Composite plant extract and use thereof, external preparation for skin

Abstract

The application discloses a kind of composite plant extract and its application, skin external preparation.The raw material of the composite plant extract includes hemerocallis fulva flower and red clover, and the weight ratio of hemerocallis fulva flower and red clover is (1:5)~(5:1).The plant extract of hemerocallis fulva flower and red clover of the application can inhibit the release of histamine, thereby reducing the occurrence of inflammation and infection;The plant extract of the application cooperates synergistically, and compared with each plant alone, the histamine inhibition effect of the plant extract of the application is significantly improved;The plant extract of the application uses pure natural plant as raw material, and has low side effect and high safety;The plant raw material used in the application is widely planted in China, and the resource is easy to obtain, the price is cheap, and the cost is low.

Description

TECHNICAL FIELD

[0001] The present application provides a composite plant extract and application thereof, and a skin external preparation. BACKGROUND

[0002] Eczema is a skin inflammatory reaction with severe itching caused by various internal and external factors. Internal factors such as chronic digestive system diseases, mental stress, insomnia, excessive fatigue, emotional changes, endocrine disorders, infections, and metabolic disorders, and external factors such as living environment, climate change, and food can affect the occurrence of eczema. In essence, eczema is a delayed type of allergic reaction caused by complex internal and external factors. Existing studies have shown that histamine is one of the causes of eczema-related itching.

[0003] Histamine is an autacoid substance that exists in the form of an inactive bound form in the granules of mast cells and basophils. In the body, histamine is formed from histidine by histidine decarboxylase (HDC), and has various biological activities, including allergic reactions, inflammatory reactions, etc. Histamine is an important chemical mediator produced by various cells, including mast cells, basophils, chromaffin cells, platelets, dendritic cells, and T cells, and its function affects various physiological functions of the body, including cell proliferation, differentiation, hematopoietic processes, embryonic development, tissue regeneration, and wound healing. In different microbial infections, histamine is widely present in various inflammatory and infectious diseases, and mainly regulates host immune responses, with positive and negative bidirectional regulation. Recent studies have found that histamine is an important regulator in immune responses.

[0004] Currently, chemical drugs still occupy a large part of the market for diseases caused by histamine, but have many side effects, such as benadryl and chlorpheniramine, which have small molecular weights and high lipid solubility, easily penetrate the blood-brain barrier into brain tissue, produce central inhibition effects such as drowsiness, sedation, hallucinations, and potential teratogenic effects. Although traditional Chinese medicines lack large brands like chemical drugs, they have certain market potential due to their high safety and the public's high trust in traditional Chinese medicine. There is a lack of traditional Chinese medicine products with significant efficacy on the market. China has rich vegetation, and it is of great significance and broad development prospects to discover or create a composition with anti-histamine efficacy from it. SUMMARY

[0005] The present application provides a composite plant extract and application thereof, and a skin external preparation. The composite plant extract of the present application can effectively inhibit the release of histamine, thereby reducing inflammation and infection, and has low side effects.

[0006] In order to achieve the above object, the present application adopts the following technical solutions:

[0007] The present application provides a kind of composite plant extract, the raw material of the composite plant extract includes herbaceous peony flower and red clover;The weight ratio of the herbaceous peony flower and the red clover is (1:5) ~ (5:1);

[0008] The preparation method of the composite plant extract includes: the raw material is sequentially extracted by alcohol and water, i.e.

[0009] In the present application, the weight ratio of the herbaceous peony flower and the red clover can be 1:1.

[0010] In the present application, the weight ratio of the herbaceous peony flower and the red clover can be 1:5.

[0011] In the present application, the weight ratio of the herbaceous peony flower and the red clover can be 5:1.

[0012] In the present application, the preparation method of the composite plant extract can include any one of the following two methods:

[0013] Method one: the herbaceous peony flower and red clover are mixed and sequentially extracted by alcohol and water, i.e.

[0014] Method two: the herbaceous peony flower is sequentially extracted by alcohol and water to obtain herbaceous peony flower extract;The red clover is sequentially extracted by alcohol and water to obtain red clover extract;The herbaceous peony flower extract and the red clover extract are combined to obtain the composite plant extract.

