Preparation method of gastrodia elata extract

Abstract

The invention belongs to the technical field of plant extraction, and provides a preparation method of a gastrodia elata extract. According to the method, the hot solvent at the temperature of 70-90 DEG C is firstly mixed with plants, then low-temperature plasma treatment is performed at the temperature of 20-30 DEG C, then countercurrent extraction is performed, the low-temperature plasma technology can enable active ingredients to be dissolved out more easily by breaking cell walls, countercurrent extraction is performed through countercurrent flowing of the solvent and materials, the extraction efficiency is improved to the maximum extent, the extraction time is shortened, and the extraction efficiency is improved to the maximum extent. The active ingredients are not damaged, the extraction rate of the active ingredients is high, the extracted ingredients are more comprehensive, a large number of organic solvents are not needed, and the method is energy-saving, environment-friendly and suitable for large-scale industrial production. The gastrodia elata extract prepared by the invention has excellent whitening and anti-oxidation effects, and can be further applied to the field of daily chemicals.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and more particularly to a method for preparing a Gastrodia elata extract. Background Art

[0002] Gastrodia elata (GASTRODIA ELATA) is a precious traditional Chinese medicinal material. The active ingredients in Gastrodia elata are primarily derived from its roots. Its chemical components include gastrodin, p-hydroxybenzaldehyde, p-hydroxybenzyl alcohol, vanillyl alcohol, gastrodin ether glycosides, and gastrodia polysaccharides. The pharmacological activities of Gastrodia elata include sedation, hypnosis, anticonvulsant effects, slowing brain aging, lowering blood pressure, antibacterial effects, anti-fatigue effects, neuroprotection, antidepressant effects, and anti-inflammatory effects. The abundant phenols, polysaccharides, organic acids, flavonoids, and terpenes in Gastrodia elata may function as effective compounds in skincare products. Gastrodin and gastrodia polysaccharides are particularly water-soluble.

[0003] The current plant extraction technologies mainly include the following: (1) Extraction method: soak the plant material in a solvent and extract the active ingredients through the penetration of the solvent. It is suitable for water-soluble and organic-soluble components. However, low-temperature soaking takes a long time and has low efficiency, while high-temperature soaking may destroy some active ingredients. In addition, the large-scale use of organic solvents may leave solvent residues, which is safe and environmentally friendly. (2) Distillation method: Utilize the characteristics of volatile components to collect active ingredients through heating evaporation and condensation, but it is limited to extracting volatile components, and high-temperature operation (generally using a high temperature above 40°C to destroy the plant cell wall) will destroy some active ingredients. (3) Ultrasonic-assisted extraction: Use the high-frequency vibration of ultrasound to destroy plant cells and accelerate the extraction process. It has the characteristics of high extraction efficiency and short extraction time. However, during the ultrasonic extraction process, local overheating may occur, which not only affects the quality of the extract but also may cause damage to the equipment. Especially in large-scale production, the power and frequency control of ultrasound is relatively complex and difficult to control. Ultrasonic blank areas may appear, resulting in uneven extraction and affecting the extraction effect. (4) Supercritical fluid extraction: By adjusting the supercritical state of the solvent, the active ingredients in the plant can be quickly extracted, but its high temperature and high pressure environment may destroy some of the active ingredients.

[0004] Therefore, there is an urgent need to develop a preparation method for Gastrodia elata extract, which has high extraction efficiency, short extraction time, can be extracted at low temperature, and will not destroy the active ingredients. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for preparing a Gastrodia elata extract. The present invention utilizes a countercurrent extraction-assisted low-temperature plasma method to extract the active ingredients from Gastrodia elata. This method not only improves extraction efficiency and shortens extraction time, but also allows for low-temperature extraction without destroying the active ingredients. This method results in a high extraction rate of the active ingredients, a more comprehensive extraction of the ingredients, and does not require the use of large amounts of organic solvents, making it more energy-efficient and environmentally friendly.

[0006] A first aspect of the present invention provides a method for preparing a Gastrodia elata extract.

