Plectranthus amboinicus extracts and method for preparing same

Abstract

A method for preparing an extract of Plectranthus amboinicus (PA) includes extracting the aerial parts of Plectranthus amboinicus with an extraction solution containing a solvent having a suitable polarity index, filtering and concentrating the resulting extract, and performing a chromatography separation step on the concentrated extract using a hydrophobic interaction chromatography resin to produce the Plectranthus amboinicus extract.

Description

[Technical Field]

[0001] Cross-reference to related applications. This application claims the benefit of U.S. Provisional Application No. 63 / 215,697, filed June 28, 2021, the entire contents of which are incorporated herein by reference. This invention relates to a method for preparing a hand-scented extract. [Previous Technology]

[0002] Plectranthus amboinicus (also known as Coleuus amboinicus Lour., Coleuus aromaticus Benth., Coleuus aromaticus auct., Plectranthus aromaticus Roxb., Plectranthus aromaticus Benth., and Plectranthus amboinicus (Lour.) Spreng.) is a perennial herbaceous plant belonging to the Lamiaceae family (also known as Labiatae), native to southern and eastern Africa. Plectranthus amboinicus is also called patchouli, Cuban oregano, Indian borage, Indian mint, Mexican mint, Mexican oregano, country borage, and Spanish thyme.

[0003] Houttuynia cordata possesses numerous health benefits, including antimicrobial, antifungal, anti-inflammatory, antidiabetic, anti-anxiety, antitumor, analgesic, antimalarial, anti-biofilm effects, diuretic, wound-healing, skin-care, respiratory disease treatment, and antiplatelet aggregation activities. It has been reported that Houttuynia cordata contains several classes of phytochemicals, including monoterpenes, diterpenes, triterpenes, sesquiterpenes, phenols, flavonoids, and esters. Different preparation methods result in different combinations of phytochemical components in Houttuynia cordata extracts at different concentrations, thus leading to different health benefits. [Summary of the Invention]

[0004] This invention is at least in part based on the development of a method for producing a *Paspalum distichum* (PA) extract having desired active ingredients (e.g., effectively promoting wound healing). This method significantly shortens the overall preparation process and has a high yield.

[0005] Therefore, the present invention provides a method for preparing a palmetto (PA) extract, the method comprising: (i) mixing the aerial parts of a palmetto with an extraction solution to produce a first palmetto extract, wherein the extraction solution contains a solvent with a polarity index of about 2.9 to 6.6; (ii) filtering and optionally concentrating the first palmetto extract to produce a second palmetto extract, which may be a concentrated palmetto extract; (iii) contacting the second palmetto extract (e.g., the concentrated palmetto extract) with a hydrophobic interaction chromatography resin; and (iv) flushing the column with an extraction solution to produce the palmetto extract, wherein the extraction solution contains a solvent with a polarity index of about 2.1 to 5.4.

[0006] In some specific embodiments, the solvent in the extraction solution in step (i) may be acetone, butyl methyl ether, ethanol, ethyl acetate, isopropanol, methanol, or a mixture thereof. In some embodiments, the solvent in the extraction solution is ethanol. In other embodiments, the solvent in the extraction solution is ethyl acetate. In other embodiments, the solvent in the extraction solution is acetone. Alternatively, the solvent in the extraction solution is isopropanol. In other embodiments, the solvent in the extraction solution may be methanol.

[0007] In some cases, step (i) may be repeated 2-4 times, optionally 2 times. In some cases, step (i) may be carried out at a temperature of about 25-80°C (e.g., about 40-80°C or about 50-80°C) for about 1-9 hours (e.g., 1-3 hours). Alternatively, step (i) may be carried out at a temperature of about 20-30°C for about 12-48 hours (e.g., 12-24 hours or 24-48 hours). In some embodiments, the weight-to-volume ratio between the aerial parts of the aralia and the extraction solution in step (i) may be about 1:10 to about 1:20 (kg / L).

[0008] In some embodiments, in step (ii), the first euphorbia extract may be concentrated to reduce its volume to about 30%-70% (e.g., about 30-50%) of its original volume to produce the concentrated euphorbia extract. In other embodiments, the first euphorbia extract may be concentrated to reduce its volume to about 2.5% to about 4% of its original volume to produce the concentrated euphorbia extract.

[0009] In other specific embodiments, step (ii) does not involve concentrating the first senna extract.

[0010] In some specific embodiments, the hydrophobic interaction chromatography resin used in step (iii) may be a nonpolar copolymer styrene-divinylbenzene adsorbent resin, a polystyrene polymer crosslinked with divinylbenzene, or a polystyrene-divinylbenzene copolymer resin. In some cases, step (iii) may be performed by loading the concentrated styrene extract onto a column containing the hydrophobic interaction chromatography resin. Alternatively, step (iii) may be performed by mixing the concentrated styrene extract with the hydrophobic interaction chromatography resin.

[0011] In some specific embodiments, the extraction solution in step (iv) may comprise a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, isopropanol, and hexane. In some cases, the extraction solution may comprise two solvents in a volume ratio of 50:50. In one embodiment, the extraction solution comprises a mixture of hexane and ethyl acetate. In another embodiment, the extraction solution comprises a mixture of ethanol and ethyl acetate. In yet another embodiment, the extraction solution comprises a mixture of acetone and ethyl acetate. In still another embodiment, the extraction solution comprises a mixture of acetone and isopropanol.

[0012] Any method disclosed herein may further include washing the hydrophobic interaction chromatography resin with an aqueous solution containing ethanol before step (iv) and after step (iii).

[0013] This document also provides a senna extract (PA) prepared by any of the methods disclosed herein. In some cases, the senna extract may contain trifurcifen, tylosin, rosmarinic acid, and carvacrol. In some embodiments, the senna extract prepared by the methods disclosed herein may contain 0.04%-0.4% trifurcifen (w / w), 0.01%-0.9% tylosin (w / w), up to 1.1% rosmarinic acid (w / w), and 0.04%-6.7% carvacrol (w / w).

