Traditional Chinese medicine composition for inhibiting hyaluronidase activity and hyaluronidase inhibitor

Abstract

The application discloses a traditional Chinese medicine composition for inhibiting hyaluronidase activity and a hyaluronidase inhibitor. The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises 1-12 parts of Mirabilis jalapa, 1-12 parts of Cnidium monnieri and 1-6 parts of Coptis chinensis by weight. The hyaluronidase inhibitor comprises 1-2 parts of Tween, 2-5 parts of lecithin, 0.1-0.5 parts of cholesterol, 40-80 parts of the traditional Chinese medicine composition and 80-100 parts of deionized water by weight. The traditional Chinese medicine composition and the hyaluronidase inhibitor can effectively inhibit the activity of hyaluronidase. The combination of Mirabilis jalapa, Cnidium monnieri and Coptis chinensis can effectively inhibit the activity of hyaluronidase. Mirabilis jalapa, Cnidium monnieri and Coptis chinensis have a remarkable synergistic effect on the activity of hyaluronidase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to a traditional Chinese medicine composition for inhibiting hyaluronidase activity and a hyaluronidase inhibitor. BACKGROUND

[0002] In recent years, allergic diseases are increasing, and the factors inducing allergic reactions are also increasing, which have become common and frequently-occurring diseases. Allergy is caused by the entry of foreign substances into the human body, and the immune system of the body recognizes such substances as foreign substances, causing the immune system to be disorderly, and causing symptoms such as allergy and inflammation. Allergic reactions can be divided into I, II, III and IV types according to their mechanisms, and most allergic diseases are type I allergic reactions related to hyaluronidase activity in the body.

[0003] Hyaluronidase refers to a class of glycosidases that can degrade hyaluronic acid and part of glycosaminoglycans into disaccharides or small molecular oligosaccharides.

[0004] Modern research shows that hyaluronidase is the main component of the extracellular matrix of connective tissue cells, and is related to most IgE-mediated type I and T cell-mediated type IV allergic reactions, and in vitro hyaluronidase inhibition experiment is an effective method for rapid screening of anti-allergic preparations in vitro.

[0005] Physalis alkekengi L. var. franchetii (Mast.) Makino, also known as hanging lantern, is the fruit of Physalis alkekengi L. var. franchetii (Mast.) Makino in Solanaceae. It tastes sour, bitter and cold, and belongs to the lung meridian. Functions: clearing heat, benefiting the throat, resolving phlegm and promoting urination. Indications: for sore throat, hoarseness, phlegm-heat cough, difficulty in urination, hot and painful urination; external treatment of pemphigus and eczema.

[0006] Cnidium, the fruit of Cnidium officinale Makino in Umbelliferae, tastes bitter and warm, and enters the kidney and spleen meridians. Functions: warming the kidney and assisting yang, dispelling wind, drying dampness and killing insects. Indications: male impotence, scrotal itching, female leukorrhagia and vaginal itching, uterine coldness and infertility, rheumatism, tinea and eczema.

[0007] Rhizoma Coptidis, the rhizome of Coptis chinensis Franch or Coptis deltoidea C. Y. Wu ex Chow in Ranunculaceae, tastes bitter and cold, and enters the heart, liver, stomach and large intestine meridians. Functions: clearing heat, purging fire, drying dampness, detoxifying and killing insects. Indications: epidemic febrile disease, cold injury, heat in the heart, distension and vomiting, bacillary dysentery, hot diarrhea, pulmonary tuberculosis, vomiting and hematochezia, diabetes, malnutrition, ascariasis, whooping cough, sore throat, aphthous ulcer, carbuncle and eczema.

[0008] Currently, there is no patent or literature that combines Physalis alkekengi L. var. franchetii (Mast.) Makino, Cnidium officinale Makino and Rhizoma Coptidis to inhibit the activity of hyaluronidase. SUMMARY

[0009] The present application provides a traditional Chinese medicine composition for inhibiting hyaluronidase activity and a hyaluronidase inhibitor, which can effectively inhibit the activity of hyaluronidase.

