

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure and can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows a schematic diagram of the flow chart for quantitative digital photographic analysis. Digital photographs of postoperative scars were taken at baseline on day 0 (month 0) and at monthly follow-up visits every three months. The photographs of the scars were divided into scar and skin portions. The color and texture characteristics of the scars were quantified using image processing techniques. Furthermore, difference ratios and relative difference ratios were calculated. [Figure 2A] 1 includes representative photographs showing postoperative scars on day 0 (month 0) after treatment with a topical formulation disclosed herein and a placebo control. The left half of the scar (from the patient's perspective) was treated with the topical formulation and the right half was treated with the placebo cream. [Figure 2B] 1 includes representative photographs showing post-operative scars at one month after treatment with a topical formulation disclosed herein and a placebo control. The left half of the scar (from the patient's perspective) was treated with the topical formulation and the right half was treated with the placebo cream. [Figure 2C] 1 includes representative photographs showing post-operative scars at two months after treatment with a topical formulation disclosed herein and a placebo control. The left half of the scar (from the patient's perspective) was treated with the topical formulation and the right half was treated with the placebo cream. [Figure 2D] 1 includes representative photographs showing post-operative scars at 3 months after treatment with a topical formulation disclosed herein and a placebo control. The left half of the scar (from the patient's perspective) was treated with the topical formulation and the right half was treated with the placebo cream. [Figure 3A]Included are figures showing the trends in difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right). CIELab-L delta ratio (left) and CIELab-L relative delta ratio (right). Eigenvalues ​​for statistically significant differences at each month are marked with an asterisk. At 3 months, there was a significant trend toward an increase in the relative difference ratio on the topical formulation-treated side, indicating a significant improvement compared to the control side. [Figure 3B] Included are figures showing the trends of difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right). CIELab-a delta ratio (left) and CIELab-a relative delta ratio (right). [Figure 3C] Included are figures showing the trends of difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right). CIELab-b delta ratio (left) and CIELab-b relative delta ratio (right). [Figure 3D] Included are figures showing the trends in difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right). RGB-R delta ratio (left) and RGB-R relative delta ratio (right). Eigenvalues ​​of statistically significant differences at each month are marked with an asterisk. At 3 months, there was a significant trend toward an increase in the relative difference ratio on the side treated with the topical formulation, indicating a significant improvement compared to the control side. [Figure 3E] Figures showing the trends in difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right) are included. RGB-G delta ratio (left) and RGB-G relative delta ratio (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. At 3 months, there was a significant trend toward an increase in the relative difference ratio on the side treated with the topical formulation, indicating a significant improvement compared to the control side. [Figure 3F]Figures showing the trends in difference ratios and relative difference ratios for the CIELab and RGB systems for the side treated with the topical formulation (left) and the side treated with the placebo cream (right). RGB-B delta ratio (left) and RGB-B relative delta ratio (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. At 3 months, there was a significant trend toward an increase in the relative difference ratio on the side treated with the topical formulation, indicating a significant improvement compared to the control side. [Figure 4A] Graphs showing the trend of the difference ratio and relative difference ratio of texture features for the side treated with the topical formulation (left) and the side treated with the placebo cream (right) are included. Contrast delta ratio (left) and contrast relative delta ratio (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. Significant trends toward the side treated with the topical formulation are indicated. [Figure 4B] Graphs showing the trend of the difference ratio and relative difference ratio of texture features for the side treated with the topical formulation (left) and the side treated with the placebo cream (right): Homogeneity Δ ratio (left) and Homogeneity Relative Δ ratio (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. Significant trends toward the side treated with the topical formulation are indicated. [Figure 4C] Graphs showing the trend of difference ratios and relative difference ratios of texture features for the side treated with topical formulation (left) and the side treated with placebo cream (right). Correlation delta ratios (left) and correlation relative delta ratios (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. Significant trends toward the side treated with topical formulation are indicated. [Figure 4D] Graphs showing the trends of the difference ratios and relative difference ratios of texture features for the side treated with the topical formulation (left) and the side treated with the placebo cream (right) are included. Entropy delta ratio (left) and entropy relative delta ratio (right). Eigenvalues ​​of statistically significant differences for each month are marked with an asterisk. Significant trends towards the side treated with the topical formulation are indicated. [Figure 5] 1 includes photographs showing improvement in scar maturation following treatment with a topical formulation disclosed herein in a female human patient with post-surgical scars. DETAILED DESCRIPTION OF THE INVENTION

[0022] After injury, the skin undergoes a wound healing process that leads to the formation of a mature scar. Wound healing refers to the body's replacement of destroyed or damaged tissue with newly produced tissue. The wound healing process involves multiple stages, including an inflammatory phase, a proliferative phase (granulation tissue development and re-epithelialization), and a remodeling phase, including maturation and scar formation (Alhajj et al., StatPearls, 2022).

[0023] Many people develop scars as a result of trauma or surgery. Scars are part of the biological healing process in the skin. During the resolution phase of wound healing, vascular cells and myofibroblasts undergo apoptosis, resulting in a significant decrease in the number of cells in granulation tissue (Darby et al., Clin Cosmet Investig Dermatology, 2014). Subsequently, ECM synthesis decreases, and type III collagen, the major collagen in granulation tissue, is replaced by type I collagen, the major dermal collagen in normal skin. Scar tissue is fibrous tissue that develops in place of normal skin after injury. In some unexpected circumstances, normal scars can transform into pathological fibrous tissue. This primarily manifests as fibroblast hyperproliferation, but also the massive deposition of ECM, including collagen and fibronectin (FN), leading to abnormal scar formation (Xue et al., Adv Wound Care, 2015). Decreased tensile strength and elasticity, as well as the absence of hair follicles, have also been observed.

[0024] Pathophysiological fibrosis is primarily caused by the excessive accumulation of extracellular matrix components (ECM), primarily collagen and fibronectin, leading to permanent scar formation (Moretti et al., J Biol Chem, 2022). Mechanical forces have been implicated as a key factor in the development of pathological fibrosis. In response to mechanical stress, fibroblasts transform into an activated form known as myofibroblasts, which exhibit a contractile phenotype (Kubow et al., Nat Commun, 2015). Myofibroblasts also overexpress and secrete transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), type I collagen, and fibronectin into the matrix (Garrett et al., Investig Ophthalmol Vis Sci, 2004).

