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WO2024129047A1 - Gel formulation with wound healing effect comprising a salvia sclarea l. and citrus aurantifolia l. - Google Patents

Gel formulation with wound healing effect comprising a salvia sclarea l. and citrus aurantifolia l. Download PDF

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Publication number
WO2024129047A1
WO2024129047A1 PCT/TR2023/051544 TR2023051544W WO2024129047A1 WO 2024129047 A1 WO2024129047 A1 WO 2024129047A1 TR 2023051544 W TR2023051544 W TR 2023051544W WO 2024129047 A1 WO2024129047 A1 WO 2024129047A1
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WIPO (PCT)
Prior art keywords
hydrogel formulation
formulation according
agents
wound
aurantifolia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/TR2023/051544
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French (fr)
Inventor
Sevde Nur BILTEKIN KALELI
Ayse Esra KARADAG
Fatih DEMIRCI
Zeynep Zisan SAKAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anadolu Universitesi
Istanbul Medipol Universitesi
Original Assignee
Anadolu Universitesi
Istanbul Medipol Universitesi
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Priority claimed from TR2022/019370 external-priority patent/TR2022019370A1/en
Application filed by Anadolu Universitesi, Istanbul Medipol Universitesi filed Critical Anadolu Universitesi
Publication of WO2024129047A1 publication Critical patent/WO2024129047A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids

