Plant composition for treating atopic dermatitis

Abstract

The invention relates to a plant composition for treating atopic dermatitis, the mixing mass ratio of baicalein-containing extract to ursolic acid-containing extract to alkannin-containing extract is (1-30): (1-20): (1-20), and the content of baicalein in the baicalein-containing extract is not less than 70%; the content of ursolic acid in the ursolic acid-containing extract is not less than 60%; the content of alkannin in the alkannin-containing extract is not less than 30%. The plant composition for treating atopic dermatitis is composed of the baicalein-containing extract, the ursolic acid-containing extract and the alkannin-containing extract, the main effective components of the plant composition are baicalein, ursolic acid and alkannin, the three components cooperate with each other to achieve a synergistic effect, and the plant composition has an inhibiting effect on biofilm development of staphylococcus aureus and can be used for treating atopic dermatitis. The staphylococcus aureus polypeptide can be used for destroying a formed biological membrane, interfering synthesis of peptidoglycan, inhibiting generation of cell walls and effectively relieving the problem of skin itch caused by staphylococcus aureus; meanwhile, inflammatory factors in the body are reduced, and skin inflammatory response is inhibited. And the plant composition is extracted from natural plants, has high effective content, and is safer and milder compared with chemical medicines.

Description

Technical Field

[0001] The invention relates to the technical field of biological products, and in particular to a plant composition for treating atopic dermatitis. Background Art

[0002] Childhood eczema, mostly referring to childhood atopic dermatitis, is a common chronic inflammatory skin disease that usually begins in infancy or early childhood and is related to factors such as poor skin barrier function, contact with allergens, and excessively dry skin. Characteristics of eczema include dry skin, erythema, blisters, exudate, and crusting. Itching is the main symptom of this disease. Severe itching causes children to lose concentration, become irritable, and have difficulty falling asleep, which seriously affects the quality of life of parents and children.

[0003] Itch is an unpleasant sensation that causes the desire to scratch, a behavioral reflex that exacerbates skin damage. Mechanical damage caused by scratching can damage the skin barrier and exacerbate inflammatory responses. Recently, scientists at Harvard Medical School demonstrated for the first time that Staphylococcus aureus can act directly on nerve cells to cause itching. The protease V8 of Staphylococcus aureus is a key mediator that causes spontaneous itching. V8 triggers itching by activating a protein called PAR1. Due to inflammation and damage to the barrier function of the skin of children with atopic dermatitis, the microbial balance that maintains skin health is broken, and Staphylococcus aureus accumulates in the epidermis and causes itching.

[0004] In recent years, the incidence of childhood eczema has increased year by year. In addition, children's skin is fragile and sensitive. Although conventional Western medicine treatment has some effect, it has obvious side effects and a high recurrence rate. Many plant extracts have been widely used in the treatment of childhood itching due to their anti-inflammatory, sedative and antibacterial properties, but they often fail to achieve the expected results. More than 90% of patients with atopic dermatitis are colonized by Staphylococcus aureus, so it is necessary to develop a plant composition to inhibit Staphylococcus aureus to improve skin lesions, itching and other symptoms in children with atopic dermatitis.

[0005] The quorum sensing (QS) system is a signal molecule called autoinducer (AI) synthesized and released by bacteria. When the critical concentration is reached, AI can bind to and activate intracellular receptors, start the expression of related genes in the bacteria, and thus regulate the physiological behavior of bacteria, including non-specific resistance mechanisms such as biofilms. Staphylococcus aureus communicates information through the LuxS / AI-2QS signaling system. The synthesis of AI-2 signaling molecules mainly depends on the LuxS protease encoded by the LuxS gene. Baicalein binds to the amino acid residues of LuxS through hydrogen bonds, which can significantly reduce the formation of biofilms and destroy mature biofilms. At the same time, it downregulates the expression of LuxS and biofilm-related gene mRNA, achieving the purpose of effectively inhibiting the growth and reproduction of Staphylococcus aureus.

[0006] Penicillin-binding proteins (PBPs) are a class of membrane proteins widely present on the surface of bacterial cell membranes and are the main targets of β-lactam drugs. PBP2a encoded by MecA has a very low affinity for β-lactam antibiotics. When other PBPs proteins are tightly bound to β-lactam drugs, PBP2a can replace these PBPs to perform functions, maintain the stability of bacterial cell peptidoglycan, and enable bacteria to grow and reproduce normally, showing drug resistance. Ursolic acid can displace PBP2, thereby interfering with the synthesis of peptidoglycan and destroying the formation of the cell wall of Staphylococcus aureus.

[0007] Intercellular adhesion protein A (icaA) and fibronectin A (fnbA) are components of the biofilm matrix of Staphylococcus aureus. Shikonin can significantly downregulate the gene expression of surface adhesion proteins such as icaA and fnbA. Shikonin can also significantly inhibit the expression of PBP2a protein and also show a significant inhibitory effect on the transcription level of mecA. Summary of the invention

[0008] The embodiment of the present invention provides a plant composition for treating atopic dermatitis. The plant composition is a mixture of baicalin extract, ursolic acid extract and shikonin extract. The components have a synergistic effect and can effectively inhibit the growth and reproduction of Staphylococcus aureus.