[0025] Myofibroblasts, the primary effector cells in scar formation, are derived from fibroblasts. Generally, exacerbated scar formation is thought to be associated with the c-Jun N-terminal kinase (JNK), TGF-β, Wnt, and Hippo pathways, which are associated with increased fibroblast migration, increased myofibroblast transition, and ECM rearrangement (Qian et al., Oxidative Med Cell Longev, 2022). Specifically, ERK-YAP activation in human cells promotes the transition of fibroblasts to a fibrosis-promoting phenotype (Chen et al., Nat Commun, 2021). In hypertrophic scars, TGF-β-induced IL-11 release is significantly upregulated, activating enrichment of CD39+ fibroblasts in the upper dermis and secreting large amounts of ECM (Huang et al., J Invest Dermatol, 2022). Keratinocytes secrete high-mobility group box chromosomal protein 1 (HMGB1), which induces the α-smooth muscle actin promoter by motivating fibroblasts (Zhao et al., J Invest Dermatol, 2018). CXCL4 stimulates myofibroblast differentiation and collagen synthesis in fibrotic tissue (Affandi et al., Cell Rep, 2022). N-cadherin has been shown to be important in injury-induced flare-ups, as well as in the migration of fascial fibroblasts, which gradually contract the skin and form scars (Jiang et al., Nat Commun, 2020).

[0026] The present disclosure provides topical formulations for use in promoting scar maturation or reducing the risk of scar formation in a subject, such as a human patient. Also provided herein is a quantitative digital photographic analysis for use in assessing the progression of scar maturation in a subject.

[0027] I. Topical Preparations The instant disclosure provides a topical formulation for use in promoting scar maturation or reducing the risk of scar formation in a subject. The topical formulation disclosed herein comprises an extract of Plectranthus amboinicus (PA) and suitable excipients and carriers as disclosed herein. In some embodiments, the topical formulation may further comprise an extract of Centella asiatica (CA).

[0028] (a) Plectranthus amboinicus extract Plectranthus amboinicus (formerly known as Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus aromaticus auct., Plectranthus aromaticus Roxb., Plectranthus aromaticus Benth., and Plectranthus amboinicus (Lour.) Spreng.) is a perennial medicinal herb in the Lamiaceae (also called Labiatae) family native to southern and eastern Africa. Plectranthus amboinicus is also known as Patchouli, Cuban oregano, Indian borage, Indian mint, Mexican mint, Mexican oregano, Country borage, and Spanish thyme.

[0029] PA extract refers to an extract obtained from a PA plant using one or more suitable solvents. In some embodiments, the PA extract is prepared using the aerial parts of the PA plant. In some cases, at least one of the solvents used to prepare the extract has a polarity index of less than 7 (e.g., less than 5). See, for example, U.S. Pat. No. 10,758,584 and International Patent Application No. PCT / CN2022 / 101441. The relevant disclosures of each are incorporated by reference in their entirety for the purposes herein. As used herein, a solvent refers to a substance or mixture of substances that dissolves other substances to form a solution. The PA extract described herein may be prepared using a single solvent. The solvent used in each extraction step to prepare the extracts described herein (including both PA extracts and CA extracts) may be a single solvent. Alternatively, the solvent may be a mixture of two or more solvents.

[0030] The PA extracts described herein may contain terpenoids (e.g., monoterpenoids, diterpenoids, triterpenoids, and / or sesquiterpenoids), flavonoids, phenols, essential oils, or combinations thereof. The PA extracts for preparing the pharmaceutical compositions disclosed herein may contain salvigenin, and optionally one or more of cirsimaritin, rosmarinic acid, and carvacrol.

[0031] The PA extract described herein may be prepared by extracting the entire PA plant or a portion thereof (e.g., the aerial parts) with one or more suitable solvents to form a solution, and then drying the solution to form the PA extract. Because the PA extract contains nonpolar molecules such as flavonoids, terpenoids (e.g., monoterpenoids, diterpenoids, triterpenoids, and / or sesquiterpenoids), phenols, or essential oils, at least one of the extraction solvents may have a relatively low polarity (e.g., a polarity index of less than 7) to facilitate dissolution of the nonpolar molecules. "Extraction" can be performed by directly contacting the PA material with a suitable solvent or by eluting the active ingredients of the PA from a resin to which the active ingredients are attached.

[0032] In some embodiments, solvents with a polarity index of less than 7 can be used to extract active ingredients from PA to produce a PA extract. Such solvents can include ethyl acetate, methyl acetate, propanol, butanol, or chloroform. Alternatively, the solvent can be a mixture of one or more solvents with different polarity indices. Examples include, but are not limited to, mixtures of ethanol and ethyl acetate, ethyl acetate and butanol, ethanol and propanol, and methyl acetate and butanol.

[0033] In some embodiments, the PA extract may be prepared by a process involving the use of a solvent, such as a solvent having a polarity index of less than 7 (e.g., < about 6.5, < about 6.0; < about 5.5, < about 5.0, < 4.9, < 4.8, < 4.7, < 4.6, or < 4.5). Examples include, but are not limited to, methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or combinations thereof.

[0034] The PA material may be the whole PA or a part thereof (e.g., an aerial part such as a leaf) and may be prepared by routine methodology. The PA material may be a fresh plant or a part thereof. Alternatively, the PA material may be in a dried form. The PA may optionally be dried to form a powder, which may be used as the PA material for preparing the PA extract.

[0035] Any PA material as described herein can be extracted one or more times with a suitable solvent to produce a crude extract. The solvent used to prepare the crude extract can be a highly polar solvent, for example, having a polarity index greater than 5 and preferably less than 7 (e.g., >5.2, >5.5, >5.8, >6, or more, and preferably less than 7). Examples include, but are not limited to, ethanol, acetone, methanol, water, or combinations thereof. If desired, the crude extract can be concentrated by conventional methods to produce a concentrated crude extract.

[0036] The crude extract can then be contacted with a suitable resin (e.g., a non-ionic absorbent resin) under suitable conditions that allow the active ingredients in the crude extract to bind to the resin. Typical resins used in preparing Plectranthus amboinicus extracts include, but are not limited to, DIAION® HP20, DIAION® HP20SS, Sepabeads® SP207, Amberlite™ XAD-2, or Amberlite™ XAD-4.

[0037] The resin can then be washed one or more times and eluted with a suitable solvent, e.g., a solvent having a polarity index of less than 7, to produce a PA extract, which can be dried by conventional methods (e.g., freeze-drying, spray-drying, or concentrator drying) to produce a dried PA extract, which can be semi-solid or pasty.

