Preparation method and application of fresh dandelion extract

Abstract

The invention relates to a preparation method and application of a fresh dandelion extract, fresh dandelion is used as an initial raw material, the fresh dandelion extract is obtained by the steps of cleaning and selecting, cutting into sections, juicing, extracting decoction dregs, filtering, concentrating and drying, when the decoction dregs are extracted, an extraction solvent is added into the decoction dregs, meanwhile, an active agent Tween 20 is added, and ultrasonic extraction is adopted. Effective components in fresh dandelion medicinal materials can be reserved to the maximum extent, the active agent Tween 20 has an amphiphilic structure, micelles can be formed and have a solubilizing effect on the effective components of natural medicines, and the leaching efficiency and the extraction rate can be improved. Main active ingredients of the fresh dandelion extract are flavonoids and phenolic acids, and the contents of the flavonoids and the phenolic acids can reach 35% or above after purification.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a fresh dandelion extract and a preparation method and application thereof. Background Art

[0002] Dandelion is the dried whole herb of Taraxacum mongolicum Hand.-Mazz., Taraxacum borealisinense Kitam. or several plants of the same genus. This product is bitter, sweet and cold. It enters the liver and stomach meridians. It has the effects of clearing away heat and detoxification, reducing swelling and dispersing nodules, and promoting diuresis and relieving stranguria. Dandelion is rich in flavonoids, phenolic acids, sesquiterpenes, and coumarins. Studies have shown that its plant extracts and some monomer compounds have pharmacological activities such as antibacterial, antioxidant, liver protection and anti-tumor. The dandelions commonly used now are all dried dandelion slices, and the processing method is: dig up when the flowers first bloom from spring to autumn, remove impurities, wash, and dry. However, the process of making the medicine into dry medicine is easy to be contaminated and moldy, and the effective ingredients will also be lost in large quantities during the drying process. The clinical efficacy is lower than that of fresh dandelions. Dried dandelion medicinal materials often use a large amount of water as a solvent, and the non-polar effective ingredients in the medicinal materials are difficult to extract. The existing extraction method focuses on extracting a type of chemical component extraction, such as CN114699438A extracting total flavonoids from dandelions, but the extraction efficiency is limited, and only dry dandelions are extracted.

[0003] At present, in clinical practice, the use rate of anti-inflammatory drugs ranks second, second only to anti-infective drugs. There are anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs) on the market. For example, there are a series of adverse reactions in the long-term use of adrenocortical hormone drugs, so there are certain limitations in the use process. Fresh dandelion, as a natural plant, has stronger activity than dried, multi-target effect, and small toxic and side effects, and can be developed and applied as a new anti-inflammatory substance. The current method of extracting dry dandelion is not suitable for the treatment of fresh dandelion. The preparation of fresh dandelion extract is only juicing, which obtains less active ingredients and has low extraction efficiency. Summary of the invention

[0004] The present invention adopts fresh dandelions to prepare dry dandelions without drying, thus effectively reducing the loss of effective components, and the adopted extraction solvents can be recycled and reused, thus reducing environmental pollution. The present invention overcomes the shortcomings of large fresh drug dosage, poor preservation, seasonal influences, etc., and realizes convenient processing and effective extraction of fresh dandelions. Two types of components are extracted at the same time, and the disadvantage of extremely low extraction efficiency of active components of single juicing is overcome by juicing and residue extraction, thereby improving the extraction efficiency of effective components of fresh dandelions, preserving the effective active components in the fresh dandelions, and improving the purity of the active components.

[0005] The invention provides a preparation method of a fresh dandelion extract. The fresh dandelion is used as a starting material, and the method comprises the steps of cleaning, segmenting, juicing, extracting medicinal residues, filtering, concentrating and drying to obtain the fresh dandelion extract. When extracting the medicinal residues, an extraction solvent is added to the medicinal residues, and an active agent Tween 20 is added at the same time, and ultrasonic extraction is adopted.

[0006] Furthermore, when extracting the medicinal residue, the amount of the extraction solvent added is 1 to 10 times the volume of the medicinal residue; the amount of the active agent Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL; when ultrasonic extraction is used, stirring is performed for 10 to 30 minutes, ultrasonic extraction is performed for 10 to 60 minutes, the remaining medicinal residue is then added with the extraction solvent and ultrasonic extraction is repeated 1 to 4 times, all fresh juices are combined, and filtered with a 40 to 120 mesh sieve to obtain fresh dandelion juice;

[0007] Preferably, the extraction solvent is alcohol or water, more preferably, the extraction solvent is ethanol, and even more preferably, the extraction solvent is ethanol with a concentration of 40 to 70%.

[0008] Preferably, the cutting is into 2-10 cm pieces.

[0009] Furthermore, during the concentration and drying, the fresh juice is concentrated under reduced pressure to 10% to 15% of the volume before extraction to form a concentrated product, and the concentrated product is dried to obtain a fresh dandelion extract, the main active component flavonoids content of which reaches more than 25%, and the phenolic acid content reaches more than 20%.

[0010] Furthermore, the method further comprises purification, wherein the concentrated product is purified by using XDA-8, D101 or AB-8 macroporous resin to purify the dandelion extract, the liquid after purification is concentrated under reduced pressure to form a purified concentrated product, the purified concentrated product is dried, crushed and sieved to obtain a purified fresh dandelion extract, wherein the content of flavonoids as the main active component reaches more than 35%, and the content of phenolic acids reaches more than 35%.

