Total flavone extract in abelmoschus manihot flowers as well as preparation method and application of total flavone extract

Abstract

The invention provides a preparation method of a general flavone extract in abelmoschus manihot flowers, which comprises the following steps: A) crushing the abelmoschus manihot flowers, extracting with an alcohol solvent, filtering, and concentrating to obtain an extracting solution; b) purifying the extracting solution by adopting macroporous resin to obtain an eluent; and C) concentrating the eluent, and drying to obtain the traditional Chinese medicine composition. According to the method, diluted alcohol extraction and a high-speed homogenization-ultrasonic-assisted liquid phase extraction method (HSH-UAE) are adopted, so that the contact area of a solvent and the abelmoschus manihot flowers is increased, full contact of medicinal materials and the solvent is guaranteed, and flavonoid substances are dissolved more sufficiently, so that the extraction speed is increased, and the extraction efficiency is improved. Macroporous adsorption resin is selected for separation and purification, the purification effect is good, operation is easy, safety, no toxicity and no pollution are achieved, reuse can be achieved after slight treatment, and the production cost is effectively reduced. Compared with a traditional method, the method has the technical advantages that the extraction speed is high, the extraction yield is high, effective components are fully extracted and reserved, and the process is simple.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, in particular to a total flavonoids extract from hibiscus flowers and a preparation method and application thereof. Background Art

[0002] Hibiscus hibiscus, also known as golden flower sunflower, belongs to the Malvaceae family and is an annual herbaceous flowering plant. The wild resources of Hibiscus hibiscus were on the verge of extinction in the 1980s, but were later discovered and cultivated by scientific researchers in the mountains. At present, the artificial cultivation of Hibiscus hibiscus is mainly distributed in Hubei, Jiangsu, Anhui, Shandong, Hainan, Guangxi and other regions.

[0003] Hibiscus hibiscus flower is used as medicine, and it is both a medicine and food, and has health-care functions. It is praised by the biological world as the "plant giant panda" and "life-saving grass". Modern medical research results show that Hibiscus hibiscus flower can significantly enhance human resistance, regulate human immunity, improve cardiovascular and cerebrovascular and microcirculatory functions, and enhance the body's ability to resist oxidation and brain tissue ischemia and hypoxia. It contains total flavonoids, full-price protein, beneficial trace elements, high polysaccharide gum, dietary fiber, etc., which have anti-inflammatory, analgesic, anti-fatigue, anti-aging, anti-cancer, and lipid-lowering effects.

[0004] Total flavonoids are natural phenolic substances that are widely present in plants in nature. They can reduce free radicals in the body and achieve the purpose of anti-oxidation. The whole plant of Hibiscus hibiscus contains flavonoid compounds, which is one of the plants with a high flavonoid content, and the content exceeds the commonly used raw materials such as soybeans and ginkgo in the current flavonoid production by dozens of times. The content of total flavonoids in different parts of Hibiscus hibiscus plants is different, with the highest content in flowers. The total flavonoid content in dried Hibiscus hibiscus flowers can be as high as 3.97%. Therefore, how to develop such compounds to effectively apply them to the treatment of tumor diseases, such as melanoma, is an important research task.

[0005] Melanoma refers to the malignant lesions of melanocyte tumors, namely malignant melanoma (MM), while benign lesions are melanocytic nevi. Melanoma originates from melanocytes of the neural crest and is caused by their uncontrolled transformation and proliferation. Melanoma is the most invasive and deadly skin cancer, and the number of deaths caused by it accounts for almost 90% of the total number of deaths related to skin tumors. What makes melanoma more dangerous than other skin cancers such as squamous cell carcinoma and basal cell carcinoma is its ability to metastasize, and even very thin tumors can metastasize. In the early stage, melanoma can spread to other organs such as the liver, lungs, and brain through lymphatic and hematogenous metastasis. In the terminal stage, brain metastasis will occur. The survival rate of patients with systemic metastasis does not exceed one year. Melanoma has the characteristics of insidious onset, rapid development, strong metastasis, poor prognosis, and high recurrence.

[0006] Traditional extraction methods often use water as the extraction solvent, which may cause a large amount of leaching of mucilage and water-soluble components such as polysaccharides and tannins in Hibiscus hibiscus flowers, which not only increases the viscosity of the extract and makes it difficult to filter, but also increases the impurity components in the extract and reduces the total flavonoid content. Even if alcohol solvents are used, there are still problems such as long extraction time, low extraction efficiency, and large solvent consumption. Conventional reflux extraction methods increase the leaching of flavonoids and improve the extraction efficiency by increasing the temperature and increasing the contact area between the medicinal material powder and the solvent. However, a high temperature environment can also cause the decomposition and deterioration of some flavonoids, resulting in the loss of effective ingredients and safety considerations under industrial production. Enzymolysis can destroy plant tissue cells through enzymolysis, promote the dissolution and release of flavonoids, and improve extraction efficiency. However, it will also cause the degradation of some flavonoids and the loss of effective monomer components, and the enzyme inactivation link of the enzymolysis process may also cause safety considerations under industrial production.