Definitions

  • the present invention relates to the wound-healing potential of phytotherapeutic forms of combinations of essential oils Salvia sclarea L., Citrus aurantifolia L.
  • the invention relates to the construction of a unique herbal wound-healing hydrogel from some essentials oils that have been proven to have a wound-healing effect and are safely used as wound-healing.
  • the wound is the deterioration of the anatomical and functional integrity of the living tissue with various factors; the restoration of this integrity through a series of processes is called wound healing.
  • Wound healing is divided into 4 phases as hemostasis, inflammation, proliferation-maturation, and remodeling.
  • Wound-healing agents exist as an important need in daily life, from simple events to more serious situations.
  • Natural products due to the side effects of chemical products have started to be preferred more recently in wound healing.
  • Dermatix® in the form of silicone gel and it can have side effects as well as its healing effect.
  • the patient may be exposed to hormonal changes or have hives while trying to treat the patient's wound.
  • Natural resources are becoming very popular in the treatment of diseases. It has been proven in various studies that said invention has a wound-healing effect and a unique herbal woundhealing hydrogel is made from some essential oils that have been safely used as wound- healing for centuries among the public. Thus, it is aimed to develop natural wound healing agents against various injuries, which is a common problem in human life, and to gain an economic and domestic value in combating this situation. It is aimed to obtain a national and standardized phytopharmaceutical product with natural content by using the essential oils of two plants grown in the flora of Tiirkiye.
  • the wound-healing effect of the plants and their essential oils is found in both the experimental literature and ethnob otani cal sources, but it is observed that in vivo and in vitro wound studies conducted with formulated combinations are not available. It is thought to obtain a result that reduces external dependence thanks to the preparation prepared within the scope of the invention. In addition, it is predicted considering the increasing war, injury, and accident situations in the world that its use will be beneficial in various areas. It is thought that this idea put forward by the invention will meet a current need from both a natural and domestic source.
  • the present invention which is based on a reliable traditional use, can be updated using different pharmaceutical forms and nanotechnological methods in this sense.
  • Salvia sclarea L., Citrus aurantifolia L. essential oils which are thought to have potential effects on wound treatments considering the literature due to the side effects of chemical drugs in wound healing, are used.
  • Different preparations of these essential oils are known to have antimicrobial and antioxidant effects among the public and in experimental studies, and some species promote healthy cell proliferation and angiogenesis.
  • Non-irritant phytotherapeutic formulations effective in wound healing are obtained from different combinations of standardized drugs within the scope of the invention.
  • the in vitro characterization properties of these essential oils and their combinations are being investigated for the first time based on evidence.
  • Essential oils are supplied in pharmacopoeial quality from a domestic company and standardization analyses are carried out with GC-MS and GC-FID.
  • cytotoxicity is investigated with the MTT experiment in the healthy cell line and its effects on wound treatment are evaluated with the scratch experiment in the in vitro model.
  • the checkerboard method is employed to determine the effective combinations of essential oils by considering their synergistic potential, and it is revealed whether there is synergistic activity by calculating FICI values and effective combined concentrations are determined.
  • the carbopol gel formulation is created by loading the standardized essential oils determined as a result of in vitro results into the carrier systems with effective doses and the in vitro effects of the loaded formulations are investigated with the same methods; the effects of the formulation on healthy cells are evaluated and wound healing potentials are studied on dermal fibroblast cells. Irritation studies on HaCaT cells of the final product are also being investigated. All analyses are studied in accordance with the literature and the results are evaluated in appropriate software. The following is a detailed description of the steps of obtaining the formulation of the invention.
  • Essential oils to be supplied from different domestic companies in appropriate quality are used.
  • Essential oils with the most appropriate results are used in GC analyses.
  • the supplied essential oils are standardized. This standardization process is based on the given literature and the major substances given in the European Pharmacopoeia. The standardization process is presented in detail with methods such as Diode Array Detector, Mass Spectrometer, etc.
  • the analysis of the essential oils is illuminated by simultaneous analyses of the GC and GC/MS systems. GC analysis is performed with the Agilent 6890N GC system. Analysis is performed using Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) and Helium (0.8 mL/min) as carrier gas.
  • the GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min.
  • the FID detector temperature is adjusted to 300°C.
  • the relative ratios of the compounds are calculated using the FID chromatogram computer.
  • the volatile compounds separated in the GC column are then identified by taking individual mass spectra of each that act as detectors.
  • the volatile compounds are divided into GC columns. Then, it is defined by taking separate mass spectra in mass spectrometry. Here, the mass spectrum is used in the detector function.
  • GC/MS analysis is performed by using the Agilent 5975 GC/MSD system.
  • Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) is used for separation of components.
  • Helium 0.8 mL/min
  • the GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min.
  • Split is applied at a rate of 1 in 40 and the energy of the mass spectra is taken as 70 eV. Mass values are recorded between m/z 35 and 450.
  • the adhesion indices (RRI) of the substances in the column are calculated with reference to n-Alkanes.
  • Component analyses are carried out using "Ba ⁇ er Essential Oil Components Library", Wiley and MassFinder 3.1 Library Scanning Software (Karaka ⁇ et al., 2019).
  • the cryotube is taken from the frozen vial in the liquid nitrogen and kept until it reaches room temperature, it is centrifuged at 1000 rpm for 5 minutes in a +4°C centrifuge by adding 1 :2 DMEM (Dulbecco Modified Eagle Medium, biowest) to the dissolved cells. Supernatant is discarded and medium (DMEM-High Glucose, FBS 10% (Fetal bovine serum), antibiotic 1% (Penicillin+Streptomycin+ Amphotericin B, Thermo Fisher Scientific) is added to the cells in the pellet.
