Preparation method and application of fresh dandelion extract

Abstract

The present application relates to a kind of preparation method and application of fresh dandelion extract, with fresh dandelion as starting material, including net selection, cutting section, juice extraction, dregs extraction, filtration, concentration, drying to obtain fresh dandelion extract, when the dregs extraction, the dregs are added to extraction solvent, active agent tween 20 is added simultaneously, and ultrasonic extraction is adopted.The present application can maximize the effective components in fresh dandelion medicinal materials, active agent tween 20 has amphiphilic structure and can form micelle, which has solubilizing effect on natural medicine effective components, can improve leaching efficiency and extraction rate.The main active ingredients of the fresh dandelion extract are flavonoids and phenolic acids, and after purification, the content of flavonoids and phenolic acids can reach more than 35%.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fresh dandelion extract, its preparation method, and its application. Background Technology

[0002] Dandelion is the dried whole herb of *Taraxacum mongolicum* Hand.-Mazz., *Taraxacum borealisinense* Kitam., or several other species in the same genus, belonging to the Asteraceae family. It is bitter, sweet, and cold in nature. It enters the liver and stomach meridians. It has the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting diuresis. Dandelion is rich in flavonoids, phenolic acids, sesquiterpenes, coumarins, and other chemical components. Studies have shown that its plant extracts and some monomeric compounds possess antibacterial, antioxidant, hepatoprotective, and antitumor pharmacological activities. Currently, the dandelion commonly used is dried dandelion slices. The processing method is as follows: harvesting from spring to autumn when the flowers first bloom, removing impurities, washing, and sun-drying. However, during the process of making the medicine into dried medicine, it is easy to be contaminated and moldy, and a large amount of effective ingredients are also lost during the drying process, resulting in lower clinical efficacy than fresh use. Dried dandelion medicine often uses a large amount of water as a solvent, making it difficult to extract the non-polar effective components in the medicine. Existing extraction methods focus on extracting one type of chemical component, such as CN114699438A for extracting total flavonoids from dandelion, which has limited extraction efficiency and only extracts from dried dandelion.

[0003] Currently, anti-inflammatory drugs rank second in clinical use, after anti-infective drugs. Commercially available anti-inflammatory drugs include non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs). However, long-term use of drugs like corticosteroids has resulted in a series of adverse reactions, thus limiting their application. Fresh dandelion, as a natural plant, has stronger activity, more multi-target effects, and fewer toxic side effects than dried dandelion, making it a potential candidate for development and application as a novel anti-inflammatory substance. Current methods for extracting dried dandelion are not suitable for processing fresh dandelion; the preparation of fresh dandelion extracts currently only involves juicing, yielding low levels of active ingredients and low extraction efficiency. Summary of the Invention

[0004] This invention utilizes fresh dandelion without drying it into dried dandelion, effectively reducing the loss of active ingredients. Furthermore, the extraction solvents used are recyclable and reusable, reducing environmental pollution. This invention overcomes the drawbacks of fresh dandelion, such as large quantities required, difficulty in preservation, and seasonality. It achieves convenient processing and effective extraction of fresh dandelion, simultaneously extracting two types of components. The use of juice extraction and residue extraction overcomes the extremely low extraction efficiency of active ingredients from single juice extraction, improving the extraction efficiency of active ingredients from fresh dandelion while preserving and increasing the purity of the active ingredients.

[0005] This invention provides a method for preparing fresh dandelion extract, using fresh dandelion as the starting material, including cleaning, cutting, juicing, extracting from the residue, filtering, concentrating, and drying to obtain fresh dandelion extract. During the residue extraction, the residue is added with an extraction solvent and the active agent Tween 20, and ultrasonic extraction is used.

[0006] Furthermore, during the extraction of the medicinal residue, the amount of extraction solvent added is 1 to 10 times the volume of the medicinal residue; the amount of activator Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL; when using ultrasonic extraction, stirring is performed for 10 to 30 min, ultrasonic extraction is performed for 10 to 60 min, and the remaining medicinal residue is added to the extraction solvent and ultrasonic extraction is repeated 1 to 4 times. All fresh juices are combined and filtered through a 40-120 mesh sieve to obtain fresh dandelion juice.

