Citron extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant extraction, and provides a citron extract as well as a preparation method and application thereof. The preparation method comprises the following steps: pre-treating to remove volatile components by using a water steam distillation method, heating, refluxing and extracting by using ethanol, filtering, concentrating, separating and purifying by using macroporous resin, concentrating, drying and the like. The extraction efficiency is high, energy is saved, the environment is protected, the production period is greatly shortened, and the citron active matter can be rapidly extracted. The prepared citron extract is stable in quality, the content of active ingredients such as naringin and the like in the extract is relatively high, volatile ingredients such as essential oil and the like are removed, and the purity of the extract and the safety of people with sensitive skin are guaranteed. The obtained citron extract has excellent skin moisturizing, anti-inflammatory, anti-allergy, emotion beautifying and anti-aging capabilities, is very friendly to people with sensitive skin, has higher stability, is suitable for large-scale production, and has important application value in preparation of large-scale healthy products and products with emotion beautifying or oral beautifying effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and particularly relates to a citron extract, a preparation method thereof, and an application thereof. Background Art

[0002] Citron (Citrus medica L.) is a small arbor plant with both medicinal and edible uses in the Rutaceae family and the Citrus genus. It is rich in various active chemical substances such as volatile oils, flavonoids, and alkaloids, and has multiple effects such as antioxidant, antibacterial, anti-diabetic, and lipid-lowering effects. Citron is widely used in Yunnan with a long history and has excellent performance in southern Yunnan medicine and ethnic minority medicine.

[0003] Currently, the extraction methods for citron are mostly water extraction with alcohol precipitation, enzymatic hydrolysis, fermentation, steam distillation method, etc.; the components obtained by water extraction with alcohol precipitation are mostly citron polysaccharide components, and the extraction methods have disadvantages such as low extraction rate, long time consumption, loss of active ingredients, and poor stability; the extracts obtained by enzymatic hydrolysis and fermentation are prone to cause skin allergies; the steam distillation method obtains citron essential oil, and the essential oil components have strong permeability, resulting in skin discomfort. Patents and literature are listed as follows:

[0004] Chinese Patent CN 113288911 A discloses an application of citron fruit polysaccharide in the preparation of anti-breast cancer drugs. After defatting with organic solvents and hot water extraction, a citron fruit polysaccharide extract is obtained through decolorization, deproteinization, dialysis, alcohol precipitation, and drying, which has good anti-breast cancer effects.

[0005] Chinese Patent CN 112759660A discloses a citron polysaccharide, its extraction method and application. The citron fruit is extracted with 20 - 80% ethanol to remove pigments and small molecule impurities, and then water extraction, alcohol extraction, deproteinization treatment, alcohol precipitation, passing through a cellulose column, and passing through a gel column to obtain citron polysaccharide, which has the effect of enhancing immunity.

[0006] Chinese Patent CN 116459186A discloses a soothing and anti-aging essential oil composition and its preparation method. 2 - 8% citron essential oil is used as part of the composition, and the main role of citron in it is to scavenge free radicals and have an antioxidant effect.

[0007] Chinese Patent CN 114129612A discloses a plant extract with the function of reducing uric acid, its prepared product and application. The flavonoids and polysaccharide components in sophora flower and sophora japonica flower are extracted by water extraction with heating under reflux method, and the obtained extract can reduce uric acid in the body, has the characteristics of safety and no side effects, and has high market competitiveness.

[0008] Yu Yue et al. published an article titled "Detection and Quality Safety Risk Analysis of Rose Essential Oil" in the journal "Fragrance and Fragrance Cosmetics", stating that essential oil ingredients have a high probability of causing allergic risks.

[0009] Therefore, there is an urgent need to develop a preparation method for citron extract that has simple process, high recovery rate, low cost, low pollution, is suitable for industrial production and has high safety and high activity. Summary of the invention

[0010] In view of the deficiencies in the prior art, the main purpose of the present invention is to propose a citron extract and a preparation method and application thereof, aiming to solve at least one of the technical problems that the existing citron extract extraction method has poor safety and environmental protection performance, the quality of the extract cannot be guaranteed, and there is currently no large-scale industrial production of citron extract for the preparation of skin care products with emotional skin care, soothing anti-inflammatory and / or anti-aging effects.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a method for preparing a citron extract, which specifically comprises the following steps:

[0013] Step S1, crushing the dried citron fruit and placing it in an above-water distillation device, the distillation temperature is 95° C. to 105° C., the distillation time is 0.5 h to 1 h, and a citron product (citron medicinal material) with volatile components removed is obtained;

[0014] Step S2, using an ethanol-water solvent to heat and reflux the citron product obtained in step S1, and filtering and concentrating the extract to obtain a citron crude extract;

[0015] Step S3, loading the citron crude extract from step S2 onto a macroporous resin column, performing separation and purification using the macroporous resin, and concentrating and drying the eluate to obtain a citron extract.

