A medicinal and edible composition for relieving or treating vaginitis and its preparation and application

Abstract

The present invention relates to the field of medicine and food compositions, and in particular to a medicine and food composition for relieving or treating vaginitis, and its preparation and application. The present invention selects dandelion and cornus officinalis as raw materials for step-by-step extraction to obtain a medicine and food composition. The two raw materials are compounded to comprehensively exert their effects, have the effects of clearing away heat and detoxification, antibacterial and anti-inflammatory, immunomodulation, etc., can relieve the symptoms of bacterial vaginitis, and have a good therapeutic effect on bacterial vaginitis. Compared with the method of using antibiotics for treatment in the prior art, the medicine and food composition provided by the present invention is applied in treatment, which is safer, will not cause the generation of bacterial resistance, will not have side effects on the human body, and can improve the quality of life of patients. The optimization of the medicine and food raw material formula and extraction method provided by the present invention can more effectively reduce the loss of effective ingredients in herbal raw materials compared with the prior art, thereby improving its therapeutic efficacy and reducing the generation of side effects.

Description

Technical Field

[0001] The present invention relates to the field of medicine-food homologous compositions, and in particular to a medicine-food homologous composition for alleviating or treating vaginitis, and a preparation method and application thereof. Background Art

[0002] Bacterial vaginosis (BV) is a common vaginal infection in women of childbearing age, which can lead to premature birth, infertility or other vaginal diseases. The specific cause of BV has not yet been elucidated, but most scholars believe that the occurrence of the disease is closely related to the structure of the vaginal microbiome. There are a variety of microbial communities distributed in the female vagina, and different microorganisms antagonize and coexist with each other to maintain the physiological function of the female reproductive tract. The flora of a healthy female vagina is mainly lactobacillus, while this type of flora is reduced in the vagina of BV patients, resulting in excessive growth of other pathogenic bacteria, such as Gardnerella, which changes the vaginal environment and causes inflammation.

[0003] Currently, the treatments for bacterial vaginitis mainly include: oral antibiotics, such as metronidazole, tinidazole, etc., which can effectively inhibit the growth and reproduction of bacteria; the use of vaginal washes and vaginal suppositories, such as metronidazole suppositories, tinidazole suppositories, etc.; or the use of Chinese herbal prescriptions or Chinese patent medicines, such as Chinese herbal decoctions that clear away heat and toxic substances, and reduce inflammation and kill bacteria, for external use as auxiliary treatments for bacterial vaginitis.

[0004] In the prevention and treatment of BV, the potential of edible and medicinal raw materials is particularly prominent. On the one hand, these raw materials are rich in a variety of active ingredients, such as polysaccharides, flavonoids, alkaloids, etc. These ingredients have biological activities such as antibacterial, anti-inflammatory, and immune enhancement, which help regulate the balance of vaginal microecology and inhibit the growth of pathogenic bacteria; on the other hand, the side effects of edible and medicinal raw materials are relatively small, and they are easily absorbed and utilized by the human body, which is in line with the modern people's pursuit of a healthy and natural lifestyle.

[0005] At present, some studies have reported the application of edible and medicinal ingredients in the prevention and treatment of BV, but most of them are used in topical dosage forms such as lotions. Therefore, this study aims to explore the application potential of edible and medicinal ingredients in the prevention and treatment of BV in oral dosage forms, and to develop edible and medicinal products for the prevention and treatment of BV that are efficient, safe and easy to use by screening and optimizing the combination of raw materials, so as to provide new treatment options for BV patients.

[0006] There are many methods for extracting and studying natural plants, such as solvent extraction (organic solvent or water) and supercritical fluid extraction. The most commonly used is solvent extraction, but traditional organic solvents, such as ethanol and ethyl acetate, have limited solubility for cellulose, resulting in a low natural product extraction rate. There are also problems such as high toxicity and volatility during the extraction process, which does not conform to the concept of green chemistry. Water extraction is not soluble for flavonoids, saponins and other components in plants, resulting in a low extraction recovery rate. Although supercritical fluid extraction is more environmentally friendly, it has a large initial investment and high operating costs, which limits its large-scale application. Therefore, finding an efficient and green extraction process to improve the extraction rate is a very meaningful research direction. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a medicine-food combination having a good therapeutic effect on bacterial vaginitis.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a method for preparing a medicine-food composition, comprising the following steps:

[0010] (1) respectively crushing and sieving dandelion and cornus officinalis to obtain dandelion powder and cornus officinalis powder;

[0011] (2) adding an extraction solvent to the dandelion powder, performing ultrasonic extraction, centrifuging, and collecting a filtrate;

[0012] The extraction solvent comprises the following components in parts by weight: 5-10 parts of betaine, 5-10 parts of fructose, 5-15 parts of xylitol, 15-40 parts of lactic acid, 15-40 parts of glycine, and 15-35 parts of water;

[0013] (3) Adding cornus officinalis powder to the filtrate obtained in step (2), performing ultrasonic extraction, centrifugation, and concentration to obtain the medicine-food composition.

