Preparation method, product and application of platycodon grandiflorum extract with soothing effect

Abstract

The present invention relates to a preparation method, product and application of a platycodon grandiflorum extract with a soothing effect, comprising the following steps: drying and pulverizing platycodon grandiflorum and mixing it with water for flash extraction; mixing the flash extraction solution and the platycodon grandiflorum residue with absolute ethanol or an ethanol aqueous solution for reflux extraction; filtering and concentrating the reflux extraction solution and then purifying and eluting it with macroporous resin, and collecting the eluate; concentrating and drying the eluate to obtain the platycodon grandiflorum extract. This preparation method combines flash extraction and reflux extraction, which not only realizes more effective utilization of platycodon grandiflorum raw materials, but also significantly improves the anti-inflammatory and soothing effects of the final product. Specifically, it inhibits the TRPV1 expression of keratinocytes, inhibits the calcium influx phenomenon of keratinocytes, and inhibits the degranulation rate of mast cells, that is, it exerts a soothing effect through multiple targets and multiple pathways. At the same time, it has high safety and will not cause skin adverse reactions, which provides a new strategy for the preparation of products with soothing effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and relates to a preparation method, a product and an application of a platycodon grandiflorum extract with a soothing effect. Background Art

[0002] In addition to the barrier protection function, the skin also has a sensory function. The reasons for skin cells to generate sensory signals are as follows: firstly, there are sensory afferent nerve fibers in the stratum corneum of the skin, and the sensory nerve fibers can perceive various external stimuli; secondly, some receptors that can sense temperature and pain will be expressed on the surface of keratinocytes, such as a large class of transmembrane receptors TRPs. TRP channels are involved in numerous physiological processes, such as intracellular calcium balance, cell chemotaxis, nerve migration, neurite extension, epithelial cell calcium transport, cell proliferation and differentiation, and cell immune response. Most members of the TRP channels are also involved in the perception of sensory stimuli, including osmotic pressure, light signal transduction, mechanical force stimulation, taste and pheromone recognition, temperature perception, pain and itching sensations, etc.

[0003] The TRPV1 channel is one of the most widely studied members of the TRP channels. The TRPV1 channel is the receptor of capsaicin and undertakes the functions of temperature and nociceptive pain perception. It was early named VR1 due to its sensitivity to capsaicin stimulation. Recent studies have found that TRPV1 plays a key role in sensitive skin. TRPV1 in sensitive skin is easily activated, leading to the opening of calcium influx channels. In addition, after the activation of TRPV1, it will induce the secretion of inflammatory factors and inflammatory mediators, resulting in symptoms such as burning and stinging. Currently, inhibiting the activation of TRPV1 is an effective measure for treating sensitive skin and is also a major focus of cosmetics R & D enterprises.

[0004] Studies have found that TRPV1 is expressed on the envelopes of various cells such as epidermal keratinocytes, sebaceous gland cells, dermal fibroblasts, dendritic cells, mast cells, and dermal vascular endothelial cells. Among them, mast cells are one of the immune cells widely distributed in the skin and mucosa. They participate in the occurrence and development of innate immunity and adaptive immunity by degranulating and releasing pro-inflammatory mediators such as histamine, TNF-α, prostaglandins, proteases, etc. Mast cells are the main effector cells in allergic diseases such as skin allergy, urticaria, allergic rhinitis, and asthma. The skin allergy participated by mast cells is manifested as itching. The activation of the TRPV1 channel causes the degranulation of mast cells and transmits the itching signal to sensory nerves.

[0005] Platycodon grandiflorus is the dried root of Platycodon grandiflorus, a perennial herbaceous plant of the genus Platycodon in the family Campanulaceae, and it belongs to the plants that can be used as both medicine and food. Its main components include saponins, flavonoids, amino acids, phenols, etc., and it has the effects of reducing phlegm and relieving cough, antibacterial and anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and hypolipidemic. In recent years, these biological effects of Platycodon grandiflorus have become a hot topic of research for domestic and foreign scientific research workers. Especially for its anti-inflammatory and anti-tumor effects, more and more research reports have described the mechanism of action of Platycodon grandiflorus extracts on various inflammations and tumors in the body. However, in the field of cosmetics, there is still relatively little research on the anti-allergic mechanism of Platycodon grandiflorus extracts for sensitive skin. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method, a product and an application of a Platycodon grandiflorus extract with a soothing effect.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a preparation method of a Platycodon grandiflorus extract with a soothing effect, and the preparation method includes the following steps:

[0009] (1) Dry and crush Platycodon grandiflorus and then mix it with water, and perform flash extraction to obtain a flash extraction solution and Platycodon grandiflorus residue;

[0010] (2) Mix the flash extraction solution and Platycodon grandiflorus residue with absolute ethanol or an ethanol aqueous solution, and perform reflux extraction to obtain a reflux extraction solution;

[0011] (3) Filter and concentrate the reflux extraction solution, and then purify and elute it with macroporous resin. The macroporous resin is a mixture of HPD100 and DM130, and collect the eluate;

[0012] (4) Concentrate and dry the eluate to obtain the Platycodon grandiflorus extract.

[0013] The present invention creatively develops a method for preparing Platycodon grandiflorus extract. By combining flash extraction and reflux extraction, it not only realizes more effective utilization of Platycodon grandiflorus raw materials, but also significantly improves the soothing effect of the final product. Specifically, it can inhibit the TRPV1 expression of keratinocytes, inhibit the calcium influx phenomenon of keratinocytes, and inhibit the degranulation rate of mast cells, that is, it exerts a soothing effect through multiple targets and multiple pathways. At the same time, it has high safety and will not cause skin adverse reactions, which provides a new strategy for preparing soothing products. At the same time, this preparation method is easy to realize industrialized scale production and has practicality.

[0014] Furthermore, the present invention creatively uses a mixed resin of HPD100 and DM130 to purify the extraction product. Compared with other types of resin combinations or single resins, HPD100 and DM130 can cooperate synergistically, and the final product obtained by separation and purification has better effects in inhibiting the expression of TRPV1 in keratinocytes and soothing.

[0015] Preferably, in step (1), the comminution is to 20 - 40 mesh, such as 20 mesh, 22 mesh, 25 mesh, 28 mesh, 30 mesh, 32 mesh, 35 mesh, 37 mesh, 38 mesh, 40 mesh, etc.

[0016] Preferably, in step (1), the mass ratio of platycodon grandiflorum to water is 1:(2 - 5), such as 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0017] Preferably, in step (1), the flash extraction is carried out in a flash extractor, and the mechanical rotation speed is 3000 - 7000 rpm, such as 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, etc.

[0018] Preferably, in step (1), the flash extraction is carried out 1 - 3 times, such as 1 time, 2 times or 3 times; each time for 1 - 5 min, such as 1 min, 2 min, 3 min, 4 min, 5 min, etc.

[0019] Preferably, in step (2), after mixing the flash extraction solution and platycodon residue with anhydrous ethanol or ethanol aqueous solution, the final concentration of ethanol is 70 - 80%, such as 70%, 73%, 75%, 78%, 80%, etc.

[0020] Compared with other final ethanol concentrations, using an ethanol aqueous solution with a final concentration of 70 - 80% for reflux extraction results in a final product with better effects in inhibiting the expression of TRPV1 in keratinocytes and soothing.

[0021] Preferably, in step (2), the mass ratio of anhydrous ethanol or ethanol aqueous solution to platycodon grandiflorum in step (1) is 1:(8 - 15), such as 1:8, 1:9, 1:10, 1:12, 1:13, 1:14, 1:15, etc.

[0022] Preferably, in step (2), the reflux extraction is carried out 1 - 3 times, such as 1 time, 2 times or 3 times; each time for 1 - 4 h, such as 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, etc.

[0023] Preferably, the mass ratio of HPD100 to DM130 is (1-4):1, such as 1:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.

[0024] Based on the synergistic cooperation relationship between HPD100 resin and DM130 resin, when the two satisfy the above specific mass ratio relationship, the final product of separation and purification has better effects in inhibiting the expression of TRPV1 in keratinocytes and soothing.

[0025] Preferably, the dosage of the macroporous resin in step (3) is 1-4 times that of the Platycodon grandiflorum in step (1), such as 1 time, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, etc.