[0011] Furthermore, the reduced pressure concentration method is to concentrate under reduced pressure at 50-80°C to a concentrated product with a relative density of 1.05-1.35.

[0012] Furthermore, during the drying, the drying method used is freeze drying or spray drying;

[0013] The freeze drying is to freeze the concentrated product of fresh dandelion extract in a refrigerator at -20 to -80°C for 24 to 48 hours, and then freeze-dry it at a pressure of 0.08 to -0.09 MPa, a temperature of -40 to -60°C, and a freezing time of 48 to 72 hours;

[0014] The spray drying is to set the air inlet temperature of the concentrated product of fresh dandelion extract to 100-190°C and the air outlet temperature to 80-100°C.

[0015] The present invention also provides a fresh dandelion extract obtained by the above-mentioned preparation method, wherein the content of flavonoids as the main active component reaches more than 25%, and the content of phenolic acids reaches more than 20%.

[0016] The present invention also provides a fresh dandelion medicinal composition, which comprises the extract prepared above or the extract described above.

[0017] The present invention also provides a use of the fresh dandelion extract prepared above or the extract described above in preparing anti-inflammatory drugs, health foods or cosmetics.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] The invention can retain the active ingredients in the fresh dandelion medicinal material to the maximum extent, and the active agent Tween 20 has an amphiphilic structure and can form micelles to solubilize the active ingredients of natural medicines, thereby improving the leaching efficiency and extraction rate. The addition of the active agent can reduce the surface tension, make the medicinal material easy to be infiltrated, thereby improving the efficiency of the solvent penetrating into the interior of the cell, and the active ingredients are also easier to diffuse and dissolve in the solvent, which can greatly improve the extraction efficiency of the active ingredients, and improve the purity of the active ingredients. At the same time, the solvent can be recycled and reused, reducing waste. The main active ingredients of the fresh dandelion extract are flavonoids and phenolic acids, and the content of flavonoids and phenolic acids after purification can reach more than 35%. The main active ingredients include caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin, and luteolin glycosides, and have significant anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of fresh dandelion extract on inflammatory response of RAW264.7 cells induced by lipopolysaccharide (LPS), (A) NO, (B) IL-1β, (C) TNF-α levels, (###p<0.001vs control group; **p<0.01 and ***p<0.001vs LPS group);

[0021] Figure 2 Effects of fresh dandelion extract on LPS-induced inflammatory response in EPH4-EV cells, (A) iNOS, (B) COX2, (C) TNF-α levels; (###P<0.001vs control group; **P<0.01 and ***P<0.001vs LPS group);

[0022] Figure 3 Effect of fresh dandelion extract on histopathological changes of LPS-induced mastitis in mice (scale bar: 100 μm), (A) control group, (B) LPS group, (CE) LPS modeling + FDE (200 mg / kg, 400 mg / kg and 800 mg / kg) group, (F) mammary tissue pathological histological score. (### indicates P<0.001 compared with the control group, *P<0.05, ***P<0.001 indicates P<0.05, P<0.001 compared with the LPS modeling group, LPS: lipopolysaccharide; CON: no treatment group);

[0023] Figure 4 Effects of fresh dandelion extract on pathological changes of DSS-induced colitis in mice (scale bar: 100 μm), (A) control group, (B) DSS group, (CE) DSS modeling + FDE (125 mg / kg, 250 mg / kg and 500 mg / kg) group, (F) pathological histological scores of mouse colon tissue, (### indicates P<0.001 compared with the control group, *P<0.05, ***P<0.001 indicates P<0.05, P<0.001 compared with the LPS modeling group, DSS: dextran sulfate sodium; CON: no treatment group);

[0024] Figure 5 Effects of fresh dandelion extract and purified fresh dandelion extract on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells, (A) NO, (B) IL-1β, and (C) TNF-α levels (###p<0.001vs control group; **p<0.01 and ***p<0.001vs LPS group).

[0025] Specific implementation method

[0026] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0027] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0028] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0029] Example 1

[0030] This embodiment provides a method for preparing a fresh dandelion extract, which uses fresh dandelions as a starting material, including cleaning, cutting, juicing, extracting medicinal residues, filtering, concentrating, and drying to obtain the fresh dandelion extract, specifically comprising the following steps:

[0031] 1) Collecting fresh whole dandelions (including their roots) during the spring and autumn harvesting period, removing impurities, cleaning, washing, cutting into small pieces, squeezing them while they are fresh with an extrusion press, filtering to obtain fresh juice, and separating the fresh juice and the medicinal residue;

[0032] 2) Adding the extraction solvent to the above-mentioned medicinal residues, adding the active agent Tween 20 at the same time, stirring, ultrasonic extraction, filtering with a filter press to obtain juice, adding the extraction solvent to the remaining medicinal residues to repeat the ultrasonic extraction, and then squeezing to obtain juice, combining all the fresh juices, filtering, and obtaining fresh dandelion juice.

[0033] 3) The fresh dandelion juice is concentrated to 10-15% of the volume before extraction to form a concentrated product, and the concentrated product is dried to obtain a fresh dandelion extract.