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

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

[0009] Furthermore, during the concentration and drying process, the fresh juice is concentrated under reduced pressure to 10%–15% of its original volume before extraction to form a concentrated product. The concentrated product is then dried to obtain fresh dandelion extract, in which the main active components, flavonoids, contain more than 25% and phenolic acids, contain more than 20%.

[0010] Furthermore, the process also includes purification. The concentrated product is purified using XDA-8, D101, or AB-8 macroporous resin to extract dandelion. After purification, the liquid is concentrated under reduced pressure to form a purified concentrated product. The purified concentrated product is dried, pulverized, and sieved to obtain a purified extract of fresh dandelion. The main active components, flavonoids, have a content of more than 35%, and phenolic acids have a content of more than 35%.

[0011] Furthermore, the vacuum concentration method described herein involves concentrating the product under reduced pressure at 50–80 °C to a relative density of 1.05–1.35.

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

[0013] The freeze-drying process involves freezing the concentrated extract of fresh dandelion in a freezer at -20 to -80 ℃ for 24 to 48 hours, followed by freeze-drying at a pressure of 0.08 to -0.09 MPa, a temperature of -40 to -60 ℃, and a freezing time of 48 to 72 hours.

[0014] The spray drying process involves setting the inlet temperature of the concentrated dandelion extract to 100–190 °C and the outlet temperature to 80–100 °C.

[0015] The present invention also provides a fresh dandelion extract, which is obtained by the preparation method described above, and the main active components, flavonoids, have a content of more than 25% and phenolic acids have a content of more than 20%.

[0016] The present invention also provides a fresh dandelion pharmaceutical composition comprising the extract prepared above or the extract described above.

[0017] The present invention also provides the application of the fresh dandelion extract prepared above or the extract described above in the preparation of anti-inflammatory drugs or cosmetics.

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

[0019] This invention can maximize the retention of active ingredients in fresh dandelion. The active agent Tween 20, with its amphiphilic structure, can form micelles that solubilize the active ingredients of natural drugs, thereby improving leaching efficiency and extraction rate. Adding the active agent reduces surface tension, making the medicinal material easier to wet, thus improving the efficiency of solvent penetration into cells. The active ingredients are also more easily diffused and dissolved in the solvent, significantly improving the extraction efficiency and purity of the active ingredients. Simultaneously, the solvent can be recycled and reused, reducing waste. The main active ingredients in the fresh dandelion extract are flavonoids and phenolic acids. After purification, the content of flavonoids and phenolic acids can reach over 35%. The main active ingredients include caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin, and luteolinoglycoside, exhibiting significant anti-inflammatory activity. Attached Figure Description

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

[0022] Figure 3 Effects of fresh dandelion extract on histopathological changes in 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) groups, (F) Histopathological scores of breast tissue. (### indicates P<0.001 compared with the control group, *P<0.05, ***P<0.001 indicates P<0.05 compared with the LPS modeling group, P<0.001, 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) groups, (F) histopathological scores of mouse colon tissue, (### indicates P<0.001 compared with the control group, *P<0.05, ***P<0.001 indicates P<0.05 compared with the LPS modeling group, P<0.001, DSS: sodium dextran sulfate; 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, (C) TNF-α levels (###p<0.001 vs control group; **p<0.01 and ***p<0.001 vs LPS group).

[0025] Specific implementation methods

[0026] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0028] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0029] Example 1

[0030] This embodiment provides a method for preparing fresh dandelion extract, using fresh dandelion as the starting material, including cleaning, cutting, juicing, extracting from the residue, filtering, concentrating, and drying to obtain fresh dandelion extract, specifically including the following steps:

[0031] 1) Collect fresh whole dandelion (including its roots) during the spring and autumn harvest seasons, remove impurities, clean, wash and cut into small pieces, press while fresh using an extrusion press, filter to obtain fresh juice, and separate the fresh juice from the dregs.

[0032] 2) Add the extraction solvent and the activator Tween 20 to the above-mentioned dregs, stir, extract with ultrasound, filter with a filter press to obtain juice, add the extraction solvent to the remaining dregs again for repeated ultrasound extraction, press to obtain juice, combine all fresh juice, filter, and obtain fresh dandelion juice.

