Extraction method of aizoon stonecrop herb flavonoid extract

Abstract

The invention belongs to the technical field of plant component extraction, and discloses an extraction method of aizoon stonecrop herb flavonoid extract, which comprises the following specific steps: step 1, raw material pretreatment; step 2, crushing treatment; step 3, preliminary filtration treatment; step 4, extracting again; 5, preparing an aqueous solution; and step 6, adsorption and elution. In a low-temperature vacuum environment, aizoon stonecrop herb plants are quickly frozen and dehydrated, so that the stability of heat-sensitive active ingredients is effectively maintained, uniform fine powder is formed, subsequent uniform permeation of a solvent is facilitated, flavonoid compounds are fully released through 70% ethanol under the high-frequency vibration action of ultrasonic waves, and the effect of removing the heat-sensitive active ingredients is achieved. Compared with a traditional extraction method, the method is operated under mild conditions, damage of high temperature to active ingredients is avoided, meanwhile, the extraction period is greatly shortened, energy consumption is reduced, and the recovery rate of total flavonoids is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant component extraction, and specifically relates to a method for extracting flavonoid extracts from Sedum aizoon L.. Background Art

[0002] Sedum aizoon L. is a perennial herbaceous plant of the genus Sedum in the Crassulaceae family. Its roots, stems, and leaves can all be used as medicine, and it has the effects of clearing heat and detoxifying, stopping bleeding and reducing swelling, etc..

[0003] The chemical components of Sedum aizoon L. are very rich, mainly including flavonoids, terpenoids, alkaloids, phenolic acids, etc.. Among them, flavonoid compounds are the main active components of Sedum aizoon L.. At present, 49 flavonoid components have been isolated from it, covering flavonols, flavones, dihydroflavones, isoflavones, dihydroflavonols, and anthocyanins, etc.. Terpenoid components mainly include monoterpenes, sesquiterpenes, pentacyclic triterpenes, and chain triterpenes. In addition, Sedum aizoon L. also contains alkaloid compounds (such as (-)-sedinine, racemic sedamine, senecionine, etc.), phenylpropanoid compounds (such as coumarin compounds and lignin compounds), phenolic compounds (such as gallic acid, methyl gallate, arbutin, salidroside, etc.), and steroidal compounds (such as 3-oxo-cholest-4-en-26-oic acid, cholesterol, daucosterol, etc.). Current research shows that the flavonoid extracts of Sedum aizoon L. have in vitro antibacterial effects, indicating that it may have certain application values in the treatment of suppurative inflammation and intestinal infectious diseases, etc..

[0004] Due to the relatively complex active components of Sedum aizoon L., traditional extraction technologies are difficult to fully extract the flavonoid substances therein, and the extraction rate and purity are relatively low. At present, there is no report on the extraction method of flavonoid active components of Sedum aizoon L.. Therefore, it is necessary to develop an efficient method for extracting total flavonoids from Sedum aizoon L. to improve the extraction efficiency of flavonoid substances, provide support for the separation and identification of active components of Sedum aizoon L., and at the same time provide a theoretical basis for the antibacterial effect of its flavonoid substances. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting flavonoid extracts from Sedum aizoon L. to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solution: A method for extracting flavonoid extracts from Sedum aizoon L., and the specific steps of this extraction method are as follows: Step 1, raw material pretreatment: Wash the Sedum aizoon L. plants and then perform pre-freezing treatment, and then dry them with a vacuum freeze dryer; Step 2, crushing treatment: After crushing the freeze-dried plant materials into fine powder, sieve them through a 60-mesh sieve to obtain freeze-dried powder of Sedum aizoon L.; Step 3, preliminary filtration treatment: First, mix the freeze-dried powder of Sedum aizoon L. with 70% ethanol at a ratio of 1:10 to obtain a mixed solvent. Then, perform ultrasonic extraction on the mixed solvent. Extract continuously according to the above steps three times, collect the supernatant, and finally filter the mixture with a Buchner funnel to collect the filtrate; Step 4, re-extraction: Rotate and evaporate the filtrate on a rotary evaporator until it becomes viscous, then dry it in an oven until it becomes an extract. Add absolute ethanol to the extract, perform ultrasonic extraction on the mixture, and finally filter it with a Buchner funnel to obtain an ethanol solution of Sedum aizoon L. extract; Step 5, preparation of aqueous solution: Freeze the ethanol solution of Sedum aizoon L. extract, then filter it with a Buchner funnel. Rotate and evaporate the filtrate on a rotary evaporator until it becomes viscous, and then add deionized water to the extract while it is hot to obtain an aqueous solution of Sedum aizoon L. extract; Step 6, adsorption and elution: Mix the aqueous solution of Sedum aizoon L. extract with AB-8 macroporous resin at a ratio of 1:10, collect the mixed macroporous resin, fill it into a resin column, and finally elute the resin with 70% ethanol to obtain the total flavonoids in Sedum aizoon L.;

