Gastrodin extract, preparation method and application thereof

Abstract

The present application relates to the technical field of plant extraction, and discloses a Gastrodia extract, a preparation method and application thereof. The method comprises the following steps: contacting Gastrodia powder with alcohol I with a water content of less than 10 vol% to perform pre-extraction, to obtain a mixture I; performing extraction treatment on the mixture I in the presence of supercritical carbon dioxide, to separate and obtain an extraction liquid; performing enrichment treatment I on the extraction liquid, to obtain an enriched substance; performing liquid-liquid extraction on the enriched substance in the presence of alcohol II and an organic solvent, to separate and obtain a lower layer extraction liquid; and performing enrichment treatment II on the lower layer extraction liquid. The method provided by the present application can improve the extraction rate of the Gastrodia extract, the obtained Gastrodia extract has good moisturizing and soothing effects, and the characteristic aroma of Gastrodia can be retained, so that the Gastrodia extract can be used in skin care products and as a flavoring.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, specifically to Gastrodia elata extract, its preparation method, and its application. Background Technology

[0002] Gastrodia elata is one of my country's precious traditional Chinese medicinal herbs, with a unique aroma that cannot be matched by industrially synthesized fragrances.

[0003] Currently, most domestic and international research on Gastrodia elata focuses on its main component, gastrodin, and its applications are primarily concentrated on efficacy studies. The commonly used extraction method for Gastrodia elata is alcohol extraction; however, this method is time-consuming and yields low extraction rates, while also resulting in some loss of aroma and active ingredients.

[0004] Supercritical carbon dioxide extraction has become a popular plant extraction method in the last decade, and it is gradually being applied in the food, fragrance, and pharmaceutical industries. It is suitable for extracting plants with high levels of fat-soluble components. However, the products extracted by supercritical carbon dioxide extraction often contain a significant amount of waxy components, requiring further purification using techniques such as molecular distillation and membrane distillation.

[0005] However, there are currently few studies on the extraction of Gastrodia elata using supercritical carbon dioxide extraction. The main components of dried Gastrodia elata are protein, starch, gastrodin, gastrodin aglycone, vanillin, organic acids, and polysaccharides. Previous studies have shown that because Gastrodia elata contains relatively few fat-soluble components, direct extraction using supercritical carbon dioxide extraction on pulverized Gastrodia elata yields an extraction rate of less than 0.1%, with significant aroma loss. Even after concentration, the extraction rate using supercritical carbon dioxide extraction with ethanol as an entrainer is still less than 0.2%.

[0006] Therefore, the existing methods for extracting Gastrodia elata generally have low extraction rates and are not suitable for large-scale production.

[0007] In recent years, due to the widespread use of agricultural films in agricultural planting, reports of plasticizers being detected in plant extracts have become frequent. Therefore, to ensure that plant extracts are free of plasticizers, it is necessary to avoid contact between the material and plastics throughout the extraction process, and further purify plant extracts containing plasticizers.

[0008] In summary, it is necessary to provide a method for extracting Gastrodia elata with a high extraction rate and without plasticizers in the extract. Summary of the Invention

[0009] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide a method for extracting Gastrodia elata with high extraction rate and no plasticizer in the extract.

[0010] To achieve the above objectives, a first aspect of the present invention provides a method for preparing Gastrodia elata extract using supercritical carbon dioxide, the method comprising:

[0011] (1) Gastrodia elata powder was contacted with alcohol I with a water content of less than 10 vol% for pre-extraction to obtain mixture I;

[0012] (2) The mixture I is extracted in the presence of supercritical carbon dioxide to obtain an extract; and the extract is enriched to obtain a concentrate.

[0013] (3) The enriched material is subjected to liquid-liquid extraction in the presence of alcohol II and organic solvent to separate the lower extract; and the lower extract is subjected to enrichment treatment II.

[0014] In step (1), the weight ratio of the gastrodia powder to the alcohol I is 1-3:1;

[0015] In step (2), the extraction process includes adding an entrainer after the extraction process has been carried out for 30 min to 75 min; the extraction conditions include a temperature of 32℃ to 60℃, a pressure of 25 MPa to 60 MPa, and a time of at least 30 min after the entrainer is added.