[0038] In some embodiments, the resin absorption step can be performed by mixing the crude extract with the resin in a vessel. In other embodiments, the resin separation step can be performed in a chromatography column setup.

[0039] In one example, a PA extract can be prepared as follows: Approximately 1.5 g of the aerial parts of PA, including leaves and / or stems, can be collected and extracted with a solvent having a polarity of less than 7 (e.g., methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or a combination thereof) at room temperature for 30 minutes to 6 hours. Alternatively, this extraction process can be carried out at a temperature of approximately 50 to 80°C. The resulting crude extract can be directly loaded onto a nonionic adsorption resin column and eluted with a solvent having a polarity of less than 6 (e.g., ethanol, ethyl acetate, butanol, dichloromethane, hexane, toluene, or a combination thereof). The eluted components can be collected and purified by extraction with a solvent having a polarity of less than 6 (e.g., those described herein). The resulting filtrate can be collected to produce a PA extract.

[0040] One specific example of a PA preparation process is provided in Example 1. In some cases, the PA extract used in any topical formulation disclosed herein may be prepared by the process described in Example 1.

[0041] (b) Centella asiatica extract Centella asiatica (formerly or alternatively known as Centella asiatica Urban, Centella asiatica (L.) Urban, Hydrocotyle asiatica L., and Trisanthus cochinchinensis Lour.) is a perennial medicinal herb of the Mackinlayaceae family, or Apiaceae (also known as Umbelliferae), subfamily Mackinlayoideae, native to Asia, Africa, and South America. Centella asiatica is also known as European water-marvel, Gotu kola, Kola, Pennywort, Indian pennywort, Marsh pennywort, Pennyweed, Indian ginseng, Horse-hoof grass, Pegaga, Mandookaparni, Tiger herbal, Spadleaf, or Tono. Centella asiatica extracts typically contain two major compounds: asiaticoside and madecassic acid.

[0042] In some embodiments, the topical formulations disclosed herein may further comprise a CA extract, which may contain an asiaticoside compound. The CA extract described herein refers to an extract obtained from the whole CA plant or a part thereof. The CA extract may contain asiaticoside and, optionally, madecassic acid.

[0043] CA extracts can be prepared according to conventional methods, such as those described in U.S. Patent Nos. 5,834,437, 6,417,349, 6,475,536, and 6,267,996, as well as U.S. Patent Nos. CN1313124, CN1089497, and CN1194154. The following is an example:

[0044] CA material can be prepared through routine procedures. Such material can be fresh CA plants or parts thereof, or dried CA. The CA material can be extracted with a suitable solvent, such as water, ethanol, or a mixture thereof, to produce a crude extract. The crude extract, which can be optionally concentrated, can be mixed with a suitable resin or loaded onto a column packed with resin. After one or more washes, the resin can be eluted with a suitable solvent. The resulting eluate can be concentrated to form a paste, which can be dried by conventional methods, such as vacuum drying, to produce a powder of CA extract. If necessary, the CA powder can be ground through a mesh (e.g., No. 100 mesh).

[0045] The CA extract may be prepared by the same or similar process as described above for producing the PA extract. The PA extract and / or the CA extract may be concentrated using a vacuum rotary evaporator.

[0046] (c) Suitable excipients and carriers In some cases, topical formulations may further comprise one or more suitable carriers or excipients, such as a viscosity increasing agent (e.g., about 1.0-10%), one or more ointment bases (e.g., one or more cream bases) which may range from about 5-30%, one or more antimicrobial preservatives (e.g., about 0.005-0.2% by weight, e.g., about 0.01-0.2% by weight), one or more emulsifiers (e.g., about 0.5-10% by weight, e.g., about 0.5-6% by weight), or combinations thereof. These ingredients may be dissolved or dispersed in a suitable solvent.

[0047] As used herein, the term "about" refers to a specific numerical boundary (including both upper and lower limits) for a specified parameter as would be understood by one of ordinary skill in the art in relation to the particular context. In some instances, the term "about" refers to ±5% (e.g., ±3% or ±2%) of the particular value.

[0048] A "viscosity-increasing agent" is an agent used to thicken a formulation. Typical viscosity-increasing agents may include, for example, cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or a combination thereof. The viscosity-increasing agent may be present in the topical composition at a concentration of about 1.0-10% (w / w). For example, the topical composition may contain about 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, or 9.5-10% (w / w) of the viscosity-increasing agent. Alternatively, the topical formulation may include about 1-5%, 2.5-7.5%, or 5-10% (w / w) of a viscosity increasing agent. In a specific example, the topical formulation may include cetostearyl alcohol as an emulsifier, which may be about 4-6% (w / w) in the topical formulation.

[0049] An "ointment base" can be any semi-solid formulation or vehicle into which an active agent may be incorporated. Typical ointment bases include, but are not limited to, oleaginous ointment bases (e.g., white petrolatum or white ointment), absorbent ointment bases (e.g., hydrophilic petrolatum, anhydrous lanolin, Aquabase™, Aquaphor®, and Polysorb®), water / oil emulsion ointment bases (e.g., cold cream, hydrous lanolin, rose water ointment, Hydrocream™, Eucerin®, and Nivea®), oil / water emulsion ointment bases (e.g., hydrophilic ointment, Dermabase™, Velvachol®, and Unibase®), and water-miscible ointment bases (polyethylene glycol (PEG) ointment, and Polybase™). The ointment base may be pharmacologically inactive but may incorporate water to provide an emollient protective film. In specific embodiments, the ointment base may be any petrolatum compound (e.g., petrolatum, white petrolatum, white soft paraffin, liquid petrolatum, liquid paraffin). In further specific embodiments, the ointment base comprises white petrolatum (e.g., CAS No. 8009-03-8). In some examples, the ointment base for use in the topical formulations disclosed herein comprises a combination of liquid petrolatum and white petrolatum. The ointment base may be present in the topical composition at a concentration of about 5-30% (w / w), e.g., 10-30% (w / w). For example, the topical composition may comprise about 5-25%, 5-20%, 5-15%, 10-15%, 15-20%, 20-25%, or 25-30% (w / w) of the ointment base. Specifically, the topical formulation may comprise about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / w) of an ointment base. In a specific example, the topical formulation may comprise about 20-30% (w / w) of a combination of liquid petrolatum and white petrolatum as the ointment base.