[0009] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0010] A plant composition for treating atopic dermatitis comprises a baicalin extract, an ursolic acid extract and a shikonin extract in a mixing mass ratio of (1-30):(1-20):(1-20), wherein the baicalin content in the baicalin extract is not less than 70%, the ursolic acid content in the ursolic acid extract is not less than 60%, and the shikonin content in the shikonin extract is not less than 30%.

[0011] Furthermore, the preparation method of the baicalein extract comprises:

[0012] Taking dried baicalein-containing plants, grinding them and sieving them to obtain baicalein-containing ground materials;

[0013] The baicalin extract is obtained by extracting the baicalin-containing powder by the immersion method in the ethanol extraction method, and then rotary distilling and concentrating the powder to obtain the baicalin extract;

[0014] The pH of the baicalein extract is adjusted to 2-4 with an acid, and then allowed to stand for 4-8 hours, then washed with water until neutral, and then the pH of the baicalein extract is adjusted to 7-9 with an alkali, and then allowed to stand for 30-60 minutes, and the supernatant is taken after centrifugation and adjusted to neutrality, and the filtrate is taken after filtering and rotary evaporation and concentration to obtain a crude baicalein product;

[0015] The crude baicalein product is subjected to enzymatic hydrolysis, alcohol precipitation and filtration in sequence, and the filtrate is subjected to rotary evaporation, concentration and vacuum drying to obtain a baicalein-containing extract.

[0016] Furthermore, the plant containing baicalein is at least one of Scutellaria baicalensis, Cyperus rotundus, Oroxylum indica, and Plantago asiatica.

[0017] Furthermore, the immersion method comprises adding no less than 50% ethanol to the baicalein-containing crushed material, extracting for no less than 2 hours and filtering, and if the extraction is performed multiple times, combining the filtrates from multiple times.

[0018] Furthermore, the enzymatic hydrolysis includes adding β-glucosidase or cellulase to the crude baicalein under the conditions of 40-60° C. and pH 6-7 for 2-3 hours.

[0019] Furthermore, the alcohol precipitation comprises adding the liquid after enzymatic hydrolysis into an ethanol solution of not less than 90% for alcohol precipitation for 1-4 hours.

[0020] Furthermore, the preparation method of the ursolic acid extract comprises:

[0021] Taking the dried ursolic acid-containing plant, crushing it and then sieving it to obtain a crushed material containing ursolic acid;

[0022] Add 30%-60% ethanol to the crushed material containing ursolic acid, heat to 50-60°C, reflux for 1-2 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0023] Add 30-70 times of ethanol not less than 90% to the ursolic acid extract, heat to 40-60°C and extract for 30-60 minutes, filter and adsorb with macroporous resin, wash and elute after saturated adsorption, collect the eluate, perform rotary evaporation, concentrate and vacuum dry to obtain an ursolic acid extract.

[0024] Furthermore, the plant containing ursolic acid is at least one of loquat, oldenlandia diffusa, privet, selfheal, and bearberry.

[0025] Furthermore, the preparation method of the shikonin-containing extract comprises:

[0026] The dried shikonin-containing plant is crushed and sieved to obtain a crushed material containing shikonin;

[0027] The shikonin-containing crushed material is extracted with CO2 supercritical fluid for 2-3 hours to obtain an oily substance;

[0028] The oily substance is soaked in petroleum ether for 2-4 hours, then heated to 40-50°C and extracted for 30-60 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain an extract containing shikonin.

[0029] The plant composition for treating atopic dermatitis according to claim 9, characterized in that the plant containing shikonin is at least one of Lithospermum officinale, Lithospermum xinjiangensis, and Lithospermum nemerongsi.

[0030] The embodiments of the present invention have the following advantages:

[0031] A plant composition for treating atopic dermatitis of the present invention is composed of a baicalin extract, an ursolic acid extract and a shikonin extract, wherein the main effective ingredients are baicalin, ursolic acid and shikonin, which cooperate with each other to have a synergistic effect, inhibit the biofilm development of Staphylococcus aureus, destroy the formed biofilm, interfere with the synthesis of peptidoglycan, inhibit the formation of cell walls, and effectively alleviate the skin itching problem caused by Staphylococcus aureus; at the same time, the inflammatory factors in the body are reduced and the skin inflammatory reaction is inhibited. Moreover, the plant composition is obtained by extracting natural plants, has a high effective content, and is safer and milder than chemical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0033] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0034] Figure 1 This is a data graph of Experiment 3 of a plant composition for treating atopic dermatitis provided by an embodiment of the present invention;

[0035] Figure 2 This is a data graph of Experiment 3 of a plant composition for treating atopic dermatitis provided by an embodiment of the present invention;

[0036] Figure 3 This is a data graph of Experiment 3 of a plant composition for treating atopic dermatitis provided by an embodiment of the present invention;