[0010] The present invention solves its technical problem by adopting the following technical solution:

[0011] A traditional Chinese medicine composition for inhibiting hyaluronidase activity, comprising, by weight, 1-12 parts Physalis alkekengi, 1-12 parts Cnidium monnieri, and 1-6 parts Coptis chinensis.

[0012] As a preferred embodiment, the traditional Chinese medicine composition with hyaluronidase activity comprises, by weight, 1-10 parts Physalis alkekengi, 1-10 parts Cnidium monnieri, and 1-5 parts Coptis chinensis.

[0013] As a preferred embodiment, the traditional Chinese medicine composition with hyaluronidase activity comprises, by weight, 5 parts Physalis alkekengi, 5 parts Cnidium monnieri, and 3 parts Coptis chinensis.

[0014] The inventors of this invention discovered in extensive research that using Physalis alkekengi, Cnidium monnieri, or Coptis chinensis alone, or any two of them, has a poor inhibitory effect on hyaluronidase. By using Physalis alkekengi, Cnidium monnieri, and Coptis chinensis in combination, the activity of hyaluronidase can be effectively inhibited. Physalis alkekengi, Cnidium monnieri, and Coptis chinensis have a significant synergistic effect on hyaluronidase activity. Furthermore, the inventors found that the components of Physalis alkekengi, Cnidium monnieri, and Coptis chinensis have a better effect under the above-mentioned weight ratios.

[0015] This invention also provides a method for preparing a traditional Chinese medicine composition that inhibits hyaluronidase activity, comprising the following steps:

[0016] S01. Weigh 1-12 parts of Physalis alkekengi, 1-12 parts of Cnidium monnieri, and 1-6 parts of Coptis chinensis according to the weight ratio, dry them, pulverize them to 60-100 mesh, mix them evenly, and obtain a mixed powder.

[0017] S02. Add the mixed powder to a 70-95% ethanol aqueous solution and soak for 10-30 hours. Transfer it to a chromatography column, compact it, elute with a 70-95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0018] As a preferred embodiment, the weight ratio of the mixed powder to the ethanol aqueous solution is 1:3~6.

[0019] The present invention also provides a hyaluronidase inhibitor, comprising, by weight, 1-2 parts Tween, 2-5 parts lecithin, 0.1-0.5 parts cholesterol, 40-80 parts traditional Chinese medicine composition, and 80-100 parts deionized water;

[0020] The inventors further discovered that Tween, lecithin, and cholesterol can effectively improve the transdermal absorption and bioavailability of the traditional Chinese medicine composition, thereby enhancing its inhibitory effect on hyaluronidase.

[0021] The traditional Chinese medicine composition comprises, by weight, 1-12 parts Physalis alkekengi, 1-12 parts Cnidium monnieri, and 1-6 parts Coptis chinensis.

[0022] As a preferred embodiment, the traditional Chinese medicine composition comprises, by weight, 1-10 parts Physalis alkekengi, 1-10 parts Cnidium monnieri, and 1-5 parts Coptis chinensis.

[0023] As a preferred embodiment, the traditional Chinese medicine composition comprises, by weight, 5 parts Physalis alkekengi, 5 parts Cnidium monnieri, and 3 parts Coptis chinensis.

[0024] This invention also provides a method for preparing a hyaluronidase inhibitor, comprising the following steps:

[0025] S11. Weigh 1-12 parts of Physalis alkekengi, 1-12 parts of Cnidium monnieri, and 1-6 parts of Coptis chinensis according to the weight, dry them, pulverize them to 60-100 mesh, mix them evenly, and obtain a mixed powder.

[0026] S12. Add the mixed powder to a 70-95% ethanol aqueous solution and soak for 10-30 hours. Transfer it to a chromatography column, compact it, elute with a 70-95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition.