[0007] Specifically, a method for preparing a Gastrodia elata extract comprises the following steps:

[0008] The plant is mixed with a solvent, subjected to low-temperature plasma treatment, and then subjected to countercurrent extraction to obtain the Gastrodia elata extract;

[0009] The temperature of the low-temperature plasma treatment is 20-30°C.

[0010] The present invention utilizes low-temperature plasma treatment to pre-treat Gastrodia elata, destroy cell walls, promote the release of active ingredients (gastrodin, Gastrodia elata polysaccharides, etc.), reduce the mass transfer resistance during the extraction process, create favorable conditions for subsequent full extraction, and carry out at 20-30 ℃, low temperature, will not destroy the active ingredients, so that the extracted ingredients are more comprehensive. Then countercurrent extraction is carried out, during which the solvent passes through the baffles and openings in the extraction equipment, and the reverse flow of the solvent and the material makes the Gastrodia elata raw material fully contact with the solvent, greatly improving the extraction efficiency of the active ingredients. Moreover, the structure of the plant cell wall after low-temperature plasma pretreatment is looser, and the space and pores in the cell increase, so that the active ingredients can be better released without a large amount of organic solvent, and the solvent residue can also be reduced. Therefore, the method of the present invention is more energy-saving and environmentally friendly, and shortens the extraction time. In addition, low-temperature plasma treatment can effectively degrade polyphenol oxidase and peroxidase in plants without using chemical reagents. These enzymes may cause oxidation and degradation of active ingredients in traditional extraction processes, and after low-temperature plasma pretreatment, the retention rate of active ingredients is significantly improved. Low-temperature plasma pretreatment not only improves the extraction rate of active ingredients, but also significantly enhances the antioxidant activity of the extract (for example, in Gastrodia elata flower extraction, this treatment method can increase the content of flavonoids and polyphenols in the extract).

[0011] Preferably, the plant is Gastrodia elata stem and / or Gastrodia elata flower.

[0012] Preferably, the solid-liquid ratio of Gastrodia elata to the solvent is 1:5-1:30, and in the solid-liquid ratio, the weight unit of Gastrodia elata is g, and the volume unit of the solvent is mL.

[0013] Preferably, the solvent is an ethanol aqueous solution.

[0014] Further preferably, the solvent is an ethanol aqueous solution with an ethanol volume fraction of 50%-70%.

[0015] Preferably, the Gastrodia elata is first crushed to an average particle size of 25-250 μm before mixing.

[0016] Further preferably, the Gastrodia elata is first crushed to an average particle size of 50-200 μm before mixing.

[0017] More preferably, the Gastrodia elata is first crushed to an average particle size of 100-200 μm before mixing.

[0018] Preferably, the low-temperature plasma treatment is performed by a low-temperature plasma generator.

[0019] Preferably, the temperature of the low-temperature plasma treatment is 10-40°C, and / or the power of the low-temperature plasma treatment is 80-160W, and / or the frequency of the low-temperature plasma treatment is 5-20MHz (radio frequency excitation), and / or the treatment time of the low-temperature plasma treatment is 5-40 minutes, and / or the gas environment of the low-temperature plasma treatment is argon (purity ≥99.99%), and / or the gas flow rate of the low-temperature plasma treatment is 3-15L / min, and / or the reaction chamber pressure of the low-temperature plasma treatment is 0.1-1bar (low pressure maintains stable discharge).

[0020] Further preferably, the temperature of the low-temperature plasma treatment is 20-30°C, and / or the power of the low-temperature plasma treatment is 100-140W, and / or the frequency of the low-temperature plasma treatment is 10-14 MHz (radio frequency excitation), and / or the treatment time of the low-temperature plasma treatment is 10-30 minutes, and / or the gas environment of the low-temperature plasma treatment is argon (purity ≥99.99%), and / or the gas flow rate of the low-temperature plasma treatment is 5-10 L / min, and / or the reaction chamber pressure of the low-temperature plasma treatment is 0.2-0.5 bar (low pressure to maintain stable discharge).