[0007] Therefore, it is very necessary to provide a method for extracting total flavonoids from hibiscus flowers with a high extraction rate. Summary of the invention

[0008] In view of this, the present invention provides a method for extracting total flavonoids from hibiscus flowers, which has a high total flavonoid extraction rate. At the same time, the extract of the present invention can treat melanoma.

[0009] The present invention provides a method for preparing a total flavonoid extract from hibiscus flowers, comprising the following steps:

[0010] A) crushing hibiscus flowers, extracting with an alcohol solvent, filtering and concentrating to obtain an extract;

[0011] B) purifying the extract using a macroporous resin to obtain an eluent;

[0012] C) The eluate is concentrated and dried to obtain the product.

[0013] Preferably, the alcohol solvent is a 50% to 90% ethanol solution, and the material-liquid ratio of the extraction is 1:25 g / mL to 1:75 g / mL.

[0014] Preferably, the hibiscus flower is dried hibiscus flower, and the crushing mesh size is 50 mesh.

[0015] Preferably, the extraction in step A) is high-speed homogenization-ultrasonic assisted extraction; the high-speed homogenization parameters are: homogenization speed 5000rpm~7000rpm, homogenization time 60min~90min;

[0016] The ultrasonic-assisted extraction parameters are: ultrasonic power 300W-700W, temperature 50-70°C, and extraction time 60min-120min.

[0017] Preferably, the macroporous resin is an AB-8 type macroporous adsorption resin;

[0018] The resin purification specifically comprises:

[0019] The extract was adjusted to pH 4-6 using acetic acid-sodium acetate buffer to obtain a loading solution;

[0020] After the sample solution is loaded, it is eluted with pure water, then with 5% ethanol, and finally with 40-90% ethanol, and the eluate with 40-90% ethanol is collected.

[0021] Preferably, the volume ratio of the sample solution to the resin is 1:1-1:2, and the adsorption rate is 1BV / h; pure water is 5-6BV of the resin; 5% ethanol solution is 0.5BV of the resin; 40-90% ethanol is 2-5BV of the resin; and the elution rate of 40-90% ethanol is 1-1.5BV / h.

[0022] Preferably, the total flavonoid extract includes hyperoside, isoquercetin, gossypol-3'-O-β-glucoside and quercetin-3'-O-glucoside.

[0023] The invention provides a total flavonoids extract from hibiscus flowers, which is prepared by the preparation method described in any one of the above technical solutions.

[0024] The present invention provides the use of the total flavonoid extract prepared by any one of the preparation methods described above in the preparation of a drug for treating melanoma.

[0025] Preferably, the total flavonoid extract includes hyperoside, isoquercetin, gossypol-3'-O-β-glucoside, and quercetin-3'-O-glucoside, and the total content is 55% or more, preferably 60% or more, and preferably 70-80%. Further, hyperoside, isoquercetin, gossypol-3'-O-β-glucoside, and quercetin-3'-O-glucoside are preferred.

[0026] The treatment includes inhibiting tumor cell growth, reducing tumor cell viability, reducing melanoma cell migration ability, inhibiting tumor cell cloning ability and reducing tumor weight.

[0027] Compared with the prior art, the present invention provides a preparation method of total flavonoid extract from hibiscus flower, comprising the following steps: A) crushing hibiscus flower, extracting with alcohol solvent, filtering and concentrating to obtain extract; B) purifying the extract with macroporous resin to obtain eluent; C) concentrating the eluent and drying. The present invention adopts dilute alcohol extraction and cooperates with high-speed homogenization-ultrasonic assisted liquid extraction (HSH-UAE) to increase the contact area between the solvent and hibiscus flower, ensure that the medicinal material and the solvent are fully in contact, and the flavonoids are more fully dissolved, thereby accelerating the extraction speed and improving the extraction efficiency. The use of macroporous adsorption resin for separation and purification has good purification effect, simple operation, safety, non-toxicity and pollution-free, and can be reused after a little treatment after use, effectively reducing the production cost. Compared with the traditional method, the technical advantages of this method are fast extraction speed, high extraction yield, sufficient extraction and retention of effective components, simple process, etc.

[0028] The hibiscus flower total flavonoids extract or the composition of the invention has the effect of treating melanoma, and provides a better treatment method for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The tumor inhibition effect of various compositions of Hibiscus tiliaceus flower extracts at different concentrations;

[0030] Figure 2 The effect of different concentrations of hibiscus flower extract on the migration ability of B16 cells;

[0031] Figure 3 The effect of different concentrations of hibiscus flower extract on the clone-forming ability of B16 cells;

[0032] Figure 4 is the average volume change of the tumor in each group after 15 days of treatment;

[0033] Figure 5 The results are H&E tissue staining. DETAILED DESCRIPTION

[0034] The present invention provides a total flavonoid extract from hibiscus flower and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0035] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0036] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0037] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0038] The present invention provides a method for preparing a total flavonoid extract from hibiscus flowers, comprising the following steps:

[0039] A) crushing hibiscus flowers, extracting with an alcohol solvent, filtering and concentrating to obtain an extract;

[0040] B) purifying the extract using a macroporous resin to obtain an eluent;

[0041] C) The eluate is concentrated and dried to obtain the product.