[0015] Among them, the method one can include the following steps:

[0016] Step (1), according to the composition ratio of raw materials, herbaceous peony flower and red clover are mixed, alcohol extraction, filtration, to obtain drug residue A and alcohol extract;

[0017] Step (2), the drug residue A is water extracted, filtered, to obtain drug residue B and water extract;

[0018] Step (3), the alcohol extract and the water extract are combined, filtered, to obtain the initial product;

[0019] Step (4), the initial product is added with stabilizer, stirred, aged, filtered, to obtain the composite plant extract;

[0020] Among them, the method two can include the following steps:

[0021] Step (1), take herbaceous peony flower, alcohol extraction, filtration, to obtain filter residue and alcohol extract;The filter residue is water extracted, filtered, to obtain water extract;The alcohol extract and the water extract are combined to obtain herbaceous peony flower extract;

[0022] Step (2), take the red clover, alcohol extract, filter, get the residue and alcohol extract; the residue is water extracted, filtered, and the alcohol extract and the water extract are combined to obtain the red clover extract; the steps (1) and (2) are not in sequence;

[0023] Step (3), the mixture of the hemerocallis flower extract and the red clover extract is added with a stabilizer, stirred, aged, filtered, and a composite plant extract is obtained.

[0024] In step (1) of the method one, the alcohol extraction process preferably uses ethanol; the concentration of the ethanol is preferably 80-100%, for example 90%.

[0025] In step (1) of the method one, the alcohol extraction process preferably uses ethanol; the concentration of the ethanol is preferably 80-100%, for example 90%.

[0026] In step (1) of the method one, the pH of the alcohol extraction process is preferably 8-12, for example 8, 10 or 12.

[0027] In step (1) of the method one, the temperature of the alcohol extraction is preferably 60-85℃, for example 60℃, 75℃ or 85℃; the time of the alcohol extraction is preferably 0.5-2 hours, for example 1 hour.

[0028] In step (1) of the method one, the filtration method can be a conventional method in the art, for example gauze filtration.

[0029] In step (2) of the method one, the water extraction process preferably uses water; the concentration of the water is preferably 80-100%, for example 90%.

[0030] In step (2) of the method one, the temperature of the water extraction is preferably 60-95℃, for example 60℃, 85℃ or 95℃; the time of the water extraction is preferably 0.5-2 hours, for example 1 hour.

[0031] In step (2) of the method one, the filtration method can be a conventional method, for example gauze filtration.

[0032] In step (3) of the method one, the filtration method can be a conventional method, preferably diatomite filtration.

[0033] In step (3) of the method one, the filtration process can further include a concentration step; the concentration method is a conventional method in the art, preferably a method under reduced pressure.

[0034] In step (4) of Method I, the stabilizer is preferably one or more of phytosterols, Tween-80, Tween-20, butylene glycol, glycerol, propylene glycol, methyl propylene glycol, and dipropylene glycol.

[0035] In step (4) of Method I, the stabilizer is preferably added in an amount of 2 to 4 times, for example 3 times, the weight of the medicinal material.

[0036] In step (4) of Method I, the stirring time is preferably 0.5 to 1.5 hours, for example 1 hour.

[0037] In step (4) of Method I, the aging temperature is preferably 2 to 8°C, for example 4°C, and the aging time is preferably 1 to 3 days, for example 1 day, 2 days, or 3 days.

[0038] In step (4) of Method I, the filtration method can be a conventional method, and is preferably diatomite filtration.

[0039] In step (4) of Method I, the filtration can further include a step of adding a solvent to the filtrate.

[0040] The solvent can be a conventional solvent in the art for dissolving smaller molecular components in the filtrate, and is preferably one or more of butylene glycol, glycerol, propylene glycol, methyl propylene glycol, and dipropylene glycol.

[0041] The solvent is preferably added in an amount of 3 to 8 times, for example 5 times, the weight of the medicinal material.

[0042] In step (1) of Method II, the alcohol extraction process preferably uses ethanol, and the concentration of the ethanol is preferably 80 to 100%, for example 90%.

[0043] In step (1) of Method II, the alcohol extraction process preferably uses ethanol, and the concentration of the ethanol is preferably 80 to 100%, for example 90%.

[0044] In step (1) of Method II, the alcohol extraction process is preferably performed at a temperature of 60 to 95°C, and for a time of 0.5 to 2 hours, for example 1 hour.

[0045] In step (1) of Method II, the filtration method after alcohol extraction can be a conventional method in the art, for example gauze filtration.

[0046] In step (1) of Method II, the water extraction process preferably uses water added in an amount of 6 to 12 times, for example 6 times, 10 times, or 12 times, the weight of the medicinal material.

[0047] In step (1) of Method Two, the temperature of the water extraction is preferably 60-95°C, such as 60°C, 85°C or 95°C; and the time of the water extraction is preferably 0.5-2 hours, such as 1 hour.

[0048] In step (1) of Method Two, the filtration method after the water extraction can be a conventional method in the art, such as gauze filtration.

[0049] In step (1) of Method Two, after the alcohol extract and the water extract are combined, a concentration step can also be included.

[0050] In step (2) of Method Two, the alcohol used in the alcohol extraction is preferably ethanol; and the concentration of the ethanol is preferably 80-100%, such as 90%.