[0007] Preferably, in the raw material pretreatment stage, the Sedum aizoon L. plants need to be frozen in an environment of -40°C to -50°C. The vacuum degree of the vacuum freeze dryer is 10 - 30 Pa, and it is necessary to maintain a constant temperature for 72 hours during drying.

[0008] Preferably, the specific steps of the preliminary filtration treatment are as follows: A1, Pour the weighed freeze-dried powder into a clean extraction container, and slowly add the pre-prepared 70% ethanol while stirring well with a stirrer; A2, Place the mixed solution container in an ultrasonic water bath, set the water bath temperature to 60°C, adjust the ultrasonic power to 120 W, then turn on the ultrasonic device, maintain ultrasonic extraction for 1 hour, turn off the ultrasonic device after extraction, let the mixed solution stand and precipitate, and finally collect the supernatant; A3, Repeat the extraction step of A2 three times; A4, Combine the extracted supernatants in the same container, and filter them with a Buchner funnel to collect 5 ml of filtrate.

[0009] Preferably, the specific steps of the re-extraction stage are as follows: B1, Transfer the clarified ethanol filtrate obtained in Step 3 to the flask of a rotary evaporator, and at the same time turn on the device, continuously observe the state of the liquid in the flask until the liquid is gradually concentrated into a viscous state; B2, First, adjust the oven temperature to 80°C, then evenly spread the viscous liquid after rotary evaporation and concentration on a high-temperature resistant container, and put the prepared sample into the oven; B3. Continuously observe the state change of the sample during baking. When it presents an extract state, stop baking. B4. Add 10 times the volume of absolute ethanol and 50 ml to the extract, and mix well. Place the mixture in an ultrasonic extraction device for ultrasonic extraction. B5. Finally, after the extraction is completed, use a Buchner funnel for suction filtration to obtain the ethanol solution of Sedum aizoon extract.

[0010] Preferably, in B1, the temperature of the rotary evaporator is set at 65 °C and the rotation speed is set at 121 rpm.

[0011] Preferably, in the aqueous solution preparation stage, the ethanol solution of Sedum aizoon extract needs to be placed in a -20 °C refrigerator and frozen for 12 h. The specific steps for preparing the aqueous solution are as follows: C1. Pour the frozen solution into a Buchner funnel and use vacuum for rapid filtration to collect the clarified filtrate. C2. Transfer the filtered low-temperature filtrate to the flask of the rotary evaporator. Set the temperature of the rotary evaporator at 65 °C and the rotation speed at 121 rpm. C3. Turn on the rotary evaporator and continuously monitor the state of the liquid in the flask until the solution gradually concentrates into a viscous state. C4. When the concentrated solution is still in a hot state, slowly add 10 - 15 times the volume of deionized water to the concentrated solution to obtain the aqueous solution of Sedum aizoon extract.

[0012] Preferably, the specific steps of the adsorption and elution stage are as follows: D1. Mix the aqueous solution of Sedum aizoon extract prepared in step 5 with the pretreated macroporous resin at a volume-to-mass ratio of 1:10 and place it in a conical flask. D2. Set the shaker rotation speed at 120 r / min and shake for 2 hours to allow the flavonoid components in the aqueous solution to fully contact and be adsorbed by the resin. D3. After the adsorption is completed, filter the mixture through a Buchner funnel and collect the macroporous resin. D4. Uniformly fill the recovered resin into a pre-prepared resin column and slowly pass water until the washing liquid is clear. D5. Then use 70% ethanol as the eluent to elute the resin column and collect the ethanol eluate. D6. Finally, concentrate and dry the ethanol eluate to obtain the total flavonoids in Sedum aizoon.

