Cydonia oblonga seed antioxidant extract as well as preparation method and application thereof

Abstract

The invention relates to a quince seed antioxidant extract as well as a preparation method and application thereof. The preparation method of the quince seed antioxidant extract comprises the following steps: crushing dried quince seeds to prepare quince seed powder; soaking the quince seed powder in a first solvent to prepare a crude extracting solution; the first solvent comprises ethanol and water; concentrating and drying the crude extracting solution to obtain a crude extract; mixing the crude extract with water to obtain an extract aqueous solution; mixing the extract aqueous solution with petroleum ether, and carrying out first extraction treatment to obtain a petroleum ether extraction part and a petroleum ether extracted aqueous solution; mixing the petroleum ether extracted aqueous solution with ethyl acetate, and carrying out second extraction treatment to obtain an ethyl acetate extracted part and an ethyl acetate extracted aqueous solution; the ethyl acetate extraction part is subjected to first concentration treatment, and a first quince seed antioxidant extract is prepared. According to the method, the flavonoid compounds can be efficiently and specifically enriched from the quince seeds.

Description

Technical Field

[0001] This invention relates to the field of extraction technology of effective components from natural plants, and in particular to quince seed antioxidant extract, its preparation method and application. Background Technology

[0002] Oxidative stress is a significant factor contributing to aging and various chronic diseases, such as cancer, cardiovascular disease, and diabetes. Currently, while synthetic antioxidants widely used in the food industry (such as BHT and BHA) are effective and legally recognized as safe at certain dosages, controversies surrounding their potential toxic side effects at high doses persist. With consumers increasingly favoring "clean label" and natural products, exploring safe, efficient, and clearly defined natural antioxidants from medicinal plant resources has become a research hotspot and inevitable trend in the functional food, pharmaceutical, and cosmetic fields.

[0003] Quince seeds are the seeds of the quince plant, a member of the Rosaceae family. Traditionally, they are used to treat coughs and dysentery. Current research focuses primarily on the nutritional components of quince fruit, while studies on the bioactivity of its seeds, particularly the different chemically polar parts, are lacking. Therefore, the high-value development and utilization of quince seed resources and the development of novel natural antioxidants are of great significance. Summary of the Invention

[0004] Therefore, it is necessary to provide quince seed antioxidant extract, its preparation method, and its applications, which can efficiently obtain antioxidant components.

[0005] One aspect of the present invention provides a method for preparing an antioxidant extract of quince seeds, comprising the following steps: pulverizing dried quince seeds to obtain quince seed powder; soaking the quince seed powder in a first solvent to obtain a crude extract; the first solvent comprising ethanol and water; concentrating and drying the crude extract to obtain a crude extract; mixing the crude extract with water to prepare an aqueous extract solution; mixing the aqueous extract solution with petroleum ether and performing a first extraction treatment to obtain a petroleum ether extract fraction and a petroleum ether extract aqueous solution; mixing the petroleum ether extract aqueous solution with ethyl acetate and performing a second extraction treatment to obtain an ethyl acetate extract fraction and an ethyl acetate extract aqueous solution; and performing a first concentration treatment on the ethyl acetate extract fraction to prepare a first quince seed antioxidant extract.

[0006] In some embodiments, the preparation method satisfies one or more of the following characteristics:

[0007] The flavonoid content of the first quince seed antioxidant extract is 10%-15% by mass;

[0008] The volume fraction of ethanol in the first solvent is 70%~80%;

[0009] The amount of the first solvent used to soak each gram of the quince powder is 30 mL to 50 mL.

[0010] In some embodiments, the step of soaking the quince seed powder in a first solvent to prepare a crude extract includes: soaking the quince seed powder in a first solvent for 20 min to 60 min, then sonicating for 5 min to 15 min, separating the solid and liquid, and collecting the crude extract.

[0011] In some embodiments, the step of mixing the crude extract with water includes: mixing the crude extract with water at a mass ratio of 1:(18-25).

[0012] In some embodiments, the preparation method satisfies at least one of the following characteristics:

[0013] (1) The volume ratio of the aqueous extract to the petroleum ether is 1:(0.8~1.5);

[0014] (2) The volume ratio of the petroleum ether extraction aqueous solution to the ethyl acetate is 1:(0.8~1.5).

[0015] (3) The first concentration process includes vacuum distillation and drying.

[0016] In some embodiments, the preparation method further includes: mixing the aqueous solution after ethyl acetate extraction with n-butanol, performing a third extraction treatment to obtain the n-butanol extract fraction and the aqueous solution after n-butanol extraction; and performing a second concentration treatment on the n-butanol extract fraction to obtain a second quince antioxidant extract.

[0017] Optionally, the volume ratio of the aqueous solution after ethyl acetate extraction to n-butanol is 1:(0.8~1.5).

[0018] Optionally, the flavonoids in the second quince antioxidant extract contain 4%-6% by mass.