[0037] Figure 4 This is a data graph of Experiment 3 of a plant composition for treating atopic dermatitis provided by an embodiment of the present invention;

[0038] Figure 5 This is a data graph of Experiment 3 of a plant composition for treating atopic dermatitis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] A plant composition for treating atopic dermatitis, the preparation method of which comprises the steps of taking dried plants containing baicalin, plants containing ursolic acid and plants containing shikonin, crushing them and then sieving them to obtain baicalin-containing crushed material, ursolic acid-containing crushed material and shikonin-containing crushed material respectively. It is worth noting that the baicalein-containing plants, ursolic acid-containing plants and shikonin-containing plants involved in the present technology do not refer to a specific part of the above-mentioned plants, but can be any one or more combinations of roots, stems, leaves and other parts, and can also be derived from chemical synthesis or plant tissue culture. For example, the baicalein-containing plant is at least one of Scutellaria baicalensis, Herba Cynomorii, Oroxylum indicum, and Plantago asiatica. In the following embodiments, the root of Scutellaria baicalensis is used as the baicalein-containing plant; the ursolic acid-containing plant is at least one of loquat, Hedyotis diffusa, Ligustrum lucidum, Prunella vulgaris, and bearberry. In the following embodiments, loquat leaves are used as the ursolic acid-containing plant; the shikonin-containing plant is at least one of Lithospermum officinale, Lithospermum xinjiang, and Lithospermum nemeronggensis. In the following embodiments, the root of Lithospermum officinale is used as the shikonin-containing plant.

[0041] Example 1

[0042] 1. The method of extracting baicalin-containing extract from the root of Scutellaria baicalensis includes:

[0043] a. Grind the root of Scutellaria baicalensis and pass it through a 40-mesh sieve to obtain a ground root of Scutellaria baicalensis;

[0044] b. According to the immersion method in the ethanol extraction method, 50% ethanol is added to the crushed root of Scutellaria baicalensis, and the mixture is extracted for 2 hours and filtered. If the extraction is repeated multiple times, the filtrates are combined and then rotary evaporated and concentrated to obtain baicalin extract. This step is used to reduce the volume of the solution to facilitate the subsequent washing process.

[0045] c. Using HCl, the pH of the baicalein extract was adjusted to 3, and then allowed to stand for 6 hours, then washed with water until neutral, and then using NaOH to adjust the pH of the baicalein extract to 8, and then allowed to stand for 30 minutes. After centrifugation, the supernatant was adjusted to neutral, and concentrated by rotary evaporation to obtain a crude baicalein product;

[0046] d. The crude baicalein product was hydrolyzed by adding β-glucosidase at 50° C. and pH 7 for 3 hours. Cellulase can also be used in this embodiment. A 90% ethanol solution was added to the hydrolyzed solution for alcohol precipitation for 3 hours, and then the filtrate was filtered and concentrated by rotary evaporation and vacuum dried to obtain a scutellaria root extract. The baicalein content in the baicalein extract in this embodiment was 91.62%.

[0047] 2. The method of extracting ursolic acid extract from loquat leaves includes:

[0048] a. Grind the loquat leaves and pass them through a 40-mesh sieve to obtain loquat leaf crushed material;

[0049] b. Add 60% ethanol to the loquat leaf powder, heat and reflux at 60° C. for 2 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0050] c. Add 70 times 90% ethanol to the ursolic acid extract, heat to 60°C and extract for 60 minutes, filter and adsorb with macroporous resin, wash with water after the macroporous resin column is saturated with adsorption, elute with 95% ethanol by volume, preferably 4BV / h by elution rate, collect the eluate for rotary evaporation, concentrate, and vacuum dry to obtain an ursolic acid extract, wherein the ursolic acid content in the ursolic acid extract is 79.67%. In this embodiment, the macroporous resin is macroporous resin S-8, preferably a macroporous resin adsorption column.

[0051] 3. The method of extracting shikonin-containing extract using lithospermum officinale root includes:

[0052] a. Grind the lithospermum officinale root and pass it through a 40-mesh sieve to obtain a lithospermum officinale root grind;

[0053] b. The ground lithospermum erythrorhizon was extracted with CO2 supercritical fluid for 2 hours to obtain an oily substance;

[0054] c. The oily substance was soaked in petroleum ether for 4 hours, then heated to 50° C. and extracted for 30 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain a shikonin-containing extract, wherein the shikonin content of the shikonin-containing extract was 40.73%.

[0055] 4. The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), thereby obtaining a plant composition for treating atopic dermatitis.

[0056] Example 2

[0057] 1. The method of extracting baicalin-containing extract from the root of Scutellaria baicalensis includes:

[0058] a. Grind the root of Scutellaria baicalensis and pass it through a 40-mesh sieve to obtain a ground root of Scutellaria baicalensis;

[0059] b. According to the immersion method in the ethanol extraction method, add no less than 60% ethanol to the crushed Scutellaria root, extract for 3 hours, and filter. If the extraction is repeated multiple times, combine the filtrates and evaporate and concentrate them to obtain baicalin extract. This step is used to reduce the volume of the solution to facilitate the subsequent washing process.