[0013] Preferably, it also includes an antibacterial experiment on the flavonoid substances of Sedum aizoon. The experimental steps are as follows: S1, Medium preparation: Prepare solid and liquid beef extract peptone media according to the formula of 3 g of beef extract, 10 g of peptone, 5 g of NaCl, 1000 mL of distilled water, and 15 - 20 g of agar. Add 0.1 mol / L NaOH dropwise to adjust the pH of the medium to 7.2 - 7.5, and then autoclave at 121 °C for 20 min and dispense into petri dishes; S2, Bacterial activation: Transfer the bacteria onto the beef extract peptone medium and culture in a 37 °C biochemical incubator for 24 h, and repeat the transfer 2 times to fully activate the bacteria; S3, Preparation of bacterial suspension: Take a 15 mL centrifuge tube, add 8 mL of LB liquid medium, dip a yellow pipette tip into the bacteria, and then directly pipette the tip into the centrifuge tube. Culture on a shaker at 37 °C and 220 rpm for 12 - 16 h. After mixing, pipette 100 μL, measure the OD value on an ELISA plate reader with a 96 - well plate, repeat 3 times, take the average value, and then culture the OD value of the bacterial suspension to 1 and dilute to an appropriate concentration; S4, Uniformly spread the bacteria on the petri dish, punch the medium into circles with a diameter of 6 mm using the punching method, inject 100 μL of Sedum aizoon flavone extract into the holes, invert the petri dish and culture in a 37 °C biochemical incubator, and observe and measure the size of the inhibition zone at 24 h, 36 h, and 48 h respectively.

[0014] The beneficial effects of the present invention are as follows: 1. In the low - temperature vacuum environment, the Sedum aizoon plants are quickly frozen and dehydrated, which not only effectively maintains the stability of thermosensitive active ingredients, forms a uniform fine powder, is conducive to the uniform penetration of subsequent solvents, and then uses 70% ethanol and the high - frequency vibration of ultrasonic waves to fully release flavonoid compounds. Compared with traditional extraction methods, this method operates under mild conditions, avoids the destruction of active ingredients by high temperature, significantly shortens the extraction cycle, saves energy consumption, and improves the recovery rate of total flavonoids; 2. By freezing and settling at - 20 °C, the present invention effectively promotes the precipitation of polysaccharides and other high - molecular impurities in the filtrate, thereby significantly reducing the impurity content. When subsequent column separation using AB - 8 macroporous resin is carried out, the adsorption load of the resin is greatly reduced, the elution process is smoother, and at the same time, the filtrate and solution after freezing and settling treatment become clearer, improving the purity of the target flavonoid and significantly increasing the separation efficiency; 3. Through steps such as vacuum freeze - drying, ultrasonic - assisted extraction, low - temperature settling, and macroporous resin separation, the Sedum aizoon total flavonoid extract obtained by the present invention has a high content and purity, and is low - cost, suitable for large - scale production and promotion. Through experimental verification, this flavonoid extract has a good antibacterial effect and can effectively inhibit the growth of various pathogenic microorganisms, providing a reliable theoretical basis and application foundation for clinical anti - inflammation, analgesia, and health food development. Brief Description of the Drawings

[0015] Figure 1 This is the extraction flow chart of total flavonoids from Sedum aizoon L. of the present invention; Figure 2 This is the experimental graph of the inhibitory effect of the total flavonoid extract from Sedum aizoon L. of the present invention on Staphylococcus aureus; Figure 3 This is the experimental graph of the inhibitory effect of the total flavonoid extract from Sedum aizoon L. of the present invention on Escherichia coli; Figure 4 This is the experimental graph of the inhibitory effect of the total flavonoid extract from Sedum aizoon L. of the present invention on Bacillus subtilis; Figure 5 This is the flow chart of the antibacterial experiment of flavonoids from Sedum aizoon L. of the present invention. Detailed Embodiments