[0016] In step (3), the organic solvent is at least one of isododecane, isohexadecane, squalane, petroleum ether, and soybean oil.

[0017] A second aspect of the present invention provides a Gastrodia elata extract prepared by the method described in the first aspect above.

[0018] A third aspect of the present invention provides the application of the Gastrodia elata extract described in the second aspect above in fragrances and skin care products.

[0019] The technical solution provided by this invention has at least the following advantages over the prior art:

[0020] 1) This invention uses alcohols with a specific water content to contact Gastrodia elata, thereby pre-extracting the non-lipid-soluble components contained in Gastrodia elata, which greatly increases the content of non-lipid-soluble components from Gastrodia elata in supercritical carbon dioxide, and thus improves the extraction rate of Gastrodia elata extract.

[0021] 2) The present invention performs enrichment treatment I on the extract obtained by supercritical carbon dioxide extraction. Preferably, the waxy components in the extract are removed by sequentially using the first concentration, freezing treatment, solid-liquid separation and second concentration, thereby improving the solubility and consistency of Gastrodia elata extract.

[0022] 3) The preparation process of the Gastrodia elata extract provided by the present invention is simple. It does not require the use of non-toxic and harmless raw materials during the preparation process, making the entire preparation process safe and pollution-free. At the same time, it can effectively remove plasticizers that may be present in the Gastrodia elata extract, and the obtained Gastrodia elata extract has a good moisturizing and soothing effect, making the Gastrodia elata extract usable in skin care products.

[0023] 4) The Gastrodia elata extract prepared by the method provided by the present invention retains the characteristic aroma of Gastrodia elata and can be used as a natural fragrance in food flavorings and daily fragrances. Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] As previously stated, a first aspect of the present invention provides a method for preparing Gastrodia elata extract using supercritical carbon dioxide, the method comprising:

[0026] (1) Gastrodia elata powder was contacted with alcohol I with a water content of less than 10 vol% for pre-extraction to obtain mixture I;

[0027] (2) The mixture I is extracted in the presence of supercritical carbon dioxide to obtain an extract; and the extract is enriched to obtain a concentrate.

[0028] (3) The enriched material is subjected to liquid-liquid extraction in the presence of alcohol II and organic solvent to separate the lower extract; and the lower extract is subjected to enrichment treatment II.

[0029] In step (2), the extraction process includes adding an entrainer after the extraction process has been carried out for 30 min to 75 min; the extraction conditions include a temperature of 32℃ to 60℃, a pressure of 25 MPa to 60 MPa, and a time of at least 30 min after the entrainer is added.

[0030] In step (3), the organic solvent is at least one of isododecane, isohexadecane, squalane, petroleum ether, and soybean oil.

[0031] Preferably, in step (1), the pre-extraction conditions include a temperature of 5℃-45℃ and a time greater than 24h. More preferably, in step (1), the pre-extraction conditions include a temperature of 25℃-35℃ and a time of 24h-96h. The inventors of this invention have found that this preferred condition results in a higher extraction rate.

[0032] Preferably, in step (1), the weight ratio of the gastrodia powder to the alcohol I is 1-3:1. The inventors of this invention have discovered that when the weight ratio of the gastrodia powder to the alcohol I exceeds 3:1, the mixture I cannot be loaded into the extraction vessel, and a portion of the alcohol I needs to be removed before extraction can proceed.

[0033] Preferably, in step (1), the average particle size of the gastrodia powder is 75μm-250μm.

[0034] Preferably, in step (2), the extraction time is up to 40 minutes after the entrainer is added.

[0035] Preferably, in step (2), the separation conditions include: being carried out in a separation vessel at a temperature of 40°C-60°C and a pressure of 5MPa-8MPa.

[0036] More preferably, the organic solvent is at least one of isododecane, isohexadecane, and squalane.

[0037] According to a preferred embodiment, in step (2), the entrainer is anhydrous ethanol, and the amount of the entrainer relative to 1 kg of the Gastrodia elata powder is 0.01 L-1 L; preferably 0.5 L-1 L. The inventors of the present invention have found that this preferred embodiment has a higher extraction rate.

