Preparation method of sophora flower flavone extract

Abstract

The invention relates to a preparation method of a sophora flower flavone extract, which comprises the following steps: adding sophora flower powder into an ethanol solution, sealing, carrying out ultrasonic-assisted water bath extraction and magnetic stirring at the same time, centrifuging, and taking supernatant to obtain a sophora flower flavone extracting solution; the sophora flower flavone extracting solution is concentrated to obtain concentrated thick paste, then water is added, the concentrated thick paste is heated and stirred, the concentrated thick paste is stored overnight at the low temperature of 4 DEG C, precipitates are taken through centrifugation, the precipitates are dried and ground, and the sophora flower flavone extract is obtained, the average kaempferol content in the extract obtained through the technology reaches 76% or above, and the extract shows excellent effects of dispelling the effects of alcohol and protecting the liver. The preparation process is reasonable, simple, convenient, stable and feasible, and the extracted sophora flower flavone extract has a commercial utilization value.

Description

Technical Field

[0001] This invention belongs to the field of extract preparation, and specifically relates to a method for preparing a Sophora japonica flavonoid extract. Background Technology

[0002] Flavonoids are a class of bioactive substances widely found in plants, primarily from fruits, vegetables, tea, and herbs. Flavonoids possess various biological activities, such as antioxidant, anti-inflammatory, anti-cancer, blood sugar regulating, and lipid-lowering effects, attracting increasing attention from researchers. Plants have relatively high flavonoid content, and their extraction processes are constantly being improved and refined. Currently, the main extraction methods for plant flavonoids include ultrasonic-assisted extraction, microwave-assisted extraction, water extraction, and ethanol reflux extraction. Among these, ultrasonic and microwave-assisted extraction are relatively new technologies, offering advantages such as fast extraction speed, high yield, high efficiency, and simple operation.

[0003] Traditional water-solvent extraction methods can only extract flavonoid glycosides, and the resulting products have a high content of impurities, such as sugar, protein, starch, etc. In addition, this method has disadvantages such as low extraction rate, complicated process and high cost, so it has been gradually replaced by other processes. Qian Xu et al. found that the optimal extraction conditions for flavonoids from Sophora flavescens by water bath ethanol extraction were 74.47% ethanol concentration, 18 times material-to-liquid ratio, 89.℃ temperature and 2.1h extraction time, with a flavonoid extraction rate of 104.59mg / g. [Title] Application of response surface methodology to optimize extraction of flavonoids from fructus sophorae [Authors] Xu Q, Shen Y, Wang H, et al [Journal] Food Chemistry, 2013, 138(4): 2122-2129. Hou Lili et al. extracted flavonoids from Sophora japonica by boiling water decoction, and the extraction rate of flavonoids under this method was 85.4mg / g.

Title

Authors

Journal

[0004] Ultrasonic-assisted extraction is a new extraction technology. During the propagation of ultrasound in the medium, the cell walls and cell membranes of plants can be broken due to cavitation, and the effective components to be extracted are quickly dissolved. There is no chemical reaction in this process, and the biological activity of the extracted substances is not changed. Therefore, it has a high extraction efficiency. Jing Jiyue et al. found that the optimal process for ultrasonic-assisted extraction of flavonoids from passion fruit peel is 67% ethanol concentration, 40 times material-to-liquid ratio, 25℃ temperature, and 0.5h extraction time. The flavonoid extraction rate is 11.56mg / g. [Title] Optimization of extraction process of total flavonoids from passion fruit peel and its antioxidant activity [Authors] Jing Jiyue, Xu Mengyuan, Tian Yongtao, et al. [Journal] China Condiments, 2022, 47(07):43-48. Zhu Xiuhong et al. used surfactant synergistic ultrasonic extraction to extract total flavonoids from paulownia flowers. The SDS mass concentration was 0.55%, the temperature was 60℃, and the ultrasonic time was 65min. Under these conditions, the total flavonoid extraction rate of paulownia flowers was 64.98mg / g.

