A method for pre-treating and extracting epimedium by irradiation

Abstract

This invention discloses a method for irradiation pretreatment-assisted extraction of Epimedium. Specifically, it discloses a method for extracting the active ingredients of Epimedium, including the following steps: A1) sealing dried Epimedium in a packaging bag, and irradiating the sealed Epimedium with an electron beam at room temperature; A2) pulverizing and sieving the irradiated Epimedium to obtain Epimedium powder; A3) soaking the Epimedium powder in a solvent, ultrasonicating, centrifuging, and collecting the supernatant to obtain Epimedium extract. The method of this invention significantly improves the extraction yield of total flavonoids, total flavonol glycosides, and active substances such as icariin and citric acid C from Epimedium. Simultaneously, the Epimedium extract also significantly enhances the ABTS free radical scavenging activity and hydroxyl free radical scavenging activity. This invention provides high-quality raw materials for the preparation of icariin monomers and the development of health products, and broadens the application scope of irradiation technology.

Description

Technical Field

[0001] This invention relates to the fields of irradiation processing of traditional Chinese medicine and extraction technology of active ingredients from traditional Chinese medicine, specifically to a method for irradiation pretreatment-assisted extraction of Epimedium. Background Technology

[0002] Epimedium is a plant belonging to the Berberidaceae family. Epimedium brevicornu Maxim., Epimedium sarcodactylis Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium brevicornu Epimedium pubescens Maxim. or Korean Epimedium Epimedium koreanum The dried leaves of Nakai (Epimedium sarmentosum) have various effects, including tonifying kidney yang, dispelling wind and dampness, and strengthening muscles and bones. As a traditional tonic in Chinese medicine, it has a long history of medicinal use. Meanwhile, as a health food, Epimedium (Epimedium sarmentosum) can enhance immunity, increase bone density, and relieve physical fatigue.

[0003] The Chinese Pharmacopoeia (2020 edition) uses the content of total flavonoids and total flavonol glycosides (icariin, cyproheptadine I, cyproheptadine A, cyproheptadine B, and cyproheptadine C) as the quality evaluation indicators for epimedium. Epimedium flavonoids have the effects of regulating the endocrine and immune systems, and also possess antioxidant, anti-inflammatory, anti-aging, anti-cancer, and antidepressant functions. Icariin is a very promising natural compound with various pharmacological effects such as anti-inflammatory, anti-tumor, and immunomodulatory effects. It has been proven to inhibit various cancer cells, such as ovarian cancer, breast cancer, and lung cancer. Its drug, icoplanin, can effectively prolong the survival of patients with cervical cancer and liver cancer. Another major component of Epimedium, Ascorbic acid C, has the same structural core as icariin and is the most abundant flavonoid glycoside in Epimedium. Its pharmacological activity is not as good as that of icariin, but it can be directionally converted into high-value icariin through certain technologies.

[0004] Currently, there are over 300 kinds of traditional Chinese medicines and health products containing Epimedium raw materials or its components, with an annual consumption exceeding 3,500 tons, gradually entering the ranks of bulk commodities. However, Epimedium is not rich in flavonoids, which poses a significant challenge to the extraction of flavonoid components from it. Therefore, improving the extraction rate of flavonoids and icariin and other active ingredients from Epimedium is of great significance for improving the utilization rate of raw materials and reducing production costs. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the extraction yield of effective components from Epimedium and / or enhance the antioxidant activity of Epimedium extract. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To address the aforementioned technical problems, this invention first provides a method for extracting the effective components of Epimedium, the method comprising the following steps: A1) Place the dried Epimedium in a sealed packaging bag and irradiate the sealed Epimedium with an electron beam; A2) The irradiated Epimedium was crushed and sieved to obtain Epimedium powder; A3) The Epimedium powder is soaked in solvent, ultrasonically treated, and the supernatant is collected by centrifugation to obtain Epimedium extract.

[0007] In the above method, the irradiation dose can be 7-10 kGy.

[0008] In the above method, the energy of the electron beam can be 10 MeV.

[0009] Furthermore, the electron beam can be generated by a high-energy electron accelerator.

[0010] Furthermore, the power of the high-energy electron accelerator can be 20 kW.

[0011] In the above method, the irradiation treatment may include single-sided irradiation treatment or double-sided irradiation treatment. When single-sided irradiation treatment is used, the thickness of the dried Epimedium in step A1) may be less than or equal to 5 cm.

[0012] In the above method, when double-sided irradiation treatment is used, the thickness of the dried Epimedium in step A1) can be less than or equal to 10 cm.