[0038] According to another preferred embodiment, in step (2), the enrichment treatment I method includes: sequentially subjecting the extract to a first concentration, a freezing treatment, a solid-liquid separation, and a second concentration. The inventors of this invention have discovered that this preferred embodiment achieves a higher extraction rate.

[0039] More preferably, in step (2), the conditions for the first concentration are controlled such that the volume ratio of the product obtained after the first concentration to the weight ratio of the extract is 0.2L-0.6L:1kg.

[0040] In a preferred embodiment, in step (2), the conditions for the first concentration and the second concentration each independently include: a temperature of 35°C-50°C and a pressure of 50Pa-100Pa.

[0041] It should be noted that the present invention does not impose any particular limitations on the solid-liquid separation method. Those skilled in the art can choose from the technical means known in the art. For example, filtration can be used.

[0042] Preferably, in step (2), the freezing conditions include a temperature of -40°C to 0°C and a time of 8-24 hours. More preferably, the temperature is -25°C to 0°C and the time is 12-24 hours. The inventors of this invention have found that the Gastrodia elata extract prepared under these preferred conditions has a better moisturizing effect.

[0043] Preferably, in step (3), the conditions for liquid-liquid extraction include: a temperature of 5°C-40°C and a time of 20 min-60 min.

[0044] In a preferred embodiment, in step (3), the weight ratio of the enriched substance to the alcohol II is 1:3-6.

[0045] Preferably, the alcohol substance II is anhydrous ethanol.

[0046] According to a preferred embodiment, in step (3), the ratio of the sum of the weights of the enriched substance and the alcohol II to the weight of the organic solvent is 1:0.7-1.3.

[0047] Preferably, in step (3), the enrichment treatment II method includes: performing a third concentration on the lower layer extract; the conditions for the third concentration include: a temperature of 35℃-50℃ and a pressure of 50Pa-100Pa.

[0048] It should be noted that the present invention does not impose any particular restrictions on the equipment used for concentration. Those skilled in the art can select equipment based on known techniques in the field. For example, rotary evaporators, pharmaceutical solvent concentration equipment, etc., can be used.

[0049] As previously stated, a second aspect of the present invention provides a Gastrodia elata extract prepared by the method described in the first aspect.

[0050] As previously stated, the third aspect of the present invention provides the application of the Gastrodia elata extract described in the second aspect in fragrances and skin care products.

[0051] The invention will now be described in detail through examples. Unless otherwise specified, room temperature refers to 25 ± 2°C.

[0052] In the following examples, unless otherwise specified, all raw materials used were commercially purchased.

[0053] In this invention, the extraction rate = (weight of product / weight of Gastrodia elata raw material) × 100%;

[0054] Gastrodia elata: originates from Zhaotong, Yunnan.

[0055] Example 1

[0056] (1) 3 kg of Gastrodia elata powder (average particle size of 180 μm) was contacted with anhydrous ethanol (alcohol I) for pre-extraction to obtain mixture I; the weight ratio of Gastrodia elata powder to alcohol I was 2:1.

[0057] The pre-extraction conditions were: room temperature and 24 hours.

[0058] (2) In the presence of supercritical carbon dioxide, mixture I was extracted in an extraction vessel to obtain an extract; the extract was then subjected to a first concentration, freezing, filtration (solid-liquid separation), and a second concentration (enrichment treatment I) to obtain an enriched product (GC-MS analysis showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was 6.1 wt%), with an extraction rate of 1.12 wt%.

[0059] The extraction method is as follows: anhydrous ethanol (entrainer) is added after 60 minutes of extraction; the extraction conditions are: temperature 45℃, pressure 45MPa, and extraction is carried out for another 30 minutes after the entrainer is added; the amount of entrainer used is 0.6L relative to 1kg of Gastrodia elata powder.

[0060] The separation conditions are as follows: the separation is carried out in a separation vessel at a temperature of 50°C and a pressure of 6 MPa.

[0061] The conditions for the first concentration were: a rotary evaporator was used, the temperature was 45℃, the pressure was 80Pa, and the concentration was carried out until the ratio of the volume of the product to the weight of the extract was 0.3L:1kg.

