Method for simultaneously extracting flavones and iridoid glycosides from eucommia ulmoides male flowers by using deep eutectic solvent and application

Abstract

This invention discloses a method and its application for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The method includes pretreatment of Eucommia ulmoides male flowers, preparation of the deep eutectic solvent, and extraction of flavonoids and iridoid glycosides from the flowers. The deep eutectic solvent provided by this invention is prepared from betaine and citric acid, and is characterized by being green, natural, safe, non-toxic, simple in preparation, and low in cost. It not only increases the extraction rate of bioactive compounds from Eucommia ulmoides male flowers, but also significantly enhances the antioxidant and anti-aging bioactivity of Eucommia ulmoides male flowers compared to organic solvents, thereby increasing the content of total flavonoids and iridoid glycosides in the extract. Therefore, it can be used as a novel solvent for the preparation of liquid formulations of Eucommia ulmoides male flower extract, enabling liquid addition.

Description

Technical Field

[0001] This invention belongs to the field of material extraction technology, specifically relating to a method and application for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. Background Technology

[0002] Eucommia ulmoides, a dioecious tree species, is a precious medicinal plant endemic to China. Its stamens, known as Eucommia ulmoides male flowers, were approved as a new food ingredient by the National Health and Family Planning Commission in 2014. Various products using Eucommia ulmoides male flowers and their extracts, including tea, health supplements, and functional beverages, are widely popular with consumers and have attracted significant attention from the pharmaceutical and cosmetic industries. Eucommia ulmoides male flowers not only possess rich nutritional value but also exhibit significant health benefits such as lowering blood pressure, blood lipids, and blood sugar. Furthermore, they possess anti-fatigue and anti-aging properties. Eucommia ulmoides male flowers contain various bioactive components, including iridoids (such as genipin (GP) and aucubin (AU)), lignans (such as pinoresinol diglucoside), and flavonoids (such as isoquercitrin, rutin, and 3-arabinoglycoside glucosylquercetin). However, most extraction methods for Eucommia ulmoides male flowers still use traditional organic solvents, which poses a certain risk factor in food and cosmetic processing. While water can replace organic solvents, its extraction efficiency is too low. Therefore, an efficient and green extraction method should be developed to extract the target compound from Eucommia ulmoides male flowers.

[0003] Flavonoids and iridoid glycosides in the male flowers of Eucommia ulmoides possess excellent antioxidant and anti-photoaging bioactivities [Liu Cong, Guo Feifei, Xiao Junping, et al. Research progress on chemical constituents and pharmacological effects of different parts of Eucommia ulmoides [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45(03):497-512.]. However, due to the different polarities of flavonoids and iridoid glycosides, the optimal conditions for extracting flavonoids from the male flowers of Eucommia ulmoides are an ethanol concentration of 60% [Bai Xiting, Zhu Wenxue, Luo Lei, et al. Eucommia ulmoides]. Research on the extraction process of flavonoids from male flowers of tea[J]. Food Research and Development, 2008, (11): 96-100.], while the extraction conditions for iridoid glycosides are 95% ethanol[Ding Yanxia, ​​Wang Tengyu, Zhang Yaowen, et al. Study on triterpenoid chemical components in male flowers of Eucommia ulmoides[J]. Chinese Journal of Traditional Chinese Medicine, 2014, 39(21): 4225-4229.]. Therefore, it is difficult to extract them simultaneously with traditional organic solvents and obtain a stable liquid formulation. Therefore, it is necessary to develop a new green extraction solvent to achieve the extraction of multiple active ingredients and form a stable solvent. The purpose of this invention is to provide a natural deep eutectic solvent that can simultaneously extract flavonoids and iridoid glycosides, and form a stable liquid solvent with antioxidant and anti-aging activities superior to organic solvent extracts.

[0004] This study aims to explore the possibility of extracting total flavonoids and iridoid glycosides from male flowers of Eucommia ulmoides using deep eutectic solvents, and to determine whether deep eutectic solvents can increase the concentration of extracted flavonoids and iridoid glycosides and exhibit similar or stronger antioxidant and anti-aging activities compared to organic solvent extraction. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method and application for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent, the method comprising:

[0008] (1) Pretreatment of Eucommia ulmoides male flowers: Wash and blanch the Eucommia ulmoides male flowers and then dry them;

[0009] (2) Extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flowers: The pretreated Eucommia ulmoides male flowers were mixed with a solution containing a deep eutectic solvent and heated to obtain a crude extract. The crude extract was centrifuged and filtered to obtain the final product.