[0014] The present invention selects two herbs, dandelion and cornus officinalis, as raw materials for step-by-step extraction to obtain a medicine-food homologous composition. Dandelion has the effects of clearing away heat and detoxification, removing dampness and clearing heat, and anti-inflammatory and sterilization, and can effectively eliminate pathogens. At the same time, removing dampness and clearing heat helps to improve the hot and humid environment in the vagina, thereby alleviating inflammatory symptoms, and has a good therapeutic effect on female bacterial vaginitis; Cornus officinalis has the characteristics of nourishing the liver and kidney, astringing and astringent, and has a good conditioning effect on women's irregular menstruation, metrorrhagia and leukorrhea, and can also invigorate qi and nourish blood, and has a good improvement effect on improving the symptoms of pale complexion, fatigue and lack of spirit caused by insufficient qi and blood in women, and plays an auxiliary role in the compound to enhance the overall effect of the compound. The composite use of these two medicine-food homologous raw materials can comprehensively play a variety of effects such as clearing away heat and detoxification, antibacterial and anti-inflammatory, and immunomodulation, thereby effectively treating bacterial vaginitis.

[0015] The present invention provides a method for extracting a medicine-food combination by adding an appropriate amount of hydrogen bond acceptors and hydrogen bond donors, extracting in steps, and applying ultrasound-assisted extraction. The supramolecular system formed by the added hydrogen bond acceptors, hydrogen bond donors, and small molecules contained in the medicine-food combination raw materials improves the extraction efficiency of the medicine-food combination, while protecting the biological activity of the functional substances in the raw materials.

[0016] A hydrogen bond donor (HBD) refers to a compound containing hydrogen atoms that are covalently bonded to highly electronegative atoms (such as oxygen, nitrogen, and fluorine), so that the hydrogen atoms have a partial positive charge and can serve as hydrogen bond donors, while a hydrogen bond acceptor (HBA) refers to a compound containing one or more highly electronegative atoms (such as oxygen, nitrogen, and fluorine) that do not share electron pairs, and such atoms can serve as hydrogen bond acceptors. For example, molecules such as betaine, lactic acid, and glycine contain exposed carboxyl oxygen atoms, which are good hydrogen bond acceptors and can form strong hydrogen bonds with hydrogen atoms in other molecules. Therefore, betaine, lactic acid, and glycine can serve as hydrogen bond acceptors, while molecules such as fructose and xylitol contain free reducing groups such as aldehydes or ketones. The hydrogen atoms on these groups can form hydrogen bonds with atoms with greater electronegativity (such as nitrogen, oxygen, and fluorine), and therefore have the characteristics of hydrogen bond donors. Mixing two or more high melting point solid compounds (hydrogen bond donors and hydrogen bond acceptors) in an appropriate molar ratio can form a transparent liquid with a lower melting point than any single component, which is called a deep eutectic solvent (DES). The eutectic system formed by natural substances and their derivative components is called a natural deep eutectic solvent (NADES). As the ionic radius of hydrogen bond acceptors and hydrogen bond donors increases, the energy decreases, thereby reducing the melting point, making the mixed extract have better permeability, and playing an effect similar to supramolecular extraction. The active ingredients in the plant will further form hydrogen bonds and van der Waals forces with the components of the natural deep eutectic solvent, and more binding forces are introduced into the system. These interaction forces help to extract the active ingredients more effectively and targetedly, thereby more efficiently extracting the effective ingredients in the mixed raw materials.

[0017] The present invention has found through a large number of experimental investigations that the various plant raw materials in the medicinal and edible composition have quite different ingredients. For example, dandelion is rich in flavonoids, which can be well extracted in natural low eutectic solvents. At the same time, flavonoids are rich in hydroxyl groups, which are very good hydrogen bond donors, and can further enhance the permeability of natural low eutectic solvents. Cornus officinalis is rich in sugars and organic acids, and has a relatively large viscosity. Therefore, the post-extraction of Cornus officinalis can ensure the fluidity of the solvent. The present invention can promote the connection of related substances through hydrogen bonds through step-by-step extraction and the properties of the extracted substances, and fully extract the effective components of each raw material. The method is environmentally friendly and low-cost. The obtained extract is lighter in color and has a unique plant flavor; the content of effective ingredients is high and the variety is complete; among them, the content of quercetin is more than 4.1%, and the content of total polysaccharides is more than 14.4%.