[0062] The freezing conditions are: temperature -20℃, time 24h;

[0063] The conditions for the second concentration were: to use a rotary evaporator, at a temperature of 45°C and a pressure of 80Pa, and to concentrate until the weight of the product no longer changed within 10 minutes.

[0064] (3) In the presence of anhydrous ethanol (alcohol II) and isododecane (organic solvent), the enriched material was subjected to liquid-liquid extraction in a separatory funnel to separate the lower reddish-brown extract; the lower extract was then concentrated for the third time (enrichment treatment II) to obtain Gastrodia elata extract T1 (GC-MS analysis showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was less than 0.1 wt%), with an extraction rate of 1.10 wt%.

[0065] The conditions for liquid-liquid extraction were: room temperature and 1 hour.

[0066] The conditions for the third concentration were: to use a rotary evaporator, at a temperature of 45°C and a pressure of 80 Pa, until the weight of the product no longer changed within 10 minutes.

[0067] Example 2

[0068] (1) 3 kg of Gastrodia elata powder (average particle size of 250 μm) was contacted with 95 vol% ethanol (alcohol I, water content of 5 vol%) for pre-extraction to obtain mixture I; the weight ratio of Gastrodia elata powder to alcohol I was 3:1.

[0069] The pre-extraction conditions were: room temperature and 48 hours.

[0070] (2) In the presence of supercritical carbon dioxide, mixture I was extracted in an extraction vessel to obtain an extract; the extract was then subjected to a first concentration, freezing, filtration (solid-liquid separation), and a second concentration (enrichment treatment I) to obtain an enriched product (GC-MS analysis showed that it contained 7.3 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP), with an extraction rate of 1.00 wt%;

[0071] The extraction method is as follows: anhydrous ethanol (entrainer) is added after 45 minutes of extraction; the extraction conditions are: temperature 35℃, pressure 40MPa, and extraction is carried out for another 30 minutes after the entrainer is added; the amount of entrainer used is 0.5L relative to 1kg of Gastrodia elata powder.

[0072] The separation conditions are: carried out in a separation vessel at a temperature of 45°C and a pressure of 6 MPa;

[0073] The conditions for the first concentration were: a rotary evaporator was used, the temperature was 45℃, the pressure was 70Pa, and the concentration was carried out until the volume ratio of the product to the weight of the extract was 0.3L:1kg.

[0074] The freezing conditions were: temperature -20℃, time 15h;

[0075] The conditions for the second concentration were: to use a rotary evaporator, at a temperature of 45°C and a pressure of 70 Pa, until the weight of the product no longer changed within 10 minutes.

[0076] (3) In the presence of anhydrous ethanol (alcohol II) and isododecane (organic solvent), the enriched material was subjected to liquid-liquid extraction in a separatory funnel to separate the lower reddish-brown extract; the lower extract was then concentrated for the third time (enrichment treatment II) to obtain Gastrodia elata extract T2 (GC-MS analysis showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was less than 0.1 wt%), with an extraction rate of 0.95 wt%.

[0077] The conditions for liquid-liquid extraction were: room temperature and 1 hour.

[0078] The conditions for the third concentration were: to use a rotary evaporator, at a temperature of 45°C and a pressure of 70 Pa, until the weight of the product no longer changed within 10 minutes.

[0079] Example 3

[0080] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0081] The pre-extraction time is 96 hours;

[0082] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T3 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.14 wt%.

[0083] Example 4

[0084] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0085] The pre-extraction time is 48 hours;

[0086] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T4 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.12 wt%.

[0087] Example 5

[0088] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0089] The pre-extraction time is 16 hours;

[0090] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T5 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 0.69 wt%.

[0091] Example 6

[0092] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0093] In the pre-extraction, the amount of anhydrous ethanol was adjusted so that the weight ratio of gastrodia powder to alcohol I was 5:1. After the pre-extraction, the mixture was filtered to remove 1 / 3 of the total weight of liquid from the mixture I before extraction.

[0094] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T6 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.13 wt%.

