Method for increasing content of flavanone in pericarpium citri reticulatae and application of pericarpium citri reticulatae extract in treatment of eczema

Abstract

The invention discloses a method for increasing the content of flavanone in pericarpium citri reticulatae and application of pericarpium citri reticulatae extract in treatment of eczema. The invention provides a method for increasing the content of flavanone in pericarpium citri reticulatae, the content of flavanone in pericarpium citri reticulatae can be remarkably increased by adopting a specific natural deep-eutectic solvent for extraction, and the extracted pericarpium citri reticulatae extract has the effect of treating eczema, can relieve symptoms of eczema, can relieve epidermal hyperkeratosis, incomplete keratosis and granular layer and spine layer fertilizer thickness, and can be used for improving the content of flavanone in pericarpium citri reticulatae. The number of mast cells is reduced, skin repair is promoted, skin itch caused by eczema is relieved, and the ointment can rapidly permeate into the skin to accelerate wound healing; the pericarpium citri reticulatae extract can be used for treating eczema, the inflammatory factor level in skin lesion tissue can be reduced, eczematous dermatitis is further reduced, the effect of the pericarpium citri reticulatae extract is equivalent to that of eczema positive drugs, and is superior to that of other pericarpium citri reticulatae extract obtained by extracting natural deep-eutectic solvents and water, and the pericarpium citri reticulatae extract can be used for developing more eczema treatment drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting active pharmaceutical ingredients, and more specifically, relates to a method for increasing the flavanone content in tangerine peel and the application of tangerine peel extract in treating eczema. Background Art

[0002] Eczema is a common inflammatory skin disease of the epidermis and superficial dermis caused by multiple internal and external factors. It has the characteristics of severe itching, polymorphic skin lesions, symmetrical distribution, exudative tendency and easy recurrence. According to the urgency of the disease, it is divided into acute eczema, chronic eczema and subacute eczema. Once the patient suffers from acute eczema, he is susceptible to microbial infection such as external viruses and bacteria, which further causes chronic eczema. Chronic eczema has a long course of disease, is stubborn, easily relapses, and is relatively difficult to treat. Severe cases can affect the patient's life and work. At present, Western medicine mainly relies on topical glucocorticoid preparations and oral antihistamines to treat this disease, but its efficacy is short-lived, easy to relapse after drug withdrawal, and long-term use has more adverse reactions.

[0003] Flavanones in tangerine peel extract are also called dihydroflavonoids, which are natural flavonoid compounds. Flavanones mainly include hesperidin, naringenin, eriodictyol and other substances. Flavonoids are a class of polyphenolic compounds with 2-phenylchromone as the basic structural unit, which are commonly found in various plants. It has a variety of pharmacological activities and has been proven to have anti-inflammatory, anti-tumor, antiviral and antioxidant properties. At present, the methods for extracting flavanones include reflux method, ultrasonic extraction method, microwave extraction method, etc., but all of them require a large amount of organic solvents and are less environmentally friendly. In addition, after ultrasonic extraction of flavanones using organic solvents as solvents for soaking, the obtained flavanone crude extract contains a large amount of organic solvent residues, which is harmful to the environment and is not good for human health after being made into medicines; while the content of flavanones prepared by water extraction method is low. Due to the large variety of flavonoids, complex structures and diverse physiological activities, most flavonoids have complex structures and similarities, resulting in low extraction efficiency, organic solvent residues, high extraction costs, and difficult separation during their extraction. Therefore, establishing a method that can quickly and selectively increase the content of active flavanones in tangerine peel extract is of great significance for preparing more drugs containing tangerine peel extract.

