Title of Invention: An Anti-burn Herbal Gel Emulsion and A Method for Preparation Thereof

[0001]|The present disclosure application claims priority from IR Patent Application, Application No 139950140003003721 , filed on 18 July, 2020, entitled “Preparation of Anti-burn Herbal Gel and Emulsion with Water-in-Oil-in-Water (W/O/W) method”, which is incorporated by reference herein in its entirety

Technical Field

[0002] The present disclosure is related to an herbal pharmaceutical composition for wound healing and a method for preparation of the composition, especially a water-in-oil-in-water emulsion using an aqueous solution of a chitosan.

Background Art

[0003] Skin regeneration and healing due to a wound or burn consist of at least three stages including inflammation, proliferation, and tissue remodeling. These three stages occurred by some biochemical and cellular events.

[0004] Various treatments are topically and systemically used to heal wounds and provide a suitable condition to help and speed the three stages of wound healing. These treatments include a variety of wound healing agents such as different antibiotics and antiseptics, debriding agents, and some substances like biological extracts, vitamins and minerals, and plant products.

[0005] Plant products like herbal extracts due to having phenolic and flavonoid compounds can improve a wound healing process through improving blood clotting, preventing the speared of the infection because of their anti-bacterial properties, and speeding up the wound repairing. Actually, the plant products accelerate the healing and manage the wound healing. Furthermore, the plant products can enhance collagen deposition and proliferation of fibroblasts and fibrocytes cells, which both contribute to a faster wound healing process.

[0006] Nowadays, healing wound caused by burn is an important issue in the medical world, so finding a drug or a substance that can heal a burn wound with minimal side effects is really invaluable. Therefore, developing an effective and anti-burn pharmaceutical composition is required that the composition may be produced through an emulsifying method that a chitosan can use as an emulsifier to provide a water-in-oil-in-water gel emulsion as the pharmaceutical composition, such that by using an acidic a-amino acid can overcome the solubility problem of the chitosan and enhance a uniform distribution of a plurality of chitosan nanoparticles containing at least one herbal extract.

Summary of Invention

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] In a general aspect, the present disclosure is directed to an exemplary method for producing a gel emulsion. The exemplary method may comprise extracting at least one herbal extract, obtaining a first aqueous solution by adding the herbal extract to a water-alcohol solution such that a ratio of water to an alcohol may be adjusted in a range of 5:95 to 15:85 (v/v). Moreover, the exemplary method may further comprises forming an oil phase by adding at least one emulsifier to at least one oil, obtaining a water-in-oil mixture by adding the first aqueous solution to the oil phase, preparing a second aqueous solution by mixing a linear polysaccharide and a water-acid solution such that the water-acid solution comprises at least one acidic a-amino acid, and producing the gel emulsion by emulsifying the water-in-oil mixture utilizing the second aqueous solution such that a ratio of the second aqueous solution to the water-in-oil mixture may be adjusted in a range of 75:25 to 85:15 v/v.

[0009] The above general aspect may have one or more of the following features. In an exemplary implementation, the method may further comprises preparing a mixture of at least two different gel emulsion by homogenizing the at least two different gel emulsion such that the at least two gel emulsion differ in a type of the extract. In an exemplary implementation, the herbal extract may comprise an extract of Calendula officinalis species, an extract of Eucalyptus globulus species, an extract of Urtica dioica species, or an extract of Rosmarinus officinalis species. In one or more exemplary implementation, the herbal extract may comprise a leave extract, a flower extract, a bud extract, a petal extract, a stem extract, or a combination thereof. In one or more exemplary implementation, the herbal extract may comprise a flower extract of Calendula officinalis, a leave extract of Eucalyptus globulus, a leave extract of Urtica dioica, a leave extract of Rosmarinus officinalis, or a combination of at least two thereof. In an exemplary implementation, a concentration of the herbal extract may be in a range of 20 to 30 % (w/v). in an exemplary implementation, the alcohol may comprise one or more of ethanol, isopropanol, or a combination thereof. In an exemplary implementation, at least one oil may comprise olive oil, pumpkin seed oil, sesame oil, black seed oil, grape seed oil, castor oil, sunflower oil, a vegetable oil, or a combination of at least two thereof. In an exemplary implementation, the emulsifier may comprise at least one poly glycerol fatty acid ester. In an exemplary implementation, at least one emulsifier may comprise polyglyceryl-6 polyricinoleate, polyglyceryl-10 decaoleate, polyglyceryl-10 pentaoleate, polyglyceryl-10 pentastearate, or a combination of at least two thereof. In an exemplary implementation, the method may further comprise filtering the first aqueous solution and the oil phase. In an exemplary implementation, a ratio of the first aqueous solution to the oil phase may be adjusted in a range of 15:85 to 25:75 (v/v). in an exemplary implementation, the first aqueous solution may have at least one hydrogen carbonate substance. In an exemplary implementation, the linear polysaccharide may comprise a chitosan. In an exemplary implementation, a concentration of the linear polysaccharide may be in a range of 8 to 12 % by weight. In an exemplary implementation, the a-amine acid may comprise aspartic acid. In an exemplary embodiment, the gel emulsion may comprise a plurality of nano-particles with a mean diameter less than 90 nm.