[0034] As a further preferred embodiment, the extraction solvent is ethanol or water, more preferably, the extraction solvent is ethanol, further preferably ethanol with a concentration of 40-70%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70% or any value between any two values.

[0035] The amount added is 1 to 10 times of the medicinal residue, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or any value between any two values.

[0036] The amount of active agent Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL, such as 0.01, 0.02, 0.03, 0.04, 0.05 or any value between any two values.

[0037] Preferably, during ultrasonic extraction, stirring is performed for 10 to 30 min, and ultrasonic extraction is performed for 10 to 60 min, for example, 10, 20, 30, 40, 50, 60 min or any value between any two values.

[0038] The remaining medicinal residue is then added with an extraction solvent and ultrasonic extraction is repeated for 1 to 4 times, more preferably 2 times. All the fresh juices are combined and filtered through a 40-120 mesh sieve, for example, a 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 mesh sieve or any value between any two values ​​to obtain fresh dandelion juice.

[0039] The present invention adopts an ultrasonic extraction method to extract fresh dandelions, which can effectively avoid the problems of a long preparation cycle of a traditional extraction and purification process, a large loss of active ingredients from fresh products to drying, incomplete extraction of active ingredients, and easy oxidation and degradation of active ingredients, and obtain a fresh dandelion extract with a high content of effective substances at a lower temperature and a shorter extraction cycle.

[0040] As a further preferred embodiment, the cutting is into segments of 2 to 10 cm, more preferably 3 to 5 cm, without crushing.

[0041] As a further preferred embodiment, during the concentration and drying, the fresh juice is concentrated under reduced pressure to 10-15% of the volume before extraction to form a concentrated product, and the concentrated product is dried to obtain a fresh dandelion extract, wherein the content of flavonoids as the main active components reaches more than 25%, and the content of phenolic acids reaches more than 20%.

[0042] As a further preferred embodiment, the preparation method further comprises purification, wherein the concentrated product is purified by using a macroporous resin to purify the fresh dandelion extract, the liquid after purification is concentrated under reduced pressure to form a purified concentrated product, the purified concentrated product is dried, crushed, and sieved to obtain a purified fresh dandelion extract, wherein the content of flavonoids as the main active component reaches more than 35%, and the content of phenolic acids reaches more than 35%.

[0043] Preferably, during purification, a macroporous resin method including XDA-8, D101 or AB-8 is used.

[0044] Preferably, the concentrated product may be a concentrated liquid or a concentrated extract.

[0045] Preferably, the purified fresh dandelion extract is in powder form.

[0046] As a further preferred embodiment, the reduced pressure concentration method is carried out at 50-80°C, more preferably 70°C, to concentrate the product under reduced pressure to a relative density of 1.05-1.35, for example 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35.

[0047] As a further preferred embodiment, during the drying, the drying method used is freeze drying or spray drying;

[0048] Preferably, the freeze drying is to freeze the concentrated product of fresh dandelion extract in a -20 to -80°C refrigerator for 24 to 48 hours, and then freeze-dry it at a pressure of 0.08 to -0.09 MPa, a temperature of -40 to -60°C, and a freezing time of 48 to 72 hours;

[0049] Preferably, the spray drying is to set the air inlet temperature of the concentrated fresh dandelion extract product to 100-190°C and the air outlet temperature to 80-100°C.

[0050] Preferably, after drying, the mixture is sieved through a 80-140 mesh sieve and vacuum packaged.

[0051] Example 2

[0052] This example provides a dandelion extract obtained by the preparation method described above, wherein the content of flavonoids as the main active component reaches more than 25%, and the content of phenolic acids reaches more than 20%; preferably, after the dandelion extract is purified, the content of flavonoids as the main active component reaches more than 35%, and the content of phenolic acids reaches more than 35%.

[0053] The invention uses high performance liquid phase tandem mass spectrometry to identify that the fresh dandelion extract contains 285 compounds, including 74 phenolic acids and 73 flavonoids. The fresh dandelion extraction method of the invention can make the flavonoid extraction rate reach more than 5%, and the phenolic acid extraction rate reach more than 15%. The total flavonoid and total phenolic acid contents are respectively more than 25% and 20%, and the flavonoid and phenolic acid contents can reach more than 35% after purification.

[0054] The flavonoids include luteolin, dendrobium, rutin, and luteolin; the phenolic acids include caffeic acid, chlorogenic acid, p-coumaric acid, and chicoric acid.

[0055] The fresh dandelion extract extraction method of the invention can obtain compounds rich in flavonoids and phenolic acids.

[0056] A fresh dandelion medicinal composition comprises the extract prepared above or the extract described above.

[0057] A use of the fresh dandelion extract as described above in preparing anti-inflammatory drugs, health foods or cosmetics.