  • DMEM Dulbecco Modified Eagle Medium, biowest
  • the cell-medium mixture is added to the flasks (2 pcs.) coated with 25cm 2 Poly-D- Lysine as 4 ml in order for the cells to reach a sufficient number.
  • the cells are removed with the help of 0.25 Trypsin-EDTA (Sigma-Aldrich) after reaching 80% content and added to 24 and 96-well plates. When the cells cover the wells by 90%, they are tested to perform MTT studies and wound models.
  • PCS-201-01 Healthy human primary dermal fibroblast cells (PCS-201-01, ATCC, USA), DMEM-High Glucose, FBS 10% (Fetal bovine serum), antibiotic 1% (Penicillin+Streptomycin+ Amphotericin B, Thermo Fisher Scientific) are grown in medium for cell viability MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test.
  • PCS-201-01 cells are kept in incubation at 37°C in 5% CO2 and humid environments. The cells are grown in 96-well flat-bottomed plates with a density of about 2 * 105 cells/well and incubated until they reach the 80% junction level.
  • the medium is washed with phosphate- buffered saline (PBS) after removal from the wells.
  • PBS phosphate- buffered saline
  • 100 pl of DMEM medium, supported separately with essential oils prepared in different concentration doses, is added to each well covered with PCS-201-01 cells.
  • the treated mixtures are removed, and the well is washed with PBS after the treated cells are incubated for an additional 24 hours.
  • 10 pl of MTT (5 mg/ml in PBS) reagent is added to each well and incubated for another 4 hours at 37°C.
  • the formazan crystals are dissolved in 100 pl of Isopropanol and the absorbance values are measured in the ELISA reader (Spectramax i3) at 450 nm. All experiments are performed in at least three replicates and their average values are analyzed statistically.
  • the cell culture wound model experiment is performed to examine cell migration and proliferation ability according to treatments.
  • PCS-201-01 cells are grown in DMEM-High glucose supplemented with 10% FBS.
  • 2 ⁇ 105 cells/well are planted in 24-well cell culture plates and incubated for 24 hours until the wells reach a density of 70-80% as a single layer.
  • An artificial linear wound is created by drawing the cellular single layer with 200 pl pipette tip without changing the medium.
  • Wells are gently washed with PBS.
  • Essential oils are added to the wells in different concentrations.
  • Wound size is observed at certain periods for 0-48 hours incubation. Scratch wound closures are observed using the Leica DFC295 inverted microscope for photography. The width of the scratch area is monitored and photographed at 0-hour, 3-hour, 6-hour, 12-hour, 18-hour, 24-hour, 30-hour and 48-hour time intervals to measure cell migration ability.
  • a double combination is designed as S. sclarea + C. Aurantifolia.
  • Each essential oil is evaluated separately and whether there is synergy tested in cell culture studies is evaluated separately according to the results of the study before the combinations are studied. These combinations are made by checkerboard method as in NCCLS (1990). This method will be modified for in vitro cell culture experiments.
  • the combinations of essential oil ratios C. aurantifolia: S. sclarea, 1 :99, 2:98, 3:97, .... 97:3, 98:2, 99: 1 are used.
  • a 5% gel is prepared to increase the exposure time of the formulation to the wound area and to make it easy to apply.
  • This gel formulation contains at least one pharmaceutically acceptable excipient as well as essential oils.
  • Excipients of the formulation may be solvents, gelling agents, emulsifiers, suspenders, thickeners, antioxidants, pH adjusters, surfactants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, humectants, absorption delaying agents, plumpers, preservatives, stabilizers, binders, disintegrants, lubricants, sweetening agents, flavoring agents, and combinations thereof.
  • the solvent of the gel formulation can be selected from a group consisting of ethanol, methanol, propanol, isopropanol, water or a binary or triple mixture thereof.
  • the surfactant of the formulation may be selected from the group consisting of triethanolamine, sodium lauryl sulfate, sodium dodecyl sulfate, polysorbates, for example, polysorbate 20, polysorbate 60, poloxamer, phosphatidylcholine, benzalkonium chloride, span 20, span 80, span 85, cetylpyridinium chloride, cetyltrimethylammonium bromide, dodecyl amine, polyoxyethylene nonylphenol, polyoxyethylene (POE) alkyl esters, sorbitan monopalmitate, sorbitan monolaurate, polyoxyethylene sorbitan monoesters.
  • POE polyoxyethylene
  • 0.5 g carbopol and 7g glycerin mixture 5 g distilled water is mixed in the first solution in the preferred embodiment of the invention.
  • 20 g of isopropyl alcohol and 0.5 g of carbopol mixture are mixed with 5 g of distilled water in the second solution.
  • these two solutions are combined and mixed with a magnetic stirrer at 25°C until the entire carbopol is dissolved in order to ensure gelation.
  • various concentrations of C. aurantifolia and S. sclarea essential oils are added to the mixture of the above reagents.
  • 3.5 g triethanolamine dissolved in 5 g water is also added to the mixture of reagents.
  • enough water is added to bring the mixture to 100 grams.
  • the essential oil combination is loaded into the gel in the range of 0.1-10%.
  • the resulting mixture is mixed with the help of a spatula until it becomes homogeneous.
  • the gel is then transferred to a clear glass bottle. And then, it is centrifuged at 112-1006 x g for 10 minutes to remove the compressed air particles.
  • a gel without essential oil is also prepared and used as a control group. Stability control of the formulations is checked whether they remain at +4°C, 25°C and 37-40°C degrees, relative humidity, and stable pH for 0-3-6 months (Karadag et al., 2020). It is observed that it remains stable at room temperature.
  • the MTT method will be applied for the cytotoxicity experiments of the formulation. A low or zero toxicity range is observed based on the tests.
  • Irritation Activity Tests of the Formulation Human keratinocyte cell line will be used for the irritation study of the formulation.
  • Dulbecco's modified Eagle's medium/high glucose (DMEM/High) medium will be cultured with 10% (v/v) heat-inactivated fetal bovine serum, L-glutamine (2mM), antibiotic- antimycotic solution (100 U/mL penicillin, 100 pg/mL streptomycin, and 0.25 pg/mL amphotericin B).
  • Irritation experiments on this cell line will be carried out as specified in the literature (Wu et al., 2020). A non-irritant or low irritation range is observed according to the tests.
  • the Scratch Assay Method will be applied to test the healing effect of the formulation in the in vitro wound model. It is observed that the wound healing effect is 90% and above.

