Optimized extraction and purification method of erigeron breviscapus general flavone extract with anti-oxidation effect

Abstract

The invention specifically discloses an optimized extraction and purification method of an erigeron breviscapus general flavone extract with an anti-oxidation effect, and belongs to the field of natural product extraction. The ultrasonic-assisted extraction technology is adopted, the response surface optimization method is combined, the total flavonoids are optimized and extracted, and the optimal extraction condition is obtained. The total flavonoids are purified by adopting a macroporous adsorption resin chromatography method, the macroporous adsorption resin is good in stability, strong in adsorption capacity, easy to elute and strong in regeneration capacity, the optimal purification condition is determined by exploring the influence of different factors such as temperature, time and pH on the adsorption process of the macroporous resin, and the antioxidant activity of the purified total flavonoids is further explored.

Description

Technical Field

[0001] The invention belongs to the field of natural substance extraction, and specifically relates to an optimized extraction and purification method of a breviscapus flavonoids extract with antioxidant effect. Background Art

[0002] Erigeron breviscapus, a perennial herb of the genus Erigeron in the Asteraceae family, is named for its shape that resembles an oil lamp. It is widely distributed in Hunan, Guangxi, Guizhou, Sichuan, Yunnan and other places in my country. It was first recorded in the "Southern Yunnan Materia Medica" that it can cure hemiplegia, crooked mouth and eyes, and injuries from falls, and has the effects of mild cold and detoxification, wind and dampness removal, blood circulation and blood stasis removal, anti-inflammatory and analgesic effects.

[0003] Studies have shown that Erigeron breviscapus contains phytosterols, phenolic acids, flavonoids, caffeic acid, volatile oils and other ingredients. It has anti-inflammatory, antioxidant, lipid-lowering, cardiovascular and cerebrovascular protection and other pharmacological effects, and is widely used in the medical field. Summary of the invention

[0004] The purpose of the present invention is to provide an optimized extraction and purification method of a total flavonoid extract of Breviscapus breviscapus with antioxidant effect, so as to extract and purify the total flavonoids in Breviscapus breviscapus to the maximum extent and explore its antioxidant activity.

[0005] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides an optimized extraction method of Breviscapus breviscapus total flavonoids extract with antioxidant effect, comprising the following steps:

[0007] ① Wash the whole plant of Erigeron breviscapus thoroughly and put it in a 50℃ forced air drying oven to dry for 12 hours;

[0008] ② Use a high-speed grinder to crush the dried Erigeron breviscapus whole plant, and sieve it through a 60-mesh sieve to obtain Erigeron breviscapus powder;

[0009] ③ Add alcohol solution to the Erigeron breviscapus powder, perform ultrasonic treatment at 50-60°C for 60-80 minutes, and obtain an extract after suction filtration;

[0010] ④ Repeat step ③ and combine the obtained extracts;

[0011] ⑤ Concentrating the above extract to obtain a concentrated solution;

[0012] ⑥ Adjust the concentration of the concentrate to 1.3-1.4 mg / mL and pH (pH = 2-3), add the concentrate to a chromatography column containing a macroporous adsorption resin for purification, elute with an alcohol solution, and collect the eluate.

[0013] ⑦ The collected eluate was concentrated by rotary evaporation and freeze-dried to obtain the total flavonoids extract of Erigeron breviscapus.

[0014] In some embodiments of the present invention, in step ③, the alcohol solution is an ethanol solution with a concentration of 50-60%, for example, it can be an ethanol solution with a concentration of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%. The temperature is 50-60°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C. The time is 60-80min, for example, it can be 80min, 85min, min, 50min, 60min, 70min, 80min, 90min, 100min, etc.

[0015] In some embodiments of the present invention, the mass ratio of Erigeron breviscapus powder to alcohol solution is 1:20-1:30, for example, it can be 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30.

[0016] In some embodiments of the present invention, in step ④, the number of repetitions is 1-3 times, preferably 2 times.

[0017] In some embodiments of the present invention, in step ⑤, the concentration is concentrated under reduced pressure, the concentration temperature is 50-55°C, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C; the vacuum degree can be -0.08-0.09mPa. After concentration, the volume of the concentrate is 1 / 3-1 / 5 of the total volume of the extract, and the ethanol is substantially evaporated.