[0058] Inflammation is a series of protective immune responses produced when the host system is stimulated by damaging factors (such as pathogens, damaged cells or other stimuli, etc.), affecting the body's microenvironment to participate in the regulation of various physiological and pathological processes of the body. Under normal circumstances, inflammation can eliminate harmful stimuli, initiate the healing process and promote repair, but the continued existence of inflammation is harmful to its organs and systemic reactions. Excessive inflammation can destroy the homeostasis of normal tissues. For example, the inflammatory factor storm caused by excessive inflammation can cause multiple organ failure and even death in severe cases. Studies have found that inflammation plays an important role in the occurrence and development of many complex diseases, such as obesity, cardiovascular disease, chronic respiratory disease, rheumatoid arthritis, and cancer. Therefore, it is very important to inhibit disordered or excessive inflammation. At present, in clinical practice, there are anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs on the market, which have a series of adverse reactions during long-term use, and therefore have certain limitations during use. The fresh dandelion of the present invention, as a natural plant, has stronger activity than that after drying, multi-target effects, and small toxic and side effects. The main effective ingredients include caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin, and luteolin, and have significant anti-inflammatory activity.

[0059] The fresh dandelion extract of the present invention can significantly reduce the release of inflammatory factor NO and the expression of inflammatory factors TNF-α and IL-1β of RAW264.7 (mouse macrophages) induced by LPS solution, and has significant anti-inflammatory effect.

[0060] The dandelion extract of the present invention can significantly reduce the expression levels of TNF-α, IL-1β and IL-6 of mouse mammary epithelial cells EPh4v induced by LPS, and has significant anti-inflammatory effect.

[0061] The fresh dandelion extract of the invention can improve mouse mastitis induced by LPS solution and reduce the inflammatory cell infiltration of the mammary gland.

[0062] The fresh dandelion extract of the present invention can inhibit the ear swelling rate of mice in a xylene-induced mouse ear swelling model and has high anti-inflammatory activity.

[0063] The purified fresh dandelion extract of the invention can improve DSS-induced colitis in mice and has high anti-inflammatory activity.

[0064] The fresh dandelion extract or the purified fresh dandelion extract of the invention is used in the fields of food, health care products and medicine.

[0065] The preparation method of fresh dandelion extract of the present invention is simple and efficient, and the extract produced is better than the juicing method, and the content of active ingredients is high. The prior art is only for the extraction of active ingredients of dry dandelions, and focuses on the crude extraction of dry dandelions or a certain class of compounds, such as the extraction of flavonoids alone. The present application uses fresh dandelions as raw materials, and does not need to be processed and processed to make dandelion slices after direct harvesting, which avoids the easy pollution and mildew that may occur during the processing process, as well as the possible large-scale loss and change of active ingredients during the drying process, improves the effect, and adopts the squeezing process to extract fresh juice, and combines the solvent and ultrasonic extraction process on the residue, and adds the active agent Tween 20 at the same time, and the fresh dandelion extract prepared realizes the convenient processing and effective extraction of fresh dandelions, and the extraction efficiency is higher, the content of active ingredients is higher, and the activity is better. And the present application also finds that after the fresh dandelion extract is purified, its anti-inflammatory effect is remarkable.

[0066] The fresh dandelion extract prepared by the present invention is a natural ingredient directly extracted from fresh plants, is rich in flavonoids and phenolic acid substances, and contains main chemical components such as caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin and luteolin after purification, which can reduce the expression of inflammatory factors and inhibit inflammatory reactions, thus providing a new method for treating inflammation.

[0067] The fresh dandelion extract of the invention uses fresh dandelion as raw material, the raw material is widely available and easy to process, and in addition to the juicing process, an active agent Tween 20 is added, and a solvent and ultrasonic extraction are combined, so that the extraction rate of effective components can be greatly improved, while the use amount of the solvent is reduced, and the solvent can be reused, and is widely used in health care products, foods and medicines, and has strong market competitiveness.

[0068] Example 3

[0069] According to the content of this application, the fresh dandelion extract of this application is specifically described. If no specific experimental steps or conditions are specified in the embodiments, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be purchased commercially.

[0070] The present invention is further described in detail below in conjunction with specific examples, which should not be construed as limiting the scope of the present invention. Among them, the water content of fresh dandelions is inconsistent, and the amount of juice after juicing is not certain, but it can be controlled within ±10%.

[0071] Test Example 1

[0072] Take 1000g of freshly collected dandelions, wash with water to remove sediment, clean and cut into 7cm small segments, and squeeze juice with an extrusion press. The obtained 780g of medicinal residues are transferred to an extraction tank, 2 volumes of 60% ethanol are added, and then the active agent Tween 20 is added at a dosage of 0.03g / mL, stirred evenly and ultrasonically extracted for 30min, and the juice is squeezed by a filter press, and the extraction is repeated twice, all fresh juices are combined, filtered with an 80-mesh sieve, and then concentrated to 290mL under reduced pressure in a 70°C water bath (equivalent to a relative density of 1.21 for the concentrated solution), and the fresh dandelion juice is concentrated to no ethanol taste to obtain a fresh dandelion concentrate, which is directly dried for 36h to obtain 238g of fresh dandelion extract, with a total flavonoid content of 27.1% and a total phenolic acid content of 25.4%.

