AU2021100567A4 - Method for extracting flavonoid compounds from hypsizygus marmoreus - Google Patents

Abstract

The present disclosure provides a method for extracting flavonoid compounds from Hypsizygus marmoreus which includes infiltrating Hypsizygus marmoreus powders with ethanol by light wave coupled with ultrasound, then extracting by light wave and microwave synergistically, and finally drying to obtain a relatively high purity of flavonoid compounds. The present disclosure strictly controls various parameters of extraction, so that extracted flavonoid compounds have a high purity and a high extraction yield. 8

Description

METHOD FOR EXTRACTING FLAVONOID COMPOUNDS FROM HYPSIZYGUS MARMOREUS TECHNICAL FIELD The present disclosure relates to the technical field of Hypsizygus marmoreus processing, and in particular, to a method for extracting flavonoid compounds from Hypsizygus marmoreus. BACKGROUND Flavonoid compounds are mostly present as free molecules or in the form of glycosides. These compounds have a variety of pharmacological effects based on their diversified complex structures. They are important in treating cardio-cerebrovascular diseases, cancers and tumors, and in regulating immune system. Moreover, flavonoids have low toxicity and have long been a hot spot in research and development of natural medicines and functional foods. Although flavonoids in fungi have not been fully and deeply studied in terms of pharmacological effects, they have received extensive concerns and become a new research focus. Hypsizygus marmoreus, also known as crab-flavored mushroom and Zhenjigu, is an important factory cultivated precious edible fungus for edible, medicinal and dietary treating uses. It is currently one of the main varieties of factory cultivated edible fungi in China. Since it is rich in vitamins, amino acids, minerals and the like, it has high nutritional value and delicious taste, which is highly accepted by consumers. In recent years, researches on Hypsizygus marmoreus have made a great progress. In terms of active substances, most researches focus on extraction and efficacy of polysaccharides while only a few are about flavonoids. Huang Min et al. explored conditions for ultrasonic extraction of total flavonoids of Hypsizygus marmoreus. The optimal extraction conditions were determined as follows: material-to-liquid ratio of 1:40, ultrasonic crushing for 4 times with 75% ethanol at 70°C and extraction time of 20 min. A total flavonoid yield was 0.061%. Lv Pengtao et al. carried out ultrasound-assisted extraction of total flavonoids in Hypsizygus marmoreus root with ethanol volume fraction of 70%, material-to-liquid ratio of 1:50 (g/mL), ultrasound treatment time of 6 min, and ultrasound power of 150 W. A total flavonoid yield was 2.178 mg/g. Current methods for extracting flavonoids from Hypsizygus marmoreus have problems such as low extraction yield. Therefore, development of a time and energy reducing and efficient extraction method is important for extraction of flavonoid compounds of Hypsizygus marmoreus. SUMMARY In view of the above problems, an objective of the present disclosure is to provide a method for extracting flavonoid compounds from Hypsizygus marmoreus with a short extraction period and high extraction efficiency. The method uses light wave-ultrasound coupling infiltration, then light wave-microwave synergistic extraction, and finally drying to obtain a relatively high purity of flavonoids. The method for extracting flavonoid compounds from Hypsizygus marmoreus of the present disclosure includes the following steps: step (1): light wave-ultrasound coupling infiltration: adding an ethanol solution having a mass concentration of 85-95% to Hypsizygus marmoreus powders, and infiltrating for 300-420 s under action of light wave at 150-250 W coupled with ultrasound at 400-500 W, so that the Hypsizygus marmoreus powders are fully infiltrated by the ethanol solution; step (2): light wave-microwave synergistic extraction: adding infiltrated material obtained in step (1) into hot water at 85-100°C, extracting by light wave at 70-120 W and microwave at 700-850 W synergistically for 110-190 s, vacuum filtering, centrifuging a filtrate and concentrating; step (3): drying: quick-freezing a concentrated filtrate, and then carrying out vacuum freeze drying. Preferably, in