[0010] The extraction method described in this invention can simultaneously extract flavonoids and iridoid glycosides.

[0011] Preferably, the deep eutectic solvent is obtained by reacting a hydrogen bond acceptor and a hydrogen bond donor.

[0012] Preferably, the hydrogen bond acceptor is selected from any one or a combination of at least two of choline chloride, acetamide, or betaine, and the hydrogen bond donor is selected from any one or a combination of at least two of propylene glycol, glycerol, urea, glycolic acid, citric acid, or L-ascorbic acid.

[0013] The deep eutectic solvent provided by this invention can effectively improve the solubility of Eucommia ulmoides male flower extract. It is safe and non-toxic, with a simple preparation process and low cost. It does not affect the stability and bioactivity of active substances in Eucommia ulmoides male flower extract. It can be used as a green solvent to provide effective support for the extraction of Eucommia ulmoides male flower extract, thereby solving the problems of simultaneous extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flower, low content of effective components in the extract, organic solvent pollution, and low water solubility.

[0014] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-6), where the specific point values ​​in (1-6) can be selected from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0015] Preferably, the reaction is carried out at 60-80℃ for 30-90 min; the temperature can be selected from 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 76℃, 78℃, 80℃, etc., and the time can be selected from 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0016] Preferably, the solution containing a deep eutectic solvent in step (2) includes a deep eutectic solvent and water.

[0017] Preferably, the volume fraction of water is 0-120% of the deep eutectic solvent, such as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0018] When the volume fraction of water is 0% of the deep eutectic solvent, it is an anhydrous deep eutectic solvent.

[0019] Adding water to the deep eutectic solvent has little effect on the extraction of total flavonoids. The amount of water added can be controlled according to the actual situation to achieve the best solubility and viscosity and reduce costs.

[0020] Preferably, step (1) of blanching includes treating the male flowers of Eucommia ulmoides at 120-180℃ for 10-30s. The temperature can be selected from 120℃, 130℃, 140℃, 150℃, 160℃, 170℃, 180℃, etc., and the time can be selected from 10s, 12s, 15s, 18s, 20s, 22s, 25s, 28s, 30s, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0021] Preferably, the drying temperature is 50-80℃, such as 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] Preferably, step (1) further includes pulverizing the male flowers of Eucommia ulmoides after drying.

[0023] Preferably, the ratio of Eucommia ulmoides male flowers to the solution containing deep eutectic solvent in step (2) is 1g:(10-30)mL, where the specific point values ​​in (10-30) can be selected from 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0024] Preferably, the heating temperature in step (2) is 60-80℃ and the heating time is 30-120min. The temperature can be selected from 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 76℃, 78℃, 80℃, etc., and the time can be selected from 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0025] Preferably, the centrifugal force in step (2) is 3000-8000g, and the time is 15-45min. The centrifugal force can be selected from 3000g, 3500g, 4000g, 4500g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g, etc., and the time can be selected from 15min, 20min, 25min, 30min, 35min, 40min, 45min, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0026] Preferably, step (2) is carried out under stirring, with a stirring speed of 300-1000 rpm, such as 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0027] Preferably, the centrifugation in step (2) is carried out at 4-20℃, such as 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, etc. Other specific point values ​​within the above range can be selected, and will not be described in detail here.

[0028] In a second aspect, the present invention provides an extract of Eucommia ulmoides male flowers prepared by the method described in the first aspect, which utilizes a deep eutectic solvent to simultaneously extract flavonoids and iridoid glycosides from Eucommia ulmoides male flowers.

[0029] Thirdly, the present invention provides the use of the Eucommia ulmoides male flower extract according to the second aspect in the preparation of pharmaceuticals or cosmetics with antioxidant and / or anti-aging effects.