[0073] Detection of the content of main chemical components of fresh dandelion extract: The detection conditions are as follows: the chromatographic column is a Waters Xbridgeshield PR C18 chromatographic column (4.6mm×250mm, 5μm), the mobile phase A phase is 0.2% formic acid water, the mobile phase B phase is acetonitrile, the detection wavelength is 330nm, the detection temperature is 35°C, the gradient elution, and the elution program is as follows (0-12min, 14%-14% B; 12-13min, 14%-18% B; 13-23min, 18%-20% B; 23-28min, 20%-40% B; 28-38min, 40%-60% B; 38-43min, 60%-14% B; 43-48min, 14%-1 4% B) injection volume 10 μL; flow rate 1.0 mL / min; column temperature 30°C; detection wavelength 330 nm, the content was calculated by external standard method, and the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, calcein and rutin in dandelion extract were obtained, wherein the content of chlorogenic acid was 3.04 mg / g, the content of caffeic acid was 10.34 mg / g, the content of luteolin was 1.32 mg / g, the content of chicoric acid was 9.82 mg / g, the content of luteolin was 2.28 mg / g, the content of calcein was 1.27 mg / g, and the content of rutin was 1.49 mg / g.

[0074] Test Example 2

[0075] Take 1000g of freshly collected dandelions, wash with water to remove the sediment, clean and cut into 5cm small segments, and squeeze the juice with an extrusion press. The obtained 785g of medicinal residues are transferred to an extraction tank, 5 times the volume of 40% ethanol is added, and then the active agent Tween 20 is added at a dosage of 0.01g / mL, stirred evenly and ultrasonically extracted for 20min, and the juice is squeezed by a filter press, and the extraction is repeated 3 times, all the fresh juices are combined, filtered with a 100-mesh sieve, and then concentrated under reduced pressure in a 50°C water bath to a relative density of 1.25. The fresh dandelion juice is concentrated to no ethanol taste, and a fresh dandelion concentrate is obtained, which is directly dried for 48h to obtain 245g of fresh dandelion extract, with a total flavonoid content of 26.3% and a total phenolic acid of 21.2%.

[0076] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were as follows: the chlorogenic acid content was 2.81 mg / g, the caffeic acid content was 7.31 mg / g, the luteolin content was 1.12 mg / g, the chicoric acid content was 7.13 mg / g, the luteolin content was 1.78 mg / g, the dendrobium content was 0.78 mg / g and the rutin content was 0.96 mg / g.

[0077] Test Example 3

[0078] Take 1000g of freshly collected dandelions, wash with water to remove the sediment, clean and cut into 3cm small segments, and squeeze the juice with an extrusion press. The obtained 815g of medicinal residues are transferred to an extraction tank, 7 times the volume of 70% ethanol is added, and then the active agent Tween 20 is added at a dosage of 0.05g / mL, stirred evenly and ultrasonically extracted for 50min, and the juice is squeezed by a filter press, and the extraction is repeated twice, all the fresh juices are combined, filtered with a 120-mesh sieve, and then concentrated under reduced pressure in a water bath at 80°C to a relative density of 1.12. The fresh dandelion juice is concentrated to no ethanol taste, and a fresh dandelion concentrate is obtained, which is directly dried for 36h to obtain 228g of fresh dandelion extract, with a total flavonoid content of 25.3% and a total phenolic acid of 20.7%.

[0079] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were as follows: the chlorogenic acid content was 2.12 mg / g, the caffeic acid content was 5.15 mg / g, the luteolin content was 0.98 mg / g, the chicoric acid content was 5.65 mg / g, the luteolin content was 1.58 mg / g, the dendrobium content was 0.65 mg / g and the rutin content was 0.55 mg / g.

[0080] Test Example 4

[0081] Take 1000g of freshly collected dandelions, wash them with water to remove mud and sand, clean them, cut them into 7cm small sections, and use an extrusion press to squeeze out the juice. The obtained 800g of the medicinal residue is transferred to an extraction tank, 2 times the volume of 60% ethanol is added, and then an active agent Tween 20 is added according to the dosage of 0.03g / mL, and the mixture is stirred evenly and ultrasonically extracted for 30min. The juice is squeezed by a filter press, and the extraction is repeated twice. All the fresh juices are combined, filtered with an 80-mesh sieve, and then concentrated to 290mL under reduced pressure in a 70°C water bath. The fresh dandelion juice is concentrated to have no ethanol taste, and a fresh dandelion concentrate is obtained; the obtained concentrate is purified by a D101 macroporous resin, the eluent is 70% ethanol, the eluent is concentrated to a relative density of 1.25, the concentrated fresh juice is frozen in a -80°C refrigerator for 24h, the frozen product is taken out, freeze-dried at a pressure of 0.08MPa and a temperature of -50°C for 36h, the dried product is crushed, passed through an 80-mesh sieve, and vacuum-packed to obtain 107g of a purified fresh dandelion extract with a total flavonoid content of 40.12% and a total phenolic acid content of 38.05%.

[0082] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were 7.51 mg / g for chlorogenic acid, 17.78 mg / g for caffeic acid, 2.07 mg / g for luteolin, 14.72 mg / g for chicoric acid, 3.53 mg / g for luteolin, 2.84 mg / g for dendrobium and 2.04 mg / g for rutin.