[0010] In another general aspect, the present disclosure is directed to an exemplary method for preparation of an anti-burn gel nano-emulsion. The exemplary method may comprise extracting at least one herbal extract such that the herbal extract may comprise a flower extract of Calendula officinalis species, a leave extract of Eucalyptus globulus species, a leave extract of Urtica dioica species, a leave extract of Rosmarinus officinalis species, or a mixture thereof, obtaining a first aqueous solution by mixing the herbal extract and a water-alcohol solution such that a concentration of the herbal extract is in a range of 20 to 30% by weight, forming an oil phase by adding at least one emulsifier to at least one oil, obtaining a water-in-oil mixture by adding the first aqueous solution to the oil phase, preparing a second aqueous solution by mixing chitosan and a water-acid solution such that the water-acid solution comprises aspartic acid, producing a water-in-oil-in-water mixture by emulsifying the first mixture utilizing the second aqueous solution such that a nano-particle diameter of the gel nano-emulsion may be less than 90 nm, and preparing the anti-burn gel nano-emulsion by mixing the at least two different water-in-oil-in water mixture such that an extract type of each nano-gel emulsion may differ.

[0011] The above general aspect may have one or more of the following features. In an exemplary implementation, a ratio of the second aqueous solution to the first mixture may be adjusted in a range of 75:25 to 85:15 (v/v). In an exemplary implementation, a ratio of the first aqueous solution to the oil phase is adjusted in a range of 15:85 to 25:75 (v/v). In an exemplary implementation, the exemplary method further comprising a filtration step for filtering the first aqueous solution and the oil phase. In an exemplary implementation, a ratio of water to an alcohol in the first aqueous solution may be adjusted in a range of 5:95 to 15:85 (v/v). In one or more exemplary implementation, the alcohol may comprise one or more of ethanol, isopropanol, or a combination thereof.

[0012] In another general aspect, the present disclosure is directed to an exemplary pharmaceutical composition. The exemplary pharmaceutical composition may comprise a water-in oil-in-water gel emulsion comprising a plurality of nanoparticles, the nanoparticles may comprise a poly glycerol fatty acid ester, a wound healing agent, and a chitosan.

[0013] The above general aspect may have one or more of the following features. In an exemplary implementation, a mean diameter of the nanoparticle may be less than 90 nm. In one or more exemplary implementation, the wound healing agent may comprise at least one herbal extract. In an exemplary implementation, the herbal extract may comprise a leave extract, a flower extract, a bud extract, a petal extract, a stem extract, or a combination thereof. In an exemplary implementation, the wound healing agent may comprise one or more extract of Calendula officinalis species, Eucalyptus globulus species, Urtica dioica species, Rosmarinus officinalis species. In an exemplary implementation, the wound healing agent may comprise a mixture of a flower extract of Calendula officinalis, a leave extract of Eucalyptus globulus, a leave extract of Urtica dioica, a leave extract of Rosmarinus officinalis. In an exemplary implementation, a concentration of the mixture may be in a range of 1.5 to 4 % by weight in accordance with total weight of the composition.

Brief Description of Drawings

[0014] The drawing figures only demonstrate one or more embodiments in accord with the present teaching, by way of example only, not by way of limitation. Therefore, the drawing figures do not limit the extent of the present disclosure. Also, reference numerals with similar numbers in the figures demonstrate similar or the same elements.

Fig.1

[0015] [Fig.1 ] illustrates a flowchart of a method for producing a gel emulsion, consistent with one or more exemplary embodiments of the present disclosure.

Fig.2

[0016] [Fig.2] illustrates a Dynamic Light Scattering (DLS) curve of a plurality of nanoparticles of a gel emulsion to determine the nanoparticles size distribution, consistent with one or more exemplary embodiments of the present disclosure.

Fig.3

[0017] [Fig.3] illustrates a Scanning Electron Microscopy (SEM) image of a plurality of nanoparticles of a gel emulsion, consistent with one or more exemplary embodiments of the present disclosure.

Fig.4

[0018] [Fig.4] illustrates a Transmission Electron Microscopy (TEM) image of a plurality of nanoparticles of a gel emulsion, consistent with one or more exemplary embodiments of the present disclosure.

Fig.5

[0019] [Fig.5] illustrates a recovered encapsulated phenolic content after releasing phenolic compounds from a plurality of nanoparticles of a gel nano-emulsion during a determined time period, consistent with one or more exemplary embodiments of the present disclosure. Fig.6A

[0020] [Fig.6A] illustrates a cell viability of the human lymphocyte cells treated with different concentrations of a gel nano-emulsion in a time interval of 24, 48,72, and 96 h, consistent with one or more exemplary embodiments of the present disclosure.

Fig.6B

[0021] [Fig.6B] illustrates a cell viability of the normal skin fibroblast cells treated with different concentrations of a gel nano-emulsion in a time interval of 24, 48,72, and 96 h, consistent with one or more exemplary embodiments of the present disclosure.

Fig.7A

[0022] [Fig.7A] illustrates a fluorescent image of the human lymphocyte cells stained with Floechst 33342 that the human lymphocyte cells treated with a gel nano emulsion for 72 h, consistent with one or more exemplary embodiments of the present disclosure.

Fig.7B

[0023] [Fig.7B] illustrates a fluorescent image of the normal skin fibroblast cells stained with Floechst 33342 that the normal skin fibroblast cells treated with a gel nano-emulsion for 72 h, consistent with one or more exemplary embodiments of the present disclosure.

Description of Embodiments

[0024] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. Flowever, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims. [0025] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0026] “Chitosan”, as used herein, refers to N-deacetylated chitin and other water- soluble derivatives of chitin.

[0027] “Wound healing agent”, as used herein, refers to herbal extract, drug, and other compounds that are useful in treating wound.