step (1), the Hypsizygus marmoreus powders may be 190-250 mesh. Preferably, in step (1), a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution maybe 1:(8-10) g/mL. Preferably, in step (2), a material-to-liquid ratio of the material to the hot water may be 1:(0.5-1.5) g/mL. Hypsizygus marmoreus flavonoids obtained by the extraction method of the present disclosure can have a purity of 40-45% and an extraction yield as high as 3.5-4.5 mg/g. The present disclosure has the following beneficial effects as compared with the prior art. (1) The present disclosure adopts light wave coupled with ultrasound to enhance penetration of a high concentration of ethanol solution as a wetting agent, so that the Hypsizygus marmoreus powders are quickly and fully infiltrated by the ethanol solution, solving the problem that Hypsizygus marmoreus powders are not easy to be infiltrated or wetted uniformly due to high hardness thereof. (2) In the present disclosure, hot water is added to extract flavonoids from Hypsizygus marmoreus powders at a relatively low concentration of ethanol solution. At the same time, light wave and microwave synergistically provide sufficient heat to maintain continuous boiling of ethanol at a low concentration. Instantaneous vaporization of ethanol causes boiling inside the Hypsizygus marmoreus, leading to faster convection and diffusion of flavonoids and other active ingredients. As a result, flavonoids enter a solvent faster, which improves yield and purity of flavonoids and shortens extraction time. (3) Microwave extraction improves extraction efficiency and reduces extraction time and consumption of material and liquid by generating high-efficient internal heat and dielectric heat. However, it has problems such as uneven heating and destruction of material structures. Light wave extraction is implemented by generating heat with a lamp, then transferring emitted heat energy to a glass panel that can tolerate high temperatures by a high-efficient reflector, and transferring the heat energy to a container which needs to be heated at a high speed through this glass panel. It has many advantages, such as fast heat transfer, uniform heating and retention of active nutrients to the greatest extent. The present disclosure uses light wave and microwave to synergistically extract flavonoids from Hypsizygus marmoreus. The light wave which results in uniformity and the microwave which shows strong selectivity are combined to avoid uneven heating and damage to material structures by microwave treatment alone, thereby improving extraction efficiency and purity of flavonoids. (4) The Hypsizygus marmoreus flavonoids extracted by the present technology have a relatively high purity and need no further purification. DETAILED DESCRIPTION The method for extracting flavonoid compounds from Hypsizygus marmoreus of the present disclosure includes the following steps: step (1): light wave-ultrasound coupling infiltration: adding an ethanol solution having a mass concentration of 85-95% to Hypsizygus marmoreus powders, and infiltrating for 300-420 s under action of light wave at 150-250 W coupled with ultrasound at 400-500 W, so that the Hypsizygus marmoreus powders are fully infiltrated by the ethanol solution; step (2): light wave-microwave synergistic extraction: adding infiltrated material obtained in step (1) into hot water at 85-100°C, extracting by light wave at 70-120 W and microwave at 700-850 W synergistically for 110-190 s, vacuum filtering, centrifuging afiltrate and concentrating; step (3): drying: quick-freezing a concentrated filtrate, and then carrying out vacuum freeze drying. Specifically, in some embodiments of the present disclosure, in step (1), the Hypsizygus marmoreus powders may be 190-250 mesh. When the Hypsizygus marmoreus powders are too fine, gelatinization can easily occur upon mixing with hot water. When the Hypsizygus marmoreus powders have excessively large fineness, the extraction efficiency can be easily reduced while extraction time can be extended. Specifically, in some embodiments of the present disclosure, in step (1), a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution may be 1:(8-10) g/mL. Specifically, in some embodiments of the present disclosure, in step (2), a material-to-liquid ratio of the material to the hot water may be 1:(0.5-1.5) g/mL.