[0083] Test Example 5

[0084] Take 1000g of freshly collected fresh dandelions, wash with water to remove sediment, clean and cut into 7cm small segments, and squeeze juice with an extrusion press. The obtained 820g of medicinal residues are transferred to an extraction tank, 2 volumes of 60% ethanol are added, and then the amount of active agent Tween 20 is added in an amount of 0.02g / mL, stirred evenly and ultrasonically extracted for 30min, and the filter press is used to squeeze juice, and the extraction is repeated twice, all fresh juices are combined, filtered with a 100-mesh sieve, and then concentrated to 290mL under reduced pressure in a 70°C water bath, and the fresh dandelion juice is concentrated to no ethanol taste to obtain a fresh dandelion concentrate; the obtained concentrate is purified with XDA-8 macroporous resin, the eluent is 70% ethanol, the eluent is concentrated to a relative density of 1.20, and the concentrate is spray-dried, and the air inlet temperature is set to 130°C and the air outlet temperature is set to 90°C. The dried product was crushed, passed through an 80-mesh sieve, and vacuum-packed to obtain 100 g of purified fresh dandelion extract with a total flavonoid content of 37.82% and a total phenolic acid content of 35.52%.

[0085] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were as follows: the chlorogenic acid content was 6.12 mg / g, the caffeic acid content was 14.12 mg / g, the luteolin content was 1.77 mg / g, the chicoric acid content was 11.31 mg / g, the luteolin content was 1.23 mg / g, the dendrobium content was 2.74 mg / g and the rutin content was 1.41 mg / g.

[0086] Comparative Example 1

[0087] Take 1000g of freshly collected dandelions, wash with water to remove the sediment, clean and cut into 7cm small segments, and squeeze the juice with an extrusion press. The obtained 800g of the residue is transferred to an extraction tank, and 2 volumes of 60% ethanol are added, and ultrasonic extraction is performed for 30min with stirring, and the juice is squeezed by a filter press, and the extraction is repeated, and all the fresh juices are combined, and concentrated to 250mL under reduced pressure in a 70℃ water bath, and concentrated to a relative density of 1.15, and the fresh dandelion juice is concentrated to no ethanol taste, and a fresh dandelion concentrate is obtained, which is directly dried for 36h to obtain 244g of fresh dandelion extract, with a total flavonoid content of 17.5% and a total phenolic acid content of 15%.

[0088] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were as follows: the chlorogenic acid content was 1.13 mg / g, the caffeic acid content was 3.26 mg / g, the luteolin content was 0.54 mg / g, the chicoric acid content was 3.52 mg / g, the luteolin content was 0.97 mg / g, the dendrobium content was 0.43 mg / g and the rutin content was 0.25 mg / g.

[0089] Comparative Example 2

[0090] Take 1000g of freshly collected dandelion, wash with water to remove sediment, clean and cut into 7cm small segments, and squeeze juice with an extrusion press. The obtained 840g of dregs are transferred to an extraction tank, 2 volumes of 60% ethanol are added, ultrasonic extraction is performed for 30min, and juice is squeezed with a filter press, and the extraction is repeated, and all fresh juices are combined, and concentrated to 250mL in a 70℃ water bath under reduced pressure. The obtained concentrate is purified with D101 macroporous resin, and the eluent is 80% ethanol. The eluent is concentrated to a relative density of 1.27, and the concentrated fresh juice is frozen in a -80℃ refrigerator for 24h, and the frozen product is taken out, and freeze-dried at a pressure of 0.08MPa and a temperature of -50℃ for 36h. The dried product is crushed, passed through an 80-mesh sieve, and vacuum-packed to obtain 72g of fresh dandelion purified extract, with a total flavonoid content of 22.24% and a total phenolic acid content of 15.48%.

[0091] Detection of the contents of main chemical components of fresh dandelion extract: The detection conditions were the same as those in Test Example 1. The detection results showed that the contents of chlorogenic acid, caffeic acid, luteolin, chicoric acid, luteolin, dendrobium and rutin in dandelion extract were as follows: the chlorogenic acid content was 1.76 mg / g, the caffeic acid content was 4.35 mg / g, the luteolin content was 0.48 mg / g, the chicoric acid content was 4.86 mg / g, the luteolin content was 1.49 mg / g, the dendrobium content was 0.60 mg / g and the rutin content was 0.50 mg / g.

[0092] Experimental Example 1 Fresh dandelion extract inhibits the inflammatory response of RAW264.7 mouse macrophages induced by LPS solution.

[0093] RAW264.7 mouse macrophages were inoculated in a 6-well plate and incubated in a constant temperature incubator at 37°C and 5% CO2 until the cell density reached 80% to 90%. FDE (fresh dandelion extract): The extract in Experimental Example 1 was used. The preparation method was to weigh 10 mg of the fresh dandelion extract in Experimental Example 1, dissolve it in 10 mL of PBS, filter it with a sterile membrane, and prepare it with a culture medium to a corresponding concentration, and then treat it as follows: LPS+FDE group (which was divided into 3 groups, 25, 50, 1 00 group), Control group, LPS group, and DEX group; among them, 1 mL of FDE with a concentration of 25, 50, and 100 μg / mL was added to the LPS+FDE group, and 1 mL of culture medium was added to the other groups. After pre-incubation for 1 hour, the culture medium was discarded, and a mixed culture medium with an LPS concentration of 1.0 μg / mL and an FDE concentration of 25, 50, and 100 μg / mL was added to the LPS+FDE group, a mixed culture medium containing 1 μg / mL dexamethasone + 1.0 μg / mL LPS was added to the DEX group, a culture medium with an LPS concentration of 1.0 μg / mL was added to the LPS group, and an equal amount of culture medium was added to the Control group. All of them were cultured for 6 hours, and the NO content was detected using a NO detection kit (Biyuntian) and the total RNA was extracted and then the expression of TNF-α and IL-1β was detected using qPCR. Figure 1 .