[0042] The total flavonoid content of the extract of the present invention includes hyperoside, rutin, isoquercetin, quercetin-3'-O-β-glucoside and quercetin-3'-O-glucoside in terms of weight percentage.

[0043] The invention extracts flavonoid functional components from hibiscus flowers, and pharmacological activity studies show that the components have an obvious therapeutic effect on melanoma.

[0044] The preparation method of the total flavonoids extract from hibiscus flowers provided by the present invention comprises the following steps: firstly crushing the hibiscus flowers, taking dried hibiscus flowers, crushing them and sieving them to obtain dark yellow powder, wherein the crushing mesh size is 50 meshes.

[0045] After crushing, alcohol solvent is used for extraction. The alcohol solvent of the present invention is 50% to 80% ethanol solution; specifically, it can be 50% ethanol solution, 60% ethanol solution, 70% ethanol solution, 80% ethanol solution; or any range between the two. The present invention finds that only the use of the above concentration of ethanol solution as an extractant has a good technical effect. Particularly preferred is 50% ethanol.

[0046] The material-liquid ratio of the extraction of the present invention is 1:25 g / mL to 1:75 g / mL; specifically, it can be 1:25 g / mL, 1:35 g / mL, 1:45 g / mL, 1:50 g / mL, 1:55 g / mL, 1:65 g / mL, 1:75 g / mL; or any range value therebetween. The most preferred range is 1:50 g / mL.

[0047] The present invention finds that the dilute alcohol extraction effect using the above-mentioned material-liquid ratio is excellent, and the above-mentioned effect cannot be achieved using other ratios.

[0048] The present invention preferably uses an alcohol extraction method. The use of alcohol solvents is conducive to the leaching of flavonoid components, while preventing the large amount of mucus leaching caused by water as a solvent, and avoiding the problem of difficulty in filtration caused by increased viscosity of the extract. Using the above-mentioned most preferred parameters, the ratio of the extract obtained can achieve a good effect in treating melanoma.

[0049] The extraction of the present invention is high-speed homogenization-ultrasonic assisted extraction; the high-speed homogenization parameters are: homogenization speed 5000rpm~7000rpm, homogenization time 60min~90min;

[0050] The homogenization speed of the present invention is 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, or any range between the two.

[0051] The homogenization time of the present invention is 60 min, 70 min, 80 min, 90 min; or any range value between the two.

[0052] The high-speed homogenization method of the present invention increases the contact area between the solvent and the hibiscus flower. The specific homogenization speed and homogenization time can ensure that the medicinal material is fully in contact with the solvent, and the flavonoids are more fully dissolved, thereby accelerating the extraction speed and improving the extraction efficiency.

[0053] The ultrasonic-assisted extraction parameters of the present invention are: ultrasonic power 300W-700W, temperature 50-70°C, and extraction time 60min-120min.

[0054] The present invention extracts power of 300W, 400W, 500W, 600W, 700W, or any range value between the two.

[0055] The temperature of the present invention is 50°C, 55°C, 60°C, 65°C, 70°C, or any range between them, and 50°C is particularly preferred.

[0056] Ultrasonic-assisted extraction method can make plant tissue cells more completely broken through the combined effects of the above power, temperature and time, which can reduce the time and increase the extraction efficiency. At the same time, the medicinal materials are extracted at the above temperature and power, and the heating temperature is low, which avoids the destruction of the structure of flavonoids due to thermal effect and cavitation effect, improves the flavonoid extraction effect, and effectively reduces the impurity content in the extract.

[0057] The invention uses the high-speed homogenization-ultrasonic-assisted liquid phase extraction method to achieve a high extraction rate of flavonoids.

[0058] The total flavonoid extract of the present invention includes but is not limited to hyperoside, isoquercetin, quercetin-3'-O-β-glucoside, and quercetin-3'-O-glucoside.

[0059] The extract was filtered to remove the solid and the filtrates were combined.

[0060] Concentration: The filtrate is concentrated under reduced pressure (below 60°C, 0.09MPa) to recover ethanol.

[0061] The extract is purified by using a macroporous resin to obtain an eluent.

[0062] The macroporous resin of the present invention is preferably pretreated by the following method: AB-8 type macroporous adsorption resin is soaked in 95% ethanol for 20 to 24 hours to fully swell, and then the column bed is washed with 95% ethanol, and then washed with distilled water until there is no alcohol taste. After soaking in 5% hydrochloric acid and NaOH solution for 2 hours, it is washed with distilled water until it is neutral, and finally washed with 95% ethanol until there is no white turbidity, and washed with water until there is no alcohol taste for use.

[0063] According to the present invention, the macroporous resin is an AB-8 type macroporous adsorption resin;

[0064] The resin purification of the present invention specifically includes:

[0065] First, the pH of the extract is adjusted to 4-6 using acetic acid-sodium acetate buffer, and impurities are removed by filtration to obtain a sample solution.

[0066] The extract was diluted with water to a concentration of 0.2 g of raw material per ml. The pH was controlled to 4-6 using acetic acid-sodium acetate buffer and impurities were removed by filtration.