Hypsizygus marmoreus flavonoids obtained by the extraction method of the present disclosure can have a purity of 40-45% and an extraction yield as high as 3.5-4.5 mg/g. The present disclosure is further described below with reference to embodiments. Example 1 A method for extracting flavonoid compounds from Hypsizygus marmoreus included the following specific steps. Step (1): light wave-ultrasound coupling infiltration: an ethanol solution having a mass concentration of 95% was added to 200 mesh Hypsizygus marmoreus powders, where a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution was 1:10 g/mL. Infiltration was carried out for 420 s under action of light wave at 250 W coupled with ultrasound at 500 W, so that the Hypsizygus marmoreus powders were fully infiltrated by the ethanol solution. Step (2): light wave-microwave synergistic extraction: infiltrated material obtained in step (1) was added into hot water at 100°C, where a material-to-liquid ratio of the material to the hot water was 1:1 g/mL. Extraction was carried out by light wave at 120 W and microwave at 850 W synergistically for 110 s, followed by vacuum filtering, centrifuging a filtrate and concentrating. Step (3): drying: a concentrated filtrate was quickly frozen, and then vacuum freeze dried. Hypsizygus marmoreus flavonoids obtained by the present disclosure had a purity of 41.6% and an extraction yield of 3.6 mg/g. Example 2 A method for extracting flavonoid compounds from Hypsizygus marmoreus included the following specific steps. Step (1): light wave-ultrasound coupling infiltration: an ethanol solution having a mass concentration of 95% was added to 250 mesh Hypsizygus marmoreus powders, where a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution was 1: 8 g/mL. Infiltration was carried out for 400 s under action of light wave at 200 W coupled with ultrasound at 450 W, so that the Hypsizygus marmoreus powders were fully infiltrated by the ethanol solution. Step (2): light wave-microwave synergistic extraction: infiltrated material obtained in step (1) was added into hot water at 100°C, where a material-to-liquid ratio of the material to the hot water was 1:0.5 g/mL. Extraction was carried out by light wave at 100 W and microwave at 800 W synergistically for 150 s, followed by vacuum filtering, centrifuging a filtrate and concentrating. Step (3): drying: a concentrated filtrate was quickly frozen, and then vacuum freeze dried. Hypsizygus marmoreus flavonoids obtained by the present disclosure had a purity of 40.2% and an extraction yield of 3.7 mg/g.

Example 3 A method for extracting flavonoid compounds from Hypsizygus marmoreus included the following specific steps. Step (1): light wave-ultrasound coupling infiltration: an ethanol solution having a mass concentration of 95% was added to 210 mesh Hypsizygus marmoreus powders, where a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution was 1:(8-10) g/mL. Infiltration was carried out for 300-420 s under action of light wave at 150-250 W coupled with ultrasound at 400-500 W, so that the Hypsizygus marmoreus powders were fully infiltrated by the ethanol solution. Step (2): light wave-microwave synergistic extraction: infiltrated material obtained in step (1) was added into hot water at 85-100°C, where a material-to-liquid ratio of the material to the hot water was 1:1.2 g/mL. Extraction was carried out by light wave at 80 W and microwave at 820 W synergistically for 110-190 s, followed by vacuum filtering, centrifuging a filtrate and concentrating.