[0095] In this embodiment, the need for further filtration adds a process step, which is not conducive to subsequent mass production.

[0096] Example 7

[0097] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0098] In the extraction process, 95 vol% ethanol was used as the entrainer;

[0099] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T7 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.02 wt%.

[0100] Example 8

[0101] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0102] In enrichment treatment I, freezing is not performed, that is, the extract is sequentially concentrated, separated into solid and liquid and concentrated.

[0103] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T8 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.30 wt%.

[0104] In this embodiment, the Gastrodia elata extract contains a high amount of wax, which is not conducive to subsequent use.

[0105] Example 9

[0106] This embodiment uses a method similar to that of Embodiment 1, except that in this embodiment:

[0107] In enrichment treatment I, the freezing temperature is -40℃;

[0108] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, T8 (which, according to GC-MS analysis, contained less than 0.1 wt% of the plasticizer di(2-ethylhexyl) phthalate (DHEP),) was obtained with an extraction rate of 1.08 wt%.

[0109] Comparative Example 1

[0110] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0111] Without pre-extraction, the Gastrodia elata powder was directly placed in the extraction vessel for extraction in the presence of supercritical carbon dioxide, and anhydrous ethanol was added after 60 minutes of extraction.

[0112] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, DT1 (analyzed by GC-MS, showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was less than 0.1 wt%), was obtained with an extraction rate of 0.09 wt%.

[0113] Comparative Example 2

[0114] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0115] Pre-extraction was performed by contacting Gastrodia elata powder with an 85% (v / v) ethanol aqueous solution (Gastrodia elata powder was severely clumped).

[0116] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, DT2 (with a content of less than 0.1 wt% of plasticizer di(2-ethylhexyl) phthalate (DHEP) as determined by GC-MS), was obtained with an extraction rate of 0.38 wt%.

[0117] Comparative Example 3

[0118] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0119] In the extraction process, anhydrous ethanol was added before the extraction process, and the extraction time was 90 min.

[0120] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, DT3 (analyzed by GC-MS, showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was less than 0.1 wt%), was obtained with an extraction rate of 0.15 wt%.

[0121] Comparative Example 4

[0122] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0123] During the extraction process, the entrainer is added before extraction for 15 minutes.

[0124] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, DT4 (analyzed by GC-MS, showed that the content of plasticizer di(2-ethylhexyl) phthalate (DHEP) was less than 0.1 wt%), was obtained with an extraction rate of 0.95 wt%.

[0125] Comparative Example 5

[0126] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0127] During the extraction process, the temperature was 70℃ and the pressure was 65MPa;

[0128] All other conditions were the same as in Example 1, and the following extract of Gastrodia elata, DT5 (with a content of less than 0.1 wt% of plasticizer di(2-ethylhexyl) phthalate (DHEP) as determined by GC-MS), was obtained with an extraction rate of 1.20 wt%.

[0129] In this comparative example, increasing the extraction temperature and pressure improved the extraction rate to some extent. However, if the pressure and temperature are too high, the requirements for the extraction equipment will be high and the cost will be expensive when mass production is carried out.

[0130] Comparative Example 6

[0131] This comparative example was conducted using a method similar to that of Example 1, except that in this comparative example:

[0132] In liquid-liquid extraction, the organic solvent is ethyl acetate;

[0133] All other conditions are the same as in Example 1.

[0134] In this comparative example, the enriched product obtained after enrichment treatment I could not be effectively separated in the presence of anhydrous ethanol and ethyl acetate, thus failing to obtain the required Gastrodia elata extract.

[0135] Test case

[0136] The Gastrodia elata extracts prepared in the examples and comparative examples were tested for their moisturizing and soothing effects. The specific test methods are as follows, and the test results are shown in Table 1:

[0137] Mix 6.6 mg of hyaluronidase with 10 mL of acetate buffer (pH 5.6) to obtain Mixture I for later use; mix 2.5 mg of sodium hyaluronate with 5 mL of acetate buffer (pH 5.6) to obtain Mixture II for later use; mix 50 mL of sodium carbonate solution (1.0 mol / L) with 3.5 mL of acetylacetone to obtain Mixture III for later use; mix 0.8 g of p-dimethylaminobenzaldehyde with 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol to obtain Mixture IV for later use.

