Preparation method, product and application of bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ

Abstract

The present invention relates to a preparation method, product and application of a Bletilla striata extract rich in bletilloside and gymnemaoside III. The preparation method of the Bletilla striata extract rich in bletilloside and gymnemaoside III includes: heating and refluxing Bletilla striata roots with an ethanol aqueous solution to obtain a crude extract; performing vacuum concentration on the crude extract to obtain a concentrated crude extract; mixing the concentrated crude extract with an alkaline reagent to adjust the pH to obtain a conversion solution; adsorbing the conversion solution with macroporous resin, and eluting to obtain an eluate; concentrating the eluate and freeze-drying. This extraction method is not only simple and easy to operate, uses environmentally friendly materials, but also can significantly increase the content of bletilloside. The extract obtained in the present invention has stable quality, no phototoxicity and sensitization, a light smell, and has good cell migration promotion activity and moisturizing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of material extraction, and relates to a preparation method of a Bletilla striata extract rich in Bletilla striata glycosides and Ginsenoside III, as well as a product and application thereof. Background Art

[0002] Bletilla striata (Bletilla striata), a terrestrial herbaceous plant of the genus Bletilla in the Orchidaceae family, is primarily grown in Yunnan, Guizhou, Sichuan, Hunan, Jiangxi, and Guangxi provinces. According to the Southern Yunnan Materia Medica, Bletilla striata is known to "treat lung qi damage caused by tuberculosis, replenish lung deficiency, relieve coughs, eliminate hemoptysis caused by tuberculosis, and astringe lung qi." Its pharmacological effects include hemostasis, wound healing, anti-inflammatory, antibacterial, and anti-tumor properties. It is clinically used to treat hemoptysis, traumatic bleeding, skin chapping, and gastrointestinal bleeding.

[0003] Currently, most research and development on Bletilla striata focuses on cultivation techniques (Guizhou Baili Biotechnology Development Co., Ltd., CN201911383481.8.2019-12-27), species identification (Guizhou University of Traditional Chinese Medicine, CN201410733438.0.2014-12-04), and determination of the main components (Sichuan Liyan Workshop Biotechnology Co., Ltd., CN202111337441.7.2021-11-08). Research on Bletilla striata components primarily focuses on the preparation of sugars and their efficacy and application (Kunming Institute of Botany, Chinese Academy of Sciences, CN202110328789.3.2021-03-27). The preparation of Bletilla striata glycosides is complex, involving multiple steps such as extraction and separation; it contains a high number of toxic organic solvents, resulting in low safety; the Bletilla striata glycoside content is low, and other components are unclear, making it difficult to mass-produce and apply Bletilla striata extracts.

[0004] Therefore, there is an urgent need for an extraction method that can increase the content of Bletilla striata glycosides in an extract while ensuring the safety of the extract. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a preparation method of a Bletilla striata extract rich in Bletilla striata glycosides and Ginsenoside III, as well as its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a Bletilla striata extract rich in Bletilla striata glycosides and Ginsenoside III, the preparation method comprising:

[0008] (1) extracting the root of Bletilla striata by heating and refluxing with an ethanol aqueous solution to obtain a crude extract;

[0009] (2) concentrating the crude extract under reduced pressure to obtain a crude concentrate;

[0010] (3) Mix the crude extract concentrate with an alkaline reagent to adjust the pH and obtain a conversion solution.

[0011] (4) Use macroporous resin to adsorb the conversion solution, elute it, and obtain an eluate.

[0012] (5) Concentrate the eluate and freeze-dry it.

[0013] The preparation method of the present invention can significantly increase the content of bletillastriol in the extract, simplify the extraction and purification of bletillastriol, obtain the extract only using ethanol and pure water as solvents, and improve the safety of the extract. It can convert impurities in bletilla striata that are similar in structure to bletillastriol into bletillastriol, or improve the solubility of bletillastriol, thereby increasing the yield and purity of the finally obtained bletillastriol. It can remove impurities with high polarity such as colloidal medium and pigment in bletilla striata and impurities with polarity less than that of bletillastriol in one step. The extract has good water solubility, is nearly clear and transparent after dissolving in water, has a light smell, is convenient for formula addition, and has good formula compatibility. It has passed the cell phototoxicity detection test and has no phototoxicity; it has passed the sensitization experiment and has no sensitization; it proves that the extract has good safety. The cell scratch experiment shows that it has good cell migration promoting activity, acts on FLG, LOR, AQP-3, has repair and anti-inflammatory activities, and can be added as an active ingredient to skin care products.

[0014] The yield of the extract obtained by using this method relative to the quality of the extracted medicinal materials is higher than 20 mg / g, the percentage content of bletillastriol in the extract is higher than 80%, the percentage content of gymnemaoside Ⅲ is lower than 10%, the ash content is lower than 0.1%, and the metal ion content is determined.