[0050] In some embodiments, the "ointment base" described herein comprises less than 20% water and volatile materials and more than 50% hydrocarbons, waxes, or polyols as a vehicle.

[0051] In some embodiments, the "ointment base" described herein is a "cream base," which contains more than 20% water and volatile substances, and / or typically less than 50% hydrocarbons, waxes, or polyols as a vehicle for the drug substance. Cream bases can be multiphase preparations containing a lipophilic phase and an aqueous phase. In some cases, cream bases are lipophilic cream bases, having a lipophilic phase as the continuous phase. Such cream bases typically contain water-in-oil emulsifiers, such as wool alcohol, sorbitan esters, and monoglycerides. In other cases, cream bases are hydrophilic cream bases, having an aqueous phase as the continuous phase. Such cream bases typically contain oil-in-water emulsifiers, such as sodium or trolamine soap, sulfated fatty alcohols, polysorbates, polyoxyl fatty acids, and fatty alcohol esters, which may be used in combination with water-in-oil emulsifiers, if necessary.

[0052] An "antimicrobial preservative" can be any compound capable of destroying microorganisms, preventing the growth or proliferation of microorganisms, or preventing the pathogenic action of microorganisms. Typical antimicrobial preservatives include, but are not limited to, paraben compounds (esters of parahydroxybenzoic acid, such as paraben, methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, benzylparaben, isobutylparaben, isopropylparaben, benzylparaben, or their sodium salts), benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercaptan nitrate, propylene glycol, and thimerosal. The antimicrobial preservative may be present in the topical composition at a concentration of about 0.005-0.2%, e.g., about 0.01-0.2% (w / w). For example, the topical composition may contain about 0.005-0.01%, 0.01-0.05%, 0.05-0.1%, 0.1-0.15%, or 0.15-0.2% (w / w) of the antimicrobial preservative. Specifically, the topical composition may comprise about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 percent (w / w) of an antimicrobial preservative. In some embodiments, the topical formulation comprises methylparaben and propylparaben, combined, at about 0.08-0.15% (w / w).

[0053] An "emulsifier" is a compound or substance that acts as a stabilizer for a mixture of two or more liquids that are normally immiscible (cannot be mixed or blended). Typical emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, propylene glycol monostearate, and polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and the like), and / or cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and the like). cellulose, hydroxypropyl methylcellulose, and methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span® 60], n® 65], glyceryl monooleate, and sorbitan monooleate [Span® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g.,Emulsifiers include, but are not limited to, polyoxyethylene lauryl ether (Brij® 30), and poly(vinylpyrrolidone)), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, and docusate sodium, and / or combinations thereof. Emulsifiers may be present in the topical composition at a concentration of about 0.5-10% (w / w), e.g., 0.5-6% (w / w). For example, a topical composition may contain about 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 5-10%, 6-10%, or 8-10% (w / w) of emulsifier. Specifically, a topical formulation may contain about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10% (w / w) emulsifier. In some embodiments, topical formulations may contain about 2-4% (w / w) sorbitan monostearate and polysorbate 60 as emulsifiers.

[0054] The topical formulations of the present invention may further comprise one or more solvents (e.g., non-aqueous solvents or water). Exemplary non-aqueous solvents include any known solvent, including, but not limited to, propylene glycol, glycol, and mixtures thereof. The non-aqueous solvent may be present in the topical composition at a concentration of about 2-65% (w / w). For example, the topical composition may comprise about 2-15%, 15-30%, 30-45%, or 45-65% (w / w) of the solvent. In some embodiments, the topical formulations as disclosed herein may also comprise water.

[0055] In some embodiments, the topical formulations of the present invention may further comprise one or more emollients, fragrances, or pigments. The topical formulations of the present invention may also be used in conjunction with wound dressings (e.g., adhesive bandages, adhesive plasters, etc.).

[0056] Non-limiting examples of solvents include cyclohexane, n-hexane, n-decane, i-octane, octane, butyl ether, carbon tetrachloride, triethylamine, i-propyl ether, toluene, p-xylene, t-butyl methyl ether, benzene, benzyl ether, dichloromethane, methylene chloride, chloroform, dichloroethane, ethylene dichloride, 1-butanol, i-butyl alcohol, tetrahydrofuran, ethyl acetate, 1-propanol, 2-propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzonitrile, 1,4-dioxane, or p-dioxane.

[0057] Additional information regarding topical formulations can be found, for example, in US Pat. No. 10,758,584, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referred to herein.

[0058] The topical formulations disclosed herein may be in any suitable form. Examples include, but are not limited to, creams, dressings, ointments, lotions, pastes, masks, and gels. In some cases, the topical formulation may be placed on a device (e.g., a patch) for application to the site of skin damage.

[0059] II. Methods for promoting scar maturation Any of the topical formulations disclosed herein can be used to promote scar maturation or reduce the risk of scar formation in a subject, such as a human. To practice the methods disclosed herein, an effective amount of a topical formulation can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as topical application to a skin site where a scar is or will form (e.g., a surgical site or site where a surgical procedure is being performed).

[0060] In some cases, the topical formulations described herein can be used to promote the maturation of postoperative scars or reduce the risk of postoperative scar formation. In some cases, the topical formulations disclosed herein can be used to promote the maturation of scars caused by trauma. In some examples, the topical formulations can be used to promote the maturation of hypertrophic scars. Hypertrophic scars (HTS) are thickened, wide, and elevated scars and remain a significant challenge in wound management after injury. Hypertrophic scars are a fibroproliferative disorder and are thought to be caused by abnormalities in myofibroblast apoptosis. Myofibroblasts produce excess collagen, which causes scars to elevate above the surrounding skin, potentially contributing to pathological scar formation (Moulin et al., J Cell Physiol, 2004).

[0061] The topical formulation may be applied to the scar site or the area at risk of scar formation according to a suitable dosage and treatment regimen. The dosage and administration regimen of the described method depend on the nature and condition of the condition being treated, the age and condition of the patient, and any prior or concomitant therapy. In some cases, the topical formulation may be applied once a week, once every other day, once a day, twice a day, three times a day, or four times a day for a suitable period of time.

[0062] The subject that is treated by the topical formulation disclosed herein can be human or non-human mammal.In some embodiments, the subject is a human patient that has scar (for example, postoperative scar, skin injury scar, keloid scar or hypertrophic scar) or is at risk of scar formation (for example, is going to undergo surgery that may cause postoperative scar formation).The subject that has scar as disclosed herein can be identified by routine medical examination.