[0067] After the sample solution is loaded, it is eluted with pure water. The volume ratio of the sample solution to the resin is 1:1-1:2, and the adsorption rate is 1BV / h; after the adsorption is completed, 5-6BV of pure water is used for rapid washing, and the effluent is discarded.

[0068] Then 5% ethanol was used for elution, the volume of 5% ethanol solution was 0.5 BV of the resin, and the effluent was discarded.

[0069] Finally, 40-90% ethanol is used for elution, and the eluate of 40-90% ethanol is collected. The volume of 40-90% ethanol is 2-5 BV of the resin; and the elution rate of 40-90% ethanol is 1-1.5 BV / h.

[0070] The eluate was collected and the ethanol was recovered under reduced pressure until there was no alcohol taste; the parameters of the reduced pressure were: below 60°C and 0.09 MPa. The solution was concentrated to dryness and dried in a vacuum oven until the water content was less than 5% to obtain a brown-yellow powder with a slightly bitter taste, which was the total flavonoid extract.

[0071] The invention adopts a method of separation and impurity removal by macroporous adsorption resin, which has good purification effect, is easy to operate, safe, non-toxic and pollution-free, and can be reused after a slight treatment after use, thereby effectively reducing production costs.

[0072] The present invention finds that only by using the above-mentioned specific macroporous adsorption resin of the present invention can the impurities of the hibiscus flower be well removed, and the total flavonoids extraction rate obtained is high. If other resins are used instead, the extraction effect is poor.

[0073] The present invention creatively adopts the method of high-speed homogenization-ultrasonic assisted extraction combined with macroporous adsorption resin separation and impurity removal. The coordinated cooperation of the above steps enables the final total flavonoid extraction rate to be high.

[0074] The invention provides a total flavonoids extract from hibiscus flowers, which is prepared by the preparation method described in any one of the above technical solutions.

[0075] The present invention can adopt colorimetry and high performance liquid chromatography for determination.

[0076] The colorimetric method is as follows:

[0077] a. Preparation of reference solution: Accurately weigh 12.5 mg of rutin standard dried to constant weight into a 25 ml volumetric flask, dissolve and dilute with 70% ethanol to prepare a standard solution with a concentration of 0.5 mg / mL.

[0078] b. Selection of measurement wavelength: Refer to the NaNO2-Al(NO3)3 spectrophotometry method, use a pipette to accurately transfer 1 mL of rutin standard solution and test solution into a brown 10 mL volumetric flask, and dilute to the same volume with 70% ethanol. Add 0.4 mL of 5% sodium nitrite, and let it stand for 6 minutes; add 0.4 mL of 10% aluminum nitrate, and let it stand for 6 minutes; finally, add 4.0 mL of 4% sodium hydroxide and 70% ethanol to the scale, shake well and let it stand for 15 minutes; at the same time, take 5.0 mL of ethanol and prepare a blank control solution in the same way, scan the absorption curve in the range of 200-600 nm, and determine its maximum absorption wavelength.

[0079] c. Preparation of standard curve: Accurately pipette 0.125, 0.25, 0.4, 0.5, 0.6, 0.8 and 1 mL of standard solution into 10 mL volumetric flasks respectively, and prepare samples according to NaNO2-Al(NO3)3 spectrophotometry. Take the first bottle as blank control solution, measure the absorbance at the maximum absorption wavelength, and draw the standard curve with absorbance (A) as the ordinate and rutin reference solution concentration (C) as the abscissa.

[0080] d. Determination and calculation of total flavonoids content: Accurately weigh the dry powder of Hibiscus hibiscus flower extract and dissolve it in 10 mL of solvent, shake well. Pipette 1 mL of liquid into a 10 mL volumetric flask, add 0.4 mL of 5% sodium nitrite, and let it stand for 6 minutes; add 0.4 mL of 10% aluminum nitrate, and let it stand for 6 minutes; finally, add 4.0 mL of 4% sodium hydroxide and 70% ethanol to the scale, shake well, let it stand for 15 minutes, and measure the absorbance at the maximum absorption wavelength to obtain the total flavonoids content (mg / g) = V×C×X / M.

[0081] Where: V—volume of the extract, mL; C—concentration of the total flavonoids sample solution, mg / mL; X—total multiple of dilution of the total flavonoids extract; M—mass of sample added each time for extraction, g

[0082] The high performance liquid chromatography method is specifically as follows: chromatographic column: C18 column (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile as A, 0.5% phosphoric acid aqueous solution as B, gradient elution method (0-40 min, 10%→17% A; 40-59 min, 17%→33% A; 59-62 min, 33% A; 62-65 min, 33%→10% A); detection wavelength: 384 nm; flow rate: 1.0 mL·min －1 ; Column temperature: 25℃; Injection volume: 20μL.

[0083] The present invention provides the use of the total flavonoid extract prepared by any one of the preparation methods described above in the preparation of a drug for treating melanoma.

[0084] The total flavonoid extract of the invention comprises hyperoside, isoquercetin, quercetin-3'-O-β-glucoside and quercetin-3'-O-glucoside.