[0013] Furthermore, the ambient temperature conditions can be 0–40°C.

[0014] In the above method, the sieving in step A2) can be sieve 40-60 mesh.

[0015] In the above method, the solvent mentioned in step A3) can be an alcohol solution.

[0016] Furthermore, the solvent may be an ethanol solution.

[0017] In the above method, the solvent in step A3) can be ethanol with a volume fraction of 65%-85%, and the soaking time can be 20-40 min.

[0018] In the above method, the ratio of Epimedium powder to solvent in step A3) can be 1 g: 60-200 mL.

[0019] Further, the ratio can be 1 g: 60-150 mL, 1 g: 60-180 mL, 1 g: 100-150 mL, 1 g: 100-180 mL or 1 g: 100-200 mL.

[0020] In the above method, the ultrasonic treatment time in step A3) can be 20-40 min, and the power can be 400-800W.

[0021] In the above method, A3) may specifically include the following steps: placing the Epimedium powder in a container, adding solvent and soaking for 20-40 min; then ultrasonically extracting for 20-40 min, cooling, and replenishing the lost weight with solvent at 4000 r·min -1 Centrifuge for 10 min, collect the supernatant to obtain Epimedium extract.

[0022] In the above method, the dried epimedium mentioned in step A1) can be dried epimedium leaves or powdered epimedium.

[0023] In the above method, the packaging bag material mentioned in step A1) includes polymer materials such as polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), polyamide (PA), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), acrylonitrile-styrene-butadiene copolymer (ABS), etc.

[0024] Furthermore, the packaging bag may be made of polyethylene (PE).

[0025] In the above method, the epimedium can be Epimedium sagittatum. The scientific name of Epimedium sagittatum is... Epimedium sagittatum (Sieb. et Zucc.)Maxim.

[0026] The present invention also provides the application of any of the methods described herein in improving the extraction yield of effective components of Epimedium or enhancing the antioxidant activity of Epimedium extract.

[0027] Furthermore, the active ingredients include total flavonoids and total flavonol glycosides. The total flavonol glycosides include icariin A, icariin B, icariin C, icariin, and cymosin I.

[0028] The active ingredients (i.e., active components) of Epimedium have a wide range of applications. Effective extraction of these active ingredients not only improves efficacy but also reduces the amount of medicinal material used and manufacturing costs, allowing for better utilization of this valuable traditional Chinese medicine resource. This invention provides a method for irradiation-assisted extraction of active ingredients (such as total flavonoids and icariin) from Epimedium. The method employs electron beam irradiation pretreatment, followed by soaking and ultrasonic extraction. The ultrasonically extracted Epimedium solution is then cooled and centrifuged to obtain the supernatant. Experiments show that this invention significantly improves the extraction yield of total flavonoids, total flavonol glycosides, and active substances such as icariin and icariin C from Epimedium. Simultaneously, the Epimedium extract also significantly enhances the ABTS and hydroxyl radical scavenging activities. This invention provides high-quality raw materials for the preparation of icariin monomers and the development of health products, and broadens the application scope of irradiation technology.

[0029] The beneficial technical effects of the present invention are as follows: I. This invention provides a method for extracting Epimedium using irradiation pretreatment. Irradiation treatment significantly improves the extraction yield of active components from Epimedium, increasing the yields of total flavonoids, total flavonol glycosides, icariin, and citric acid C by 12.92%-21.90%, 11.5%-25.98%, 16.51%-24.51%, and 16.23%-37.68%, respectively. Simultaneously, the Epimedium extract also significantly enhances the ABTS free radical scavenging activity and hydroxyl free radical scavenging activity.

[0030] Second, this invention provides high-quality raw materials for the preparation of icariin monomers and the development of health products, and broadens the application scope of irradiation technology. Attached Figure Description

[0031] Figure 1 This invention relates to the effect of different irradiation doses on the scavenging rate of hydroxyl radicals in Epimedium.

[0032] Figure 2 This invention investigates the effect of different irradiation doses on the ABTS free radical scavenging rate of Epimedium. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] The packaging bags in the following examples are made of polyethylene (PE).

[0036] The Epimedium used in the following examples are all dried leaves of Epimedium sagittatum.