[0019] In some embodiments, the preparation method further includes: subjecting the aqueous solution after n-butanol extraction to a third concentration treatment to obtain a third quince antioxidant extract;

[0020] Optionally, the flavonoid content in the third quince antioxidant extract is 0.3%-0.5% by mass.

[0021] In some embodiments, the preparation method further includes: subjecting the petroleum ether extract fraction to a fourth concentration treatment to obtain a fourth quince antioxidant extract;

[0022] Optionally, the flavonoid content in the fourth quince antioxidant extract is 0.05%-0.07% by mass.

[0023] In a second aspect, this application provides a quince seed antioxidant extract, prepared by any of the above-described preparation methods.

[0024] In a third aspect, this application provides the use of the quince seed antioxidant extract as described above as an antioxidant.

[0025] The researchers in this application used a systematic solvent extraction method combined with in vitro antioxidant evaluation to perform activity-directed separation and screening of quince seed extract. They successfully discovered and confirmed that after soaking the crude extract in an aqueous solution with a specific solvent, petroleum ether extraction effectively separated low-polarity impurities such as chlorophyll and lipids from the extract's aqueous solution. Further extraction with ethyl acetate revealed that the ethyl acetate extract was the core effective fraction with the strongest antioxidant activity and the highest total flavonoid content. This breakthrough overcomes the limitation of existing technologies that only assess the activity of quince seeds at the crude extract level. Based on this, the aforementioned method for preparing quince seed antioxidant extract can efficiently and specifically enrich flavonoids with high antioxidant activity from quince seeds. Attached Figure Description

[0026] Figure 1 This is a linear relationship graph showing the DPPH scavenging rates of different extracts and vitamin C at different concentrations in Example 1.

[0027] Figure 2 This is a linear relationship graph of the total reducing power of different extracts and Vc at different concentrations in Example 1. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0031] In this document, when referring to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of selectable values ​​within a numerical interval is considered continuous, and includes the two endpoints (i.e., the minimum and maximum values) of the numerical interval, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, and other numerical interval types.

[0032] In this document, for methods involving multiple steps, unless otherwise explicitly stated herein, there is no strict order constraint on the execution of these steps; they may be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but may be executed at different times, and their execution order is not necessarily sequential, but may be executed in turn, alternately, or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0033] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0034] The term "quince seed" refers to the seed of the quince (Cydonia oblonga Mill.), a plant belonging to the genus Cydonia in the family Rosaceae. Quince twigs are slender and cylindrical; winter buds are ovate, pubescent, and purplish-brown; leaves are simple, alternate, ovate or oblong, with a rounded or nearly cordate base, glabrous or sparsely pubescent when young on the upper surface, densely pubescent on the lower surface, and entire; flowers are solitary, with white, obovate petals; pedicels are densely pubescent; bracts are membranous, ovate, and deciduous; fruit is pear-shaped, yellow, and fragrant, with persistent, reflexed sepals; fruit stalks are short and stout; flowering occurs from April to May, and fruiting in October.

[0035] The term "total flavonoids" refers to a class of naturally occurring compounds widely found in plants, possessing a variety of biological and pharmacological activities. They are a general term for flavonoids, typically existing in plants as glycosides or aglycones. The basic structure of flavonoids is a benzopyranone, consisting of two benzene rings (A and B rings) linked by a three-carbon chain (C6-C3-C6 structure).

[0036] Currently, solvent extraction is commonly used to enrich specific active components from natural plants. However, the selection of solvents and the design of extraction processes depend on a thorough understanding of the chemical properties of the target plant. For quince seeds, there is currently no clear and effective technical solution for determining which solvent can most effectively enrich their antioxidant active ingredients. Therefore, this application provides at least one quince seed antioxidant extract, its preparation method, and its applications.

[0037] According to a typical embodiment of this application, a method for preparing an antioxidant extract of quince seeds is provided, comprising the following steps: pulverizing dried quince seeds to obtain quince seed powder; soaking the quince seed powder in a first solvent to obtain a crude extract; the first solvent includes ethanol and water; concentrating and drying the crude extract to obtain a crude extract; mixing the crude extract with water to obtain an aqueous extract solution; mixing the aqueous extract solution with petroleum ether and performing a first extraction treatment to obtain a petroleum ether extract fraction and a petroleum ether extract aqueous solution; mixing the petroleum ether extract aqueous solution with ethyl acetate and performing a second extraction treatment to obtain an ethyl acetate extract fraction and an ethyl acetate extract aqueous solution; and performing a first concentration treatment on the ethyl acetate extract fraction to prepare a first quince seed antioxidant extract.

[0038] The researchers in this application used a systematic solvent extraction method combined with in vitro antioxidant evaluation to perform activity-directed separation and screening of quince seed extract. They successfully discovered and confirmed that after soaking the crude extract in an aqueous solution with a specific solvent, petroleum ether extraction effectively separated low-polarity impurities such as chlorophyll and lipids from the extract's aqueous solution. Further extraction with ethyl acetate revealed that the ethyl acetate extract was the core active fraction with the strongest antioxidant activity and the highest total flavonoid content. This breakthrough overcomes the limitation of existing technologies that only assess the activity of quince seeds at the crude extract level. Based on this, the aforementioned method for preparing quince seed antioxidant extract can efficiently and specifically enrich flavonoids with high antioxidant activity from quince seeds.