[0014] Details of one or more specific embodiments of the present invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of several specific embodiments, as well as from the appended claims.

Implementation Method

[0016] Phyllanthus praecox is a medicinal plant with various pharmacological properties. Different preparation methods result in different combinations of phytochemical components in Phyllanthus praecox (PA) extracts at different concentrations, leading to different biological activities and medicinal values.

[0017] This document provides a method for producing a senna extract containing phytochemicals having desired therapeutic activity (e.g., wound healing). Such a method exhibits one or more advantageous features, such as high yield and reduced preparation time. Therefore, the preparation method disclosed herein can efficiently produce senna extract for use in preparing pharmaceutical compositions having desired therapeutic efficacy (e.g., promoting wound healing).

[0018] I. Preparation of PA extract

[0019] In some aspects, the present invention provides a method for preparing a senna extract using the aerial parts of the senna plant as a starting material. The aerial parts may include leaves, stems, flowers, or combinations thereof. In some embodiments, the entire aerial part of the senna plant may be used as the starting material. Fresh aerial parts of the senna plant may be used. Alternatively, dried aerial parts of the senna plant may be used. In some cases, the aerial parts of the senna plant may be dried to form a powder, which may be used as a starting material for preparing the senna extract disclosed herein, also within the scope of the present invention.

[0020] Any preparation method disclosed herein may include at least the following steps: (i) an extraction step, (ii) a filtration and concentration step, (iii) a separation step by hydrophobic interaction chromatography, and (iv) an extraction step. In some cases, the method may further include a washing step between the separation step (iii) and the extraction step (iv).

[0021] (a) Extraction steps

[0022] The extraction step can be performed by mixing the above-ground parts of the *Hedyotis diffusa* plant (e.g., those disclosed above) with an extraction solution containing a solvent having a suitable polarity index. The polarity index of a solvent is a relative measure of the degree of interaction between a solvent and various polarity test solutes. Table 1 below lists commonly used solvents and their polarity indices (Snyder polarity indices).

[0023] Table 1. Exemplary solvents and their polarity indices solvent Polarity Index (Snyder) Cyclohexane 0 n-Hexane 0 n-Decane 0.3 Isooctane 0.4 Octane 0.4 Butyl ether 1.7 Carbon tetrachloride 1.7 Triethylamine 1.8 Isopropyl ether 2.2 Toluene 2.3 p-xylene 2.4 tert-butyl methyl ether 2.9 benzene 3 benzyl ether 3.3 dichloromethane 3.4 dichloromethane 3.4 chloroform 3.4-4.4 dichloroethane 3.7 dichloroethane 3.7 1-Butanol 3.9 Isobutanol 3.9 Tetrahydrofuran 4.2 Ethyl acetate 4.3 1-Propanol 4.3 2-Propanol 4.3 Methyl acetate 4.4 Cyclohexanone 4.5 Methyl ethyl ketone (methyl ethyl ketone, MEK) 4.5 Nitrobenzene 4.5 Benzonitrile 4.6 1,4-Dioxane 4.8 p-Dioxane 4.8 ethanol 5.2 Nitroethane 5.3 Pyridine 5.3 acetone 5.4 benzyl alcohol 5.5 2-Methoxyethanol 5.7 acetic acid 6.2 Acetonitrile 6.2 N,N-Dimethylformamide 6.4 dimethyl monoxide 6.5 methanol 6.6 Methionine 7.3 water 9

[0024] In some cases, a solvent with a polarity index of about 2.9 to about 6.6 may be used in the extraction step. In some cases, the solvent may be a single solvent (i.e., containing a homogeneous group of solvent compounds, such as ethanol). In other cases, the solvent may be a co-solvent (i.e., a mixture of multiple solvent compounds, such as a mixture of ethanol and acetone).

[0025] Suitable solvents for the extraction step may be any solvent or mixture thereof with a polarity index of 2.9 to 6.6 in Table 1. In some embodiments, the solvent may be ethanol. In other embodiments, the solvent may be acetone. In still other embodiments, the solvent may be a mixture of ethanol and acetone (e.g., a 1:1 volume ratio). Other suitable solvents include tert-butyl methyl ether, ethyl acetate, isopropanol, methanol, or mixtures thereof.

[0026] In some cases, the aerial parts of the *Hedyotis diffusa* plant may be mixed with any suitable solvent disclosed herein at a ratio of about 1:10 to 1:20 (kg / L). For example, 1 kg of the aerial parts of the *Hedyotis diffusa* plant may be placed in 10-20 liters of solvent. The mixture may be stirred at a suitable temperature (e.g., room temperature to 80°C) for a suitable period of time (e.g., 1 hour to 24 hours). In some embodiments, the extraction step may be carried out at an elevated temperature, for example, at 60 ± 5°C. In this case, the extraction time may be shortened, for example, about 1 hour to 2 hours (e.g., about 90 minutes). Alternatively, the extraction step may be carried out at room temperature, for example, at about 20-25°C. In this case, the extraction time may require about 12 hours to 24 hours. In other embodiments, the extraction step may be carried out at about 30-40°C for about 3-8 hours (e.g., 4 hours).

[0027] Any extraction step disclosed herein may be repeated multiple times, for example, twice.

[0028] (b) Filtration and selective concentration steps

[0029] In some cases, the crude extract obtained from the extraction steps disclosed herein may be filtered to remove solid components.

[0030] In some specific embodiments, the crude extract or the resulting filtrate can be concentrated by conventional methods, such as vacuum concentration. In some embodiments, the volume of the concentrated extract can be reduced to about 1%-70% of the original volume (crude extract or filtrate). In some embodiments, the crude extract or filtrate can be lightly concentrated, for example, the volume of the concentrated extract can be about 30%-70% (e.g., about 30-50%) of the volume before concentration (original volume). In other embodiments, the crude extract or filtrate can be highly concentrated, for example, reduced to about 2%-10% of the volume. In some embodiments, the volume of the concentrated extract can be about 2-4% of the volume before concentration (original volume).