Step (3): drying: a concentrated filtrate was quickly frozen, and then vacuum freeze dried. Hypsizygus marmoreus flavonoids obtained by the present disclosure had a purity of 41.2-44.3% and an extraction yield of 3.7-4.3 mg/g. Example 4 A method for extracting flavonoid compounds from Hypsizygus marmoreus included the following specific steps. Step (1): light wave-ultrasound coupling infiltration: an ethanol solution having a mass concentration of 95% was added to 190 mesh Hypsizygus marmoreus powders, where a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution was 1: 9 g/mL. Infiltration was carried out for 320 s under action of light wave at 180 W coupled with ultrasound at 430 W, so that the Hypsizygus marmoreus powders were fully infiltrated by the ethanol solution. Step (2): light wave-microwave synergistic extraction: infiltrated material obtained in step (1) was added into hot water at 100°C, where a material-to-liquid ratio of the material to the hot water was 1:1.5 g/mL. Extraction was carried out by light wave at 70W and microwave at 700W synergistically for 110 s, followed by vacuum filtering, centrifuging a filtrate and concentrating. Step (3): drying: a concentrated filtrate was quickly frozen, and then vacuum freeze dried. Hypsizygus marmoreus flavonoids obtained by the present disclosure had a purity of 43.1% and an extraction yield of 4.4 mg/g. Example 5 A method for extracting flavonoid compounds from Hypsizygus marmoreus included the following specific steps. Step (1): light wave-ultrasound coupling infiltration: an ethanol solution having a mass concentration of 95% was added to 250 mesh Hypsizygus marmoreus powders, where a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution was 1:10 g/mL. Infiltration was carried out for 350 s under action of light wave at 200 W coupled with ultrasound at 500 W, so that the Hypsizygus marmoreus powders were fully infiltrated by the ethanol solution. Step (2): light wave-microwave synergistic extraction: infiltrated material obtained in step (1) was added into hot water at 90°C, where a material-to-liquid ratio of the material to the hot water was 1:0.8 g/mL. Extraction was carried out by light wave at 120 W and microwave at 700W synergistically for 120 s, followed by vacuum filtering, centrifuging a filtrate and concentrating. Step (3): drying: a concentrated filtrate was quickly frozen, and then vacuum freeze dried. Hypsizygus marmoreus flavonoids obtained by the present disclosure had a purity of 41.6% and an extraction yield of 3.9 mg/g. Example 6 Effect of different extraction methods on flavonoids of Hypsizygus marmoreus Different extraction methods such as ethanol extraction, microwave extraction, light wave extraction, and light wave-microwave synergistic extraction were selected to investigate effect of different extraction methods on extraction yield and purity of flavonoids of Hypsizygus marmoreus. Results were shown in Table 1. Table 1 Effect of different extraction methods on flavonoids of Hypsizygus marmoreus

Extraction with 60% Extraction by microwave with Extraction by light wave with Example Indicator ethanol 60% ethanol 60% ethanol 2

Yield 2.7 3.2 3.1 3.7 (mg/g) Purity (%) 31.6% 32.6% 33.5% 40.2%

Herein, several embodiments are used for illustration of the principles and implementations of the present disclosure. The description of the above embodiments is used to help illustrate the method of the present disclosure and the core principles thereof. In addition, those of ordinary skill in the art can make various modifications in terms of specific implementations and scope of application in accordance with the teachings of the present disclosure. In conclusion, the content of the present specification shall not be construed as a limitation to the present disclosure.

Claims (5)

1. What is claimed is: 1. A method for extracting flavonoid compounds from Hypsizygus marmoreus, comprising the following steps: step (1): light wave-ultrasound coupling infiltration: adding an ethanol solution having a mass concentration of 85-95% to Hypsizygus marmoreus powders, and infiltrating for 300-420 s under action of light wave at 150-250 W coupled with ultrasound at 400-500 W, so that the Hypsizygus marmoreus powders are fully infiltrated by the ethanol solution; step (2): light wave-microwave synergistic extraction: adding infiltrated material obtained in step (1) into hot water at 85-100°C, extracting by light wave at 70-120 W and microwave at 700-850 W synergistically for 110-190 s, vacuum filtering, centrifuging a filtrate and concentrating; step (3): drying: quick-freezing a concentrated filtrate, and then carrying out vacuum freeze drying.
2. 2. The method for extracting flavonoid compounds from Hypsizygus marmoreus according to claim 1, wherein in step (1), the Hypsizygus marmoreus powders are 190-250 mesh.
3. 3. The method for extracting flavonoid compounds from Hypsizygus marmoreus according to claim 1, wherein in step (1), a material-to-liquid ratio of the Hypsizygus marmoreus powders to the ethanol solution is 1:(8-10) g/mL.
4. 4. The method for extracting flavonoid compounds from Hypsizygus marmoreus according to claim 1, wherein in step (2), a material-to-liquid ratio of the material to the hot water is 1:(0.5-1.5) g/mL.
5. 5. The method for extracting flavonoid compounds from Hypsizygus marmoreus according to claim 1, wherein extracted flavonoid compounds of Hypsizygus marmoreus have an extraction yield of 3.5-4.5 mg/g and a purity of 40-45%.