[0039] In some embodiments, dried quince seeds are pulverized using a pulverizer to prepare quince seed powder. Optionally, the particle size of the quince seed powder is 30 to 200 mesh to facilitate the leaching of its active ingredients and improve the yield of antioxidant extracts.

[0040] In some embodiments, the volume fraction of ethanol in the first solvent is 70% to 80%. The crude extract obtained after soaking in the first solvent with this composition, combined with the subsequent extraction method of this application, is beneficial for separating quince antioxidant extracts with higher total flavonoid content. Non-limitingly, the volume fraction of ethanol in the first solvent can be 70%, 72%, 74%, 75%, 76%, 78%, 80%, etc. Optionally, the volume fraction of water in the first solvent is 20% to 30%, for example, 20%, 25%, 30%, etc.

[0041] In some embodiments, the amount of the first solvent used to soak each gram of quince seed powder is 30 mL to 50 mL. Non-limitingly, the amount of the first solvent used to soak each gram of quince seed powder is 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, etc.

[0042] In some embodiments, the step of soaking quince seed powder in a first solvent to prepare a crude extract includes: soaking quince seed powder in the first solvent for 20 min-60 min (e.g., 20 min, 30 min, 40 min, 50 min, 60 min, etc.), followed by ultrasonic treatment for 5 min-15 min (e.g., 5 min, 10 min, 15 min, etc.), solid-liquid separation, separating the quince seed solid powder, and collecting the crude extract; soaking and ultrasonic treatment in the first solvent helps to fully extract flavonoids from the quince seed powder. Optionally, solid-liquid separation can be performed by methods such as filtration or centrifugation to separate the soaked quince seed solid powder.

[0043] Optionally, the quince seed powder can be soaked in the first solvent multiple times, that is, the separated quince seed solid powder can be soaked again to further improve the leaching rate of flavonoids in the quince seed powder. The extracts obtained after multiple soaking treatments can be combined or concentrated and dried separately to prepare crude extracts.

[0044] In some embodiments, the quince seed solid powder collected after solid-liquid separation is soaked and separated again, and the collected liquid is used as crude extract.

[0045] In some embodiments, the crude extract is concentrated and dried by vacuum distillation to obtain a crude extract. Optionally, the concentration and drying temperature is 30°C to 70°C to prevent the antioxidants in the extract from deteriorating. Non-limitingly, the concentration and drying temperature is independently 30°C, 40°C, 50°C, 60°C, 70°C, etc.

[0046] The crude extract is mixed with water to dissolve it, thus preparing an aqueous extract solution for subsequent extraction processes.

[0047] In some embodiments, the step of mixing the crude extract with water includes mixing the crude extract with water at a mass ratio of 1:(18-25), which can effectively dissolve the crude extract while improving the yield of subsequent extraction processes. Non-limitingly, the mass ratio of crude extract to water can be 1:18, 1:20, 1:22, 1:24, 1:25, etc. Optionally, the system formed by mixing the crude extract with water can be filtered to remove insoluble matter before extraction with petroleum ether or the like.

[0048] In some embodiments, the volume ratio of the extract aqueous solution to petroleum ether is 1:(0.8~1.5), for example, it can be 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.5, etc. Optionally, the above-described first extraction treatment, including extraction 2-5 times according to the above-described amount of petroleum ether, can more effectively remove impurities such as chlorophyll and oils from the crude extract.

[0049] In some embodiments, the volume ratio of the aqueous solution to ethyl acetate after petroleum ether extraction is 1:(0.8~1.5), for example, it can be 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.5, etc., which is beneficial for the full extraction of flavonoids, while taking into account the high yield of antioxidant components and solvent loss.

[0050] Optionally, the second extraction process includes: extracting the petroleum ether-extracted aqueous solution 2-5 times according to the amount of ethyl acetate used as described above.

[0051] In some embodiments, the first concentration process described above includes vacuum distillation and drying to remove the solvent from the ethyl acetate extract. Optionally, the concentration and drying processes are performed at temperatures ranging from 30°C to 70°C to prevent the antioxidants in the extract from deteriorating. Non-limitingly, the concentration and drying processes are performed at temperatures independently of 30°C, 40°C, 50°C, 60°C, 70°C, etc.

[0052] In some embodiments, the flavonoid content of the first quince antioxidant extract obtained after the first concentration treatment is 10%-15% by mass; non-limitingly, it can be 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0053] In some embodiments, the preparation method of the above-mentioned quince antioxidant extract further includes: mixing the aqueous solution after ethyl acetate extraction with n-butanol, and performing a third extraction treatment to obtain the n-butanol extract fraction and the aqueous solution after n-butanol extraction; and performing a second concentration treatment on the n-butanol extract fraction to obtain the second quince antioxidant extract.