[0031] Alternatively, the crude extract or the resulting filtrate may be substantially unconcentrated (volume reduction of less than 20%, for example less than 15%, 10%, or 5%) or not concentrated at all.

[0032] (c) Separation steps

[0033] The concentrated crude extract obtained from the filtration and concentration steps can then be separated by hydrophobic interaction chromatography, which separates the components in the concentrated crude extract based on differences in surface hydrophobicity. This step can be performed by contacting the concentrated solution with a hydrophobic interaction chromatography resin, which can be any nonionic and hydrophobic resin. Examples include, but are not limited to, nonpolar copolymer styrene-divinylbenzene adsorbent resins (e.g., DIAION® HP20, DIAION® HP20SS, AMBERLITE® XAD-2, or AMBERLITE® XAD-4), a nonionic and crosslinked polymer adsorbent resin (SEPLITE® LX20), or polystyrene-divinylbenzene copolymer resins (e.g., AmberliteTMXAD1180N). Other examples include methacrylate-based resins, silicone, and nonionic polystyrene-divinylbenzene polymer resins (e.g., SEPABEAD® SP27).

[0034] In some embodiments, the hydrophobic interaction chromatography resin may be packed in a column that has been pre-washed to equilibrate before loading the material for separation. The concentrated solution can then be loaded onto and flow through the column. In some embodiments (e.g., high-concentration crude extract), the column may be sequentially washed with one or more solutions after loading. The solutions used for sequential washing may contain solvents with high to low polarity indices. For example, the column may be washed first with water, then with 50% ethanol (dissolved in water), and then with ethanol. Alternatively, the resin may be washed with a solvent with a low polarity index (e.g., hexane). In other embodiments, this washing step may not be necessary, for example, for low-concentration crude extracts.

[0035] In other embodiments, the concentrated solution may be mixed with a hydrophobic interaction chromatography resin. After stirring for a suitable period of time, the mixture may be filtered to remove the supernatant. In some cases (e.g., highly concentrated crude extracts), the resin may be sequentially washed with one or more solutions after loading by mixing and stirring the resin in the solution. The solutions used for sequential washing may contain solvents with high to low polarity indices. For example, the resin may first be washed with water, then with 50% ethanol (dissolved in water), and then with ethanol. Alternatively, the resin may be washed with a solvent with a low polarity index (e.g., hexane). In other embodiments, this washing step may not be necessary, for example, for low-concentration crude extracts.

[0036] (d) Rinse-off step

[0037] The resin packed in the column or mixed with the concentrated crude extract can then be flushed with a suitable extraction solution to provide the target gentian (PA) extract. The extraction solution may contain a solvent with a suitable polarity index, for example, from about 2.1 to about 5.4. In some embodiments, a solvent with a polarity index of 4.3-5.4 may be used. In some embodiments, the solvent may be a single solvent. In others, the solvent may be a co-solvent. Examples of solvents used for the extraction solution include, but are not limited to, mixtures of ethanol and ethyl acetate (e.g., at a volume ratio of 1:1 or 2:1), mixtures of acetone and ethyl acetate (e.g., at a volume ratio of 1:1 or 2:1), and mixtures of hexane and ethyl acetate (e.g., at a volume ratio of 1:1). Other solvents with similar polarity indices to these embodiments may also be used in the extraction solution.

[0038] (e) Exemplary extraction method

[0039] Table 2 below provides exemplary solvents and conditions for each step of the extraction method disclosed herein. These exemplary conditions are provided for illustrative purposes only.

[0040] Table 2. Exemplary solvents and conditions used in the preparation of *Hedyotis diffusa* extract. step Reagents and conditions Example extraction Solvent: Polarity index (PI) is approximately 2.9 to 6.6. Ethanol (PI: 5.2) Ethyl acetate (PI: 4.3) ˙Acetone (PI: 5.4) Isopropanol (PI: 4.3) Methanol (PI: 6.6) Temperature: 25-80ºC 25 ± 2ºC 40 ± 2ºC 60 ± 5ºC Time: 1-9 hours 1 hour 2 hours 3 hours Number of times: 2-4 ˙1 time ˙2 times ˙3 times concentrate yes Up to 6 times concentrated no Separation Resin: Non-polar copolymer styrene-divinylbenzene adsorption resin, non-ionic cross-linked polymer adsorption resin, polystyrene-divinylbenzene copolymer resin, or SiO2 HP20 LX-20 ˙XAD1180N Silicone Separation methods load ˙Combination Stirring Pour Extraction solution: a solvent with a PI of approximately 2.1-5.4. hexane and ethyl acetate · Ethanol and ethyl acetate ˙Acetone and ethyl acetate Acetone and Isopropanol

[0041] Fractions extracted using any of the extraction solutions disclosed herein may be collected, which are euphorbia extracts containing phytochemicals (e.g., trifleurone) with desired biological activity (e.g., promoting wound healing).

[0042] II. A pharmaceutical composition containing Paeonia lactiflora (PA) extract

[0043] *Paspalum discolor* (PA) extracts prepared by any of the methods disclosed herein may be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in the treatment disclosed herein. "Acceptable" means that the carrier must be compatible with (and preferably, able to stabilize) the active ingredient of the composition and harmless to the individual to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, *Remington: The Science and Practice of Pharmacy*, 20th edition (2000), edited by Lippincott Williams and Wilkins, KE Hoover.

[0044] The pharmaceutical composition used in this method may contain pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized or aqueous forms. (Remington: The Science and Practice of Pharmacy, 20th edition (2000), edited by Lippincott Williams and Wilkins, KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and may contain buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin. Gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, asparagine, histamine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEENTM, PLURONICTM, or polyethylene glycol (PEG).

[0045] Pharmaceutical compositions intended for internal administration must be sterile. This is easily achieved, for example, through filtration with a sterile filter membrane. Therapeutic compositions are typically placed in containers with a sterile access port, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0046] The pharmaceutical compositions described herein may be unit dosage forms for oral, parenteral, rectal or topical administration, such as tablets, pills, capsules, powders, granules, solutions, suspensions or suppositories.