[0030] Fourthly, the present invention provides the use of the extract of Eucommia ulmoides male flowers according to the second aspect in the preparation of hyaluronidase inhibitors, collagenase inhibitors or elastase inhibitors.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Compared with existing technologies, this invention utilizes a deep eutectic solvent prepared from betaine and citric acid to effectively extract flavonoids and iridoid glycosides from Eucommia ulmoides male flowers. This is because betaine and citric acid molecules contain hydroxyl, carboxyl, and amino groups, which can form hydrogen bonds with each other. When the molar ratio is preferably 1:1, hydrogen bonds can form not only between these two molecules but also with the groups on the flavonoid and iridoid glycoside molecules in Eucommia ulmoides male flowers, thus forming a large and stable hydrogen bond network. This improves extraction efficiency and produces a deep eutectic extract of Eucommia ulmoides male flowers with more significant antioxidant and anti-aging activities, which can be used to prepare antioxidant and anti-aging health foods and cosmetics. Furthermore, since betaine and citric acid are derived from natural plants, the extraction solvent is non-toxic and pollution-free, making it a novel green and environmentally friendly solvent. Attached Figure Description

[0033] Figure 1 The main active compounds in the extract of Eucommia ulmoides male flowers extracted according to the methods described in Application Example 1 and Comparative Application Example 1 are: 1. genipin, 2. maleic acid, 3. guaiacol, 4. oxalic acid, 5. aucubin, 6. chlorogenic acid, 7. quercetin 3,4'-diglucoside, 8. genipin, 9. 3-[(4-O-glucopyranosyl-glucopyranosyl)oxy]-4,5-dihydroxybenzoic acid, 1 0. Luteolin, 11. Pinoresinol diglucoside, 12. Hesperidin B, 13. 3-Arabinose-glucosylquercetin, 14. Isorhamnetin 3,7-O-diglucoside, 15. Pinoresinol vanillyl ether glucoside, 16. Eugenol-β-eugenol ether monosaccharide, 17. Eugenol vanillyl ether diglucoside, 18. Quercetin, 19. Pinoresinol vanillyl ether glucoside, 20. Kaempferol.

[0034] Figure 2 The inhibition rate of hyaluronidase, collagenase, and elastase by the extract of Eucommia ulmoides male flowers extracted according to the method described in Application Example 1 and Comparative Application Example 1.

[0035] Figure 3 The figure shows the results of the inhibitory effect of Eucommia ulmoides male flower extract, extracted according to the method described in Application Example 1 and Comparative Application Example 1, on β-gal-induced senescence of human skin fibroblasts (HFF). Detailed Implementation

[0036] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0037] The polyethylene glycol was purchased from Guangzhou Hongyun Chemical Co., Ltd. under the trade name Dow.

[0038] The male flowers of Eucommia ulmoides were purchased from Yunnan Yousheng Traditional Chinese Medicine Technology Co., Ltd.

[0039] Example 1

[0040] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and citric acid in a molar ratio of 1:1 at 70°C for 1 hour using a magnetic stirrer.

[0041] Example 2

[0042] This embodiment provides a deep eutectic solvent, which is prepared by heating choline chloride and ethylene glycol in a molar ratio of 1:3 at 60°C for 1 hour using a magnetic stirrer.

[0043] Example 3

[0044] This embodiment provides a deep eutectic solvent, which is prepared by heating choline chloride and glycerol in a molar ratio of 1:6 at 80°C for 1 hour using a magnetic stirrer.

[0045] Example 4

[0046] This embodiment provides a deep eutectic solvent, which is prepared by heating choline chloride and L-ascorbic acid in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0047] Example 5

[0048] This embodiment provides a deep eutectic solvent, which is prepared by heating acetamide and propylene glycol in a molar ratio of 1:1 at 70°C for 1 hour using a magnetic stirrer.

[0049] Example 6

[0050] This embodiment provides a deep eutectic solvent, which is prepared by heating choline chloride and acetylpropionic acid in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0051] Example 7

[0052] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and polyethylene glycol in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0053] Example 8

[0054] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and glycolic acid in a molar ratio of 1:4 at 70°C for 1 hour using a magnetic stirrer.

[0055] Example 9

[0056] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and glycerol in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0057] Example 10

[0058] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and urea in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0059] Example 11

[0060] This embodiment provides a deep eutectic solvent, which is prepared by heating betaine and L-ascorbic acid in a molar ratio of 1:2 at 70°C for 1 hour using a magnetic stirrer.

[0061] Example 12

[0062] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 5:1.

[0063] The preparation method is as follows: mix the deep eutectic solvent and water to obtain the product.

[0064] Example 13

[0065] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 5:2.

[0066] The preparation method is the same as in Example 12.

[0067] Example 14

[0068] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 5:3.

[0069] The preparation method is the same as in Example 12.

[0070] Example 15

[0071] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 5:4.