[0018] In some embodiments of the present invention, in step ⑥, the macroporous adsorption resin used is selected from one of DM-16X, NKA-II, DM130, HPD100, S-8, and AB-8, and the concentration and pH of the concentrated solution are adjusted to 1.3-1.4 mg / mL and pH=2-3, because the inventors found that the concentration and pH of the sample solution have an effect on the adsorption of total flavonoids by the macroporous resin. The concentration of the concentrated solution is 1.3-1.4 mg / mL, for example, it can be 1.31 mg / mL, 1.32 mg / mL, 1.33 mg / mL, 1.34 mg / mL, 1.35 mg / mL, 1.36 mg / mL, 1.37 mg / mL, 1.38 mg / mL, 1.39 mg / mL, 1.40 mg / mL. The pH can be 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc.

[0019] In some embodiments of the present invention, in step ⑥, the alcohol solution used for elution is a 60-70% ethanol solution, for example, it can be 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, etc.

[0020] The second aspect of the present invention is to provide a Breviscapus breviscapus total flavonoids extract with antioxidant effect, which is obtained by the above extraction method.

[0021] The third aspect of the present invention also provides a use of the above-mentioned Breviscapus breviscapus total flavonoids extract with antioxidant effect in cosmetics.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention optimizes the extraction and purification of total flavonoids in Erigeron breviscapus by combining ultrasound-assisted extraction with response surface optimization and macroporous adsorption resin adsorption purification. The process of this method is simple, which not only significantly improves the extraction efficiency and makes the total flavonoids extraction more sufficient, but also effectively removes impurities and improves the purification effect through precise adsorption and separation of macroporous adsorption resin.

[0024] 2. Compared with traditional extraction and purification methods, the present invention ensures that flavonoids are effectively extracted and purified under optimal conditions through meticulous parameter optimization, such as ultrasonic temperature, ultrasonic time, ethanol concentration, resin type, selection of elution conditions, etc., thereby improving the quality and purity of the final product.

[0025] 3. The total flavonoids extract of Breviscapus breviscapus prepared by the present invention has a high total flavonoid yield and purity, has good antioxidant effect, and can be used in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0027] in:

[0028] Figure 1 This is the standard curve of the absorbance value and concentration of the total flavonoid standard rutin.

[0029] Figure 2 This is the effect of different extraction times on the total flavonoid yield;

[0030] Figure 3 This is the effect of different ethanol concentrations on the total flavonoid yield;

[0031] Figure 4 This is the effect of different extraction temperatures on the yield of total flavonoids;

[0032] Figure 5 This is the effect of different material-liquid ratios on the total flavonoid yield;

[0033] Figure 6 This is the ABTS free radical scavenging rate of the total flavonoids extract of Breviscapus breviscapus before and after purification;

[0034] Figure 7 This is the scavenging rate of DPPH free radicals by the total flavonoids extract of Breviscapus breviscapus before and after purification;

[0035] Figure 8 The total reducing power diagram of the total flavonoids extract of Breviscapus breviscapus before and after purification;

[0036] Fig. 9 This is a graph showing the scavenging rate of superoxide free radicals by the total flavonoids extract of Erigeron breviscapus before and after purification;

[0037] Fig.10 This is the effect of purified total flavonoids from Erigeron breviscapus on the proliferation activity of L929 cells;

[0038] Fig.11 The adsorption / desorption diagram of total flavonoids by different macroporous adsorption resins. Fig.12 This is the hemolysis rate diagram of the purified total flavonoids from Breviscapus breviscapus. DETAILED DESCRIPTION

[0039] Example 1

[0040] An optimized extraction method of Breviscapus breviscapus total flavonoids extract comprises the following steps:

[0041] S1: After washing and drying the whole plant of Erigeron breviscapus, crush it with a high-speed grinder and pass it through a 60-mesh sieve to obtain Erigeron breviscapus powder.

[0042] S2: Weigh 10 g of powder, add 300 mL of 50% L-alcohol solution, and ultrasonicate at 50°C for 80 min. After the extract is filtered, the filtrate is obtained, and the ethanol is removed by rotary evaporator at 50°C, 110 rpm, and -0.09 mPa to obtain a concentrated solution.

[0043] S3: adjusting the concentration and pH of the concentrate to 1.3-1.4 mg / mL and pH=2-3, adding the concentrate to a chromatography column containing a macroporous adsorption resin for purification, eluting with an alcohol solution, and collecting the eluate.