[0033] 3) Concentrate the fresh dandelion juice to 10-15% of its original volume to form a concentrated product. Dry the concentrated product to obtain the fresh dandelion extract.

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

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

[0036] The amount of activator Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL, for example, 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 10–30 min, and ultrasonic extraction is performed for 10–60 min, for example, 10, 20, 30, 40, 50, 60 min or any value between any two values.

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

[0039] This invention employs ultrasonic extraction to extract fresh dandelion, which effectively avoids the problems of long preparation cycles, significant loss of active ingredients during the drying process from fresh product to finished product, incomplete extraction of active ingredients, and easy oxidation and degradation of active ingredients in traditional extraction and purification processes. It obtains fresh dandelion extract with high content of effective substances at lower temperatures and shorter extraction cycles.

[0040] As a further preferred embodiment, the segment is cut into 2-10 cm pieces, more preferably 3-5 cm pieces, without the need for crushing.

[0041] As a further preferred embodiment, during the concentration and drying process, the fresh juice is concentrated under reduced pressure to 10-15% of its original volume to form a concentrated product. The concentrated product is then dried to obtain a fresh dandelion extract, the main active components of which contain flavonoids at a content of more than 25% and phenolic acids at a content of more than 20%.

[0042] As a further preferred embodiment, the above preparation method also includes purification, wherein the concentrated product is purified by using macroporous resin to purify the fresh dandelion extract, and the purified liquid is concentrated under reduced pressure to form a purified concentrated product. After drying, the purified concentrated product is pulverized and sieved to obtain the purified fresh dandelion extract, wherein the content of its main active components, flavonoids, 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 can be a concentrated liquid or a concentrated extract.

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

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

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

[0048] Preferably, the freeze-drying process involves freezing the concentrated product of fresh dandelion extract in a refrigerator at -20 to -80 ℃ for 24 to 48 hours, followed by freeze-drying at a pressure of 0.08 to -0.09 MPa, a temperature of -40 to -60 ℃, and a freezing time of 48 to 72 hours.

[0049] Preferably, the spray drying process involves setting the inlet temperature of the concentrated fresh dandelion extract to 100–190 °C and the outlet temperature to 80–100 °C.

[0050] Preferably, after drying, the product is passed through an 80-140 mesh sieve and then vacuum-packed.

[0051] Example 2

[0052] This example provides a dandelion extract, obtained by the preparation method described above, wherein the main active components, flavonoids, contain more than 25% and phenolic acids, contain more than 20%; preferably, after purification, the dandelion extract contains more than 35% of the main active components, flavonoids, and more than 35% of phenolic acids.

[0053] This invention identified 285 compounds in fresh dandelion extract using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), including 74 phenolic acids and 73 flavonoids. The extraction method for fresh dandelion described in this invention can achieve an extraction rate of over 5% for flavonoids and over 15% for phenolic acids. The total flavonoid and total phenolic acid contents reached over 25% and 20%, respectively, and after purification, the contents of both flavonoids and phenolic acids reached over 35%.

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

[0055] The extraction method for fresh dandelion extract described in this invention can yield compounds rich in flavonoids and phenolic acids.

[0056] A fresh dandelion pharmaceutical composition comprising the extract prepared above or the extract described above.

[0057] An application of the above-described fresh dandelion extract in the preparation of anti-inflammatory drugs or cosmetics.

[0058] Inflammation is a series of protective immune responses produced by the host system when stimulated by damaging factors (such as pathogens, damaged cells, or other stimuli). It affects various cytokines in the body's microenvironment, which participate in regulating various physiological and pathological processes. Under normal circumstances, inflammation can eliminate harmful stimuli, initiate the healing process, and promote repair. However, the persistence of inflammation is harmful to organ and systemic responses. Excessive inflammation can disrupt the homeostasis of normal tissues. For example, a cytokine storm caused by excessive inflammation can lead to multiple organ failure and even death in severe cases. Studies have found that inflammation plays an important role in the development of many complex diseases, such as obesity, cardiovascular disease, chronic respiratory diseases, rheumatoid arthritis, and cancer. Therefore, it is crucial to suppress dysregulation or excessive inflammation. Currently, in clinical practice, there are already anti-inflammatory drugs on the market, such as non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SSAIDs). However, these drugs have a series of adverse reactions during long-term use, thus limiting their application. The fresh dandelion of this invention, as a natural plant, has stronger activity, multi-target effects, and fewer toxic side effects than dried dandelion. Its main effective components include caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin, and luteolinoglycoside, which have significant anti-inflammatory activity.