[0016] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] As Figures 1 to 5 shown, the embodiments of the present invention provide an extraction method for flavonoid extracts from Sedum aizoon L., and the specific steps of the extraction method are as follows: Step 1, raw material pretreatment: Wash the Sedum aizoon L. plants, perform pre-freezing treatment, and then dry them with a vacuum freeze dryer; Step 2, pulverization treatment: After crushing the freeze-dried plant materials into fine powder, sieve them through a 60-mesh sieve to obtain freeze-dried powder of Sedum aizoon L.; Step 3, preliminary filtration treatment: First, mix the freeze-dried powder of Sedum aizoon L. with 70% ethanol at a ratio of 1:10 (mass: volume) to obtain a mixed solvent, perform ultrasonic extraction on the mixed solvent, continuously extract three times according to the above steps, collect the supernatant, and finally filter the mixture with a Buchner funnel to collect the filtrate; Step 4, re-extraction: Rotate and evaporate the filtrate on a rotary evaporator until it becomes viscous, then dry it in an oven until it becomes an extract paste, add absolute ethanol to the extract paste, perform ultrasonic extraction on the mixture, and finally filter it with a Buchner funnel to obtain an ethanol solution of the Sedum aizoon L. extract; Step 5, aqueous solution preparation: Freeze-treat the ethanol solution of the Sedum aizoon L. extract, then filter it with a Buchner funnel, rotate and evaporate the filtrate on a rotary evaporator until it becomes viscous, and then add deionized water to the extract while it is hot to obtain an aqueous solution of the Sedum aizoon L. extract; Step 6, Adsorption and Elution: Mix the aqueous solution of Sedum aizoon extract with AB-8 macroporous resin at a ratio of 1:10. Collect the mixed macroporous resin, fill it into a resin column, and finally elute the resin with 70% ethanol to obtain the total flavonoids in Sedum aizoon.

[0018] Among them, in the raw material pretreatment stage, the Sedum aizoon plants need to be frozen in an environment of -40°C to -50°C. The vacuum degree of the vacuum freeze dryer is 10 - 30 Pa, and it needs to maintain a constant temperature for 72 hours during drying.

[0019] Low-temperature pre-freezing helps the crystallization of water in plant cells, destroys the cell wall, makes water more easily migrate during the subsequent freeze-drying process, and can maximize the stability of thermosensitive active ingredients. The drying treatment can improve the subsequent crushing and extraction efficiency.

[0020] Among them, the specific steps of the preliminary filtration treatment are as follows: A1, Pour the weighed freeze-dried powder into a clean extraction container, and slowly add the pre-prepared 70% ethanol while stirring well with a stirrer. A2, Place the mixed solution container in an ultrasonic water bath, set the water bath temperature to 60°C, adjust the ultrasonic power to 120W, then turn on the ultrasonic device, keep ultrasonic extraction for 1 hour, turn off the ultrasonic device after extraction, let the mixed solution stand for precipitation, and finally collect the supernatant. Ultrasonic waves can break plant cell walls and accelerate the release of active ingredients into the solvent; setting the temperature at 60°C helps to improve the extraction rate but avoids the degradation of active ingredients caused by high temperature. A3, Repeat the extraction steps of A2 three times. A4, Combine the extracted supernatants in the same container, and perform suction filtration with a Buchner funnel to collect 5 ml of filtrate.

[0021] Continuous extraction three times can improve the total extraction rate, ensure that as much of the extracted flavonoid components as possible are recovered, and at the same time remove undissolved plant residues through filtration to obtain a clear extract, which is beneficial to subsequent extraction operations.

[0022] Among them, the specific steps of the re-extraction stage are as follows: B1, Transfer the clear ethanol filtrate obtained in Step 3 to the flask of a rotary evaporator, and turn on the device at the same time. Continuously observe the state of the liquid in the flask until the liquid is gradually concentrated into a viscous state. Use low-temperature rotary evaporation technology to remove most of the solvent under mild conditions and concentrate the liquid into a viscous state. B2, First, adjust the oven temperature to 80°C, then evenly spread the viscous liquid after rotary evaporation concentration in a high-temperature resistant container, and put the prepared sample into the oven. B3. Continuously observe the state change of the sample during baking. When it presents an extract state, stop baking; this is convenient for subsequent dissolution operations. B4. Add anhydrous ethanol with a volume 10 times that of the extract, and 50 ml, and mix well. Place the mixture in an ultrasonic extraction device for ultrasonic extraction. Utilize the strong dissolution ability of anhydrous ethanol to ensure the full dissolution of flavonoids and other target components. B5. Finally, after the extraction is completed, use a Buchner funnel for suction filtration to obtain the ethanol solution of Sedum aizoon L. extract. Obtain a stable ethanol extract, laying a foundation for the subsequent preparation of the aqueous solution.

[0023] Among them, in B1, the temperature of the rotary evaporator is set at 65 °C, and the rotation speed is set at 121 rpm.

