Application of total flavonoids of branches and trunks of sea-buckthorn in preparation of anti-inflammatory drugs

Abstract

The invention discloses application of total flavonoids of branches and trunks of hippophae rhamnoides in preparation of anti-inflammatory drugs, and belongs to the technical field of biological medicines. The total flavonoids are prepared by taking the branches and trunks of the sea-buckthorn as raw materials, the total flavonoids have good antioxidant and anti-inflammatory effects, and the contents of TNF-alpha, IL-6 and CYP2E1 secreted by cells can be reduced by the total flavonoids of the branches and trunks of the sea-buckthorn with different concentrations, so that inflammation is relieved. Meanwhile, tests prove that the total flavonoids of the branches and trunks of the sea-buckthorn and the purple corn extract have a synergistic effect when being used together, the anti-inflammatory effect can be remarkably improved, and the composition has an excellent anti-inflammatory protection effect on alcoholic liver injury.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of total flavonoids from seabuckthorn branches and trunks in the preparation of anti-inflammatory drugs. Background Art

[0002] Seabuckthorn is rich in nutrients and is considered a widely used food and medicine in my country. The crop yield is about 4-5 tons per hectare, and in some cases 20-25 tons per hectare. The harvesting method usually adopts the cutting method. After harvesting, the young branches with berries are frozen at -38℃, and the berries are separated from the young branches without damage, and then collected and processed. Its fruit has a thin skin, juicy flesh, sweet and sour taste, and a short shelf life. It is usually preserved by freezing or processed directly into seabuckthorn puree after harvest. It can also be dried and processed into products such as dried seabuckthorn fruit and freeze-dried seabuckthorn powder. The seeds can be used to extract oil, the leaves can be used to make tea, and the branches can be used as fuel and to make works of art after being dried. Foods, cosmetics, beauty care products and health products with seabuckthorn extracts can now be seen all over the world.

[0003] Seabuckthorn total flavonoids are widely used in the pharmaceutical field and health care industry as raw materials. People usually extract seabuckthorn total flavonoids from seabuckthorn pomace. In recent years, with the extremely hot market of seabuckthorn puree, seabuckthorn raw materials have changed significantly. People no longer separate seabuckthorn pomace from seabuckthorn fruit, but directly make beverages from fresh seabuckthorn fruit together with pomace after squeezing and pulping to provide them to the market for consumption. The consumption of seabuckthorn puree in the market has become a trend, so seabuckthorn pomace has become less and less, the price has skyrocketed, and the raw materials are difficult to obtain, and there is a trend of disappearing from the market. Therefore, it is crucial to develop new sources of seabuckthorn total flavonoids and preparation methods of anti-inflammatory drugs. Summary of the invention

[0004] The purpose of the present invention is to provide an application of total flavonoids from seabuckthorn branches and trunks in the preparation of anti-inflammatory drugs, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing total flavonoids from seabuckthorn branches and trunks, comprising the following steps:

[0007] (1) crushing and defatting the stems of seabuckthorn, and then ultrasonically extracting them using ethanol to obtain a crude extract of total flavonoids from the stems of seabuckthorn;

[0008] (2) The crude extract of total flavonoids from seabuckthorn branches and trunks is purified and eluted by X-5 macroporous resin, with ethanol as the eluent, and the elution fraction with an ethanol volume fraction of 40% is collected, which is the total flavonoids from seabuckthorn branches and trunks.

[0009] The second technical solution of the present invention is the total flavonoids of seabuckthorn branches and trunks prepared by the preparation method.

[0010] The third technical solution of the present invention is the use of the total flavonoids of seabuckthorn branches and trunks in the preparation of anti-inflammatory drugs.

[0011] The fourth technical solution of the present invention is an anti-inflammatory drug, comprising the total flavonoids of seabuckthorn branches and trunks and purple corn extract.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The present invention uses seabuckthorn branches as raw materials to prepare total flavonoids, which have good antioxidant and anti-inflammatory effects. Different concentrations of total flavonoids from seabuckthorn branches can reduce the content of TNF-α, IL-6 and CYP2E1 secreted by cells, thereby reducing inflammation. At the same time, the present invention also confirms through experiments that the combined use of total flavonoids from seabuckthorn branches and purple corn extract has a synergistic effect, can significantly improve the anti-inflammatory effect, and has an excellent anti-inflammatory protective effect on alcoholic liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a graph showing the concentration of rutin.