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

[0060] The dandelion extract of this invention can significantly reduce TNF in LPS-induced EPh4v of mouse mammary epithelial cells. α, IL 1β,IL The expression level of 6 has a significant anti-inflammatory effect.

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

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

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

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

[0065] This invention provides a simple and efficient method for preparing fresh dandelion extract, targeting fresh medicinal materials. The resulting extract is superior to that produced by juicing, exhibiting a higher content of active ingredients. Existing technologies primarily focus on extracting active ingredients from dried dandelion, often emphasizing crude extraction or extraction of specific compounds, such as flavonoids. This application uses fresh dandelion as raw material, eliminating the need for processing into dried dandelion slices after direct harvesting. This avoids potential contamination and mold growth during processing, as well as significant loss and alteration of active ingredients during drying, thus improving efficacy. The method employs a pressing process to extract fresh juice, followed by solvent and ultrasonic extraction of the residue, with the addition of the active agent Tween 20. This method achieves convenient processing and effective extraction of fresh dandelion, resulting in higher extraction efficiency, higher content of active ingredients, and better activity. Furthermore, this application also reveals that the purified fresh dandelion extract exhibits significant anti-inflammatory effects.

[0066] The fresh dandelion extract obtained by this invention is a natural component directly extracted from fresh plants. It is rich in flavonoids and phenolic acids. After purification, the main chemical components it contains are caffeic acid, chlorogenic acid, p-coumaric acid, chicoric acid, luteolin, and luteolinoglycoside. It can reduce the expression of inflammatory factors and inhibit inflammatory responses, providing a new method for treating inflammation.

[0067] The fresh dandelion extract of this invention uses fresh dandelion as raw material, which is widely available and easy to process. In addition to the juicing process, the active agent Tween 20 is added, and combined with solvent and ultrasonic extraction, the extraction rate of effective ingredients can be greatly improved, while reducing the amount of solvent used. The solvent can be reused and is widely used in health products, food and medicine, and has strong market competitiveness.

[0068] Example 3

[0069] Based on the content of this application, the extraction of fresh dandelion extract is described in detail. Where specific experimental steps or conditions are not specified in the examples, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0070] The present invention will be further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention. The water content of fresh dandelion varies, and the amount of juice extracted after juicing is not fixed, but can be controlled within ±10%.

[0071] Experimental Example 1

[0072] Take 1000 g of freshly collected dandelion, wash with water to remove mud and sand, clean, cut into 7 cm sections, and press to extract juice using a squeeze press. Transfer the resulting 780 g of residue to an extraction tank, add 2 times the volume of 60% ethanol, then add the active agent Tween 20 at a dosage of 0.03 g / mL, stir evenly, and ultrasonically extract for 30 min. Press to extract juice using a filter press, repeat the extraction twice, combine all fresh juice, filter through an 80-mesh sieve, and then concentrate under reduced pressure in a 70 ℃ water bath to 290 mL (equivalent to a relative density of 1.21 for the concentrate). Concentrate the fresh dandelion juice until there is no ethanol odor, and obtain fresh dandelion concentrate. Dry directly for 36 h to obtain 238 g of fresh dandelion extract, with a total flavonoid content of 27.1% and a total phenolic acid content of 25.4%.

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

[0074] Experimental Example 2

[0075] 1000 g of freshly collected dandelion was washed to remove mud and sand, cleaned, and cut into 5 cm sections. The juice was extracted using a squeeze press. 785 g of the resulting residue was transferred to an extraction tank, and 5 times its volume of 40% ethanol was added. Then, Tween 20 was added at a dosage of 0.01 g / mL. The mixture was stirred and ultrasonically extracted for 20 min. The juice was then extracted using a filter press. This extraction was repeated three times. All the juice was combined, filtered through a 100-mesh sieve, and then concentrated under reduced pressure in a 50 ℃ water bath until the relative density was 1.25. The fresh dandelion juice was concentrated until no ethanol odor remained, yielding a concentrated fresh dandelion extract. This extract was directly dried for 48 h to obtain 245 g of fresh dandelion extract, with a total flavonoid content of 26.3% and a total phenolic acid content of 21.2%.