[0037] Example 1: Method for extracting Epimedium Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out in a 5 cm thick layer, irradiated on one side, with a dose of 7.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 40-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 20 mL of 70% ethanol was added, the weight was confirmed, and the mixture was soaked for 20 min. The mixture was then sonicated (500 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0038] Example 2, Method 2 for extracting Epimedium Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out in a 4 cm thick layer, irradiated on one side, with a dose of 8.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 50-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 30 mL of 75% ethanol was added, the weight was confirmed, and the mixture was soaked for 25 min. The mixture was then sonicated (600 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0039] Example 3, Method 3 for extracting Epimedium Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out to a thickness of 8 cm and irradiated on both sides at a dose of 9.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 50-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 30 mL of 75% ethanol was added, the weight was confirmed, and the mixture was soaked for 25 min. The mixture was then sonicated (600 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0040] Example 4, Method 4 for extracting Epimedium Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out to a thickness of 4 cm, irradiated on one side, with a dose of 10.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 60-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 30 mL of 80% ethanol was added, the weight was confirmed, and the mixture was soaked for 30 min. The mixture was then sonicated (700 W, 40 kHz) for 40 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0041] Comparative Example 1, Method 5 for Extracting Epimedium Take dried Epimedium and seal it in a packaging bag, then pulverize it and pass it through a 50-mesh sieve; weigh 0.2g of Epimedium powder and place it in a container, add 30 mL of 75% ethanol, weigh it, soak for 25 min, sonicate (600 W power, 40 kHz frequency) for 30 min, cool it, make up the weight with 70% ethanol, and sonicate at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0042] Comparative Example 2, Epimedium Extraction Method 6 Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out in a 5 cm thick layer, irradiated on one side, with a dose of 5.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 40-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 20 mL of 70% ethanol was added, the weight was confirmed, and the mixture was soaked for 20 min. The mixture was then sonicated (500 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0043] Comparative Example 3, Epimedium Extraction Method 7 Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread out in a 6 cm thick layer, irradiated on one side, with a dose of 8.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 40-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 20 mL of 70% ethanol was added, the weight was confirmed, and the mixture was soaked for 20 min. The mixture was then sonicated (500 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0044] Comparative Example 4, Epimedium Extraction Method 8 Dried Epimedium was placed in a sealed packaging bag and irradiated using a high-energy electron accelerator (10 MeV, 20 kW). The Epimedium was spread in a 12 cm thick layer and irradiated on both sides at a dose of 8.0 kGy. After irradiation, the Epimedium was pulverized and passed through a 40-mesh sieve. 0.2 g of Epimedium powder was weighed and placed in a container, 20 mL of 70% ethanol was added, the weight was confirmed, and the mixture was soaked for 20 min. The mixture was then sonicated (500 W, 40 kHz) for 30 min, cooled, and the weight was replenished with 70% ethanol at 4000 r·min. -1 Centrifuge for 10 minutes and collect the supernatant to obtain the Epimedium extract.

[0045] Experimental Example 1 In this experiment, Epimedium was treated in each group according to the methods of Examples 1-4 and Comparative Examples 1-4, and the contents of total flavonoids, total flavonol glycosides, icariin, and citric acid C in the Epimedium extracts of each group were determined. The relevant determination methods are as follows: (1) Total flavonoid content Accurately weigh an appropriate amount of icariin reference standard and prepare reference solutions with concentrations of 2, 5, 10, 15, 30, and 60 μg / mL using 70% ethanol. Accurately measure 0.2, 0.5, 1.0, 1.5, 3.0, and 6.0 mL of the reference solution into separate 10 mL volumetric flasks, add 70% ethanol to the mark, and mix well. Using 70% ethanol as a blank, measure the absorbance at 270 nm using UV-Vis spectrophotometry (General Chapter 0401 of the 2020 Chinese Pharmacopoeia). Plot a standard curve with absorbance (Y) on the ordinate and concentration (X, μg / mL) on the abscissa: Y = 0.0213X + 0.0016 (2.00–60.00 μg / mL, r = 0.9997). Read the concentration (μg / mL) of the test solution from the standard curve.

[0046] (2) Determination of total flavonol glycoside content Preparation of reference solutions: Accurately weigh icariin A, icariin B, icariin C, icariin, and cymosin I, and add methanol to prepare solutions with concentrations of 0.12, 0.30, 0.20, 0.33, and 0.16 mg·mL, respectively. -1 A mixture of reference standards.

[0047] Chromatographic conditions: Column: Agilent 5 TC-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: 0.1% formic acid aqueous solution (A) ~ acetonitrile (B); Flow rate: 0.8 mL·min -1 Column temperature: 30℃; Injection volume: 10 µL; Detection wavelength: 270nm; Elution gradient: 21%B → 30%B from 0 to 25 min, 30%B → 70%B from 25 to 35 min, 70%B → 90%B from 35 to 45 min, and 90%B from 45 to 50 min.