[0021] Preferably, during the countercurrent extraction, Gastrodia elata and solvent are continuously added to perform continuous countercurrent extraction. Through continuous countercurrent extraction, multi-stage extraction and solvent recycling, the amount of solvent used can be significantly reduced.

[0022] Preferably, the countercurrent extraction is carried out in a countercurrent extraction device, the extraction temperature of the countercurrent extraction is 35-45℃ (water bath circulation temperature control), and / or the time of the countercurrent extraction is 40-60 minutes (single countercurrent cycle), and / or the solvent flow rate of the countercurrent extraction is 0.5-1.5 L / min, and / or the countercurrent extraction is 3-5 stages (series extraction tower), and / or the stirring mode of the countercurrent extraction is low-speed spiral stirring (20-50 rpm).

[0023] Preferably, the rotation speed of the low-speed spiral stirring is 20-50 rpm. The low-speed spiral stirring is to avoid damaging the cell structure.

[0024] Preferably, after the countercurrent extraction, a post-treatment step is further included, the post-treatment includes filtering to obtain a filtrate, and discharging from a liquid outlet.

[0025] Compared with the prior art, the beneficial effects of the present application are as follows:

[0026] (1) The present application mixes the solvent with the plant, first carries out low-temperature plasma treatment at 20-30℃, and then carries out countercurrent extraction. This low-temperature plasma treatment-countercurrent extraction combined method uses low-temperature plasma technology to damage the cell wall, so that the active ingredients are more easily dissolved out, providing strong conditions for subsequent extraction, and low-temperature treatment is beneficial to protect the effective components. Then, the countercurrent extraction is carried out through the reverse flow of the solvent and the material, which maximizes the extraction efficiency. The present application combines the high-efficiency crushing effect of low-temperature plasma and the high-mass transfer efficiency of countercurrent extraction, and cooperatively improves the extraction rate of active ingredients (such as gastrodin and gastrodiploside) in Gastrodia elata. Overcomes the problems of low extraction rate and long extraction time of traditional methods (such as decoction method, immersion method, and reflux extraction method). In addition, low-temperature plasma treatment may have limited effect on the extraction of some components, and after combining with countercurrent extraction, various active ingredients in Gastrodia elata can be more comprehensively extracted. Therefore, the Gastrodia elata extraction method of the present application has high extraction efficiency, shortens the extraction time, has high extraction rate of active ingredients, and extracts more comprehensive components.

[0027] (2) The present application adopts low-temperature plasma treatment, which is carried out at a low temperature of 20-30℃, avoiding the damage of high-temperature treatment to heat-sensitive active ingredients, so as to not destroy the active ingredients, making the extracted components more comprehensive, overcoming the problem of degradation or volatilization of active ingredients caused by too high treatment temperature (as high as 80℃) in traditional heat extraction methods (such as reflux extraction).

[0028] (3) The present application uses low-temperature plasma treatment to improve the extraction efficiency and shorten the extraction time. Low-temperature extraction saves energy consumption, and does not need to use a large amount of organic solvent, which is energy-saving and environmentally friendly, and also reduces the use amount of solvent and the generation of waste.

[0029] (4) The countercurrent treatment of the present invention can be automated and is suitable for large-scale industrial production. Compared with traditional batch extraction methods, continuous countercurrent extraction can improve production efficiency and reduce labor costs.

[0030] (5) The Gastrodia elata extract prepared by the present invention has excellent whitening and antioxidant effects and can be further applied to the field of daily chemical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a photo of the freeze-dried powder of Gastrodia elata extract prepared in Example 1 of the present invention;

[0032] Figure 2 The HPLC chromatogram of the Gastrodia elata extract prepared in Example 1 of the present invention is shown;

[0033] Figure 3 This is a standard curve of gastrodin in the Gastrodia elata extract prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0035] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0036] The raw materials used in the present invention are as follows:

[0037] Gastrodia elata raw materials: Gastrodia elata freshly picked locally in Zhaotong, Yunnan.