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

[0077] Experimental Example 3

[0078] 1000 g of freshly collected dandelion was washed to remove mud and sand, cleaned, and cut into 3 cm pieces. The juice was extracted using a squeeze press. 815 g of the resulting residue was transferred to an extraction tank, and 7 times its volume of 70% ethanol was added. Then, Tween 20 was added at a dosage of 0.05 g / mL. The mixture was stirred and ultrasonically extracted for 50 min. The juice was then extracted using a filter press. This extraction was repeated twice. All the juice was combined and filtered through a 120-mesh sieve. The extract was then concentrated under reduced pressure in an 80 ℃ water bath until a relative density of 1.12 was achieved. The fresh dandelion juice was concentrated until no ethanol odor remained, yielding a concentrated fresh dandelion extract. This extract was directly dried for 36 h to obtain 228 g of fresh dandelion extract, with a total flavonoid content of 25.3% and a total phenolic acid content of 20.7%.

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

[0080] Test Example 4

[0081] Take 1000g of freshly collected dandelion, wash it with water to remove mud and sand, clean it, cut it into 7cm sections, and press it with a squeeze press to extract the juice. The obtained 800 g of residue was transferred to an extraction tank, and 2 times the volume of 60% ethanol was added. Then, Tween 20 activator was added at a dosage of 0.03 g / mL. The mixture was stirred and ultrasonically extracted for 30 min. The juice was then extracted by pressing with a filter press. The extraction was repeated twice. All the fresh juice was combined and filtered through an 80-mesh sieve. The juice was then concentrated to 290 mL under reduced pressure in a water bath at 70 ℃. The fresh dandelion juice was concentrated until there was no ethanol odor, resulting in a concentrated fresh dandelion extract. The concentrated extract was purified using D101 macroporous resin. The eluent was 70% ethanol and concentrated to a relative density of 1.25. The concentrated fresh juice was frozen in a -80 ℃ freezer for 24 h. The frozen product was then freeze-dried at a pressure of 0.08 MPa and a temperature of -50 ℃ for 36 h. The dried product was pulverized, passed through an 80-mesh sieve, and vacuum-packed to obtain 107 g of purified fresh dandelion extract with a total flavonoid content of 40.12% and a total phenolic acid content of 38.05%.

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

[0083] Experimental Example 5

[0084] Take 1000 g of freshly collected dandelion, wash with water to remove mud and sand, clean, cut into 7 cm sections, and press to extract juice using a squeeze press. Transfer the resulting 820 g of residue to an extraction tank, add 2 times the volume of 60% ethanol, then add the active agent Tween 20 at a dosage of 0.02 g / mL, stir evenly, and ultrasonically extract for 30 min. Press to extract juice using a filter press, repeat the extraction twice, combine all fresh juice, filter through a 100-mesh sieve, and then concentrate under reduced pressure in a 70 ℃ water bath to 290 mL. Concentrate the fresh dandelion juice until there is no ethanol odor to obtain fresh dandelion concentrate. The obtained concentrate is purified using XDA-8 macroporous resin, with 70% ethanol as the eluent. The eluent is concentrated to a relative density of 1.20. The concentrate is then spray-dried with the inlet temperature set at 130 ℃ and the outlet temperature at 90 ℃. The dried product was pulverized, passed through an 80-mesh sieve, and vacuum-packed to obtain 100g of purified dandelion extract, with a total flavonoid content of 37.82% and a total phenolic acid content of 35.52%.