[0024] Among them, in the stage of preparing the aqueous solution, first place the ethanol solution of Sedum aizoon L. extract in a -20 °C refrigerator and freeze it for 12 h. The specific steps for preparing the aqueous solution are as follows: C1. Pour the frozen solution into a Buchner funnel and use vacuum rapid filtration to collect the clarified filtrate. This process helps some components precipitate or form crystals at low temperature, laying a foundation for subsequent separation. C2. Transfer the low-temperature filtrate obtained by filtration to the flask of the rotary evaporator. Set the temperature of the rotary evaporator at 65 °C and the rotation speed at 121 rpm. C3. Turn on the rotary evaporator and continuously monitor the state of the liquid in the flask until the solution gradually concentrates into a viscous state. C4. When the concentrated solution is still in a hot state, slowly add deionized water with a volume 10 - 15 times that of the concentrated solution to the concentrated solution to obtain the aqueous solution of Sedum aizoon L. extract.

[0025] This step can change the system polarity, which is beneficial for subsequent resin adsorption and separation; at the same time, by adding water in a hot state, part of the flavonoid substances can precipitate or exist in an aqueous solution state, which is more suitable for further purification.

[0026] Among them, the specific steps of the adsorption and elution stage are as follows: D1. Mix the aqueous solution of Sedum aizoon L. extract prepared in step 5 with the pretreated macroporous resin at a volume - to - mass ratio of 1:10, and place it in a conical flask. D2. Set the shaker rotation speed at 120 r / min and shake for 2 hours to enable the flavonoid components in the aqueous solution to fully contact and be adsorbed by the resin. D3. After the adsorption is completed, filter the mixture through a Buchner funnel and collect the macroporous resin. D4. Uniformly fill the recycled resin into a pre-prepared resin column, and slowly pass water until the washing liquid becomes clear to remove free impurities and dissolved substances; D5. Then use 70% ethanol as the eluent to elute the resin column, and collect the ethanol eluate; D6. Finally, concentrate and dry the ethanol eluate to obtain the total flavonoids in Sedum aizoon L.

[0027] 70% ethanol can effectively disrupt the hydrophobic interaction between flavonoids and the resin, so ethanol is selected as the eluent.

[0028] Example 1: (1) Wash the Sedum aizoon L. plants, pre-freeze them in an environment of -40 to -50 °C, and then dry them with a vacuum freeze dryer at a vacuum of 10 - 30 Pa and keep the temperature constant for 72 hours; (2) Crush the dried Sedum aizoon L. and pass it through a 60-mesh sieve to obtain the freeze-dried powder of Sedum aizoon L.; (3) Mix 10 g of the Sedum aizoon L. powder evenly with 100 mL of 70% ethanol to obtain a mixture. Ultrasonically extract the mixture (60 °C, 120 W, 1 h), take the supernatant, continuously extract three times according to the above steps, collect the supernatant, and finally filter the mixture with a Buchner funnel to collect 5 mL of the filtrate; (4) Rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until it becomes viscous, dry it in an oven (80 °C) until it becomes an extract paste. Add about 50 mL of absolute ethanol to the extract paste, ultrasonically extract the mixture to completely dissolve the ethanol and the extract, and filter it with a Buchner funnel to obtain the ethanol solution of the Sedum aizoon L. extract; (5) Place the ethanol solution of the Sedum aizoon L. extract in a -20 °C refrigerator and freeze it for 12 h, filter it with a Buchner funnel, rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until it becomes viscous, and add 25 mL of deionized water to the extract while it is hot to obtain an aqueous solution of the Sedum aizoon L. extract; (6) Mix 25 mL of the aqueous solution of the Sedum aizoon L. extract with 250 g of AB-8 macroporous resin, place it in a conical flask, shake it on a shaker for 2 h at 120 r / min, and then filter it with a Buchner funnel to collect the macroporous resin; (7) Fill the macroporous resin into a resin column, continuously wash the resin with water until the liquid becomes clear, use 70% ethanol to elute the resin, collect the ethanol eluate, and concentrate and dry the ethanol eluate to obtain the total flavonoids in Sedum aizoon L.