[0072] The preparation method is the same as in Example 12.

[0073] Example 16

[0074] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 1:1.

[0075] The preparation method is the same as in Example 12.

[0076] Example 17

[0077] This embodiment provides a deep eutectic solution, which includes the deep eutectic solvent described in Example 1 and water, with a volume ratio of 5:6.

[0078] The preparation method is the same as in Example 12.

[0079] Comparative Example 1

[0080] This comparative example provides an extraction solvent, which is 60% ethanol.

[0081] Application Example 1

[0082] This application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The extraction method includes:

[0083] (1) Pretreatment of Eucommia ulmoides male flowers: The enzymes in Eucommia ulmoides male flowers were inactivated twice at 150℃ for 20s each time, and dried at 65℃ until the weight of the sample remained constant. Then, the sample was pulverized and passed through a 50-mesh sieve to obtain Eucommia ulmoides male flower powder, which was stored in a -20℃ refrigerator for later use.

[0084] (2) Extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flowers: 1g of Eucommia ulmoides male flower powder was mixed with 20mL of the deep eutectic solvent described in Example 1 and extracted for 75min in a constant temperature magnetic stirrer with a stirring speed of 700rpm and a heating temperature of 70℃. Finally, the mixture was centrifuged at 5000g for 30min and the Eucommia ulmoides male flower extract was obtained using a 0.45μm polytetrafluoroethylene microfilter.

[0085] Application Example 2

[0086] This application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The extraction method includes:

[0087] (1) Pretreatment of Eucommia ulmoides male flowers: The enzymes in Eucommia ulmoides male flowers were inactivated twice at 120℃ for 30s each time, and dried at 50℃ until the weight of the sample remained constant. Then, the sample was pulverized and passed through a 50-mesh sieve to obtain Eucommia ulmoides male flower powder, which was stored in a -20℃ refrigerator for later use.

[0088] (2) Extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flowers: 1g of Eucommia ulmoides male flower powder was mixed with 30mL of the deep eutectic solvent described in Example 1 and extracted for 2h in a constant temperature magnetic stirrer with a stirring speed of 300rpm and a heating temperature of 60℃. Finally, the mixture was centrifuged at 3000g for 45min and the Eucommia ulmoides male flower extract was obtained using a 0.45μm polytetrafluoroethylene microfilter.

[0089] Application Example 3

[0090] This application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The extraction method includes:

[0091] (1) Pretreatment of Eucommia ulmoides male flowers: The enzymes in Eucommia ulmoides male flowers were inactivated twice at 180℃ for 10s each time, and dried at 80℃ until the weight of the sample remained constant. Then, the sample was pulverized and passed through a 50-mesh sieve to obtain Eucommia ulmoides male flower powder, which was stored in a -20℃ refrigerator for later use.

[0092] (2) Extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flowers: 1g of Eucommia ulmoides male flower powder was mixed with 10mL of the deep eutectic solvent described in Example 1 and extracted for 30min in a constant temperature magnetic stirrer with a stirring speed of 1000rpm and a heating temperature of 80℃. Finally, the mixture was centrifuged at 8000g for 15min and the Eucommia ulmoides male flower extract was obtained using a 0.45μm polytetrafluoroethylene microfilter.

[0093] Application Example 4-10

[0094] This application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The only difference between this method and Application Example 1 is that the deep eutectic solvent described in Example 1 is replaced in equal amounts with the deep eutectic solvents described in Examples 3-5 and 8-11, while other operations remain unchanged.

[0095] Application Example 11-16

[0096] This application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The only difference between this method and Application Example 1 is that the deep eutectic solvent described in Example 1 is replaced in equal amounts with the deep eutectic solutions described in Examples 12-17, while other operations remain unchanged.

[0097] Comparative Application Example 1

[0098] This comparative application example provides a method for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent. The only difference between this method and Application Example 1 is that the deep eutectic solvent described in Example 1 is replaced in equal amounts with the extraction solvent described in Comparative Example 1, while all other operations remain unchanged.