[0028] "healing" in relation to a wound, as used herein, refers to the process of repairing a wound, such as the formation of a scar.

[0029] "Herbal extract", as used herein, refers to any extract obtainable from a plant or any portion thereof. Typically, the herbal extract comprises the active ingredient (s) of the plant.

[0030] Therefore, the plant extract can be obtained from the fruit, from the peel or bark of the fruit, the seeds, the bark, the leaves, the roots, the rhizome, the flower, the bark of the root or the stem of a plant, or a combination thereof.

[0031] “Plant” or “plants’, as used herein, may refer to Calendula officinalis species, Eucalyptus globulus species, Urtica dioica species, and/or Rosmarinus officinalis species.

[0032] Fig.1 illustrates an exemplary flowchart of a method 100 for producing a gel emulsion, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, as illustrated in Fig.1 , in step 102, at least one herbal extract may be extracted and in the next step (104), a first aqueous solution may be obtained by adding the herbal extract to a water-alcohol solution. In an exemplary embodiment, the water-alcohol solution may comprise a ratio of water to an alcohol in a range of 5:95 to 15:85 by volume. Step 106 may comprise forming an oil phase by adding at least one emulsifier to at least one oil and step 108 may comprise obtaining a water-in-oil mixture by adding the first aqueous solution to the oil phase. Step 110 may comprise preparing a second aqueous solution by mixing a linear polysaccharide and a water-acid solution. In an exemplary embodiment the water-acid solution may comprise at least one acidic a-amino acid. Furthermore, the exemplary method may further comprise step 112 that the step 112 may comprise producing the gel emulsion by emulsifying the water-in-oil mixture utilizing the second aqueous solution. In an exemplary embodiment, a ratio of the second aqueous solution to the water-in-oil mixture may be adjusted in a range of 75:25 to 85:15 v/v.

[0033] In one or more exemplary embodiments, the exemplary method 100 may further comprise filtering the first aqueous solution and the oil phase to separate unwanted impurity. In an exemplary embodiment, the second aqueous solution may also filter to remove impurity.

[0034] In an exemplary embodiment, the exemplary method 100 may further comprise preparing a mixture of at least two different gel emulsion by homogenizing the at least two different gel emulsion such that an extract type of each gel emulsion may differ. In an exemplary embodiment, the extract type may be chosen from at least one extract of Calendula officinalis species, Eucalyptus globulus species, Urtica dioica species, Rosmarinus officinalis species and/or a mixture thereof.

[0035] In one or more exemplary embodiments of the present disclosure, to prepare the herbal extract, different parts of plants comprising at least one or a mixture of fruits, leaves, stems, roots, flowers, buds, petals, dermal tissue, gum, resin, or a mixture of the different parts may be used.

[0036] In one or more exemplary embodiments, a flower of Calendula officinalis species to prepare the herbal extract may be used. [0037] In one or more exemplary embodiments of the present disclosure, a leave of Eucalyptus globulus species may be used to prepare the herbal extract.

[0038] In one or more exemplary embodiments, a leave extract of Urtica dioica may be prepared utilizing a leave of Urtica dioica species.

[0039] In one or more exemplary embodiments, a leave of Rosmarinus officinalis species may be gathered to prepare the herbal extract.

[0040] In one or more exemplary embodiments, the herbal extract may comprise at least one extract of a flower extract of Calendula officinalis, a leave extract of Eucalyptus globulus, a leave extract of Urtica dioica, a leave extract of Rosmarinus officinalis, or a combination of at least two thereof.

[0041] In an exemplary embodiment, a concentration of the herbal extract, for preparing the first aqueous solution in step 102, may be in a range of 20 to 30 percent by weight per volume.

[0042] In an exemplary embodiment, the herbal extract may comprise an extract that may have a wound healing effect.

[0043] In an exemplary embodiment, the alcohol may comprise ethanol, isopropanol, and/or a combination thereof.

[0044] In one or more exemplary embodiments, the emulsifier may comprise at least one poly glycerol fatty acid ester. In one or more exemplary embodiments, the emulsifier may comprise polyglyceryl-6 polyricinoleate, polyglyceryl-10 decaoleate, polyglyceryl-10 pentaoleate, polyglyceryl-10 pentastearate, or a combination of at least two thereof.

[0045] In an exemplary embodiment, the at least one oil may comprise any type of vegetable oil, for example but not limited to, olive oil, pumpkin seed oil, sesame oil, black seed oil, grape seed oil, castor oil, sunflower oil, a combination of at least two thereof, or other vegetable oils that are well known to those skilled in the art.

[0046] In one or more exemplary embodiments, to prepare the water-in-oil mixture (step 108), the first aqueous solution may be added into the oil phase in a ratio of 25:75 to 15:85 (v/v). In one or more exemplary embodiments, the ratio of the first aqueous solution to the oil phase may be adjusted in a range of 20:80 to 15:85 by volume. In an exemplary embodiment, the ratio of the first aqueous solution to the oil phase may be adjusted at 20:80 (v/v).

[0047] In one or more exemplary embodiments, the first aqueous solution may include at least one hydrogen carbonate substance. In this exemplary embodiment, when the water-in-oil mixture and the second aqueous solution was mixed to prepare the gel emulsion, using the hydrogen carbonate substance may cause more solubility of the chitosan as well as more uniformity of the plurality of nanoparticles because of producing CO2 gas through reaction of the acidic a- amino acid and hydrogen carbonate substance. In one or more exemplary embodiments, the hydrogen carbonate substance may comprise sodium hydrogen carbonate, potassium hydrogen carbonate, cesium hydrogen carbonate, magnesium hydrogen carbonate, calcium hydrogen carbonate, ammonium hydrogen carbonate, a combination thereof and/or other hydrogen carbonate substances that are well known for those skilled in the art.