[0038] Example 1

[0039] A method for preparing a Gastrodia elata extract comprises the following steps:

[0040] (1) An ethanol-water solution (volume ratio of ethanol to water = 3:1) with a volume of 5 times (i.e., the material-liquid ratio of Gastrodia elata to solvent is 1:5, the weight unit of Gastrodia elata is g, and the volume unit of solvent is mL) the volume of Gastrodia elata material is used as solvent and transported to the extraction tank through the solvent inlet.

[0041] (2) Grind the raw material of Gastrodia elata to an average particle size of 150±50 μm and add it into the extraction tank through the top feeding port.

[0042] (3) Low-temperature plasma pretreatment: Start the low-temperature plasma generator at 25°C, with a power of 120W, a frequency of 13.56MHz, an argon flow rate of 10L / min, and a reaction chamber pressure of 0.5bar, and perform low-temperature plasma treatment on the Gastrodia elata raw material in the extraction tank for 15min.

[0043] (4) Turn on the stirring paddle of the countercurrent extraction equipment, set the temperature to 40°C, the single cycle time to 45 minutes, the solvent flow rate to 1 L / min, the countercurrent stage to 3, and start low-speed stirring at 40 rpm. The solvent passes through the baffle and opening in the extraction tank to form a countercurrent extraction with the Gastrodia elata raw material, so that the Gastrodia elata and the solvent are fully in contact. During the extraction process, the Gastrodia elata raw material and solvent are continuously added to achieve continuous countercurrent extraction.

[0044] (5) The extract is filtered through the filter and discharged from the liquid outlet. If the filter is clogged, it can be directly removed and cleaned without affecting the extraction process. After the extraction is completed, the stirring blade and the feed port are closed. Washing water is introduced through the washing water inlet to clean the extraction tank and discharge the residue. For the convenience of storage, freeze drying is performed to obtain the powder of the Gastrodia elata extract ( Figure 1 ).

[0045] Examples 2-7

[0046] Example 2-7 provides a method for preparing Gastrodia elata extract, which differs from Example 1 in that some operating parameters are different, as shown in Table 1.

[0047] Comparative Example 1 (low-temperature plasma extraction method alone)

[0048] A method for preparing a Gastrodia elata extract differs from Example 1 in that countercurrent extraction is not performed. The specific steps are as follows: an ethanol-water solution (ethanol to water volume ratio = 3:1) 5 times the amount of Gastrodia elata material is delivered to an extraction tank through the solvent inlet. The Gastrodia elata raw material is crushed to an average particle size of 150±50 μm and added to the extraction tank through the top feed port. A low-temperature plasma generator is activated at a power of 120 W, a frequency of 13.56 MHz, a treatment time of 15 minutes, and an argon flow rate of 10 L / min. The reaction chamber pressure is 0.5 bar to pretreat the Gastrodia elata raw material in the extraction tank to destroy cell walls and promote the release of active ingredients. After extraction is completed, the stirring paddle and feed port are closed. Wash water is introduced through the wash water inlet to clean the extraction tank, remove residue, filter, and remove impurities, color, and flavor to obtain the Gastrodia elata extract. For convenient storage, the Gastrodia elata extract is freeze-dried to obtain a powder.

[0049] Comparative Example 2 (single countercurrent extraction method)

[0050] A method for preparing a Gastrodia elata extract, which differs from Example 1 in that low-temperature plasma extraction is not performed, and the specific steps are as follows:

[0051] An ethanol-water solution (ethanol:water volume ratio = 3:1) was delivered to the extraction tank through the solvent inlet. The raw Gastrodia elata was crushed to an average particle size of 150 ± 50 μm and added to the extraction tank through the top feed port. The countercurrent extraction equipment's agitator was activated, and the temperature was set to 40°C, the single cycle time to 45 minutes, the solvent flow rate to 1 L / min, the number of countercurrent stages to 3, and low-speed stirring at 40 rpm to ensure thorough contact between the raw Gastrodia elata and the solvent. The solvent passed through the baffles and openings within the extraction tank, creating a countercurrent extraction with the raw Gastrodia elata. The extract was filtered through a filter and discharged through the outlet. During the first extraction, the raw Gastrodia elata and solvent were continuously added to achieve continuous countercurrent extraction. If the filter became clogged, it could be removed and cleaned without affecting the extraction process. After the extraction was completed, the agitator and feed port were closed. Wash water was introduced through the wash water inlet to clean the extraction tank. Residue was removed, filtered, and impurities, color, and flavor removed to obtain the Gastrodia elata extract. For convenient storage, the extract was freeze-dried to obtain a powdered Gastrodia elata extract.

[0052] Comparative Example 3

[0053] A method for preparing a Gastrodia elata extract differs from Example 1 in that step (4) is different, specifically: the countercurrent extraction in step (4) is replaced by an extraction method, wherein the extraction temperature is 40° C. and the extraction time is 135 min.

[0054] Comparative Example 4

[0055] A method for preparing a Gastrodia elata extract, which differs from Example 1 in that step (3) is different, specifically: the low-temperature plasma extraction in step (3) is replaced by an ultrasound-assisted high-temperature extraction method, wherein the extraction temperature is 60°C, the extraction time is 15 minutes, and the ultrasonic frequency is 20kHz.

[0056] Some operating parameters of Examples 1-7 of the present invention are shown in Table 1 below.

[0057] Table 1 Key operating parameters of each embodiment

[0058]

[0059] Product effect testing

[0060] 1. Test of Gastrodin and Gastrodia Polysaccharide Content

[0061] (1) Test method for gastrodin content: High performance liquid chromatography method, specific parameters are as follows:

[0062] Chromatographic column: AQ-C18 4.6*100mm, 2.7μm;

[0063] Injection volume: 10 μL;

[0064] Mobile phase: acetonitrile: water (0.1% acetic acid, volume fraction) = 3:97; flow rate = 1.0 mL / min;

[0065] Column temperature: 35 °C;

[0066] Detection wavelength: 270 nm.

[0067] (2) Test method for the content of Gastrodia elata polysaccharide: test the content of Gastrodia elata polysaccharide by phenol-sulfuric acid colorimetry

[0068] Test principle: the phenol-sulfuric acid colorimetry is based on the principle that sugar substances can be oxidized to furfural substances under acidic conditions. The color depth of the generated furfural substances is proportional to the content of sugar substances. Therefore, the content of Gastrodia elata polysaccharide in the sample can be determined by observing the color depth of the reaction solution.

[0069] Test method: prepare a standard curve: accurately pipette 0.2 mg / mL glucose standard solution 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16 and 0.18 mL, respectively, each to 0.20 mL with water, add 5% phenol 0.40 mL, then quickly add concentrated sulfuric acid (analytical pure, d = 1.84 g / cm 3 ) 2.0 mL, stand for 10 minutes, shake well, and stand at room temperature for 20 minutes. Measure the absorbance value: measure the absorbance value at 490 nm on a UV-visible spectrophotometer, and use distilled water instead of the sugar solution as a blank control. Calculate the sugar content: take the sugar content as the abscissa and the absorbance value as the ordinate to obtain the standard curve. Further, measure the absorbance value of the sample, and calculate the content of Gastrodia elata polysaccharide in the sample using the standard curve.

[0070] (3) Test results

[0071] In the Gastrodia elata extract of Example 1, the detection of Gastrodia elata element with a residence time of 5.930 min Figure 2 ) and Figure 3 (the residence time of this Gastrodia elata element under this HPLC test method is about 5.905 min) basically coincides (the residence time allows for a small error in the decimal point), and the content of Gastrodia elata element in the extract is obtained by calculating the area of the peak of Gastrodia elata element.

[0072] Table 2 Test results of the mass content of Gastrodia elata element and Gastrodia elata polysaccharide in each example and comparative example

[0073]

[0074] As shown in the above table, the content of gastrodin in the ginseng of the embodiments 1-7 is as high as 0.74-1.03 wt%, and the content of ginseng polysaccharide is as high as 23.24-27.75 wt%, wherein the extraction rate of gastrodin and total ginseng polysaccharide in the ginseng of the embodiment 1 is the highest, and the mass fraction can reach 1.03% and 27.75%, respectively.