[0051] In step (2) of Method Two, the weight of the alcohol solution added in the alcohol extraction is preferably 6-15 times the weight of the medicinal material, such as 6 times, 10 times or 15 times.

[0052] In step (2) of Method Two, the pH of the system in the alcohol extraction is preferably 8-12, such as 8, 10 or 12.

[0053] In step (2) of Method Two, the temperature of the alcohol extraction is preferably 60-85°C; and the time of the alcohol extraction is preferably 0.5-2 hours, such as 1 hour.

[0054] In step (2) of Method Two, the filtration method after the alcohol extraction can be a conventional method in the art, such as gauze filtration.

[0055] In step (2) of Method Two, the weight of the water added in the water extraction is preferably 6-12 times the weight of the medicinal material, such as 6 times, 10 times or 12 times.

[0056] In step (2) of Method Two, the temperature of the water extraction is preferably 60-95°C, such as 60°C, 85°C or 95°C; and the time of the water extraction is preferably 0.5-2 hours, such as 1 hour.

[0057] In step (2) of Method Two, the filtration method after the water extraction can be a conventional method in the art, such as gauze filtration.

[0058] In step (2) of Method Two, after the alcohol extract and the water extract are combined, a concentration step can also be included.

[0059] Preferably, the stabilizer in step (3) of the second method is one or more of dipropylene glycol, phytosterols, Tween-80, Tween-20, butanediol, glycerol, propylene glycol, methyl propylene glycol and dipropylene glycol.

[0060] Preferably, the stabilizer in step (3) of the second method is added in an amount of 2-4 times, for example 3 times, the weight of the medicinal material.

[0061] Preferably, the stirring time in step (3) of the second method is 0.5-1.5 hours, for example 1 hour.

[0062] Preferably, the aging temperature in step (3) of the second method is 2-8℃, for example 4℃, and the aging time is 1-3 days, for example 1 day, 2 days or 3 days.

[0063] Preferably, the filtering method in step (3) of the second method is diatomite filtration.

[0064] Preferably, the method further comprises the step of adding a solvent to the filtrate after filtering.

[0065] Preferably, the solvent is one or more of butanediol, glycerol, propylene glycol, methyl propylene glycol and dipropylene glycol.

[0066] Preferably, the solvent is added in an amount of 3-8 times, for example 5 times, the weight of the medicinal material.

[0067] The present application also provides a composite plant extract, wherein the raw material of the composite plant extract comprises an extract of Hemerocallis citrina Baroni flowers and an extract of Trifolium pratense L.

[0068] In the present application, the preparation method of the extract of Hemerocallis citrina Baroni flowers comprises the following steps: taking Hemerocallis citrina Baroni flowers, alcohol extraction, filtration to obtain filter residue and alcohol extract; water extraction of the filter residue, filtration to obtain water extract; and combining the alcohol extract and the water extract to obtain the extract of Hemerocallis citrina Baroni flowers.

[0069] In the present application, the preparation method of the extract of Trifolium pratense L. comprises the following steps: taking Trifolium pratense L., alcohol extraction, filtration to obtain medicinal residue and alcohol extract; water extraction of the medicinal residue, filtration to obtain water extract; and combining the alcohol extract and the water extract to obtain the extract of Trifolium pratense L.

[0070] The present application also provides the use of the aforementioned composite plant extract as a component for inhibiting histamine in a skin external preparation.

[0071] The application also provides a skin external preparation comprising the aforementioned composite plant extract.

[0072] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining preferred examples of the application.

[0073] The reagents and raw materials used in the application are commercially available.

[0074] The positive progress effect of the application is that:

[0075] The plant extracts of Hemerocallis and Trifolium pretense of the application can inhibit the release of histamine, thereby reducing the occurrence of inflammation and infection; the plant extracts of the application have a synergistic effect, and the histamine inhibition effect of the plant extracts of the application is significantly improved compared with each plant alone.

[0076] The plant extracts of the application use pure natural plants as raw materials, have low side effects, and are highly safe.

[0077] The plant raw materials used in the application are widely planted in China, are easy to obtain, are cheap, and have low cost. DETAILED DESCRIPTION

[0078] The application will be further described below by way of examples, but the application is not limited in the scope of the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0079] The Hemerocallis used in the following examples and comparative examples is obtained from the flower parts of Hemerocallis fulva (L.) L. of the Liliaceae family and Hemerocallis genus; the Trifolium pretense is the whole grass of Trifolium pretense L. of the Leguminosae family and the butterfly flower subfamily; the honeysuckle is obtained from the dried flower buds or the initially opened flowers of Lonicera japonica Thunb of the Caprifoliaceae family and the same genus plant.