[0026] Preferably, the process of purification and elution is as follows: first, elute with 3-8 BV (such as 3 BV, 4 BV, 5 BV, 6 BV, 7 BV, 8 BV, etc.) of pure water or 0-10% (such as 2%, 5%, 8%, 10%, etc.) ethanol aqueous solution, and then elute with 3-8 BV (such as 3 BV, 4 BV, 5 BV, 6 BV, 7 BV, 8 BV, etc.) of 60-70% (such as 60%, 63%, 67%, 70%, etc.) ethanol aqueous solution, and collect the ethanol eluate.

[0027] Preferably, the drying method in step (4) includes freeze-drying.

[0028] In a second aspect, the present invention provides a Platycodon grandiflorum extract prepared by the preparation method according to the first aspect.

[0029] In a third aspect, the present invention provides the use of the Platycodon grandiflorum extract according to the second aspect in the preparation of a product having a soothing and / or antipruritic effect.

[0030] In a fourth aspect, the present invention provides the use of the Platycodon grandiflorum extract according to the second aspect in the preparation of an inhibitor of TRPV1 expression in keratinocytes.

[0031] In a fifth aspect, the present invention provides the use of the Platycodon grandiflorum extract according to the second aspect in the preparation of an inhibitor of calcium influx in keratinocytes.

[0032] In a sixth aspect, the present invention provides the use of the Platycodon grandiflorum extract according to the second aspect in the preparation of an inhibitor of mast cell degranulation.

[0033] Other specific point values not listed in the above numerical ranges involved in the present invention are all within the protection scope of the present invention. For the sake of brevity of description and space, they will not be elaborated one by one here.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention creatively develops a method for preparing platycodon grandiflorum extract, which combines flash extraction and reflux extraction. It not only realizes more effective utilization of platycodon grandiflorum raw materials, but also significantly improves the soothing effect of the final product. Specifically, it inhibits the TRPV1 expression of keratinocytes, inhibits the calcium influx phenomenon of keratinocytes, and inhibits the degranulation rate of mast cells, that is, it exerts a soothing effect through multiple targets and multiple pathways. At the same time, it has high safety and will not cause skin adverse reactions, which provides a new strategy for preparing soothing products. At the same time, this preparation method is easy to realize industrial scale-up production and has practicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a process schematic diagram of the method for preparing platycodon grandiflorum extract involved in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0038] Example 1

[0039] This example provides a platycodon grandiflorum extract, and its preparation method is as follows (the process schematic diagram is as Figure 1 shown):

[0040] (1) Dry and crush platycodon grandiflorum to 30 mesh, then mix it with 3 times the feeding amount of water, place it in a flash extractor for flash extraction once, the extraction time is 3 min, and the mechanical rotation speed is 5000 rpm to obtain a flash extract and platycodon grandiflorum residue;

[0041] (2) Mix the flash extract and platycodon grandiflorum residue with 10 times the feeding amount of high-concentration ethanol aqueous solution to make the final concentration of ethanol 75%, carry out reflux extraction for 1.5 h, and repeat the above reflux extraction operation once, and combine the two reflux extracts;

[0042] (3) Filter, concentrate the reflux extract, and then purify and elute it with macroporous resin. The macroporous resin is a mixture of HPD100 resin and DM130 resin with a mass ratio of 2:1. The process of purification and elution is as follows: first elute with 5 BV of pure water, and then elute with 4 BV of 65% ethanol aqueous solution, and collect the alcohol eluate;

[0043] (4) Concentrate and lyophilize the eluate to obtain the platycodon grandiflorum extract.

[0044] Example 2

[0045] This example provides a platycodon grandiflorum extract, and its preparation method is as follows:

[0046] (1) Dry the platycodon root and crush it to 40 mesh, then mix it with water at 4 times the feeding amount, place it in a flash extractor for flash extraction twice, with the extraction time of 2 min and the mechanical rotation speed of 6000 rpm to obtain the flash extract and platycodon root residue;

[0047] (2) Mix the flash extract and platycodon root residue with a high-concentration ethanol aqueous solution at 8 times the feeding amount to make the final concentration of ethanol 80%, carry out reflux extraction for 2 h, and repeat the above reflux extraction operation once, then combine the two reflux extracts;

[0048] (3) Filter and concentrate the reflux extract, and then purify and elute it with macroporous resin. The macroporous resin is a mixture of HPD100 resin and DM130 resin with a mass ratio of 1:1. The process of purification and elution is as follows: first elute with 7 BV of pure water, then elute with 5 BV of 60% ethanol aqueous solution, and collect the alcohol eluate;

[0049] (4) Concentrate the eluate and freeze-dry it to obtain the platycodon root extract.