[0054] Optionally, the volume ratio of the aqueous solution after ethyl acetate extraction to n-butanol is 1:(0.8~1.5). For example, the volume ratio of the aqueous solution after ethyl acetate extraction to n-butanol can be 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.5, etc.

[0055] Optionally, the third extraction process described above includes extraction 2-5 times according to the amount of n-butanol used described above.

[0056] In some embodiments, the flavonoid content in the second quince antioxidant extract is 4%-6% by mass; non-limitingly, it can be 4%, 5%, 6%, etc.

[0057] In some embodiments, the above preparation method further includes: subjecting the aqueous solution after n-butanol extraction to a third concentration treatment to obtain a third quince antioxidant extract; optionally, the mass percentage content of flavonoids in the third quince antioxidant extract is 0.3%-0.5%.

[0058] In some embodiments, the preparation method further includes: subjecting the petroleum ether extract to a fourth concentration treatment to obtain a fourth quince seed antioxidant extract. Optionally, the flavonoid content in the fourth quince seed antioxidant extract is 0.05%-0.07% by mass.

[0059] In some embodiments, the specific process methods for the second, third, and fourth concentration treatments described above each independently include vacuum distillation and drying treatment, and can be referred to the method of the first concentration treatment, which will not be described in detail here.

[0060] According to another typical embodiment of this application, a quince seed antioxidant extract is provided, which is prepared by any of the above preparation methods.

[0061] The main components of the quince seed antioxidant extract prepared by the above method are flavonoids, which have high DPPH free radical scavenging rate and reducing properties. They can be used to prepare highly active, standardized natural antioxidants and related functional products.

[0062] According to another typical embodiment of this application, the application of the quince seed antioxidant extract as described above as an antioxidant is provided. The quince seed antioxidant extract described above contains abundant flavonoids, exhibiting high DPPH free radical scavenging rate and reducing properties, and thus possesses high activity and safety as a natural antioxidant.

[0063] In some embodiments, the application may be for therapeutic purposes. For example, as an antioxidant in pharmaceuticals.

[0064] In some embodiments, the quince antioxidant extract described above is provided for use as an antioxidant in pharmaceuticals.

[0065] In some embodiments, the application can also be for non-diagnostic and non-therapeutic purposes. For example, it can be used as an antioxidant in food, cosmetics, skincare products, etc. The application can include clinical use for treating diseases, as well as non-therapeutic research purposes, such as as a reference reagent.

[0066] In some embodiments, the use of the quince antioxidant extract as described above in the preparation of compositions or formulations with antioxidant effects is provided.

[0067] According to another typical embodiment of this application, a composition or preparation having antioxidant effects is provided, comprising the quince seed antioxidant extract as described above and acceptable excipients.

[0068] In this application, unless otherwise specified, "acceptable" means a ligand, reagent, material, substance, or composition that, within the scope of reasonable judgment in the relevant field, is suitable for administration to a subject and is proportionate to a reasonable benefit / risk ratio, based on the intended purpose of the composition or formulation having antioxidant efficacy. For example, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for administration to a patient and are proportionate to a reasonable benefit / risk ratio.

[0069] In some embodiments, the composition with antioxidant properties may be a pharmaceutical composition, a health supplement composition, or a cosmetic composition. Cosmetic compositions may include skincare compositions.

[0070] In some embodiments, the formulation with antioxidant properties may be a pharmaceutical preparation or a drug. In other embodiments, the formulation with antioxidant properties may be a health supplement or a cosmetic. Cosmetics may include skin care products.

[0071] In some embodiments, either the pharmaceutical composition or the pharmaceutical formulation may independently include quince antioxidant extract and pharmaceutically acceptable excipients. In other embodiments, either the health supplement composition or the health supplement may independently include quince antioxidant extract and health supplement excipients. In still other embodiments, either the cosmetic composition or the cosmetic product may include quince antioxidant extract and cosmetic excipients.

[0072] In some embodiments, an antioxidant method is provided, comprising the step of administering an effective amount of the above-described quince antioxidant extract to a subject.

[0073] In this application, "effective amount" refers to the amount of a pharmaceutical active ingredient that will elicit a biological or medical response in an individual, such as the amount of a pharmaceutical active ingredient that brings about positive physiological and / or pharmacological effects in an individual, including but not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.

[0074] In this application, unless otherwise specified, "subject" refers to an animal to which the target ingredient or substance is administered. For example, a subject may be an animal taking a composition or preparation with antioxidant effects, or a patient taking a pharmaceutical composition, a consumer of a health supplement, or a user for cosmetic purposes. In this application, unless otherwise specified, "patient" refers to an animal, preferably a mammal. In some embodiments, the subject is a human.

[0075] Differences among subjects may lead to variations in the selection range of other ingredients in the aforementioned pharmaceutical compositions, pharmaceutical preparations, drugs, foods, health products, and other compositions or preparations with antioxidant effects.