[0080] Example 1

[0081] (1) 1 part of Hemerocallis and 1 part of Trifolium pretense are mixed, 95% ethanol is added in an amount of 6 times the amount of medicinal materials, the pH is adjusted to 8, and reflux extraction is performed at 60°C for 1 hour, and then gauze filtration is performed to obtain an alcohol extract and a residue;

[0082] (2) The residue is added with water in an amount of 6 times the amount of medicinal materials, and extraction is performed at 60°C for 1 hour, and then gauze filtration is performed to obtain a water extract and a residue;

[0083] (3) The alcohol extract and the water extract are combined, and diatomite filtration is performed;

[0084] (4) The filtrate is concentrated under reduced pressure to obtain an extract.

[0085] (5) Add stabilizer to 3 times the amount of medicinal materials, stir for 1 hour; the stabilizer is a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0086] (6) Store at 4°C for 1 day;

[0087] (7) Filter through diatomite, and supplement the obtained filtrate with dipropylene glycol to 5 times the amount of medicinal materials;

[0088] (8) Sterilize and cool to obtain an extract;

[0089] (9) Filter the extract through a 0.45 μm organic membrane, and detect and fill.

[0090] Example 2

[0091] (1) Mix 1 part of Hemerocallis citrina Baroni flowers and 5 parts of Trifolium pratense, add 95% ethanol to 15 times the amount of medicinal materials, adjust the pH to 12, and reflux extract at 85°C for 1 hour, filter through gauze to obtain an alcohol extract and residue;

[0092] (2) Add water to the residue to 10 times the amount of medicinal materials, and extract at 85°C for 1 hour, filter through gauze to obtain a water extract and residue;

[0093] (3) Combine the alcohol extract of (1) and the water extract of (2), and filter through diatomite;

[0094] (4) Concentrate under reduced pressure to a extract;

[0095] (5) Add stabilizer to 3 times the amount of medicinal materials, stir for 1 hour; the stabilizer is a mixture of Tween-80 and butanediol in a weight ratio of 1:12;

[0096] (6) Store at 4°C for 2 days;

[0097] (7) Filter through diatomite, and supplement the obtained filtrate with butanediol to 5 times the amount of medicinal materials;

[0098] (8) Sterilize and cool to obtain an extract;

[0099] (9) Filter the extract through a 0.45 μm organic membrane, and detect and fill.

[0100] Example 3

[0101] (1) Mix 5 parts of Hemerocallis citrina Baroni flowers and 1 part of Trifolium pratense, add 95% ethanol to 10 times the amount of medicinal materials, adjust the pH to 10, and reflux extract at 75°C for 1 hour, filter through gauze to obtain an alcohol extract and residue;

[0102] (2) Add water to the residue to 12 times the amount of medicinal materials, and extract at 95°C for 1 hour, filter through gauze to obtain a water extract and residue;

[0103] (3) combine the alcohol extract of (1) and the water extract of (2), filter through diatomite;

[0104] (4) concentrate under reduced pressure to a extract;

[0105] (5) add stabilizer to 3 times the amount of medicinal material, stir for 1 hour; the stabilizer is a mixture of Tween-20 and propylene glycol in a weight ratio of 1:12;

[0106] (6) store at 4°C for 3 days;

[0107] (7) filter through diatomite, and make up with propylene glycol to 5 times the amount of medicinal material;

[0108] (8) sterilize and cool to obtain the extract;

[0109] (9) filter the extract through a 0.45 μm organic membrane, test, and fill;

[0110] Example 4

[0111] (1) take 1 part of Hemerocallis citrina Baroni flowers, add 6 times the amount of medicinal material of 95% ethanol, reflux extract at 60°C for 1 hour, filter through gauze to obtain an alcohol extract and a residue;

[0112] (2) add 6 times the amount of medicinal material of water to the residue, extract at 60°C for 1 hour, filter through gauze to obtain a water extract and a residue;

[0113] (3) combine the alcohol extract of (1) and the water extract of (2), filter through diatomite, concentrate to a extract to obtain Hemerocallis citrina Baroni flower extract;

[0114] (4) take 1 part of red clover, add 6 times the amount of medicinal material of 95% ethanol, adjust the pH to 8, reflux extract at 60°C for 1 hour, filter through gauze to obtain an alcohol extract and a residue;

[0115] (5) add 6 times the amount of medicinal material of water to the residue, extract at 60°C for 1 hour, filter through gauze to obtain an alcohol extract and a residue;

[0116] (6) combine the alcohol extract of (1) and the water extract of (2), filter through diatomite, concentrate to a extract to obtain red clover extract;

[0117] (7) mix the Hemerocallis citrina Baroni flower extract and the red clover extract in a weight ratio of 1:1, then add stabilizer to 3 times the amount of medicinal material, stir for 1 hour; the stabilizer is a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0118] (8) store at 4°C for 1 day;

[0119] (9) filter through diatomite, and make up with dipropylene glycol to 5 times the amount of medicinal material;

[0120] (10) sterilize and cool to obtain the extract;

[0121] (11) Extract 0.45 μm organic membrane filtration, detection, filling.