Title

Authors

Journal

[0005] In the ethanol reflux method, volatile solvents such as ethanol can be used in the extraction process. After the extraction solvent evaporates when heated, it is condensed through a condenser and refluxed back into the extractor. This process is repeated to ensure that the useful substances in the raw material are fully extracted. Ethanol is the main extraction solvent in the reflux method. Yang Rili used the ethanol reflux method to extract total flavonoids from Sophora japonica. The experimental results showed that when the material-liquid ratio was 1:8 and the temperature was raised to 80℃, refluxed three times, and 60% ethanol was used, the extraction rate of total flavonoids reached the highest level, which was 15.03%. [Title] Orthogonal Experiment to Optimize the Extraction Process of Total Flavonoids from Sophora japonica [Author] Yang Rili [Journal] Guangzhou Chemical Industry, 2012, 40(12):75-76. As one of the traditional methods for extracting flavonoids, the reflux method has many disadvantages, such as requiring a long time, high cost, requiring a large amount of extraction reagents and easily causing waste of reagents.

[0006] Microwave-assisted extraction refers to the extraction of raw materials using the thermal and perturbation effects of microwaves. Microwave radiation can simultaneously heat the inside and outside of the raw materials, while accelerating molecular collision efficiency, thereby speeding up the dissolution rate of the extract, significantly shortening the extraction time, and improving the extraction effect. Hu Xun et al. used microwave technology to extract total flavonoids from water hyacinth. Through orthogonal experiments, they determined the process conditions for microwave-assisted extraction of total flavonoids from water hyacinth: using 40% ethanol, an extraction time of 4 minutes, a material-to-liquid ratio of 1:80 (g / mL), and a medium microwave power. The total flavonoid yield was 0.881%. [Title] Extraction, Separation, and Study of Some Bioactivities of Total Flavonoids from Water Hyacinth [Author] Hu Xun [Journal] Fujian Normal University, 2008: 170-271.

[0007] Currently, there is limited research on flavonoid extracts from Sophora japonica flowers, and literature on Sophora japonica flowers for relieving hangovers is even scarcer. The main component extracted from Sophora japonica flowers using the process described in this invention is kaempferol, which differs from rutin reported in previous studies and exhibits extremely superior hangover-relieving and liver-protecting effects. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for preparing Sophora japonica flavonoid extract, which overcomes the shortcomings of existing hangover relief and liver protection products and obtains a safe and effective food and medicine homology product.

[0009] A method for preparing a Sophora japonica flavonoid extract according to the present invention includes:

[0010] (1) After drying the locust flowers, remove the impurities and crush them to obtain locust flower powder;

[0011] (2) Add the Sophora japonica powder to the ethanol solution, use ultrasound to perform water bath extraction and magnetic stirring at the same time, centrifuge and take the supernatant to obtain Sophora japonica flavonoid extract;

[0012] (3) The flavonoid extract of Sophora japonica was concentrated to obtain a concentrated paste. Then water was added, the mixture was heated and stirred, stored at 4°C overnight, the precipitate was collected by centrifugation, dried and ground to obtain the flavonoid extract of Sophora japonica.

[0013] The preferred embodiment of the above preparation method is as follows:

[0014] In step (2), the concentration of the ethanol solution is 50-80% (V / V); the ratio of the sophora japonica powder to the ethanol solution is 1:10-40 (W / V).

[0015] In step (2), the sealing method is tin foil sealing; the extraction temperature is 30-70℃, the extraction time is 30-90min; the centrifugation speed is 9000-12000rpm, and the centrifugation time is 5-20min.

[0016] In step (2), the content of flavonoids in the Sophora japonica flavonoid extract was determined by ultraviolet-visible spectrophotometry with rutin standard as a reference.

[0017] The method for determining the flavonoid content in the extract of Sophora japonica flowers includes the following steps: (a) Prepare rutin standard solutions of 2, 4, 6, 8, and 10 mg / ml. Take 0.1 ml of rutin standard solutions of different concentrations, add 0.4 ml of 60% ethanol (V / V) and 0.03 ml of 5% sodium nitrite solution (W / V), mix and stir and let stand for 6 min. Then add 0.03 ml of 10% aluminum nitrate solution (W / V), mix and stir and let stand for 6 min. Finally, add 0.4 ml of 4% sodium hydroxide solution (W / V) and 0.04 ml of 60% ethanol, stir and mix well and let stand for 20 min. Add 9 ml of deionized water, and use 0.1 ml of deionized water instead of rutin standard solution as a blank tube to measure the absorbance at 510 nm. Plot the rutin standard curve to determine the total flavonoid content.