[0075] The comparative example 1 only uses the low-temperature plasma extraction method for extraction without implementing the countercurrent extraction, so that the content of gastrodin and ginseng polysaccharide is greatly reduced.

[0076] The comparative example 2 only uses the countercurrent extraction without implementing the low-temperature plasma extraction, so that the content of gastrodin and ginseng polysaccharide is greatly reduced.

[0077] The content of gastrodin and ginseng polysaccharide obtained by the extraction method of the comparative example 3 is not only lower than that of the embodiments 1-7, but also the lowest among the four comparative examples, which is because the natural solvent soaking of the extraction method is difficult to completely break the cell wall and extract the cell membrane of the plant, so that the extraction efficiency is low, but the cost performance of the method is high.

[0078] Although the content of the active substance extracted by the ultrasonic-assisted high-temperature extraction method of the comparative example 4 is higher than that of the comparative examples 1-3, the extraction rate of the comparative example 4 is still lower compared with the embodiments 1-7, so the advantages of the extraction method of the present application can be fully shown.

[0079] 2, Test of the inhibition effect on tyrosinase

[0080] (1) Test method

[0081] The present application uses the spectrophotometry to test the inhibition effect on tyrosinase, and the test principle is that tyrosinase can catalyze L-dopa to generate dopa pigment, and the characteristic absorption peak of the product is at 475 nm, the difference between the light absorption properties of the substrate and the product is utilized, the change of the light absorption value of the reaction mixture is measured to characterize the change of the tyrosinase activity, and the change of the light absorption value can characterize the change of the tyrosinase activity.

[0082] Test concentration: the aqueous solution with the solute mass fraction of 10% of the ginseng extract is selected for efficacy test.

[0083] (2) Test results

[0084] Table 3: Test results of the inhibition effect of the ginseng extract of each embodiment and comparative example on tyrosinase

[0085]

[0086] As shown in the above table, the inhibition rate of the embodiments 1-7 on tyrosinase is as high as 25-42%, wherein the inhibition rate of the embodiment 1 is the highest, reaching 42%.

[0087] Comparative Example 1 only used low-temperature plasma extraction method for extraction without countercurrent extraction, resulting in a significant decrease in the contents of gastrodin and gastrodia polysaccharide.

[0088] Comparative Example 2 only used countercurrent extraction without low-temperature plasma extraction, resulting in a significant decrease in the contents of gastrodin and gastrodia polysaccharide.

[0089] Comparative Example 3 exhibited the lowest tyrosinase inhibition rate; the extract obtained by ultrasound-assisted high-temperature extraction in Comparative Example 4 exhibited lower in vitro tyrosinase inhibition rates than those in Examples 1-7. This is directly proportional to the aforementioned active ingredient content: lower active ingredient content corresponds to lower tyrosinase inhibition rates.

[0090] 3. ABTS free radical scavenging rate test

[0091] (1) Test method

[0092] Test principle: The ABTS free radical scavenging ability test method is an in vitro test method to evaluate the ability of antioxidant samples to scavenge ABTS free radicals.

[0093] Test steps:

[0094] (a) Reagent Preparation: Prepare 7.4 mmol / L ABTS solution and 2.6 mmol / L potassium persulfate solution using PBS buffer (pH 7.3) as the solvent. Mix the ABTS solution and potassium persulfate solution in a 1:1 volume ratio to prepare a working solution. Incubate in the dark at room temperature for approximately 12 hours.

[0095] (b) Sample treatment: The mixed working solution was diluted 40 times with PBS buffer.

[0096] (c) Determine absorbance: Mix the working solution with the sample solution, and measure the absorbance of the mixed solution before and after the reaction.

[0097] (d) Data processing: The scavenging rate of the sample on ABTS free radicals was calculated based on the absorbance change.