[0016] The preparation method of the present invention comprises pretreatment by steam distillation on water to remove volatile components, ethanol heating reflux extraction, filtration, vacuum concentration, macroporous resin separation and purification, vacuum concentration, drying, etc. The preparation method has high extraction efficiency, energy saving and environmental protection, easy industrialization, greatly shortens the production cycle, can achieve rapid extraction of citron active substances, the prepared citron extract has stable quality, and the content of active ingredients in the extract such as naringin is relatively high. At the same time, volatile components such as essential oils are removed to ensure the purity of the extract and the safety of sensitive skin groups. The obtained citron extract has excellent skin moisturizing, anti-inflammatory, soothing, emotional beauty and anti-aging abilities, and is very friendly to sensitive skin groups. It also has high stability, is suitable for large-scale production, can be used as a raw material for the production of large health products, and has important application value for preparing products with emotional beauty or oral beauty effects.

[0017] Preferably, in step S1, after pulverization, it is sieved through a 50-100 mesh sieve, and the mass ratio of citron powder to distilled water is 1:(5-9).

[0018] Among them, the above "50-100 mesh" can specifically take values such as: 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, 100 mesh, etc.; the above "1:(5-9)" can specifically take values such as: 1:5, 1:6, 1:7, 1:8, 1:9, etc.

[0019] Preferably, the above distillation temperature is 95°C - 105°C, such as 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, etc.

[0020] Preferably, the above distillation time is 0.5h - 1h, such as 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0021] Preferably, in step S2, the mass ratio of the citron product to the ethanol-water solvent is 1:(10-15); the mass concentration of ethanol in the ethanol-water solvent is 45% - 65%; the extraction times are 1-3 times, and the extraction time for each time is 1h - 2h. In the present invention, the concentration of ethanol in the ethanol-water solvent is the mass concentration.

[0022] Among them, the above "1:(10-15)" can specifically take values such as: 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0023] Among them, the above "45% - 65%" can specifically take values such as: 45%, 50%, 55%, 65%, etc.

[0024] Among them, the above "1-3" can specifically take values such as: 1, 2, 3, etc.; the above "1h - 2h" can specifically take values such as: 1, 1.5, 2h, etc.

[0025] Preferably, in step S3, the macroporous resin is any one of XAD-16N type, HPD-300 type, HPD-100 type, LD-605 type or Amberlite XAD-7HP type, preferably XAD-16N type or HPD-300 type.

[0026] Preferably, the procedure for separation and purification using macroporous resin is: first, elute with water for 2-5 column volumes to remove impurities, then elute with 35% concentration ethanol for 1-3 column volumes to remove impurities, and then elute with 65% concentration ethanol for 7-10 column volumes to collect the eluate.

[0027] The use of the above-mentioned specific three-stage extraction and purification procedure can significantly increase the contents of total flavonoids and naringin in Citrus medica, which has a positive effect on enhancing the final moisturizing, anti-inflammatory, anti-allergic, emotional beauty and anti-aging effects of the product.

[0028] Preferably, the drying method in step S3 includes any one or a combination of at least two of freeze-drying, vacuum drying or spray drying.

[0029] In a second aspect, the present invention provides a Citrus medica extract, which is prepared by the preparation method of the Citrus medica extract described in the first aspect; the content of naringin in the Citrus medica extract is not less than 20%, for example, it can be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, etc., and other specific point values within this numerical range can be selected, and will not be elaborated one by one here; the content of total flavonoids is not less than 35%, for example, it can be 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, etc., and other specific point values within this numerical range can be selected, and will not be elaborated one by one here.

[0030] Using rutin as a reference substance, the content of total flavonoids in the Citrus medica extract prepared by the present invention is determined by the sodium nitrite-aluminum nitrate method.

[0031] In a third aspect, the present invention provides the application of the Citrus medica extract described in the second aspect in the preparation of large health products with moisturizing, anti-inflammatory, anti-allergic, emotional beauty and anti-aging effects; the large health products are skin care products or foods.

[0032] The present invention discloses a preparation method of a Citrus medica extract with high safety and having moisturizing, anti-inflammatory, anti-aging and emotional beauty effects. After crushing and sieving the Citrus medica fruit, the volatile components are removed by steam distillation method, and then ethanol heating reflux extraction method is used for extraction, macroporous resin separation and purification, reduced pressure concentration and drying to obtain the Citrus medica extract, and the process is optimized with the contents of total flavonoids and naringin as indexes. This preparation method has the advantages of high extraction efficiency, energy saving, simple operation, easy industrialization, good efficacy of the obtained extract and high safety.

[0033] Compared with the prior art, it has at least the following beneficial effects:

[0034] (1) The preparation method of a Citrus medica extract of the present invention creatively uses steam distillation on water to remove volatile components and then uses ethanol-water heating reflux extraction and macroporous resin separation and purification technology, ensuring that the obtained extract is safe and non-allergenic, has excellent moisturizing, anti-inflammatory, anti-allergic, emotional beauty and anti-aging effects, and the preparation method has simple operation, easy industrialization, high extraction efficiency and environmental protection, providing a basis for large-scale industrial production and multi-scenario application of Citrus medica.

[0035] (2) The preparation method of a citron extract of the present invention has improved extraction rate, total flavonoid content, and naringin content compared with ultrasonic extraction, and the moisturizing, anti-aging, and anti-inflammatory effects of the extracted product are more excellent; the volatile components that are prone to cause allergies are removed, and it has more excellent safety.

[0036] (3) The citron extract obtained in the present invention is rich in naringin and flavonoids, among which the naringin content is not less than 20%; the total flavonoid content is not less than 35%.