[0016] Figure 2 is the relationship between ethanol concentration and extraction rate.

[0017] Figure 3 is the relationship between extraction time and extraction rate.

[0018] Figure 4 It is the relationship between liquid-to-solid ratio and extraction rate.

[0019] Figure 5 is the relationship between extraction temperature and extraction rate.

[0020] Figure 6 This is the effect of total flavonoids from seabuckthorn branches and trunks on the content of tumor necrosis factor (TNF-α).

[0021] Figure 7 This study is about the effect of total flavonoids from seabuckthorn branches on the content of interleukin-6 (IL-6).

[0022] Figure 8 The effect of total flavonoids from seabuckthorn branches and trunks on the content of cytochrome p450 isoenzyme (CYP2E1). DETAILED DESCRIPTION

[0023] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0029] The embodiment of the present invention provides a method for preparing total flavonoids from seabuckthorn branches and trunks, comprising the following steps:

[0030] (1) crushing and defatting the stems of seabuckthorn, and then ultrasonically extracting them using ethanol to obtain a crude extract of total flavonoids from the stems of seabuckthorn;

[0031] (2) The crude extract of total flavonoids from seabuckthorn branches and trunks is purified and eluted by X-5 macroporous resin, with ethanol as the eluent, and the elution fraction with an ethanol volume fraction of 40% is collected, which is the total flavonoids from seabuckthorn branches and trunks.

[0032] In some specific embodiments, the conditions of ultrasonic extraction include: the volume concentration of the ethanol is 60% to 61%, the liquid-to-solid ratio of the ethanol to the defatted sea buckthorn branches is (23 to 24): 1 mL / g, the extraction temperature is 55 to 56°C, and the extraction time is 1.5 to 2 h.

[0033] The embodiment of the present invention also provides total flavonoids of seabuckthorn branches and trunks prepared by the preparation method.

[0034] The embodiment of the present invention also provides the use of the total flavonoids of seabuckthorn branches and trunks in the preparation of anti-inflammatory drugs.

[0035] The embodiment of the present invention further provides an anti-inflammatory drug, comprising the total flavonoids of seabuckthorn branches and trunks and purple corn extract.

[0036] In some specific embodiments, the mass ratio of the total flavonoids of seabuckthorn branches and trunks to the purple corn extract is 1:(1-2).

[0037] In some specific embodiments, the anti-inflammatory drug is used to treat the inflammatory response caused by alcoholic liver injury.

[0038] In some specific embodiments, the anti-inflammatory drug is used to reduce TNF-α, IL-6 and CYP2E1 levels.

[0039] Example 1

[0040] 1 Determination of total flavonoids content in seabuckthorn branches

[0041] The seabuckthorn branches used in this embodiment are from Xinjiang.

[0042] The stems of sea buckthorn were preliminarily dried (50°C, 72h), crushed with a Chinese medicine grinder, and defatted by Soxhlet extraction. 1.00g of the defatted sample was weighed, added with 25mL of 60% ethanol-water, mixed, and ultrasonically extracted for 1.5h. Centrifuged to obtain the supernatant. Then 25mL of 60% ethanol-water was added, mixed, and ultrasonically extracted for the same time, and this was repeated three times. The three supernatants were combined to obtain the crude extract of total flavonoids from sea buckthorn stems, and the volume was adjusted to 100mL with 60% ethanol for later use.

[0043] The total flavonoids content was determined by the aluminum chloride method, and a standard curve was drawn using rutin as the standard. Take 0.010g of rutin, dissolve it with a small amount of 60% ethanol-water, add 60% ethanol-water to make up to 100mL, and mix evenly to obtain a rutin standard solution with a concentration of 0.1mg / mL. Accurately pipette 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6mL of rutin standard solution into an EP tube, add 0.5mL of 5% aluminum chloride solution, mix well, and make up to 5mL with 60% ethanol-water. Shake well and let stand at room temperature for 10min. Use the blank control without adding rutin standard solution, measure the absorbance at a wavelength of 400nm, use the absorbance value as the vertical coordinate, and the mass of rutin in the color developing solution as the horizontal coordinate to draw a standard curve as shown below. Figure 1 shown.