[0047] To prepare a solid composition, such as a tablet, the main active ingredient may be mixed with a pharmaceutical carrier, such as a conventional tableting ingredient, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and other pharmaceutical diluents, such as water, to form a solid preform composition containing a homogeneous mixture of the compounds of the present invention, or a non-toxic, pharmaceutically acceptable salt thereof. When these preform compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, such that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The solid preform composition is then subdivided into unit dosage forms of the type described above, containing 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition may be encapsulated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, a tablet or pill may comprise an inner dosage form and an outer dosage form component, the latter being encapsulated in the form of the former. These two components are separated by an enteric coating, which resists disintegration in the stomach and allows the inner component to enter the duodenum intact or with delayed release. A variety of materials can be used for such enteric coatings or coatings, including various polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0048] Suitable surfactants particularly include nonionic agents such as polyoxyethylene sorbitan (e.g., TweenTM 20, 40, 60, 80, or 85) and other sorbitans (e.g., SpanTM 20, 40, 60, 80, or 85). Compositions containing surfactants will conveniently contain between 0.05% and 5% of the surfactant, and may be between 0.1% and 2.5%. It is understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if desired.

[0049] In certain embodiments, the pharmaceutical compositions disclosed herein containing *Hedyotis diffusa* extract may be topical formulations that can be used to promote wound healing (e.g., wound healing in diabetic patients). Such topical formulations may contain about 0.0001% to about 0.5% (w / w) of trifleurone and optionally asiaticoside in an amount of about 0.05% to about 5% (w / w). In certain embodiments, asiaticoside may be contained in *Centella asiatica* extract, or both in an amount of about 0.1-30% (w / w). See, for example, U.S. Patent No. 10,758,584, the relevant disclosure of which is incorporated herein by reference for the purposes and intent of this document.

[0050] Topical formulations may also contain one or more carriers or excipients, including one or more thickeners (e.g., about 1.0-10%), one or more ointment bases (e.g., one or more cream bases) ranging from about 5% to 30%, one or more antimicrobial preservatives (e.g., about 0.005-0.2% by weight), one or more emulsifiers (about 0.5-10% by weight), or combinations thereof. These components may be soluble or dispersed in a suitable solvent.

[0051] The topical formulation also includes one or more carriers and excipients, including thickeners, ointment bases (e.g., cream bases), antimicrobial preservatives, emulsifiers, and / or solvents.

[0052] "Thickening agent" is an agent used to increase the viscosity of a formulation. Exemplary thickening agents may include, for example, cetearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, beeswax, stearic acid, cetyl alcohol, or combinations thereof. The thickening agent may be present in the topical formulation at a concentration of about 1.0-10% (w / w). For example, the topical formulation may contain about 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, or 9.5-10% (w / w) of the thickening agent. Alternatively, the topical formulation may contain about 1-5%, 2.5-7.5%, or 5-10% (w / w) of a thickener.

[0053] The "ointment base" may be any semi-solid formulation or carrier incorporating active agents. Exemplary ointment bases include, but are not limited to, oily ointment bases (e.g., white petrolatum or white ointment), absorbent ointment bases (e.g., hydrophilic petrolatum, anhydrous lanolin, Aquabase™, Aquaphor®, and Polysorb®), water / oil emulsion ointment bases (e.g., cold cream, hydrous lanolin, rose water ointment, Hydrocream™, Eucerin®, and Nivea®), oil / water emulsion ointment bases (e.g., hydrophilic ointment, Dermabase™, Velvachol®, and Unibase®), and water-miscible ointment bases (e.g., polyethylene glycol (PEG) ointment and Polybase™). The ointment base may be pharmacologically inert but may retain moisture to provide a moisturizing protective film. In one specific embodiment, the ointment base may be any petrolatum compound (e.g., petrolatum, white petrolatum, white nephrite, liquid petrolatum, liquid paraffin). In another specific embodiment, the ointment base is white petrolatum (CAS No. 8009-03-8). The ointment base may be present in the topical formulation at a concentration of about 5-30% (w / w), for example, 10-30% (w / w). For example, the topical formulation may contain about 5-25%, 5-20%, 5-15%, 5-15%, 10-15%, 15-20%, 20-25%, or 25-30% (w / w) of the ointment base. Specifically, the topical formulation may contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / w) of an ointment base.

[0054] In some specific embodiments, the "ointment matrix" described herein contains less than 20% water and volatiles, and more than 50% hydrocarbons, waxes, or polyols as a carrier. In some specific embodiments, the "ointment matrix" described herein is a "cream matrix" containing more than 20% water and volatiles and / or typically containing less than 50% hydrocarbons, waxes, or polyols as a carrier for the pharmaceutical substance. The cream matrix may be a multiphase formulation comprising a lipophilic phase and an aqueous phase. In some cases, the cream matrix is ​​a lipophilic cream matrix having a lipophilic phase as a continuous phase. Such cream matrices typically contain water-in-oil emulsifiers, such as lanolin, sorbitan esters, and monoglycerides. In other cases, the cream matrix is ​​a hydrophilic cream matrix having an aqueous phase as a continuous phase. This type of cream base typically contains oil-in-water emulsifiers, such as sodium soap or triethanolamine soap, sulfated fatty alcohols, polysorbate esters, and polyoxyethylated fatty acids and fatty alcohol esters, which may be combined with water-in-oil emulsifiers if desired.

[0055] An "antimicrobial preservative" may be any compound capable of destroying microorganisms, preventing the reproduction or growth of microorganisms, or preventing the pathogenicity of microorganisms. Exemplary antimicrobial preservatives include, but are not limited to, parabens (parabens; for example, parabens, methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, benzylparaben, isobutylparaben, isopropylparaben, benzylparaben, or sodium salts thereof), benzalkonium chloride, benzyl chloride, benzyl alcohol, boric acid, bromonitrile, hexadecyltrimethylammonium bromide, hexadecylpyridine chloride, chlorhexidine, chlorobutanol, chlorocresol, dichlorophenol, cresol, ethanol, glycerol, hexamethylenetetramine, imidurea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Antimicrobial preservatives may be present in topical formulations at concentrations of about 0.005-0.2%, for example, about 0.01-0.2% (w / w). For example, topical formulations may contain about 0.005-0.01%, 0.01-0.05%, 0.05-0.1%, 0.1-0.15%, or 0.15-0.2% (w / w) of antimicrobial preservatives. Specifically, a topical formulation may contain about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2% (w / w) of an antimicrobial preservative.