[0018] (b) Replace the rutin standard solution with 0.1 ml of Sophora japonica flavonoid extract, add reagents according to the steps in (a), use 0.1 ml of deionized water instead of rutin standard solution as a blank tube, measure absorbance at 510 nm, and calculate the flavonoid content in the extract by substituting into the rutin standard curve.

[0019] In step (3), the concentration is carried out by vacuum concentration and the ethanol solution is recovered as raw material, and then concentrated by boiling water bath at normal pressure; wherein the vacuum concentration temperature is 70-90℃.

[0020] In step (3), the volume of water is 4-6 times that of the concentrated paste.

[0021] In step (3), the centrifugation speed is 9000-12000 rpm and the centrifugation time is 5-20 min; the drying temperature is 50-80℃.

[0022] The specific step (3) involves concentrating the extract under reduced pressure and recovering the ethanol solution, then concentrating it again under normal pressure to obtain a concentrated paste; adding excess water to the concentrated paste, boiling it to dissolve it, storing it at low temperature overnight, centrifuging it to collect the precipitate, drying it, and grinding it to obtain the final product.

[0023] The sophora japonica flavonoid extract prepared by the method of the present invention contains an average kaempferol content of 76% or more.

[0024] The present invention relates to the application of the sophora japonica flavonoid extract in the preparation of hangover relief and liver protection products.

[0025] Note: All W / V values ​​are in g / mL.

[0026] Beneficial effects

[0027] Compared with existing plant flavonoid extraction processes, this invention shows that under the extraction conditions, the extraction rate of Sophora japonica flavonoids is higher and the extraction effect is better.

[0028] Compared with the ethanol reflux method and microwave-assisted extraction method, the present invention requires simpler equipment and less extraction solvent.

[0029] This invention has good repeatability, easy product quality control, and stable product effects, giving the product stable multiple effects such as promoting hangover relief and protecting the liver.

[0030] The Sophora japonica flowers used in this invention belong to the category of plants that are both food and medicine. Compared with plants that are toxic, such as Sophora flavescens, they are safer as raw materials for extracting flavonoids. Attached Figure Description

[0031] Figure 1 Line graphs showing the blood alcohol concentration in rats at 0.0h, 0.5h, 1.0h, 1.5h, 2.0h, and 3.0h after administration of alcohol.

[0032] Figure 2 Liver tissue section of rats with acute alcohol poisoning. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0034] Example 1

[0035] After drying the locust flowers, impurities were removed, and the mixture was pulverized to obtain locust flower powder. 20.00g of the powder was weighed and added to a 50% ethanol solution at a powder-to-ethanol ratio of 1:10 (V / V). The mixture was then added to a rotor and sealed with tin foil. The mixture was then subjected to an ultrasonic-assisted water bath at 30.0℃ for 30 minutes, followed by centrifugation at 10000 rpm for 10 minutes. The supernatant was collected to obtain the locust flower flavonoid extract. The extract was then concentrated under reduced pressure using a rotary evaporator at the boiling point of ethanol (78℃) to obtain a highly concentrated and viscous locust flower flavonoid concentrate. The concentrated flavonoid solution of Sophora japonica was concentrated in a boiling water bath under normal pressure to obtain a concentrated flavonoid paste. Five times the volume of deionized water was added to the concentrated paste and heated to boiling with stirring to dissolve. After standing overnight in a refrigerator at 4°C, it was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the precipitate was dried in an oven at 70°C. It was then ground into Sophora japonica flavonoid extract powder using a glass grinding bowl. The extracted flavonoids amounted to 2.16 g, and the flavonoid extraction rate was 108.23 mg / g. The sample was tested by Wuhan Huasite Industrial Biological Development Co., Ltd., and the kaempferol content reached 76.4%.

[0036] The prepared Sophora japonica flavonoid extract powder was used as a hangover remedy and liver protectant. It was divided into low (80 mg / kg), medium (160 mg / kg), and high (320 mg / kg) dose groups according to the dosage. It was compared with the positive control drug Haiwang Jinzun to determine its hangover remedy and liver protectant effect on rats with acute alcohol poisoning.