[0138] 40 μL of a certain concentration of Gastrodia elata extract was mixed with 40 μL of the above mixture I and reacted at 37℃ for 20 min. Then, 8 μL of calcium chloride aqueous solution (2.5 mol / L) was added and reacted at 37℃ for 20 min. Next, 40 μL of the above mixture II was added and reacted at 37℃ for 40 min, followed by incubation at room temperature for 10 min. Then, 40 μL of distilled water, 8 μL of sodium hydroxide aqueous solution (5.0 mol / L), and 40 μL of the above mixture III were added sequentially, followed by boiling water bath for 15 min, ice bath for 10 min, and incubation at room temperature for 10 min. Finally, 80 μL of the above mixture IV was added, and the mixture was incubated at room temperature for 30 min to obtain the sample group.

[0139] 40 μL of a certain concentration of Gastrodia elata extract (including 0.01 wt%, 0.02 wt%, 0.08 wt%, and 0.12 wt%) was mixed with 40 μL of acetate buffer solution with pH = 5.6 and reacted at 37 °C for 20 min. Then, 8 μL of calcium chloride aqueous solution (2.5 mol / L) was added and reacted at 37 °C for 20 min. Next, 40 μL of the above mixture II was added and reacted at 37 °C for 40 min, followed by incubation at room temperature for 10 min. Then, 40 μL of distilled water, 8 μL of sodium hydroxide aqueous solution (5.0 mol / L), and 40 μL of the above mixture III were added sequentially, followed by boiling water bath for 15 min, ice bath for 10 min, and incubation at room temperature for 10 min. Finally, 80 μL of the above mixture IV was added, and the mixture was incubated at room temperature for 30 min to obtain the sample control group.

[0140] 40 μL of potassium glycyrrhizate (1 mg / mL) was mixed with 40 μL of the above mixture I and reacted at 37 °C for 20 min. Then, 8 μL of calcium chloride aqueous solution (2.5 mol / L) was added and reacted at 37 °C for 20 min. Next, 40 μL of the above mixture II was added and reacted at 37 °C for 40 min, followed by incubation at room temperature for 10 min. Then, 40 μL of distilled water, 8 μL of sodium hydroxide aqueous solution (5.0 mol / L), and 40 μL of the above mixture III were added sequentially, followed by boiling water bath for 15 min, ice bath for 10 min, and incubation at room temperature for 10 min. Finally, 80 μL of the above mixture IV was added, and the mixture was incubated at room temperature for 30 min to obtain the positive control group.

[0141] After mixing 40 μL of distilled water with 40 μL of the above mixture I, the mixture was reacted at 37 °C for 20 min. Then, 8 μL of calcium chloride aqueous solution (2.5 mol / L) was added, and the mixture was reacted at 37 °C for 20 min. Next, 40 μL of the above mixture II was added, and the mixture was reacted at 37 °C for 40 min and then placed at room temperature for 10 min. Then, 40 μL of distilled water, 8 μL of sodium hydroxide aqueous solution (5.0 mol / L), and 40 μL of the above mixture III were added sequentially, followed by boiling water bath for 15 min, ice bath for 10 min, and then placed at room temperature for 10 min. Finally, 80 μL of the above mixture IV was added, and the mixture was placed at room temperature for 30 min to obtain the model control group.

[0142] 40 μL of distilled water and 40 μL of acetate buffer solution with pH = 5.6 were mixed and reacted at 37°C for 20 min. Then, 8 μL of calcium chloride aqueous solution (2.5 mol / L) was added and reacted at 37°C for 20 min. Next, 40 μL of acetate buffer solution with pH = 5.6 was added and reacted at 37°C for 40 min, followed by incubation at room temperature for 10 min. Then, 40 μL of distilled water, 8 μL of sodium hydroxide aqueous solution (5.0 mol / L), and 40 μL of the above mixture III were added sequentially, followed by boiling water bath for 15 min, ice bath for 10 min, and incubation at room temperature for 10 min. Finally, 80 μL of the above mixture IV was added, and the mixture was incubated at room temperature for 30 min to obtain the blank control group.