[0018] The present invention uses herbal formulas to replace traditional antibiotics for treatment, which can avoid bacterial resistance and side effects on the human body caused by long-term use of antibiotics. At the same time, the herbal formula can also regulate human immunity and enhance the body's disease resistance, thereby achieving the purpose of preventing and treating female bacterial vaginitis.

[0019] Preferably, the extraction solvent comprises the following components in parts by weight: 5-10 parts of betaine, 5-10 parts of fructose, 5-15 parts of xylitol, 20-30 parts of lactic acid, 15-30 parts of glycine, and 25-30 parts of water.

[0020] More preferably, the extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water.

[0021] Preferably, the weight ratio of the dandelion powder to the cornus officinalis powder is (10-90): (10-90).

[0022] Preferably, in step (2), the weight volume ratio of dandelion powder to extraction solvent is dandelion powder: extraction solvent = 1 g: (20-200) mL.

[0023] More preferably, in step (2), the weight volume ratio of dandelion powder to extraction solvent is dandelion powder: extraction solvent = 1 g: (20-40) mL.

[0024] Preferably, in steps (2) and (3), the conditions for ultrasonic extraction are: ultrasonic power 100-500 W, extraction temperature 20-60° C., and extraction time 10-60 min.

[0025] The present invention adopts ultrasound-assisted extraction. When ultrasound acts on liquid, a large number of small bubbles will be generated in the liquid, thereby increasing the contact area between the substance and the extractant. At the same time, during the extraction process, ultrasound will cause a certain degree of damage to the cell walls of plant cells, accelerating the infiltration of the components to be extracted from the cell fluid and speeding up the mass transfer rate, thereby enhancing the transport and extraction of active compounds and improving the extraction rate.

[0026] More preferably, in steps (2) and (3), the conditions for ultrasonic extraction are: ultrasonic power 250-300 W, extraction temperature 30-40° C., and extraction time 15-30 min.

[0027] Preferably, in step (3), the centrifugal speed is 8000 r / min.

[0028] Preferably, the concentration method is reduced pressure concentration.

[0029] Preferably, the conditions for the reduced pressure concentration are a temperature of 60-65° C. and a vacuum degree of 0.08-0.1 MPa.

[0030] In a second aspect, the present invention provides a medicine-food composition prepared by the above-mentioned preparation method.

[0031] In a third aspect, the present invention provides the use of the above-mentioned medicine-food composition in the preparation of a preparation or medicine for alleviating or treating vaginitis.

[0032] Preferably, the vaginitis comprises bacterial vaginitis.

[0033] Preferably, the preparations include oral preparations and external preparations.

[0034] In a fourth aspect, the present invention provides a preparation or medicine for alleviating or treating vaginitis, comprising the above-mentioned medicine-food composition.

[0035] The beneficial effects of the present invention are:

[0036] The present invention selects two herbs, dandelion and cornus officinalis, as raw materials for step-by-step extraction to obtain a medicine-food combination. The combined use of these two medicine-food raw materials can comprehensively exert multiple effects such as clearing away heat and detoxification, antibacterial and anti-inflammatory, and immune regulation, has a good therapeutic effect on female bacterial vaginitis, and can alleviate the symptoms of female bacterial vaginitis. Compared with the antibiotic treatment in the prior art, the herbal formula of the present invention is safer, will not cause the generation of bacterial resistance, and will not have side effects on the human body, thereby improving the quality of life of patients.

[0037] In addition, the present invention finds that by optimizing the extraction method of the two raw materials, using appropriate amounts of hydrogen bond acceptors and hydrogen bond donors to form an extraction solvent, extracting in steps, and applying ultrasound-assisted extraction, the effective components in the raw materials can be extracted. The extraction method can effectively retain the effective components in the herbal medicine, reduce the loss of effective components, and improve the bioavailability of the drug. At the same time, it also avoids the use of organic solvents, thereby reducing production costs and reducing environmental pollution.

[0038] Compared with the prior art, the optimization of the medicine-food raw material formula and extraction method of the present invention can more effectively reduce the loss of active ingredients in the herbal medicine, thereby improving the efficacy of the drug and reducing the occurrence of side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A typical graph of the fluorescence intensity of Gardnerella vaginalis in the zebrafish urogenital tract after sample treatment.