[0037] Line graphs showing blood alcohol concentrations in rats at 0.0h, 0.5h, 1.0h, 1.5h, 2.0h, and 3.0h after administration of alcohol are shown below. Figure 1 As shown, after ingesting alcohol, the blood alcohol concentration in rats continuously increases, transitioning from the latency period to the maintenance period. The rats reach their deepest state of intoxication at the peak alcohol concentration, after which the blood alcohol concentration gradually decreases, and the rats gradually recover from their intoxicated state to a normal state, exhibiting a righting reflex. Figure 1 The results showed that the blood alcohol concentration in the treatment group was significantly lower than that in the alcohol model group, indicating that Sophora japonica flowers have the ability to promote alcohol detoxification and accelerate alcohol metabolism in rats.

[0038] Liver tissue sections of rats with acute alcohol poisoning, as shown in the figure Figure 2 As shown, by Figure 2 It can be seen that the liver lobule structure of normal rats in the blank control group was intact and uniform; while in the alcohol model group, many liver lobule cells were damaged, resulting in inflammatory response and structural damage, with many large fat droplets; in the low-dose Sophora japonica extract group, the liver lobules were partially damaged, with many fat droplets; in the medium-dose, high-dose, and Haiwang Jinzun groups, the inflammatory response in the liver tissue was very mild, and the liver structure basically returned to normal, with only a few fat droplets. This indicates that excessive alcohol consumption can damage liver cells in rats, affecting normal liver metabolism, and causing a large accumulation of fat droplets in the liver, easily leading to alcoholic fatty liver. Medium and high doses of Sophora japonica extract can effectively alleviate alcohol-induced liver damage, reducing liver cell damage, edema, and other lesions.

[0039] Example 2

[0040] After drying the locust flowers, impurities were removed, and the flowers were pulverized to obtain locust flower powder. 16.61g of the powder was weighed and added to a 70% ethanol solution (V / V) at a powder-to-ethanol-solution ratio of 1:20 (W / V). The mixture was then added to a rotor and sealed with tin foil. The mixture was then subjected to ultrasonic-assisted water bath treatment at 51.0℃ for 50.9 min, followed by centrifugation at 10000 rpm for 10 min. The supernatant was collected to obtain the locust flower flavonoid extract. The extract was then concentrated under reduced pressure using a rotary evaporator at the boiling point of ethanol (78℃) to obtain a highly concentrated and viscous locust flower flavonoid concentrate. The concentrated flavonoid solution of Sophora japonica was concentrated in a boiling water bath under normal pressure to obtain a concentrated flavonoid paste. Five times the volume of deionized water was added to the concentrated paste and heated to boiling with stirring to dissolve. After standing overnight in a refrigerator at 4°C, it was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the precipitate was dried in a 70°C oven. It was then ground into Sophora japonica flavonoid extract powder using a glass grinding bowl. The extracted flavonoid content was 1.84 g, and the flavonoid extraction rate was 110.65 mg / g. The sample was tested by Wuhan Huasite Industrial Biological Development Co., Ltd., and the kaempferol content reached 78.0%.

[0041] Example 3

[0042] After drying the locust flowers, impurities were removed, and the flowers were pulverized to obtain locust flower powder. 13.50g of the powder was weighed and added to a 68.6% ethanol solution (V / V) at a ratio of 1:24.55 (W / V). The mixture was then added to a rotor and sealed with tin foil. The mixture was then subjected to an ultrasonic-assisted water bath at 51.0℃ for 50.9 min, followed by centrifugation at 10000 rpm for 10 min. The supernatant was collected to obtain the locust flower flavonoid extract. The extract was then concentrated under reduced pressure using a rotary evaporator at the boiling point of ethanol (78℃) to obtain a highly concentrated and viscous locust flower flavonoid concentrate. The concentrated flavonoid solution of Sophora japonica was concentrated in a boiling water bath under normal pressure to obtain a concentrated flavonoid paste. Five times the volume of deionized water was added to the concentrated paste and heated to boiling with stirring to dissolve. After standing overnight in a refrigerator at 4°C, it was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the precipitate was dried in a 70°C oven. It was then ground into Sophora japonica flavonoid extract powder using a glass grinding bowl. The extracted flavonoid content was 1.42 g, and the flavonoid extraction rate was 105.11 mg / g. The results were tested by Wuhan Huasite Industrial Biological Development Co., Ltd., and the kaempferol content reached 77.3%.