[0004] Traditional Chinese medicine is used for treating eczema with external treatment, which has a long history, good curative effect, low recurrence rate and few adverse reactions, and has certain advantages. At present, more and more clinical drugs are prepared by different Chinese medicine prescriptions, and Chinese medicine has better safety than Western medicine, and is used to prepare medicines for treating eczema, and its side effect is low and the recurrence probability is low. As disclosed in the prior art, a Chinese medicine composition for treating eczema contains Sophora flavescens, Cortex Phellodendri, Folium Artemisiae Argyi, White Ringworm Peel, Menthol, Pollen Typhae Carbon, Fructus Gardeniae, Beeswax, Flos Chrysanthemi, Periostracum Cicadae, Folium Citri Reticulatae, Fructus Kochiae, Flower of the Pipe, Herba Taraxaci, Borneolum Syntheticum, etc., and the existing Chinese medicine prescription composition is complex, and the components are more, and it is necessary to play its best effect through reasonable compatibility, and the drug effect is played slowly, and the cost is also higher. In the existing research, there is a compounding method using dried tangerine peel to treat eczema, but there is no research on directly using dried tangerine peel extract for the treatment of eczema, and there are few eczema medicines directly prepared by using Chinese medicine dried tangerine peel extract. Therefore, it is urgent to develop more eczema treatment drugs with better therapeutic effects, containing tangerine peel extracts, which are natural, safe, and have no side effects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above defects and shortcomings, and provide a method for increasing the flavanone content in tangerine peel and the use of tangerine peel extract in the preparation of a drug for treating eczema.

[0006] The first object of the present invention is to provide a method for increasing the flavanone content in tangerine peel.

[0007] The second object of the present invention is to provide application of the above method.

[0008] The third object of the present invention is to provide a tangerine peel extract.

[0009] The fourth object of the present invention is to provide the application of tangerine peel extract.

[0010] The fifth object of the present invention is to provide a medicine.

[0011] The sixth object of the present invention is to provide a method for extracting flavanones from tangerine peel.

[0012] The seventh object of the present invention is to provide tangerine peel flavanone.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The invention provides a method for increasing the flavanone content in tangerine peel. The tangerine peel is crushed and uniformly mixed with a natural low eutectic solvent, and ultrasonic extraction is performed at 40-70°C for 20-40 minutes to obtain a tangerine peel extract. The natural low eutectic solvent is prepared by heating and dissolving a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is selected from one of triethylene glycol, 1,3-butanediol, glycerol, citric acid, and ethylene glycol, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(1-5); and the mass ratio of the tangerine peel to the natural low eutectic solvent is 1:(10-30).

[0015] The present invention provides a method for increasing the content of flavanones in tangerine peel, using a specific natural deep eutectic solvent, under the assistance of ultrasound, extraction can significantly increase the content of flavanones in tangerine peel, and the tangerine peel extract obtained by the extraction has the effect of treating eczema, can relieve eczema symptoms, reduce epidermal hyperkeratosis, parakeratosis and granular layer, stratum spinosum hypertrophy thickness, and reduce the number of mast cells, promote skin repair, reduce the skin itching caused by eczema, and can quickly penetrate the skin to accelerate wound healing; it can also reduce the level of inflammatory factors in skin lesions, further reduce the occurrence of eczematous dermatitis, and its effect is comparable to eczema positive drugs, and is better than other natural deep eutectic solvents and water extraction obtained tangerine peel extract, can be used to develop more stable and lasting can reduce the skin itching caused by eczema and quickly penetrate the skin to accelerate wound healing eczema treatment drugs. The method steps provided by the present invention are simple and fast, and have a high extraction efficiency.

[0016] The invention extracts the tangerine peel extract by using a natural low eutectic solvent. Compared with the traditional extraction method, hydrogen bonds are formed between hydrogen bond donors and acceptors in the natural low eutectic solvent and the effective components of the tangerine peel, thereby enhancing the solubility of the effective components in the natural low eutectic solvent. The hydroxyl groups on the flavanones form hydrogen bonds with the hydrogen bond donors, which can dissolve the flavanones well, help improve the dispersibility of the flavanones, reduce the potential risks of irritation and toxicity, and have the characteristics of being green, safe and environmentally friendly. Compared with organic solvents, the components of the natural low eutectic solvents are usually derived from natural products. The hydrogen bond donors used in the invention are polyols, and the hydrogen bond acceptors are choline compounds, which are widely present in nature, are non-toxic and biodegradable, and are environmentally friendly materials.

[0017] Preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(3-5).

[0018] Preferably, the hydrogen bond donor is triethylene glycol.

[0019] Furthermore, the ultrasonic power is 300-450W.

[0020] Preferably, the ultrasonic conditions are: temperature 40°C, time 30min, power 330W.

[0021] The present invention provides application of the method in increasing the flavanone content in tangerine peel.

[0022] The invention provides a tangerine peel extract prepared by the method.