[0027] S13. Add 1-2 parts by weight of Tween, 2-5 parts by weight of lecithin, 0.1-0.5 parts by weight of cholesterol, and 40-80 parts by weight of the traditional Chinese medicine composition into a reaction vessel, heat to 50-60°C, stir evenly, and then put into a rotary evaporator for rotary evaporation to remove ethanol; then add 80-100 parts by weight of deionized water, stir evenly, and then homogenize at 300-400 bar pressure using a high-pressure homogenizer for 8-15 minutes to obtain the hyaluronidase inhibitor.

[0028] As a preferred embodiment, the weight ratio of the mixed powder to the ethanol aqueous solution is 1:3~6.

[0029] The beneficial effects of the present invention are as follows: The traditional Chinese medicine composition and hyaluronidase inhibitor described in the present invention can effectively inhibit the activity of hyaluronidase. By using Physalis alkekengi, Cnidium monnieri, and Coptis chinensis in combination, the activity of hyaluronidase can be effectively inhibited. Physalis alkekengi, Cnidium monnieri, and Coptis chinensis have a significant synergistic effect on the activity of hyaluronidase. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] In this invention, unless otherwise stated, all parts are by weight.

[0032] In this invention, the Physalis alkekengi is a Physalis alkekengi medicinal material, Cnidium monnieri is a Cnidium monnieri medicinal material, and Coptis chinensis is a Coptis chinensis medicinal material.

[0033] In this invention, the chromatography column was purchased from Huabo Technology Co., Ltd., with an outer diameter of 120 mm and a length of 1000 mm, and a collection bottle of 2 L.

[0034] Example 1

[0035] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 5 parts Physalis alkekengi, 5 parts Cnidium monnieri, and 3 parts Coptis chinensis.

[0036] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0037] S01. Weigh out 5 parts of Physalis alkekengi, 5 parts of Cnidium monnieri, and 3 parts of Coptis chinensis according to the weight, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0038] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0039] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0040] Example 2

[0041] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 10 parts Physalis alkekengi, 1 part Cnidium monnieri, and 5 parts Coptis chinensis.

[0042] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0043] S01. Weigh 10 parts of Physalis alkekengi, 1 part of Cnidium monnieri, and 5 parts of Coptis chinensis according to the weight, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0044] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0045] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0046] Example 3

[0047] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 1 part Physalis alkekengi, 10 parts Cnidium monnieri, and 1 part Coptis chinensis.

[0048] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0049] S01. Weigh out 1 part Physalis alkekengi, 10 parts Cnidium monnieri, and 1 part Coptis chinensis according to the weight, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0050] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0051] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0052] Example 4

[0053] A hyaluronidase inhibitor, characterized in that, by weight, it comprises 1 part Tween, 5 parts lecithin, 0.1 part cholesterol, 80 parts traditional Chinese medicine composition, and 80 parts deionized water;

[0054] The traditional Chinese medicine composition comprises, by weight, 5 parts Physalis alkekengi, 5 parts Cnidium monnieri, and 3 parts Coptis chinensis.

[0055] The method for preparing the hyaluronidase inhibitor includes the following steps:

[0056] S11. Weigh out 5 parts of Physalis alkekengi, 5 parts of Cnidium monnieri, and 3 parts of Coptis chinensis according to the weight ratio, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0057] S12. Add the mixed powder to a 95% ethanol aqueous solution and soak for 24 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition.

[0058] S13. Add 1 part Tween 60, 5 parts lecithin, 0.1 part cholesterol, and 80 parts of the traditional Chinese medicine composition to a reaction vessel according to the weight ratio. Heat to 55°C, stir evenly, and put into a rotary evaporator for rotary evaporation to remove ethanol. Then add 80 parts deionized water, stir evenly, and then homogenize at 350 bar pressure for 10 min using a high-pressure homogenizer to obtain the hyaluronidase inhibitor.