[0037] (4) The yield of the citron extract obtained in the present invention is relatively high, and the yield is not less than 15%.

[0038] (5) The citron extract obtained in the present invention can significantly increase the content of β-endorphin in keratinocytes, indicating that it may have the effect of emotional skin care.

[0039] (6) The citron extract obtained in the present invention can significantly decrease the expression level of the HSD1 gene and significantly increase the expression level of the HSD2 gene, indicating that it may have the effect of emotional skin care.

[0040] (7) The citron extract obtained in the present invention has excellent emotional beauty effects. Description of the Drawings

[0041] Figure 1 It is the HPLC result diagram of the product prepared in Example 1 and the naringin standard measured by an ultra-high performance liquid chromatograph;

[0042] Figure 2 It is the headspace injection GC-MS detection result diagram of the citron extracts prepared in Example 1 and Comparative Example 3;

[0043] Figure 3 It is the experimental result diagram of the inhibition of TRPV1 by the citron extract prepared in Example 1, where BC is the blank control, NC is the model group, and PC is the positive control;

[0044] Figure 4 It is the inhibition situation of the PGE2 factor by the citron extract prepared in Example 1, where BC is the blank control, NC is the model group, and PC is the positive control. Detailed Embodiments

[0045] The technical solutions of the present invention will be further described below with reference to the drawings and through specific embodiments. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0046] Unless otherwise specified, the raw materials and materials used in the embodiments of the present invention are purchased through general commercial channels.

[0047] The sources of the active ingredients contained in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown, and the necessary excipient ingredients contained in other commercially available raw materials are not elaborated):

[0048] The raw material of Citrus medica is a dried product purchased from Kunming, Yunnan Province.

[0049] The macroporous resin of XAD-16N type is a product named macroporous adsorption resin XAD-16N purchased from Beijing Ruida Henghui Technology Development Co., Ltd.

[0050] The macroporous resin of HPD-300 type is a product named macroporous resin HPD300 purchased from Beijing Ruida Henghui Technology Development Co., Ltd.

[0051] Example 1

[0052] This example provides a preparation method of Citrus medica extract, and the preparation method includes the following steps:

[0053] Step S1: Take 50 g of the raw material of dried and pulverized Citrus medica that has passed through an 80-mesh sieve, put it into a water distillation device, with a distillation temperature of 100 °C, a distillation time of 0.7 h, and a material-liquid ratio of 1:7, to obtain a Citrus medica product (Citrus medica medicinal material) from which volatile components are removed;

[0054] Step S2: Use an ethanol-water solvent with a concentration of 55% to perform heat reflux extraction on the Citrus medica product obtained in Step S1 to obtain an extract, with a material-liquid ratio of 1:12, an extraction time of 1.5 h, and extract twice; the extract passes through a 400-mesh sieve, and the filtrate is concentrated under reduced pressure at a pressure of 125 mbar to obtain a crude Citrus medica extract;

[0055] Step S3: Measure 100 g of the activated XAD-16N type macroporous resin and load it into a column; load the crude Citrus medica extract onto the column, perform static adsorption for 15 h, with a sample loading speed of 1 BV / h. After loading, use 200 mL of water to elute and remove impurities from the resin column at a speed of 3 BV / h, and after the water washing is completed, use 100 mL of 35% ethanol to elute and remove impurities from the resin column at a speed of 3 BV / h, and then use 450 mL of 65% ethanol to elute at a flow rate of 2 BV / h, collect the eluate, concentrate it under reduced pressure, and freeze-dry it into a powder to obtain the Citrus medica extract.

[0056] Example 2

[0057] This example provides a preparation method of Citrus medica extract, and the preparation method includes the following steps:

[0058] Step S1: Take 50 g of the raw material of dried and pulverized Citrus medica that has passed through a 50-mesh sieve, put it into a water distillation device, with a distillation temperature of 95 °C, a distillation time of 0.5 h, and a material-liquid ratio of 1:9, to obtain a Citrus medica product (Citrus medica medicinal material) from which volatile components are removed;

[0059] Step S2: Use an ethanol - water solvent with a concentration of 45% to heat - reflux extract the citron product obtained in Step S1 to obtain an extract. The material - liquid ratio is 1:10, the extraction time is 1 h, and the extraction is carried out 3 times. The extract is filtered through a 600 - mesh sieve, and the filtrate is concentrated under reduced pressure at a pressure of 100 mbar to obtain a crude citron extract.

[0060] Step S3: Measure 100 g of activated HPD - 300 macroporous resin and pack it into a column. Load the crude citron extract onto the column and perform static adsorption for 15 h with a sample - loading speed of 1 BV / h. After loading, wash the resin column with 100 mL of water at a speed of 3 BV / h to remove impurities. After the water washing is completed, wash the resin column with 50 mL of 35% ethanol at a speed of 3 BV / h to remove impurities. Then, elute with 350 mL of 65% ethanol at a flow rate of 2 BV / h, collect the eluate, concentrate it under reduced pressure, and freeze - dry it into a powder to obtain the citron extract.

[0061] Example 3

[0062] This example provides a preparation method of a citron extract, and this preparation method includes the following steps:

[0063] Step S1: Take 50 g of the raw material of dried and pulverized citron that has passed through a 100 - mesh sieve, put it into a water - distillation device, the distillation temperature is 105 °C, the distillation time is 1 h, and the material - liquid ratio is 1:5 to obtain a citron product (citron medicinal material) after removing volatile components.