[0015] Preferably, the bletilla striata root in step (1) is dry tuber powder, and the powder passes through a 10-100 mesh sieve, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0016] Preferably, the mass ratio of the ethanol aqueous solution to the bletilla striata root is (5-20):1, and the specific point values in (5-20) can be selected as 5, 6, 7, 8, 9, 10, 11, 12,  13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0017] When the ethanol aqueous solution and the bletilla striata root are mixed and extracted at the above specific mass ratio, the extraction effect is the best.

[0018] Preferably, the temperature for heating under reflux extraction is 65 - 100°C, and the time is 0.5 - 4 h. The temperature for heating under reflux extraction can be selected as 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, etc., and the time for heating under reflux extraction can be selected as 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0019] Preferably, the mass percentage content of ethanol in the ethanol aqueous solution is 80 - 99%, such as 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0020] Preferably, the number of times of heating under reflux extraction is 1 - 5 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0021] Preferably, the pressure for vacuum concentration is 50 - 200 mbar, such as 50 mbar, 80 mbar, 100 mbar, 120 mbar, 150 mbar, 180 mbar, 200 mbar, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0022] Preferably, the temperature for vacuum concentration is 40 - 60°C, such as 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0023] Preferably, the rotation speed for vacuum concentration is 20 - 50 rpm, such as 20 rpm, 25 rpm, 30 rpm, 35 rpm, 40 rpm, 45 rpm, 50 rpm, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0024] The obtained concentrated solution is brown, with a slightly acidic smell. Adding a small amount of water forms a light brown suspension with brown viscous substances inside.

[0025] Preferably, the alkaline reagent includes sodium citrate and / or sodium hydroxide.

[0026] Preferably, the pH of the conversion solution is 4 - 7, such as 4, 4.5, 5, 5.5, 6, 6.5, 7, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0027] Preferably, after adjusting the pH, static settling or stirring is further included;

[0028] Preferably, the temperature for the static settling or stirring is 10 - 70°C, and the time is 1 - 72 h.

[0029] The temperature for the static settling or stirring can be selected as 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, etc., and the time can be selected as 1 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h, 72 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0030] Preferably, the macroporous resin includes ADS-1, ADS-7, ADS-8, DM130, DM301, HP100, HP2MG, NKA-9, XAD-2 or XAD-8.

[0031] Preferably, the mass of the macroporous resin is 1 - 5 times that of the Bletilla striata roots, such as 1 time, 2 times, 3 times, 4 times, 5 times, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0032] Preferably, the adsorption time in step (4) is 2 - 24 h, such as 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0033] Preferably, the elution includes eluting successively with pure water, 25 - 35% ethanol aqueous solution, and 45 - 55% ethanol aqueous solution.

[0034] The 45 - 55% ethanol eluate is a light yellow transparent eluate, and the eluate has a sweet and fragrant smell.

[0035] The mass percentage content of ethanol in the 25 - 35% ethanol aqueous solution can be selected as 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, etc., and the mass percentage content of ethanol in the 45 - 55% ethanol aqueous solution can be selected as 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0036] Preferably, the volume ratios of the pure water, 25 - 35% ethanol aqueous solution, 45 - 55% ethanol aqueous solution to the macroporous resin are independently 5 - 15, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0037] Preferably, the pressure for concentration in step (5) is 50 - 200 mbar and the temperature is 40 - 60 °C.

[0038] The pressure for concentration can be selected as 50 mbar, 80 mbar, 100 mbar, 120 mbar, 150 mbar, 180 mbar, 200 mbar, etc., and the temperature can be selected as 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0039] Preferably, the lyophilization includes pre - freezing and drying.

[0040] Preferably, the temperature for pre - freezing is - 80 to - 20 °C and the time is 4 - 24 h.

[0041] The temperature for pre - freezing can be selected as - 80 °C, - 75 °C, - 70 °C, - 65 °C, - 60 °C, - 55 °C, - 50 °C, - 45 °C, - 40 °C, - 35 °C, - 30 °C, - 25 °C, - 20 °C, etc., and the time can be selected as 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0042] Preferably, the temperature for drying is - 80 to - 20 °C and the time is 24 - 72 h.

[0043] The temperature for drying can be selected as - 80 °C, - 75 °C, - 70 °C, - 65 °C, - 60 °C, - 55 °C, - 50 °C, - 45 °C, - 40 °C, - 35 °C, - 30 °C, - 25 °C, - 20 °C, etc., and the time can be selected as 24 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h, 72 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0044] In the second aspect, the present invention provides a Bletilla striata extract prepared by the preparation method of the Bletilla striata extract rich in bletillastriatins and gymnemaoside Ⅲ according to the first aspect.