[0122] Comparative Example 1

[0123] (1) Take 10 parts of Hemerocallis citrina Baroni and 1 part of Trifolium pratense L, add 95° ethanol in an amount of 20 times the medicinal material, adjust the pH to 7, and reflux extract at 50°C for 1 hour, and filter with gauze;

[0124] (2) Add water in an amount of 5 times the medicinal material to the residue, and extract at 50°C for 1 hour, and filter with gauze;

[0125] (3) Combine (1) and (2), and filter with diatomite;

[0126] (4) Concentrate under reduced pressure to a extract;

[0127] (5) Add a stabilizer in an amount of 3 times the medicinal material, and stir for 1 hour; the stabilizer is dipropylene glycol.

[0128] (6) Filter with diatomite, and add dipropylene glycol in an amount of 5 times the medicinal material;

[0129] (8) Sterilize, cool, filter with a 0.45 μm organic membrane, and detect and fill.

[0130] Comparative Example 2

[0131] (1) Take 1 part of Hemerocallis citrina Baroni and 8 parts of Trifolium pratense L, add 95° ethanol in an amount of 5 times the medicinal material, adjust the pH to 5, and reflux extract at 60°C for 1 hour, and filter with gauze;

[0132] (2) Add water in an amount of 15 times the medicinal material to the residue, and extract at 40°C for 1 hour, and filter with gauze;

[0133] (3) Combine (1) and (2), and filter with diatomite;

[0134] (4) Concentrate under reduced pressure to a extract;

[0135] (5) Add a stabilizer in an amount of 3 times the medicinal material, and stir for 1 hour; the stabilizer is a mixture of phytosterols and butanediol in a weight ratio of 1:12;

[0136] (6) Store at 4°C for 2 days;

[0137] (7) Filter with diatomite, and add butanediol to make up to an amount of 5 times the medicinal material;

[0138] (8) Sterilize, cool, filter with a 0.45 μm organic membrane, and detect and fill.

[0139] Comparative Example 3

[0140] (1) Take 1 part of Hemerocallis citrina Baroni flower and 1 part of Trifolium pratense L, add 6 times the amount of 95° ethanol of the medicinal material, adjust the pH to 8, and reflux extract at 60°C for 1 hour, filter;

[0141] (2) Concentrate under reduced pressure to extract;

[0142] (3) Add stabilizer to 3 times the amount of the medicinal material, stir for 1 hour; the stabilizer is a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0143] (4) Store at 4°C for 1 day;

[0144] (5) Filter through diatomite, and supplement dipropylene glycol to 5 times the amount of the medicinal material;

[0145] (6) Sterilize, cool, filter through 0.45 μm organic membrane, and detect and fill.

[0146] Comparative Example 4

[0147] (1) Take 1 part of Hemerocallis citrina Baroni flower and 1 part of Trifolium pratense L, add 6 times the amount of 95° ethanol of the medicinal material, adjust the pH to 8, and reflux extract at 60°C for 1 hour, filter;

[0148] (2) Concentrate under reduced pressure to extract;

[0149] (3) Add stabilizer to 3 times the amount of the medicinal material, stir for 1 hour; the stabilizer is a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0150] (4) Store at 4°C for 1 day;

[0151] (5) Filter through diatomite, and supplement dipropylene glycol to 5 times the amount of the medicinal material;

[0152] (6) Sterilize, cool, filter through 0.45 μm organic membrane, and detect and fill.

[0153] Comparative Example 5

[0154] (1) Take Hemerocallis citrina Baroni flower, add 10 times the amount of 95° ethanol of the medicinal material, adjust the pH to 10, and reflux extract at 60°C for 1 hour, filter through gauze;

[0155] (2) Add the residue to 10 times the amount of water of the medicinal material, extract at 95°C for 1 hour, and filter through gauze;

[0156] (3) Combine (1) and (2), and filter through diatomite;

[0157] (4) Concentrate under reduced pressure to extract;

[0158] (5) Add stabilizer to 3 times the amount of the medicinal material, stir for 1 hour; the stabilizer is a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0159] (6) 4°C refrigeration for 3 days;

[0160] (7) diatomite filtration, complete with dipropylene glycol to 5 times the amount of medicinal materials;

[0161] (8) sterilization, cooling, 0.45 μm organic membrane filtration, detection and filling.