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

[0086] Comparative Example 1

[0087] 1000 g of freshly collected dandelion was washed to remove mud and sand, cleaned, and cut into 7 cm sections. The juice was extracted using a squeeze press. 800 g of the resulting residue was transferred to an extraction tank, and twice the volume of 60% ethanol was added. The mixture was stirred and ultrasonically extracted for 30 min. The juice was then extracted using a filter press. This extraction process was repeated, and all the juices were combined. The extract was concentrated under reduced pressure in a 70 ℃ water bath to 250 mL. The concentration was further reduced to a relative density of 1.15. The dandelion juice was concentrated until no ethanol odor remained, yielding a concentrated dandelion extract. This extract was directly dried for 36 h to obtain 244 g of dandelion extract, with a total flavonoid content of 17.5% and a total phenolic acid content of 15%.

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

[0089] Comparative Example 2

[0090] 1000 g of freshly collected dandelion was washed to remove mud and sand, cleaned, and cut into 7 cm sections. The juice was extracted using a squeeze press. 840 g of the resulting residue was transferred to an extraction tank, and twice the volume of 60% ethanol was added. The mixture was stirred and ultrasonically extracted for 30 min. The juice was then extracted again using a filter press. The extraction process was repeated, and all the juices were combined. The extract was concentrated to 250 mL under reduced pressure in a 70 ℃ water bath. The concentrated extract was purified using D101 macroporous resin, with 80% ethanol as the eluent. The eluent was concentrated to a relative density of 1.27. The concentrated juice was frozen at -80 ℃ for 24 h. The frozen product was then freeze-dried at 0.08 MPa and -50 ℃ for 36 h. The dried product was pulverized, passed through an 80-mesh sieve, and vacuum-packed to obtain 72 g of purified dandelion extract, with a total flavonoid content of 22.24% and a total phenolic acid content of 15.48%.

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

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

[0093] RAW264.7 mouse macrophages were cultured and seeded in 6-well plates. Incubation was carried out at 37 ℃ in a 5% CO2 incubator until the cell density reached 80%–90%. FDE (fresh dandelion extract) was prepared using the extract from Example 1. 10 mg of the fresh dandelion extract from Example 1 was weighed and dissolved in 10 mL of PBS. The solution was filtered through a sterile membrane and prepared with culture medium to the appropriate concentration. The following treatments were then established: an LPS+FDE group (divided into three groups: 25, 50, and 100 μg / mL), a Control group, an LPS group, and a DEX group. In the LPS+FDE groups, 1 mL of FDE at concentrations of 25, 50, and 100 μg / mL was added, while 1 mL of culture medium was added to the other groups. After pre-incubation for 1 h, the culture medium was discarded. In the LPS+FDE groups, LPS at a concentration of 1.0 μg / mL and FDE at concentrations of 25, 50, and 100 μg / mL were added. The DEX group was cultured in a mixed medium containing 1 μg / mL dexamethasone and 1.0 μg / mL LPS, the LPS group was cultured in a medium containing 1.0 μg / mL LPS, and the Control group was cultured in a medium containing the same amount of medium. All groups were cultured for 6 h. NO levels were detected using a NO detection kit (Beyotime), and TNF-α and IL-1 levels were detected by qPCR after total RNA extraction. 1β expression level as Figure 1 .

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

[0095] EPH4-EV mouse mammary epithelial cells were cultured and seeded in 6-well plates. Incubation was carried out at 37°C with 5% CO2 until the cell density reached 80%–90%. FDE (fresh dandelion extract): The extract from Example 1 was used. 10 mg of fresh dandelion extract from Example 1 was weighed and dissolved in 10 mL of PBS. The solution was filtered through a sterile membrane and prepared with culture medium to the appropriate concentration. The treatment method was the same as in Example 1. LPS+FDE group (divided into 3 groups: 25, 50, and 100 mg), LPS group, DEX group, and control group were set up. All cells were cultured for 12 h. Total RNA was extracted and qPCR was used to detect the expression levels of iNOS, COX-2, and TNF-α. Figure 2 .

[0096] Experimental Example 3: Dandelion can inhibit the ear swelling rate of a xylene-induced mouse ear swelling model, demonstrating high anti-inflammatory activity.