[0064] Step S2: Use an ethanol - water solvent with a concentration of 65% to heat - reflux extract the citron product obtained in Step S1 to obtain an extract. The material - liquid ratio is 1:15, the extraction time is 2 h, and the extraction is carried out 1 time. The extract is filtered through a 200 - mesh sieve, and the filtrate is concentrated under reduced pressure at a pressure of 150 mbar to obtain a crude citron extract.

[0065] Step S3: Measure 100 g of activated XAD - 16N macroporous resin and pack it into a column. Load the crude citron extract onto the column and perform static adsorption for 15 h with a sample - loading speed of 1 BV / h. After loading, wash the resin column with 250 mL of water at a speed of 3 BV / h to remove impurities. After the water washing is completed, wash the resin column with 150 mL of 35% ethanol at a speed of 3 BV / h to remove impurities. Then, elute with 500 mL of 65% ethanol at a flow rate of 2 BV / h, collect the eluate, concentrate it under reduced pressure, and freeze - dry it into a powder to obtain the citron extract.

[0066] Example 4

[0067] This example provides a preparation method of a citron extract, and the difference between this preparation method and that of Example 1 is only that the distillation time in Step S1 is changed to 0.2 h.

[0068] Example 5

[0069] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the distillation time in step S1 is changed to 1.5 h.

[0070] Example 6

[0071] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the material-liquid ratio in step S2 is changed to 1:5.

[0072] Example 7

[0073] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the material-liquid ratio in step S2 is changed to 1:20.

[0074] Example 8

[0075] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the ethanol concentration ratio in step S2 is changed to 85%.

[0076] Example 9

[0077] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the ethanol concentration ratio in step S2 is changed to 40%.

[0078] Example 10

[0079] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the concentration of ethanol for impurity removal in step S3 is changed from 35% to 20%.

[0080] Example 11

[0081] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that the concentration of ethanol for elution and collection in step S3 is changed from 65% to 45%.

[0082] Comparative Example 1

[0083] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that step S1 is cancelled and steps S2 and S3 are directly carried out.

[0084] Comparative Example 2

[0085] This example provides a preparation method of citron extract. The difference between this preparation method and that of Example 1 is only that step S3 is cancelled and the crude citron extract obtained in step S2 is directly freeze-dried into powder to obtain the citron extract.

[0086] Comparative Example 3

[0087] This comparative example provides a method for preparing citron extract, and the preparation method is ultrasonic extraction method, which specifically includes the following steps:

[0088] (1) Take 50 g of dried and pulverized citron raw materials passed through an 80-mesh sieve, according to the solid-liquid ratio of 1:10, use 50% ethanol aqueous solution for ultrasonic extraction, the power of ultrasonic is 600 W, the ultrasonic time is 40 minutes, the extraction solution is passed through a 200-mesh sieve, and the filtrate is concentrated under reduced pressure at a pressure of 150 mbar to obtain the crude citron extract.

[0089] (2) Measure 100 g of activated XAD-16N macroporous resin and pack it into a column; load the crude citron extract onto the column, perform static adsorption for 15 h, the sample loading speed is 1 BV / h, after loading, use 200 mL of water to elute and remove impurities from the resin column at a speed of 3 BV / h, after the water washing is completed, use 100 mL of 35% ethanol to elute and remove impurities from the resin column at a speed of 3 BV / h, and then use 450 mL of 65% ethanol to elute at a flow rate of 2 BV / h, collect the eluate, concentrate under reduced pressure, and freeze-dry into powder to obtain the citron extract.

[0090] Test Example 1

[0091] This test example conducts sensitization tests on the citron extracts prepared in Examples 1 to 11 and Comparative Examples 1 to 3.

[0092] Test method: Using human acute monocytic leukemia cell line THF-1, the sensitization of the obtained citron extract is tested through H-CLAT cell experiment. Among them, CD86 refers to CD86 protein, which belongs to the immunoglobulin superfamily and is a molecule expressed on antigen-presenting cells; CD54 refers to intercellular adhesion molecule 1, "-" indicates negative expression of CD86 / CD54, when the expression levels of surface markers CD86 and CD54 of the citron extract do not exceed the threshold at a concentration of 0.1 mg / mL, the H-CLAT test is judged as negative, otherwise it is judged as positive; "negative" means there is no sensitization risk, "positive" means there is a sensitization risk, and the results are shown in Table 1.

[0093] Table 1. Results of sensitization test

[0094]

[0095]

[0096] According to the data in Table 1, the test results of Examples 1-3, 5-11 and Comparative Example 2 of the present invention are all negative, and the test results of Comparative Examples 1 and 3 are all positive, indicating that the steam distillation method is significantly effective in removing volatile components that are likely to cause skin allergies; the positive result of Example 4 indicates that if the steam distillation time is not higher than 0.2 h, the removal of volatile components will be incomplete, resulting in a risk of sensitization.

[0097] The citron extract prepared by the preparation method provided in Examples 1-3 of the present invention is safe and non-irritating, has no risk of sensitization, and can be safely applied to the field of skin care products. And it fully complies with the various indicators in the "Technical Specifications for Cosmetics Safety (2015 Edition)" promulgated by the state, is non-irritating to the skin, and is safe and reliable.