[0040] Figure 2 A typical graph of the fluorescence intensity of Staphylococcus aureus in the zebrafish urogenital tract after sample treatment. DETAILED DESCRIPTION

[0041] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0042] In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0043] Embodiment 1:

[0044] An embodiment of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0045] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively for later use;

[0046] (2) Add 2000 mL of extraction solvent to 50 g of dandelion powder, perform ultrasonic extraction at 40° C. for 20 min with an ultrasonic power of 300 W; after the extraction, centrifuge and collect the filtrate;

[0047] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 10 parts of fructose, 15 parts of xylitol, 30 parts of lactic acid, 40 parts of glycine, and 15 parts of water;

[0048] (3) Add 50 g of Cornus officinalis powder to the filtrate obtained in step (2), and perform ultrasonic extraction at 40° C. for 20 min with an ultrasonic power of 300 W. After the extraction, centrifuge and concentrate the extract to obtain the edible and medicinal composition.

[0049] Embodiment 2-8:

[0050] Examples 2-8 are the medicinal and edible compositions of the present invention, and the preparation methods thereof differ from those of Example 1 only in the number of extraction solvent components, as shown in Table 1.

[0051] Table 1 Components of the extraction solvents in each embodiment

[0052] Example Betaine fructose Xylitol lactic acid Glycine water Example 1 5 10 15 30 40 15 Example 2 10 10 10 40 20 15 Example 3 5 10 5 40 20 25 Example 4 5 5 5 30 30 25 Example 5 10 5 15 20 20 30 Example 6 10 10 15 20 15 30 Example 7 10 10 10 15 20 35 Example 8 10 10 15 15 15 35

[0053] Embodiment 9-17:

[0054] Examples 9-17 are the edible and medicinal compositions of the present invention, and the preparation methods thereof differ from those of Example 4 only in that the weight ratios of dandelion powder and cornus powder are different, as shown in Table 2.

[0055] Table 2 Weight ratio of dandelion powder and cornus powder in each embodiment

[0056]

[0057]

[0058] Embodiment 18:

[0059] An embodiment of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0060] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively for later use;

[0061] (2) Add 2000 mL of extraction solvent to 100 g of dandelion powder, perform ultrasonic extraction at 30° C. for 30 min with an ultrasonic power of 250 W; after the extraction, centrifuge and collect the filtrate;

[0062] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water;

[0063] (3) Add 100 g of Cornus officinalis powder to the filtrate obtained in step (2), and perform ultrasonic extraction at 30° C. for 30 min with an ultrasonic power of 250 W. After the extraction, centrifuge and concentrate the extract to obtain the edible and medicinal composition.

[0064] Embodiment 19:

[0065] An embodiment of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0066] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively for later use;

[0067] (2) Add 1500 mL of extraction solvent to 50 g of dandelion powder, perform ultrasonic extraction at 35° C. for 20 min with an ultrasonic power of 250 W; after the extraction, centrifuge and collect the filtrate;

[0068] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water;

[0069] (3) Add 50 g of Cornus officinalis powder to the filtrate obtained in step (2), and perform ultrasonic extraction at 35° C. for 20 min with an ultrasonic power of 250 W. After the extraction, centrifuge and concentrate the extract to obtain the edible and medicinal composition.

[0070] Embodiment 20:

[0071] An embodiment of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0072] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively for later use;

[0073] (2) Add 8000 mL of extraction solvent to 200 g of dandelion powder, perform ultrasonic extraction at 30° C. for 15 min with an ultrasonic power of 300 W; after the extraction, centrifuge and collect the filtrate;

[0074] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water;

[0075] (3) Add 100 g of Cornus officinalis powder to the filtrate obtained in step (2), and perform ultrasonic extraction at 30° C. for 15 min with an ultrasonic power of 300 W. After the extraction, centrifuge and concentrate the extract to obtain the edible and medicinal composition.

[0076] Comparative Examples 1-6:

[0077] Comparative Examples 1-6 are the medicinal and edible compositions of the present invention, and the preparation methods thereof differ from those of Example 1 only in that the formula of the extraction solvent is different, as shown in Table 3.

[0078] Table 3 Components of the extraction solvents in each comparative example

[0079] Comparative Example Betaine fructose Xylitol lactic acid Glycine water Comparative Example 1 0 5 5 30 30 30 Comparative Example 2 5 0 5 30 30 30 Comparative Example 3 5 5 0 30 30 30 Comparative Example 4 5 5 10 0 40 35 Comparative Example 5 5 5 10 40 0 35 Comparative Example 6 5 5 10 40 40 0

[0080] Comparative Example 7:

[0081] A comparative example of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0082] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively, taking 50 g of each powder and mixing them evenly to obtain a mixed powder;

[0083] (2) adding 2000 mL of extraction solvent to the mixed powder obtained in step (1), and ultrasonically extracting at 40° C. for 20 min with an ultrasonic power of 300 W; after the extraction, centrifuging and collecting the filtrate;

[0084] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water;

[0085] (3) After the extraction is completed, the extract is centrifuged and concentrated to obtain the medicinal and edible composition.