[0097] Xylene-induced mouse ear swelling test: Thirty mice were randomly divided into 5 groups. Group A was the blank control group, given 0.2 mL / 20g physiological saline. Groups B, C, and D were the experimental groups, given 0.1 mL / 20g, 0.2 mL / 20g, and 0.4 mL / 20g of fresh dandelion extract solution (prepared by weighing 10 mg of fresh dandelion extract from Example 4, dissolving it in 10 mL of PBS, and filtering through a sterile membrane), respectively, for 5 consecutive days. Group E was the positive control group, given a single dose of 0.2 mL / 20g hydrodexamethasone injection. One hour after the last administration, xylene was applied to the right ear of the mice to induce inflammation. One hour later, the mice were sacrificed, both ears were cut off, and ear pieces were punched out from the same location using a 9 mm diameter punch, weighed, and recorded. Table 1 shows that there was no significant difference in the low-dose group compared with the blank control group; however, the high-dose and medium-dose groups showed a significantly reduced degree of ear swelling compared with the blank control group. The flavonoids extracted from fresh dandelion could inhibit ear swelling in mice, and the inhibitory effect was dose-dependent.

[0098]

[0099] Experiment Example 4: Fresh dandelion extract alleviates LPS-induced mastitis in mice

[0100] A mouse mastitis model was established using intramammary infusion of LPS. Forty-eight female BALB / c mice were used before modeling and divided into six groups of eight mice each: a blank control group (CON) receiving solvent via gavage; a mastitis model (LPS) group receiving intramammary infusion of LPS; a fresh dandelion extract group receiving FDE-L (extract from Example 1), FDE-M (extract from Example 5), and FDE-H (extract from Example 4) via oral gavage for seven consecutive days at 400 mg / kg; and a dexamethasone group (DEX group) receiving intraperitoneal injection of dexamethasone at 10 mg / kg 12 h and 1 h before modeling. Except for the blank control group, all other groups received intramammary infusion of LPS (50 μL, concentration 0.2 mg / mL). Mammary lesions of the mice were observed. Figure 3 As shown, compared with the LPS group, FDE administration significantly reduced the damage to pathological tissues. The degree of inflammatory cell infiltration decreased significantly with increasing concentration, and the swollen acinar wall structure gradually returned to normal.

[0101] Experimental Example 5: Purified extract of fresh dandelion alleviated and improved DSS-induced colitis in mice.

[0102] Forty-eight C57BL / 6 mice were acclimatized for one week and then divided into seven groups of eight mice each: a control group (CON), a DSS model group (3% DSS), a positive control group (mesalazine MS), and high, medium, and low dose FDE (extract from Experiment 4) groups (FDE-C, FDE-B, and FDE-A). Based on 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 The low-dose (FDE-A) is 125 mg / kg. -1 The dosage of mesalazine is 10 mg / kg. -1 Except for the control group which had free access to food, mice in the other groups had free access to 3% DSS for 7 days, and were administered the corresponding dose of the drug by gavage for 7 days. The remaining groups were administered distilled water by gavage and intraperitoneal injection of inhibitors once daily. At the end of the experiment, mice were sacrificed, and colon tissue was collected and fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining. Figure 4 The control group showed no histopathological damage, the colonic mucosa was intact, goblet cells were neatly arranged, and crypts were intact. The model group showed a large number of inflammatory cell infiltrations and bleeding in the mucosa and submucosa, with a significant reduction in goblet cells and absence of crypts. After drug treatment, the colonic mucosal damage in all groups was significantly improved, the number of goblet cells increased, the crypts were basically intact, and there was only a small amount of inflammatory cell infiltration.

[0103] Experimental Example 6

[0104] FDEa group: The extract from test example 1 was used; FDEb group: The extract from test example 4 was used. The preparation method and other experimental conditions were the same as in Experiment 1. Two groups were set up: an LPS+FDE group (divided into two subgroups: 25 μg / mL and 100 μg / mL), a blank control group (Control group), an LPS group, and a positive control group (DEX group). The FDEa group was supplemented with 1 mL of FDEa (extract from Experiment 1) at concentrations of 25 μg / mL and 100 μg / mL, respectively; the FDEb group was supplemented with 1 mL of FDEb (extract from Experiment 4) at concentrations of 25 μg / mL and 100 μg / mL, respectively; and the other groups were supplemented with 1 mL of culture medium. After pre-incubation for 1 h, the culture medium was discarded. The FDE group was supplemented with a mixed culture medium containing 1.0 μg / mL LPS and corresponding FDE concentrations of 25 μg / mL and 100 μg / mL, respectively; the DEX group was supplemented with a mixed culture medium containing 1 μg / mL dexamethasone and 1.0 μg / mL LPS; the LPS group was supplemented with culture medium containing 1.0 μg / mL LPS; and the Control group was supplemented with an equal volume of culture medium. The cultures were then incubated for 6 hours. h, NO content was detected using a NO detection kit, and TNF-α and IL were detected by qPCR after total RNA extraction. 1β expression level as Figure 5 .