[0098] Test Example 2

[0099] The yields, total flavonoid contents, total polysaccharide contents and naringin contents of the citron extracts prepared in Examples 1-3 and Comparative Examples 1-3 were detected.

[0100] The calculation formula for the yield is:

[0101] Yield (%) = (M1 / M2) × 100%, where M1 is the weight of the citron extract and M2 is the feeding amount of the citron extract. The test results are shown in Table 3.

[0102] Detection method for total flavonoid content: The sodium nitrite-aluminum nitrate method was used. Specifically:

[0103] Take rutin reference substance and prepare a standard solution of 0.5 mg / mL. Dilute it by the half-dilution method to obtain rutin standard solutions with different concentrations; prepare the citron extracts prepared in Examples 1-3 and Comparative Examples 1-3 into solutions of 0.5 mg / mL as the samples to be tested. Take 1.0 mL of the standard solution and the sample to be tested into a 10 mL volumetric flask, add 0.4 mL of 5% sodium nitrite solution to each, mix well, let stand for 6 min, then add 0.4 mL of 1% aluminum nitrate solution to each, mix well, let stand for 6 min, then add 2.0 mL of 4% sodium hydroxide solution to each, mix well, make up the volume, let stand at room temperature for 15 min, and measure the absorbance at a wavelength of 500 nm.

[0104] According to the absorbance values and concentrations, a standard curve was drawn, and the standard curve y = 0.3993x + 0.0120, R 2 = 0.99; substitute the absorbance values of the citron extracts prepared in Examples 1-3 and Comparative Examples 1-3 into the standard curve to calculate the total flavonoid content in the extracts. The test results are shown in Table 3.

[0105] (2) Determination method for total polysaccharide content: The sulfuric acid-phenol method was used for determination, specifically as follows:

[0106] ① Preparation of standard solution: Prepare a 0.1 mg / mL glucose standard solution. Sequentially pipette 0, 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL, 1.6 mL, and 2 mL into test tubes, and add ddH 2 O to each test tube to make up 2 mL for later use.

[0107] ② Preparation of sample solution: Accurately weigh the sample powder, add ddH 2 O to prepare a 0.1 mg / mL sample solution for testing. Preparation of 5% phenol solution: Accurately weigh 2.5 g of solid phenol powder, add 47.5 mL of ddH 2 O and dissolve it at 75 °C, and store it in the dark.

[0108] ③ Reaction of standard solution and sample solution: Sequentially pipette 1 mL of the prepared standard solution and sample solution into test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution for reaction, gently shake, use ddH 2 O as the blank control, and measure the absorbance at 490 nm using a microplate reader. Plot a standard curve with concentration as the abscissa and absorbance as the ordinate. Substitute the absorbance of the sample solution into the standard curve for calculation. The results of the total polysaccharide content determination are shown in Table 3.

[0109] The detection of naringin content uses ultra-high performance liquid chromatography, as follows:

[0110] The naringin standard product was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. An Agilent 1290 Infinity II ultra-high performance liquid chromatograph was used, and the chromatographic column was an Agilent ZORBA×SB-AQ column (3.0×100, 3.5 μm). 0.1% formic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The flow rate was 0.4 mL / min, the detection wavelength was 319 nm, the column temperature was 25 °C, and the elution program was as shown in Table 2. The standard naringin was detected according to the serial dilution method at 5 concentrations (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL) to obtain the naringin standard curve: y = 7169.8x + 203.65, R 2 = 0.99; The HPLC results of the product prepared in Example 1 and the naringin standard product are as Figure 1 shown. Similarly, the products prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to HPLC detection. The obtained results were substituted into the naringin standard curve, and the obtained results were divided by the injection concentration. The results are shown in Table 3.

[0111] Table 2. Elution program

[0112]

[0113] Table 3. Yield, total flavonoids, total polysaccharides and naringin content

[0114] Yield Total flavonoids (%) Total polysaccharides (%) Naringin (%) Example 1 18.73 46.75 2.31 29.91 Example 2 15.62 42.77 1.25 27.41 Example 3 16.32 45.11 1.26 28.62 Comparative Example 1 17.17 30.22 1.05 18.72 Comparative Example 2 25.88 15.29 1.11 8.15 Comparative Example 3 6.33 9.99 0.53 6.28

[0115] It can be seen from the data results in Table 3 that the citron extracts prepared by the preparation methods involved in Examples 1 to 3 of the present invention have high yields and high contents of the active ingredients total flavonoids, total polysaccharides and naringin; Comparative Example 1 has a high yield because it removes step S1 and does not remove the easily sensitizing volatile components, but there is a risk of sensitization; Comparative Example 2 has a high crude extraction yield because it removes the separation and purification in step S3, but the contents of its active ingredients total flavonoids, total polysaccharides and naringin are low. Comparative Example 3 uses conventional ultrasonic extraction, and its yield and the contents of the active ingredients total flavonoids, total polysaccharides and naringin are all low, and there is a risk of sensitization.

[0116] Test Example 3

[0117] The moisturizing, anti-aging and anti-allergic activities of the citron extracts prepared in Examples 1 to 11 and Comparative Examples 1 to 3 were detected.