[0076] There are no particular restrictions on the method of application, such as oral administration or topical application. Depending on the method of application, the quince antioxidant extract can be delivered to the appropriate site on the subject in a suitable dosage form.

[0077] The type of formulation with antioxidant activity is not particularly limited; for example, it can be a solid dosage form or a liquid dosage form. For example, the type of formulation with antioxidant activity may include at least one of capsules, pills, tablets, granules, liquids, ointments, aerosols, sprays, etc. Depending on the type of formulation, the "acceptable excipients" can be selected from excipients suitable for formulating the corresponding dosage form.

[0078] In some embodiments, the use of the above-described quince seed antioxidant extract in the preparation of a pharmaceutical product containing antioxidant components is provided. Further, the above-described pharmaceutical product containing antioxidant components comprises any of the above-described quince seed antioxidant extracts or quince seed antioxidant extracts prepared by any of the above-described preparation methods. Further, the above-described pharmaceutical product containing antioxidant components may also contain pharmaceutically acceptable excipients.

[0079] In some implementations, the types of pharmaceutically acceptable excipients are not particularly limited, as long as they enable the drug to be formulated into an easily administered dosage form. It is understood that different dosage forms may lead to different ranges and combinations of excipients.

[0080] In some embodiments, pharmaceutically acceptable excipients may include, but are not limited to, one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, isotonic or isotropic modifiers.

[0081] In some embodiments, the aforementioned diluent may be selected from, but is not limited to, starches, sugars, celluloses, and inorganic salts; one or a combination of multiple diluents may be selected.

[0082] In some embodiments, the aforementioned wetting agent may be selected from, but is not limited to, water and ethanol, and one or a combination of multiple agents may be selected.

[0083] In some embodiments, the aforementioned adhesive may be selected from, but is not limited to, starch paste, dextrin, sugar, cellulose derivatives, and gelatin; one or a combination of multiple adhesives may be selected.

[0084] In some embodiments, the aforementioned disintegrant is selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, croscarmellose, surfactants, and effervescent disintegrants; one or a combination of multiple disintegrants may be selected.

[0085] In some embodiments, the aforementioned lubricant may be selected from, but is not limited to, talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, and polyethylene glycol, and may be selected from one or a combination of multiple substances.

[0086] In some embodiments, the aforementioned color, flavor and aroma modifiers may be selected from, but are not limited to, pigments, fragrances, sweeteners, adhesives and odorants, and one or a combination of multiple can be selected.

[0087] In some embodiments, the aforementioned solvent may be selected from, but is not limited to, water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, liquid paraffin, fatty oil, and ethyl acetate. One or a combination of multiple solvents may be selected.

[0088] In some embodiments, the aforementioned solubilizer may be selected from, but is not limited to, Tween compounds, maltose compounds, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates; one or a combination of multiple solubilizers may be selected.

[0089] In some embodiments, the aforementioned cosolvent may be selected from, but is not limited to, organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone, and glycerol, and may be selected from one or a combination of multiple types.

[0090] In some embodiments, the aforementioned emulsifier may be selected from, but is not limited to, Span, Tween, Mize, benzyl esters, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, and bentonite. One or a combination of multiple emulsifiers may be selected.

[0091] In some embodiments, the aforementioned antioxidants may be selected from, but are not limited to, sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and their esters, and one or a combination of multiple antioxidants may be selected.

[0092] In some embodiments, the aforementioned metal complexing agent may be selected from, but is not limited to, disodium ethylenediaminetetraacetate and polycarboxylic acid compounds; one or a combination of multiple compounds may be selected.

[0093] In some embodiments, the aforementioned inert gas may be selected from, but is not limited to, nitrogen and carbon dioxide, and one or a combination of multiple gases may be selected.

[0094] In some embodiments, the aforementioned preservatives are selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils; one or a combination of multiple preservatives may be selected.

[0095] In some embodiments, the aforementioned local analgesic is selected from benzyl alcohol, chlorobutanol, lidocaine, and procaine, and one or a combination of multiple agents may be selected.

[0096] In some embodiments, the aforementioned pH adjuster may be selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. One or a combination of multiple substances may be selected.

[0097] In some embodiments, the aforementioned isotonic or isotropic regulator is selected from glucose, sodium chloride, sodium citrate, sorbitol, and xylitol, and one or a combination of multiple substances can be selected.

[0098] In some embodiments, the above application further includes administering an effective amount of quince antioxidant extract to the subject. When administered to the subject as a pharmaceutical or health supplement, the "effective amount" is, correspondingly, a therapeutically effective amount or a health-promoting effective amount.

[0099] In this application, unless otherwise specified, "therapeutic effective amount" refers to the amount of a pharmaceutical active ingredient that will elicit a biological or medical response in an individual, such as the amount of a pharmaceutical active ingredient that brings about positive physiological and / or pharmacological effects in an individual, including but not limited to reducing or inhibiting enzyme or protein activity, improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease. In this application, unless otherwise specified, "health-care effective amount" refers to the amount of a health supplement active ingredient that will elicit a biological or physiological response in an individual.