[0086] Comparative Example 8:

[0087] A comparative example of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0088] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively for later use;

[0089] (2) Add 2000 mL of extraction solvent to 50 g of Cornus officinalis powder, perform ultrasonic extraction at 40° C. for 20 min with an ultrasonic power of 300 W; after the extraction, centrifuge and collect the filtrate;

[0090] The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water;

[0091] (3) Add 50 g of dandelion powder to the filtrate obtained in step (2), and perform ultrasonic extraction at 40° C. for 20 min with an ultrasonic power of 300 W. After the extraction, centrifuge and concentrate the extract to obtain the edible and medicinal composition.

[0092] Comparative Example 9:

[0093] A comparative example of the medicine-food combination of the present invention; the preparation method of the medicine-food combination of the present invention comprises the following steps:

[0094] (1) Pretreatment of the edible and medicinal raw materials: washing and dusting the dried dandelion and cornus officinalis raw materials, drying them, crushing them and sieving them to obtain dandelion powder and cornus officinalis powder respectively, taking 50 g of each powder and mixing them evenly to obtain a mixed powder;

[0095] (2) adding 3000 mL of pure water to the mixed powder obtained in step (1), and ultrasonically extracting at 65° C. for 2 h with an ultrasonic power of 300 W; after the extraction, centrifuging and collecting the filtrate;

[0096] (3) After the extraction is completed, centrifuge to obtain the filter residue, add 3000 mL of pure water, and perform ultrasonic extraction at 65° C. for 2 h. After the extraction is completed, centrifuge and concentrate to obtain the extract, which is the medicinal and edible composition.

[0097] Test Example 1: Active ingredient detection

[0098] Test samples: the medicinal and edible compositions (extracts) prepared in Examples 1-20 and Comparative Examples 1-9.

[0099] 1. Detect the polysaccharide content in the test sample by spectrophotometry. The specific method is as follows:

[0100] 1. Draw a standard curve

[0101] Accurately measure 0mL, 0.40mL, 0.80mL, 1.20mL, 1.60mL, and 1.80mL of glucose standard stock solution and place them in 25mL stoppered test tubes, accurately add water to 2.00mL, shake well, add 1mL of 5% phenol solution and 10mL of concentrated sulfuric acid to each test tube in turn, mix on a rotary mixer, boil in a boiling water bath for 15min, take out, mix on a rotary mixer, let stand for 30min to cool to room temperature, and within 90min, use the corresponding reagent as blank, measure the absorbance at a wavelength of 485nm by UV-visible spectrophotometry, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0102] 2. Prepare the test solution

[0103] Weigh 0.6 g of the extract, accurately weigh it, put it in a 150 mL volumetric flask, accurately add 50 mL of water with a pipette, weigh it, ultrasonically treat it for 20 min (250 W, 40 kHz), take it out, make up the lost weight with water, shake it well, filter it, take 1 mL of the filtrate, put it in a 15 mL centrifuge tube, add 8 mL of anhydrous ethanol, shake it gently, place it at 4 ° C for 2 h, centrifuge it at 4000 r / min for 20 min, discard the supernatant, add an appropriate amount of boiling water to dissolve the precipitate, transfer it to a 20 mL volumetric flask, make up to the scale with water, shake it well, and you will get the test solution.

[0104] 3. Determination of polysaccharide content

[0105] Accurately measure 2.0 mL of the test solution and place it in a 25 mL stoppered test tube. Use the reagent blank to adjust the zero value, and use a 10 mm optical diameter cuvette to measure the absorbance at a wavelength of 485 nm in a UV-visible spectrophotometer. Use D-anhydrous glucose as a control and calculate the polysaccharide content in the standard curve.

[0106] The polysaccharide content is expressed as mass fraction w, the value is expressed in g / 100g, and is calculated according to formula (1).

[0107]

[0108] In the above formula:

[0109] w——polysaccharide content, in grams per 100 grams (g / 100g);

[0110] C——Calculate the concentration of polysaccharide in the sample from the standard curve in micrograms (μg);

[0111] m——the weight of the sample to be tested, in grams (g);

[0112] V1——the fixed volume of the test solution, in milliliters (mL);

[0113] V2——the volume of diluent used, in milliliters (mL);

[0114] V3——the fixed volume of the diluent, in milliliters (mL);

[0115] V4——The volume of the test sample solution on the machine, in milliliters (mL).