[0105] In the experimental case, the effect of the fresh dandelion extract group became more and more significant with the increase of FDE concentration, eventually approaching the effect of the DEX group. This indicates 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 use. Fresh dandelion, as a natural plant, has stronger activity, multi-target action, and less toxic side effects than dried dandelion, and can be developed and applied as a new type of anti-inflammatory substance.

[0106] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a fresh dandelion extract, using fresh dandelion as the starting material, characterized in that, Includes the following steps: 1) During the spring and autumn harvest seasons of fresh dandelion, collect the whole fresh herb, including its roots, remove impurities, clean and select, wash, cut into sections, press while fresh using an extrusion press, filter to obtain fresh juice, and separate the fresh juice from the dregs. 2) Add the extraction solvent to the above-mentioned residue, and simultaneously add the activator Tween 20. The amount of activator Tween 20 added relative to the extraction solvent is 0.01-0.05 g / mL. When using ultrasonic extraction, stir for 10-30 min, ultrasonically extract for 10-60 min, filter with a filter press to obtain juice, add the remaining residue to the extraction solvent again for repeated ultrasonic extraction, and then press to obtain juice. Combine all the fresh juices, filter, and obtain fresh dandelion juice. The extraction solvent is ethanol with a concentration of 40-70%. 3) Fresh dandelion juice is concentrated to 10% to 15% of its original volume before extraction to form a concentrated product. The concentrated product is dried to obtain fresh dandelion extract, whose main active components, flavonoids, have a content of more than 25% and phenolic acids have a content of more than 20%.

2. The preparation method according to claim 1, characterized in that, During the extraction of the medicinal residue, the amount of extraction solvent added is 1 to 10 times the volume of the medicinal residue; the remaining medicinal residue is then added to the extraction solvent and the ultrasonic extraction is repeated 1 to 4 times. All the fresh juice is combined and filtered through a 40 to 120 mesh sieve to obtain fresh dandelion juice.

3. The preparation method according to claim 1, characterized in that, The segments are cut into 2-10 cm pieces.

4. The preparation method according to claim 1, characterized in that, The process also includes purification, wherein the concentrated product is purified using XDA-8, D101 or AB-8 macroporous resin to purify the dandelion extract. After purification, the liquid is concentrated under reduced pressure to form a purified concentrated product. After drying, the purified concentrated product is pulverized and sieved to obtain a purified dandelion extract, the main active components of which contain more than 35% flavonoids and more than 35% phenolic acids.

5. The preparation method according to claim 1 or 4, characterized in that, The vacuum concentration method described above involves concentrating the product under reduced pressure at 50–80°C to a relative density of 1.05–1.

35.

6. The preparation method according to claim 1, characterized in that, During the drying process, the drying method used is freeze drying or spray drying; The freeze-drying process involves freezing the concentrated extract of fresh dandelion in a freezer at -20 to -80°C for 24 to 48 hours, followed by freeze-drying at a pressure of 0.08 to -0.09 MPa and a temperature of -40 to -60°C for a duration of [duration missing]. 48～72 h； The spray drying process involves setting the inlet temperature of the concentrated dandelion extract to 100–190 °C and the outlet temperature to 80–100 °C.

7. A fresh dandelion extract, obtained by the preparation method described in claim 1, wherein the main active components, flavonoids, contain more than 25% and phenolic acids, contain more than 20%.

8. A fresh dandelion medicinal composition, characterized in that, It includes the extract prepared according to any one of claims 2-6 or the extract according to claim 7.

9. The use of a fresh dandelion extract prepared according to any one of claims 2-6 or the extract according to claim 7 in the preparation of anti-inflammatory drugs or cosmetics.

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