[0060] c. Using HCl to adjust the pH of the baicalein extract to 2 and then standing for 8 hours, then washing with water until neutral, then using NaOH to adjust the pH of the baicalein extract to 8 and standing for 60 minutes, centrifuging and adjusting the supernatant to neutral, and concentrating by rotary evaporation to obtain a crude baicalein product;

[0061] d. The crude baicalein product was hydrolyzed by adding β-glucosidase at 40°C and pH 7 for 2 hours. Cellulase can also be used in this embodiment. A 90% ethanol solution was added to the hydrolyzed solution for alcohol precipitation for 2 hours, and then the filtrate was filtered and concentrated by rotary evaporation and vacuum dried to obtain a scutellaria root extract. The baicalein content in the baicalein extract in this embodiment was 87.15%.

[0062] 2. The method of extracting ursolic acid extract from loquat leaves includes:

[0063] b. Grinding the loquat leaves and passing them through a 40-mesh sieve to obtain loquat leaf pulverizer;

[0064] b. Add 30% ethanol to the loquat leaf powder, heat and reflux at 50° C. for 1.5 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0065] c. Add 50 times 90% ethanol to the ursolic acid extract, heat to 40°C and extract for 30 minutes, filter and adsorb with macroporous resin, wash with water after the macroporous resin column is saturated with adsorption, elute with 98% ethanol by volume, preferably at a rate of 4 BV / h, collect the eluate for rotary evaporation, concentration, and vacuum drying to obtain an ursolic acid extract, wherein the ursolic acid content in the ursolic acid extract is 73.16%. In this embodiment, the macroporous resin is macroporous resin S-8, preferably a macroporous resin adsorption column.

[0066] 3. The method of extracting shikonin-containing extract using lithospermum officinale root includes:

[0067] a. Grind the lithospermum officinale root and pass it through a 40-mesh sieve to obtain a lithospermum officinale root grind;

[0068] b. The ground lithospermum erythrorhizon was extracted with CO2 supercritical fluid for 3 hours to obtain an oily substance;

[0069] c. The oily substance was soaked in petroleum ether for 3 hours, then heated to 40° C. and extracted for 60 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain a shikonin-containing extract, wherein the shikonin content of the shikonin-containing extract was 38.19%.

[0070] 4. The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), thereby obtaining a plant composition for treating atopic dermatitis.

[0071] Example 3

[0072] 1. The method of extracting baicalin-containing extract from the root of Scutellaria baicalensis includes:

[0073] a. Grind the root of Scutellaria baicalensis and pass it through a 60-mesh sieve to obtain a ground root of Scutellaria baicalensis;

[0074] b. According to the immersion method in the ethanol extraction method, 50% ethanol is added to the crushed root of Scutellaria baicalensis, and the mixture is extracted for 2 hours and filtered. If the extraction is repeated multiple times, the filtrates are combined and then rotary evaporated and concentrated to obtain baicalin extract. This step is used to reduce the volume of the solution to facilitate the subsequent washing process.

[0075] c. Using HCl, the pH of the baicalein extract was adjusted to 4, and then allowed to stand for 4 hours, then washed with water until neutral, and then using NaOH to adjust the pH of the baicalein extract to 9, and then allowed to stand for 30 minutes. After centrifugation, the supernatant was adjusted to neutral, and concentrated by rotary evaporation to obtain a crude baicalein product;

[0076] d. The crude baicalein product was hydrolyzed by adding β-glucosidase at 60° C. and pH 6 for 3 hours. Cellulase can also be used in this embodiment. A 90% ethanol solution was added to the hydrolyzed solution for alcohol precipitation for 1 hour, and then the filtrate was filtered and concentrated by rotary evaporation and vacuum dried to obtain a scutellaria root extract. The baicalein content in the baicalein extract in this embodiment was 84.11%.

[0077] 2. The method of extracting ursolic acid extract from loquat leaves includes:

[0078] c. Grinding the loquat leaves and passing them through a 60-mesh sieve to obtain loquat leaf crushed material;

[0079] b. Add 50% ethanol to the loquat leaf powder, heat and reflux at 60° C. for 2 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0080] c. Add 50 times 95% ethanol to the ursolic acid extract, heat to 40°C and extract for 60 minutes, filter and adsorb with macroporous resin, wash with water after the macroporous resin column is saturated with adsorption, elute with 95% ethanol by volume, preferably at a rate of 4 BV / h, collect the eluate for rotary evaporation, concentration, and vacuum drying to obtain an ursolic acid extract, wherein the ursolic acid content in the ursolic acid extract is 69.84%. In this embodiment, the macroporous resin is macroporous resin S-8, preferably a macroporous resin adsorption column.