[0045] The Bletilla striata extract obtained by using the preparation method of the present invention has no phototoxicity and sensitization, and is safe and reliable.

[0046] In a third aspect, the present invention provides an application of the Bletilla striata extract according to the second aspect in the preparation of a product having a repair effect.

[0047] The Bletilla striata extract of the present invention has a significant effect of promoting cell migration, acts on genes related to FLG, LOR, and AQP-3, and can be added as a repair effect ingredient to the skin care product formula.

[0048] In a fourth aspect, the present invention provides an application of the Bletilla striata extract according to the second aspect in the preparation of a product having anti-inflammatory and moisturizing effects.

[0049] Preferably, the product includes cosmetics, skin care products or health products.

[0050] Preferably, the mass percentage content of the Bletilla striata extract in the product is 0.001-3%, such as 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0051] The Bletilla striata extract can be directly added to the product or dissolved in a 1,3-butanediol or pentanediol solution with a mass percentage content of 10-40% and then added to the skin care product formula.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The preparation method of the present invention can significantly increase the content of bletillastriatins in the extract.

[0054] (2) The preparation method of the present invention can simplify the extraction and purification of bletillastriatins. Only ethanol and pure water are used as solvents to obtain the extract, which can improve the safety of the extract.

[0055] (3) The preparation method of the present invention can convert impurities with structures similar to bletillastriatins in Bletilla striata into bletillastriatins, or improve the solubility of bletillastriatins, thereby increasing the yield and purity of the finally obtained bletillastriatins.

[0056] (4) The preparation method of the present invention can remove polar impurities such as gum medium and pigment in Bletilla striata and impurities with polarity less than bletillaside in one step.

[0057] (5) The extract prepared by the present invention has good water solubility. After being dissolved in water, the color is nearly clear and transparent, the smell is light, it is convenient for formula addition, and it has good formula compatibility.

[0058] (6) The extract obtained by using the preparation method of the present invention has passed the cell phototoxicity detection test and has no phototoxicity; it has passed the sensitization experiment and has no sensitization; it proves that the extract has good safety.

[0059] (7) The extract obtained by using the preparation method of the present invention shows good cell migration promoting activity in the cell scratch experiment. Acting on FLG, LOR, and AQP-3, it has repair and anti-inflammatory activities and can be added as an active ingredient to skin care products.

[0060] (8) For the extract obtained by using the preparation method of the present invention, quality control is carried out on two monomer compounds of the extract to ensure the stability, safety, and effectiveness between batches, and it can be applied to large-scale production and commercial applications. Description of the Drawings

[0061] Figure 1 It is a flow chart for the preparation of the extract.

[0062] Figure 2 It is a chromatographic analysis chart of bletillaside after hydrolysis in an acidic environment under high temperature and high pressure conditions.

[0063] Figure 3 It is a result chart of the peak area of bletillaside after hydrolysis conversion of the crude extract at different pH values and different temperatures. Among them, Figure A is the peak area of bletillaside after hydrolysis conversion at different pH values, and Figure B is the peak area of bletillaside after hydrolysis conversion at different temperatures.

[0064] Figure 4 It is a result chart of the peak area of bletillaside under different hydrolysis conditions.

[0065] Figure 5 It is the analysis result of the components in different elution parts of the extract.

[0066] Figure 6 It is the chemical structure formula of the extract. Among them, Figure A is the chemical structure formula of bletillaside, and Figure B is the chemical structure formula of gymnemaoside Ⅲ.

[0067] Figure 7 It is the LC-ELSD analysis chromatogram of bletillaside in the Bletilla striata extract.

[0068] Figure 8 It is the result of the scratch experiment of the extract. Detailed implementation manners

[0069] The technical solution of the present invention will be further described below through specific implementation manners. 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 on the present invention.

[0070] Preparation Example 1

[0071] This preparation example provides a preparation method of a crude extract concentrate of Bletilla striata, and the preparation method includes:

[0072] (1) Weigh 100 g of dried Bletilla striata stems, powder them, then pass through a 40-mesh sieve, put them into an extraction bag, and perform reflux extraction 3 times with 95% ethanol. The mass ratio of the ethanol aqueous solution to the Bletilla striata roots is 10:1, the extraction temperature is 80 °C, and the time is 1 h. Filter the obtained extraction solution, and combine the three filtrates to obtain a crude extract;

[0073] (2) Under the conditions of a pressure of 80 mbar and a temperature of 50 °C, heat and concentrate the crude extract under reduced pressure to remove ethanol, and obtain a brown crude extract concentrate.