[0099] Test Example 1

[0100] Preliminary screening of deep eutectic solvents

[0101] Table 1

[0102] sample State at 20℃ Example 1 viscous transparent liquid Example 2 Dark yellow transparent liquid Example 3 Transparent and highly liquid Example 4 Transparent and highly liquid Example 5 Transparent and highly liquid Example 6 light yellow transparent liquid Example 7 Precipitation Example 8 viscous transparent liquid Example 9 Transparent and highly liquid Example 10 viscous transparent liquid Example 11 Transparent and highly liquid

[0103] As shown in Table 1, the formation of deep eutectic solvents is affected by the properties and proportions of the components, as well as the state of the components themselves. Therefore, when preparing deep eutectic solvents, it is appropriate to select liquid single components or increase the proportion of liquid components to increase their fluidity, so as to better conduct dissolution experiments.

[0104] Test Example 2

[0105] Standard curves of total flavonoids and iridoid glycosides

[0106] Total flavonoids test method:

[0107] Accurately measure 1.0 mL of the test solution and the reference solution into 10 mL volumetric flasks, respectively. Accurately add 0.4 mL of 5% sodium nitrite solution to each flask, mix well, and let stand for 6 min. Then, accurately add 0.4 mL of 1% aluminum nitrate solution to each flask, mix well, and let stand for 6 min. Next, add 2.0 mL of 4% sodium hydroxide solution to each flask, mix well, and dilute to the mark with 95% ethanol. Mix well and let stand at room temperature for 15 min. After the reaction is complete, pipette 200 μL of each sample into a 96-well plate, using the reagent as a blank, and measure the absorbance at a wavelength of 500 ± 2 nm. Plot a standard curve based on the absorbance values ​​and the concentration of the rutin reference solution.

[0108] Methods for testing iridoid glycosides:

[0109] Place 1 mL of Eucommia ulmoides male flower test solution into a 10 mL volumetric flask, then add 2 mL of 1 mol / L hydrochloric acid solution, shake well, place in a 90℃ water bath, boil for 15 min, then let stand at room temperature to cool, then add 0.5 mL of dinitrophenylhydrazine ethanol solution, shake well, place in a 90℃ water bath for 25 min, then let stand at room temperature for 15 min to cool, finally add 3 mL of 1 mol / L NaOH 70% ethanol solution, shake well, let stand at room temperature for 1 h, using 70% ethanol as a blank control, and measure the absorbance at 462 nm.

[0110] The standard curves for total flavonoids and iridoid glycosides were obtained by linear regression with their concentration (x) on the x-axis and absorbance value (y) on the y-axis.

[0111] Table 2

[0112] Eucommia male flower extract Linear range Regression equation <![CDATA[R 2 ]]> Total flavonoids 0.03125-1 mg / mL y = 0.9311x + 0.0095 0.9996 Total iridoid glycosides 9.2-36.8 μg / mL y = 0.0661x - 0.1305 0.9963

[0113] According to Table 2, R 2All values ​​were above 0.99, indicating a good linear relationship within this concentration range, which can be used for subsequent quantitative analysis.

[0114] Test Example 3

[0115] The total flavonoid content in the extracts of Eucommia ulmoides male flowers extracted according to the methods described in Application Examples 1-16 and Comparative Application Example 1 was determined using the sodium nitrite-aluminum nitrate method, with the same testing method as in Test Example 2. After acid hydrolysis, the total iridoid content was determined by colorimetric analysis using dinitrophenylhydrazine ethanol solution and NaOH-70% ethanol solution, with the same testing method as in Test Example 2.

[0116] Table 3

[0117] serial number Total flavonoid content (mg / g) Total iridoid content (mg / g) Application Example 1 31.89 24.88 Application Example 2 30.23 22.12 Application Example 3 30.11 17.09 Application Example 4 24.24 15.23 Application Example 5 —(Vc affects the results of total flavonoid determination) 6.48 Application Example 6 26.99 14.84 Application Example 7 25.38 14.07 Application Example 8 26.87 10.11 Application Example 9 34.07 19.63 Application Example 10 —(Vc affects the results of total flavonoid determination) 17.25 Application Example 11 30.93 21.16 Application Example 12 27.39 21.88 Application Example 13 27.25 17.25 Application Example 14 29.79 13.23 Application Example 15 31.16 11.16 Application Example 16 31.83 11.88 Comparative Application Example 1 27.50 4.27

[0118] The extract of Eucommia ulmoides male flowers mainly includes flavonoids, iridoid glycosides, chlorogenic acid, amino acids, and other nutrients and functional elements. Among them, flavonoids and iridoid glycosides are the main chemical components that give Eucommia ulmoides male flowers their unique pharmacological activities. Therefore, the results of deep eutectic solvent extraction and traditional organic solvent extraction can be compared to evaluate the efficiency of the deep eutectic solvent extraction method and to use it for the quality control of Eucommia ulmoides male flowers, their extracts, and herbal medicine preparations.