[0085] The type and content of the extract prepared by the extraction method of the present invention ensure good therapeutic effect on the melanoma.

[0086] The treatment includes inhibiting tumor cell growth, reducing tumor cell viability, reducing melanoma cell migration ability, inhibiting tumor cell cloning ability and reducing tumor weight.

[0087] Compared with the traditional solvent extraction method, the advantages of the present invention are that it adopts dilute alcohol extraction, applies high-speed homogenization-ultrasonic assisted liquid phase extraction (HSH-UAE), and selects macroporous adsorption resin for separation and purification. The use of alcohol solvents is conducive to the leaching of flavonoid components, and at the same time prevents the large amount of mucus leaching caused by water as the solvent, and avoids the problem of filtration difficulties caused by the increase in the viscosity of the extract. The high-speed homogenization method increases the contact area between the solvent and the hibiscus flower, ensures that the medicinal material and the solvent are fully in contact, and the flavonoids are dissolved more fully, thereby accelerating the extraction speed and improving the extraction efficiency. The ultrasonic assisted extraction method makes the plant tissue cells more completely broken by mechanical vibration, which can reduce the time and increase the extraction efficiency. At the same time, the medicinal material is extracted under a mild dynamic environment, and the heating temperature is low, which avoids the destruction of the structure of the flavonoids due to thermal effect and cavitation effect, and effectively reduces the impurity content in the extract. In the further purification step, the method of separation and impurity removal using macroporous adsorption resin has good purification effect, simple operation, safety, non-toxicity and pollution-free, and can be reused after a little treatment after use, effectively reducing the production cost. Compared with traditional methods, the technical advantage of this method lies in the combined application of high-speed homogenization-ultrasonic assisted liquid extraction (HSH-UAE), which has the advantages of fast extraction speed, high extraction yield, sufficient extraction and retention of effective ingredients, and simple process.

[0088] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0089] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0090] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0091] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0092] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0093] To further illustrate the present invention, a total flavonoids extract from hibiscus flowers provided by the present invention, and a preparation method and application thereof are described in detail below in conjunction with examples.

[0094] Example 1

[0095] A method for preparing a hibiscus flower total flavonoids extract comprises the following steps:

[0096] S1: Weigh and crush dried hibiscus flowers, pass through a 50-mesh No. 3 sieve, and add them to a 50% ethanol solution; the mass volume ratio of hibiscus flowers to ethanol solution is 1 g:50 mL;

[0097] S2: high-speed homogenization at 5000 rpm for 60 min, ultrasonic extraction at 50 °C for 90 min to obtain a crude extract;

[0098] S3: The crude extract is filtered to obtain filtrate A, and ethanol is recovered under reduced pressure at 45°C and 0.09 MPa until it is exhausted, diluted with water, and acetic acid-sodium acetate buffer is added to control the pH to 5;

[0099] S4: pre-treating the AB-8 macroporous resin by soaking it in 95% ethanol by volume for 24 hours, soaking it in 5% hydrochloric acid and sodium hydroxide for 2 hours respectively, and washing it with pure water until it is neutral; using the AB-8 macroporous resin column for adsorption extraction, washing impurities with pure water, and then eluting with ethanol to obtain an eluate;

[0100] S5: the eluate is concentrated and dried at 45°C and 0.09 MPa to obtain a total flavonoid extract of hibiscus flowers, which is crushed to obtain a brown-yellow powder with a bitter taste;

[0101] Example 2

[0102] A method for preparing a hibiscus flower total flavonoids extract comprises the following steps:

[0103] S1: Weigh and crush dried hibiscus flowers, pass through a 50-mesh No. 3 sieve, and add them to a 50% ethanol solution; the mass volume ratio of hibiscus flowers to ethanol solution is 1 g:50 mL;

[0104] S2: high-speed homogenization at 5000 rpm for 60 min, ultrasonic extraction at 60 °C for 120 min to obtain a crude extract;

[0105] S3: The crude extract is filtered to obtain filtrate A, and ethanol is recovered under reduced pressure at 45°C and 0.09 MPa until it is exhausted, diluted with water, and acetic acid-sodium acetate buffer is added to control the pH to 5;

[0106] S4: pre-treating the AB-8 macroporous resin by soaking it in 95% ethanol by volume for 24 hours, soaking it in 5% hydrochloric acid and sodium hydroxide for 2 hours respectively, and washing it with pure water until it is neutral; using the AB-8 macroporous resin column for adsorption extraction, washing impurities with pure water, and then eluting with ethanol to obtain an eluate;

[0107] S5: the eluate is concentrated and dried at 45°C and 0.09 MPa to obtain a total flavonoid extract of hibiscus flowers, which is crushed to obtain a brown-yellow powder with a bitter taste;

[0108] Example 3

[0109] A method for preparing a hibiscus flower total flavonoids extract comprises the following steps:

[0110] S1: Weigh and crush dried hibiscus flowers, pass through a 50-mesh No. 3 sieve, and add them to a 50% ethanol solution; the mass volume ratio of hibiscus flowers to ethanol solution is 1 g:50 mL;