[0048] In one or more exemplary embodiments, the linear polysaccharide may comprise a chitosan that may be used to prepare the second aqueous solution. In these exemplary embodiments, the acidic a-amino acid may be used to adjust a pH value of the second aqueous solution and also improve a solubility degree of the linear polysaccharide. In an exemplary embodiment, the pH value may be adjusted in a range of 3.5 to 5.5.

[0049] In one or more exemplary embodiments, the acidic a-amino acid may comprise aspartic acid, glutamic acid, aspartic acid derivatives, glutamic acid derivatives, and/or a combination thereof. In one or more exemplary embodiments, other type of acids, such as acetic acid, oxalic acid, a combination thereof, or other acids that are well known for those skilled in the art, may be used to adjust the pH value of the second solution as well as improve the solubility degree of the linear polysaccharide.

[0050] In one or more exemplary embodiments, a concentration of the linear polysaccharide may be in a range of 8 to 12 percent by weight.

[0051] In one or more exemplary embodiments, the method that disclosed in the present disclosure may applied to prepare a pharmaceutical composition. In one or more exemplary embodiments, the pharmaceutical composition may comprise a plurality of particles such that the particles may comprise the poly glycerol fatty acid ester, a wound healing agent, and the linear polysaccharide. In one or more exemplary embodiments, the particles may comprise a diameter less than lOOOnm, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 100 nm. In one or more exemplary embodiments, the diameter of particles may be less than 90 nm. In an exemplary embodiment, the diameter of particles may be in a range of 70 to 100 nm.

[0052] In one or more exemplary embodiments, the wound healing agent may comprise an herbal extract such that the herbal extract may have a good effect on treatment of a wound. In one or more exemplary embodiments, the herbal extract may comprise a leave extract, a flower extract, a bud extract, a petal extract, a stem extract, and/ or a combination thereof. In one or more exemplary embodiments, the wound healing agent may comprise an extract of Calendula officinalis species, an extract of Eucalyptus globulus species, an extract of Urtica dioica species, an extract of Rosmarinus officinalis species, and/or a mixture of at least two thereof. In one or more exemplary embodiments, the wound healing agent may comprise a flower extract of Calendula officinalis, a leave extract of Eucalyptus globulus, a leave extract of Urtica dioica, a leave extract of Rosmarinus officinalis, and/or a mixture of at least two thereof. In an exemplary embodiment, a concentration of the herbal extract may be in a range of 1.5 to 4 percent by weight in accordance with a total weigh of the pharmaceutical composition.

[0053] In one or more exemplary embodiments, typical subjects that can be treated according to this aspect of the present invention may include mammals, such as humans or domesticated animals that include, but are not limited to, horses (i.e., horses), cattle, goats, sheep, pigs, dogs, Cats, camels, alpacas, llamas and yaks, male or female, at any age who need treatment of a burn injury or a wound.

Examples

[0054] EXAMPLE 1 : HERBAL EXTRACT PREPARATION [0055] The plant species, including Eucalyptus globulus species, Urtica dioica species, Rosmarinus officinalis species, and Calendula officinalis species were first obtained and separately dried.

[0056] About 400-500 g of dried flower of Calendula officinalis species were weighted and grinded to obtain a grinded powder. Following that, 800-1200 liters of ethanol (80%) were added to the grinded powder and incubated for 24 to 72 hours. After that, an extract of Calendula officinalis species was obtained utilizing a press apparatus. Following that, residual solvents were evaporated utilizing an evaporator in a vacuum condition, so that a pure extract in a powder state was obtained.

[0057] The above steps were applied to dried leave of Eucalyptus globulus species, dried leave of Urtica dioica species, or dried leave of Rosmarinus officinalis species, so that an extract of Eucalyptus globulus species, an extract of Urtica dioica species, or an extract of Rosmarinus officinalis species was obtained

[0058] EXAMPLE 2: ANTI-BURN NANO-EMULSION PREPARATION

[0059] In Example 2, an exemplary method for preparation of an anti-burn nano emulsion was carried out consistent with the teachings of exemplary embodiments of the present disclosure. In this example, the extract of Calendula officinalis was added to a water-ethanol solution (5-15% v/v) and a first aqueous solution was obtained. The first aqueous may include a concentration of the extract in a range of 20-30 % w/v. Furthermore, polyglycerol polyricinoleate was added to olive oil to obtain an oil phase such that a concentration of polyglycerol polyricinoleate was in a range of 7 to 15 % v/v. following that, prepared solutions were filtered and unwanted impurities were removed. Afterward, the first aqueous solution was slowly added to the oil phase in a ratio of 20:80 and totally mixed for about 3 to 5 minutes, as a result a water-in-oil solution was prepared. Afterward, a second aqueous solution was prepared by adding 1 g chitosan to 10 ml deionized water and pH of solution was adjusted around 5 using aspartic acid to improve the solubility of chitosan. Then, the second aqueous solution was added to the water-in-oil solution in a ratio of 75:25 or 80:20 and homogenized for 2-5 minutes under a pressure about 100-230 bar. As a result, a nano-emulsion containing Calendula officinalis extract was prepared. The method applied separately for three extracts comprising the extract of Eucalyptus globulus species, the extract of Urtica dioica species, and the extract of Rosmarinus officinalis to prepared three nano-emulsions containing each of the extracts. Following that, the all nano-emulsions were mixed together under a pressure about 80 to 120 bar and as a result, an anti-burn nano-emulsions was prepared.

