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

Abstract

The invention relates to a preparation method of a platycodon grandiflorum extract with a soothing effect as well as a product and application of the platycodon grandiflorum extract. The preparation method comprises the following steps: drying and crushing platycodon grandiflorum, mixing with water, and carrying out flash extraction; mixing the flash extracting solution and the platycodon grandiflorum residues with absolute ethyl alcohol or an ethanol water solution, and carrying out reflux extraction; filtering and concentrating the reflux extracting solution, purifying and eluting by adopting macroporous resin, and collecting eluent; and concentrating and drying the eluent to obtain the platycodon grandiflorum extract. According to the preparation method, flash extraction and reflux extraction are combined, so that not only is a platycodon grandiflorum raw material more effectively utilized, but also the anti-inflammatory and soothing effects of a final product are remarkably improved, and the anti-inflammatory and soothing effects are specifically shown as inhibition of TRPV1 expression of keratinocytes, inhibition of a calcium influx phenomenon of the keratinocytes and inhibition of the degranulation rate of mast cells; in other words, the soothing effect is achieved in a multi-target and multi-way mode. Meanwhile, the safety is high, untoward effects of the skin cannot be caused, and a new strategy is provided for preparing products with relieving effects.

Description

Technical Field

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

[0002] In addition to its barrier protection function, the skin also has sensory functions. The reasons why skin cells generate sensory signals are: first, there are sensory afferent nerve fibers in the stratum corneum of the skin, which can sense various external stimuli; second, the surface of keratinocytes expresses some receptors that can sense temperature and pain, such as a large class of transmembrane receptors TRPs. TRP channels are involved in many physiological processes, such as intracellular calcium balance, cell-derived chemotaxis, nerve migration, neurite extension, epithelial cell calcium transport, cell proliferation and differentiation, and cellular immune response. Most members of the TRP channel are also involved in the perception of sensory stimuli, including osmotic pressure, light signal transmission, mechanical force stimulation, taste and pheromone recognition, temperature perception, pain and itching perception, etc.

[0003] TRPV1 channel is one of the most widely studied members of TRP channel. TRPV1 channel is the receptor of capsaicin, which is responsible for the perception of temperature and noxious pain. Because of its ability to perceive the stimulation of pepper, it was named VR1 in the early days. 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 TRPV1 is activated, it will induce the secretion of inflammatory factors and inflammatory mediators, resulting in symptoms such as burning and stinging. At present, inhibiting TRPV1 activation is an effective measure to treat sensitive skin, and it is also a hot spot for cosmetics research and development companies.

[0004] Studies have found that TRPV1 is expressed on the membranes of epidermal keratinocytes, sebaceous gland cells, dermal fibroblasts, dendritic cells, mast cells, dermal vascular endothelial cells, and other cells. Mast cells are one of the immune cells widely distributed in the skin and mucous membranes. They release proinflammatory mediators such as histamine, TNF-α, prostaglandins, and proteases through degranulation, participate in the occurrence and development of innate immunity and adaptive immunity, and are the main effector cells of allergic diseases such as skin allergies, urticaria, allergic rhinitis, and asthma. Skin allergies involving mast cells are manifested as itching. TRPV1 channel activation causes mast cell degranulation and transmits itching signals to sensory nerves.

[0005] Platycodon grandiflorus is the dried root of Platycodon grandiflorus, a perennial herb of the Campanulaceae family. It is a plant with both medicinal and edible properties. Its main components include saponins, flavonoids, amino acids, phenols, etc. It has the effects of expectoration and cough relief, antibacterial and anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and hypolipidemic. In recent years, these biological effects of Platycodon grandiflorus have become a hot topic for researchers at home and abroad, especially the anti-inflammatory and anti-tumor effects. More and more studies have reported the mechanism of action of Platycodon grandiflorus extract on various inflammations and tumors in the body. However, in the field of cosmetics, there are few studies on the anti-allergic mechanism of Platycodon grandiflorus extract for sensitive skin. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a preparation method of a Platycodon grandiflorum extract with soothing effect, and its product and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a Platycodon grandiflorum extract having a soothing effect, the preparation method comprising the following steps:

[0009] (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;

[0010] (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;

[0011] (3) filtering and concentrating the reflux extract, and then purifying and eluting it with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 and DM130, and collecting the eluate;

[0012] (4) Concentrating and drying the eluate to obtain the Platycodon grandiflorum extract.