[0081] 3. The method of extracting shikonin-containing extract using lithospermum officinale root includes:

[0082] a. Grind the lithospermum officinale root and pass it through a 60-mesh sieve to obtain a pulverized lithospermum officinale root;

[0083] b. The ground lithospermum erythrorhizon was extracted with CO2 supercritical fluid for 2 hours to obtain an oily substance;

[0084] c. The oily substance was soaked in petroleum ether for 2 hours, then heated to 50° C. and extracted for 30 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain a shikonin-containing extract, wherein the shikonin content of the shikonin-containing extract was 37.05%.

[0085] 4. The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), thereby obtaining a plant composition for treating atopic dermatitis.

[0086] Example 4

[0087] 1. The method of extracting baicalin-containing extract from the root of Scutellaria baicalensis includes:

[0088] a. Grind the root of Scutellaria baicalensis and pass it through a 60-mesh sieve to obtain a ground root of Scutellaria baicalensis;

[0089] b. According to the immersion method in the ethanol extraction method, 50% ethanol is added to the crushed root of Scutellaria baicalensis, and the mixture is extracted for 2 hours and filtered. If the extraction is repeated multiple times, the filtrates are combined and then rotary evaporated and concentrated to obtain baicalin extract. This step is used to reduce the volume of the solution to facilitate the subsequent washing process.

[0090] c. Using HCl, the pH of the baicalein extract was adjusted to 4, and then allowed to stand for 4 hours, then washed with water until neutral, and then using NaOH to adjust the pH of the baicalein extract to 9, and then allowed to stand for 30 minutes. After centrifugation, the supernatant was adjusted to neutral, and concentrated by rotary evaporation to obtain a crude baicalein product;

[0091] d. The crude baicalein product was hydrolyzed by adding β-glucosidase at 60° C. and pH 7 for 2 hours. Cellulase can also be used in this embodiment. A 90% ethanol solution was added to the hydrolyzed solution for alcohol precipitation for 4 hours, and then the filtrate was filtered and concentrated by rotary evaporation and vacuum dried to obtain a scutellaria root extract. The baicalein content in the baicalein extract in this embodiment was 83.92%.

[0092] 2. The method of extracting ursolic acid extract from loquat leaves includes:

[0093] d. Grinding the loquat leaves and passing them through a 60-mesh sieve to obtain loquat leaf pulverized material;

[0094] b. Add 60% ethanol to the loquat leaf powder, heat and reflux at 60° C. for 2 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0095] c. Add 70 times 90% ethanol to the ursolic acid extract, heat to 60°C and extract for 60 minutes, filter and adsorb with macroporous resin, wash with water after the macroporous resin column is saturated with adsorption, elute with 95% ethanol by volume, preferably 4BV / h by elution rate, collect the eluate for rotary evaporation, concentrate, and vacuum dry to obtain an ursolic acid extract, wherein the ursolic acid content in the ursolic acid extract is 68.31%. In this embodiment, the macroporous resin is macroporous resin S-8, preferably a macroporous resin adsorption column.

[0096] 3. The method of extracting shikonin-containing extract using lithospermum officinale root includes:

[0097] a. Grind the lithospermum officinale root and pass it through a 60-mesh sieve to obtain a pulverized lithospermum officinale root;

[0098] b. The ground lithospermum erythrorhizon was extracted with CO2 supercritical fluid for 3 hours to obtain an oily substance;

[0099] c. The oily substance was soaked in petroleum ether for 2 hours, then heated to 40° C. and extracted for 60 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain a shikonin-containing extract, wherein the shikonin content of the shikonin-containing extract was 35.19%.

[0100] 4. The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), thereby obtaining a plant composition for treating atopic dermatitis.

[0101] Example 5

[0102] 1. The method of extracting baicalin-containing extract from the root of Scutellaria baicalensis includes:

[0103] a. Grind the root of Scutellaria baicalensis and pass it through an 80-mesh sieve to obtain a ground root of Scutellaria baicalensis;

[0104] b. According to the immersion method in the ethanol extraction method, 60% ethanol is added to the crushed root of Scutellaria baicalensis, and the mixture is extracted for 3 hours and filtered. If the extraction is repeated multiple times, the filtrates are combined and then rotary evaporated and concentrated to obtain baicalin extract. This step is used to reduce the volume of the solution to facilitate the subsequent washing process.

[0105] c. Using HCl, the pH of the baicalein extract was adjusted to 3, and then allowed to stand for 4 hours, then washed with water until neutral, and then using NaOH to adjust the pH of the baicalein extract to 8, and then allowed to stand for 60 minutes. After centrifugation, the supernatant was adjusted to neutral, and concentrated by rotary evaporation to obtain a crude baicalein product;

[0106] d. The crude baicalein product was hydrolyzed by adding β-glucosidase at 40° C. and pH 7 for 3 hours. Cellulase can also be used in this embodiment. A 90% ethanol solution was added to the hydrolyzed solution for alcohol precipitation for 3 hours, and then the filtrate was filtered and concentrated by rotary evaporation and vacuum dried to obtain a scutellaria root extract. The baicalein content in the baicalein extract in this embodiment was 78.94%.