[0111] S2: high-speed homogenization at 6000 rpm for 90 min, ultrasonic extraction at 50 °C for 90 min to obtain a crude extract;

[0112] S3: The crude extract is filtered to obtain filtrate A, and ethanol is recovered under reduced pressure at 45°C and 0.09 MPa until it is exhausted, diluted with water, and acetic acid-sodium acetate buffer is added to control the pH to 5;

[0113] S4: pre-treating the AB-8 macroporous resin by soaking it in 95% ethanol by volume for 24 hours, soaking it in 5% hydrochloric acid and sodium hydroxide for 2 hours respectively, and washing it with pure water until it is neutral; using the AB-8 macroporous resin column for adsorption extraction, washing impurities with pure water, and then eluting with ethanol to obtain an eluate;

[0114] S5: the eluate is concentrated and dried at 45°C and 0.09 MPa to obtain a total flavonoid extract of hibiscus flowers, which is crushed to obtain a brown-yellow powder with a bitter taste;

[0115] Example 4

[0116] A method for preparing a hibiscus flower total flavonoids extract comprises the following steps:

[0117] S1: Weigh and crush dried hibiscus flowers, pass through a 50-mesh No. 3 sieve, and add them to a 70% ethanol solution; the mass volume ratio of hibiscus flowers to ethanol solution is 1 g:75 mL;

[0118] S2: high-speed homogenization at 5000 rpm for 60 min, ultrasonic extraction at 50 °C for 90 min to obtain a crude extract;

[0119] S3: The crude extract is filtered to obtain filtrate A, and ethanol is recovered under reduced pressure at 45°C and 0.09 MPa until it is exhausted, diluted with water, and acetic acid-sodium acetate buffer is added to control the pH to 5;

[0120] S4: pre-treating the AB-8 macroporous resin by soaking it in 95% ethanol by volume for 24 hours, soaking it in 5% hydrochloric acid and sodium hydroxide for 2 hours respectively, and washing it with pure water until it is neutral; using the AB-8 macroporous resin column for adsorption extraction, washing impurities with pure water, and then eluting with ethanol to obtain an eluate;

[0121] S5: the eluate is concentrated and dried at 45°C and 0.09 MPa to obtain a total flavonoid extract of hibiscus flowers, which is crushed to obtain a brown-yellow powder with a bitter taste;

[0122] Example 5

[0123] The difference between this embodiment and embodiment 1 is that the 50% ethanol solution in S1 is replaced by a 90% ethanol solution.

[0124] Example 6

[0125] The difference between this embodiment and embodiment 1 is that the 50% ethanol solution in S1 is replaced by 80% ethanol solution.

[0126] Example 7

[0127] The difference between this embodiment and embodiment 1 is that the 50% ethanol solution in S1 is replaced by a 70% ethanol solution.

[0128] Example 8

[0129] The difference between this embodiment and embodiment 1 is that the 50% ethanol solution in S1 is replaced by a 60% ethanol solution.

[0130] Example 9

[0131] The difference between this embodiment and embodiment 1 is that the ultrasonic temperature in S2 is changed from 50°C to 70°C.

[0132] Example 10

[0133] The difference between this embodiment and embodiment 1 is that the ultrasonic temperature in S2 is changed from 50°C to 60°C.

[0134] Embodiment 11

[0135] The difference between this embodiment and embodiment 1 is that 5000 rpm in S2 is replaced by 7000 rpm.

[0136] Example 12

[0137] The difference between this comparative example and Example 1 is that 5000 rpm in S2 is replaced by 6000 rpm.

[0138] Example 13

[0139] The difference between this embodiment and embodiment 1 is that the high-speed homogenization time of 60 minutes in S2 is changed to 90 minutes.

[0140] Embodiment 14

[0141] The difference between this embodiment and embodiment 1 is that the high-speed homogenization time of 60 minutes in S2 is changed to 70 minutes.

[0142] Embodiment 15

[0143] The difference between this embodiment and embodiment 4 is that the high-speed homogenization time of 60 minutes in S2 is changed to 80 minutes.

[0144] Comparative Example 1

[0145] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the S-8 type macroporous resin.

[0146] Comparative Example 2

[0147] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the D101 type macroporous resin.

[0148] Comparative Example 3

[0149] The difference between this comparative example and Example 1 is that the AB-8 macroporous resin is replaced by HPD600 macroporous resin.

[0150] Comparative Example 4

[0151] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the XDA-1 type macroporous resin.

[0152] Comparative Example 5

[0153] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the LSA-10 type macroporous resin.

[0154] Comparative Example 6

[0155] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the NKA-9 type macroporous resin.

[0156] Comparative Example 7

[0157] The difference between this comparative example and Example 1 is that the AB-8 type macroporous resin is replaced by the X-5 type macroporous resin.

[0158] Comparative Example 8

[0159] The difference between this comparative example and Example 1 is that the mass volume ratio of hibiscus flower and water in S1 is changed from 1 g:50 mL to 1 g:10 mL.

[0160] Comparative Example 9

[0161] The difference between this comparative example and Example 1 is that the mass volume ratio of hibiscus flower and ethanol solution in S1 is changed from 1 g:50 mL to 1 g:5 mL.