[0013] The present invention creatively develops a method for preparing Platycodon grandiflorum extract, combining flash extraction with reflux extraction, which not only realizes more effective utilization of Platycodon grandiflorum raw materials, but also significantly improves the soothing effect of the final product, which is specifically manifested in: inhibiting TRPV1 expression of keratinocytes, inhibiting calcium influx phenomenon of keratinocytes, and inhibiting mast cell degranulation rate, that is, exerting soothing effects in multiple targets and multiple pathways. At the same time, it is highly safe and will not cause adverse skin reactions, which provides a new strategy for preparing soothing products. At the same time, the preparation method is easy to realize industrialized scale-up production and is practical.

[0014] Furthermore, the present invention creatively uses a mixed resin of HPD100 and DM130 to purify the extracted product. Compared with other types of resin combinations or single resins, HPD100 and DM130 can work together, and the final product of their separation and purification is more effective in inhibiting the expression of TRPV1 in keratinocytes and soothing.

[0015] Preferably, the crushing in step (1) is to 20-40 mesh, for example, 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), for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

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

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

[0019] Preferably, in step (2), after mixing the flash extract and Platycodon grandiflorum residue with anhydrous ethanol or ethanol aqueous solution, the final concentration of ethanol is 70-80%, for example, 70%, 73%, 75%, 78%, 80%, etc.

[0020] Compared with other final ethanol concentrations, the final product prepared by reflux extraction with an ethanol aqueous solution having a final concentration of 70-80% is more effective in inhibiting TRPV1 expression in keratinocytes and soothing the skin.

[0021] Preferably, 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), for example, 1:8, 1:9, 1:10, 1:12, 1:13, 1:14, 1:15, etc.

[0022] Preferably, the reflux extraction in step (2) is performed 1-3 times, for example, 1 time, 2 times or 3 times; each time for 1-4 h, for example, 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, for example, 1:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.

[0024] Based on the synergistic relationship between HPD100 resin and DM130 resin, when the two satisfy the above-mentioned specific mass ratio relationship, the final product of their separation and purification is more effective in inhibiting keratinocyte TRPV1 expression and soothing.

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

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

[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 according to the preparation method described in the first aspect.

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

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

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

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

[0033] Other specific point values ​​not listed in the above numerical range involved in the present invention are all within the protection scope of the present invention. Based on considerations of length and brevity of description, they are not described 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, combining flash extraction with reflux extraction, which not only realizes more effective utilization of Platycodon grandiflorum raw materials, but also significantly improves the soothing effect of the final product, which is specifically manifested in: inhibiting TRPV1 expression of keratinocytes, inhibiting calcium influx phenomenon of keratinocytes, and inhibiting mast cell degranulation rate, that is, exerting soothing effects in multiple targets and multiple pathways. At the same time, it is highly safe and will not cause adverse skin reactions, which provides a new strategy for preparing soothing products. At the same time, the preparation method is easy to realize industrialized scale-up production and is practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention is a schematic diagram of the process for preparing the Platycodon grandiflorum extract. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0038] Example 1

[0039] This embodiment provides a Platycodon grandiflorum extract, and the preparation method thereof is as follows (the schematic diagram is as follows Figure 1 shown):

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

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

[0042] (3) Filtering and concentrating the reflux extract, and then purifying and eluting it with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 resin and DM130 resin in a mass ratio of 2:1. The purification and elution process is: first eluting with 5 BV of pure water, then eluting with 4 BV of 65% ethanol aqueous solution, and collecting the alcohol washing liquid;

[0043] (4) Concentrating the eluate and freeze-drying it to obtain the Platycodon grandiflorum extract.

[0044] Example 2

[0045] This embodiment provides a Platycodon grandiflorum extract, and the preparation method thereof is as follows:

[0046] (1) Dry and crush Platycodon grandiflorum to 40 mesh, mix with 4 times the amount of water, place in a flash extractor for flash extraction twice, the extraction time is 2 min, the machine speed is 6000 rpm, and obtain flash extract and Platycodon grandiflorum residue;

[0047] (2) Mix the flash extract and Platycodon grandiflorum residue with 8 times the amount of high-concentration ethanol aqueous solution to make the final concentration of ethanol 80%, and perform reflux extraction for 2 h. Repeat the above reflux extraction operation once, and combine the two reflux extracts;

[0048] (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 resin and DM130 resin in a mass ratio of 1:1. The purification and elution process is: first eluting with 7 BV of pure water, then eluting with 5 BV of 60% ethanol aqueous solution, and collecting the alcohol washing liquid;

[0049] (4) Concentrating the eluate and freeze-drying it to obtain the Platycodon grandiflorum extract.