[0094] Experimental Example 2 Fresh dandelion extract inhibits LPS-induced inflammatory response in the mammary epithelium of EPH4-EV mice.

[0095] Culture EPH4-EV mouse mammary epithelial cells were inoculated in a 6-well plate and incubated in a constant temperature incubator at 37°C and 5% CO2 until the cell density reached 80% to 90%. FDE (fresh dandelion extract): The extract in Experimental Example 1 was used. The preparation method was to weigh 10 mg of the fresh dandelion extract in Experimental Example 1, dissolve it in 10 mL of PBS, filter it with a sterile membrane, and configure it to the corresponding concentration with culture medium. The treatment method was the same as that in Experimental Example 1, and LPS+FDE group (which was divided into 3 groups, namely 25, 50, and 100 groups), LPS group, DEX group, and control group were set up respectively; finally, they were all cultured for 12 hours, and the expression of iNOS, COX-2, and TNF-α was detected by qPCR after total RNA extraction. Figure 2 .

[0096] Experimental Example 3: Dandelion can inhibit the ear swelling rate of mice in the xylene-induced mouse ear swelling model and has high anti-inflammatory activity.

[0097] Xylene causes mouse auricle swelling test: 30 mice were randomly divided into 5 groups. Group A was a blank control group, which gave 0.2mL / 20g normal saline, and groups B, C, and D were experimental groups, which gave fresh dandelion extract solution (using fresh dandelion extract in test example 4, preparation method weighed fresh dandelion extract 10mg in test example 4, dissolved in 10mL PBS, aseptic membrane filtration) 0.1mL / 20g, 0.2mL / 20g and 0.4mL / 20g, respectively, for continuous administration for 5d. Group E was a positive drug control group, which was given 0.2mL / 20g dexamethasone injection at a disposable rate. The last administration was 1h, and the right auricle of mice was coated with xylene to cause inflammation. After 1h, mice were put to death, and ears were punched with a puncher with a diameter of 9mm at the same position, weighed and recorded. As shown in Table 1, there was no significant difference in the low-dose group compared with the blank control group, while the ear swelling of the high-dose group and the medium-dose group was significantly reduced compared with the blank control group. The flavonoids of fresh dandelion extract can inhibit the ear swelling of mice, and the inhibitory effect is dose-dependent.

[0098] Table 1 Effect of fresh dandelion extract on xylene-induced ear swelling in mice

[0099]

[0100] Experimental Example 4 Fresh dandelion extract alleviates LPS-induced mastitis in mice

[0101] A mouse mastitis model was established by intramammary infusion of LPS. Before modeling, 48 female BALB / c mice were divided into 6 groups, with 8 mice in each group, including a blank control group (CON): given solvent by gavage; a mastitis model (LPS) group: intramammary infusion of LPS; a fresh dandelion extract group: FDE-L (the extract in Experimental Example 1), FDE-M (the extract in Experimental Example 5), and FDE-H (the extract in Experimental Example 4), each of which was orally gavaged at 400 mg / kg for 7 consecutive days; a dexamethasone group (DEX group), in which mice were given an intraperitoneal injection of dexamethasone at 10 mg / kg 12 h and 1 h before modeling; except for the blank control group, the other groups were required to be intramammary infused with LPS (50 μL, concentration of 0.2 mg / mL); and the mammary pathological sections of the mice were observed. Figure 3 As shown, compared with the LPS group, FDE administration can significantly reduce the damage of pathological tissues. It can be seen that the degree of inflammatory cell infiltration gradually decreases with the increase of concentration, and the swollen alveolar wall structure gradually returns to normal.

[0102] Experimental Example 5: Purified fresh dandelion extract alleviates and improves DSS-induced colitis in mice.

[0103] After 1 week of adaptive feeding, 48 C57BL / 6 mice were divided into 7 groups, 8 mice in each group, namely, blank group (CON), DSS model group (3% DSS), positive drug group (mesalazine MS), FDE (the extract in Test Example 4) high, medium and low dose groups (FDE-C, FDE-B, FDE-A). According to the literature and preliminary experimental results, the high dose of fresh dandelion extract (FDE-C) was 500 mg kg -1 The medium dose (FDE-B) is 250 mg kg -1 , low dose (FDE-A) is 125 mg kg -1 The dose of mesalazine is 10 mg kg -1 Except for the blank group, which was free to eat, the mice in other groups drank 3% DSS freely for 7 days and were given corresponding doses by gavage for 7 days. The mice in other groups were given distilled water by gavage and inhibitors were injected intraperitoneally once a day. At the end of the experiment, the mice were killed and the colon tissues were fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining. Figure 4 There was no histopathological damage in the blank group, the colon mucosa was intact, the goblet cells were neatly arranged, and the crypts were intact; in the model group, there was a large amount of inflammatory cell infiltration and bleeding in the mucosa and submucosal layer, the goblet cells were significantly reduced, and the crypts were missing; after drug treatment, the colon mucosal damage in each group was significantly improved, the goblet cells increased, the crypts were basically intact, and only a small amount of inflammatory cell infiltration was present.