[0056] An "emulsifier" is a compound or substance that acts as a stabilizer in a mixture of two or more normally immiscible (immiscible or non-mixable) liquids. Exemplary emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginate, sodium alginate, tragacanth gum, chondroitin, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, and high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetyl monostearate, ethylene glycol). Distearate, glyceryl monostearate, propylene glycol monostearate, and polyvinyl alcohol), carbomer (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers, and carboxylated vinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and methyl cellulose), sorbitol fatty acid esters (e.g., polyoxyethylene dehydrated sorbitol monolaurate [Tween®]). 20], polyoxyethylene sorbitol [Tween® 60], polyoxyethylene sorbitol monooleate [Tween® 80], sorbitol monopalmitate [Span® 40], sorbitol monostearate [Span® 60], sorbitol tristearate [Span® 65], glyceryl monooleate, and sorbitol monooleate [Span® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj® 45] Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij®]), and poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl lauryl ether, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, hexadecylpyridine chloride, benzalkonium chloride, and sodium docusate, and / or combinations thereof. The emulsifier may be present in the topical formulation at a concentration of about 0.5-10% (w / w), for example 0.5-6% (w / w). For example, the topical formulation may contain about 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 5-10%, 6-10%, or 8-10% (w / w) of emulsifier.Specifically, the topical formulation may contain about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% (w / w) of emulsifier.

[0057] The topical formulations disclosed herein may also contain one or more solvents (e.g., non-aqueous solvents or water). Exemplary non-aqueous solvents may include, but are not limited to, any known solvents, including propylene glycol, ethylene glycol, and mixtures thereof. The non-aqueous solvent may be present in the topical formulation at a concentration of about 2-65% (w / w). For example, the topical formulation may contain about 2-15%, 15-30%, 30-45%, or 45-65% (w / w) of solvent. In some specific embodiments, the topical formulations of the present invention may also contain water.

[0058] In some specific embodiments, the topical formulation may also contain one or more emollients, fragrances, or pigments. The topical formulation may also be used in conjunction with wound dressings (e.g., adhesive bandages, plaster patches, etc.) (e.g., cyclohexane, n-hexane, n-decane, isooctane, octane, butyl ether, carbon tetrachloride, triethylamine, isopropyl ether, toluene, p-xylene, tert-butyl methyl ether, benzyl ether, dichloromethane, chloroform, dichloroethane, vinyl dichloride, 1-butanol, isobutanol, tetrahydrofuran, ethyl acetate, 1-propanol, 2-propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzyl nitrile, 1,4-dioxane, or p-dioxane).

[0059] III. Therapeutic use of pharmaceutical compositions containing Paeonia lactiflora (PA) extract

[0060] Any pharmaceutical composition (e.g., a topical preparation) containing an effective amount of *Hedyotis diffusa* extract prepared by the methods disclosed herein may be used to treat a target disease or condition in an individual in need of treatment, such as promoting wound healing, by means of an appropriate route, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or local routes.

[0061] As used herein, "effective amount" means the amount of each active agent, alone or in combination with one or more other active agents, required to impart a therapeutic effect to an individual. It will be apparent to those skilled in the art that determining whether a given amount of *Hedyotis diffusa* extract achieves a therapeutic effect is readily apparent. As is known to those skilled in the art, the effective amount depends on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concurrent treatments (if any), the specific route of administration, and similar factors within the knowledge and expertise of a healthcare professional. These factors are well known to those skilled in the art and can be resolved through experiments not exceeding the usual methods. Generally, it is preferable to use the maximum dose of the individual component or its combination, i.e., the highest safe dose based on reasonable medical judgment.

[0062] Empirical considerations, such as half-life, often help determine dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the extract or the active ingredient therein. Generally, but not always, the frequency of administration can be determined and adjusted during treatment based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / symptom.

[0063] In some specific embodiments, the topical preparations disclosed herein may be applied to the wound site following an appropriate dosage and treatment regimen. The dosage and administration regimen of the method will depend on the nature and condition of the wound being treated, the patient's age and condition, and any prior or concurrent treatments. In some cases, the topical preparation may be applied once a week, every other day, once daily, twice daily, three times daily, or four times daily for a suitable period of time. Treatment may be discontinued when the wound has healed. If necessary, for example, if the wound recurs, treatment may be resumed.

[0064] The term "wound" refers to damage to living tissue caused by cutting, striking, or other impact (e.g., caused by a medical condition such as a dermatological disease), typically in which the skin is cut or ruptured. Wounds may be associated with medical conditions, such as dermatological diseases. The term "wound healing" refers to the dynamic and complex process of replacing inactive or missing cellular structures and / or tissue layers. The term "promotion of wound healing" or "promoting wound healing" refers to an increased degree or rate of induction of replacement of inactive or missing cellular structures and / or tissue layers. As an example, promotion of wound healing may be expressed by partial or complete closure of ulcers or an increased rate of ulcer healing (including, but not limited to, more rapid changes in ulcer size, area, or severity compared to control ulcers treated with placebo, faster ulcer closure, and / or an increased percentage change in ulcer size, area, or severity relative to baseline).

[0065] The recipient of this topical preparation may be a human or a non-human mammal. In some specific embodiments, the individual is a human patient with an open wound, which is defined as damage or injury that destroys living tissue (e.g., skin) and causes a disruption of the normal continuity of biological structures. Open wounds may include, but are not limited to, abrasions, incisions, lacerations, punctures, avulsions, cuts, or other similar injuries.