[0100] The following are detailed embodiments. The embodiments described below illustrate the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application.

[0101] Example 1

[0102] 1.1 Preparation of materials and reagents

[0103] The quince seeds were purchased from Xinjiang Xinlvbao Pharmaceutical Co., Ltd. in November 2024. The product batch number is WPZ-YP-241113, and the production date is November 13, 2024.

[0104] 1,1-Diphenyl-2-trinitrophenylhydrazine, trichloroacetic acid, K3[Fe(CN)6] (Shanghai Maclean Biochemical Technology Co., Ltd.), anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.), vitamin C reference standard (purity 100%, batch number: 100080-202012) were purchased from the National Institutes for Food and Drug Control, petroleum ether, ethyl acetate and n-butanol, FeCl3 (Tianjin Zhiyuan Chemical Reagent Co., Ltd.), sodium dihydrogen phosphate, and disodium hydrogen phosphate (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0105] 1.2 Instruments and Equipment

[0106] DFY-500 pulverizer (Wenling Linda Machinery Co., Ltd.); Evolution One UV spectrophotometer (Thermo Fisher Scientific (Shanghai) Co., Ltd.); AS2310193 ultrasonic cleaner (Tianjin Autosense Instruments Co., Ltd.); MS205DU / A analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); PX822ZH / E analytical balance (Ohaus Instruments (Changzhou) Co., Ltd.); LC-LX-L40B benchtop low-speed centrifuge (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); LC-DZF-6090AB vacuum drying oven (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.).

[0107] 1.3 Preparation of extracts with different polarities

[0108] 1.3.1 Preparation of crude extract of quince seeds

[0109] Take 500 g of dried quince seeds and pulverize them using a pulverizer to obtain quince seed powder (particle size 30 to 200 mesh), and set aside. Take 40 g of quince seed powder, place it in a 1 L Erlenmeyer flask, add 800 mL of 75% ethanol (v / v) and soak for 30 min, then ultrasonically extract for 10 min, filter, and perform two extractions. Combine the two extracts, concentrate under reduced pressure at 50 ℃ to 200 mL, and then concentrate and dry at 50 ℃ to obtain a 75% ethanol extract (E5, i.e., crude extract).

[0110] 1.3.2 Preparation of quince seed extracts by petroleum ether, ethyl acetate, n-butanol, and water extraction

[0111] Take 5g of crude extract, dissolve it in 100ml of water, and filter the filtrate. Then extract the filtrate sequentially with 100mL of petroleum ether, ethyl acetate, and n-butanol, respectively. After shaking in the same direction, allow it to stand and separate into layers. The lower layer is the aqueous suspension extract, and the upper layer is the organic solvent layer. Extract three times with different polar solvents, collect the extracts of each phase, concentrate under reduced pressure, and dry to obtain the petroleum ether extract (E1), ethyl acetate extract (E2), n-butanol extract (E3), and aqueous extract (E4).

[0112] Extraction yield of each polar fraction = (mass of the obtained extract / mass of quince powder weighed) * 100%.

[0113] Determination of total flavonoids

[0114] Accurately weigh 6.58 mg of rutin reference standard and place it in a 25 ml volumetric flask. Dilute to the mark with 70% ethanol to prepare a rutin standard solution with a concentration of 0.2426 mg / ml. Accurately pipette 0, 0.5, 1.0, 2.0, 4.0, 5.0, and 6.0 ml of the rutin standard solution into 25 ml volumetric flasks, add 1.0 ml of 5% sodium nitrite solution, shake well, and let stand for 6 min. Then add 1.0 ml of 10% aluminum nitrate solution, shake well, and after 6 min, add 10 ml of 4% sodium hydroxide solution, shake well, and dilute to the mark with 70% ethanol solution. Let stand for 15 min. Using a reagent blank as a reference, take a known total flavonoid standard solution and scan it at wavelengths of 400–700 nm. Observe the absorbance at each wavelength. The results show that the absorbance value reaches the peak at a wavelength of 505 nm. The total flavonoid determination in this study was performed at 505 nm. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa. Accurately weigh appropriate amounts of different concentrations (0.31 g for m, E2, E3, and E5; 1.25 g for E1; and 1.45 g for E4), and dilute to volume (V) with 70% ethanol. Take a certain amount (V) from each concentration. 吸 The sample solution was placed in a 25 mL volumetric flask (V 显 The absorbance values ​​were measured using the same method as for the standard solution, and the total flavonoid concentration (C) was calculated using the standard working curve equation; Total flavonoid content (mg / g) = (C × V × V) 显 ) / (V 吸 ×m).