[0059] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0060] Example 5

[0061] The hyaluronidase inhibitor, by weight, comprises 2 parts Tween, 2 parts lecithin, 0.5 parts cholesterol, 40 parts traditional Chinese medicine composition, and 100 parts deionized water;

[0062] The traditional Chinese medicine composition comprises, by weight, 5 parts Physalis alkekengi, 5 parts Cnidium monnieri, and 3 parts Coptis chinensis.

[0063] The method for preparing the hyaluronidase inhibitor includes the following steps:

[0064] S11. Weigh out 5 parts of Physalis alkekengi, 5 parts of Cnidium monnieri, and 3 parts of Coptis chinensis according to the weight ratio, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0065] S12. Add the mixed powder to a 95% ethanol aqueous solution and soak for 24 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition.

[0066] S13. Add 2 parts by weight of Tween 60, 2 parts by weight of lecithin, 0.5 parts by weight of cholesterol and 40 parts by weight of traditional Chinese medicine composition into a reaction vessel, heat to 55°C, stir evenly, and put into a rotary evaporator for rotary evaporation to remove ethanol; then add 100 parts by weight of deionized water, stir evenly, and then homogenize at 350 bar pressure using a high-pressure homogenizer for 10 min to obtain hyaluronidase inhibitor.

[0067] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0068] Comparative Example 1

[0069] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses an equal amount of a single Physalis alkekengi to replace Physalis alkekengi, Cnidium monnieri, and Coptis chinensis, while all other aspects are the same. Here it is referred to as Physalis alkekengi extract.

[0070] The preparation method of the Physalis extract includes the following steps:

[0071] S01. Weigh 13 parts of Physalis according to the weight, dry them, and grind them to 80 mesh to obtain Physalis powder;

[0072] S02. Soak the Physalis powder in 95% ethanol aqueous solution for 18 hours, transfer it into a chromatography column, compact it, elute with 95% ethanol aqueous solution, and collect the eluent to obtain the Physalis extract.

[0073] The weight ratio of the Physalis alkekengi powder to the ethanol aqueous solution is 1:4.

[0074] Comparative Example 2

[0075] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 uses an equal amount of a single Cnidium monnieri to replace Physalis alkekengi, Cnidium monnieri, and Coptis chinensis, while all other aspects are the same. Here it is referred to as Cnidium monnieri extract.

[0076] The preparation method of the Cnidium monnieri extract includes the following steps:

[0077] S01. Weigh 13 parts of Cnidium monnieri according to the weight, dry them, and grind them to 80 mesh to obtain Cnidium monnieri powder;

[0078] S02. Soak Cnidium monnieri powder in 95% ethanol aqueous solution for 18 hours, transfer it into a chromatography column, compact it, elute with 95% ethanol aqueous solution, and collect the eluent to obtain Cnidium monnieri extract.

[0079] The weight ratio of Cnidium monnieri powder to ethanol aqueous solution is 1:4.

[0080] Comparative Example 3

[0081] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses an equal amount of a single Coptis chinensis to replace Physalis alkekengi, Cnidium monnieri, and Coptis chinensis, while all other aspects are the same. Here it is referred to as Coptis chinensis extract.

[0082] The preparation method of the Physalis extract includes the following steps:

[0083] S01. Weigh 13 parts of Coptis chinensis according to the weight, dry them, and grind them to 80 mesh to obtain Coptis chinensis powder;

[0084] S02. Soak Coptis chinensis powder in 95% ethanol aqueous solution for 18 hours, transfer it into a chromatography column, compact it, elute with 95% ethanol aqueous solution, and collect the eluent to obtain Coptis chinensis extract.

[0085] The weight ratio of Coptis chinensis powder to ethanol aqueous solution is 1:4.

[0086] Comparative Example 4

[0087] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain Coptis chinensis, but all other aspects are the same.

[0088] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 6.5 parts Physalis alkekengi and 6.5 parts Cnidium monnieri.