[0162] Comparative Example 10

[0163] The difference between this comparative example and Example 1 is that the mass volume ratio of hibiscus flower and ethanol solution in S1 is changed from 1 g:50 mL to 1 g:100 mL.

[0164] Comparative Example 11

[0165] The difference between this comparative example and Example 1 is that the ultrasonic temperature in S2 is changed from 50°C to 40°C.

[0166] Experiment 1: Colorimetric determination of total flavonoids content

[0167] Principle of the test: The flavonoid nucleus contains alkaline oxygen atoms, which generally have phenolic hydroxyl groups and can produce yellow complexes with aluminum ions. Sodium nitrite and sodium hydroxide are added to make it red in alkaline solution. The solution has maximum absorption at 510nm, and the color reaction is stable within 60 minutes. Rutin is used as a reference substance, and aluminum nitrate is used as a color developer for the colorimetric determination of flavonoids. The absorbance is linearly related to the concentration of rutin, and the total flavonoid content is determined by spectrophotometry.

[0168] Preparation of reference solution:

[0169] Accurately weigh 12.5 mg of rutin standard dried to constant weight into a 25 ml volumetric flask, dissolve and dilute with 70% ethanol to prepare a standard solution with a concentration of 0.5 mg / mL.

[0170] Selection of measurement wavelength:

[0171] Referring to the NaNO2-Al(NO3)3 spectrophotometric method, use a pipette to accurately transfer 1 mL of the rutin standard solution and the test solution into a brown 10 mL volumetric flask, and dilute to the same volume with 70% ethanol. Add 0.4 mL of 5% sodium nitrite, and let it stand for 6 minutes; add 0.4 mL of 10% aluminum nitrate, and let it stand for 6 minutes; finally, add 4.0 mL of 4% sodium hydroxide and 70% ethanol to the scale, shake well and let it stand for 15 minutes; at the same time, take 5.0 mL of ethanol and prepare a blank control solution in the same way, scan the absorption curve in the range of 200-600 nm, and determine its maximum absorption wavelength.

[0172] Preparation of standard curve:

[0173] Accurately pipette 0.125, 0.25, 0.4, 0.5, 0.6, 0.8 and 1 mL of the standard solution into 10 mL volumetric flasks, and prepare samples according to the NaNO2-Al(NO3)3 spectrophotometric method. Take the first bottle as the blank control solution, measure the absorbance at the maximum absorption wavelength, and draw a standard curve with the absorbance (A) as the ordinate and the concentration of the rutin reference solution (C) as the abscissa.

[0174] Determination and calculation of total flavonoids content:

[0175] Accurately weigh the dry powder of Hibiscus hibiscus flower extract and dissolve it in 10mL of solvent, and shake it well. Pipette 1mL of liquid into a 10mL volumetric flask, add 0.4mL of 5% sodium nitrite, and let it stand for 6 minutes; add 0.4mL of 10% aluminum nitrate, and let it stand for 6 minutes; finally, add 4.0mL of 4% sodium hydroxide and 70% ethanol to the scale line, shake well, let it stand for 15 minutes, and measure the absorbance at 510nm to obtain the total flavonoid content (mg / g) = V×C×X / M. Where: V-volume of the extract, mL; C-concentration of the total flavonoid determination sample solution, mg / mL; X-total multiple of dilution of the total flavonoid extract; M-mass of sample added each time for extraction, g. The results are shown in Table 1:

[0176]

[0177]

[0178] By comparing the data of Example 1 and Comparative Examples 1 to 7, it is found that the flavonoid content of the total flavonoid extract of hibiscus flower in Example 1 is higher than that of Comparative Examples 1 to 7, indicating that the flavonoid content of the total flavonoid extract of hibiscus flower can be increased by adsorbing flavonoid compounds in the filtrate using AB-8 type macroporous resin;

[0179] The flavonoid content of the total flavonoid extract of hibiscus flower in Example 1 is higher than that in Example 4 and Comparative Examples 8 to 10, indicating that the leaching effect of hibiscus flower flavonoids is best when the mass volume ratio of hibiscus flower to water is 1 g:50 mL;

[0180] The flavonoid content of the total flavonoid extract of hibiscus flower in Example 1 is higher than that in Examples 4 to 8, indicating that the increase in the water content of the extract can increase the flavonoid content of the total flavonoid extract of hibiscus flower; by comparing the data of Examples 1 to 2, Examples 9 to 10 and Comparative Example 11, it is found that when the temperature of ultrasonic-assisted extraction is 50° C. and the time is 90 min, the extraction efficiency of total flavonoids of hibiscus flower is the highest;

[0181] The flavonoid content of the total flavonoid extract of hibiscus flower in Example 1 is higher than that in Example 3 and Examples 11-12, indicating that when the high-speed homogenization rate is 5000rpm, the flavonoid content of the total flavonoid extract of hibiscus flower can be increased; by comparing the data of Example 1 and Example 3 and Examples 13-15, it is found that the flavonoid content of the total flavonoid extract of hibiscus flower in Examples 1 and 3 is higher than that in Examples 13-15, indicating that when the high-speed homogenization time is 60min, the flavonoid content of the total flavonoid extract of hibiscus flower is the highest.