[0050] Example 3

[0051] This example provides a platycodon root extract, and its preparation method is as follows:

[0052] (1) Dry the platycodon root and crush it to 20 mesh, then mix it with water at 2 times the feeding amount, place it in a flash extractor for flash extraction once, with the extraction time of 4 min and the mechanical rotation speed of 4000 rpm to obtain the flash extract and platycodon root residue;

[0053] (2) Mix the flash extract and platycodon root residue with a high-concentration ethanol aqueous solution at 8 times the feeding amount to make the final concentration of ethanol 70%, carry out reflux extraction for 2 h, and repeat the above reflux extraction operation once, then combine the two reflux extracts;

[0054] (3) Filter and concentrate the reflux extract, and then purify and elute it with macroporous resin. The macroporous resin is a mixture of HPD100 resin and DM130 resin with a mass ratio of 4:1. The process of purification and elution is as follows: first elute with 5 BV of pure water, then elute with 3 BV of 70% ethanol aqueous solution, and collect the alcohol eluate;

[0055] (4) Concentrate the eluate and freeze-dry it to obtain the platycodon root extract.

[0056] Example 4

[0057] This example provides a platycodon root extract, and the difference between its preparation method and that of Example 1 is only that in step (2), the flash extract and platycodon root residue are mixed with a high-concentration ethanol aqueous solution at 10 times the feeding amount to make the final concentration of ethanol 90%, and other conditions remain unchanged.

[0058] Example 5

[0059] This example provides a platycodon grandiflorum extract. The difference in its preparation method from that of Example 1 is only that in step (2), the flash extraction solution and the platycodon grandiflorum residue are mixed with a high-concentration ethanol aqueous solution with a feeding amount of 10 times, so that the final concentration of ethanol is 60%, and other conditions remain unchanged.

[0060] Example 6

[0061] This example provides a platycodon grandiflorum extract. The difference in its preparation method from that of Example 1 is only that in step (3), the process of purification and elution is as follows: first, elute with 5 BV of pure water, and then elute with 4 BV of 75% ethanol aqueous solution, and collect the alcohol eluate.

[0062] Comparative Example 1

[0063] This comparative example provides a platycodon grandiflorum extract. The difference in its preparation method from that of Example 1 is only that in step (3), the macroporous resin is a mixture of D101 resin and AB-8 resin with a mass ratio of 2:1, and other conditions remain unchanged.

[0064] Comparative Example 2

[0065] This comparative example provides a platycodon grandiflorum extract. The difference in its preparation method from that of Example 1 is only that in step (3), the macroporous resin is a single HPD100 resin, and other conditions remain unchanged.

[0066] Comparative Example 3

[0067] This comparative example provides a platycodon grandiflorum extract. The difference in its preparation method from that of Example 1 is only that in step (3), the macroporous resin is a single DM130 resin, and other conditions remain unchanged.

[0068] Comparative Example 4

[0069] This comparative example provides a platycodon grandiflorum extract, and its preparation method is as follows:

[0070] (1) Dry and crush the platycodon grandiflorum to 30 meshes, then mix it with a 75% ethanol aqueous solution with a feeding amount of 10 times, carry out reflux extraction for 2 h, and repeat the above reflux extraction operation once, and combine the two reflux extraction solutions;

[0071] (2) Filter and concentrate the reflux extraction solution, and then use macroporous resin for purification and elution. The macroporous resin is a mixture of HPD100 resin and DM130 resin with a mass ratio of 2:1. The process of purification and elution is as follows: first, elute with 5 BV of pure water, and then elute with 4 BV of 65% ethanol aqueous solution, and collect the alcohol eluate;

[0072] (3) Concentrate and freeze-dry the eluate to obtain the platycodon grandiflorum extract.