[0098] Evaluation method: Calculate the inhibition rate of each sample in an aqueous solution with a solute mass fraction of 10% to evaluate its antioxidant activity. Generally speaking, the higher the inhibition rate, the stronger the antioxidant activity of the sample.

[0099] (2) Test results

[0100] Table 4 Test results of ABTS free radical scavenging rate of the extracts of each embodiment and comparative example

[0101]

[0102] As can be seen from the above table, Examples 1-7 of the present invention have ABTS free radical scavenging rates as high as 37.77-48.13%, indicating that the Gastrodia elata extract of the present invention has high antioxidant activity. Among them, Example 1 has the best inhibition rate, indicating the best effect in scavenging ABTS free radicals.

[0103] Comparative Example 1 only uses low-temperature plasma extraction method for extraction without countercurrent extraction, resulting in a decrease in the ABTS free radical scavenging rate.

[0104] Comparative Example 2 only used countercurrent extraction without low-temperature plasma extraction, which resulted in a lower ABTS free radical scavenging rate.

[0105] The ABTS free radical scavenging rates of the extraction method of Comparative Example 3 and the ultrasound-assisted high-temperature extraction method of Comparative Example 4 were also low, among which the scavenging rate of Comparative Example 3 was the lowest.

[0106] 4. Test of the inhibitory effect on B16 cell melanin

[0107] (1) Test method

[0108] Basic Principle: Mouse B16 melanoma cells are a commonly used cell model for melanin research, useful for studying the effects of whitening cosmetics on intracellular tyrosinase and its impact on cellular melanin synthesis. Tyrosinase catalysis primarily occurs in two reactions: the conversion of tyrosine to dopa and the conversion of dopa to dopaquinone. Therefore, most whitening agents work by inhibiting tyrosinase activity, reducing melanin production and achieving whitening.

[0109] Concentration screening: The results of the cytotoxicity test showed that the cell survival rate of the sample was >85% when the concentration was ≥3% (sample: the Gastrodia elata extract powder prepared in each embodiment and comparative example was prepared into an aqueous solution as the sample, and "3%" here refers to the solute mass percentage of the sample). Therefore, the solute mass percentage of the sample was selected as 3wt% as the test concentration.

[0110] Test method: Using mouse melanoma cells (B16) that highly express melanin as a cell model, the OD values ​​of each sample were measured after adding a solute with a concentration of 3 wt% to the cell culture medium. 405nm The change in melanin production was obtained by measuring the absorbance at the control group. The blank control group was treated with PBS phosphate buffer solution (pH 7.3), with the melanin content set to a reference value of 100%. The positive control group was treated with vitamin C (Vc) and vitamin E (Ve) at concentrations of 200 μg / mL and 20 μg / mL, respectively, yielding relative melanin contents of 73.71% and 83.52%, respectively. The relative melanin content of each example and comparative example sample was calculated using the above method.

[0111] (2) Test results

[0112] Table 5 Inhibitory effect test of each embodiment and comparative example on B16 cell melanin

[0113]

[0114] As can be seen from the table above, after treating B16 melanoma cells with Examples 1-7 of the present invention, the relative value of melanin content decreased to 52.34-69.83%, with a high inhibition rate. Moreover, at the same concentration, the sample of Example 1 had the best effect on clearing melanin from B16 cells.

[0115] The inhibition efficiency of Comparative Examples 1-4 is lower than that of Examples 1-7.

[0116] 5. Skin soothing effect test

[0117] (1) Test method

[0118] Test basis: "Group Standard T / SPMA 003-2023 Cosmetic Soothing Efficacy Test Method Based on the Determination of Nitric Oxide Content in Lipopolysaccharide-Induced Macrophage Inflammatory Cell Model".