[0116] 2. Quercetin content detection

[0117] 1. Chromatographic conditions.

[0118] Chromatographic column: octadecyl bonded silica gel column (150 mm × 4.6 mm, 3.5 μm) or other similar type chromatographic columns; mobile phase: methanol-0.2% phosphoric acid solution (38:62, volume ratio); wavelength: 360 nm; column temperature: 35 ° C; flow rate: 1.0 mL / min; injection volume: 5 μL.

[0119] 2. Drawing of standard curve.

[0120] Accurately weigh 10 mg (accurate to 0.1 mg) of quercetin reference substance, dissolve it with methanol and make up to 250 mL, shake well, and store it in a refrigerator at 0℃-4℃. Accurately pipette 0μL, 50μL, 125μL, 250μL, 375μL, and 500μL of quercetin reference substance stock solution into 1mL volumetric flasks, make up to volume with methanol, shake well, and perform liquid chromatography analysis under the specified chromatographic conditions. Perform qualitative analysis by retention time, make a standard curve by using the peak area of ​​the sample versus the concentration of the standard solution, and obtain the linear regression equation.

[0121] 3. Prepare the test solution.

[0122] Take 0.2g of the extract, accurately weigh it, place it in a 150mL stoppered conical flask, add 25mL of a mixed solution of methanol-hydrochloric acid (4:1, volume ratio), stopper it tightly, shake it until the sample is dissolved on the inner wall and bottom of the conical flask, add a few zeolites to prevent violent boiling, weigh it, place it in an 85℃ water bath and heat it under reflux for 1h (keep it slightly boiling), cool it, weigh it again, add methanol to make up the lost weight, shake it well, centrifuge it in a centrifuge at 4000r / min for 10min, accurately measure 5mL of the supernatant, place it in a 50mL volumetric flask, add methanol to the mark, shake it well, filter it through a 0.45μm organic phase filter, take the filtrate to obtain the test solution.

[0123] 4. Sample measurement.

[0124] The multi-point calibration external standard method was used for quantification. 5 μL of the standard curve working solution and the test solution were accurately aspirated and injected into the high performance liquid chromatograph for determination and recording of the chromatogram. If the response value of quercetin in the test solution exceeds the linear range, it should be appropriately diluted before analysis.

[0125] The quercetin content is expressed as mass fraction w, and the value is expressed as g / 100g, calculated according to formula (2).

[0126]

[0127] In the above formula:

[0128] w——quercetin content, in grams per 100 grams (g / 100g);

[0129] C——Calculate the concentration of quercetin in the sample from the standard curve in micrograms per milliliter (μg / mL);

[0130] V1——the fixed volume of the test solution, in milliliters (mL); V2——the used volume of the test sample extract, in milliliters (mL); V3——the fixed volume of the test sample extract, in milliliters (mL); m——the sample weight of the test sample, in grams (g);

[0131] ω——water content of the test sample, %.

[0132] The results of polysaccharide and quercetin content detection are shown in Table 4.

[0133] Table 4 Total polysaccharide and quercetin content test results

[0134]

[0135]

[0136] Test Example 2: Functional in vitro experiment - anti-vaginal pathogen infection

[0137] 1. Test sample preparation: 1 g of the edible-medicinal composition (extract) prepared in Examples 9-19 and Comparative Example 9 was dissolved in 10 mL of sterile water, centrifuged at 4°C, 4500×g for 10 min, the supernatant was concentrated 3 times, and stored at 4°C for later use.

[0138] 2. Activation of vaginal Gardnerella

[0139] The frozen tube of vaginal Gardnerella was taken out, 1 mL of bacterial solution was inoculated into 100 mL of TSB medium + 5% defibrinated sheep blood, and cultured in a micro-anaerobic gas production bag (5% CO2) environment at 37°C for 24 hours.

[0140] 3. Antibacterial experiment of medicine-food combination

[0141] Take vaginal Gardnerella liquid (100 μL, concentration is about 1×10 8 CFU / mL) was spread on an agar plate, a sterilized filter paper was placed on the plate, four were placed in parallel, 20 μL of the prepared edible and medicinal composition sample experimental solution (Examples 17-27, Comparative Example 28) was taken and placed on the filter paper, sterile water was used as a blank control, the plate was allowed to rest for 20 min, then the plate was slowly placed in a 37°C micro-anaerobic gas production bag (5% CO2) and cultured for 48 h, the inhibition zone was observed after culture, and the diameter of the inhibition zone was measured. The results are shown in Table 5.