[0089] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0090] S01. Weigh 6.5 parts of Physalis alkekengi and 6.5 parts of Cnidium monnieri according to the weight ratio, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder;

[0091] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0092] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0093] Comparative Example 5

[0094] The difference between Comparative Example 5 and Example 1 is that Comparative Example 4 does not contain Cnidium monnieri, but all other aspects are the same.

[0095] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 7.5 parts Physalis alkekengi and 5.5 parts Coptis chinensis.

[0096] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0097] S01. Weigh 7.5 parts of Physalis alkekengi and 5.5 parts of Coptis chinensis according to the weight ratio, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder.

[0098] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0099] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0100] Comparative Example 6

[0101] The difference between Comparative Example 6 and Example 1 is that Comparative Example 6 does not contain broccoli, but all other aspects are the same.

[0102] The traditional Chinese medicine composition for inhibiting hyaluronidase activity comprises, by weight, 7.5 parts of Cnidium monnieri and 5.5 parts of Coptis chinensis.

[0103] The method for preparing the traditional Chinese medicine composition that inhibits hyaluronidase activity includes the following steps:

[0104] S01. Weigh 7.5 parts of Cnidium monnieri and 5.5 parts of Coptis chinensis according to the weight ratio, dry them, grind them to 80 mesh, mix them evenly, and obtain a mixed powder;

[0105] S02. Add the mixed powder to a 95% ethanol aqueous solution and soak for 18 hours. Transfer it to a chromatography column, compact it, elute with a 95% ethanol aqueous solution, and collect the eluent to obtain the traditional Chinese medicine composition that inhibits hyaluronidase activity.

[0106] The weight ratio of the mixed powder to the ethanol aqueous solution is 1:4.

[0107] To further demonstrate the effectiveness of the present invention, the following test methods are provided:

[0108] 1. Experiment 1: Hyaluronidase is a specific lysin of hyaluronic acid and a participant in allergic reactions, showing a strong correlation with histamine release from mast cells. Whether a substance possesses anti-allergic activity can be determined using the hyaluronidase activity inhibition rate; a higher hyaluronidase activity inhibition rate indicates a stronger anti-allergic effect, and vice versa.

[0109] Hyaluronidase can non-selectively hydrolyze the 1,4-bond between β-N-acetylglucosamine and D-glucuronate in hyaluronic acid to obtain β-N-acetylglucosamine. β-N-acetylglucosamine can condense with acetylacetone under alkaline conditions to generate the chromogen 2-methyl-3-diacetylpyrrole derivative. The chromogen reacts with p-dimethylaminobenzaldehyde in concentrated hydrochloric acid and ethanol to produce color.

[0110] 1. Experimental Preparation

[0111] 1.1 Reagents

[0112] Anhydrous calcium chloride, hyaluronidase, sodium hyaluronate, sodium hydroxide, acetylacetone, anhydrous sodium carbonate, p-dimethylaminobenzaldehyde, concentrated hydrochloric acid, anhydrous ethanol, glacial acetic acid, and sodium acetate crystals.

[0113] 1.2 Reagent Preparation

[0114] 1.2.1 Acetic acid buffer solution (pH=5.6)

[0115] Measure 1.155 mL of glacial acetic acid, dilute to 100 mL, mix well, and take 4.8 mL of this solution as solution A; weigh 2.72 g of sodium acetate crystals, dissolve in water, and bring the volume to 100 mL, mix well, and take 45.2 mL of this solution as solution B; mix solutions A and B, and bring the volume to 100 mL with water. Accurately measure the pH value and adjust it to 5.6 using either solution A or B.

[0116] 1.2.2 Hyaluronidase solution

[0117] Hyaluronidase was dissolved in acetate buffer solution, and the final working concentration was 1250 U / mL.

[0118] 1.2.3 Sodium hyaluronate solution

[0119] Weigh 5 mg of sodium hyaluronate and place it in a beaker. Add 10 mL of acetic acid buffer solution to obtain a 0.5 mg / mL sodium hyaluronate solution.

[0120] 1.2.4 Ehrlich's reagent

[0121] Weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol.