[0044] S4: The collected eluate is concentrated by rotary evaporation and freeze-dried to obtain the total flavonoids extract of Erigeron breviscapus.

[0045] Determination of total flavonoids content

[0046] The total flavonoids content was determined by NaNO2-Al(NO)3 colorimetric method. The absorbance was measured at a wavelength of 510 nm. Rutin was used as the standard to establish a standard curve. Figure 1 As shown, the total flavonoid content was calculated according to formula (1):

[0047] Total flavonoid content (%) = c*V*n / m*100% (1)

[0048] Wherein, c is the total flavonoid concentration (mg / mL) calculated according to the standard curve;

[0049] V is the total volume of the extract (mL);

[0050] n is the dilution multiple;

[0051] m is the mass of Erigeron breviscapus powder (g).

[0052] Example 2

[0053] A total flavonoids extract of Erigeron breviscapus with antioxidant effect, the specific scheme is as follows:

[0054] Efficacy Test

[0055] 1. DPPH free radical scavenging rate

[0056] DPPH free radicals usually appear violet in solution and have a specific absorption spectrum in the visible light region, with the maximum absorption peak usually located near 517nm. The antioxidant properties of antioxidants can be evaluated by measuring their ability to scavenge DPPH free radicals.

[0057] ABTS free radical scavenging rate

[0058] ABTS free radicals are usually blue-green in solution and have a specific absorption spectrum in the visible light region, with the maximum absorption peak usually located near 734nm. The ability of the antioxidant to scavenge ABTS free radicals is determined by the degree to which the antioxidant causes the ABTS free radical solution to fade.

[0059] Hydroxyl radical scavenging rate

[0060] Hydroxyl free radical is a highly active oxygen free radical with strong oxidative ability. It can react with various molecules in the body, causing cell damage and dysfunction. Therefore, the hydroxyl free radical scavenging ability is also an important means to evaluate the antioxidant effect.

[0061] Superoxide radical scavenging rate

[0062] Superoxide radicals do not directly show obvious color in the solution. Pyrogallol rapidly self-oxidizes under alkaline conditions, releasing superoxide anion radicals and generating colored intermediates. When an antioxidant is added to a system containing pyrogallol, the antioxidant reacts with superoxide radicals, thereby reducing the amount of colored intermediates generated. By measuring the color change of the solution after the reaction, the ability of the antioxidant to scavenge superoxide radicals can be indirectly reflected.

[0063] Total reduction power

[0064] The determination of total reducing power usually relies on the reduction of higher valence ions (such as Fe 3+ ) is reduced to a low valence state (such as Fe 2+ ) capacity, by measuring the Fe 2+ The concentration change can indirectly reflect the total reducing power of the antioxidant.

[0065] Specifically, it can be measured by referring to the group standard "T / ZGKSL 001-2019".

[0066] Depend on Figure 6-9 It can be seen that the antioxidant effect of the purified total flavonoids extract of Breviscapus breviscapus was significantly improved, and to a certain extent, it was similar to the antioxidant effect of VC.

[0067] Example 3

[0068] The toxicity test of the total flavonoids extract of Breviscapus breviscapus is as follows:

[0069] The MTT experiment was performed using mouse fibroblast L929 as a cell model to evaluate the cytotoxicity of the extract. MTT is a yellow dye. The succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTT to produce a water-insoluble blue-purple crystalline formazan that is deposited in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its absorbance value at 490nm is measured by an enzyme marker. The cell viability is calculated by a formula, which can indirectly reflect the toxicity of the sample to the cells.

[0070] 1. Cell Culture

[0071] L929 cells were cultured in DMEM medium containing 5% fetal bovine serum in a constant temperature incubator at 37°C and 5% CO2.

[0072] 2. Cell Viability Detection

[0073] The cell proliferation inhibition rate was detected using the MTT method. Cells in the logarithmic growth phase were prepared to a cell density of 1×10 4cell / mL cell suspension, 200μL per well was inoculated into a 96-well plate and placed in a constant temperature incubator for culture. After 24 hours, the old culture medium was aspirated, 20μL of total flavonoids extract of Erigeron breviscapus was added to the example group, 200μL of complete culture medium was added to the blank group, and 20μL of phenol was added to the control group. After 24 hours of culture, 20μL of 5mg / mL MTT solution was added, and after incubation at 37℃ for 4 hours, the supernatant was discarded, 200μL DMSO was added to each well, and its absorbance was detected at 490nm to calculate the cell proliferation rate.