[0143] The absorbance of the sample group, sample control group, positive control group, model control group, and blank control group at a wavelength of 530 nm was measured using an ELISA reader. The inhibition rate of Gastrodia elata extract against hyaluronidase was calculated according to the following formula to evaluate the moisturizing and soothing effect of Gastrodia elata extract:

[0144] Inhibition rate (%) = (1 - (absorbance of sample group - absorbance of sample control group) / (absorbance of model control group - absorbance of blank control group)) × 100%.

[0145] Table 1

[0146]

[0147]

[0148] As can be seen from the results in Table 1, the Gastrodia elata extract provided by this invention has a high inhibitory effect on hyaluronidase, indicating that it has a good moisturizing and soothing effect on the skin and can be used as an active ingredient in skin care products.

[0149] In addition, GC-MS analysis of the Gastrodia elata extracts prepared in Examples 1-9 showed that they contained natural fragrance components such as furfural, pyranone, nicotinamide, vanillin, and cinnamic acid, and had the characteristic aroma of Gastrodia elata, thus making them suitable as natural fragrance raw materials.

[0150] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing Gastrodia elata extract using supercritical carbon dioxide, characterized in that, The method includes: (1) Gastrodia elata powder was pre-extracted by contacting it with alcohol I with a water content of less than 10 vol% to obtain mixture I; (2) In the presence of supercritical carbon dioxide, the mixture I is extracted to obtain an extract; and the extract is enriched to obtain a concentrate. (3) In the presence of alcohol II and organic solvent, the enriched product is subjected to liquid-liquid extraction to separate the lower extract; and the lower extract is subjected to enrichment treatment II. In step (2), the extraction process includes adding an entrainer after 30-75 minutes of extraction; the extraction conditions include a temperature of 32°C-60°C, a pressure of 25MPa-60MPa, and a time of at least 30 minutes after the entrainer is added. In step (1), the alcohol I is ethanol; In step (3), the organic solvent is at least one of isododecane, isohexadecane, and squalane; In step (3), the alcohol substance II is anhydrous ethanol.

2. The method according to claim 1, wherein, In step (1), the pre-extraction conditions include: a temperature of 5℃-45℃ and a time greater than 24h; and / or, In step (1), the weight ratio of the gastrodia powder to the alcohol I is 1-3:1; and / or, In step (1), the average particle size of the gastrodia powder is 75µm-250µm.

3. The method according to claim 1, wherein, In step (2), the separation conditions include: being carried out in a separation vessel at a temperature of 40℃-60℃ and a pressure of 5MPa-8MPa.

4. The method according to any one of claims 1-3, wherein, In step (2), the entrainer is anhydrous ethanol, and the amount of the entrainer is 0.01L-1L relative to 1kg of Gastrodia elata powder.

5. The method according to any one of claims 1-3, wherein, In step (2), the enrichment treatment I method includes: sequentially subjecting the extract to a first concentration, freezing treatment, solid-liquid separation, and a second concentration; and / or, In step (2), the conditions of the first concentration are controlled such that the volume ratio of the product obtained after the first concentration to the weight ratio of the extract is 0.2L-0.6L:1kg.

6. The method according to claim 5, wherein, In step (2), the conditions for the first concentration and the second concentration each independently include: a temperature of 35°C-50°C and a pressure of 50Pa-100Pa; and / or, In step (2), the freezing conditions include a temperature of -40°C to 0°C and a time of 8h to 24h.

7. The method according to any one of claims 1-3, wherein, In step (3), the conditions for liquid-liquid extraction include: a temperature of 5°C-40°C and a time of 20 min-60 min; and / or, In step (3), the weight ratio of the enriched substance to the alcohol II is 1:3-6; and / or, In step (3), the ratio of the sum of the weights of the enriched substance and the alcohol II to the weight of the organic solvent is 1:0.7-1.

3.

8. Gastrodia elata extract prepared by the method according to any one of claims 1-7.

9. The application of the Gastrodia elata extract according to claim 8 in fragrances and skin care products.

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