[0122] 1.2.5 Sodium carbonate solution

[0123] Weigh 5.3 g of anhydrous sodium carbonate and place it in a beaker. Dilute to 50 mL with pure water to obtain a 1 mol / L sodium carbonate solution.

[0124] 1.2.6 Acetylacetone solution

[0125] Dissolve 3.5 mL of acetylacetone in 50 mL of 1.0 mol / L sodium carbonate solution. Prepare this solution before use.

[0126] 1.3 Test Instruments and Equipment

[0127] 1.3.1 Equipment

[0128] Balance, water bath, shaker, ultraviolet spectrophotometer.

[0129] 1.3.2 Tools

[0130] Pipettes (100 μL-1000 μL), pipettes (1000 μL-5000 μL), timer.

[0131] 1.3.3 Glassware

[0132] Beaker, volumetric flask, 10 mL glass test tube.

[0133] 3. Experimental Procedure

[0134] 3.1 Test Procedure

[0135] Take 0.1 mL of CaCl2 solution (0.25 mmol / L) and 0.5 mL of hyaluronidase solution and incubate at 37 °C for 20 min in a water bath; add 0.5 mL of the test sample solution (the test samples are the traditional Chinese medicine compositions or extracts described in Examples 1-3 and Comparative Examples 1-6, and the hyaluronidase inhibitors described in Examples 4 and 5, respectively, and each sample is prepared as a 5% aqueous solution with deionized water; each group is measured three times and the average value is taken), and continue incubation for 20 min; then add 0.5 mL of sodium hyaluronate solution (0.5 mg / mL), incubate at 37 °C for 30 min, remove and place at room temperature for 5 min; add 0.1 mL of NaOH solution (0.4 mol / L) and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 15 min, and immediately transfer to an ice water bath to cool for 5 min; add 1.0 mL of Ehrlich reagent and dilute with 3.0 mL of anhydrous ethanol, and place at room temperature for 20 min. The color was developed at 0 min, and the absorbance value at a wavelength of 540 nm was measured using a spectrophotometer.

[0136] 3.2 Results Analysis and Presentation

[0137] The formula for calculating the inhibition rate of hyaluronidase on the sample is as follows:

[0138]

[0139] Where: A—absorbance value of the control solution (acetic acid buffer solution is used instead of the sample solution);

[0140] B—Absorbance value of the control blank solution (using acetate buffer solution instead of sample solution and enzyme solution);

[0141] C—Absorbance value of the sample solution;

[0142] D—Absorbance value of blank sample solution (using acetic acid buffer solution instead of enzyme solution);

[0143] The test results are shown in Table 1.

[0144] Table 1

[0145]

[0146] As can be seen from Table 1, the traditional Chinese medicine composition of the present invention can effectively inhibit the activity of hyaluronidase.

[0147] Comparing Examples 1-3, it can be seen that the ratio of different Chinese herbal medicine compositions can significantly affect the activity of hyaluronidase, with Example 1 being the optimal ratio.

[0148] Comparative examples 1-3 show that Tween 80, lecithin, and cholesterol in the hyaluronidase inhibitor of the present invention can effectively improve the transdermal absorption and bioavailability of the traditional Chinese medicine composition, thereby further improving the inhibition rate of hyaluronidase.

[0149] Comparing Example 1 with Comparative Examples 1-6, it can be seen that the Chinese herbal composition contains Physalis alkekengi, Cnidium monnieri, and Coptis chinensis, which have a significant synergistic effect in inhibiting hyaluronidase activity. The combination of the three herbs can inhibit hyaluronidase activity more effectively than using any one or two of them.

[0150] 2. Anti-allergy test.

[0151] Ten volunteers, half male and half female, aged 20-25, were selected. 20 μL of 1% histamine phosphate was applied to a 3×3 cm area of ​​the skin, spread evenly, and the volunteers sat quietly and waited for the skin to redden and itch, recording the time. Then, 20 μL of 0.1% diluted test sample (the traditional Chinese medicine composition described in Example 1, the hyaluronic acid inhibitor described in Example 4, the hyaluronic acid inhibitor described in Example 5, and an equal volume of distilled water for the control group) was applied to the test area, and the time for the redness and itching to subside was recorded.