[0063] As used herein, "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. Determining whether an amount of a composition achieves a therapeutic effect would be apparent to one of ordinary skill in the art. As recognized by those skilled in the art, an effective amount will vary depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concomitant treatment (if any), the specific route of administration, and similar factors within the knowledge and expertise of a medical professional. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is usually preferable to use the maximum dose of each component or combination thereof, i.e., the safest dose according to sound medical judgment.

[0064] Usually, empirical considerations such as half-life contribute to the determination of dosage.Dosage frequency can be determined and adjusted during the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of target disease / disorder.Alternatively, sustained continuous release formulation of composition may be suitable.Various formulations and means for achieving sustained release are known in the art.

[0065] In one embodiment, dosages of the compositions described herein may be empirically determined in individuals who have received one or more doses of the composition. The individual is given increasing doses of the agonist. Disease / disorder indicators may be followed to assess the effectiveness of the agonist.

[0066] Generally, when administering any of the compositions described herein, an initial candidate amount can be administered to the subject. Depending on the symptoms, repeated administration over a period of several days or longer can be continued until the desired symptomatic suppression occurs, or until a therapeutic level sufficient to alleviate the target disease or disorder or its symptoms is achieved. The specific administration regimen, i.e., dosage, timing and repetition, depends on the specific individual and their medical history, as well as the characteristics of each individual drug (e.g., drug half-life, and other considerations known in the art).

[0067] For purposes of this disclosure, the appropriate dosage of the compositions described herein will depend on the particular active agent(s) contained therein, the type and severity of the disease / disorder, whether the composition is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to treatment, and the discretion of the attending physician. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in disease severity (e.g., as represented by a disease score). Methods for determining whether a dosage has produced the desired result will be apparent to those of skill in the art. Administration of any of the compositions disclosed herein can be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and / or other factors known to those of skill in the art. Administration of the composition can be essentially continuous over a preselected period of time, or can be administered continuously at spaced intervals, e.g., either before, during, or after the onset of the target disease or disorder.

[0068] As used herein, the term "treating" refers to the application or administration of a composition containing one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, cure, alleviate, relieve, alter, correct, ameliorate, improve, or affect the disease, symptom of a disease, or predisposition to a disease / disorder.

[0069] Alleviating a target disease / disorder includes slowing the progression or progression of the disease, reducing the severity of the disease, or prolonging survival. Alleviating a disease or prolonging survival does not necessarily require a curative outcome. As used herein, "delaying" the progression of a target disease or disorder means suspending, hindering, slowing, preventing, stabilizing, and / or postponing the progression of the disease. This delay can be for various lengths of time, depending on the disease being treated and / or the medical history of the individual. A method of "delaying" or alleviating disease progression or delaying the onset of a disease is a method that reduces the probability of one or more symptoms of the disease progressing in a given time frame and / or reduces the severity of symptoms in a given time frame compared to the absence of the method. Such comparisons are usually based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0070] "Progression" or "progression" of a disease refers to the initial symptoms of the disease and / or its subsequent progression. Disease progression can be detected and assessed using standard clinical techniques, as is well known in the art. However, progression also refers to progression that may not be detectable. For purposes of this disclosure, progression or progression refers to the biological course of symptoms. "Progression" includes onset, recurrence, and manifestation. As used herein, "manifestation" or "development" of a target disease or disorder includes initial onset and / or recurrence.

[0071] The particular dosing regimen used in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history.

[0072] In some embodiments, any of the compositions disclosed herein may be used in combination with one or more additional therapeutic agents to treat a target disease. In some cases, the additional therapeutic agents may serve to enhance and / or complement the effectiveness of the compositions disclosed herein.

[0073] The efficacy of treatment for a target disease / disorder can be assessed by methods well known in the art.

[0074] III. Assessment of scar maturation In some aspects, provided herein are evaluation methods for monitoring scar maturation in a subject who may be the subject of a treatment (e.g., comprising any of the topical formulations as disclosed herein) to improve scar maturation. For example, evaluation assays may be performed before, during, and / or after treatment with a topical formulation to monitor efficacy and progression of scar maturation in the subject. Evaluation may involve evaluation of the skin condition of the scar site and, optionally, of an adjacent normal skin site over the course of treatment. Such skin condition may include pigmentation, vascularity, suppleness, scar height, hue (color) characteristics, texture characteristics, or a combination thereof.

[0075] In some embodiments, assessment methods may include semi-quantitative scar assessment, quantitative digital photographic analysis, or a combination thereof. Semi-quantitative scar assessment assays may include the Vancouver Scar Scale (VSS) and / or modified Vancouver Scar Scale (mVSS) instrumentation, as are well known in the art. See also Example 2 below.

[0076] In some instances, the assessment methods disclosed herein involve quantitative digital photographic analysis, which involves acquiring digital images of a skin site and processing those images to obtain changes in skin characteristics (e.g., color and / or texture characteristics) over the course of treatment. Comparison of such characteristics between scar and normal skin sites may be involved to provide further information regarding treatment efficacy / progression of scar maturation. Scar color is thought to be related to scar maturation, with CEILab and hue being colorimetric values ​​for quantification and communication (Draaijers et al., Burns, 2004). Texture can be divided into color, size, and shape, which generate perceptions of density, coarseness, fineness, and smoothness (Kwak et al., Expert Syst Appl, 2015).

[0077] In some instances, the digital photograph analysis disclosed herein may include analysis of contrast, correlation, homogeneity, and / or entropy of texture information.

[0078] In some embodiments, digital photograph analysis may include: (a) obtaining digital images of one or more scar regions and one or more normal skin regions at a skin site in need thereof (e.g., where a treatment such as a topical preparation is applied); (b) processing the digital images to calculate hue and texture characteristics of the scar region and normal skin region; and (c) comparing the hue (color) and texture characteristics of the scar region with those of the normal skin region to assess scar maturation.

[0079] IV. Kits for use in promoting scar maturation The present disclosure also provides kits for use in promoting scar maturation in a subject in need thereof. Such kits may include one or more containers containing an extract of Plectranthus amboinicus, optionally in combination with an extract of Centella asiatica, or any of the topical formulations as described herein, including one or more active agents thereof, such as those disclosed herein, and one or more suitable excipients and carriers, also as disclosed herein.

[0080] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering a topical formulation to promote scar maturation or reduce the risk of scar formation according to any of the methods described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has a scar or is at risk of scar formation.