[0162] Comparative Example 6

[0163] (1) red clover was weighed, 95% ethanol was added to 10 times the amount of medicinal materials, the pH was adjusted to 10, and reflux extraction was performed at 60°C for 1 hour, and gauze filtration was performed;

[0164] (2) the residue was added to 10 times the amount of water, 95°C extraction was performed for 1 hour, and gauze filtration was performed;

[0165] (3) (1) and (2) were combined, and diatomite filtration was performed;

[0166] (4) reduced pressure concentration to extract;

[0167] (5) a stabilizer was added to 3 times the amount of medicinal materials, and stirring was performed for 1 hour; the stabilizer was a mixture of phytosterols and dipropylene glycol in a weight ratio of 1:12;

[0168] (6) 4°C refrigeration for 3 days;

[0169] (7) diatomite filtration, complete with dipropylene glycol to 5 times the amount of medicinal materials;

[0170] (8) sterilization, cooling, 0.45 μm organic membrane filtration, detection and filling.

[0171] Comparative Example 7

[0172] (1) 1 part of day lily flowers and 1 part of honeysuckle were mixed, 95% ethanol was added to 6 times the amount of medicinal materials, the pH was adjusted to 8, reflux extraction was performed at 60°C for 1 hour, gauze filtration was performed, and an alcohol extract and residue were obtained;

[0173] (2) the residue was added to 6 times the amount of water, 60°C extraction was performed for 1 hour, gauze filtration was performed, and a water extract and residue were obtained;

[0174] (3) the alcohol extract and water extract were combined, and diatomite filtration was performed;

[0175] (4) the filtrate was reduced pressure concentrated to extract;

[0176] (5) a stabilizer was added to 3 times the amount of medicinal materials, and stirring was performed for 1 hour; the stabilizer was the same as in Example 1;

[0177] (6) 4°C refrigeration for 1 day;

[0178] (7) diatomite filtration, the obtained filtrate, complete with dipropylene glycol to 5 times the amount of medicinal materials;

[0179] (8) sterilization, cooling, to obtain the extract;

[0180] (9) 0.45 pm organic membrane filtration of the extract, detection, filling.

[0181] Comparative Example 8

[0182] (1) 1 part of red clover and 1 part of honeysuckle were mixed, 95% ethanol was added in an amount of 6 times the amount of medicinal materials, the pH was adjusted to 8, and reflux extraction was performed at 60°C for 1 hour. Filtration was performed with gauze to obtain an alcohol extract and a residue;

[0183] (2) The residue was added with water in an amount of 6 times the amount of medicinal materials, and extraction was performed at 60°C for 1 hour. Filtration was performed with gauze to obtain a water extract and a residue;

[0184] (3) The alcohol extract and the water extract were combined and filtered with diatomite;

[0185] (4) The filtrate was concentrated under reduced pressure to obtain an extract;

[0186] (5) A stabilizer was added in an amount of 3 times the amount of medicinal materials, and stirring was performed for 1 hour. The stabilizer was the same as in Example 1;

[0187] (6) Cold storage was performed at 4°C for 1 day;

[0188] (7) Filtration was performed with diatomite, and the obtained filtrate was supplemented with dipropylene glycol in an amount of 5 times the amount of medicinal materials;

[0189] (8) Sterilization, cooling, to obtain the extract;

[0190] (9) 0.45 pm organic membrane filtration of the extract, detection, filling.

[0191] Table 1 Process Comparison Table

[0192]

[0193]

[0194] Example 1

[0195] The final products prepared in the examples and comparative examples were divided into 4 parts (50 g each, sealed in transparent PETE vials), and were placed in a 4°C refrigerator, a 48°C constant temperature incubator, normal temperature and light, and normal temperature and light conditions, respectively. The appearance, odor, color comparison and observation of the samples were performed on the 7th day, the 14th day, the 21st day and 1 month, respectively.

[0196] Table 2 Stability Test Table

[0197]

[0198]

[0199]

[0200] Example 2

[0201] Determination of mast cell histamine release

[0202] 1. Experimental materials

[0203] PB85 mast cells (Cell Bank of Chinese Academy of Sciences), RPMI1640 medium (Gibco), fetal bovine serum (Gibco), Trypsin-EDTA (0.25%) phenol red (Gibco), CCK8 detection kit (Bi Yun Tian), (ELISA) Histamine (HA) (Cloud-Clone Corp.), C48 / 80 compound (SIGMA).

[0204] 2. Experimental instruments

[0205] Pipette (eppendorf), CO2 incubator, clean bench, high-pressure sterilization pot, high-speed centrifuge, refrigerator, microplate reader.

[0206] 3. Experimental method

[0207] 3.1 Cell culture

[0208] P815 mast cells were cultured in 1640 complete medium (1640 basal medium, 10% FBS, 1% double antibody) at 37°C, 5% CO2 cell incubator, P815 semi-suspended growth, cell density reached 70%-80%, the cells were scraped off the culture bottle with a cell scraper, and the cells were subcultured at a ratio of 1:3. After 2-3 days of conventional culture, the logarithmic growth phase cells were used for experiment.