[0023] The invention provides application of tangerine peel extract in preparing medicine for treating eczema.

[0024] The invention also provides an eczema medicine containing the tangerine peel extract.

[0025] At the same time, the present invention also provides a method for extracting flavanones from tangerine peel, wherein the tangerine peel flavanones are obtained by separating and purifying the crude tangerine peel extract obtained by molecular imprinting technology. The method has a strong recognition ability for target molecules and can specifically recognize and bind to target molecules from a complex mixture, just like an "artificial key" is only suitable for a specific "lock". It has the advantages of safety, high efficiency, stability and directionality, expands the source and separation method of flavanones, and also provides a reference for extracting more active ingredients from tangerine peel.

[0026] The present invention provides a method for extracting flavanones from tangerine peel, comprising the following steps:

[0027] S1. Preparation of flavanone molecular imprinted polymer: using flavanone as a template molecule, adding a functional monomer, a crosslinking agent and an initiator, reacting under inert gas conditions, stirring evenly, standing at a constant temperature, washing, and drying to obtain a flavanone molecular imprinted polymer; the molar ratio of the template molecule, the functional monomer, the crosslinking agent and the initiator is 2:(5-10):(10-15):(0.5-1); the functional monomer is methacrylic acid, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile;

[0028] S2. The polymer of S1 is crushed and loaded into a solid phase extraction column, and then the tangerine peel extract prepared according to claim 1 is added. After all impurities are removed, the flavanone is combined with the molecularly imprinted polymer, eluted, and rotary evaporated to obtain a solid, which is the flavanone.

[0029] Preferably, the molar ratio of the flavanone, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 2:5:10:1.

[0030] Preferably, in S1, the stirring speed is 300-450 r / min, the standing time is 20-30 h, the temperature is 50-70°C, the drying time is 10-12 h, and the drying temperature is 50-70°C.

[0031] More preferably, the conditions for preparing the flavanone molecular imprinted polymer are: stirring speed of 450 r / min, standing time of 24 h, temperature of 50° C.; drying time of 10 h, drying temperature of 60° C.

[0032] The invention provides tangerine peel flavanone, which is prepared by the method.

[0033] The present invention has the following beneficial effects:

[0034] The method provided by the present invention can improve the flavanone content in tangerine peel extract, and the tangerine peel extract prepared by the tangerine peel extract can be used to treat eczema, relieve eczema symptoms, and reduce epidermal hyperkeratosis, parakeratosis, and granular layer and spinous layer fat thickness, and mast cells are significantly reduced in number, promote skin repair, and can reduce the skin itching caused by eczema, and can quickly penetrate the skin to accelerate wound healing; it can also reduce IL-4, TNF-α, IL-2, IL-6, IL-1β and IFN-γ water in inflammatory factors in skin lesions, effectively inhibit inflammatory factors, and further reduce the occurrence of eczematous dermatitis. Its effect is comparable to positive drugs, and is superior to tangerine peel extracts prepared by other natural low eutectic solvents (DESs-4 and DESs-5 groups), and is expected to be used to develop more eczema drugs, as well as to treat contact dermatitis and neurodermatitis.

[0035] At the same time, the present invention also provides a method for extracting flavanones from tangerine peel. The crude tangerine peel extract obtained by extraction is separated and purified by molecular imprinting technology to obtain tangerine peel flavanones, which expands the source and separation method of flavanones and also provides a reference for extracting more active ingredients from tangerine peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Comparison pictures of the skin of KM mice in each group before and after treatment.

[0037] Figure 2 Shown are the observation results of skin tissue pathology of KM mice in each group.

[0038] Figure 3 Statistical results of epidermal thickness of KM mice in each group.

[0039] Figure 4 The results of toluidine blue staining of skin lesion tissues of KM mice in each group.

[0040] Figure 5 This is a comparison chart of the number of mast cells in the skin lesion tissue of KM mice in each group. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] The damaged part of the tangerine peel used in the invention is removed before use, and then foreign matters on the surface are washed away by running clean water, and the tangerine peel is repeatedly washed until the foreign matters are completely washed away, and then dried for standby use.