[0107] 2. The method of extracting ursolic acid extract from loquat leaves includes:

[0108] e. Grinding the loquat leaves and passing them through an 80-mesh sieve to obtain loquat leaf pulverizer;

[0109] b. Add 60% ethanol to the loquat leaf powder, heat and reflux at 50° C. for 1 hour, and then concentrate by rotary evaporation to obtain ursolic acid extract;

[0110] c. Add 60 times 90% ethanol to the ursolic acid extract, heat to 60°C and extract for 30 minutes, filter and adsorb with macroporous resin, wash with water after the macroporous resin column is saturated with adsorption, elute with 95% ethanol by volume, preferably 4BV / h by elution rate, collect the eluate for rotary evaporation, concentrate, and vacuum dry to obtain an ursolic acid extract, wherein the ursolic acid content in the ursolic acid extract is 65.04%. In this embodiment, the macroporous resin is macroporous resin S-8, preferably a macroporous resin adsorption column.

[0111] 3. The method of extracting shikonin-containing extract using lithospermum officinale root includes:

[0112] a. Grind the lithospermum officinale root and pass it through an 80-mesh sieve to obtain a pulverized lithospermum officinale root;

[0113] b. The ground lithospermum erythrorhizon was extracted with CO2 supercritical fluid for 2 hours to obtain an oily substance;

[0114] c. The oily substance was soaked in petroleum ether for 3 hours, then heated to 50° C. and extracted for 30 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain a shikonin-containing extract, wherein the shikonin content of the shikonin-containing extract was 32.76%.

[0115] 4. The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), thereby obtaining a plant composition for treating atopic dermatitis.

[0116] Experiment 1

[0117] Taking the above Example 1 as an example, the micro-checkerboard dilution method is used to detect the MIC of the active ingredient in a plant composition for treating atopic dermatitis. The specific steps are as follows:

[0118] Preparation of components: Take 256 mg of baicalin, 256 mg of ursolic acid, and 256 mg of shikonin, dissolve them in 10 ml of DMSO to form a stock solution, mix them thoroughly with a vortex instrument until the liquid is clear, and store them in a -20°C refrigerator for later use.

[0119] Preparation of bacterial solution: Take the frozen Staphylococcus aureus out of the -20℃ refrigerator and place it on a sterile operating table at room temperature to completely thaw. After it is completely thawed, mix it evenly with a vortexer, then use a pipette to draw 100μL of bacterial solution and add it to 20mL of freshly prepared MH broth medium, shake it evenly in a 37℃ constant temperature shaker and culture it overnight. Use a bacterial turbidity meter to measure the bacterial turbidity and dilute the bacterial turbidity to 1×10 6 CFU / mL.

[0120] Single-component MIC value determination: Add serially diluted drug stock solution to a 96-well plate, add 100 μL of drug stock solution to each well and the final concentration is 5×10 5 CFU / mL bacterial suspension 100μL, each drug concentration for 3 replicates. Select three other wells, one well with 1% DMSO as a control, one well with 200μL blank culture medium as a control, one well with 200μL bacterial solution and blank culture medium as a positive control. After culturing in a 37℃ incubator for 18h, put it in an ELISA reader to read the results, use the ELISA reader to read the MIC endpoint combined with naked eye observation, the single component results are as follows:

[0121] The MIC value of baicalein was 64 μg / mL;

[0122] The MIC value of ursolic acid was 16 μg / mL;

[0123] The MIC value of shikonin was 32 μg / mL.

[0124] Determination of the combined MIC value of the two components: set the final concentration gradient of 2, 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32 and 1 / 64 MIC for the two drugs respectively. Add a final concentration of 5×10 5 CFU / mL, 100 μL of 8×8 combined bacterial suspension was added, 50 μL of various concentrations of A solution was added, and finally 50 μL of various concentrations of B solution was added. In addition, to avoid contamination, six wells were selected, three wells were used as negative control wells (200 μL of blank culture medium), and the other three wells were used as positive control wells (100 μL of the final concentration of 5×10 5 CFU / mL bacterial solution and 100μL blank culture medium). Finally, the 96-well plate was placed on a shaker and shaken for 5 minutes to fully mix the drug solution and bacterial solution. After culturing in a 37°C incubator for 18 hours, the plate was placed in an ELISA reader to read the results. The MIC endpoint of the combination of the two drugs was determined by the ELISA reader and combined with visual observation. Finally, the concentration of the two drugs that produced the best effect was determined, and the partial inhibitory concentration index (FICI) was calculated. Each group of experiments was repeated three times, and the results of the combination of the two components were as follows:

[0125] The MIC values ​​of baicalin combined with ursolic acid were 16 μg / mL and 4 μg / mL, respectively;

[0126] The MIC values ​​of baicalin and shikonin combined were 8 μg / mL and 4 μg / mL, respectively;

[0127] The MIC values ​​of ursolic acid combined with shikonin were 2μg / mL and 4μg / mL, respectively.

[0128] Determination of MIC value of three-component combination: Using the same two-component combination method, the MIC values ​​of baicalin, ursolic acid and shikonin combined were 4μg / mL, 1μg / mL and 1μg / mL, respectively.