[0060] EXAMPLE 3: PHYSIOCHEMICAL CHARACTERIZATION OF THE GEL NANO-EMULSION

Particle Size, Zeta-Potential, and Polvdispersitv Index Determination

[0061] A mean particle size, zeta-potential and polydispersity index of the nanoparticles of the gel nano-emulsion were determined using Malvern zetasizer (Malvern instrument, Worcestershire, UK) apparatus. Fig.2 illustrates a particle size distribution of the nanoparticles. As illustrated in Fig.2, the nanoparticles have a mean diameter about 80.10± 0.25 nm.

A physical stability and uniformity of the nanoparticles were evaluated by the polydispersity index and zeta potential that are two important indicators that show the stability of the nanoparticles in the gel nano-emulsion. Table.1 shows the zeta potential and polydispersity index of the nanoparticles of the gel nano-emulsion. The polydispersity index values more than 0.5 indicate a poor uniformity of nanoparticles, while the polydispersity values less than 0.5 show an acceptable uniformity (Cheng et al., “Synthesis of glycyrrhetinic acid-modified chitosan 5- fluorouracil nanoparticles and its inhibition of liver cancer characteristics in vitro and in vivo. Mar Drugs,” 11: 3517-3536 (2013), which is hereby incorporated by reference), so the polydispersity index value 0.412 indicates that the nanoparticles have the acceptable uniformity. Furthermore, the nanoparticles show a zeta potential value about positive 40.1 mV. An acceptable zeta potential value is about ±25 mV and the nanoparticles with zeta potential more than +30 mV or more than -30 mV are considerably stable (Abdel-Rehim et al., “Investigation of the Tribological Behavior of Mineral Lubricant Using Copper Oxide Nano Additives,” Lubricants; 9(2):16 (2021), and Tech Note: “Zeta potential - An introduction in 30 minutes," which are hereby incorporated by references). Therefore, the result of zeta potential illustrates that the nanoparticles also considered stable. Table 1 : Zeta Potential and Polydispersity Index of the Gel Nano-emulsion

[0062] Surface Morphology and Particle Size Investigation

[0063] A surface morphology and a particle size of the nanoparticles of the gel nano emulsion were investigated using Scanning Electron Microscopy (SEM Phenom Pro-X, Phenom-World, Netherlands) and Transmission Electron Microscopy (TEM EM 900, Zeiss, Germany).

[0064] Fig.3 and Fig.4 illustrate a SEM image and a TEM image of nanoparticles, respectively. As illustrated in Fig.3 and Fig.4, the nanoparticles have a uniform spherical shape with a relatively monodispersed size distribution.

Determination of Antioxidant Activity

[0065] An antioxidant activity of each extract and the gel nano-emulsion was analyzed using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. The DPPH solution was prepared in methanol. Then, the DPPH solution was subsequently added to the various concentrations of each extract or gel nano-emulsion (5, 15, 30, 45, and 60 ppm). The absorbance changes were measured at 517 nm. Synthesized Butylated hydroxytoluene (BHT) was used as a positive control. These measurements were performed in triplicate and the IC50 values were calculated using a linear regression analysis and used to indicate the antioxidant activity.

[0066] Table.2 shows the IC50 values of the extracts and the gel nano-emulsion. The lower IC50 value indicated a higher antioxidant activity. The antioxidants have a beneficial effect on a treatment of burn patients. The materials with the antioxidant activities cause reduction of a rate of a wound infection and a healing time. Also, these materials affect a mortality rate. (Ann Burns Fire Disasters.

2010 Dec 31 ; 23(4): 199-205). So, as illustrated in Table.2, the gel nano emulsion has a good antioxidant activity in compared to the extracts of Calendula officinalis species, Urtica dioica species, and Eucalyptus globulus species. Therefore, the gel nano-emulsion has a suitable efficiency in a treatment of a wound due to the high antioxidant activity. Table.2: IC ₅₀ Value of the Extracts and the Gel Nano-emulsion

Determination of Total Polyphenolic, tal Flavonoid, and Total Alkaloid Contents

[0067] A total polyphenols content of the extracts and gel nano-emulsion was measured by UV spectrophotometrically according to the Folin-Ciocalteu method using gallic acid as a standard with slightly modification (Chandra et al., "Assessment of Total Phenolic and Flavonoid Content, Antioxidant Properties, and Yield of Aeroponically and Conventionally Grown Leafy Vegetables and Fruit Crops: A Comparative Study," Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 253875, 9 pages (2014), which is hereby incorporated by reference). Briefly, 0.1 ml of the extract or gel nano-emulsion was mixed with 0.5 ml of Folin- Ciocalteu reagent in a test tube and volume was made up to the 3 ml with distilled water. After 3 minutes of incubation, 2 ml of 20% sodium carbonate (Na2CC>3) solution was added and mixed thoroughly. The resulting mixture was incubated for 5 minutes at 50 °C and cooled at room temperature. Absorbance of the mixture was measured at 765 nm against the reagent blank. All measurements were carried out in triplicate. A Content of phenolic compounds was expressed as mg of gallic acid equivalents (GAE)/g of each extract or gel nano-emulsion using the linear equation obtained from calibration curve of the standard gallic acid graph. The coefficient of determination ( R ² ) was 0.9962. Table.3 shows the total phenolic content of the extracts and gel nano-emulsion. Table.3: Total Phenolic Content (TPC) of Extracts and Gel nano-emulsion

[0068] Isolated polyphenolic substances in herbal extracts have a good effect on the wound treatment (Nasiri et al., “Effect of Malva sylvestris cream on burn injury and wounds in rats". Avicenna J. Phytomed., 5(4):341-354 (2015), which is hereby incorporated by reference). The gel nano-emulsion containing all four herbal extracts has a total phenolic content about 147 mg/g that indicates a good amount of polyphenolic substance is exist. Therefore, the healing activity of the prepared gel nano-emulsion, at least in part, can be attributed to its high polyphenolic content.