[0043] Example 4

[0044] After drying the locust flowers, impurities were removed, and the flowers were pulverized to obtain locust flower powder. 12.11g of the powder was weighed and added to an 80% ethanol solution (V / V) at a powder-to-ethanol-solution ratio of 1:40 (W / V). The mixture was then added to a rotor and sealed with tin foil. The mixture was then subjected to an ultrasonic-assisted water bath at 70℃ for 90 minutes, followed by centrifugation at 10000 rpm for 10 minutes. The supernatant was collected to obtain the locust flower flavonoid extract. The extract was then concentrated under reduced pressure using a rotary evaporator at the boiling point of ethanol (78℃) to obtain a highly concentrated and viscous locust flower flavonoid concentrate. The concentrated flavonoid solution of Sophora japonica was concentrated in a boiling water bath under normal pressure to obtain a concentrated flavonoid paste. Five times the volume of deionized water was added to the concentrated paste and heated to boiling with stirring to dissolve. After standing overnight in a refrigerator at 4°C, it was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the precipitate was dried in an oven at 70°C. It was then ground into Sophora japonica flavonoid extract powder using a glass grinding bowl. The extracted flavonoid content was 1.30 g, and the flavonoid extraction rate was 107.39 mg / g, of which the kaempferol content reached 77.1%.

Claims

1. A method for preparing a flavonoid extract from Sophora japonica, comprising: (1) After drying the locust flowers, remove the impurities and crush them to obtain locust flower powder; (2) Add the Sophora japonica powder to the ethanol solution, seal it, use ultrasound to assist in water bath extraction and simultaneously perform magnetic stirring, centrifuge to obtain the Sophora japonica flavonoid extract; (3) The flavonoid extract of Sophora japonica was concentrated to obtain a concentrated paste. Then water was added, the mixture was heated and stirred, stored at 4°C overnight, the precipitate was collected by centrifugation, dried and ground to obtain the flavonoid extract of Sophora japonica.

2. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ethanol solution is 50-80% (V / V); the ratio of the sophora japonica powder to the ethanol solution is 1:10-40 (W / V).

3. The preparation method according to claim 1, characterized in that, In step (2), the sealing method is tin foil sealing; the extraction temperature is 30-70℃, the extraction time is 30-90min; the centrifugation speed is 9000-12000rpm, and the centrifugation time is 5-20min.

4. The preparation method according to claim 1, characterized in that, In step (2), the content of flavonoids in the Sophora japonica flavonoid extract was determined by ultraviolet-visible spectrophotometry with rutin standard as a reference.

5. The preparation method according to claim 4, characterized in that, The method for determining the flavonoid content in the extract of Sophora japonica flavonoids includes the following steps: (a) Prepare rutin standard solutions of 2, 4, 6, 8, and 10 mg / ml. Take 0.1 ml of rutin standard solutions of different concentrations, add 0.4 ml of 60% ethanol (V / V) and 0.03 ml of 5% sodium nitrite solution (W / V), mix and stir and let stand for 6 min. Then add 0.03 ml of 10% aluminum nitrate solution (W / V), mix and stir and let stand for 6 min. Finally, add 0.4 ml of 4% sodium hydroxide solution (W / V) and 0.04 ml of 60% ethanol, stir and mix well and let stand for 20 min. Add 9 ml of deionized water, and use 0.1 ml of deionized water instead of rutin standard solution as a blank tube to measure the absorbance at 510 nm. Plot the rutin standard curve to determine the total flavonoid content. (b) Replace the rutin standard solution with 0.1 ml of Sophora japonica flavonoid extract, add reagents according to the steps in (a), use 0.1 ml of deionized water instead of rutin standard solution as a blank tube, measure absorbance at 510 nm, and calculate the flavonoid content in the extract by substituting into the rutin standard curve.

6. The preparation method according to claim 1, characterized in that, In step (3), the concentration is carried out by vacuum concentration and the ethanol solution is recovered as raw material, and then concentrated by boiling water bath at normal pressure; wherein the vacuum concentration temperature is 70-90℃.

7. The preparation method according to claim 1, characterized in that, In step (3), the volume of water is 4-6 times that of the concentrated paste.

8. The preparation method according to claim 1, characterized in that, In step (3), the centrifugation speed is 9000-12000 rpm and the centrifugation time is 5-20 min; the drying temperature is 50-80℃.

9. A sophora japonica flavonoid extract prepared by the method of claim 1, wherein the extract obtained by this process has a kaempferol content of more than 76%, indicating an excellent hangover relief and liver protection effect.

Images