[0209] 3.2 Cytotoxicity test

[0210] Experimental grouping: blank control group, model group (C48 / 80), experimental group (different drugs).

[0211] P815 cells were cultured in RPMI-1640 medium containing 10% FBS in a 37°C, 5% CO2 incubator, and the cells were subcultured when the cell fusion reached 80%. The cells were scraped off the culture bottle with a cell scraper, and the cells were inoculated in a 96-well plate at a cell density of about 5x10 3 After 24 hours of culture, the sample group was added with several samples at a concentration of 1%, and the control group (no sample with cells), blank hole (no sample without cells) and sample blank hole (with sample without cells) were set. Each well was set with 3 replicate wells. After 24 hours of culture, 20 μL of CCK-8 solution was added to each well, and after 4 hours of continuous culture, the absorbance of each well at 450 nm was measured by microplate reader.

[0212] Cell viability (%) = (sample OD value - sample blank group OD value) / (control group OD value - blank group OD value)

[0213] 3.3 Mast cell histamine release assay

[0214] Experimental grouping: blank control group, model group (C48 / 80), experimental group (different drugs).

[0215] P815 cells were cultured in RPMI-1640 medium containing 10% FBS at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were passaged and seeded in a 6-well plate at a density of 5 x 10 5 The cells were cultured for 24 hours, then 1 ml of the extract of Examples 1-4 and Comparative Examples 1-8 was added to each well at a concentration of 0.002%, and the plate was incubated at 37°C in a 5% CO2 incubator for 30 min. C48 / 80 was added to stimulate the cells for 15 min, and then the cells were placed in ice water to terminate the treatment for 10 min. The supernatant was centrifuged at 10,000 rpm for 5 min at 4°C.

[0216] The amount of histamine released by the cells was measured according to the instructions of the histamine ELISA kit. The amount of histamine was calculated based on the standard curve.

[0217] 3.4 Statistical analysis

[0218] The OD values of the test results are expressed as mean ± standard deviation (x ± s). The t-test was used for statistical analysis, and the two-tailed test was used with a significance level of α = 0.05.

[0219] 3.5 Experimental results

[0220] 3.5.1 Test results of the effect of the sample on the cell viability of P815 cells

[0221] Table 3 shows the effect of the extract of Examples and Comparative Examples on the survival rate of P815 mast cells. According to Table 3, the extract of Examples and Comparative Examples had no effect on the cell viability of P815 cells.

[0222] Table 3

[0223] Cell survival rate (%) Example 1 101 Example 2 99.6 Example 3 104 Example 4 101 Comparative Example 1 99.8 Comparative Example 2 96.1 Comparative Example 3 98.9 Comparative Example 4 100.2 Comparative Example 5 98.9 Comparative Example 6 102 Comparative Example 7 99.5 Comparative Example 8 100.1

[0224] 3.5.2 Effect of the sample on the release of histamine by P815 mast cells

[0225] The effect of the extracts obtained in the examples and comparative examples on the histamine release rate of P815 mastocytes is shown in Table 4. As can be seen from Table 4, the histamine inhibitory effect of all the examples is superior to that of the comparative examples.

[0226] Table 4

[0227]

[0228]

Claims

1. A compound plant extract, characterized in that, The raw materials for the compound plant extract are daylily flowers and red clover; the weight ratio of daylily flowers to red clover is (1:5) to (5:1). The preparation method of the compound plant extract includes the following steps: Step (1): Mix daylily flowers and red clover according to the proportion of the raw material composition, extract with alcohol, filter, and obtain residue A and alcohol extract; the alcohol extraction process uses ethanol, the weight of the alcohol solution added in the alcohol extraction process is 6 to 15 times the weight of the medicinal material, the pH of the alcohol extraction system is 8 to 12, and the temperature of the alcohol extraction is 60 to 85℃. Step (2): The residue A is subjected to water extraction and filtration to obtain residue B and water extract; the weight of water added during the water extraction process is 6 to 12 times the weight of the medicinal material, and the water extraction temperature is 60 to 95°C. Step (3): Combine the alcohol extract and the water extract, filter, and obtain the initial product; Step (4): A stabilizer is added to the initial product, stirred, aged, and filtered to obtain a compound plant extract; the stabilizer is one or more of phytosterols, Tween-80, Tween-20, butylene glycol, glycerin, propylene glycol, methyl propylene glycol and dipropylene glycol; the weight of the stabilizer added is 2 to 4 times the weight of the medicinal material.

2. The compound plant extract as described in claim 1, characterized in that, The weight ratio of the daylily flowers to the red clover is 1:1; Alternatively, the weight ratio of the daylily flowers to the red clover is 1:5; Alternatively, the weight ratio of the daylily flowers to the red clover is 5:

1.