[0044] Example 1 Preparation of Tangerine Peel Extract and Determination of Flavanone Content

[0045] Preparation of natural deep eutectic solvents (NADESs): The NADESs were prepared by heating the hydrogen bond donor and the hydrogen bond acceptor at 80°C with constant stirring until the liquid solvent was uniform, and the NADESs were prepared as shown in Table 1.

[0046] Preparation of tangerine peel: Select dry, mold-free, high-quality tangerine peel, wash away foreign matter in the tangerine peel with clean water, dry it and crush it into powder.

[0047] Tangerine peel and a natural low eutectic solvent are mixed in a mass ratio of 1:25, and extraction is performed under the conditions of ultrasonic power of 330 W, time of 30 min, and extraction temperature of 40° C. The obtained extract is tangerine peel extract.

[0048] Liquid chromatography-mass spectrometry was then used to detect the content of flavanones in the extract. The specific method is: 20 μL of the tangerine peel extract enters the liquid chromatography system for separation, and the separated components enter the mass spectrometer for detection. The column temperature is maintained at 25°C, and the ultraviolet detection wavelength is 280nm. According to the characteristic peaks and intensities of each ion in the mass spectrum combined with the information of the standard, the flavanones are analyzed. The flavanone content is analyzed based on the obtained information. Rutin is used as a standard (0-150 μg) to draw a standard curve. 20 μL is brought into the standard curve to calculate the content of flavanones.

[0049] The determination results are shown in Table 1, which show that there are significant differences in the content of flavanones in the extracts of tangerine peel after extraction with different NADESs, and the content of flavanones extracted with choline chloride and citric acid is extremely low; when other conditions in the extraction method are the same, the tangerine peel extract obtained by extracting with choline chloride and triethylene glycol as hydrogen bond acceptors and hydrogen bond donors has the highest content of flavanones, and the content of flavanones in the extract reaches 2.1064 mg / g.

[0050] Table 1 Ratios of different natural deep eutectic solvents

[0051]

[0052]

[0053] Example 2 Preparation of Tangerine Peel Extract

[0054] The hydrogen bond donor and the hydrogen bond acceptor are mixed and heated with choline chloride and triethylene glycol (molar ratio 1:3), and stirred continuously at 80° C. until the liquid solvent is uniform, thereby preparing a natural low eutectic solvent for use.

[0055] Take tangerine peel powder and natural low eutectic solvent in a mass ratio of 1:20, extract at an ultrasonic power of 330 W, a time of 30 min, and an extraction temperature of 40° C. The extracted solution is the tangerine peel extract.

[0056] Example 3 Preparation of Tangerine Peel Extract

[0057] The hydrogen bond donor and the hydrogen bond acceptor are mixed and heated with choline chloride and triethylene glycol (molar ratio 1:3), and stirred continuously at 80° C. until the liquid solvent is uniform, thereby preparing a natural low eutectic solvent for use.

[0058] Take tangerine peel powder and natural low eutectic solvent, according to the mass ratio of 1:30, under the condition of ultrasonic power of 330W, time of 30min, extraction temperature of 40°C, and the extracted solution is tangerine peel extract.

[0059] Example 4

[0060] The extraction preparation method is the same as in Example 1, except that the ultrasonic power is 300W.

[0061] Example 5

[0062] The extraction preparation method is the same as in Example 1, except that the ultrasonic power is 450W.

[0063] Example 6

[0064] The extraction preparation method is the same as in Example 1, except that the ultrasonic time is 20 min.

[0065] Example 7

[0066] The extraction preparation method is the same as in Example 1, except that the ultrasonic time is 40 min.

[0067] Example 8 A method for extracting flavanones using dried tangerine peel

[0068] The tangerine peel extract (crude extract) prepared in Example 2 was extracted using molecular imprinting technology, and flavanone was added as a template molecule to the functional monomer methacrylic acid, the cross-linking agent ethylene glycol dimethacrylate and the initiator azobisisobutyronitrile in a molar ratio of 2:5:10:1. Under nitrogen conditions, the mixture was stirred at a speed of 450 r / min, and allowed to stand at a constant temperature for 24 hours. Finally, the mixture was washed with an ethanol solution, and the dried solid was dried at 60°C for 10 hours to obtain a flavanone molecular imprinting polymer.