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Abstract

The invention relates to the wound-healing potential of phytotherapeutic formulations of combinations of Salvia sclarea and Citrus aurantifolia essential oils.

Description

GEL FORMULATION WITH WOUND HEALING EFFECT COMPRISING A SALVIA SCLAREA L. AND CITRUS AURANTIFOLIA L.
Technical Field
The present invention relates to the wound-healing potential of phytotherapeutic forms of combinations of essential oils Salvia sclarea L., Citrus aurantifolia L. The invention relates to the construction of a unique herbal wound-healing hydrogel from some essentials oils that have been proven to have a wound-healing effect and are safely used as wound-healing.
State of the Art (Background)
The wound is the deterioration of the anatomical and functional integrity of the living tissue with various factors; the restoration of this integrity through a series of processes is called wound healing. Wound healing is divided into 4 phases as hemostasis, inflammation, proliferation-maturation, and remodeling. Wound-healing agents exist as an important need in daily life, from simple events to more serious situations. Natural products due to the side effects of chemical products have started to be preferred more recently in wound healing. There are many products on the market for wound treatment. One of them is Dermatix® in the form of silicone gel and it can have side effects as well as its healing effect. The patient may be exposed to hormonal changes or have hives while trying to treat the patient's wound. Such side effects of chemical drugs lead people to use natural products and there are products available on the market used in herbal wound treatment. The fact that important woundhealing preparations used in the market are of herbal origin, but that they are synthetic, directs the studies in the literature to the development of natural products.
Brief Description and Objects of the Invention
Natural resources are becoming very popular in the treatment of diseases. It has been proven in various studies that said invention has a wound-healing effect and a unique herbal woundhealing hydrogel is made from some essential oils that have been safely used as wound- healing for centuries among the public. Thus, it is aimed to develop natural wound healing agents against various injuries, which is a common problem in human life, and to gain an economic and domestic value in combating this situation. It is aimed to obtain a national and standardized phytopharmaceutical product with natural content by using the essential oils of two plants grown in the flora of Tiirkiye.
The wound-healing effect of the plants and their essential oils is found in both the experimental literature and ethnob otani cal sources, but it is observed that in vivo and in vitro wound studies conducted with formulated combinations are not available. It is thought to obtain a result that reduces external dependence thanks to the preparation prepared within the scope of the invention. In addition, it is predicted considering the increasing war, injury, and accident situations in the world that its use will be beneficial in various areas. It is thought that this idea put forward by the invention will meet a current need from both a natural and domestic source. The present invention, which is based on a reliable traditional use, can be updated using different pharmaceutical forms and nanotechnological methods in this sense.
Detailed Description of the Invention
Salvia sclarea L., Citrus aurantifolia L. essential oils which are thought to have potential effects on wound treatments considering the literature due to the side effects of chemical drugs in wound healing, are used. Different preparations of these essential oils are known to have antimicrobial and antioxidant effects among the public and in experimental studies, and some species promote healthy cell proliferation and angiogenesis. Non-irritant phytotherapeutic formulations effective in wound healing are obtained from different combinations of standardized drugs within the scope of the invention. The in vitro characterization properties of these essential oils and their combinations are being investigated for the first time based on evidence. Essential oils are supplied in pharmacopoeial quality from a domestic company and standardization analyses are carried out with GC-MS and GC-FID. Firstly, cytotoxicity is investigated with the MTT experiment in the healthy cell line and its effects on wound treatment are evaluated with the scratch experiment in the in vitro model. The checkerboard method is employed to determine the effective combinations of essential oils by considering their synergistic potential, and it is revealed whether there is synergistic activity by calculating FICI values and effective combined concentrations are determined. The carbopol gel formulation is created by loading the standardized essential oils determined as a result of in vitro results into the carrier systems with effective doses and the in vitro effects of the loaded formulations are investigated with the same methods; the effects of the formulation on healthy cells are evaluated and wound healing potentials are studied on dermal fibroblast cells. Irritation studies on HaCaT cells of the final product are also being investigated. All analyses are studied in accordance with the literature and the results are evaluated in appropriate software. The following is a detailed description of the steps of obtaining the formulation of the invention.