[0066] In other embodiments, the individual is a human patient with a chronic wound, which may be damage or impairment of living tissue (e.g., skin) that disrupts the normal continuity of a biological structure and fails to heal in a series of orderly stages and / or within a predictable timeframe. Chronic wounds may include, but are not limited to, surgical wounds, traumatic wounds, pressure sores, venous ulcers, or diabetic ulcers. In other embodiments, chronic wounds may be associated with diseases or conditions such as cancer, burns, bedsores, or skin conditions such as atopic dermatitis.

[0067] In one embodiment, the individual is a human patient with foot ulcers associated with diabetes (e.g., type I or type II). Diabetes mellitus (also known as diabetes) is a group of metabolic diseases that result in prolonged high blood glucose levels. Diabetes may be caused by the pancreas' inability to produce enough insulin or by the body's cells not responding correctly to the insulin produced. The three main types of diabetes are type I (also known as "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes"; caused by the pancreas' inability to produce enough insulin), type II (also known as "non-insulin-dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes"; caused by cells not responding correctly to insulin), and gestational diabetes (high blood glucose observed during pregnancy in individuals with no prior history of diabetes). Many serious complications have been observed in patients with diabetes, including, but not limited to, chronic wounds such as diabetic foot ulcers (also known as diabetic ulcers).

[0068] In some specific embodiments, the individual to be treated by the methods described herein has a severe wound, for example, an ulcer with an area greater than 2 cm² (e.g., 3 cm², 4 cm², or 5 cm²). In some embodiments, the individual has one or more plantar ulcers.

[0069] No further details are needed; those skilled in the art will be able to fully utilize the invention based on the above description. Therefore, the following specific embodiments should be interpreted as illustrative only and not as limiting other parts of the invention in any way. All publications cited herein for the purposes or subject matter are incorporated herein by reference.

[0070] Example 1: Exemplary method for preparing hand incense A

[0071] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 2.5%-4% of the original volume. The concentrated solution was diluted with water and loaded onto a column packed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin). The column was washed sequentially with water, 50% ethanol, and ethanol. The column was then flushed with an extraction solution containing ethanol and ethyl acetate at a ratio of approximately 1:1 (v / v). The flushed portion was collected and dried to obtain the *Hedyotis diffusa* extract (yield: 1.0%).

[0072] Example 2: Exemplary method for preparing hand-held incense B

[0073] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice. The resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 2.5%-4% of the original volume. The concentrated solution was diluted with water and then mixed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin), and then packed into a column. The column was washed sequentially with water, 50% ethanol, and ethanol. The solid fraction was then extracted with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The extracted fraction was collected and dried to obtain *Hedyotis diffusa* extract (yield: 1.0%).

[0074] Example 3: Exemplary method for preparing hand-held incense C

[0075] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 2.5%-4% of the original volume. The concentrated solution was diluted with water and then loaded onto a column packed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin). The column was washed sequentially with water, 50% ethanol, and ethanol. The column was then flushed with an extraction solution containing acetone and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 1.1%).

[0076] Example 4: Exemplary method for preparing hand-held incense D

[0077] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 2.5%-4% of the original volume. The concentrated solution was diluted with water and then mixed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin), and then packed into a column. The column was washed sequentially with water, 50% ethanol, and ethanol, and then flushed with an extraction solution of acetone and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 1.1%).

[0078] Example 5: Exemplary method for preparing hand-held incense E

[0079] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 30-70% of the original volume. Without dilution, the concentrated solution was loaded onto a column packed with DIAION® HP-20 resin (a nonpolar copolymer styrene-divinylbenzene adsorbent resin). The column was flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 1.2%).

[0080] Example 6: Exemplary method for preparing hand-held incense F

[0081] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at room temperature (e.g., about 20-25°C) for 24 hours. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was about 2.5%-4% of the original volume. The concentrated solution was diluted with water and then loaded onto a column packed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin). The column was washed sequentially with water, 50% ethanol, and ethanol. The column was then flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of about 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 0.4%).

[0082] Example 7: Exemplary method for preparing hand-held incense G

[0083] During extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated to dryness under vacuum. The dried *Hedyotis diffusa* extract was then loaded onto a silicone-filled column. The column was washed with hexane. The column was then flushed with an extraction solution containing hexane and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce the *Hedyotis diffusa* extract (yield: 1.4%).

[0084] Example 8: Exemplary method for preparing hand-held incense H

[0085] During extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated to dryness under vacuum. The dried *Hedyotis diffusa* extract was then loaded onto a column packed with HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin). The column was washed with hexane. The column was then flushed with an extraction solution of hexane and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce the *Hedyotis diffusa* extract (yield: 1.0%).

[0086] Example 9: Exemplary Hand Incense Preparation Process I

[0087] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 30-70% of the original volume. Without dilution, the concentrated solution was then loaded onto a column packed with LX-20 resin (a nonionic, hydrophobic polystyrene polymer crosslinked with divinylbenzene). The column was flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 1.2%).

[0088] Example 10: Exemplary method for preparing hand-held incense J

[0089] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. This step was repeated twice, and the resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 30-70% of the original volume. Without dilution, the concentrated solution was then loaded onto a column packed with XAD1180N resin (polystyrene-divinylbenzene copolymer resin). The column was flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to obtain *Hedyotis diffusa* extract (yield: 1.1%).

[0090] Example 11: Exemplary method for preparing hand-held incense K

[0091] For extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90-180 minutes. The resulting mixture was filtered and concentrated using rotary evaporation until the volume of the concentrated extract was approximately 30-70% of the original volume. The concentrated solution was mixed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin) and then packed into a column. The column was flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to produce *Hedyotis diffusa* extract (yield: 1.0%).

[0092] Example 12: Exemplary method for preparing hand-held incense L

[0093] During extraction, 1 kg of *Hedyotis diffusa* aerial parts were stirred in 10-20 liters of ethanol at 60 ± 5°C for 90 minutes. The resulting mixture was filtered and concentrated under vacuum until the volume of the concentrated extract was approximately 30%-70% of the original volume. The concentrated solution was then loaded onto a column packed with DIAION® HP-20 resin (a non-polar copolymer styrene-divinylbenzene adsorbent resin). The column was flushed with an extraction solution containing ethanol and ethyl acetate in a volume ratio of approximately 1:1. The flushed portion was collected and dried to obtain *Hedyotis diffusa* extract (yield: 1.3%).