[0073] Comparative Example 5

[0074] This comparative example provides a platycodon grandiflorum extract, and its preparation method is as follows:

[0075] (1) Dry and crush platycodon grandiflorum to 30 mesh, then mix it with 10 times the feeding amount of 75% ethanol aqueous solution, carry out reflux extraction for 1.5 h, and repeat the above reflux extraction operation 2 times, and combine the three reflux extracts;

[0076] (2) Filter and concentrate the reflux extract, and then purify and elute it with macroporous resin. The macroporous resin is a mixture of HPD100 resin and DM130 resin with a mass ratio of 2:1. The process of purification and elution is as follows: first elute with 5 BV of pure water, then elute with 4 BV of 65% ethanol aqueous solution, and collect the alcohol eluate;

[0077] (3) Concentrate and lyophilize the eluate to obtain the platycodon grandiflorum extract.

[0078] Test Example 1

[0079] Effect on the expression level of TRPV1 in keratinocytes:

[0080] The test method is as follows: Use keratinocytes to evaluate the soothing effect of the platycodon grandiflorum extract by detecting the relative expression level of the capsaicin receptor (TRPV1) protein through immunofluorescence (IF).

[0081] Seed cells at 4.5×10 4 / well into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2, 95% RH). When the cell confluence rate in the 24-well plate reaches 40%-60%, administer the drugs. Among them, the blank control group: do not add trans-4-tert-butylcyclohexanol and there is no capsaicin stimulation; the model control group: do not add trans-4-tert-butylcyclohexanol and there is capsaicin stimulation; the positive control group: add trans-4-tert-butylcyclohexanol with a concentration of 15.6 μg / mL and there is capsaicin stimulation; the sample group: add the diluted solutions of the platycodon grandiflorum extracts prepared in Examples 1-6 and Comparative Examples 1-5 with a concentration of 0.0002 mg / mL each and there is capsaicin stimulation.

[0082] After the administration was completed, the 24-well plates were placed in an incubator and cultured for 24 h. After the culture ended, the supernatant of the culture medium was aspirated and discarded, and the cells were washed 3 times with PBS and the PBS was aspirated and discarded. Then, 200 μL of goat serum was added dropwise to each well and blocked at room temperature for 60 min. The goat serum blocking solution was aspirated and discarded. Then, the diluted primary antibody Anti-VR1 antibody (200 μL / well) (diluted with goat serum at 1:150) was added dropwise to each well and incubated overnight at 4°C. The primary antibody was aspirated and discarded, and the cells were washed 3 times with PBS for 5 min each time. The residual PBS liquid on the coverslips was blotted dry with absorbent paper. Then, the diluted fluorescent secondary antibody Goat pAb to Rb IgG (Alexa Fluor® 488) (200 μL / well) (diluted with PBS at 1:500) was added dropwise to each well and incubated in the dark at room temperature for 1 h. The secondary antibody was aspirated and discarded, and the cells were washed 3 times with PBS for 5 min each time. The residual PBS liquid on the coverslips was blotted dry with absorbent paper. Then, Hochest33342 (200 μL / well) (diluted with PBS at 1:500) was added dropwise to each well and incubated at room temperature for 5 min. The Hochest33342 was aspirated and discarded, and the cells were washed 3 times with PBS for 5 min each time. The residual PBS liquid on the coverslips was blotted dry with absorbent paper. The coverslips were picked out with a needle, a drop of anti-quenching agent was added to the glass slide and the coverslips were placed upside down on the glass slide and sealed (the anti-quenching agent was aspirated with a cut yellow pipette tip); detected by fluorescence microscopy.

[0083] The detection and statistical results of the relative expression levels of the transient receptor potential vanilloid 1 (TRPV1) in each group are shown in Table 1.

[0084] Table 1

[0085]

[0086] From the data results in Table 1, it can be seen that compared with the model control group, the platycodon grandiflorum extract involved in the present invention can significantly down-regulate the expression level of TRPV1 in keratinocytes, and thus has the effects of soothing, anti-allergic and analgesic. Compared with the platycodon grandiflorum extract in the comparative example, the platycodon grandiflorum extract in the example combines flash extraction and reflux extraction and selects a specific macroporous resin for elution, which significantly improves the above effects of the final product.