[0069] The total flavonoid content of the extracts or gel nano-emulsion was determined according to aluminum chloride method using quercetin as standard (Chandra et al., " Assessment of Total Phenolic and Flavonoid Content, Antioxidant Properties, and Yield of Aeroponically and Conventionally Grown Leafy Vegetables and Fruit Crops: A Comparative Study,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 253875, 9 pages (2014), which is hereby incorporated by reference). A volume of 0.5 ml of aluminum chloride (AlC ) ethanol solution (2%) was added to 0.5 ml of sample solution. Each sample (each extract or gel nano-emulsion) was evaluated at a final concentration of 0.1 mg/ml. After 1 h of incubation at room temperature, an absorbance was measured at 420 nm. All measurements were carried out in triplicate. The total flavonoid content was calculated as mg of quercetin equivalents (QE)/g of each extract or gel nano-emulsion using the linear equation obtained from calibration curve of the standard quercetin graph. The coefficient of determination ( R ² ) was 0.9812. [0070] The total alkaloid content of the extracts and gel nano-emulsion was measured as described in Ajanal et al., “Estimation of total alkaloid in Chitrakadivati by UV-Spectrophotometer,” Anc. Sci. Life, 31 (4):198-201 (2012), which is hereby incorporated by reference. The Atropine was used as standard solution and an Atropine calibration curve was prepared to determine the total alkaloid content. Briefly, aliquots of standard solution were transferred to different separator funnels. Then 5 ml of pH 4.7 phosphate buffer and 5 ml of bromocresol green (BCG) solution was taken and the mixture was shaken with each extract or gel nano-emulsion with 1 , 2, 3, and 4 ml of chloroform. The extracts or gel nano emulsion were then collected in 10 ml volumetric flask and then diluted to adjust solution with chloroform. The absorbance of the complex in chloroform was measured at spectrum of 470 nm in UV-Spectrophotometer (Shimadzu, Japan) against the blank prepared as above but without Atropine. The coefficient of determination (R ² ) was 0.9851.

Table.4: Total Flavonoid Content (TFC) of Extracts and Gel nano-emulsion

[0071] The total alkaloid content of the extracts and gel nano-emulsion was measured as described in Ajanal et al., “Estimation of total alkaloid in Chitrakadivati by UV-Spectrophotometer,” Anc. Sci. Life, 31 (4):198-201 (2012), which is hereby incorporated by reference. The Atropine was used as standard solution and an Atropine calibration curve was prepared to determine the total alkaloid content. Briefly, aliquots of standard solution were transferred to different separator funnels. Then 5 ml of pH 4.7 phosphate buffer and 5 ml of bromocresol green (BCG) solution was taken and the mixture was shaken with each extract or gel nano-emulsion with 1 , 2, 3, and 4 ml of chloroform. The extracts or gel nano emulsion were then collected in 10 ml volumetric flask and then diluted to adjust solution with chloroform. The absorbance of the complex in chloroform was measured at spectrum of 470 nm in UV-Spectrophotometer (Shimadzu, Japan) against the blank prepared as above but without Atropine. The coefficient of determination (R ² ) was 0.9851.

Table. 5: Total alkaloid Content of Extracts and Gel nano-emulsion

[0072] The gel nano-emulsion represented an alkaloid content about 15 mg/g

(Table.5). This amount of the alkaloid confirmed that the gel nano-emulsion has a suitable antibacterial efficacy and it affects the wound healing process (Cushnie et al. , “Alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities," Int. J. Antimicrob. Agents, 44(5):377-86 (2014), which is hereby incorporated by reference).

Determination of Encapsulation Efficiency

[0073] An encapsulation efficacy of the phenolic compounds in the nanoparticles of the gel nano-emulsion was determined as a method described in Othman et.al., “Synthesis and optimization of chitosan nanoparticles loaded with l-ascorbic acid and thymoquinone,” Nanomaterials, 8(11 ):920 (2018), which is hereby incorporated by reference, with some modification. Briefly, the nanoparticles were separated from the aqueous suspension by centrifugation at 48,000 g for 30 min and a supernatant was collected and analyzed to determine a free phenolic content. The encapsulation efficiency (EE) was calculated as follows:

%EE= [(T-F)/T] x100

Where T is the total phenolic content and F is the free phenolic content. [0074] The results indicated that the encapsulation efficiency of the nanoparticles was 79.00 % ± 1.30 that confirmed that up to 80 % of the total phenolic content of the gel nano-emulsion encapsulated by nanoparticles.