[0094] Example 13: Characteristic Analysis of Hand-Scented Fragrant Extract

[0095] The main phytochemicals contained in the *Hedyotis diffusa* extract prepared in the above examples were analyzed by high performance liquid chromatography (HPLC). The results are shown in Table 3 below.

[0096] The extracts prepared by methods E, I, and J have similar phytochemical compositions, indicating that different hydrophobic resins will yield similar extracts of *Hedyotis diffusa*.

[0097] The extracts prepared by methods B and D have similar phytochemical compositions, indicating that similar extracts of *Hedyotis diffusa* can be obtained by using extraction solutions of 50% acetone / 50% ethyl acetate and 50% ethanol / 50% ethyl acetate.

[0098] In addition, the extracts prepared by methods E and K have similar phytochemical components, while the extracts prepared by methods B, D, E, and L using different chromatographic separation methods also have similar phytochemical components.

[0099] Characterization results also show that using a high extraction temperature (e.g., 60°C ± 5°C) can increase yield and shorten extraction time. Furthermore, compared to high concentration (e.g., volume reduced to approximately 2.5-4% of the original volume), low concentration after extraction (e.g., volume reduced to approximately 30-70% of the original volume) significantly reduces the separation time for chromatography, thereby substantially reducing the total time for extract preparation. For example, compared to method F (approximately 57 hours, including 48 hours of extraction and 9 hours of concentration), method E significantly reduces the total preparation time before the chromatography separation step (approximately 6 hours, including 3 hours of extraction and 3 hours of concentration). Method E also features a shorter chromatography separation step (6 hours vs. 16 hours) compared to method F.

[0100] In addition, compared with chromatography separation procedures performed in the form of a column, chromatography separation procedures performed through resin mixing and filtration steps produce extracts with higher concentrations of active ingredients (compare method A and method B, or compare method C and method D).

[0101] Table 3. Characteristic description of the extract of *Hypericum perforatum* method * Yield % Element(%) Trifoliol Citric acid Rosemary acid apigenin A 1.0% 0.3 0.02 0.01 0.1 B 1.0% 0.3 0.2 0.03 0.5 C 1.1% 0.2 0.01 0.01 0.04 D 1.1% 0.2 0.2 0.02 0.4 E 1.2% 0.2 0.2 0.1-0.4 0.8-1.2 F 0.4% 0.2 0.01 ND 0.05 G 1.4% 0.3 0.7 ND 6.7 H 1.0% 0.4 0.9 1.1 0.1 I 1.2% 0.3 0.2 0.1 1.6 J 1.1% 0.2 0.2 0.1 1.5 K 1.0% 0.2 0.2 0.2 1.4-1.6 L 1.3% 0.3 0.3 0.04 1.1 *×100%

[0102] Example 14. Other exemplary preparation methods for producing hand-scented extracts

[0103] This embodiment provides additional exemplary preparation methods for producing hand fragrance extracts, all of which are within the scope of this invention.

[0104] In these exemplary preparation processes, the aerial parts of *Hedyotis diffusa* are extracted for 1-3 hours at 25-60°C using a solvent with a polarity index of 4.3-6.6. The extraction step is repeated 1-3 times. The resulting crude extract is concentrated 6-fold or 50-fold. Alternatively, the crude extract is not concentrated. The concentrated or undiluted extract is then contacted with a suitable hydrophobic interaction chromatography resin (e.g., HP20, LX-20, or XAD1180N), for example, by loading it onto a resin-containing column or by mixing it with the resin to bind (in some cases, by stirring the mixture). The extract is then flushed off the resin using an extraction solution with a polarity index of 4.3-5.4.

[0105] Table 4 below summarizes the reagents and conditions for each step in the exemplary preparation process. Table 5 summarizes the results of these preparation processes. The *Hedyotis diffusa* extract prepared by the exemplary preparation method contains approximately 0.04%-0.2% trifurcifen, approximately 0.1%-0.3% tylosin, approximately 0-0.3% rosmarinic acid, and approximately 0.3%-1.7% carvacrol.

[0106] Table 4. Exemplary preparation process of senna extract

[0107] Table 5 Characteristic description of the hand fragrance extract prepared by the exemplary method *ND: Not detected

[0108] Other embodiments

[0109] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be used to replace alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0110] Based on the above description, those skilled in the art can easily determine the essential features of the present invention, and various changes and modifications can be made to the present invention without departing from its spirit and scope to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the patent application.

[0111] equivalent to

[0112] Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing functions and / or obtaining results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on the specific application or use of the teachings of this invention. Those skilled in the art will recognize or be able to determine many equivalents of the particular inventive embodiments described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are presented as examples only, and that embodiments of the invention may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The inventive embodiments of the invention are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, sets, and / or methods is included within the scope of the invention as described herein, provided that such features, systems, articles, materials, sets, and / or methods do not contradict each other.

[0113] All definitions defined and used herein should be understood to go beyond dictionary definitions, definitions incorporated through reference in other documents, and / or the general meaning of the defined terms.

[0114] All references, patents and patent applications disclosed herein are incorporated by way of citation for each cited subject, and in some cases, the subject may cover the entire document.

[0115] The indefinite articles “a” and “an” used in the specification and the scope of the patent application shall be understood as “at least one” unless the opposite is explicitly stated.

[0116] The phrase "and / or" as used herein in the specification and claims should be understood to mean "one or two" of the elements so combined, that is, elements that exist together in some cases and separately in others. Multiple elements listed with "and / or" should be interpreted in the same way, that is, "one or more" of the elements so combined. Other elements may optionally exist in addition to those specifically identified by the "and / or" clause, whether or not they are related to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "including," in one embodiment a reference to "A and / or B" may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so on.