[0087] Test Example 2

[0088] Effect on the calcium ion influx phenomenon in keratinocytes:

[0089] The test method was as follows: Keratinocytes were used, and the soothing and sedative effects of the platycodon grandiflorum extract were evaluated by detecting the change in the fluorescence intensity of calcium ions in keratinocytes through immunofluorescence (IF). According to 2×10 4Inoculate cells at a density of [number of cells] per well into a 96-well cell culture plate and culture overnight at 37°C and 5% CO₂. When the cell confluence reaches 80%, remove the culture medium, wash the cells 3 times with PBS, then add 100 μL of culture medium containing 1 μmol / L Fluo4 / AM to each well and culture at 37°C and 5% CO₂ for 30 min. Remove the supernatant, add 200 μL of PBS to each well, and culture at 37°C and 5% CO₂ for 20 min. Add the test substances of each group. Set 3 replicate wells for each group and add 100 μL of culture medium containing test substances at different concentrations to each well. Among them, the blank control group: does not add trans-4-tert-butylcyclohexanol and has no capsaicin stimulation; the model control group: does not add trans-4-tert-butylcyclohexanol and is stimulated with capsaicin (30 μM); the positive control group: adds trans-4-tert-butylcyclohexanol at a concentration of 7.8 μg / mL and is stimulated with capsaicin; the sample group: adds the diluted solution of the platycodon grandiflorum extract prepared in Example 1 at a concentration of 0.0002 mg / mL and is stimulated with capsaicin.

[0090] After the administration is completed, culture the 96-well plate at 37°C and 5% CO₂ for 30 min. Remove the supernatant, wash the cells 3 times with PBS, and detect with a fluorescence microplate reader under light-proof conditions, and record the fluorescence intensity value. The detection and statistical results of the fluorescence intensity during calcium ion influx in each group are shown in Table 2.

[0091] Table 2

[0092]

[0093] From the data results in Table 2, it can be seen that compared with the model control group, the platycodon grandiflorum extract involved in the present invention can significantly inhibit the phenomenon of calcium ion influx in keratinocytes, and thus has the efficacy of soothing and calming.

[0094] Test Example 3

[0095] Effect on the degranulation rate of mast cells:

[0096] The test method is as follows: Collect P815 mast cells with good cell morphology and in the logarithmic growth phase, and prepare them to a concentration of [concentration value] per 1 mL with complete culture medium 5Cells were seeded in 24-well cell culture plates and cultured at 37°C, 5% CO2 and saturated humidity. After 16 h, the original medium was removed and the test substances of each group were added. Each group had 3 replicate wells, and 900 μL of medium containing test substances at different concentrations was added to each well. Among them, the blank control group: did not add sodium cromoglycate and had no C48 / 80 stimulation; the model control group: did not add sodium cromoglycate and had C48 / 80 stimulation; the positive control group: added sodium cromoglycate at a concentration of 1 mg / mL and had C48 / 80 stimulation; the sample group: added the diluted solution of the platycodon grandiflorum extract prepared in Example 1 at a concentration of 0.0002 mg / mL and had C48 / 80 stimulation.

[0097] After the administration was completed, the 24-well plates were placed in an incubator (37°C, 5% CO2, 95% RH) and cultured for 2 h. After the samples were incubated for 2 h, according to the experimental grouping, the blank control group was supplemented with 100 μL of normal medium, and each well of the other groups was supplemented with 100 μL of 100 μg / mL C48 / 80 stock solution. After C48 / 80 stimulation for 1 h, the reaction was terminated by ice bath. After the culture was completed, the degranulation rate of the cells was statistically calculated using IPP software under an inverted microscope. The statistical results are shown in Table 3.

[0098] Table 3

[0099]

[0100] From the data results in Table 3, it can be seen that compared with the model control group, the platycodon grandiflorum extract involved in the present invention can significantly reduce the degranulation rate of mast cells, and thus has the efficacy of relieving sensitivity and itching.