[0048] Assay: Accurately pipette 10 µL of the reference solution and the test solution obtained according to the methods of Examples 1-4 and Comparative Examples 1-4, respectively, and inject them into the liquid chromatograph. Measure the content in triplicate. Plot the average peak area (Y) on the ordinate and the reference concentration (X, μg·mL⁻¹) on the ordinate. -1 Using the x-axis as the abscissa, a standard curve was plotted to obtain the regression equation. Based on the regression equation, the contents (mg / g) of icariin A, icariin B, icariin C, icariin, and cymoside I in Epimedium were calculated.

[0049] The test results are shown in Table 1.

[0050] Table 1. Content of active ingredients (mg / g)

[0051] As shown in Table 1, the detection results of Examples 1-4 and Comparative Examples 1-4 show that, compared with Comparative Example 1 without irradiation treatment, the extraction yields of total flavonoids, total flavonol glycosides, icariin and citric acid C from Epimedium extracted according to the method described in this invention are significantly improved (P<0.05).

[0052] (3) Effect of irradiation preparation of Epimedium extract on hydroxyl radical scavenging activity and ABTS radical scavenging activity ABTS free radical scavenging ability assay: Prepare ABTS solution according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd., catalog number BC4775); add 10 μL of different concentrations of test solution and 190 μL of ABTS solution to a 96-well plate, equilibrate at 30℃ for 30 min, and measure the absorbance at 734 nm using a microplate reader, which is recorded as A. 测定 Distilled water is used instead of the test solution, denoted as A. 空白 Solvent replacing ABTS solution, denoted as A. 对照 Each sample was measured in triplicate, and the average value was taken. The ABTS free radical scavenging rate was calculated according to formula (1).

[0053] Hydroxyl radical scavenging capacity determination: 0.5 mL of extract was mixed with 1 mL of 6 mmol / L ferrous sulfate, 2 mL of 6 mmol / L hydrogen peroxide, and 1 mL of 6 mmol / L salicylic acid. The mixture was placed in a 37℃ water bath for 30 min. The chamber was zeroed with distilled water, and the absorbance was measured at 510 nm, denoted as A. 测定 Distilled water is used as a substitute for the extract, denoted as A. 空白 Distilled water replacing hydrogen peroxide is denoted as A. 对照 Each sample was measured in triplicate, and the average value was taken. The hydroxyl radical scavenging rate was calculated according to formula (1).

[0054] Clearance rate (%) = [A 空白 - (A 测定 - A 对照 )] / A 空白 × 100% formula (1) The results are as follows Figure 1 and Figure 2 As shown, the antioxidant activity of Epimedium extract was reflected by measuring two indicators: hydroxyl radical scavenging rate and ABTS radical scavenging ability. The results showed that, compared with the unirradiated group (0 kGy), the antioxidant activity of Epimedium extract prepared by the method of this invention was significantly increased (P<0.05).

[0055] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for improving the extraction yield of effective components from Epimedium and enhancing the antioxidant activity of Epimedium extract, the method comprising the following steps: A1) Place the dried Epimedium in a sealed packaging bag and irradiate the sealed Epimedium with an electron beam; A2) The irradiated Epimedium was crushed and sieved to obtain Epimedium powder; A3) Soak the Epimedium powder in a solvent, sonicate it, and centrifuge to collect the supernatant to obtain Epimedium extract; The irradiation dose is 7-10 kGy; the energy of the electron beam is 10 MeV; The active ingredients of Epimedium are total flavonoids, total flavonol glycosides, epimedin and citric acid C; The antioxidant activity is ABTS free radical scavenging activity and hydroxyl free radical scavenging activity; The irradiation treatment includes single-sided irradiation or double-sided irradiation. When using single-sided irradiation, the thickness of the dried Epimedium in step A1) is less than or equal to 5 cm; when using double-sided irradiation, the thickness of the dried Epimedium in step A1) is less than or equal to 10 cm. The irradiation treatment is carried out at room temperature. The sieving in step A2) is through a 40-60 mesh sieve. The solvent in step A3) is ethanol with a volume fraction of 65%-85%, and the soaking time is 20-40 min. The ratio of Epimedium powder to solvent in step A3) is 1 g: 60-200 mL. The ultrasonic treatment in step A3) is 20-40 min, and the power is 400-800 W.

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