[0081] Instructions for use of a topical formulation typically include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided in kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions embodied on a magnetic or optical storage disk) are also acceptable.

[0082] The label or package insert indicates that the topical formulation is used to promote scar maturation. Instructions may be provided for practicing any of the methods described herein.

[0083] The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Optionally, the kit may provide additional components, such as interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the present invention provides a product comprising the contents of the kit described above.

[0084] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0085] General Technology The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press; Culture(RIFreshney,ed.1987);Introuction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.1993-8)J.Wiley and Sons;Methods in Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (lRL Press, (1986); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.) etc. are fully described in the literature.

[0086] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0087] Example 1: Typical Plectranthus amboinicus extract preparation process For extraction, 1 kg of the aerial parts of Plectranthus amboinicus was stirred in 10–20 L of ethanol at 60±5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 30–70% of the original volume. Without dilution, the concentrated solution was loaded onto a column packed with DIAION® HP-20 resin (a non-polar copolymeric styrene-divinylbenzene adsorption resin). The column was eluted with an eluent containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The eluted fractions were collected and dried to obtain the PA extract (yield: 1.2%).

[0088] Example 2. Validation of a topical formulation containing herbal extracts for post-surgical scar cosmetology using quantitative digital photographic analysis Unsightly scars after surgery remain a problem. This study aimed to evaluate the effectiveness of a topical formulation containing a combination of Plectranthus amboinicus (PA) extract and Centella asiatica (CA) extract to prevent and / or reduce scar formation after skin incision. This study also aimed to develop a digital analysis system as a sensitive means of assessing scar improvement.

[0089] This was a prospective, placebo-controlled study in patients with postoperative transverse scars. Each patient received a topical formulation on the left scar (from the patient's perspective) and a placebo cream on the right scar (from the patient's perspective). Digital photographs were used for monthly scar analysis using subjective VSS, mVSS, and VAS scores, as well as colorimetric analyses such as CIELab and hue, and contrast, correlation, homogeneity, and entropy as texture information.

[0090] Forty-six patients (mean age 52 years) participated in this study. All subjective evaluation parameters were superior in the topical formulation-treated side, with significant differences observed in pigmentation, vascularity, softness, height, itching, and patient satisfaction (p = 0.043, 0.013, 0.026, 0.002, 0.039, and 0.012, respectively). Hue and texture trends showed significant differences in most eigenvalues ​​in the topical formulation-treated side, with increased relative difference ratios. These included CIELab-L (p < 0.0001), Hue-R, B, G (p = 0.034, 0.001, and 0.011), contrast (p < 0.0001), homogeneity (p < 0.0001), correlation (p = 0.011), and entropy (p < 0.0001). The results demonstrated the excellent efficacy of topical formulations in promoting postoperative scar maturation, not only by subjective assessment but also by objective quantitative analysis. The results also demonstrated that the digital photographic quantitative analysis system is an ideal tool for quantifying scar appearance.

[0091] method research design This prospective, double-blind, placebo-controlled study enrolled 46 patients with transverse scars and investigated the relationship between a topical preparation containing herbal extract and postoperative scarring based on subjective and objective assessments. Each patient received the topical preparation (cream) on the left scar and a placebo cream on the right scar, starting at the midpoint of the scar. Treatment was administered twice daily for 84 days. Scarring was assessed clinically and photographed monthly for three months after the initial topical application on days 0, 28, 56, and 84. Clinical assessments included the Vancouver Scar Scale (VSS) and modified Vancouver Scar Scale (mVSS), quantitative digital photographic analysis established before and after treatment improvement, and a final satisfaction questionnaire combined with a visual analogue scale (VAS) at the end of the study period.

[0092] Inclusion and exclusion criteria Patients with transverse scars in the neck or abdomen after elective surgery, including thyroid and parathyroid surgery, neck mass removal, inguinal hernia repair, or any neck or abdominal surgery that could be managed with primary closure of a clean surgical wound, were included.

[0093] Exclusion criteria included a history of trauma or radiation therapy affecting the surgical incision site, a history of chemotherapy or targeted therapy for any reason, pregnancy, abnormal laboratory values ​​at screening, such as anemia, sepsis, autoimmune disease, liver function tests greater than three times the upper limit of normal, kidney function tests greater than two times the upper limit of normal, chronic alcohol or drug abuse problems, or the presence of any clinically significant condition that may compromise the subject's medication compliance or interfere with wound healing.

[0094] The study enrolled 46 patients of either sex, aged 20 to 92 years.

[0095] Preparation of topical cream formulations The topical preparation contained 1.25% (w / w) herbal extracts, including Plectranthus amboinicus extract (0.25%) and Centella asiatica extract (1%). The placebo cream contained the same excipients and carriers as the topical formulation, but without the PA and CA extracts. Both samples were prepared to have the same viscosity and yellow-green to light green appearance and were packaged in the same dispenser, but labeled left and right, respectively. The topical cream or placebo cream was applied twice daily to the transverse scar area of ​​the patient for up to three months (84 days).

[0096] Scar assessment (a) Subjective scoring of scar quality with semiquantitative assessment At the end of the study period (Month 3, Day 84), three independent observers—an otolaryngologist, a gynecologist, and a plastic surgeon—performed objective clinical evaluations of both scars using the VSS. The VSS, developed by Sullivan et al. in 1990, defines four parameters for clinician assessment, including scar pigmentation, vascularity, pliability, and height (Sullivan et al., J Burn Care Rehab, 1990). Additionally, the principal investigator utilized the mVSS developed by Nedelec et al. to evaluate the scars using additional indices of subjective symptoms, such as pain and itching, as well as modified criteria for pigmentation (generally, higher VSS and mVSS scores indicate more disfiguring scars) (Nedelec et al., J Burn Care Rehab, 2000).

[0097] In addition, patients completed a final satisfaction questionnaire using a VAS (numeric rating scale from 0 to 10) to quantify their satisfaction with the cosmetic state of their scars and the impact of scar appearance on their quality of life (QoL).