[0050] Example 3

[0051] This embodiment provides a Platycodon grandiflorum extract, and the preparation method thereof is as follows:

[0052] (1) Dry and crush Platycodon grandiflorum to 20 mesh, mix with 2 times the amount of water, place in a flash extractor for flash extraction once, the extraction time is 4 min, the machine speed is 4000 rpm, and flash extract and Platycodon grandiflorum residue are obtained;

[0053] (2) Mix the flash extract and Platycodon grandiflorum residue with a high-concentration ethanol aqueous solution of 8 times the amount of the feed, so that the final concentration of ethanol is 70%, and perform reflux extraction for 2 h. Repeat the above reflux extraction operation once, and combine the two reflux extracts;

[0054] (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 resin and DM130 resin in a mass ratio of 4:1. The purification and elution process is: first eluting with 5 BV of pure water, then eluting with 3 BV of 70% ethanol aqueous solution, and collecting the alcohol washing liquid;

[0055] (4) Concentrating the eluate and freeze-drying it to obtain the Platycodon grandiflorum extract.

[0056] Example 4

[0057] This embodiment provides a Platycodon grandiflorum extract, the preparation method of which is different from that of Example 1 only in that in step (2), the flash extract and Platycodon grandiflorum residue are mixed with a high-concentration ethanol aqueous solution having a 10-fold amount of the feed amount, so that the final concentration of ethanol is 90%, and other conditions remain unchanged.

[0058] Example 5

[0059] This embodiment provides a Platycodon grandiflorum extract, the preparation method of which is different from that of Example 1 only in that in step (2), the flash extract and Platycodon grandiflorum residue are mixed with a high-concentration ethanol aqueous solution having a 10-fold amount of the feed amount, so that the final concentration of ethanol is 60%, and other conditions remain unchanged.

[0060] Example 6

[0061] This embodiment provides a Platycodon grandiflorum extract, and the preparation method thereof is different from that of Embodiment 1 only in that the purification and elution process in step (3) is: first eluting with 5 BV of pure water, then eluting with 4 BV of 75% ethanol aqueous solution, and collecting the alcohol washing liquid.

[0062] Comparative Example 1

[0063] This comparative example provides a Platycodon grandiflorum extract, the preparation method of which is different from that of Example 1 only in that the macroporous resin in step (3) is a mixture of D101 resin and AB-8 resin in 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 preparation method of which is different from that of Example 1 only in that the macroporous resin in step (3) is a single HPD100 resin, and other conditions remain unchanged.

[0066] Comparative Example 3

[0067] This comparative example provides a Platycodon grandiflorum extract, the preparation method of which is different from that of Example 1 only in that the macroporous resin in step (3) 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 Platycodon grandiflorum to 30 mesh, then mix with 10 times the amount of 75% ethanol aqueous solution, perform reflux extraction for 2 h, repeat the above reflux extraction operation once, and combine the two reflux extracts;

[0071] (2) filtering and concentrating the reflux extract, and then purifying and eluting with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 resin and DM130 resin in a mass ratio of 2:1. The purification and elution process is: first eluting with 5 BV of pure water, then eluting with 4 BV of 65% ethanol aqueous solution, and collecting the alcohol washing liquid;

[0072] (3) Concentrating the eluate and freeze-drying it 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 with 10 times the amount of 75% ethanol aqueous solution, perform reflux extraction for 1.5 h, repeat the above reflux extraction operation twice, and combine the three reflux extracts;

[0076] (2) filtering and concentrating the reflux extract, and then purifying and eluting with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 resin and DM130 resin in a mass ratio of 2:1. The purification and elution process is: first eluting with 5 BV of pure water, then eluting with 4 BV of 65% ethanol aqueous solution, and collecting the alcohol washing liquid;

[0077] (3) Concentrating the eluate and freeze-drying it to obtain the Platycodon grandiflorum extract.