[0044] The total flavonoid content in the extract was calculated based on the absorbance results and the standard curve, which is the mass of rutin in the sample. The total flavonoid content in the seabuckthorn branches was finally measured to be 0.635%. This may be because the fruits and leaves are organs that store nutrients, so the total flavonoid content in the branches is relatively small, and most of it is lignin and cellulose that are difficult to decompose.

[0045] 2 Single factor experiment for optimization of total flavonoid extraction conditions

[0046] Four factors, extraction time, extraction temperature, solid-liquid ratio and ethanol concentration, were selected for the experiment.

[0047] 2.1 Weigh 1.00 g of defatted seabuckthorn stem powder, set the ethanol concentration to 20%, 40%, 60%, 80%, 100%, extract for 2 h, extract at 50 °C, and extract at a liquid-to-solid ratio of 20:1. Centrifuge and filter the resulting solution to determine the total flavonoid yield.

[0048] 2.2 Weigh 1.00 g of defatted seabuckthorn stem powder, set the extraction time to 0.5, 1, 1.5, 2, 2.5, 3 h, ethanol concentration 60%, extraction temperature 50 ° C, liquid-to-solid ratio 20: 1. The obtained solution was centrifuged and filtered, and the total flavonoid yield was determined.

[0049] 2.3 Weigh 1.00 g of defatted seabuckthorn stem powder, set the liquid-to-solid ratio at 10:1, 15:1, 20:1, 25:1, 30:1 (mL / g), 60% ethanol concentration, 50°C extraction temperature, and 2 h extraction time. Centrifuge the resulting solution and determine the total flavonoid yield.

[0050] 2.4 The extraction temperature was set at 40, 45, 50, 55, 60°C, the ethanol concentration was 60%, the liquid-to-solid ratio was 20:1, and the extraction time was 2h. The obtained solution was centrifuged and filtered, and the total flavonoid yield was determined.

[0051] The results of the single factor experiment are as follows Figure 2-5 As shown, combined with the single factor experimental results, three factors, namely ethanol concentration, liquid-to-solid ratio, and extraction temperature, were selected to carry out the design and experiment of response surface methodology.

[0052] The results showed that the optimal extraction process was as follows: extraction temperature 55.36℃, liquid-to-solid ratio 23.12, ethanol concentration 60.77%, extraction time 2h, and the expected final total flavonoid yield was 4.07mg / g. Further verification was carried out by three parallel experiments using the optimized conditions, which were: extraction temperature 55℃, liquid-to-solid ratio 23:1, ethanol concentration 61%, and extraction time 2h. The extraction yield was 4.19mg / g, and the theoretical error value was 4.2%, indicating that the experimental results were in good agreement with the model and achieved the highest yield in the experimental process, indicating that this response surface model is feasible and has practical value.

[0053] 3 Study on the purification, composition and antioxidant activity of total flavonoids from seabuckthorn branches

[0054] 3.1 X-5 macroporous resin was selected to purify the crude extract of total flavonoids from seabuckthorn branches. The crude extract was concentrated by rotary evaporation, and 5 times the volume of ethanol was added. The polysaccharides and proteins were removed by alcohol precipitation, and then concentrated and freeze-dried to form powder. Before loading, it was dissolved in pure water and diluted to a concentration of 1 mg / mL by adding a little ethanol. Wet column loading, 30 mL of sample, adsorption for 1.5 hours, first rinsed with 4 times column pure water, and then eluted with 2 times column volume fractions of 20%, 40%, 60%, 80%, and 100% ethanol. The eluate was collected, concentrated and freeze-dried, and the total flavonoid content was detected.

[0055] The total flavonoid content in the crude extract is 1.244%. The eluents with ethanol volume fractions of 20%, 40%, and 60% have obvious colors, and the eluents with higher concentrations of ethanol are colorless to the naked eye, and the total flavonoid content is extremely low. The 40% column sample has the darkest color, followed by 20%, and the 60% is the lightest. From Table 1, it can be seen that the total flavonoid content in the eluent with ethanol volume fraction of 40% is the highest, and 60% is the lowest. The same is true for the crude extract column yield and the total flavonoid content of the freeze-dried sample.

[0056] Table 1 Total flavonoids content of total flavonoids in seabuckthorn branches

[0057]

[0058] 3.2 The eluted fractions were tested by HPLC, and the scavenging effects of each fraction on DPPH, ABTS free radicals, hydroxyl free radicals and superoxide anions were analyzed. Finally, the eluted fraction with the best free radical scavenging effect was selected for thin layer chromatography analysis.