[0098] (b) Quantitative digital photo analysis processing Image Processing One hundred eighty-four standardized anterior / posterior photographs of the scar (four per patient) were taken before and after 3 months of treatment. All photographs were taken with a Canon EOS 850D camera, with image quality set to the highest level to capture scar details. The subject-to-lens distance was standardized at 30 cm. Digital photographs were taken and processed in MATLAB. Figure 1 shows the typical image processing pathway used in this study to quantify scar changes using color and texture features. Photographs of these scars were taken and segmented into the scar area and adjacent skin areas. The difference between the scar area and the adjacent skin areas was calculated and expressed as delta (Δ), with the photograph taken on day 0 (month 0, M0) as the baseline. Meanwhile, difference ratios and relative difference ratios were calculated. The difference ratio represents the difference between the scar and the rest of the skin compared to the skin (Δ ratio = Δ(scar-skin) / skin). Using the M0 data as the baseline, the relative Δ ratio represents the difference between the Δ ratios of Mx and M0 compared to the baseline (relative Δ ratio = (Mx Δ ratio - M0 Δ ratio) / M0 Δ ratio).

[0099] Hue characteristics and CIELab Red, green, and blue (RGB) values ​​were collected for both the scar and skin regions from each postoperative scar photograph. The RGB color space is computationally fast and does not require coordinate transformations. CIELab is a color space modeled on human vision and designed to be perceptually uniform across human perception. CIELab coordinates are L, a, and b, where L* represents the color lightness (L* = 0 indicates black, L* = 100 indicates diffuse white), a* represents the position between red and green (negative values ​​represent green, positive values ​​represent red), and b* represents the position between yellow and blue (negative values ​​represent blue, positive values ​​represent yellow). The conversion between RGB and CIELab is a two-step process following the formula from previous research (Kryjak et al., J Real Time Image Process, 2014).

[0100] Texture characteristics Skin texture, which is the perception of roughness and directionality in human vision, has a significant impact on scar cosmetology, which can be quantified and calculated. The gray level co-occurrence matrix (GLCM) describes the characteristics of the spatial distribution of gray levels between adjacent pixels in an image. In this study, we utilized second-order statistics based on the GLCM to quantify scar-induced skin changes, including four different eigenvalues: contrast, homogeneity, correlation, and entropy. Normalization was performed before extracting the GLCM eigenvalues, and the sum of the GLCM elements was calculated as 1. The eigenvalues ​​used are described below: Contrast This eigenvalue is used to measure the amount of local tone variation within an image, the intensity contrast between adjacent pixels within each region. Greater tone differences represent larger GLCM Con values. ·Homogeneity (Hom) It reflects the similarity of the image texture and measures the local variations in the texture of the image. A higher homogeneity value indicates less variation within the region and a locally uniform gray color in the image. Correlation (Cor) Correlation is used to measure the linear dependency of tones in an image. For example, if the number of textures in the horizontal direction is higher than in other directions, the value of the correlation feature will be higher along this direction. Entropy (ENT) This term comes from thermodynamics and reflects the unevenness or randomness of the tone distribution in image texture processing: the more scattered the tone distribution, the higher the entropy value.

[0101] statistical analysis The differences between the results of the topical preparation-treated and control sides were analyzed by unpaired t-test using SPSS version 22.0 (SPSS Inc.), with statistical significance set at p<0.05.

[0102] result A total of 46 human patients (57% female, mean age 52 years) were included in this study. Table 1 shows the overall distribution and characteristics of all participating patients.

[0103] [Table 1]

[0104] No side effects such as maceration or infection were observed. All patients reported that both creams were easy to apply. 67% of patients reported no itching or pain in the postoperative scar. Fifteen patients reported mild itching on the right side, and seven of them also reported itching on the left side. Excellent responses to topical treatment, including reduced redness and improved skin texture, were observed in all cases. Typical results are shown in Figures 2A-2D.

[0105] At the end of the study, clinical evaluation using either the VSS or mVSS was performed on each patient, and all parameters showed significantly lower values ​​(indicating improved scarring) on ​​the side treated with the topical formulation compared to the placebo cream, including significant differences in pigmentation, vascularity, softness, height, and itching. Patients also rated their overall satisfaction, with results indicating a significantly higher VAS on the side treated with the topical formulation, and a relatively modest effect on QoL. The results are summarized in Table 2 below.

[0106] [Table 2]

[0107] Quantitative analysis of texture revealed a decreasing trend in the difference ratio and the relative difference ratio, respectively (see Table 3 below). These results are consistent with the scar maturation process, demonstrating that quantitative analysis using digital photography is an ideal tool for discriminating between different scars. Quantitative color analysis included calculation of characteristic values, differences, and relative difference ratios from the CIELab and RGB systems, as well as graphing of trends (Figures 3A–3F). Results showed a significant improvement in the relative difference ratio of color lightness ("L" in CIELab) at 3 months in the topical formulation-treated side (p<0.0001), suggesting less pigmentation than in the control side. By the end of the study, the Fespixon-treated side showed a significant trend toward an increase in the relative difference ratio, with significant differences observed in all RGB trends (R, p=0.034; G, p=0.001; B, p=0.011). The immature scars appeared redder, suggesting relatively accelerated scar maturation compared to the control side.

[0108] [Table 3]

[0109] Texture characteristics were quantified using the Con, Hom, Cor, and Ent of the GLCM, differences and relative difference ratios were derived, and trends were graphed. The results are shown in Figures 4A-4D.

[0110] At 3 months, the difference ratio for Ent was significantly reduced in the topical formulation-treated side (p = 0.010), indicating less disruption and closer to intact, unbroken skin compared with the control side. At the end of the study, statistically significant improvements in the relative difference ratio were observed in the topical formulation-treated side for all texture eigenvalues ​​(Con, p < 0.0001; Hom, p < 0.0001; Cor, p = 0.011; Ent, p < 0.0001), reflecting increased uniformity and color linearity with decreased complexity compared with the control side. The color and texture trends showed significant increases in the relative difference ratio for most eigenvalues ​​in the topical formulation-treated side, corresponding to earlier scar maturation.

[0111] Overall, the results reported herein suggested that the assay could be discriminatory, with excellent validation of topical formulations in scar cosmetology.

[0112] Example 3: Case study on the use of a topical formulation to promote scar maturation This example provides a case study showing that the topical formulations disclosed herein successfully promoted scar maturation in human patients.

[0113] A 29-year-old female patient had a post-surgical (Cesarean section) hypertrophic scar for one year. The patient was treated with the topical formulation disclosed herein (see Example 2 above). Briefly, the topical formulation was applied to the scarred area. Scar evaluation was performed before treatment and 50 days after treatment, and the results are shown in Table 4 below. See also Figure 5.

[0114] [Table 4]

[0115] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0116] From the above description, those skilled in the art can easily grasp the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the appended claims.