[0104] Experimental Example 6

[0105] FDEa group: the extract in Experimental Example 1 was used; FDEb group used the extract in Experimental Example 4. The preparation method and other experimental conditions were the same as those in Experimental Example 1. The two groups were respectively set up with LPS+FDE group (which was divided into 2 groups, 25 and 100 groups, respectively), blank control group (Control group), LPS group, and positive drug (DEX) group; 1 mL of FDEa (the extract in Experimental Example 1) with a concentration of 25 and 100 μg / mL was added to the FDEa group, and 1 mL of FDEb (the extract in Experimental Example 4) with a concentration of 25 and 100 μg / mL was added to the FDEb group, and 1 mL of culture medium was added to the other groups. After pre-incubation for 1 hour, the culture medium was discarded, and the FDE group was added with a mixed culture medium with a concentration of 1.0 μg / mL LPS and the corresponding FDE concentrations of 25 and 100 μg / mL, respectively, and the DEX group was added with a concentration of 1 μg / mL dexamethasone + 1.0 μg / mL The LPS mixed culture medium was added with LPS concentration of 1.0 μg / mL, and the control group was added with the same amount of culture medium. After 6 h of culture, the NO detection kit was used to detect the NO content, and the total RNA was extracted and the qPCR was used to detect the expression of TNF-α and IL-1β. Figure 5 .

[0106] In the experimental example, the effect of the fresh dandelion extract group became more and more significant with the increase of FDE concentration, and finally approached the effect of the DEX group, indicating that FDE has a significant anti-inflammatory effect, and the effect is better with the increase of concentration and purity. Since hormone drugs have a series of adverse reactions during long-term use, they have certain limitations in their use. Fresh dandelion, as a natural plant, has stronger activity than dried, multi-target effects, and less toxic side effects. It can be developed and applied as a new anti-inflammatory substance.

[0107] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a fresh dandelion extract, which uses fresh dandelions as starting materials, and comprises cleaning, cutting, juicing, extracting medicinal residues, filtering, concentrating, and drying to obtain the fresh dandelion extract, characterized in that: When extracting the medicinal residues, the medicinal residues are added with an extraction solvent and an active agent Tween 20, and ultrasonic extraction is adopted.

2. The preparation method according to claim 1, characterized in that: When extracting the medicinal residue, the amount of the extraction solvent added is 1 to 10 times the volume of the medicinal residue; the amount of the active agent Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL; when ultrasonic extraction is used, stirring is performed for 10 to 30 minutes, ultrasonic extraction is performed for 10 to 60 minutes, and the remaining medicinal residue is then added with the extraction solvent to repeat ultrasonic extraction for 1 to 4 times, and all fresh juices are combined and filtered with a 40 to 120 mesh sieve to obtain fresh dandelion juice; Preferably, the cutting is into 2-10 cm pieces.

3. The preparation method according to claim 1, characterized in that: The extraction solvent is ethanol or water. Preferably, the extraction solvent is ethanol with a concentration of 40-70%.

4. The preparation method according to claim 1, characterized in that: During the concentration and drying, the fresh juice is concentrated under reduced pressure to 10% to 15% of the volume before extraction to form a concentrated product, and the concentrated product is dried to obtain a fresh dandelion extract, the main active component flavonoids content of which reaches more than 25%, and the phenolic acid content reaches more than 20%.

5. The preparation method according to claim 1, characterized in that: The method also includes purification, wherein the concentrated product is purified by using XDA-8, D101 or AB-8 macroporous resin to purify the dandelion extract, the liquid after purification is concentrated under reduced pressure to form a purified concentrated product, the purified concentrated product is dried, crushed and sieved to obtain a purified fresh dandelion extract, wherein the content of flavonoids, the main active component, reaches more than 35%, and the content of phenolic acids reaches more than 35%.

6. The preparation method according to claim 4 or 5, characterized in that: The reduced pressure concentration method is to concentrate under reduced pressure at 50-80° C. to obtain a concentrated product with a relative density of 1.05-1.

35.

7. The preparation method according to claim 1, characterized in that: During the drying, the drying method used is freeze drying or spray drying; The freeze drying is to freeze the concentrated product of fresh dandelion extract in a refrigerator at -20 to -80°C for 24 to 48 hours, and then freeze-dry it at a pressure of 0.08 to -0.09 MPa, a temperature of -40 to -60°C, and a freezing time of 48 to 72 hours; The spray drying is to set the air inlet temperature of the concentrated product of fresh dandelion extract to 100-190°C and the air outlet temperature to 80-100°C.

8. A fresh dandelion extract obtained by the preparation method according to any one of claims 1 to 7, wherein the content of flavonoids as the main active components reaches more than 25%, and the content of phenolic acids reaches more than 20%.

9. A fresh dandelion medicinal composition, characterized in that: It comprises the extract prepared by any one of claims 1 to 7 or the extract according to claim 8.

10. Use of the fresh dandelion extract prepared according to any one of claims 1 to 7 or the extract according to claim 8 in the preparation of anti-inflammatory drugs, health foods or cosmetics.

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