[0029] Example 2: (1) Wash the Sedum aizoon L. plants, pre-freeze them in an environment of -40 to -50 °C, and then dry them with a vacuum freeze dryer at a vacuum of 10 - 30 Pa and keep the temperature constant for 72 hours; (2)Crush the dried Sedum aizoon L. and sieve it through a 60-mesh sieve to obtain Sedum aizoon L. freeze-dried powder; (3)Mix 10 g of Sedum aizoon L. powder evenly with 100 mL of 70% ethanol, soak for 24 h to obtain a mixture, take the supernatant, extract three times continuously according to the above steps, collect the supernatant, and finally filter the mixture with a Buchner funnel to collect 5 mL of filtrate; (4)Rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until viscous, dry it in an oven (80 °C) until it becomes an extract paste, add about 50 mL of absolute ethanol to the extract paste, mix and stir evenly to completely dissolve the ethanol and the extract, filter with a Buchner funnel to obtain an ethanol solution of Sedum aizoon L. extract; (5)Place the ethanol solution of Sedum aizoon L. extract in a -20 °C refrigerator and freeze for 12 h, filter with a Buchner funnel, rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until viscous, and add 25 mL of deionized water to the extract while it is hot to obtain an aqueous solution of Sedum aizoon L. extract; (6)Mix 25 mL of the aqueous solution of Sedum aizoon L. extract with 250 g of AB-8 macroporous resin evenly, place it in a conical flask, shake it on a shaker for 2 h at 120 r / min, then filter it with a Buchner funnel to collect the macroporous resin; (7)Pack the macroporous resin into a resin column, continuously wash the resin with water until the liquid is clear, elute the resin with 70% ethanol, collect the ethanol eluate, and concentrate and dry the ethanol eluate to obtain the total flavonoids in Sedum aizoon L.

[0030] Example 3: (1)Wash the Sedum aizoon L. plants and dry them in an oven at 80 °C; (2)Crush the dried Sedum aizoon L. and sieve it through a 60-mesh sieve to obtain Sedum aizoon L. freeze-dried powder; (3)Mix 10 g of Sedum aizoon L. powder evenly with 100 mL of 70% ethanol, soak for 24 h to obtain a mixture, take the supernatant, extract three times continuously according to the above steps, collect the supernatant, and finally filter the mixture with a Buchner funnel to collect 5 mL of filtrate; (4)Rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until viscous, dry it in an oven (80 °C) until it becomes an extract paste, add about 50 mL of absolute ethanol to the extract paste, mix and stir evenly to completely dissolve the ethanol and the extract, filter with a Buchner funnel to obtain an ethanol solution of Sedum aizoon L. extract; (5)Place the ethanol solution of Sedum aizoon L. extract in a -20 °C refrigerator and freeze for 12 h, filter with a Buchner funnel, rotate and evaporate the filtrate on a rotary evaporator (65 °C, 121 rpm) until viscous, and add 25 mL of deionized water to the extract while it is hot to obtain an aqueous solution of Sedum aizoon L. extract; (6) Mix 25 mL of the aqueous solution of Sedum aizoon extract with 250 g of AB-8 macroporous resin, place it in a conical flask, shake it on a shaker for 2 h at 120 r / min, then filter it with a Buchner funnel to collect the macroporous resin; (7) Pack the macroporous resin into a resin column, continuously wash the resin with water until the liquid is clear, elute the resin with 70% ethanol, collect the ethanol eluate, and concentrate and dry the ethanol eluate to obtain the total flavonoids in Sedum aizoon.

[0031] Item Total flavonoid extraction volume (mL) Total flavonoid concentration (mg / mL) Example 1 5 2.0 Example 2 3 0.5 Example 3 3.5 0.8 As can be seen from the above table, after extracting the total flavonoids in Sedum aizoon by the method of Example 1, a relatively large amount of total flavonoids is obtained, and the concentration of total flavonoids in the final product is relatively high, indicating that this method has good extraction efficiency for the total flavonoids in Sedum aizoon; In Example 2, the Sedum aizoon extract was not treated with ultrasonic-assisted extraction, so substances that are difficult to separate by the AB-8 macroporous resin column are retained in the extract, affecting the content of total flavonoids in the product; In Example 3, Sedum aizoon was not freeze-dried, and physical drying destroyed the active ingredients in Sedum aizoon, resulting in a relatively low quality of the final total flavonoids obtained.