[0118] Test samples: The citron extracts prepared in Examples 1 to 11 and Comparative Examples 1 to 3 groups were respectively configured with DMSO-H 2 O solution into a solution with a concentration of 100 μg / mL for testing.

[0119] Test method:

[0120] (1) Detection of hyaluronidase activity: Determined according to the Elson Morgan method. Sodium hyaluronate was used as the substrate, and hyaluronidase hydrolyzed hyaluronic acid to generate β-N-acetylglucosamine. This substance condensed with p-dimethylaminobenzaldehyde under alkaline conditions to form a chromogen 2-methyl-3-diacetylpyrrole derivative (purple), and the inhibitory activity of the citron extract on hyaluronidase was detected at 585 nm.

[0121] (2) Determination of collagenase activity: N-[3-(2-furyl)acryloyl]-L-leucine-glycine-L-proline-L-alanine was used as the substrate, and type I collagenase derived from Clostridium histolyticum was selected as the reaction enzyme. The inhibitory activity of the citron extract on collagenase was detected at 330 nm. The results are shown in Table 4.

[0122] Table 4. Results of moisturizing, anti-aging and anti-allergic activities

[0123] Hyaluronidase inhibition rate (%) Collagenase inhibition rate (%) Example 1 51.74 67.99 Example 2 46.51 58.91 Example 3 44.32 60.82 Example 4 25.31 36.66 Example 5 17.01 21.58 Example 6 22.22 35.32 Example 7 31.89 40.83 Example 8 7.31 15.13 Example 9 3.74 12.44 Example 10 9.21 18.93 Example 11 20.03 26.32 Comparative Example 1 4.11 9.52 Comparative Example 2 0 3.64 Comparative Example 3 0 0

[0124] In vitro inhibition tests of hyaluronidase and collagenase can be used as rapid screening indicators for anti-aging and anti-allergic efficacy raw materials. It can be seen from the data in Table 4 that:

[0125] (1) It can be seen from Examples 1 to 3 that the preparation method of the citron extract provided by the present invention has a very significant inhibitory effect on hyaluronidase and collagenase, proving that it has excellent anti-aging and anti-allergy effects.

[0126] (2) By comparing Examples 1 to 3 with Examples 4 to 5, it can be known that when the steam distillation time is outside the preferred range, the anti-aging and anti-allergy effects of the prepared citron extract are inferior to those of Examples 1 to 3.

[0127] By comparing Examples 1 to 3 with Examples 6 to 9, it can be known that when the material-liquid ratio and ethanol concentration of ethanol-water heating reflux extraction are outside the preferred range (the mass ratio of citron product to ethanol-water is 1:(10 - 15); the ethanol concentration is 45% - 65%), the anti-aging and anti-allergy effects of the prepared citron extract are inferior to those of Examples 1 - 3. Therefore, it can be known that the heating reflux extraction technology with a specific material-liquid ratio and ethanol concentration range can further improve the extraction effect and the biological activity of the extract product.

[0128] (4) By comparing Examples 1 to 3 with Examples 10 to 11, it can be known that when the impurity removal and elution collection with a specific concentration of ethanol solution are not used in the macroporous resin separation and purification, the anti-aging and anti-allergy effects of the prepared citron extract are inferior to those of Examples 1 to 3. Therefore, it can be known that the impurity removal and elution collection with a specific concentration of ethanol solution can further improve the purification effect and the biological activity of the extract product.

[0129] (5) By comparing Example 1 with Comparative Example 1, it can be known that the steam distillation method adopted by the present invention can remove the easily sensitizing volatile components and can more effectively remove the impurities in the product, obtaining a citron extract product with better anti-aging and anti-allergy activities.

[0130] (6) By comparing Example 1 with Comparative Example 2, it can be known that when the macroporous resin is not used for separating and purifying the citron extract, the prepared citron extract has no inhibitory effect on hyaluronidase, and the inhibition rate on collagenase is also very low, which is 3.64%. It can be seen that the present invention further purifies the active components of the citron extract by the method of XAD-16N macroporous resin adsorption and elution.

[0131] (7) By comparing Example 1 with Comparative Example 3, it can be known that the anti-aging and anti-allergy effects of the citron extract obtained by the conventional ultrasonic extraction method are inferior to those of Example 1 of the present invention.

[0132] Test Example 4

[0133] The volatile components of the citron extracts prepared in Example 1 and Comparative Example 3 were detected. The test method was:

[0134] The method for detecting volatile components by GC-MS (gas chromatography-mass spectrometry) is an efficient and sensitive analytical technique, applicable to the determination of volatile components in various samples. The citron extracts of Example 1 and Comparative Example 3 were placed in a dynamic headspace device, and the program is shown in Table 5. The results are as Figure 2 shown.

[0135] Table 5. GC-MS program

[0136]

[0137] Figure 2 The results showed that the volatile components in Comparative Example 3 were significantly more than those in Example 1, and contained limonene, an easily sensitizing component in citron essential oil. While Example 1 basically did not contain volatile components and did not contain limonene. Therefore, by adopting the above specific three-stage extraction and purification procedure, the volatile components in the citron extract can be greatly reduced, and the sensitization risk can be lowered.