[0059] HPLC test results show that the area of ​​rutin in the column samples and crude extracts of 20%, 40%, and 60% ethanol accounts for 0%, 7.1%, 0%, and 4.5% of the total peak area, while that of quercetin is 0%, 0.8%, 0%, and 4.5%. Therefore, most of the total flavonoids such as rutin and quercetin are enriched in the elution process of 40% ethanol, resulting in the darkest color of the 40% ethanol eluate.

[0060] From the four in vitro antioxidant experiments of DPPH, ABTS, hydroxyl radicals and superoxide anions, it was found that the free radical scavenging effects of the three extracts were relatively consistent, with the 40% ethanol elution fraction > 20% ethanol elution fraction > 60% ethanol elution fraction. This shows that the total flavonoid active ingredients contained in the 40% ethanol elution fraction are the richest. From the results of thin layer chromatography, it was found that the 40% ethanol elution fraction contained at least two main active ingredients, and their polarity was greater than that of quercetin. Therefore, the 40% ethanol elution fraction of the seabuckthorn branch extract with the best antioxidant effect was selected for the next experiment.

[0061] Example 2

[0062] Anti-inflammatory protective effect of total flavonoids from seabuckthorn branches on alcoholic liver damage in HepG2 cells

[0063] HepG2 cells, also known as human liver cancer cells, were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0064] 1 The alcohol injury model of HepG2 cells was established to study the antioxidant, anti-inflammatory and hepatoprotective activities of the purified extract of seabuckthorn branches and silymarin as a reference drug. When the cells were in the logarithmic growth phase and grew to a density of about 90%, they were digested and passaged, blown into single cell suspensions, counted, and prepared into 1×10 4 Cell suspension was prepared in 96-well plates at 37°C and 5% CO2 incubator for 24 h, and 20 μL of anhydrous ethanol with a gradient of 50, 100, 200, 400, and 800 mM was added to injure the cells for 24 h. All the solution in the plate was aspirated, and 100 μL of 10% CCK8 culture medium dilution was added to each well. After 1 h, the absorbance was measured at 450 nm to detect the cell survival rate.

[0065] The results showed that when the ethanol concentration was 400 mM, the cell survival rate was 52.31 ± 0.87, which was statistically significant. Therefore, this ethanol concentration was selected as the concentration for successful modeling.

[0066] 2. When the cells grow to about 90% in the logarithmic growth phase, they are digested and passaged, blown into single-cell suspension, and inoculated into a culture dish with a diameter of 100 mm. 9 mL of suspension is added to each dish, about 3×10 6cells, and the culture dish was placed in a 37°C, 5% CO2 incubator for 24 hours. 1 mL of PBS was added to the blank group and the model group, and 1 mL of silymarin with a concentration of 20 μg / mL was added to the positive control well, which was also first dissolved with DMSO and then diluted to 0.1% with PBS. 1 mL of 40% ethanol elution fraction of seabuckthorn branch extract with a concentration of 5, 20, and 80 μg / mL was added to the dosing well, and the culture dish was placed in a 37°C, 5% CO2 incubator for 24 hours, and 1 mL of ethanol modeling solution was added, and incubated for another 24 hours under the same conditions. The culture dish was taken out and placed on ice, all the liquid in the dish was discarded, PBS was washed 5 times, RIPA high-efficiency cell lysis buffer was added, lysis was carried out on ice for 30 minutes, and refrigerated centrifugation was performed for ten minutes at a speed of 12000r / min, and the white precipitate was discarded.

[0067] The kits were purchased and tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and cytochrome P4502E1 (CYP2E1) were detected using an enzyme reader and an ultraviolet spectrophotometer.

[0068] The results are as follows Figure 6-8 As shown in the figure, compared with the blank group, the TNF-α content in the model group increased sharply, proving that the model was successfully established. The positive drug silymarin can significantly reduce the TNF-α content. The low and medium doses of total flavonoids from seabuckthorn branches and stems had no obvious protective effect on cells, while the high dose could significantly reduce the TNF-α content. The IL-6 content in the model group was significantly increased compared with the blank group, indicating that the model was successfully established. The positive drug silymarin can significantly reduce the IL-6 content. The low dose of total flavonoids from seabuckthorn branches and stems had no obvious protective effect on cells, while the medium and high doses could significantly reduce the IL-6 content. The CYP2E1 content in the model group was significantly increased compared with the blank group, indicating that the model was successfully established. The positive drug silymarin can significantly reduce the CYP2E1 content. The low dose of total flavonoids from seabuckthorn branches and stems had no obvious protective effect on cells, while the medium and high doses could significantly reduce the CYP2E1 content.