[0069] Then the polymer is crushed to obtain uniform particles, which are loaded into an empty solid phase extraction column to obtain a flavanone molecularly imprinted polymer column. The crude extract of tangerine peel extract is poured into the flavanone molecularly imprinted polymer column. After all impurities have flowed away, the flavanones are combined with the molecularly imprinted polymer, and the flavanones of the polymer are eluted with an ethanol solution. The eluate is then subjected to rotary evaporation at 60°C. The solid obtained is determined to be flavanone.

[0070] Comparative Example 1 Traditional water extraction method

[0071] Take 25g of dried tangerine peel, add 250mL of distilled water, set the ultrasonic power to 330W, and perform ultrasonic extraction for 30min. After the extraction is completed, the extraction temperature is 40℃, filter, and concentrate the filtrate under reduced pressure to 70mL.

[0072] Test Example 1 Effect of different extraction conditions on the content of flavanones in tangerine peel extract

[0073] The flavanone contents in the tangerine peel extracts prepared in Examples 4-7 and Comparative Example 1 were measured respectively by the same method as in Example 1. The measurement results are shown in Table 2. The results show that the ultrasonic extraction conditions affect the flavanone content in tangerine peel. The flavanone content in the tangerine peel extract of DESs-1 obtained by the extraction method of Example 1 can reach up to 3.1064 mg / g. This shows that the flavanone content in the tangerine peel extract can be increased only under specific conditions.

[0074] Table 2 Contents of flavanones in different tangerine peel extracts under different ultrasonic conditions

[0075]

[0076] Test Example 2DNCB-Skin Eczema Model

[0077] Adaptive feeding: Experimental SPF grade KM mice (5 weeks old), male, were purchased from Guangdong Experimental Animal Center. The mice were adaptively fed at 22°C with a 12-h light / dark cycle for six days and randomly divided into A: blank control group (Control): 6 mice; B: model group 2,4-dinitrochlorobenzene (DNCB): 6 mice; C: 0.5% DESs-1 group of Example 1: 6 mice; D: 0.5% DESs-4 group of Example 1: 6 mice; E: 0.5% DESs-5 group of Example 1: 6 mice; F: positive control group-dexamethasone ointment: 6 mice.

[0078] Skin preparation: One day before the experiment, the hair on the back of the mouse was cut short, and the depilatory agent was applied to the back of the mouse. After 3 minutes, the back was washed with warm water. The back of the mouse was considered clean when it was smooth, without any damage or residual hair. The depilatory area was about 4cm×4cm.

[0079] Skin eczema modeling: The experiment was divided into a 6-day modeling phase and a 4-day drug administration phase. During the modeling phase, 2% DNCB acetone-propylene glycol solution (acetone: propylene glycol = 1:4 volume ratio) (100 μL / mouse) was applied to the back skin of each mouse in the other groups except the blank group from 1 to 4 days. On the 6th day, 0.5% DNCB acetone-propylene glycol solution (acetone: propylene glycol = 1:4 volume ratio) (100 μL / mouse) was applied to the back skin of each mouse in the other groups except the blank group.

[0080] Administration: After modeling, mice were administered once a day. The model group and the blank control group were smeared with acetone-propylene glycol solution (acetone:propylene glycol = 1:4 volume ratio) (200 μL / mouse) on the back hair removal site, the positive control group was smeared with dexamethasone cream (100 mg / mouse) on the back hair removal site, the 0.5% DESs-1 group of mice in Example 1 were smeared with the corresponding drug (200 μL / mouse) on the back hair removal site, the 0.5% DESs-4 group of mice were smeared with the corresponding drug (200 μL / mouse) on the back hair removal site, and the 0.5% DESs-5 group of mice were smeared with the corresponding drug (200 μL / mouse) on the back hair removal site. On day 11, the back skin of mice was photographed to record the appearance, and then the mice were killed by cervical dislocation, and the back skin tissue was fixed and frozen in paraformaldehyde (two tubes of tissue each) for subsequent index determination.

[0081] 1. Skin lesion scoring

[0082] The skin lesions were recorded and scored. The clinical manifestations of skin lesions were divided into five categories: erythema, hard swelling or edema / papule, epidermal exfoliation, exudation or crusting, and lichenification. The severity of each skin lesion was scored from 0 to 3 points: 0 = none, after careful observation, this sign is still uncertain; 1 = mild, this sign does exist, but requires careful inspection to observe; 2 = moderate, this sign can be observed immediately; 3 = severe, this sign is obvious. In the scoring of various symptoms, half a grade, that is, 0.5, can be recorded.