[0129] According to the MIC value of the combined use of the three drugs obtained above, the final ratio of baicalin extract, ursolic acid extract and shikonin extract is 7:2:4. Baicalin extract, ursolic acid extract and shikonin extract, butanediol, 1,2-pentanediol and 1,2-hexanediol are mixed in a certain weight ratio, deionized water is added to 100%, and the mixture is stirred and dissolved evenly to obtain the plant composition.

[0130] A botanical composition for treating atopic dermatitis in this embodiment uses the checkerboard dilution method to accurately reflect the interaction between the effective ingredients of the three plant extracts. The combined inhibition concentration of the three drugs is much lower than the inhibition concentration of each drug alone, and Staphylococcus aureus is inhibited at an optimal low concentration, avoiding drug toxicity caused by high-dose concentrations.

[0131] Experiment 2

[0132] The plant composition for treating atopic dermatitis in Example 1 was used as a model group to test the antibacterial rate of Staphylococcus aureus. The specific test results are as follows:

[0133] (1) preparing a Staphylococcus aureus suspension;

[0134] (2) Take 0.1 ml of Staphylococcus aureus suspension and add it to 9.9 ml PBS (0.05 mol / L phosphate buffer solution) for dilution. The final concentration of Staphylococcus aureus is 5 × 10 5 CFU / mL;

[0135] (3) The model group was diluted with sterile water to different concentrations (5%, 2%, 1%, and 0.5%);

[0136] (4) Pipette 5 ml of the plant composition of different concentrations into sterilized test tubes;

[0137] (5) Pipette 0.1 ml of Staphylococcus aureus solution into the above test tube and shake well;

[0138] (6) After 30 min, take 0.5 ml of the mixture and add it to a test tube containing 4.5 ml of sterilized PBS and mix thoroughly;

[0139] (7) Pipette 1 ml of the above sample solution into a sterile culture dish, inoculate three culture dishes with each sample solution, and culture in a 37°C incubator for 72 h before counting the number of live bacterial colonies;

[0140] (8) PBS was used instead of the above plant extracts for control experiments;

[0141] (9) The experiment was repeated three times.

[0142] Calculation formula

[0143] Antibacterial rate (%) = (A1-A2) / A1×100%

[0144] A1: Average colony count of the control group

[0145] A2: Average colony count in the model group

[0146] Table 1 Antibacterial rate of Staphylococcus aureus of plant compositions at different concentrations

[0147]

[0148] Experiment 3

[0149] The plant composition for treating atopic dermatitis in Example 1 was used as a model group for the experiment. The specific experimental process and experimental data are as follows:

[0150] 1. Animal modeling and drug administration

[0151] After 1 week of adaptive feeding, 40 6-week-old male BALB / c mice were randomly divided into 4 groups, each with 10 mice, namely, blank group, model group, low-dose drug group and high-dose drug group. The back hair (3cm×3cm) was removed with a razor and depilatory cream 1 day before the experiment. On the 1st, 4th and 7th days of the experiment, 100μL of 1% DNCB solution (DNCB dissolved in acetone: olive oil = 3:1, v / v) was applied to the depilated area on the back of the model group and the drug group for sensitization; on the 14th, 17th, 19th, 22nd, 24th, 27th and 29th days of the experiment, 100μL of 0.5% DNCB solution was applied to the same part, and 20μL was applied to each ear; the blank group was applied with the same amount of acetone olive oil solvent at the same time point. When the mice showed severe itching, obvious eczematous changes and dry skin symptoms, the model was successfully established. After successful modeling, the mice were smeared with 200 μL of 1% plant composition (dissolved in saline) in the low-dose group, 200 μL of 2% plant composition (dissolved in saline) in the high-dose group, and the blank group and model group were smeared with the same amount of saline. The application was repeated twice a day for 7 days.

[0152] 2. Scoring of skin lesions on the back of mice

[0153] The severity of inflammation in the skin lesions of mice was scored. After the experiment, the severity of the back eczema symptoms (erythema, excoriation, scaling, edema) was scored as 0 (none), 1 (mild), 2 (moderate) and 3 (severe). The final score was the sum of the four symptoms. The higher the score, the more severe the skin damage.

[0154] Mouse scratching frequency measurement

[0155] Two hours after the last administration, the mice were returned to the observation cage, and the number of times each mouse scratched its own ears and back within 30 minutes was recorded by a camera, with continuous scratching counted as one time.

[0156] 4. Serum inflammatory factor levels

[0157] After the observation experiment, the mice were enucleated and blood was collected. The blood was then centrifuged at 3000 rpm / min for 15 min and the serum was collected. The levels of IL-4, IL-6 and IgE in the serum were determined according to the ELISA kit operating procedures.