[0101] Test Example 4

[0102] Skin irritation evaluation:

[0103] The test method was as follows: Using the EpiSkin artificial skin model as the test model and SDS as the induction condition, the skin irritation of the samples was detected according to the OECD TG439 method. Among them, the blank control group: treated with 10 μL of PBS for 15 min; the model control group: treated with 10 μL of 5% SDS for 15 min; the sample group: treated with 10 μL of the diluted solution of the platycodon grandiflorum extract prepared in Example 1 at a concentration of 5% for 15 min. The results are shown in Table 4.

[0104] Table 4

[0105]

[0106] From the data results in Table 4, it can be seen that the platycodon grandiflorum extract involved in the present invention has no irritation to the skin, has a mild feeling and high safety.

[0107] Test Example 5

[0108] Patch test:

[0109] The test method is as follows: 30 eligible social volunteers aged 20 - 45 years old were randomly divided into 3 groups, and each group was respectively tried out with 2 different products in Examples 1 - 6. Put them into the small chamber of the patch tester, and no treatment was done to the control hole. The patch tester with the test substance was applied to the inner side of the forearm of the subject with a low - sensitization tape, and gently pressed with the palm to make it evenly adhere to the skin for 48 h. The skin reactions were observed according to the standard in Table 5 at 30 min, 24 h, and 48 h respectively after removing the patch tester with the test substance.

[0110] Table 5

[0111]

[0112] The results showed that the products in Examples 1 - 6 of the present invention were all negative reactions after the trial, indicating that the platycodon grandiflorum extract involved in the present invention is safe and mild, and will not cause adverse reactions such as skin irritation and sensitization.

[0113] The applicant declares that the present invention uses the above - mentioned examples to illustrate the technical solution of the present invention, but the present invention is not limited to the above - mentioned examples, that is, it does not mean that the present invention must rely on the above - mentioned examples to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above - mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0115] In addition, it should be noted that in the various specific technical features described in the above - mentioned specific embodiments, they can be combined in any suitable way without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for preparing a platycodon grandiflorum extract having a soothing effect, characterized in that: The preparation method comprises the following steps: (1) drying and crushing the platycodon grandiflorum, mixing the mixture with water, and performing flash extraction to obtain a flash extract and platycodon grandiflorum residue; (2) mixing the flash extract and the platycodon grandiflorum residue with anhydrous ethanol or an ethanol-water solution, and performing reflux extraction to obtain a reflux extract; (3) filtering and concentrating the reflux extract, and then purifying and eluting with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 and DM130 in a mass ratio of (1-4):1, and the purification and elution process is: first eluting with 3-8 BV of pure water or 0-10% ethanol aqueous solution, and then eluting with 3-8 BV of 60-70% ethanol aqueous solution, and collecting the alcohol washing liquid; (4) Concentrating and drying the eluate to obtain the Platycodon grandiflorum extract.

2. The preparation method according to claim 1, characterized in that: The crushing in step (1) to a size of 20-40 mesh; The mass ratio of Platycodon grandiflorum to water in step (1) is 1:(2-5); The flash extraction in step (1) is carried out in a flash extractor, the mechanical speed is 3000-7000 rpm, and the flash extraction is carried out 1-3 times, each time for 1-5 min.

3. The preparation method according to claim 1, characterized in that: In step (2), the flash extract and platycodon grandiflorum residue are mixed with anhydrous ethanol or ethanol aqueous solution to obtain a final ethanol concentration of 70-80%; The mass ratio of the anhydrous ethanol or ethanol aqueous solution in step (2) to the platycodon grandiflorum in step (1) is 1:(8-15); The reflux extraction in step (2) is performed 1-3 times, each time for 1-4 hours.

4. The Platycodon grandiflorum extract obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the Platycodon grandiflorum extract according to claim 4 in the preparation of products with soothing and / or antipruritic effects.

6. Use of the Platycodon grandiflorum extract according to claim 4 in the preparation of a keratinocyte TRPV1 expression inhibitor.

7. Use of the Platycodon grandiflorum extract according to claim 4 in the preparation of a keratinocyte calcium influx inhibitor.

8. Use of the Platycodon grandiflorum extract according to claim 4 in the preparation of a mast cell degranulation inhibitor.

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