[0083] The skin of mice in each group before and after treatment Figure 1 As shown, compared with group B, group C and group F were only accompanied by slight redness and swelling after one week of application, indicating that the DESs-1 group of Example 1 and the positive control drug can relieve eczema and have the effect of treating eczema. Compared with group C and group F, group D and group E were still accompanied by slight redness and swelling and rough epidermis after one week of application, indicating that the effect of treating eczema was worse than that of group C and group F, and the scoring results are shown in Table 3. Combined with the above research, it is shown that the high content of flavanones in tangerine peel extract has a good effect on treating eczema. It is known that the tangerine peel extract prepared by the present invention can have a therapeutic effect on eczema, can stably and persistently reduce the skin itching caused by eczema, and can quickly penetrate the skin to accelerate wound healing.

[0084] Table 3 Skin lesion scoring results

[0085]

[0086]

[0087] 2. HE staining

[0088] Each group of mice was anesthetized by intraperitoneal injection of 2% sodium pentobarbital at a dose of 2 mL / (kg / mouse). The skin of the eczema area was cut off and then the tissue blocks were fixed in 10% neutral formaldehyde fixative, dehydrated, paraffin-embedded, and baked in an oven at 65°C for 3.5 h and at 37°C overnight. After dewaxing and dehydration, conventional HE staining was performed and the morphological changes of skin tissue were observed under a light microscope.

[0089] HE staining was used to characterize and statistically analyze the epidermal thickness of mice after drug administration. Figure 2 As shown, the epidermis and dermis structures of mice in the blank group were intact and no abnormalities were found; in the model group, the mouse epidermis showed hyperkeratosis and incomplete keratosis, and the stratum granulosum and stratum spinosum were significantly thickened. The epidermal thickness of mice in the DESs-1 group and the dexamethasone ointment group of Example 1 showed that the hyperkeratosis, incomplete keratosis, and the thickness of the stratum granulosum and stratum spinosum of the epidermis were reduced. Compared with DESs-1, the epidermis of mice in the DESs-4 and DESs-5 groups showed hyperkeratosis and incomplete keratosis, and the stratum granulosum and stratum spinosum were significantly thickened. The statistical results of epidermal thickness are shown in Figure 1. Figure 3As shown, it can be more intuitively seen that the skin tissue thickness of Example 1 is significantly thinner than that of other groups.

[0090] 3. Toluidine blue staining to observe the number of mast cells

[0091] Fresh skin tissue was fixed with 10% formalin solution, and routine dehydration and embedding were performed. Paraffin tissue sections with a thickness of 4 μm were sliced. After normal dewaxing, ethanol dewaxing was performed, toluidine blue staining was performed for 1 to 5 minutes, and ddH2O was repeatedly washed. Glacial acetic acid differentiation was performed for about 25 to 30 seconds. The biological microscope must be clear in the field of view. The particles were dehydrated in a series of gradient environments of alcohol. The xylene reagent was transparent and the slides were fixed with gum.

[0092] Toluidine blue staining results Figure 4 As shown in Figure 2, compared with the model group, the number of mast cells expressed in the tissue of the DESs-1 surface treatment group in Example 1 was significantly reduced. The statistical results of the number of mast cells in the skin lesion tissue of each group of mice are shown in Figure 2. Figure 5 As shown, compared with the blank group, the number of mast cells in the model group increased significantly, compared with the model group, the number of mast cells in the DESs-1 and dexamethasone ointment groups of Example 1 decreased significantly; compared with DESs-1, the number of mast cells in the DESs-4 and DESs-5 groups increased significantly. Therefore, it is shown that the tangerine peel extract prepared by the present invention has a certain promoting effect on the expression of collagen fibers and can promote skin repair.

[0093] 4. ELISA test

[0094] The levels of inflammatory factors IL-4, TNF-α, IL-2, IL-6, IL-1β and IFN-γ in mouse skin lesion tissues were tested by immunohistochemistry and fluorescence quantitative PCR. The detection method was strictly carried out according to the instructions of the kit.