[0074] Example 1

[0075] This example provides a preparation method of a Bletilla striata extract, and the preparation method includes:

[0076] (1) Weigh 100 g of dried Bletilla striata stems, powder them, then pass through a 40-mesh sieve, put them into an extraction bag, and perform reflux extraction 3 times with 95% ethanol. The mass ratio of the ethanol aqueous solution to the Bletilla striata roots is 10:1, the extraction temperature is 100 °C, and the time is 1 h. Filter the obtained extraction solution, and combine the three filtrates to obtain a crude extract;

[0077] (2) Under the conditions of a pressure of 80 mbar and a temperature of 50 °C, heat and concentrate the crude extract under reduced pressure to remove ethanol, and obtain a brown crude extract concentrate;

[0078] (3) Mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 5, and stir at 50 °C for 36 h;

[0079] (4) Take 200 g of macroporous resin HP2MGL for activation and column packing. Adsorb the crude extract concentrate on the column for 20 h, then wash with 1 kg of pure water to remove impurities, then wash with 1.5 kg of 30% ethanol to remove impurities, then wash with 0.5 kg of 50% ethanol, and then collect with 0.5 kg of 50% ethanol. The elution speed is 2 BV / h to obtain a light yellow transparent eluate containing bletillastemoside;

[0080] (5) Concentrate the separated and purified eluate, pre-freeze at -80 °C for 8 h, and then freeze-dry for 48 h to obtain the product.

[0081] Example 2

[0082] This example provides a preparation method of Bletilla striata extract, and the preparation method includes:

[0083] (1) Weigh 101 g of dried Bletilla striata stems, powder them, then pass through a 40-mesh sieve, put them into an extraction bag, and perform reflux extraction with 80% ethanol 5 times. The mass ratio of the ethanol aqueous solution to Bletilla striata roots is 5:1, the extraction temperature is 65 °C, and the time is 4 h. Filter the obtained extraction solution, and combine the five filtrates to obtain a crude extract;

[0084] (2) Under the conditions of a pressure of 200 mbar and a temperature of 60 °C, heat and reduce the pressure to concentrate the crude extract to remove ethanol, and obtain a brown crude extract concentrate;

[0085] (3) Mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 4, and stir at 70 °C for 1 h;

[0086] (4) Take 400 g of macroporous resin HP2MGL for activation and column packing. Load the crude extract concentrate onto the column for adsorption for 24 h, then wash with 2 kg of pure water to remove impurities, then wash with 3 kg of 25% ethanol to remove impurities, then elute with 1 kg of 45% ethanol, and then elute and collect with 1 kg of 45% ethanol. The elution speed is 2 BV / h to obtain a light yellow transparent eluate containing bletillaside;

[0087] (5) Concentrate the separated and purified eluate, pre-freeze at -20 °C for 24 h, and then freeze-dry for 72 h to obtain the product.

[0088] Example 3

[0089] This example provides a preparation method of Bletilla striata extract, and the preparation method includes:

[0090] (1) Weigh 101 g of dried Bletilla striata stems, powder them, then pass through a 40-mesh sieve, put them into an extraction bag, and perform reflux extraction with 99% ethanol 2 times. The mass ratio of the ethanol aqueous solution to Bletilla striata roots is 20:1, the extraction temperature is 100 °C, and the time is 0.5 h. Filter the obtained extraction solution, and combine the two filtrates to obtain a crude extract;

[0091] (2) Under the conditions of a pressure of 50 mbar and a temperature of 60 °C, heat and reduce the pressure to concentrate the crude extract to remove ethanol, and obtain a brown crude extract concentrate;

[0092] (3) Mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 7, and let it stand at 10 °C for 72 h;

[0093] (4) Take 101 g of macroporous resin HP2MGL for activation and column packing. Load the crude extract concentrate onto the column for adsorption for 2 h, then elute with 500 g of pure water to remove impurities, then elute with 500 kg of 35% ethanol to remove impurities, then elute with 250 g of 55% ethanol, and then collect with 250 g of 55% ethanol. The elution speed is 2 BV / h to obtain a light yellow transparent eluate containing bletillastemarin.

[0094] (5) Concentrate the separated and purified eluate, pre-freeze at -50 °C for 4 h and then lyophilize for 24 h to obtain.

[0095] Example 4

[0096] This example provides a method for preparing bletilla striata extract. The difference from Example 1 is only that step (3) is "mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 9, and stir at 50 °C for 36 h", and other operations remain unchanged.

[0097] Example 5

[0098] This example provides a method for preparing bletilla striata extract. The difference from Example 1 is only that step (3) is "mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 5, and stir at 50 °C for 80 h", and other operations remain unchanged.

[0099] Example 6

[0100] This example provides a method for preparing bletilla striata extract. The difference from Example 1 is only that step (3) is "mix the crude extract concentrate obtained in step (2) with an alkaline reagent, adjust the pH to 5, and stir at 50 °C for 0.5 h", and other operations remain unchanged.