[0032] Among them, it also includes the antibacterial experiment of the flavonoid substances in Sedum aizoon, and its experimental steps are as follows: S1, Medium preparation: Prepare nutrient broth peptone solid and liquid media according to the formula of 3 g of beef extract, 10 g of peptone, 5 g of NaCl, 1000 mL of distilled water and 15 - 20 g of agar (not added for liquid medium), adjust the pH of the medium to 7.2 - 7.5 by dropping 0.1 mol / L NaOH or 1% HCl, and then autoclave at 121 °C for 20 min and dispense into petri dishes; S2, Bacterial activation: Transfer the bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, etc.) to the nutrient broth peptone medium and culture them in a 37 °C biochemical incubator for 24 h, and repeat the transfer 2 times to fully activate the bacteria; S3, Preparation of bacterial suspension: Take a 15 mL centrifuge tube, add 8 mL of LB liquid medium, dip a yellow pipette tip into the bacteria, and then directly inject the pipette tip into the centrifuge tube. Culture it on a shaker at 37 °C and 220 rpm for 12 - 16 h. After mixing, pipette 100 μL, measure the OD value on an ELISA reader with a 96-well plate, repeat 3 times, take the average value, and then culture the OD value of the bacterial suspension to 1 and dilute it to an appropriate concentration; S4, Spread the bacteria evenly on the petri dish, punch the medium into circles with a diameter of 6 mm by the punching method, and inject 100 μL of the Sedum aizoon flavonoid extract into the holes (such as Figure 2 , Figure 3 andFigure 4 ), Invert the petri dish and culture it in a 37°C biochemical incubator. Observe and measure the size of the inhibition zone at 24 h, 36 h, and 48 h respectively.

[0033] In this experiment, the punching method was used. Common Gram-positive and Gram-negative bacteria were selected to study the antibacterial activity of the flavonoid extract of Sedum aizoon L. The research results showed that the obtained flavonoid extract of Sedum aizoon L. had obvious antibacterial or bactericidal activity, with less dosage and more obvious effect, and had good antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis.

[0034] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0035] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting flavonoids from Sedum notoginseng, characterized in that: The specific steps of the extraction method are: Step 1, raw material pretreatment: wash the Sedum notoginseng plants and pre-freeze them, and then dry them with a vacuum freeze dryer; Step 2, crushing treatment: crushing the freeze-dried plant material into fine powder, and sieving it through a 60-mesh sieve to obtain Sedum notoginseng freeze-dried powder; Step 3, preliminary filtration treatment: first, the freeze-dried powder of Sedum notoginseng is mixed with 70% ethanol in a ratio of 1:10 to obtain a mixed solvent, and then the mixed solvent is subjected to ultrasonic extraction, and the extraction is performed three times in succession according to the above steps, and the supernatant is collected, and finally the mixture is filtered with a Buchner funnel to collect the filtrate; Step 4, extracting again: the filtrate is rotary evaporated on a rotary evaporator until it becomes viscous, and then dried in an oven to an extract form, anhydrous ethanol is added to the extract, the mixture is ultrasonically extracted, and finally filtered with a Buchner funnel to obtain an ethanol solution of the Sedum notoginseng extract; Step 5, preparing an aqueous solution: freezing the ethanol solution of the Sedum notoginseng extract, then filtering it with a Buchner funnel, rotary evaporating the filtrate on a rotary evaporator until it becomes viscous, and then adding deionized water to the extract while it is hot to obtain an aqueous solution of the Sedum notoginseng extract; Step six, adsorption and elution: Mix the aqueous solution of Sedum notoginseng extract and AB-8 macroporous resin in a ratio of 1:10, collect the mixed macroporous resin, and fill it into a resin column, and finally use 70% ethanol to elute the resin to obtain the total flavonoids in Sedum notoginseng.

2. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: During the raw material pretreatment stage, the Sedum notoginseng plants need to be frozen at -40°C to -50°C. The vacuum degree of the vacuum freeze dryer is 10-30Pa, and the temperature needs to be kept constant for 72 hours during drying.

3. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: The specific steps of the preliminary filtration process are: A1, pour the weighed lyophilized powder into a clean extraction container, and slowly add the pre-prepared 70% ethanol, stirring thoroughly with a stirrer; A2, place the mixed solution container in an ultrasonic water bath, set the water bath temperature to 60°C, adjust the ultrasonic power to 120W, then turn on the ultrasonic equipment, maintain ultrasonic extraction for 1 hour, turn off the ultrasonic equipment after the extraction is completed, let the mixed solution stand and settle, and finally collect the supernatant; A3, repeat the extraction steps of A2 three times; A4, combine the extracted supernatants in the same container, filter with a Buchner funnel, and collect 5 ml of the filtrate.

4. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: The specific steps of the re-extraction stage are: B1, transfer the clarified ethanol filtrate obtained in step 3 to the flask of the rotary evaporator, turn on the equipment at the same time, and continuously observe the state of the liquid in the flask until the liquid is gradually concentrated into a viscous state; B2, first adjust the oven temperature to 80°C, then evenly spread the viscous liquid concentrated by rotary evaporation in a high-temperature resistant container, and put the spread sample into the oven; B3, continue to observe the state changes of the sample during baking, and stop baking when it appears to be an extract state; B4, add 10 times the volume of anhydrous ethanol and 50 ml to the extract, mix thoroughly, and place the mixture in an ultrasonic extraction device for ultrasonic extraction; B5. Finally, after the extraction is completed, use a Buchner funnel to filter and obtain an ethanol solution of Sedum notoginseng extract.

5. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 4, characterized in that: The temperature of the rotary evaporator in B1 was set to 65°C and the rotation speed was set to 121 rpm.

6. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: The aqueous solution preparation stage requires first placing the ethanol solution of Sedum notoginseng extract in a -20°C refrigerator for 12 hours. The specific steps for preparing the aqueous solution are: C1, pour the frozen solution into a Buchner funnel, filter quickly using vacuum, and collect the clear filtrate; C2, transfer the low-temperature filtrate obtained by filtration to the flask of a rotary evaporator, set the temperature of the evaporator to 65°C and the speed to 121 rpm; C3, turn on the rotary evaporator and continue to monitor the liquid state in the flask until the solution gradually concentrates into a viscous state; C4, while the concentrated solution is still hot, slowly add 10-15 times the volume of deionized water dropwise into the concentrated solution to obtain an aqueous solution of the Sedum notoginseng extract.

7. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: The specific steps of the adsorption and elution stages are: D1, mixing the aqueous solution of the Sedum notoginseng extract prepared in step 5 with the pretreated macroporous resin in a volume-to-mass ratio of 1:10, and placing the mixture in a conical flask; D2, set the shaking speed to 120r / min and shake for 2 hours to allow the flavonoid components in the aqueous solution to fully contact and be adsorbed by the resin; D3, after the adsorption is completed, the mixture is filtered through a Buchner funnel and the macroporous resin is collected; D4, evenly fill the recovered resin into the pre-prepared resin column, and slowly pass water until the washing liquid is clear; D5, use 70% ethanol as eluent to elute the resin pile, and collect the ethanol eluate; D6, finally concentrating and drying the ethanol eluate to obtain the total flavonoids in Sedum notoginseng.

8. The method for extracting the flavonoid extract of Sedum notoginseng according to claim 1, characterized in that: It also includes the antibacterial experiment of flavonoids of Sedum notoginseng, and the experimental steps are as follows: S1, medium preparation: beef extract peptone solid and liquid culture medium were prepared according to the formula of 3g beef extract, 10g peptone, 5g NaCl, 1000mL distilled water and 15-20g agar, 0.1mol / L NaOH was added dropwise to adjust the pH of the culture medium to 7.2-7.5, and then sterilized by high pressure at 121°C for 20min and dispensed into culture dishes; S2, bacterial activation: transfer the bacteria to beef extract peptone medium and culture them in a biochemical incubator at 37°C for 24 hours, repeat the transfer twice to fully activate the bacteria; S3, preparation of bacterial suspension: take a 15mL centrifuge tube, add 8mL of LB liquid culture medium, dip the yellow pipette tip into the bacteria, and then directly hit the pipette tip into the centrifuge tube, culture on a shaker at 37°C and 220rpm for 12-16h, after mixing, draw 100μL, measure the OD value on a 96-well plate on an enzyme reader, repeat 3 times, take the average value, then culture the bacterial suspension OD value to 1, and dilute to the appropriate concentration; S4, spread the bacteria evenly on the culture dish, punch the culture medium into a circle with a diameter of 6 mm by the punching method, inject 100 μL of Sedum notoginseng flavonoids extract into the hole, and invert the culture dish in a 37℃ biochemical incubator for culture. Observe and measure the size of the inhibition zone at 24h, 36h, and 48h.

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