[0115] Antioxidant activity assay

[0116] DPPH free radical scavenging rate

[0117] Accurately weigh vitamin C (abbreviated as Vc) standard, and prepare positive control solutions with a mass concentration of 0.01–0.05 mg / mL using 75% ethanol as the solvent. Prepare test solutions of appropriate mass concentrations for different extracts. Accurately pipette 2.0 mL of each sample solution, add 2.0 mL of DPPH radical solution (0.2 mmol / L), shake well, and let stand in the dark for 30 min. Measure the absorbance of the solution at a wavelength of 517 nm (A1). Calculate the DPPH scavenging rate of the extract based on the absorbance value, using the 75% ethanol solution as a blank reference (A0).

[0118] DPPH clearance rate (%) = (A0-A1 / A0) × 100%.

[0119] Determination of total reducing power

[0120] Accurately weigh vitamin C standard and use ultrapure water as solvent to prepare a positive control solution with a mass concentration of 0.01~0.05 mg / mL. Prepare test solutions of appropriate mass concentrations for different extracts. Pipette 2.5 mL of 1 mg / mL sample solution into a stoppered test tube, add 2.5 mL of 0.2 mol / L phosphate buffer (pH 6.6), then add 2.5 mL of 1% K3[Fe(CN)6], react in a 50℃ water bath for 20 min, then rapidly cool to room temperature under running water, and add another 2.5 mL of 10% trichloroacetic acid. After centrifugation, take 2.5 mL of the supernatant, add 2.5 mL of ultrapure water and 0.5 mL of 0.1% FeCl3 solution, shake well, let stand for 10 min, and measure the absorbance at 700 nm (A2). Use 2.5 mL of ultrapure water instead of the sample solution as a blank (A3).

[0121] Total reducing force = A2 - A3.

[0122] Data was summarized and organized using Excel, and analyzed and plotted using GraphPad Prism 10.1.2. In GraphPad, a nonlinear fitting was performed with the logarithm of concentration as the x-axis and the free radical scavenging rate as the y-axis to obtain the IC50. 50 The value of .

[0123] Results Analysis

[0124] Extract yield

[0125] Extraction yields of different polarities were obtained through three parallel experiments. The yield of crude extract (E5) was 16.92%, petroleum ether extract (E1) was 2.29%, ethyl acetate extract (E2) was 0.46%, n-butanol extract (E3) was 1.84%, and aqueous extract (E4) was 4.88%. The extraction yields of different polarities were: aqueous extract > n-butanol > petroleum ether > ethyl acetate.

[0126] In the determination of different total flavonoid contents in quince seeds, a standard curve was plotted using rutin as the standard, and the standard curve was obtained as y = 12.904x - 0.008, R0. 2=0.9997. The total flavonoid content in different parts of quince seeds was calculated using this equation. The total flavonoid contents were as follows: 75% ethanol extract (E5) 11.09 mg / g (approximately 1.1% by mass), petroleum ether (E1) 0.66 mg / g (approximately 0.066% by mass), ethyl acetate (E2) 129.11 mg / g (approximately 12.9% by mass), n-butanol (E3) 52.38 mg / g (approximately 5.2% by mass), and aqueous layer (E4) 4.34 mg / g (approximately 0.4% by mass). The total flavonoid content from highest to lowest was E2 > E3 > E5 > E4 > E1.

[0127] The DPPH free radical scavenging capacity of five different extracts was determined, and the results were... Figure 1 As shown, extracts of different polarities exhibited a relatively good dose-response relationship within the concentration range set in the experiment, and the inhibition rate increased with increasing concentration, in the order of largest to smallest: E2>E3>E5>E4>E1. The remaining extracts all showed strong free radical scavenging ability, especially extract E2, which showed no significantly weaker DPPH free radical scavenging ability than VC at a mass concentration of 0.4–1.0 mg / mL.

[0128] The total reducing power of different extracts is shown in the following results. Figure 2 The higher the total reducing power absorbance value, the stronger the antioxidant capacity. As shown in the figure, unlike the scavenging rate of DPPH, flavonoid molecules aggregate at high concentrations, affecting the scavenging rate of DPPH, but the total reducing power still maintains a high antioxidant activity.

[0129] To further investigate the antioxidant activity of different extracts of quince seeds, IC50 values ​​were calculated using Graphpad Prism, which more clearly indicates the strength of the antioxidant capacity of different extracts. As shown in Table 1, the lower the IC50 value, the stronger the ability to scavenge free radicals; the lower the A0.5 value, the stronger the total reducing power of the sample. The IC50 values ​​of the ethyl acetate extract (E2), n-butanol extract (E3), and 75% ethanol extract (E5) were all less than 1.000 mg / mL, indicating that the main antioxidant components in quince seeds have moderate polarity. Similarly, the ethyl acetate extract (E2) had the lowest A0.5 value. Overall, E2 showed significantly better activity than other polar fractions (E1, E3, E4, E5), and successfully enriched the antioxidant components in the crude extract by more than three times.

[0130] Table 1

[0131]

[0132] Comparative Example 1

[0133] The difference from Example 1 is that in step 1.3.1, anhydrous ethanol is used instead of 75% ethanol to prepare the crude extract, thus obtaining the ethanol extract (F5, i.e., the crude extract).