[0101] Comparative Example 1

[0102] This comparative example provides a method for preparing bletilla striata extract. The difference from Example 1 is only that step (3) is "adjust the pH of the crude extract concentrate obtained in step (2) to 2, and stir at 50 °C for 36 h", and other operations remain unchanged.

[0103] Comparative Example 2

[0104] This comparative example provides a method for preparing bletilla striata extract. The difference from Example 1 is only that step (3) is not included, the pH of the crude extract is 3 by itself, and other operations remain unchanged.

[0105] Test Example 1

[0106] Test on static adsorption rate and desorption rate of resin

[0107] The crude extract described in Preparation Example 1 was equally divided into 10 groups. Weigh 2 g each of macroporous resins HP100, ADS-8, ADS-1, NKA-9, ADS-7, XAD-8, XAD-2, DM130, DM301, and HP2MG for cleaning and activation, put them into conical flasks, and add 20 g of the crude extract concentrate from the reduced-pressure concentration step respectively. Shake at room temperature for 24 h; after adsorption is completed, filter to obtain the adsorption solution for standby; then wash the resin with pure water and filter to obtain the washing solution for standby; add a certain amount of ethanol, shake at room temperature for 2 h, and filter to obtain the desorption solution for standby.

[0108] Testing method: Use LC to analyze the adsorption solution, washing solution, desorption solution of the above 10 kinds of macroporous resins, and the extraction concentrate, and calculate the static adsorption rate and desorption rate of different resins. The results are shown in Table 1.

[0109] Calculation method:

[0110] Static adsorption rate (%) = [(A 吸附前 - A 吸附后 ) / A 吸附前 × 100%

[0111] Desorption rate (%) = [A 解吸附后 / (A 吸附前 - A 吸附后 )] × 100%

[0112] Table 1

[0113]

[0114] As can be seen from the above table, the adsorption rates of HPD100, ADS-7, XAD-2, DM130, and HP2MGL are all greater than 90%, so these five resins are selected for dynamic elution.

[0115] Testing Example 2

[0116] Desorption rate of dynamic adsorption of bletilla striata glycoside on five macroporous resins

[0117] Weigh 30 g each of macroporous resins HPD100, ADS-7, XAD-2, DM130, and HP2MGL for cleaning and activation, then pack them into columns. Dilute the crude extract concentrate by 2 times and load the sample. Adsorb for 24 h, and then elute successively with 75 g of pure water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, and collect the eluate; concentrate the separated and purified eluate, pre-freeze at -80 °C for 8 hours and then freeze-dry for 48 h to obtain the freeze-dried powder of the fraction in the separation and purification step.

[0118] Prepare the freeze-dried powder from the freeze-drying step into a solution of 0.5 mg / mL, and use LC to analyze the content of bletilla striata glycoside in each fraction. The results are shown in Table 2.

[0119] HPLC test method: Chromatographic column: C 18 , Mobile phase: water, acetonitrile, column oven at 30 °C, measure the absorbance value under the condition of 223 nm.

[0120] Table 2

[0121] Pure water 10% ethanol 30% ethanol 50% ethanol 70% ethanol 95% ethanol HPD100 0 0 16.62％ 64.38％ 51.67％ 8.70％ ADS-7 0 0 15.89％ 56.93％ 66.96％ 37.01％ XAD-2 0 0 10.73％ 63.4％ 41.85％ 6.15％ DM130 0 0 16.92％ 66.48％ 58.82％ 9.06％ HP2MGL 0 0 24.22％ 82.87％ 25.35％ 7.17％

[0122] It can be seen from the above table that in the 50% fraction of HP2MGL, bletilla striata glycoside was eluted in large amounts. Compared with other resins, HP2MGL has a better separation effect on bletilla striata glycoside.

[0123] Test Example 3

[0124] Chromatographic analysis of the content of bletilla striata glycoside

[0125] Divide the crude extract described in Preparation Example 1 into 5 equal groups. The NC group was not treated, and the experimental groups were respectively added with 0.1%, 1%, 2%, and 5% citric acid, heated at 120 °C for 15 min, and chromatographically analyzed the content of bletilla striata glycoside in each group.

[0126] Test method: The same as the HPLC test method in Test Example 2

[0127] The results are as Figure 2 shown. It can be seen from the above figure that under the condition of acid hydrolysis and heating at 120 °C, bletilla striata glycoside was hydrolyzed, and the degree of hydrolysis was concentration-dependent. Therefore, the acid hydrolysis condition cannot increase the content of bletilla striata glycoside in the crude extract.