3. The compound plant extract as described in claim 1, characterized in that, In step (1), the concentration of ethanol is 80-100%; And / or, in step (1), the alcohol extraction time is 0.5 to 2 hours; And / or, in step (1), the filtration method is gauze filtration; And / or, in step (2), the water extraction time is 0.5 to 2 hours; And / or, in step (2), the filtration method is gauze filtration; And / or, in step (3), the filtration method is diatomaceous earth filtration; And / or, in step (3), the filtration process further includes a concentration step; And / or, in step (4), the stirring time is 0.5 to 1.5 hours; And / or, in step (4), the aging temperature is 2~8℃ and the aging time is 1~3 days; And / or, in step (4), the filtration method is diatomaceous earth filtration; And / or, in step (4), the filtration process further includes adding a solvent to the filtrate.

4. The compound plant extract as described in claim 3, characterized in that, In step (3), the concentration is performed using a reduced pressure method; And / or, in step (4), the solvent is one or more of butanediol, glycerol, propylene glycol, methyl propylene glycol and dipropylene glycol; And / or, in step (4), the solvent added is 3 to 8 times the weight of the medicinal material.

5. A compound plant extract, characterized in that, The raw materials of the compound plant extract are daylily flower extract and red clover extract, and the weight ratio of daylily flower extract and red clover extract is (1:5) to (5:1). The preparation method of the compound plant extract includes the following steps: Step (1): Take daylily flowers, extract with alcohol, filter to obtain filter residue and alcohol extract; extract the filter residue with water, filter to obtain water extract; combine the alcohol extract and the water extract to obtain daylily flower extract; the alcohol extraction process uses ethanol, the weight of the alcohol solution added in the alcohol extraction process is 6 to 15 times the weight of the medicinal material, and the temperature of the alcohol extraction is 60 to 95°C; the weight of water added in the water extraction process is 6 to 12 times the weight of the medicinal material, and the temperature of the water extraction is 60 to 95°C; Step (2): Take red clover, extract with alcohol, filter to obtain residue and alcohol extract; extract the residue with water, filter to obtain water extract; combine the alcohol extract and the water extract to obtain red clover extract; the alcohol extraction process uses ethanol, the weight of the alcohol solution added in the alcohol extraction process is 6 to 15 times the weight of the medicinal material, the pH of the alcohol extraction system is 8 to 12, and the temperature of the alcohol extraction is 60 to 85°C; the weight of water added in the water extraction process is 6 to 12 times the weight of the medicinal material, and the temperature of the water extraction is 60 to 95°C. The steps (1) and (2) are not sequential; Step (3): The daylily flower extract and the red clover extract are mixed according to the proportion of the raw material composition, then a stabilizer is added, stirred, aged, and filtered to obtain a compound plant extract; the stabilizer is one or more of dipropylene glycol, phytosterols, Tween-80, Tween-20, butylene glycol, glycerin, propylene glycol, methyl propylene glycol and dipropylene glycol; the weight of the stabilizer added is 2 to 4 times the weight of the medicinal material.

6. The compound plant extract as described in claim 5, characterized in that, In step (1), the concentration of ethanol is 80-100%; And / or, in step (1), the alcohol extraction time is 0.5 to 2 hours; And / or, in step (1), the post-extraction filtration method is gauze filtration; And / or, in step (1), the water extraction time is 0.5 to 2 hours; And / or, in step (1), the water extraction and filtration method is gauze filtration; And / or, in step (1), after combining the alcohol extract and the water extract, a concentration step is further included; And / or, in step (2), the concentration of the ethanol is 80~100%; And / or, in step (2), the alcohol extraction time is 0.5 to 2 hours; And / or, in step (2), the post-extraction filtration method is gauze filtration; And / or, in step (2), the water extraction time is 0.5 to 2 hours; And / or, in step (2), the water extraction and filtration method is gauze filtration; And / or, in step (2), after combining the alcohol extract and the water extract, a concentration step is also included; And / or, in step (3), the stirring time is 0.5 to 1.5 hours; And / or, in step (3), the aging temperature is 2~8℃; the aging time is 1~3 days; And / or, in step (3), the filtration method is diatomaceous earth filtration; And / or, in step (3), the filtration process further includes adding a solvent to the filtrate.

7. The compound plant extract as described in claim 6, characterized in that, In step (1), the concentration is a method of reducing pressure; And / or, in step (2), the concentration is a method of decompression; And / or, in step (3), the solvent is one or more of butanediol, glycerol, propylene glycol, methyl propylene glycol and dipropylene glycol; And / or, in step (3), the weight of the solvent added is 3 to 8 times the weight of the medicinal material.

8. A topical skin preparation, characterized in that, The topical skin preparation includes the compound plant extract as described in any one of claims 1 to 7.

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