[0117] equivalent While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining the results described herein and / or obtaining one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application in which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0118] All definitions defined and used herein should be understood to control dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0119] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, including, in some cases, the entire document.

[0120] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly stated to the contrary, should be understood to mean "at least one."

[0121] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted similarly, i.e., to mean "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0122] As used in this specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of a number or list of elements, but also including one or more, and optionally further including unlisted items. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. Generally, as used herein, the term "or" should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by exclusive terms such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0123] In this specification and claims, when used in reference to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") refers, in one embodiment, to at least one A, optionally including one or more, without the presence of B (and optionally including elements other than B); in another embodiment, to at least one B, optionally including one or more, without the presence of A (and optionally including elements other than A); in yet another embodiment, to at least one A, optionally including one or more, and at least one B, optionally including one or more (and optionally including other elements); etc.

[0124] Also, unless expressly stated to the contrary, in methods claimed herein that include one or more steps or acts, it should be understood that the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

Claims

1. 1. A method for promoting scar maturation or reducing the risk of scar formation in a subject, the method comprising administering to the subject at a skin site in need thereof an effective amount of a topical formulation comprising an extract of Plectranthus Amboinicus (PA) and one or more excipients.

2. 2. The method of claim 1, wherein the PA extract comprises salvigenin, cirsimaritin, rosmarinic acid, carvacrol, or a combination thereof.

3. The PA extract is (i) mixing a portion of PA with an extraction solvent to form a first PA extract; (ii) filtering and concentrating the first PA extract to produce a concentrated PA extract; (iii) contacting the concentrated PA extract with a hydrophobic interaction chromatography resin; and (iv) eluting the column with an eluent to produce the PA extract.

3. The method of claim 1 or claim 2, wherein the method is prepared by a process comprising:

4. The method according to claim 3, wherein the part of the PA in step (i) is an aerial part.

5. 5. The method of claim 3 or claim 4, wherein the extraction solvent is acetone, butyl methyl ether, ethanol, ethyl acetate, isopropyl alcohol, methanol, or a mixture thereof.

6. The method of any one of claims 3 to 5, wherein the eluent comprises a solvent having a polarity index of about 2.1 to 5.

4.

7. 7. The method of any one of claims 3 to 6, wherein the eluent comprises a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, and hexane.

8. The method of any one of claims 1 to 7, wherein the topical formulation further comprises an extract of Centella asiatica (CA).

9. 9. The method of claim 8, wherein the CA extract comprises asiaticoside.

10. 10. The method of claim 8 or claim 9, wherein the topical formulation comprises a PA extract and a CA extract in a weight ratio of 1:1 to 1:4, optionally 1:

4.

11. The method of claim 10, wherein the topical formulation comprises about 0.25% (w / w) of PA extract and / or about 1% (w / w) of CA extract.

12. 12. The method of any one of claims 1 to 11, wherein the one or more excipients in the topical formulation comprise: (a) a viscosity increasing agent, (b) an ointment base, (c) an antibacterial agent, and (d) an emulsifier.

13. In said topical formulation, the viscosity increasing agent is about 1.0-10% (w / w); the ointment base is about 5 to 30% (w / w); the antimicrobial agent is about 0.01-0.2% (w / w); and / or the emulsifier is about 0.5-6% (w / w); The method of claim 12.

14. the viscosity increasing agent comprises cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or a combination thereof, optionally wherein the viscosity increasing agent comprises cetostearyl alcohol; the ointment base comprises one or more petrolatum compounds, optionally liquid petrolatum and white petrolatum; the antimicrobial agent comprises one or more paraben compounds, optionally methylparaben and propylparaben; and / or the emulsifier comprises sorbitan and / or polysorbate, optionally sorbitan monostearate and polysorbate 60; The method of claim 13.

15. 15. The method of any one of claims 12 to 14, wherein the one or more excipients further comprise one or more solvents, optionally propylene glycol and water.

16. 9. The method of claim 8, wherein the topical formulation comprises a total of about 0.5-5% (w / w) of Plectranthus amboinicus extract and Centella asiatica extract, about 1.0-10% (w / w) of cetostearyl alcohol, about 5-30% (w / w) of a combination of white petrolatum and liquid petrolatum, about 0.01-0.2% (w / w) of a combination of methylparaben and propylparaben, and about 0.5-6% (w / w) of a combination of sorbitan monostearate and polysorbate 60.

17. The method according to any one of claims 1 to 16, wherein the scar is a post-surgical scar or a scar caused by skin injury.

18. The method according to any one of claims 1 to 17, wherein the scar is a keloid scar or a hypertrophic scar.

19. The method of any one of claims 1 to 18, wherein the subject is a human patient.

20. 20. The method of claim 18 or claim 19, wherein the scar is a post-surgical scar and the subject has undergone or will undergo a surgical procedure at the skin site.

21. 18. The method of any one of claims 1 to 17, wherein the topical formulation is in the form of a cream, gel, dressing, spray formulation, ointment, paste, patch, mask, or lotion, optionally in the form of a cream.

22. 22. The method of any one of claims 1 to 21, wherein the topical formulation is administered to the skin site 1 to 4 times daily.

23. 23. The method of any one of claims 1 to 22, further comprising assessing the skin condition of the skin site to which the topical formulation has been applied before, during, and / or after treatment.

24. 24. The method of claim 23, wherein the skin condition comprises pigmentation, vascularity, suppleness, scar height, color characteristics, texture characteristics, or a combination thereof.

25. 25. The method of claim 23 or claim 24, wherein the assessing step comprises semi-quantitative scar assessment, quantitative digital photographic analysis, or a combination thereof.

26. the evaluating step: (a) obtaining digital images of one or more scar areas and one or more skin areas adjacent to the scar areas at the skin site to which the topical formulation has been applied; (b) processing the digital images to calculate color and texture features of the scar area and the skin area; and (c) comparing the color and texture characteristics of the scar area with the color and texture characteristics of the skin area to assess scar maturity; 26. The method of claim 25.

27. 1. A method for assessing scar maturation during treatment, comprising: (a) obtaining digital images of one or more scar areas and one or more skin areas adjacent to said scar areas at a skin site to be treated before and during the course of treatment; (b) processing the digital images to calculate color and texture features of the scar area and the skin area; and (c) comparing the color and texture characteristics of the scar area with the color and texture characteristics of the skin area to assess scar maturity; The method.

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