[0128] Test Example 4

[0129] Divide the crude extract described in Preparation Example 1 into three equal groups. The BC group was not treated, and the experimental groups were respectively added with appropriate amounts of citric acid and sodium citrate to adjust the pH to 2 and 5, stirred at 25 °C for 24 h, and chromatographically analyzed the content of bletilla striata glycoside in each group. The results are as Figure 3 shown in Figure A in;

[0130] Divide the crude extract concentrate obtained in the reduced pressure concentration step into four equal groups. The BC group was not treated, and the experimental group was added with sodium citrate to adjust the pH to 5, hydrolyzed at 4 °C, 25 °C, 40 °C, and 120 °C, and chromatographically analyzed the content of bletilla striata glycoside in each group. The results are as Figure 3 shown in Figure B in.

[0131] Divide the crude extract described in Preparation Example 1 into 4 equal groups. The BC group was not treated, and the experimental groups were respectively added with glucosidase and sodium citrate. The addition amount of glucosidase was 1%, and sodium citrate was added to pH 5, stirred at 25 °C for 24 h, and chromatographically analyzed the content of bletilla striata glycoside in each group. The results are as Figure 4 shown.

[0132] Test method: same as the HPLC test method in Test Example 2

[0133] Test Example 5

[0134] Load 200 g of activated HP2MGL macroporous resin into a column, then load the crude extract concentrate obtained in Preparation Example 1 onto the column for adsorption for 15 hours. Then elute with 1 kg of pure water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol respectively and collect the eluates. The elution speed is 2 BV / h; the eluates of pure water and 10% ethanol are turbid light brown or yellow, and the eluates of 30% ethanol - 95% ethanol are light yellow transparent solutions. Analyze using LC countercurrent fractionation, and its chromatogram is as Figure 5 shown, and the purity changes of bletilla striata glycoside in each fraction are shown in Table 3.

[0135] Test method: same as the HPLC test method in Test Example 2

[0136] Load 200 g of activated HP2MGL macroporous resin into a column, then load the crude extract concentrate obtained in Preparation Example 1 onto the column for adsorption for 15 hours. Remove impurities with 1 kg of pure water and 1 kg of 10% ethanol; elute with 1.5 kg of 30% ethanol, and collect 2 mL of the final fraction of every 0.1 kg of eluate for chromatographic analysis; elute with 0.1 - 1 Kg of 30% ethanol and 50% ethanol respectively, and collect 2 mL of the fractions after every 0.5 kg and 1 kg of eluate for chromatographic analysis.

[0137] Analyze using LC countercurrent fractionation, and the purity changes of bletilla striata glycoside in each fraction are shown in Table 4.

[0138] Table 3

[0139] Fraction Purity of bletilla striata glycoside % Pure water 0 10% ethanol 2.5 30% ethanol 35.38 50% ethanol 69.66 70% ethanol 66.43 95% ethanol 18.55

[0140] Table 4

[0141]

[0142]

[0143] From the data in Table 3 and Table 4, it can be seen that when purifying with macroporous resin, use 30% ethanol aqueous solution to wash away impurities, and then use 50% ethanol aqueous solution to collect 5 BV, and bletilla striata glycoside extract with a purity of 82 - 88% of bletilla striata glycoside can be obtained.

[0144] Test Example 6

[0145] Determination of extract content

[0146] The Bletilla striata extract obtained by the methods described in Examples 1-6 and Comparative Examples 1-2 was formulated into a 0.5 mg / mL solution, filtered through a 0.22 μm filter membrane, and then analyzed by LC-MS. Compounds were speculated based on the mass spectrometry diagram, and then the reference substance of the compound was analyzed under the same chromatographic conditions. The peak results were consistent. The structural formula of the extract is as follows Figure 6 shown, where Figure A is the chemical structural formula of bletilloside, and Figure B is the chemical structural formula of gymnemaoside Ⅲ.

[0147] A series of concentration reference substance solutions of the extract were prepared, an absorbance-concentration standard curve was plotted, and the contents of bletilloside and gymnemaoside Ⅲ in different batches of Bletilla striata were calculated. The results are shown in Table 5.

[0148] Table 5

[0149]

[0150]

[0151] Test Example 7

[0152] Component analysis

[0153] The ELSD detector can well detect components without ultraviolet absorption in natural products, such as glucose, polysaccharides, etc. Bletilla striata is rich in carbohydrates. The purified Bletilla striata extract was studied using the ELSD detector. The results showed that the percentage content of bletilloside could reach 98%, indicating that no other components could be detected except bletilloside and gymnemaoside Ⅲ, and there were fewer carbohydrate impurities in the extract of the present invention. The chromatogram is shown in Figure 7 .

[0154] Test Example 8

[0155] Analysis of ignition residue and metal ion content

[0156] The Bletilla striata extract obtained in Example 1 was incinerated in a muffle furnace until constant weight, and the residue was determined. The mass of the residue was 0.084% of the total mass of the extract, and the metal ion components of the residue were analyzed. The results are shown in Table 6.