[0119] The determination of total flavonoids and total iridoid glycosides in the extract of Eucommia ulmoides male flowers showed that the deep eutectic solvent obtained by the reaction of betaine and citric acid had a good extraction effect. Furthermore, both betaine and citric acid are natural food additives with a certain degree of safety, and can be further prepared into liquid dosage forms. The water content of the deep eutectic solvent described in Example 1 was optimized. Adding water to the deep eutectic solvent had little effect on the extraction of total flavonoids, but it did have a certain impact on the amount of total iridoid glycosides. To achieve the desired effect in practical applications, the amount of water added should be controlled according to cost and solvent viscosity to achieve optimal solubility and viscosity.

[0120] Test Example 4

[0121] Compound analysis of Eucommia ulmoides male flower extract

[0122] HPLC-MS / MS was performed using an Agilent 6545 system. The mobile phase consisted of 0.1% formic acid solution (A) and acetonitrile (B). Gradient elution times were: 0–5 min, 5% B; 5–25 min, 5%–30% B; 25–45 min, 30%–90% B; 45–50 min, 90% B; 50–52 min, 90%–5% B; 52–57 min, 5% B. The column temperature was maintained at 30 °C, and the injection volume was 2 μL. All MS data were acquired using Analyst 1.6.2 software to ensure accuracy and repeatability.

[0123] The established HPLC-QTOF-MS / MS method was used for the comprehensive quality evaluation of the Eucommia ulmoides male flower extract obtained according to the method described in Comparative Application Example 1 and the extract obtained according to the method described in Application Example 1. Twenty compounds from the different extracts were identified, including pinoresinol diglucoside, aucubin, genipin, genipin glycoside, oxalic acid, chlorogenic acid, quercetin, and kaempferol, which are abundant in Eucommia ulmoides male flowers and have clearly defined pharmacological activities. Figure 1 The peak area of ​​the compounds indicates that the extract of Eucommia ulmoides male flowers extracted according to the method described in Application Example 1 can significantly increase the content of geniposide, arbutin, and aucubin, and also increase the amount of compounds such as pinoresinol diglucoside, chlorogenic acid, quercetin, and kaempferol.

[0124] Test Example 5

[0125] Evaluation of the DPPH free radical scavenging activity of Eucommia ulmoides male flower extract

[0126] Test method: The 96-well plate was divided into a sample group, a control group, and a control group, with two replicates in each group; blank controls were set up in both the sample group and the control group, and DPPH solution was added at a 1:1 volume ratio; Vitamin C solution was used as a positive control in the sample group; the amount added to each well and the distribution in the 96-well plate are as follows (note that the operation should be carried out in the dark; after loading the plate, the reaction should be carried out at room temperature in the dark for 30 min, shaken to mix, and the absorbance was measured at 517 nm):

[0127] Sample (sample set): 150 μL sample solution + 150 μL DPPH alcohol solution

[0128] Blank group: 150 μL sample solution + 150 μL anhydrous ethanol

[0129] Control group: 150 μL DPPH solution + 150 μL sample solvent

[0130] The DPPH scavenging rates of the Eucommia ulmoides male flower extracts extracted according to the methods described in Application Examples 1-16 and Comparative Application Example 1 were determined, and curves were plotted with vitamin C as a positive control. The IC50 values ​​of the two extracts were calculated. 50 The results were compared and analyzed. The results are shown in Table 4.

[0131] Table 4

[0132]

[0133]

[0134] As shown in Table 4, the extract obtained using the extraction method described in this invention has a strong DPPH free radical scavenging rate and strong antioxidant capacity.

[0135] Test Example 6

[0136] Hyaluronidase, collagenase, and elastase inhibition rate determination

[0137] Hyaluronidase activity assay: Hyaluronidase activity was determined using sodium hyaluronate as a substrate and different extracts according to the Elson-Morgan method.

[0138] Collagenase activity assay: This method uses N-[3-(2-furanyl)acryloyl]-L-leucine-glycine-L-proline-L-alanine (also known as FALGPA) as the substrate and selects type I collagenase derived from Bacillus histolyticus as the reaction enzyme. The inhibitory effect of Eucommia ulmoides male flower extract on this enzyme in different solvents is investigated to evaluate the protective effect of the extract on human skin.