[0078] Test Example 1

[0079] Effects on TRPV1 expression in keratinocytes:

[0080] The test method is: using keratinocytes, the relative expression of capsaicin receptor (TRPV1) protein is detected by immunofluorescence (IF) to evaluate the soothing effect of Platycodon grandiflorum extract.

[0081] Press 4.5×10 4 / well inoculated cells into a 24-well plate and incubated overnight in an incubator (37°C, 5% CO2, 95% RH). When the cell plating rate in the 24-well plate reached 40%-60%, the drug was administered, wherein the blank control group: no trans-4-tert-butyl cyclohexanol was added, and no capsaicin stimulation; the model control group: no trans-4-tert-butyl cyclohexanol was added, and capsaicin stimulation; the positive control group: trans-4-tert-butyl cyclohexanol was added at a concentration of 15.6 μg / mL, and capsaicin stimulation; the sample group: the dilution of the Platycodon grandiflorum extract prepared in Examples 1-6 and Comparative Examples 1-5 was added at a concentration of 0.0002 mg / mL, and capsaicin stimulation.

[0082] After administration, the 24-well plate was placed in an incubator for 24 h. After the incubation, the culture supernatant was discarded, the plate was washed with PBS three times, the PBS was discarded, 200 μL / well of goat serum was added to each well, and the plate was blocked at room temperature for 60 min; the goat serum blocking solution was discarded, and the diluted primary antibody Anti-VR1 antibody (200 μL / well) (1:150 dilution of goat serum) was added to each well, and the plate was incubated overnight at 4°C; the primary antibody was discarded, the plate was washed with PBS three times, 5 min / time, and the residual PBS liquid on the plate was blotted with absorbent paper, and the diluted fluorescent secondary antibody Goat pAb to Rb IgG (Alexa Fluor® 488) (200 μL / well) (1:500 dilution of PBS) was added to each well, and the plate was incubated at room temperature in the dark for 1 h; the secondary antibody was discarded, the plate was washed with PBS three times, 5 min / time, and the residual PBS liquid on the plate was blotted with absorbent paper, and Hochest33342 (200 μL / well) (PBS 1:500 dilution), incubate at room temperature for 5 min; discard Hochest33342, wash 3 times with PBS, 5 min / time. Use absorbent paper to absorb the residual PBS liquid on the slide, pick up the slide with a needle, add a drop of antifade agent on the slide and put the slide upside down on the slide, seal the slide (use the cut yellow tip to absorb the antifade agent); detect under fluorescence microscope.

[0083] The statistical results of the detection of the relative expression of capsaicin receptor (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 downregulate the expression of TRPV1 in keratinocytes, and thus has the effects of soothing, anti-allergic and analgesic. Compared with the Platycodon grandiflorum extract of the comparative example, the Platycodon grandiflorum extract of the embodiment combines flash extraction with reflux extraction, and selects a specific macroporous resin for elution, which significantly improves the above-mentioned effects of the final product.

[0087] Test Example 2

[0088] Effects on calcium influx in keratinocytes:

[0089] The test method is: using keratinocytes, the changes in the fluorescence intensity of calcium ions in keratinocytes are detected by immunofluorescence (IF) to evaluate the soothing and sedative effects of Platycodon grandiflorum extract. 4Cells were inoculated into 96-well cell culture plates at 37°C and 5% CO2 overnight. When the cell plating rate reached 80%, the culture medium was removed, and the cells were washed 3 times with PBS. 100 μL of culture medium containing 1 μmol / L Fluo4 / AM was added to each well, and the cells were cultured at 37°C and 5% CO2 for 30 min. The supernatant was removed, and 200 μL of PBS was added to each well, and the cells were cultured at 37°C and 5% CO2 for 20 min. The test substances of each group were added. Each group had 3 replicate wells, and 100 μL of culture medium containing different concentrations of the test substances was added to each well. Among them, the blank control group: no trans-4-tert-butyl cyclohexanol was added, and there was no capsaicin stimulation; the model control group: no trans-4-tert-butyl cyclohexanol was added, and capsaicin (30 μM) was stimulated; the positive control group: trans-4-tert-butyl cyclohexanol was added at a concentration of 7.8 μg / mL, and capsaicin was stimulated; the sample group: the Platycodon grandiflorum extract dilution prepared in Example 1 was added at a concentration of 0.0002 mg / mL, and capsaicin was stimulated.