[0095] The results are shown in Table 4. The results show that compared with group A, the expression levels of IL-4, TNF-α, IL-6, and IL-1β in group B were significantly increased, and the expression levels of IFN-γ and IL-2 were significantly decreased. The difference was statistically significant (P < 0.01); compared with group B, the expression of IL-4, TNF-α, IL-6, and IL-1β in groups C and F were all decreased, and the expression levels of IFN-γ and IL-2 were significantly decreased. The differences were statistically significant (P < 0.01); compared with group C, the expression levels of IL-4, TNF-α, IL-6, and IL-1β in groups D and E were significantly increased, and the expression levels of IFN-γ and IL-2 were significantly decreased.

[0096] Table 4 Comparison of IL-4, TNF-α, IL-2, IL-6, IL-1β and IFN-γ levels in skin lesions of mice in each group

[0097]

[0098] In summary, the results show that the method provided by the present invention can increase the flavanone content in tangerine peel extract, and the tangerine peel extract prepared by the method can be used to treat eczema, relieve eczema symptoms, and reduce epidermal hyperkeratosis, parakeratosis, and granular layer and spinous layer fat thickness, and the number of mast cells is significantly reduced, which promotes skin repair, can reduce the skin itching caused by eczema, and can quickly penetrate the skin to accelerate wound healing; it can also reduce IL-4, TNF-α, IL-2, IL-6, IL-1β and IFN-γ water in inflammatory factors in skin lesions, effectively inhibit inflammatory factors, and further reduce the occurrence of eczematous dermatitis. Its effect is comparable to that of positive drugs, and is superior to tangerine peel extracts prepared by other natural low eutectic solvents (DESs-4 and DESs-5 groups), and is expected to be used to develop more eczema drugs, as well as to treat contact dermatitis and neurodermatitis.

[0099] At the same time, the present invention also provides a method for extracting flavanones from tangerine peel. The crude tangerine peel extract obtained by extraction is separated and purified by molecular imprinting technology to obtain tangerine peel flavanones, which expands the source and separation method of flavanones and also provides a reference for extracting more active ingredients from tangerine peel.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for increasing the flavanone content in dried orange peel, characterized in that: The tangerine peel is crushed and mixed evenly with a natural deep eutectic solvent, and ultrasonically extracted at 40 to 70° C. for 20 to 40 minutes to obtain a tangerine peel extract; The natural deep eutectic solvent is prepared by heating and dissolving a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is selected from one of triethylene glycol, 1,3-butanediol, glycerol, citric acid, and ethylene glycol, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(1-5); The mass ratio of the tangerine peel to the natural deep eutectic solvent is 1:(10-30).

2. The method according to claim 1, characterized in that: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(3-5).

3. The method according to claim 1, characterized in that: The hydrogen bond donor is triethylene glycol.

4. Application of the method according to claim 1 in increasing the flavanone content in dried tangerine peel.

5. The tangerine peel extract prepared by the method of claim 1.

6. Use of the tangerine peel extract according to claim 5 in the preparation of a drug for treating eczema.

7. An eczema drug, characterized in that: Containing the tangerine peel extract according to claim 5.

8. A method for extracting flavanones from dried tangerine peel, characterized in that: The following steps are involved: S1. Preparation of flavanone molecular imprinted polymer: using flavanone as a template molecule, adding a functional monomer, a crosslinking agent and an initiator, reacting under inert gas conditions, stirring evenly, standing at a constant temperature, washing, and drying to obtain a flavanone molecular imprinted polymer; the molar ratio of the template molecule, the functional monomer, the crosslinking agent and the initiator is 2:(5-10):(10-15):(0.5-1); the functional monomer is methacrylic acid, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile; S2. The polymer of S1 is crushed and loaded into a solid phase extraction column, and then the tangerine peel extract obtained by the method of claim 1 is added. After all impurities have flowed away, the flavanones are combined with the molecularly imprinted polymer, eluted, and rotary evaporated to obtain a solid that is the flavanones.

9. The method according to claim 8, characterized in that: In S1, the stirring speed is 300-450 r / min, the standing time is 20-30 h, the temperature is 50-70°C, the drying time is 10-12 h, and the drying temperature is 50-70°C.

10. A tangerine peel flavanone, characterized in that: Prepared by the method of claim 8 or 9.

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