[0138] Test Example 5

[0139] The anti-inflammatory and anti-allergic activities of the citron extract prepared in Example 1 were detected.

[0140] Test method:

[0141] (1) TRPV1: Using a keratinocyte soothing effect test model, the soothing effect of the citron extract prepared in Example 1 on TRPV1 was evaluated by detecting the protein content of TRPV1 by immunofluorescence (IF). A blank control group, a model group, a positive control group, and a sample group were set up. The blank control group was a normal group without any treatment, and the model group was an inflammatory state group with abnormal elevation of TRPV1 expression after stimulation with 15 μM CAP. Using 15.6 μg / mL of trans-4-tert-butylcyclohexanol as the positive control and keratinocytes as the detection model, the TRPV1 inhibition results are as Figure 3 shown. Figure 3 Among them, BC represents the blank control group, and NC represents the model group.

[0142] From Figure 3It can be seen that after CAP stimulation, compared with the BC group (blank control group), the TRPV1 expression in the NC group (model group) increased and its fluorescence signal enhanced; after adding the positive drug trans-4-tert-butylcyclohexanol, the green fluorescence signal in the PC group (positive control group) weakened compared with the NC group, the TRPV1 expression was normal and close to that of the BC group; the green fluorescence signal in the sample group (Example 1) also weakened compared with the NC group, the TRPV1 expression was normal and close to that of the BC group; indicating that the citron extract prepared in Example 1 has an inhibitory effect on TRPV1. Using the image analysis software (ImageJ) to quantify the fluorescence intensity, the experimental results show that when the concentration of the citron extract is 37.5 μg / mL, the inhibition rate of TRPV1 is 26.42%, having a good inhibitory effect on TRPV1. The citron extract prepared in Example 1 has a certain degree of soothing and anti-inflammatory efficacy, and can be added as a natural ingredient to related products such as cosmetics to relieve allergic symptoms such as redness and stinging caused by inflammation.

[0143] (2) PGE2: Using the keratinocyte soothing effect test model, the inhibitory effect of the citron extract prepared in Example 1 on the inflammatory factor PGE2 was evaluated by enzyme-linked immunosorbent assay (ELISA) as the detection method, and the blank control group, model group, positive control group and sample group were set up. The blank control group was the normal group without any treatment, and the model group was established with the abnormal increase of the inflammatory factor PGE2 after stimulating keratinocytes with 300 mJ / cm 2 of UVB. Using 0.01% concentration of dexamethasone as the positive control, the PGE2 inhibition results are as Figure 4 shown. Figure 4 Among them, BC represents the blank control group and NC represents the model group.

[0144] From Figure 4 it can be seen that after UVB stimulation, the concentration of the inflammatory factor PGE2 in the NC group (model group) was significantly higher than that in the BC group (blank control group), and the PC group (positive control group) and the sample group (Example 1) could significantly reduce the concentration of the inflammatory factor PGE2. When the concentration of the citron extract was 37.5 μg / mL, the inhibition rate of PGE2 was 18.32%, having a good inhibitory effect on PGE2. The citron extract prepared in Example 1 has a certain degree of soothing and anti-inflammatory efficacy, and can be added as a natural ingredient to related products such as cosmetics to relieve inflammation.

[0145] Test Example 6

[0146] The emotional skin care efficacy of the citron extract prepared in Example 1 was detected. As is well known, the skin is a highly innervated organ. The skin neural network is distributed from the dermis to the outermost superficial layer of the epidermis except the stratum corneum, and is closely related to our sensations such as touch, pain, itch, temperature, pressure, etc. There are multiple interactions between the skin nervous system and skin cells, and some scientific evidence has been obtained for the information transmission between nerve cells and skin cells. When skin cells produce positive substances such as endorphins, the skin is in a comfortable state, the emotional pleasure is enhanced, promoting the skin to be moist, delicate, fair, and shiny, and keeping the skin healthy, young, and beautiful. When skin cells produce negative substances such as inflammatory factors, the skin shows discomfort, the bad mood is aggravated, and the skin appears problems such as dryness, roughness, sensitivity, dullness, and fine line proliferation. Based on the research of skin neurophysiology, concepts such as "Neuro-cosmetics", "emotional beauty", and "emotional skin care" have been proposed in the beauty field to describe the beauty methods that regulate, cure skin beauty injuries or disorders related to nerves and emotions through the related effects between the nervous system and the skin.

[0147] β-endorphin is generated by the post-translational modification of the precursor protein POMC by the hormone precursor converting enzyme PCs, is expressed in keratinocytes cultured in vitro, and has the functions of regulating immunity, promoting cell growth and migration, and promoting wound healing. β-endorphin is produced in certain neurons in the central and peripheral nervous systems, but is also produced in the skin, especially in response to ultraviolet radiation. β-endorphin exerts local and global functions. The overall functions of this neuropeptide include pain management, reward effect, behavioral stability, stress response, immune regulation, and reduction of physical stress.

[0148] HSD is the abbreviation of Hydroxysteroid Dehydrogenase, which is responsible for the conversion between cortisol and the inactive precursor (corticosterone) and regulates the local glucocorticoid concentration. HSD1 catalyzes the conversion of glucocorticoids to the active form; HSD2 catalyzes the inactivation of glucocorticoids to prevent them from binding to the mineralocorticoid receptor (MR), thus ensuring the high-affinity relationship between MR and aldosterone. The balance between HSD1 and HSD2 in the skin is crucial for maintaining skin homeostasis. When the activities of HSD1 and HSD2 are balanced, the skin can normally respond to external stimuli and stress and maintain a healthy state.