[0158] Experimental results:

[0159] It can be seen from Table 2 that Figure 1-5 As shown in Table 3, compared with the model group, the symptoms of atopic dermatitis on the back of mice in the drug administration group were improved, and the skin lesion score was significantly reduced (p < 0.01); after drug administration, the number of scratching times of mice in the drug administration group was significantly reduced (p < 0.01), indicating that the plant composition of the present invention has a good improvement effect on the skin lesions and itching and scratching symptoms of mice with atopic dermatitis. As shown in Table 3, after drug administration, the inflammatory factors in the serum of mice with atopic dermatitis were significantly reduced (p < 0.01), indicating that the plant composition of the present invention can improve the inflammatory response of mice with atopic dermatitis. The therapeutic effect of the high-dose administration group is better than that of the low-dose administration group.

[0160] Table 2 Comparison of skin lesion scores and scratching times in mice (n=10)

[0161]

[0162] Note: Compared with the blank group: ** p<0.01; compared with the model group, ## p<0.01.

[0163] Table 3 IL-4, IL-6, and IgE levels in mouse serum (n=10)

[0164]

[0165] Note: Compared with the blank group: ** p<0.01; compared with the model group, ## p<0.01.

[0166] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A plant composition for treating atopic dermatitis, characterized in that: The mixing mass ratio of the baicalin extract, the ursolic acid extract and the shikonin extract is (1-30):(1-20):(1-20), the baicalin content in the baicalin extract is not less than 70%; the ursolic acid content in the ursolic acid extract is not less than 60%; the shikonin content in the shikonin extract is not less than 30%.

2. A plant composition for treating atopic dermatitis according to claim 1, characterized in that: The preparation method of the baicalein extract comprises: Taking dried baicalein-containing plants, grinding them and sieving them to obtain baicalein-containing ground materials; The baicalin extract is obtained by extracting the baicalin-containing powder by the immersion method in the ethanol extraction method, and then rotary distilling and concentrating the powder to obtain the baicalin extract; The pH of the baicalein extract is adjusted to 2-4 with an acid, and then allowed to stand for 4-8 hours, then washed with water until neutral, and then the pH of the baicalein extract is adjusted to 7-9 with an alkali, and then allowed to stand for 30-60 minutes, and the supernatant is taken after centrifugation and adjusted to neutrality, and the filtrate is taken after filtering and rotary evaporation and concentration to obtain a crude baicalein product; The crude baicalein product is subjected to enzymatic hydrolysis, alcohol precipitation and filtration in sequence, and the filtrate is subjected to rotary evaporation, concentration and vacuum drying to obtain a baicalein-containing extract.

3. A plant composition for treating atopic dermatitis according to claim 2, characterized in that: The baicalein-containing plant is at least one of Scutellaria baicalensis, Cyperus rotundus, Oroxylum indica, and Plantago asiatica.

4. A plant composition for treating atopic dermatitis according to claim 2, characterized in that: The immersion method comprises adding no less than 50% ethanol to the baicalein-containing crushed material, extracting for no less than 2 hours and filtering, and if the extraction is repeated for multiple times, combining the filtrates of the multiple times.

5. A plant composition for treating atopic dermatitis according to claim 2, characterized in that: The enzymatic hydrolysis comprises adding beta-glucosidase or cellulase to the crude baicalein under the conditions of 40-60° C. and pH 6-7 for 2-3 hours.

6. A plant composition for treating atopic dermatitis according to claim 2, characterized in that: The alcohol precipitation comprises adding the liquid after enzymatic hydrolysis into an ethanol solution of not less than 90% for alcohol precipitation for 1-4 hours.

7. A plant composition for treating atopic dermatitis according to claim 1, characterized in that: The preparation method of the ursolic acid extract comprises: Taking the dried ursolic acid-containing plant, crushing it and then sieving it to obtain a crushed material containing ursolic acid; Add 30%-60% ethanol to the crushed material containing ursolic acid, heat to 50-60°C, reflux for 1-2 hours, and then concentrate by rotary evaporation to obtain ursolic acid extract; Add 30-70 times of ethanol not less than 90% to the ursolic acid extract, heat to 40-60°C and extract for 30-60 minutes, filter and adsorb with macroporous resin, wash and elute after saturated adsorption, collect the eluate, perform rotary evaporation, concentrate and vacuum dry to obtain an ursolic acid extract.

8. A plant composition for treating atopic dermatitis according to claim 7, characterized in that: The plant containing ursolic acid is at least one of loquat, oldenlandia diffusa, privet, selfheal and bearberry.

9. A plant composition for treating atopic dermatitis according to claim 1, characterized in that: The preparation method of the shikonin-containing extract comprises: The dried shikonin-containing plant is crushed and sieved to obtain a crushed material containing shikonin; The shikonin-containing crushed material is extracted with CO2 supercritical fluid for 2-3 hours to obtain an oily substance; The oily substance is soaked in petroleum ether for 2-4 hours, then heated to 40-50°C and extracted for 30-60 minutes, and then filtered, rotary evaporated, concentrated, and vacuum dried to obtain an extract containing shikonin.

10. A plant composition for treating atopic dermatitis according to claim 9, characterized in that: The shikonin-containing plant is at least one of lithospermum officinale, lithospermum officinale from Xinjiang and lithospermum officinale from Inner Mongolia.

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