[0117] The terms "about" or "approximately" refer to a specific value within an acceptable range of error as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation, depending on actual operation in the art. Alternatively, "about" may mean a range of a given value of at most ±20%, preferably at most ±10%, more preferably at most ±5%, and even more preferably at most ±1%. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, preferably within twice the value. Where a specific value is described in the specification and claims, the term "about" is implied unless otherwise stated and herein represents within an acceptable range of error for the specific value.

[0118] As used herein in the specification and claims, “or” shall be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as inclusive, that is, including at least one, but also including several elements or more than one in a list of elements, and optionally, other unlisted items. “Comprising…” will refer to including several elements or an exact one in a list of elements only when the opposite terms are explicitly indicated, such as “only one” or “exactly one”, or when used in the claims. In general, when preceded by an exclusive term, such as “any,” “one of,” “only one,” or “exactly one,” the term “or” as used herein shall only be interpreted as indicating an exclusive alternative (i.e., “one or the other, but not both”). “Substantially comprising…” as used in the claims shall have the general meaning used in the field of patent law.

[0119] As used herein in the specification and claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but does not necessarily include at least one of each element specifically listed in the list, nor exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether or not they are related to those specifically identified elements. Therefore, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B"), in one embodiment, may refer to at least one, optionally including more than one type of A, with no B (and optionally including elements other than B); in another embodiment, may refer to at least one, optionally including more than one type of B, with no A (and optionally including elements other than A); in yet another embodiment, may refer to at least one, optionally including more than one type of A, and at least one, optionally including more than one type of B (and optionally including other elements); and so on.

[0120] It should also be understood that, unless there is an express instruction to the contrary, in any method that includes more than one step or action claimed herein, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described. [Simplified Explanation of the Diagram]

[0015] None [Biomaterial Storage]

[0122] None

Claims

1. A method for preparing an extract of Plectranthus amboinicus (PA), the method comprising: (i) The aerial parts of *Hedyotis diffusa* are mixed with an extraction solution to produce a first *Hedyotis diffusa* extract, wherein the extraction solution contains a solvent with a polarity index of about 2.9 to 6.6; (ii) The first *Hedyotis diffusa* extract is filtered to produce a second *Hedyotis diffusa* extract; (iii) The second *Hedyotis diffusa* extract is contacted with a hydrophobic interaction chromatography resin; and (iv) The column is flushed with an extraction solution to produce the *Hedyotis diffusa* extract, wherein the extraction solution contains a solvent with a polarity index of about 2.1 to 5.4, wherein the solvent in the extraction solution is acetone, butyl methyl ether, ethanol, isopropanol, methanol, or a mixture thereof; Step (i) is carried out at a temperature of about 25-80°C; and the hydrophobic interaction chromatography resin is selected from the group consisting of: nonpolar copolymer styrene-divinylbenzene adsorbent resin, polystyrene polymer crosslinked with divinylbenzene, and polystyrene-divinylbenzene copolymer resin.

2. The method of claim 1, wherein step (ii) further comprises concentrating the first euphorbia extract to produce the second euphorbia extract, the second euphorbia extract being a concentrated euphorbia extract.

3. The method of claim 1, wherein step (ii) does not include concentrating the first arugula extract.

4. The method of claim 1, wherein the solvent in the extraction solution is acetone, ethanol, isopropanol, methanol, or a mixture thereof.

5. The method of claim 4, wherein the solvent in the extraction solution is ethanol, acetone, or methanol.

6. The method of any of the requests 1 to 5, wherein step (i) is repeated 2 to 4 times, or optionally 2 times.

7. The method of any of the requests 1 to 5, wherein step (i) is performed for approximately 1 to 9 hours.

8. The method of any one of claims 1 to 5, wherein step (i) is performed at a temperature of about 50-80°C.

9. The method of any one of claims 1 to 5, wherein step (i) is performed at a temperature of about 50-80°C for about 1-9 hours.

10. The method of claim 9, wherein step (i) is performed at a temperature of about 50-80°C for about 1-3 hours.

11. The method of any one of claims 1 to 5, wherein in step (i), the weight-to-volume ratio of the arborvitae to the extract solution is 1:10 to 1:20 (kg / L).

12. The method of claim 1, 2, 4 or 5, wherein in step (ii), the first euphorbia extract is concentrated to produce the concentrated euphorbia extract, and wherein the volume of the concentrated euphorbia extract is about 30% to 70% of the volume of the first euphorbia extract.

13. The method of claim 1, 2, 4 or 5, wherein in step (ii), the first euphorbia extract is concentrated to produce the concentrated euphorbia extract, and wherein the volume of the concentrated euphorbia extract is 2.5% to 4% of the first euphorbia extract.

14. The method of any one of claims 1 to 5, wherein step (iii) is performed by loading the concentrated senna extract onto a column containing the hydrophobic interaction chromatography resin.

15. The method of any one of claims 1 to 5, wherein step (iii) comprises mixing the concentrated senna extract with the hydrophobic interaction chromatography resin to bind the components of the concentrated senna extract with the hydrophobic interaction chromatography resin.

16. The method of claim 15, wherein step (iii) further comprises stirring the concentrated senna extract with the hydrophobic interaction chromatography resin.

17. The method of any one of claims 1 to 5, wherein the extraction solution comprises a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, isopropanol, and hexane.

18. The method of claim 17, wherein the extraction solution comprises two solvents in a volume ratio of 50:

50.

19. The method of claim 18, wherein the extraction solution comprises a mixture of: (a) hexane and ethyl acetate, (b) ethanol and ethyl acetate, (c) acetone and ethyl acetate, or (d) acetone and isopropanol.

20. The method of any one of claims 1 to 5, wherein the method further comprises washing the hydrophobic interaction chromatography resin with an aqueous solution containing ethanol before step (iv) and after step (iii).

21. A PA extract prepared by the method shown in any one of claims 1 to 20.

22. The sage extract of claim 21, which contains trifleurone, citric acid, rosmarinic acid, and apigenin.