[0157] Table 6

[0158]

[0159]

[0160] From the data in Table 6, it can be seen that the mass percentage of the ash other than bletilloside and gymnemaoside Ⅲ in the bletilloside extract is 0.084%, and the inorganic elements contained in the ash are aluminum, boron, barium, calcium, copper, chromium, potassium, lithium, magnesium, phosphorus, sodium, strontium, zinc, iron, antimony, silicon, tin, titanium, sulfur.

[0161] Test Example 9

[0162] Repair activity test

[0163] A scratch test was performed using human keratinocytes HaCaT to test the repair activity of the extract obtained in Example 1 of the present invention.

[0164] Test method: A scratch model was established using HaCaT cells. Three replicate wells were set up and cultured with blank medium, EGF, and bletilla striata extract respectively. The scratch repair was observed and photographed at 0, 16, 24, and 48 h.

[0165] The results are as Figure 8 shown. The results of the scratch test indicate that the extract has the activity of promoting cell migration and can repair the skin barrier function.

[0166] Test Example 10

[0167] Keratin-related gene test

[0168] To study the regulatory effect of the extract on FLG, LOR, and AQP-3 related genes in human keratinocytes HaCaT cells and verify the action targets of the extract obtained by the preparation method of the present invention to exert the repair effect, the blank control BC group was not treated, and the positive control group PC was picrinic acid. The results show that the content of bletilla striata glycoside and the extract of gymnema sylvestre III promote keratinocyte differentiation and exert the repair effect by upregulating FLG, LOR, and AQP-3 related genes, as shown in Table 7.

[0169] Test method: Cells were inoculated into a 6-well plate at an inoculation density of 2.6×10 5 cells / well and incubated overnight in an incubator (37 °C, 5% CO2). When the cell confluence rate in the 6-well plate reached 60%, grouping and drug administration were carried out. The drug dosage per well was 2 mL, and three replicate wells were set up in each group. Incubate in an incubator (37 °C, 5% CO2) for 24 h. Discard the old liquid, wash twice with PBS, add 1 mL of RNAisoPlus to each well, blow and lyse the cells, and then collect the samples.

[0170] Gene expression detection: RNA was extracted, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed. The statistical software was used to calculate the promotion rate of gene-related expression levels.

[0171] The average promotion rate of gene-related expression levels is shown in Table 7.

[0172] Table 7

[0173] FLG LOR AQP-3 Example 1 32％ 45％ 34％ Example 2 30％ 40％ 33％ Example 3 31％ 39％ 29％ Example 4 29％ 31％ 25％ Example 5 26％ 35％ 29％ Example 6 27％ 33％ 28％ Comparative Example 1 23％ 25％ 12％ Comparative Example 2 25％ 27％ 23％

[0174] As can be seen from the data in Table 7, the extract of the present invention can up-regulate the expression of genes related to repair and moisturizing target proteins to play a moisturizing and repairing role. The concentrations of bletilla striata glycoside and gymnema sylvestre glycoside are very important for the exertion of the moisturizing and repairing effect.

[0175] Test Example 11

[0176] Anti-inflammatory activity test

[0177] An inflammatory model was established by inducing RAW264.7 cells with lipopolysaccharide (LPS), and the effect of the extract obtained by the preparation method of the present invention on the secretion of IL-6 induced by LPS was detected by ELISA method. The blank control NC group was not treated, and the positive control group was dexamethasone (DEX).

[0178] Test method: RAW264.7 cells in the logarithmic growth phase with good morphology were selected and inoculated into 24-well plates and incubated in an incubator for 24 h. A blank control group, an LPS-induced stimulation group, a positive control group (dexamethasone, dex), and a bletilla striata glycoside extract at 120 μg / mL were set up and incubated in an incubator at 37 °C and 5% CO2 for 2 h. Except for the blank control group, LPS was added to each well and incubated in the incubator for 24 h. The cell supernatant was detected according to the instructions of the ELISA kit and the nitric oxide detection kit, and the contents of NO and IL-6 in the collected cell supernatant were detected respectively. The experimental results were analyzed by data processing software, and the relative expression levels of NO and IL-6 in the sample group were calculated with the relative expression level of the LPS positive control group as 1.

[0179] Table 8

[0180] NO IL-6 Example 1 0.520 0.750 Example 2 0.635 0.789 Example 3 0.657 0.803 Example 4 0.787 0.792 Example 5 0.803 0.809 Example 6 0.802 0.805 Comparative Example 1 0.890 0.896 Comparative Example 2 0.814 0.807 NC 0.001 0.306 LPS 1.000 1.000 DEX 0.654 0.450

[0181] As can be seen from the data in Table 8, the extract can significantly inhibit the inflammatory factor IL-6 and also has a certain inhibitory effect on NO, and exerts an anti-inflammatory effect by inhibiting the inflammatory factors IL-6 and NO.