[0074] Cell proliferation rate = (sample OD - blank group OD) / (control group OD - blank group OD) × 100%

[0075] Depend on Fig.10 It can be seen that the total flavonoids extract of Breviscapus breviscapus has no obvious toxicity to cells.

[0076] Example 4

[0077] The irritation test of the total flavonoids extract of Breviscapus breviscapus is as follows:

[0078] As an in vitro experimental method, the sheep red blood cell hemolysis test is simple to operate and low in cost, and is used as an alternative to the traditional rabbit eye irritation test.

[0079] Take a certain amount of red blood cells, wash them with PBS buffer 1-3 times until the supernatant is clear, remove the supernatant, add a certain amount of PBS buffer to make a 4% red blood cell suspension, mix the sample solution and the red blood cell suspension in equal volumes, place them in a 37℃ constant temperature water bath oscillator and incubate them for 1 hour. After the incubation, centrifuge them at 8000rpm for 2 minutes, measure the absorbance value of the supernatant at a wavelength of 540nm, and calculate the hemolysis rate by the formula. The blank group and the control group were replaced with equal amounts of PBS and 0.1% TritonX-100 instead of the sample solution.

[0080] Hemolysis rate = (sample group OD - blank group OD) / (control group OD - blank group OD) × 100%

[0081] Depend on Fig.12 It can be seen that the total flavonoids extract of Erigeron breviscapus has no obvious irritation.

[0082] In summary, the optimized extraction and purification method of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect provided by the present invention can maximize the extraction of total flavonoids of Breviscapus breviscapus. The obtained total flavonoids extract has excellent antioxidant effect, is non-toxic and non-irritating, and has the potential to be used in cosmetics.

[0083] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that several improvements and modifications may be made without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for optimizing the extraction and purification of a total flavonoid extract of Breviscapus breviscapus with antioxidant effect, comprising the following steps: S1: Wash the whole plant of Erigeron breviscapus thoroughly and put it into a forced air drying oven at 50°C to dry for 12 hours; S2: using a high-speed grinder to crush the dried Breviscapus breviscapus whole plant, and sieving it through a 60-mesh sieve to obtain Breviscapus breviscapus powder; S3: adding alcohol solution to the Erigeron breviscapus powder, ultrasonicating at 50-60° C. for 60-80 min, and filtering to obtain an extract; S4: repeat step S3 to combine the obtained extracts; S5: concentrating the extract to obtain a concentrated solution; S6: adjusting the concentration of the concentrate to 1.3-1.4 mg / mL and the pH (pH=2-3), adding the concentrate to a chromatography column containing a macroporous adsorption resin for purification, eluting with an alcohol solution, and collecting the eluate. S7: The collected eluate is concentrated by rotary evaporation and freeze-dried to obtain the Erigeron breviscapus total flavonoids extract.

2. The method for optimizing the extraction and purification of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect according to claim 1, characterized in that: The concentration of the alcohol solution is 50-60% ethanol solution, and the ratio of Erigeron breviscapus to the alcohol solution is 1:20-1:

30.

3. The method for optimizing the extraction and purification of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect according to claim 1, characterized in that: The extraction is repeated 1-3 times, preferably 2 times.

4. The method for optimizing the extraction and purification of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect according to claim 1, characterized in that: The concentration is vacuum concentration with a concentration temperature of 50-55° C. and a vacuum degree of -0.08-0.09 mPa. After concentration, the volume of the concentrate is 1 / 3-1 / 5 of the volume of the extract, and the ethanol is substantially evaporated.

5. The method for optimizing the extraction and purification of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect according to claim 1, characterized in that: The macroporous adsorption resin used is one of DM-16X, NKA-II, DM130, HPD100, S-8, and AB-8. The concentration of the loading solution is 1.3-1.4 mg / mL, and the pH is 2-3.

6. The method for optimizing the extraction and purification of the total flavonoids extract of Breviscapus breviscapus with antioxidant effect according to claim 1, characterized in that: The alcohol solution of the eluent is a 60-70% ethanol solution.

7. An application of a total flavonoid extract of Breviscapus breviscapus with antioxidant effect, characterized in that: The invention comprises a Breviscapus flavonoids extract extracted by the method for extracting a Breviscapus flavonoids extract with antioxidant effect as described in claims 1 to 6, and the Breviscapus flavonoids extract is used as an antioxidant in cosmetics.

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