A) Supply of Herbal Materials
Essential oils to be supplied from different domestic companies in appropriate quality are used. Essential oils with the most appropriate results are used in GC analyses.
B) Standardization
The supplied essential oils are standardized. This standardization process is based on the given literature and the major substances given in the European Pharmacopoeia. The standardization process is presented in detail with methods such as Diode Array Detector, Mass Spectrometer, etc. The analysis of the essential oils is illuminated by simultaneous analyses of the GC and GC/MS systems. GC analysis is performed with the Agilent 6890N GC system. Analysis is performed using Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) and Helium (0.8 mL/min) as carrier gas. The GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min. The FID detector temperature is adjusted to 300°C. The relative ratios of the compounds are calculated using the FID chromatogram computer. The volatile compounds separated in the GC column are then identified by taking individual mass spectra of each that act as detectors. The volatile compounds are divided into GC columns. Then, it is defined by taking separate mass spectra in mass spectrometry. Here, the mass spectrum is used in the detector function. GC/MS analysis is performed by using the Agilent 5975 GC/MSD system. Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) is used for separation of components. Helium (0.8 mL/min) is used as the carrier gas. The GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min. Split is applied at a rate of 1 in 40 and the energy of the mass spectra is taken as 70 eV. Mass values are recorded between m/z 35 and 450. The adhesion indices (RRI) of the substances in the column are calculated with reference to n-Alkanes. Component analyses are carried out using "Ba§er Essential Oil Components Library", Wiley and MassFinder 3.1 Library Scanning Software (Karaka§ et al., 2019).
C) Subculturing Studies of Cells
The cryotube is taken from the frozen vial in the liquid nitrogen and kept until it reaches room temperature, it is centrifuged at 1000 rpm for 5 minutes in a +4°C centrifuge by adding 1 :2 DMEM (Dulbecco Modified Eagle Medium, biowest) to the dissolved cells. Supernatant is discarded and medium (DMEM-High Glucose, FBS 10% (Fetal bovine serum), antibiotic 1% (Penicillin+Streptomycin+ Amphotericin B, Thermo Fisher Scientific) is added to the cells in the pellet. The cell-medium mixture is added to the flasks (2 pcs.) coated with 25cm2 Poly-D- Lysine as 4 ml in order for the cells to reach a sufficient number. The cells are removed with the help of 0.25 Trypsin-EDTA (Sigma-Aldrich) after reaching 80% content and added to 24 and 96-well plates. When the cells cover the wells by 90%, they are tested to perform MTT studies and wound models.
D) Cytotoxicity Activity Tests by MTT Method
Healthy human primary dermal fibroblast cells (PCS-201-01, ATCC, USA), DMEM-High Glucose, FBS 10% (Fetal bovine serum), antibiotic 1% (Penicillin+Streptomycin+ Amphotericin B, Thermo Fisher Scientific) are grown in medium for cell viability MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test. PCS-201-01 cells are kept in incubation at 37°C in 5% CO2 and humid environments. The cells are grown in 96-well flat-bottomed plates with a density of about 2 * 105 cells/well and incubated until they reach the 80% junction level. The medium is washed with phosphate- buffered saline (PBS) after removal from the wells. 100 pl of DMEM medium, supported separately with essential oils prepared in different concentration doses, is added to each well covered with PCS-201-01 cells. The treated mixtures are removed, and the well is washed with PBS after the treated cells are incubated for an additional 24 hours. 10 pl of MTT (5 mg/ml in PBS) reagent is added to each well and incubated for another 4 hours at 37°C. Finally, the formazan crystals are dissolved in 100 pl of Isopropanol and the absorbance values are measured in the ELISA reader (Spectramax i3) at 450 nm. All experiments are performed in at least three replicates and their average values are analyzed statistically.
E) Determination of Wound Healing Effects by Scratch Assay Method