[0149] Test method:

[0150] The emotional skin care activities of the citron extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were detected.

[0151] Test samples: The citron extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 3 groups were respectively mixed with DMSO-H2 The O solution was prepared into a solution with a concentration of 60 μg / mL for testing.

[0152] (1) Detection of β-endorphin promoting effect: NHEK cells were incubated with extracts of Citrus medica L. var. sarcodactylis (Noot.) Swingle at different concentrations for 24 hours, and the content of β-endorphin in the cell culture medium was detected by the Elisa method to evaluate whether it has the effect of emotional skin care. If the content of β-endorphin increases, it indicates that the test substance may have the effect of emotional skin care. The results are as Figure 2 shown.

[0153] (2) Gene expression of HSD1 and HSD2: NHEK cells were incubated with extracts of Citrus medica L. var. sarcodactylis (Noot.) Swingle at different concentrations for 24 hours, and the gene expression of HSD1 and HSD2 was detected by qPCR method to evaluate whether it has the effect of emotional skin care. If the expression of HSD1 decreases and the expression of HSD2 increases, it indicates that the test substance may have the effect of emotional skin care. The results are shown in Table 6.

[0154] Table 6. Results of emotional skin care activity detection

[0155] β - endorphin content increase (%) HSD1 decrease (%) HSD2 increase (%) Example 1 47.67 66.42 32.17 Example 2 35.17 45.31 14.81 Example 3 38.53 39.57 15.82 Comparative Example 1 2.53 0 1.19 Comparative Example 2 0 0 0 Comparative Example 3 0 0 0

[0156] The results of Examples 1 to 3 in Table 6 show that by adopting the above specific three-stage extraction and purification procedure, extracts of Citrus medica L. var. sarcodactylis (Noot.) Swingle with a significant increase in β-endorphin, a significant decrease in HSD1, and a significant increase in HSD2 can be obtained, indicating that it has a significant effect on emotional skin care; Comparative Example 1 shows that the extract obtained without removing volatile components by steam distillation does not have a significant effect on emotional skin care, Comparative Example 2 shows that the extract of Citrus medica L. var. sarcodactylis (Noot.) Swingle without macroporous resin purification also does not have the effect of emotional skin care, and Comparative Example 3 shows that the extract of Citrus medica L. var. sarcodactylis (Noot.) Swingle obtained by conventional ultrasonic extraction also does not have the effect of emotional skin care. Only the above specific three-stage extraction and purification procedure can obtain extracts of Citrus medica L. var. sarcodactylis (Noot.) Swingle with a significant effect on emotional skin care.

[0157] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing a citron extract, characterized in that: The steps include: Step S1, crushing the dried citron fruit and placing it in an above-water distillation device for distillation to obtain a citron product from which volatile components have been removed; Step S2, using an ethanol-water solvent to heat and reflux extract the citron product, and filtering and concentrating the extract to obtain a citron crude extract; Step S3, loading the crude citron extract onto a macroporous resin column, performing separation and purification using the macroporous resin, and concentrating and drying the eluate to obtain a citron extract.

2. The method for preparing the citron extract according to claim 1, characterized in that: In step S1, the dried citron fruit is crushed through a 50-100 mesh sieve and then placed in an above-water distillation device, the distillation temperature is 95° C. to 105° C., the distillation time is 0.5 h to 1 h, and the mass ratio of citron powder to distilled water is 1:(5-9).

3. The method for preparing the citron extract according to claim 1, characterized in that: In step S2, the mass ratio of the citron product to the ethanol-water solvent is 1:(10-15); the concentration of ethanol in the ethanol-water solvent is 45%-65%, and the extraction time is 1h-2h.

4. The method for preparing the citron extract according to claim 1, characterized in that: In step S3, the macroporous resin is any one of XAD-16N, HPD-300, HPD-100, LD-605 or Amberlite XAD-7HP.

5. The method for preparing the citron extract according to claim 4, characterized in that: The macroporous resin is XAD-16N type or HPD-300 type.

6. The method for preparing the citron extract according to claim 1, characterized in that: In step S3, the procedure for separation and purification using a macroporous resin is: first, eluting with water for 2-5 column volumes to remove impurities, then eluting with 35% ethanol for 1-3 column volumes to remove impurities, and then eluting with 65% ethanol for 7-10 column volumes to collect the eluate.

7. The method for preparing the citron extract according to claim 1, characterized in that: The drying method in step S3 includes any one of freeze drying, vacuum drying or spray drying, or a combination of at least two of them.

8. A citron extract, characterized in that The citron extract is prepared by the preparation method of the citron extract according to any one of claims 1 to 7, wherein the naringin content in the citron extract is not less than 20%, and the total flavonoid content is not less than 35%.

9. A use of a citron extract in the preparation of a health product with moisturizing, anti-inflammatory, soothing, emotional beauty and anti-aging effects, characterized in that: The citron extract is the citron extract according to claim 8.

10. The use according to claim 9, characterized in that The health product is a skin care product or a food.

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