[0142] Table 5 Antibacterial test results of the medicinal and edible composition samples on vaginal Gardnerella

[0143]

[0144]

[0145] As can be seen from the above table, each example group and the comparative example group all have antibacterial circles, indicating that the medicinal and edible combination samples provided by each example group and the comparative example group have antibacterial effects on vaginal Gardnerella. Compared with the comparative example, the diameter of the antibacterial circle of the example is larger, and the difference is significant (p < 0.05), indicating that the medicinal and edible combination extract prepared within the scope of the present invention has a better antibacterial effect.

[0146] Compared with the antibacterial effects of Examples 9-19, Example 9-17 adjusted the weight ratio of dandelion powder to Cornus officinalis powder, which had a greater impact on the antibacterial effect, among which Example 13 had the largest antibacterial circle diameter; Example 18-19 adjusted the amount of extraction solvent added, but the antibacterial effect was not much different from that of Example 13, indicating that Example 13 is the optimal embodiment of the present invention and has the best antibacterial effect, that is, when the weight ratio of dandelion powder to Cornus officinalis powder is 50:50, the prepared extract has the best antibacterial effect, so Example 13 was selected for subsequent animal experiments for verification.

[0147] Test Example 3: Animal experiment - Anti-vaginal pathogen infection and improvement of inflammation test

[0148] Test sample: the medicine-food composition (extract) prepared in Example 13.

[0149] 1. Experiment on anti-zebrafish infection of medicine-food combination samples

[0150] According to the results of the previous concentration exploration, the No Observed Adverse Effect Level (NOAEL) of the edible and medicinal composition samples on normal zebrafish is 1000μg / mL. The low, medium and high dose groups are set at 1 / 4, 1 / 2 and 1 of the NOAEL dose, that is, the mass concentrations of the edible and medicinal composition samples are 250μg / mL, 500μg / mL and 1000μg / mL.

[0151] (1) Anti-Staphylococcus aureus infection experiment

[0152] Wild-type AB zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each well (experimental group). The sample of the medicinal and edible composition was dissolved in the fish farming water according to the preset concentration, and the zebrafish in the model control group were treated only with fish farming water, and the volume of each well was 3mL. After continuous treatment at 28℃ for 18h, Staphylococcus aureus labeled with red fluorescent CM-Dil was injected into the intestinal cavity of zebrafish in each experimental group, and the solution was changed and continued to be treated at 28℃ for 3h to establish a zebrafish Staphylococcus aureus infection model. Whether the modeling was successful was judged based on whether there was red fluorescence in the intestinal cavity of zebrafish.

[0153] (2) Anti-Gardnerella vaginalis infection experiment

[0154] Wild-type AB zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each well (experimental group). The sample of the medicinal and edible composition was dissolved in the fish farming water according to the preset concentration, and the zebrafish in the model control group were treated with fish farming water only, with a volume of 3 mL per well. After continuous treatment at 28°C for 18 hours, each experimental group was injected with vaginal Gardnerella labeled with green fluorescent CM-DiI into the intestinal cavity of zebrafish, and the solution was changed and continued to be treated at 28°C for 3 hours to establish a zebrafish vaginal Gardnerella infection model, and the success of the model was judged by whether there was green fluorescence in the intestinal cavity.

[0155] After the above experiments, 10 zebrafish were randomly selected from each group and photographed using a fluorescence microscope. The data were analyzed and collected using NIS-ElementsD 3.20 advanced image processing software to analyze the fluorescence intensity and antibacterial rate of pathogenic bacteria in the intestinal cavity of zebrafish, which were used to evaluate the anti-infection efficacy of the medicine-food combination samples. The antibacterial rate was calculated according to formula (3).

[0156] Inhibition rate = (F model - F experiment) / F model × 100% - Formula (3)

[0157] Table 6 Detection results of fluorescence intensity of pathogenic bacteria in zebrafish intestinal cavity and antibacterial rate of medicinal and edible composition samples on pathogenic bacteria

[0158]

[0159] Note: Values ​​are expressed as mean ± SD. Different letters in the same column indicate significant differences.

[0160] The results are shown in Table 6. Figure 1 , Figure 2 As shown. Compared with the model control group, the fluorescence intensity of pathogenic bacteria in zebrafish was reduced after 24 hours of administration of the water-soluble medicine-food combination sample. The medicine-food combination sample has antibacterial ability against Staphylococcus aureus and Gardnerella vaginalis, and the antibacterial effect is good. The low concentration group of 250μg / mL has shown an antibacterial effect, and the inhibition rate against Staphylococcus aureus is 38.95% (P<0.0001). As the concentration increases, the inhibition rate has an increasing trend, with a maximum of 48.85% (P<0.0001); the inhibition rate against Gardnerella vaginalis is 12.68% (P<0.0.05), and as the concentration increases, the inhibition rate has an increasing trend, with a maximum of 30.98% (P<0.0001).