[0152] Samples to be tested: Sample 1 - Anti-allergy repair peptide, Control sample 2 - GPI, Control sample 3 - Deminshu.

[0153] Test site: Inner side of the arm

[0154] Test environment: Temperature: 20℃~22℃, Humidity: 40%~60%

[0155] Test basis: (1) GB17149.2-1997 Determination of the degree of reaction of the test site;

[0156] (2) Tang Yueqiong, Qu Li, Jiang Weixia, et al. Assessment and countermeasures of pruritus in patients with skin diseases [J]. Journal of PLA Nursing, 2007, 024(005): 76-76;

[0157] The reference standards for skin erythema and itching are shown in Table 2, and the test results are shown in Table 3.

[0158] Table 2

[0159]

[0160] Table 3

[0161]

[0162] As can be seen from Table 3, the traditional Chinese medicine composition and hyaluronidase inhibitor described in this invention can effectively resist allergies and have good anti-allergic effects. The Tween 80, lecithin and cholesterol in the hyaluronidase inhibitor can effectively improve the transdermal absorption and bioavailability of the traditional Chinese medicine composition, thereby further improving the anti-allergic effect.

[0163] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A Chinese medicine composition for inhibiting hyaluronidase activity, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

2. The traditional Chinese medicine composition for inhibiting hyaluronidase activity according to claim 1, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

3. The traditional Chinese medicine composition for inhibiting hyaluronidase activity according to claim 1, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

4. A method for preparing a traditional Chinese medicine composition for inhibiting hyaluronidase activity, characterized in that, The method comprises the following steps: S01, 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis are weighed by weight, dried, crushed to 60-100 meshes, mixed uniformly, and a mixed powder is obtained; S02, the mixed powder is soaked in 70-95% ethanol aqueous solution for 10-30 hours, transferred into a chromatographic column, compacted, eluted with 70-95% ethanol aqueous solution, and the eluate is collected to obtain the hyaluronidase inhibitor.

5. The preparation method of the traditional Chinese medicine composition for inhibiting hyaluronidase activity according to claim 4, characterized in that, The weight ratio of the mixed powder to the ethanol aqueous solution is 1:3-6.

6. Hyaluronidase inhibitors, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight. The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

7. The hyaluronidase inhibitor according to claim 6, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

8. The hyaluronidase inhibitor according to claim 6, characterized in that, The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

9. A process for the preparation of a hyaluronidase inhibitor, characterized in that, The method comprises the following steps: S11, 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis are weighed by weight, dried, crushed to 60-100 meshes, mixed uniformly, and a mixed powder is obtained; S12, the mixed powder is soaked in 70-95% ethanol aqueous solution for 10-30 hours, transferred into a chromatographic column, compacted, eluted with 70-95% ethanol aqueous solution, and the eluate is collected to obtain the hyaluronidase inhibitor; S13, 1-2 parts of Tween, 2-5 parts of lecithin, 0.1-0.5 parts of cholesterol, and 40-80 parts of the traditional Chinese medicine composition are added into a reaction kettle by weight, heated to 50-60°C, stirred uniformly, and then evaporated in a rotary evaporator; 80-100 parts of deionized water is added, stirred uniformly, and then homogenized at a pressure of 300-400 bar for 8-15 minutes by using a high-pressure homogenizer to obtain the hyaluronidase inhibitor.

10. The method of claim 9, wherein the hyaluronidase inhibitor is prepared by, The weight ratio of the mixed powder to the ethanol aqueous solution is 1:3-6. The hyaluronidase inhibitor is prepared from 1-12 parts of Physostigma, 1-12 parts of Cnidium, and 1-6 parts of Coptis by weight.

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