The cell culture wound model experiment is performed to examine cell migration and proliferation ability according to treatments. PCS-201-01 cells are grown in DMEM-High glucose supplemented with 10% FBS. 2^ 105 cells/well are planted in 24-well cell culture plates and incubated for 24 hours until the wells reach a density of 70-80% as a single layer. An artificial linear wound is created by drawing the cellular single layer with 200 pl pipette tip without changing the medium. Wells are gently washed with PBS. Essential oils are added to the wells in different concentrations. Wound size is observed at certain periods for 0-48 hours incubation. Scratch wound closures are observed using the Leica DFC295 inverted microscope for photography. The width of the scratch area is monitored and photographed at 0-hour, 3-hour, 6-hour, 12-hour, 18-hour, 24-hour, 30-hour and 48-hour time intervals to measure cell migration ability.
F) Checkerboard Method
A double combination is designed as S. sclarea + C. Aurantifolia. Each essential oil is evaluated separately and whether there is synergy tested in cell culture studies is evaluated separately according to the results of the study before the combinations are studied. These combinations are made by checkerboard method as in NCCLS (1990). This method will be modified for in vitro cell culture experiments. The combinations of essential oil ratios C. aurantifolia: S. sclarea, 1 :99, 2:98, 3:97, .... 97:3, 98:2, 99: 1 are used.
G) Design of Carbopol Gel Formulation
A 5% gel is prepared to increase the exposure time of the formulation to the wound area and to make it easy to apply. This gel formulation contains at least one pharmaceutically acceptable excipient as well as essential oils. Excipients of the formulation may be solvents, gelling agents, emulsifiers, suspenders, thickeners, antioxidants, pH adjusters, surfactants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, humectants, absorption delaying agents, plumpers, preservatives, stabilizers, binders, disintegrants, lubricants, sweetening agents, flavoring agents, and combinations thereof. The solvent of the gel formulation can be selected from a group consisting of ethanol, methanol, propanol, isopropanol, water or a binary or triple mixture thereof. The surfactant of the formulation may be selected from the group consisting of triethanolamine, sodium lauryl sulfate, sodium dodecyl sulfate, polysorbates, for example, polysorbate 20, polysorbate 60, poloxamer, phosphatidylcholine, benzalkonium chloride, span 20, span 80, span 85, cetylpyridinium chloride, cetyltrimethylammonium bromide, dodecyl amine, polyoxyethylene nonylphenol, polyoxyethylene (POE) alkyl esters, sorbitan monopalmitate, sorbitan monolaurate, polyoxyethylene sorbitan monoesters. 0.5 g carbopol and 7g glycerin mixture 5 g distilled water is mixed in the first solution in the preferred embodiment of the invention. 20 g of isopropyl alcohol and 0.5 g of carbopol mixture are mixed with 5 g of distilled water in the second solution. Then, these two solutions are combined and mixed with a magnetic stirrer at 25°C until the entire carbopol is dissolved in order to ensure gelation. Separately, various concentrations of C. aurantifolia and S. sclarea essential oils are added to the mixture of the above reagents. 3.5 g triethanolamine dissolved in 5 g water is also added to the mixture of reagents. And finally, enough water is added to bring the mixture to 100 grams. The essential oil combination is loaded into the gel in the range of 0.1-10%. The resulting mixture is mixed with the help of a spatula until it becomes homogeneous. The gel is then transferred to a clear glass bottle. And then, it is centrifuged at 112-1006 x g for 10 minutes to remove the compressed air particles. A gel without essential oil is also prepared and used as a control group. Stability control of the formulations is checked whether they remain at +4°C, 25°C and 37-40°C degrees, relative humidity, and stable pH for 0-3-6 months (Karadag et al., 2020). It is observed that it remains stable at room temperature.
H) Cytotoxicity Activity Tests of the Formulation by MTT Method
The MTT method will be applied for the cytotoxicity experiments of the formulation. A low or zero toxicity range is observed based on the tests.
I) Irritation Activity Tests of the Formulation Human keratinocyte cell line (HaCaT) will be used for the irritation study of the formulation. Dulbecco's modified Eagle's medium/high glucose (DMEM/High) medium will be cultured with 10% (v/v) heat-inactivated fetal bovine serum, L-glutamine (2mM), antibiotic- antimycotic solution (100 U/mL penicillin, 100 pg/mL streptomycin, and 0.25 pg/mL amphotericin B). Irritation experiments on this cell line will be carried out as specified in the literature (Wu et al., 2020). A non-irritant or low irritation range is observed according to the tests. J) Determination of Wound Healing Effect of the Formulation by Scratch Assay Method
The Scratch Assay Method will be applied to test the healing effect of the formulation in the in vitro wound model. It is observed that the wound healing effect is 90% and above.