[0161] 2. Experiment on improving inflammation in zebrafish using samples of medicine-food combination

[0162] Transgenic neutrophil green fluorescent MPX strain zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each group (normal control group, model control group, and low, medium, and high dose groups), for a total of 150 fish. The sample of the medicinal and edible composition was dissolved in fish farming water, and the concentration was the same as that of the anti-zebrafish infection experiment of the medicinal and edible composition sample. The zebrafish in the normal control group and the model control group were only treated with fish farming water, and the volume of each well was 3 mL. After continuous treatment at 28°C for 18 hours, except for the normal control group, the remaining experimental groups were injected with Staphylococcus aureus into the intestinal cavity of zebrafish to establish a zebrafish inflammatory infection model, and the solution was changed and continued to be treated at 28°C for 3 hours. After the experiment, 10 zebrafish were randomly selected from each group and photographed with a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils (N) in the intestinal cavity of zebrafish and the inflammation inhibition rate, which were used to evaluate the inflammation inhibition rate of the medicinal and edible composition sample. The inflammation inhibition rate was calculated according to formula (4).

[0163] Inflammation inhibition rate = (N model control group average value - N experimental group) / (N model control group average value - N normal control group average value) × 100% - Formula (4)

[0164] Table 7 Detection results of the number of neutrophils in the intestinal cavity of zebrafish and the inhibition rate of zebrafish intestinal inflammation by the medicinal and edible composition samples

[0165]

[0166] Note: Values ​​are expressed as mean ± SD. Different letters in the same column indicate significant differences.

[0167] The results are shown in Table 7. The number of neutrophils in the intestinal cavity of the normal control group was significantly lower than that of the model control group (P < 0.0001), indicating that the modeling was successful. The inflammation inhibition rates of the 250 μg / mL, 500 μg / mL and 1000 μg / mL groups were 24.48% (P < 0.0001), 53.53% (P < 0.0001), and 56.43% (P < 0.0001), respectively. As the concentration increased, the inflammation inhibition rate had an upward trend, which was consistent with the anti-Staphylococcus aureus infection effect of the medicine-food homologous composition sample. It is speculated that it may achieve anti-inflammatory effect by resisting Staphylococcus aureus infection.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a medicine-food composition, characterized in that: The steps include: (1) respectively crushing and sieving dandelion and cornus officinalis to obtain dandelion powder and cornus officinalis powder; (2) adding an extraction solvent to the dandelion powder, performing ultrasonic extraction, centrifuging, and collecting a filtrate; The extraction solvent comprises the following components in parts by weight: 5-10 parts of betaine, 5-10 parts of fructose, 5-15 parts of xylitol, 15-40 parts of lactic acid, 15-40 parts of glycine, and 15-35 parts of water; (3) Adding cornus officinalis powder to the filtrate obtained in step (2), performing ultrasonic extraction, centrifugation, and concentration to obtain the medicine-food composition.

2. The preparation method according to claim 1, characterized in that The extraction solvent comprises the following components in parts by weight: 5-10 parts of betaine, 5-10 parts of fructose, 5-15 parts of xylitol, 20-30 parts of lactic acid, 15-30 parts of glycine, and 25-30 parts of water.

3. The preparation method according to claim 2, characterized in that: The extraction solvent comprises the following components in parts by weight: 5 parts of betaine, 5 parts of fructose, 5 parts of xylitol, 30 parts of lactic acid, 30 parts of glycine, and 25 parts of water.

4. The preparation method according to claim 1, characterized in that: The weight ratio of the dandelion powder to the cornus officinalis powder is (10-90): (10-90).

5. The preparation method according to claim 1, characterized in that: In the step (2), the weight volume ratio of dandelion powder to extraction solvent is dandelion powder: extraction solvent = 1 g: (20-200) mL.

6. The preparation method according to claim 1, characterized in that: In the steps (2) and (3), the conditions for ultrasonic extraction are: ultrasonic power 100-500W, extraction temperature 20-60°C, and extraction time 10-60min.

7. The medicine-food composition prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the medicine-food composition as claimed in claim 7 in the preparation of a preparation or medicine for alleviating or treating vaginitis.

9. The use according to claim 8, characterized in that The vaginitis includes bacterial vaginitis.

10. A preparation or medicine for relieving or treating vaginitis, characterized in that: Including the medicine-food composition as described in claim 7.

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