[0134] The preparation of the petroleum ether, ethyl acetate extract (F2), n-butanol extract (F3), and aqueous layer (F4) of quince seeds was carried out in the same manner as in Example 1.3.2.

[0135] The yield and total flavonoid content were calculated using the same method as in Example 1.

[0136] The yield of the ethanol extract (F5, i.e., crude extract) was 7.96%, the yield of the ethyl acetate extract (F2) was 0.24%, the yield of the n-butanol extract (F3) was 1.5%, and the yield of the aqueous layer (F4) was 4.88%.

[0137] The total flavonoid content of the ethanol extract (F5) was 0.48%, the total flavonoid content of the ethyl acetate extract (F2) was 1.7%, the total flavonoid content of the n-butanol extract was 0.5%, and the total flavonoid content of the aqueous layer was 0.3%.

[0138] Comparative Example 2

[0139] The difference from Example 1 is that in step 1.3.2, after the crude extract is dissolved in water in the same way, it is directly extracted with ethyl acetate and n-butanol in the same way as in Example 1 to obtain the ethyl acetate extract (G2), the n-butanol extract (G3) and the aqueous layer (G4).

[0140] The yield and total flavonoid content were calculated using the same method as in Example 1. The yield of the ethyl acetate extract (G2) was 2.23%, and the total flavonoid content (by mass) of the ethyl acetate extract (G2) was 0.6%; the yield of the n-butanol extract (G3) was 1.17%, and the total flavonoid content (by mass) was 0.36%; and the yield of the aqueous layer (G4) was 3.73%, and the total flavonoid content (by mass) was 0.28%.

[0141] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing an antioxidant extract of quince seeds, characterized in that, The steps include the following: The dried quince seeds are pulverized to prepare quince seed powder; The quince seed powder is soaked in a first solvent to prepare a crude extract; the first solvent includes ethanol and water. The crude extract was concentrated and dried to obtain a crude extract. The crude extract was mixed with water to prepare an aqueous extract solution. The aqueous extract and petroleum ether were mixed and subjected to a first extraction process to obtain the petroleum ether extract fraction and the aqueous extract after petroleum ether extraction. The petroleum ether extraction aqueous solution was mixed with ethyl acetate and subjected to a second extraction treatment to obtain the ethyl acetate extract fraction and the ethyl acetate extraction aqueous solution; the ethyl acetate extract fraction was subjected to a first concentration treatment to prepare the first quince antioxidant extract.

2. The preparation method according to claim 1, characterized in that, It meets one or more of the following characteristics: (1) The mass percentage of flavonoids in the first quince seed antioxidant extract is 10%-15%; (2) The volume fraction of ethanol in the first solvent is 70%~80%; (3) The amount of the first solvent used to soak each gram of the quince powder is 30 mL to 50 mL.

3. The preparation method according to claim 2, characterized in that, The step of preparing a crude extract by soaking the quince seed powder in a first solvent includes: soaking the quince seed powder in a first solvent for 20 min - 60 min, then sonicating for 5 min - 15 min, separating the solid and liquid, and collecting the crude extract. Optionally, the quince seed solid powder collected after solid-liquid separation is soaked and separated again, and the collected liquid is used as the crude extract.

4. The preparation method according to claim 1, characterized in that, The step of mixing the crude extract with water includes: mixing the crude extract with water at a mass ratio of 1:(18-25).

5. The preparation method according to claim 1, characterized in that, It meets at least one of the following characteristics: (1) The volume ratio of the aqueous extract to the petroleum ether is 1:(0.8~1.5); (2) The volume ratio of the petroleum ether extraction aqueous solution to the ethyl acetate is 1:(0.8~1.5). (3) The first concentration process includes vacuum distillation and drying.

6. The preparation method according to claim 1, characterized in that, The preparation method further includes: mixing the aqueous solution after ethyl acetate extraction with n-butanol, and performing a third extraction treatment to obtain the n-butanol extract fraction and the aqueous solution after n-butanol extraction; and performing a second concentration treatment on the n-butanol extract fraction to obtain a second quince antioxidant extract. Optionally, the volume ratio of the aqueous solution after ethyl acetate extraction to n-butanol is 1:(0.8~1.5). Optionally, the flavonoids in the second quince antioxidant extract contain 4%-6% by mass.

7. The preparation method according to claim 6, characterized in that, The preparation method further includes: subjecting the aqueous solution after n-butanol extraction to a third concentration treatment to obtain a third quince antioxidant extract; Optionally, the flavonoid content in the third quince antioxidant extract is 0.3%-0.5% by mass.

8. The preparation method according to claim 7, characterized in that, The preparation method further includes: subjecting the petroleum ether extract fraction to a fourth concentration treatment to obtain a fourth quince antioxidant extract; Optionally, the flavonoid content in the fourth quince seed antioxidant extract is 0.05%-0.07% by mass.

9. A quince seed antioxidant extract, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the quince seed antioxidant extract as described in claim 9 as an antioxidant.

Images