[0069] Example 3

[0070] Anti-inflammatory protective effect of total flavonoids from seabuckthorn branches and purple corn extract (purchased from Xi'an An'ao Biotechnology Co., Ltd.) on alcoholic liver damage in HepG2 cells

[0071] The total flavonoids from seabuckthorn branches and purple corn extract were mixed in mass ratios of 2:1, 1:1 and 1:2, respectively.

[0072] HepG2 cell culture and modeling were the same as in Example 2. 1 mL of PBS was added to the blank group and the model group. 20 μg / mL of total flavonoids from seabuckthorn branches was used as a control. The experimental groups were added with mixtures of 2:1, 1:1, and 1:2 in mass ratios, and the added concentration was 20 μg / mL.

[0073] Tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and cytochrome P4502E1 (CYP2E1) were detected.

[0074] The results showed that compared with the blank group, the TNF-α content in the model group increased sharply, proving that the model was successfully established. The total flavonoids of seabuckthorn branches in the control group had no obvious effect on reducing the content of TNF-α, while the experimental groups with a mass ratio of 2:1, 1:1 and 1:2 could significantly reduce the content of TNF-α, among which the group with a mass ratio of 1:2 had the best reduction effect. The IL-6 content in the model group was significantly increased compared with the blank group, indicating that the model was successfully established. The total flavonoids of seabuckthorn branches in the control group had no obvious effect on reducing IL-6, while the experimental groups with a mass ratio of 2:1, 1:1 and 1:2 could significantly reduce the content of IL-6, among which the group with a mass ratio of 1:1 had the best reduction effect. The CYP2E1 content in the model group was significantly increased compared with the blank group, indicating that the model was successfully established. The total flavonoids of seabuckthorn branches and trunks in the control group did not significantly reduce the content of CYP2E1, while the experimental groups with a mass ratio of 2:1, 1:1 and 1:2 could significantly reduce the content of CYP2E1, among which the group with a mass ratio of 1:2 had the best reduction effect.

[0075] It can be seen that when the dosage was 20 μg / mL, the combined use of total flavonoids from seabuckthorn branches and purple corn extract had a significantly better anti-inflammatory protective effect on alcoholic liver injury than the total flavonoids from seabuckthorn branches alone, and the two had a synergistic effect.

[0076] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing total flavonoids from seabuckthorn branches, characterized in that: The following steps are involved: (1) crushing and defatting the stems of seabuckthorn, and then ultrasonically extracting them using ethanol to obtain a crude extract of total flavonoids from the stems of seabuckthorn; (2) The crude extract of total flavonoids from seabuckthorn branches and trunks is purified and eluted by X-5 macroporous resin, with ethanol as the eluent, and the elution fraction with an ethanol volume fraction of 40% is collected, which is the total flavonoids from seabuckthorn branches and trunks.

2. The preparation method according to claim 1, characterized in that: The ultrasonic extraction conditions include: the volume concentration of the ethanol is 60% to 61%, the liquid-to-solid ratio of the ethanol to the defatted seabuckthorn branches is (23 to 24): 1 mL / g, the extraction temperature is 55 to 56° C., and the extraction time is 1.5 to 2 hours.

3. The total flavonoids of seabuckthorn branches and trunks prepared by the preparation method according to claim 1 or 2.

4. Use of total flavonoids from seabuckthorn branches and trunks as claimed in claim 3 in the preparation of anti-inflammatory drugs.

5. An anti-inflammatory drug, characterized in that: The method comprises the total flavonoids of seabuckthorn branches and trunks as claimed in claim 3 and purple corn extract.

6. The anti-inflammatory drug according to claim 5, characterized in that The mass ratio of the total flavonoids of seabuckthorn branches and trunks to the purple corn extract is 1:(1-2).

7. The anti-inflammatory drug according to claim 5, characterized in that The anti-inflammatory drug is used for treating the inflammatory response caused by alcoholic liver damage.

8. The anti-inflammatory drug according to claim 5, characterized in that The anti-inflammatory drug is used to reduce TNF-α, IL-6 and CYP2E1 levels.

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