In Vitro Release of Phenolic Compounds

[0075] A released phenolic compounds content from the nanoparticles were measured using a method described in Gharib et al. , “Preparation, characterization and in vitro efficacy of magnetic nanoliposomes containing the artemisinin and transferrin," DARU. J. Pharm. Sci. ,22:44 (2014), which is hereby incorporated by reference. Briefly, a cellulose membrane (molecular weight cut off of 8000 kDa) was placed between a donor section and a receptor section a donor medium contains 1 ml of the gel nano-emulsion and a receptor medium contains 10 ml of citrate-phosphate buffer (0.1 M, pH 7.4). During the dialysis, a temperature was kept at 37 °C. At predetermined time intervals, between 2h to 12 h, the released phenolic compounds content was analyzed utilizing a spectrophotometry as described above for determination of the phenolic content.

[0076] Fig.5 illustrated the total encapsulated phenolic compounds contents (%) after releasing the phenolic compounds from the nanoparticles of the gel nano emulsion during the predetermined time intervals (at 37 °C). As illustrated in Fig.5, the total encapsulated phenolic compounds content after 12 h was about 72.30 % ± 1.75. This result suggest that the nanoparticles could be stable at the body temperature (at 37 °C) and the encapsulated phenolic compounds would be released slowly at a wound site.

[0077] EXAMPLE 4: IN TOOCYTOTOXYCITY ASSESSMENT In Vitro Cell Viability Assay

[0078] Cell viability of normal human skin fibroblast cell line (HSF-PI-16) and human lymphocyte was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT) assay as described previously. Briefly, 5 c 10 ³ of each cell was incubated in 96 well plates in the presence of different concentrations of the gel nano-emulsion (0, 100, 200, 300, and 400 pg/ml) for 24, 48, 72, and 96 h in a final volume of 200 pi. At the end of each time, 20 mI aliquots of MTT solution (5 mg/ml in PBS) was added to each well, and the plates were incubated at 37 °C. Subsequently, the purple-blue MTT formazan precipitate was dissolved in triplicate. As illustrated in Fig.6A and Fig.6B, the human lymphocyte cells and the normal skin fibroblast cells after 24, 48, 72, and 96 h treatment with different concentration of the gel nano-emulsion show a viability percentage above 97% that confirmed the gel nano-emulsion had no significant cytotoxic activity against the human lymphocyte and normal skin fibroblast cells.

Nuclear Staining with Hoechst 33342

[0079] The human lymphocyte cells were incubated with 400 pg/ml of the gel nano emulsion for 72 h and washed with PBS buffer. After fixing in a freshly prepared ice-cold paraformaldehyde (0.1 %) for 10 min, then the cells were stained with 50 pg/ml of Hoechst 33342 for 1 min and the morphologic changes of the human lymphocyte were observed under a fluorescent microscope. The same method was applied to the normal skin fibroblast cells and the morphology changes of the cells were observed under the fluorescent microscope. Fig.7A and Fig.7B illustrate the morphology changes of the human lymphocyte cells and normal skin fibroblast cells treated by the gel nano-emulsion, respectively. As indicated in Fig.7A and Fig.7B, the both type of cells show no significant characteristic apoptotic features such as s nuclear fragmentation and a chromatin condensation.

[0080] EXAMPLE 5: IN VIVO ASSESSMENTS Determination of the Gel Nano-emulsion Efficacy

[0081] The in vivo therapeutic efficacies of gel nano-emulsion containing Eucalyptus globulus species, Urtica dioica species, Rosmarinus officinalis species, and Calendula officinalis species extract were tested by a method as described in Gharib et al., “Therapeutic efficacy of epigallocatechin gallate-loaded nanoliposomes against burn wound infection by methicillin-resistant Staphylococcus aureus,” Skin Pharmacol Physiol., 26(2):68-75 (2013), which is hereby incorporated by reference, with some modification. In brief, Wistar rats (200 g) were obtained from the National Institute of Pasture, Iran. All rats were handled according to the national guidelines for laboratory animals. The animals were kept in cages in a controlled environment (22±2 °C, 50% humidity, and 12h light/dark cycle) with water and food ad libitum. The animals were anesthetized intraperitoneally with 0.2 ml/100g of ketamine and 0.05 ml/100g of xylazine, and their backs were shaved. To induce burn wounds on the backs of the rats a method applied as described in Souza et al., “Heterogeneity of mast cells and expression of Annexin A 1 protein in a second degree burn model with silver sulfadiazine treatment,” PLoS One, 12(3): e0173417 (2017), which is hereby incorporated by reference. Briefly, a metal block with dimensions of 1 x1cm ² was heated in boiling water for 15 min and applied to the animal’s shaved back for 50 s. Also, a sterile biopsy punch equipment (Revolving punch pliers, Germany) was used to create full thickness skin wounds (1 x1cm ² ) on the backs of the rats that the back skin area was cleaned with 70% ethanol (Ponrasu et al., “Efficacy of Annona squamosa on wound healing in streptozotocin-induced diabetic rats,” Int. Wound J., 9(6):613-623 (2012), which is hereby incorporated by reference).