[0139] Elastase inhibition assay: Porcine pancreatic elastase stock solution (0.015625 mg / mL) was prepared in sterile water. 0.1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline buffer was used as the substrate. 70 μL of 0.015625 mg / mL enzyme solution, 10 μL of different concentrations of sample and positive control (sodium cevelex) solution were added to each well. After incubation at 37°C for 10 min, 100 μL of 100 μmol / L substrate was added, for a total reaction volume of 230 μL. A 100% enzyme activity control was included, and a blank control of 130 μL was replaced with an equal volume of PBS. The reaction was incubated at 37°C for 15 min, and the absorbance at 405 nm was measured.

[0140] As Figure 3 As shown, the in vitro inhibitory activities of different extracts on hyaluronidase, collagenase, and elastase were evaluated. The results showed that the different extracts exhibited the strongest inhibitory effect on hyaluronidase, followed by collagenase, and the weakest inhibitory effect on elastase. The Eucommia ulmoides male flower extract prepared according to the method described in Application Example 1 generally showed stronger inhibitory activity against the three enzymes than the Eucommia ulmoides male flower extract prepared according to the method described in Comparative Application Example 1, indicating that the Eucommia ulmoides male flower extract prepared according to the method described in Application Example 1 has stronger activity.

[0141] Test Example 7

[0142] HFF cell anti-aging activity test of Eucommia ulmoides male flower extract

[0143] β-galactosidase is the most widely used biomarker for highlighting the cellular senescence process. HFF cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and incubated overnight at 37°C with a mixture of SA-β-GAL staining solution. Senescent cells (staining blue with SA-β-GAL) were observed and counted under a Leica microscope, and the percentage of SA-β-GAL positive cells was determined. The results are shown below. Figure 3 As shown.

[0144] Through the establishment of an in vitro cell anti-aging model, it was observed that 60 μg / mL Eucommia ulmoides male flower extract could effectively inhibit the expression of SA-β-gal, and the inhibitory effect of Eucommia ulmoides male flower extract prepared according to the method described in Application Example 1 was more obvious (SA-β-gal stained positive cells are blue).

[0145] The applicant declares that the present invention illustrates a method and application for simultaneously extracting flavonoids and iridoid glycosides from Eucommia ulmoides male flowers using a deep eutectic solvent through the above embodiments. However, the present invention is not limited to the above embodiments, i.e., it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0146] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0147] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of Eucommia ulmoides male flower extract in the preparation of hyaluronidase inhibitors, collagenase inhibitors, or elastase inhibitors, characterized in that, The Eucommia ulmoides male flower extract was prepared by a method comprising the following steps: (1) Pretreatment of Eucommia ulmoides male flowers: After washing and blanching the Eucommia ulmoides male flowers, they are dried. The blanching includes treating the Eucommia ulmoides male flowers at 120-180℃ for 10-30 seconds. (2) Extraction of flavonoids and iridoid glycosides from Eucommia ulmoides male flowers: The pretreated Eucommia ulmoides male flowers were mixed with a solution containing a deep eutectic solvent at a ratio of 1 g:(10-30) mL, heated to obtain a crude extract, which was then centrifuged and filtered to obtain the crude extract. The deep eutectic solvent is obtained by reacting hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:(1-6); The hydrogen bond acceptor is betaine, and the hydrogen bond donor is citric acid; The solution containing the deep eutectic solvent in step (2) includes the deep eutectic solvent and water, wherein the volume fraction of water is 0-20% of the deep eutectic solvent.

2. The application according to claim 1, characterized in that, The reaction was carried out at 60-80℃ for 30-90 minutes.

3. The application according to claim 1, characterized in that, The drying temperature is 50-80℃.

4. The application according to claim 1, characterized in that, Step (1) after drying also includes pulverizing the male flowers of Eucommia ulmoides.

5. The application according to claim 1, characterized in that, The heating temperature in step (2) is 60-80℃, and the heating time is 30-120 min.

6. The application according to claim 1, characterized in that, In step (2), the centrifugal force is 3000-8000g and the time is 15-45min.

7. The application according to claim 1, characterized in that, Step (2) is carried out with stirring at a speed of 300-1000 rpm.

8. The application according to claim 1, characterized in that, The centrifugation in step (2) is carried out at 4-20℃.

Images