Claims

CLAIMS A hydrogel formulation, characterized in that it comprises the following:
• standardized C. aurantifolia and S. sclarea essential oils as drug substances and
• at least one pharmaceutically acceptable excipient. The hydrogel formulation according to Claim 1, characterized in that it comprises 0.1- 10% by weight of C. aurantifolia and S. sclarea essential oil. The hydrogel formulation according to Claim 1, characterized in that said excipient is selected from solvents, gelling agents, emulsifiers, suspenders, thickeners, antioxidants, pH adjusters, surfactants, preservatives, isotonic agents, humectants, absorption retarding agents, plumpers, preservatives, stabilizers, binders, disintegrants, lubricants, sweetening agents, flavoring agents, or combinations thereof. The hydrogel formulation according to Claim 1 or 3, characterized in that the solvent is selected from ethanol, methanol, propanol, isopropanol, water or a binary or triple combination thereof. The hydrogel formulation according to Claim 1, 3 or 4, characterized in that the solvent is isopropyl alcohol. The hydrogel formulation according to Claim 1 or 3, characterized in that the thickener is carbomer and glycerin. The hydrogel formulation according to Claim 1 or 3, characterized in that the surfactant is selected from triethanolamine, sodium lauryl sulfate, sodium dodecyl sulfate, polysorbates; for example, polysorbate 20, polysorbate 60, poloxamer, phosphatidylcholine, benzalkonium chloride, span 20, span80, span 85, cetylpyridinium chloride, cetyltrimethylammonium bromide, dodecyl amine, polyoxyethylene nonylphenol, polyoxyethylene (POE) alkyl esters, sorbitan monopalmitate, sorbitan monolaurate, polyoxyethylene sorbitan monoesters or combinations thereof. The hydrogel formulation according to Claim 1, 3 or 7, characterized in that the surfactant is triethanolamine. The hydrogel formulation according to any one of the preceding claims for woundhealing use.
PCT/TR2023/051544 2022-12-15 2023-12-14 Gel formulation with wound healing effect comprising a salvia sclarea l. and citrus aurantifolia l. Ceased WO2024129047A1 (en)

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TR2022/019370 TR2022019370A1 (en) 2022-12-15 WOUND HEALING EFFECTIVE GEL FORMULATION CONTAINING SALVIA SCLAREA L. AND CITRUS AURANTIFOLIA L.
TR2022019370 2022-12-15

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2015143386A1 (en) * 2014-03-21 2015-09-24 The Regents Of The University Of Michigan Topical nanoemulsion therapy for wounds
EP3097905A1 (en) * 2015-05-28 2016-11-30 Symrise AG Cosmetic compositions

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Publication number Priority date Publication date Assignee Title
WO2015143386A1 (en) * 2014-03-21 2015-09-24 The Regents Of The University Of Michigan Topical nanoemulsion therapy for wounds
EP3097905A1 (en) * 2015-05-28 2016-11-30 Symrise AG Cosmetic compositions

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Title
EKAWATI, EVY R.; SUNGGING PRADANA, MUHAMMAD; DARMANTO, WIN: "Lime (Citrus aurantifolia) peel as natural antibacteria for wound skin infection caused by staphylococcus aureus", INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH, ASSOCIATION OF INDIAN PHARMACISTIND, vol. 11, no. 1, 1 March 2019 (2019-03-01), pages 363 - 366, XP009555725, ISSN: 0975-2366, DOI: 10.31838/ijpr/2019.11.01.042 *
SREEPIAN APICHAI, POPRUK SUPALUK, NUTALAI DARANEE, PHUTTHANU CHAYANIT, SREEPIAN PREEYAPORN M.: "Antibacterial Activities and Synergistic Interaction of Citrus Essential Oils and Limonene with Gentamicin against Clinically Isolated Methicillin-Resistant Staphylococcus aureus", THE SCIENTIFIC WORLD JOURNAL, HINDAWI LIMITED, vol. 2022, 28 February 2022 (2022-02-28), pages 1 - 12, XP093185277, ISSN: 2356-6140, DOI: 10.1155/2022/8418287 *

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