Then, 5mI of the bacterial inoculum of clinical isolated resistant pseudomonas aeruginosa and staphylococcus aureus (containing 1 x 10 ⁵ colony-forming unit (CFUJ of each bacterium) was applied subcutaneously into the site of wounds area (1 x1 cm ² ) on the animal’s back. The burned and skin wounds were divided into 7 groups. All groups were treated topically as follows: control group (C) received physiological saline (1 ml/kg/12 h); first burn group (G1) received the gel nano-emulsion (100 mg/kg/12h); first skin wound group (G2) received the gel nano-emulsion (100 mg/kg/12h); second burn group (G3) received Silvamax ointment (100 mg/kg/12h); second skin wound group (G4) received Silvamax ointment (100 mg/kg/12h); third burn group (G5) received Burnova Gel ^® containing aloe vera and cucumber extracts (100 mg/kg/12h); and third skin wound group (G6) received Burnova Gel ^® containing aloe vera and cucumber extracts (100 mg/kg/12h). The treatment was for 12 days starting from the 3rd day after infection. Two days after the last dose the surviving rats were anesthetized and sacrificed by cervical dislocation and the CFU was counted to determine bacterial count at wound area of each group. The results and the survival rate of animal in each group was represented in Table.6. Table.6: Wound Area Bacterial Count and Animal’s Survival Rate

The Data are expressed as Mean ± Standard error of mean from three separate experiments. Analysis of variance of one-way classification between the treatment means was heterogeneous and the t-test values (two-tailed) were significant, ^* p < 0.05.

[0082] As showed in Table.6, treatment of rats with the gel nano-emulsion resulted in 100% survival rate and completes eradication of two resistant bacteria from the skin of infected animals, whereas the group of rats treated with Silvamax and Burnova Gel® as commercial brands showed an increase in survival rate for the burned and wounded animals of 50 and 90%, respectively. Therefore, the gel nano-emulsion represented a better antibacterial property compared to the both commercial brands. The antibacterial property may be provided by the extracts of the gel nano-emulsion and due to an interaction between the bacterial cells and the nanoparticles containing the extracts in the burned or wounded area.

Measurement of Wound Contraction

[0083] Wound healing was monitored on days 4, 8 and 12 post treatments, so a wound contraction and wound area were obtained via measurement of relic wound size utilizing a caliper and a graphing paper (Table.7). Wound size was expressed as the percentage of the wound area determined on every post treatment day, compared with the original wound area:

%wound contraction = (Ao - At)/Ao c100

Where Ao is the original wound area and At is the area of wound at the time of post treatments (on day 4, 8 and 12 accordingly). Table.7: Wound Contraction and Wound Area

The Data are expressed as Meant Standard error of mean from three separate experiments, *p < 0.05.

[0084] As represented in Table.7, the groups were treated by the gel nano-emulsion showed a wound contraction about 100%. Also, due to this excellent wound healing, the wound area cannot be measured. These results confirmed that the gel nano-emulsion contain a great and suitable phenolic and flavonoid content that encapsulated in nanoparticles of the gel nano-emulsion. These compounds provide a good wound healing potency owing to their astringent and antibacterial activity.

Measurement of Tensile Strength and Hvdroxyproline Estimation of Healing Skin

[0085] A tensile strength and hydroxyproline of a healing skin wound were measured as methods described in Nagar et al., “Pharmacological investigation of the wound healing activity of oestrum nocturnum (L.) ointment in wistar albino," Rats. J. Pharm., vol 2016: 9249040 (2016), which is hereby incorporated by reference.

[0086] A degree of wound healing is obtained by the tensile strength of a healing skin wound. This tensile strength represents how much the healed tissue resists to breaking under tension and may identify the quality of healing tissue. At the end of treatment period, the animals were anesthetized and the healed tissue was excised from all animals. To measure of the tensile strength, a tensiometer was applied on the excised tissue.

[0087] All rats were analyzed for the estimation of hydroxyproline. In brief, healing tissues of skin were dried in a hot air oven at 60 °C to constant weight and were hydrolyzed in 6N HCI for 3h at 130 °C. The hydrolysates were then neutralized to pH 7.0 and were subjected to Chloramine-T oxidation for 20 min. After 5 min, the reaction was terminated by the addition of 0.4M perchloric acid and developed color with Ehrlich reagent at 60 °C. After thorough stirring the samples were analyzed at 575 nm in UV-Visible Spectrophotometer (Shimadzu, Japan). The hydroxyproline content in the tissue samples was calculated using a standard curve of the pure L-hydroxyproline. The coefficient of determination ( R ² ) was 0.9991.

[0088] Table.8 shows the tensile strength and Hydroxyproline content of healing skin.

Table.8: Tensile Strength and Hydroxyproline of Healing Skin in the Treatments Groups

The Data are expressed as Mean ±Standard error of mean and the experiments were done in triplicates, *p < 0.01, **p < 0.05 and *** Non applicable, no rats survived.

[0089] Collagen as a main extracellular protein plays an important role in wound healing and integrity of tissue matrix and a degree of wound healing depends on an amount of new collagen secretion and deposition (Hassan et al., “pharmacological and toxicological properties of leaf extracts of Kingelia africana (Bignoniaceae),” J. Pharmacol. Toxicol., 6(2):124-132 (2011), which is hereby incorporated by reference). Evaluation of a level of hydroxyproline that is a biochemical marker for assessment of a collagen turnover is represented in Table.8. As represented in Table.8, the groups that were treated with the gel nano-emulsion indicated a significant hydroxyproline level. These results confirmed that an excellent level of new collagen was dispositioned on the wounded or burned area and result in a swift wound healing that may be occurred due to presence of the herbal extracts active compounds in the nanoparticles of the gel nano-emulsion.

[0090] While particular aspects of the present subject matter described herein have been shown and described, it will be apparent to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this subject matter described herein and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this subject matter described herein. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” to introduce claim recitations. Flowever, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).

[0091] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first, second, and third and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” “include,” “including,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, apparatus, or device. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or device that comprises the element. Moreover, “may” and other permissive terms are used herein for describing optional features of various embodiments. These terms likewise describe selectable or configurable features generally, unless the context dictates otherwise.