[0119] Test principle: The occurrence of skin irritation mainly involves the physiological process of skin barrier-neurovascular-immune inflammation. Skin irritation problems can be improved by relieving skin inflammatory response. NO (nitric oxide) participates in the physiological process of skin inflammatory response as an immunomodulatory factor. By inhibiting the release of excessive NO by activated immune cells, the immune response of the skin can be reduced, thereby improving the irritation of the skin. Bacterial lipopolysaccharide (LPS) was used to induce the release of NO from the mouse mononuclear macrophage leukemia cell line RAW264.7. The NO concentration in the cell culture supernatant was determined by the Griess method. The inhibition rate of the test substance on the release of NO by mouse macrophages was calculated to evaluate the soothing effect of the test substance.

[0120] Cell source: RAW264.7 mouse mononuclear macrophage leukemia cell line, purchased from ATCC TIB-7.1.

[0121] Stimulation conditions: LPS exposure dose was 1 μg / mL, and cells were stimulated for 18 hours.

[0122] Safety test concentration: The solute mass fraction of the aqueous solution of Gastrodia elata extract is 3%, the cell viability is >85%, and it is safe for RAW264.7 cells.

[0123] The change rate of NO concentration compared with the model group was recorded. The specific results are shown in Table 6 below.

[0124] (2) Test results

[0125] Table 6 Soothing efficacy test results of various examples and comparative examples (negative numbers indicate a decrease in NO concentration compared to the model group)

[0126]

[0127] In the above table, the model group was compared with the blank group, P<0.01, indicating that the stimulation was effective.

[0128] As can be seen from the table above, the NO concentrations of Examples 1-7 of the present invention decreased by 26.68-38.91%, among which Example 1 had the best effect on relieving the inflammation model of RAW264.7 cells stimulated by LPS, with the NO concentration decreasing by 38.91%.

[0129] The inflammation model relief effects of Comparative Examples 1-4 were all worse than those of Examples 1-7.

Claims

1. A method for preparing a Gastrodia elata extract, characterized in that: The steps include: The plant is mixed with a solvent, subjected to low-temperature plasma treatment, and then subjected to countercurrent extraction to obtain the Gastrodia elata extract; The temperature of the low-temperature plasma treatment is 20-30°C.

2. The preparation method according to claim 1, characterized in that The plant is Gastrodia elata stem and / or Gastrodia elata flower.

3. The preparation method according to claim 1, characterized in that The solid-liquid ratio of the Gastrodia elata and the solvent is 1:5-1:30, wherein the weight unit of the Gastrodia elata is g, and the volume unit of the solvent is mL.

4. The preparation method according to claim 1, characterized in that The solvent is an ethanol aqueous solution.

5. The preparation method according to claim 1, characterized in that The Gastrodia elata is firstly pulverized to an average particle size of 25-250 μm before mixing.

6. The preparation method according to claim 1, characterized in that The low-temperature plasma treatment is performed by a low-temperature plasma generator.

7. The preparation method according to claim 6, characterized in that The temperature of the low-temperature plasma treatment is 10-40°C, and / or the power of the low-temperature plasma treatment is 80-160W, and / or the frequency of the low-temperature plasma treatment is 5-20MHz, and / or the treatment time of the low-temperature plasma treatment is 5-40 minutes, and / or the gas environment of the low-temperature plasma treatment is argon, and / or the gas flow rate of the low-temperature plasma treatment is 3-15L / min, and / or the reaction chamber pressure of the low-temperature plasma generator is 0.1-1bar.

8. The preparation method according to claim 1, characterized in that During the countercurrent extraction process, Gastrodia elata and solvent are continuously added to perform continuous countercurrent extraction.

9. The preparation method according to claim 1, characterized in that The countercurrent extraction is carried out in a countercurrent extraction device, the extraction temperature of the countercurrent extraction is 35-45°C, and / or the countercurrent extraction time is 40-60 minutes, and / or the solvent flow rate of the countercurrent extraction is 0.5-1.5 L / min, and / or the countercurrent stage number of the countercurrent extraction is 3-5 stages, and / or the stirring mode of the countercurrent extraction is low-speed spiral stirring.

10. The preparation method according to claim 9, characterized in that The rotation speed of the low-speed spiral stirring is 20-50 rpm.

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