[0090] After the administration, the 96-well plate was incubated at 37°C and 5% CO2 for 30 min. The supernatant was removed, the cells were washed three times with PBS, and the fluorescence intensity was recorded using a fluorescence microplate reader under light-proof conditions. The statistical results of the fluorescence intensity detection during calcium ion influx in each group are shown in Table 2.

[0091] Table 2

[0092]

[0093] It can be seen from the data results in Table 2 that, compared with the model control group, the Platycodon grandiflorum extract of the present invention can significantly inhibit the influx of calcium ions into keratinocytes, and thus has a soothing and calming effect.

[0094] Test Example 3

[0095] Effects on mast cell degranulation rate:

[0096] The test method is: collect P815 mast cells with good cell morphology and in the logarithmic growth phase, and use complete culture medium to make the concentration of 0.5×10 5cells, inoculated in a 24-well cell culture plate, cultured at 37°C, 5% CO2 and saturated humidity, removed the original culture medium after 16 hours, and added the test substances of each group. Each group has 3 replicate wells, and 900 μL of culture medium containing different concentrations of the test substances is added to each well. Among them, the blank control group: no addition of disodium cromoglycate, and no C48 / 80 stimulation; the model control group: no addition of disodium cromoglycate, and C48 / 80 stimulation; the positive control group: adding disodium cromoglycate at a concentration of 1 mg / mL, and C48 / 80 stimulation; the sample group: adding the dilution of the Platycodon grandiflorum extract prepared in Example 1 with a concentration of 0.0002 mg / mL, and C48 / 80 stimulation.

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

[0098] Table 3

[0099]

[0100] It can be seen from the data results in Table 3 that, compared with the model control group, the Platycodon grandiflorum extract of the present invention can significantly reduce the degranulation rate of mast cells, and thus has the effect of soothing allergies and relieving itching.

[0101] Test Example 4

[0102] Skin irritation evaluation:

[0103] The test method is: using the EpiSkin artificial skin model as the test model, SDS as the induction condition, and detecting the skin irritation of the sample according to the OECDTG439 method. Among them, the blank control group: treated with 10 μL PBS for 15 min; the model control group: treated with 10 μL 5% SDS for 15 min; the sample group: treated with 10 μL of the Platycodon grandiflorum extract dilution prepared in Example 1 with a concentration of 5% for 15 min. The results are shown in Table 4.

[0104] Table 4

[0105]

[0106] It can be seen from the data results in Table 4 that the Platycodon grandiflorum extract of the present invention is non-irritating to the skin, has a mild feel and is highly safe.

[0107] Test Example 5

[0108] Patch test:

[0109] The test method is as follows: 30 social volunteers who meet the requirements, aged 20-45 years old, are selected and randomly divided into 3 groups, and each group is respectively tested with 2 different products in Examples 1-6. Place the tester in a small chamber, and the control well is not treated in any way. The tester with the test substance added is applied to the inner side of the forearm of the subject with a low-allergenic tape, and the palm is gently pressed to make it evenly applied to the skin, and the test is continued for 48 hours. The skin reaction is observed according to the standards in Table 5 at 30 minutes, 24 hours and 48 hours after the test substance spot is removed.

[0110] Table 5

[0111]

[0112] The results showed that the products of Examples 1-6 of the present invention all had negative reactions after trial use, indicating that the Platycodon grandiflorum extract of 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 illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of 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] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

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 it with a macroporous resin, wherein the macroporous resin is a mixture of HPD100 and DM130, and collecting the eluate; (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 preparation method according to claim 1, characterized in that: The mass ratio of HPD100 to DM130 in step (3) is (1-4):

1.

5. The preparation method according to claim 1, characterized in that: The purification and elution process in step (3) is: firstly elute with 3-8 BV of pure water or 0-10% ethanol aqueous solution, then elute with 3-8 BV of 60-70% ethanol aqueous solution, and collect the alcohol washing liquid.

6. The Platycodon grandiflorum extract obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the Platycodon grandiflorum extract according to claim 6 in preparing a product with soothing and / or antipruritic effects.

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

9. Use of the Platycodon grandiflorum extract according to claim 6 in the preparation of a keratinocyte calcium influx inhibitor.

10. Use of the Platycodon grandiflorum extract according to claim 6 in the preparation of a mast cell degranulation inhibitor.

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