[0182] Test Example 12

[0183] The extract obtained by the preparation method of the present invention was subjected to a phototoxicity detection test in accordance with the "Technical Specifications for Cosmetics Safety" (2015 edition), and the results are shown in Table 9.

[0184] Table 9

[0185] Batch number Example 1 Example 2 Example 3 Phototoxicity test results No phototoxicity No phototoxicity No phototoxicity Allergenicity test results No allergenicity No allergenicity No allergenicity Pesticide residue test results None None None Hormone test results None None None Antibiotic test results None None None

[0186] As can be seen from the results in Table 9, the bletilla striata extract obtained by using the method of the present invention does not have phototoxicity at a concentration of 4 mg / mL.

[0187] The applicant declares that the present invention illustrates a preparation method, product and application of a Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ through the above embodiments. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments 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 disclosure scope of the present invention.

[0188] 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 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.

[0189] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A preparation method of Bletilla striata extract rich in bletilloside and gymnemaoside Ⅲ, characterized in that, The preparation method includes: (1) Heating and refluxing the Bletilla striata roots with an ethanol aqueous solution to obtain a crude extract; (2) Performing vacuum concentration on the crude extract to obtain a concentrated crude extract; (3) Mixing the concentrated crude extract with an alkaline reagent to adjust the pH and obtain a conversion solution; (4) Adsorbing the conversion solution with macroporous resin, and eluting to obtain an eluate; (5) Concentrating the eluate and freeze-drying; The macroporous resin is HP2MGL; The pH of the conversion solution is 4 - 7; The mass percentage of ethanol in the ethanol aqueous solution is 80 - 99%; After adjusting the pH, it further includes standing or stirring; The temperature of the standing or stirring is 10 - 70°C, and the time is 1 - 72 h.

2. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The mass ratio of the ethanol aqueous solution to the Bletilla striata roots is (5 - 20):

1.

3. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The temperature of the heating and refluxing extraction is 65 - 100°C, and the time is 0.5 - 4 h.

4. The preparation method of the Bletilla striata extract rich in bletillastriatins and gymnemaoside Ⅲ according to claim 1, characterized in that, The number of times of the heating and refluxing extraction is 1 - 5 times.

5. The preparation method of the Bletilla striata extract rich in bletilloside and gymnemaoside Ⅲ according to claim 1, characterized in that, The pressure of the vacuum concentration is 50 - 200 mbar.

6. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The temperature of the vacuum concentration is 40 - 60°C.

7. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The alkaline reagent includes sodium citrate and / or sodium hydroxide.

8. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that The mass of the macroporous resin is 1 - 5 times that of the Bletilla striata roots.

9. The preparation method of the Bletilla striata extract rich in bletilloside and gymnemaoside Ⅲ according to claim 1, characterized in that, The adsorption time in step (4) is 2 - 24 h.

10. The preparation method of the Bletilla striata extract rich in bletillastriatins and gymnemaoside Ⅲ according to claim 1, characterized in that, The elution includes sequentially eluting with pure water, a 25 - 35% ethanol aqueous solution, and a 45 - 55% ethanol aqueous solution.

11. The preparation method of the Bletilla striata extract rich in bletilloside and gymnemaoside Ⅲ according to claim 10, characterized in that, The volume ratio of the pure water, the 25 - 35% ethanol aqueous solution, and the 45 - 55% ethanol aqueous solution to the macroporous resin is independently 5 - 15.

12. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The pressure of the concentration in step (5) is 50 - 200 mbar, and the temperature is 40 - 60°C.

13. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 1, characterized in that, The freeze-drying includes pre-freezing and drying.

14. The preparation method of the Bletilla striata extract rich in bletilloside and gymnemaoside Ⅲ according to claim 13, characterized in that, The temperature of the pre-freezing is -80 to -20°C, and the time is 4 - 24 h.

15. The preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to claim 13, characterized in that, The temperature of the drying is -80 to -20°C, and the time is 24 - 72 h.

16. The Bletilla striata extract prepared by the preparation method of the Bletilla striata extract rich in bletillaside and gymnemaoside Ⅲ according to any one of claims 1 - 15.

17. The application of the Bletilla striata extract according to claim 16 in the preparation of a product with a repair effect.

18. The application of the Bletilla striata extract according to claim 16 in the preparation of a product with anti-inflammatory and moisturizing effects.

19. The application according to claim 17 or 18, characterized in that, The mass percentage of the Bletilla striata extract in the product is 0.001 - 3%.

Images