[0182] Experiment 2: Determination of monomer flavonoid content by HPLC:

[0183] Chromatographic column: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile as A, 0.5% phosphoric acid aqueous solution as B; detection wavelength: 365 nm; flow rate: 1.0 mL / min; column temperature: 25 ° C; injection volume: 20 μL

[0184] The Examples and Comparative Examples in which the total flavonoids content in the obtained hibiscus flower total flavonoids extract was higher than 60% were selected to measure the content percentage of the main monomers in the total flavonoids. The results are shown in Table 2 below:

[0185]

[0186]

[0187] Experiment 3: Cell viability assay

[0188] The drugs used in the following experimental examples are the four hibiscus flower extracts with the highest percentage of each main monomer flavonoid in the total flavonoids measured in Experiment 2, and the cell viability detection related experiments were carried out.

[0189] Cell culture and grouping: Mouse melanoma cells (B16) were cultured in 1640 medium containing 20% ​​fetal bovine serum in a cell culture incubator, and cells in the logarithmic growth phase were prepared into suspension for the experiment. The cells were divided into 10 groups, namely control group, blank group, and drug-treated group (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 mg / ml). Each group of cells was set up with 5 replicate wells, and after drug administration, they were inoculated in a 96-well plate. After the corresponding culture time, 10ul CCK-8 solution was added to each well and incubated in an incubator.

[0190] Cell viability was detected using the CCK-8 colorimetric method: the 96-well plate was placed in an incubator at 5% CO and 37°C. After culturing the cells for 24, 48, and 72 hours, 10 microliters of CCK-8 working solution was added to each well, and the cells were further incubated at 37°C for 4 hours. Then, each incubated plate was placed in a microplate spectrophotometer to measure the optical density (OD) at a wavelength of 450nm. The OD values ​​of the blank wells (culture medium and CCK-8 working solution) and the control wells (untreated cells, culture medium, and CCK-8 working solution) were measured at the same time. According to the formula: cell activity = absorbance value of the experimental group - calculated cell viability. Cell inhibition rate (%) = (cell control group A-experimental group A) / (cell control group A-blank control group A) × 100% 1, IC50 was calculated by origin8.5 statistical software.

[0191] Figure 1 .Tumor inhibition effects of various compositions of Hibiscus tiliaceus flower extracts at different concentrations

[0192]

[0193] The experimental results show that the total flavonoids of hibiscus flower prepared in Example 1 has the strongest inhibitory effect on tumor cells. After the total flavonoids of hibiscus flower acted on tumor cells for 48 hours, the cell growth was inhibited to a certain extent, and the viability of melanoma cells in the drug administration group was significantly reduced. As the drug concentration decreased, the cell viability increased. And as the concentration of total flavonoids of hibiscus flower increased, the cell growth inhibition rate increased, showing a good dose-effect relationship (n=3), showing good anti-tumor activity.

[0194] Experiment 4: Cell migration and clone formation assay

[0195] The drug used in the following experimental examples is the hibiscus flower extract prepared according to the method described in Example 1.

[0196] Cell migration assay: Use a 1 ml pipette tip to scratch the wound perpendicular to the marking line of the six-well plate, and wash the cells with PBS to remove debris. Observe the wound healing under an optical microscope after 24, 48, and 72 hours.

[0197] Clone formation experiment: adjust the cell concentration, inoculate cells in a 6-well plate, terminate the culture when the cell colony is greater than 50, and perform fixation, staining, and photography.

[0198] Figure 2 Effects of different concentrations of hibiscus flower extract on the migration ability of B16 cells

[0199] Figure 3 .Effects of different concentrations of hibiscus flower extract on the clone formation ability of B16 cells (*P<0.05, **P<0.01, ****P<0.001)

[0200] The results of the scratch test showed that compared with the control group, the migration ability of B16 melanoma cells was significantly reduced after being treated with high, medium and low doses of the drug groups. Compared with 0h, the scratch area of ​​cells treated with hibiscus flower total flavonoids decreased more significantly at 48h and 72h, and the cell migration rate was lower, which showed that hibiscus flower total flavonoids can effectively reduce the migration ability of melanoma cells ( Figure 2 ). The results of treating B16 with 0, 0.25, 0.5, and 0.75 mg / ml respectively to detect the effect on the cloning ability of melanoma cells indicate that total flavonoids from hibiscus flowers can significantly inhibit the cloning ability of cancer cells ( Figure 3 )

[0201] Experiment 5: Animal Experiment

[0202] The drug used in the following experimental examples is the hibiscus flower extract prepared according to the method described in Example 1.

[0203] Experimental modeling: 30 male mice were selected and distinguished by ear tags. On day 0, melanoma cells were inoculated subcutaneously on the left back of the mice, with an inoculation number of 6×105. Five days after inoculation, the tumor grew to 30mm3. The mice were randomly divided into five groups (blank group, control group, TFH25, 50, 75 mg / kg), with five mice in each group. From the fifth day, the mice were gavaged and the weight and tumor size of the mice were monitored every 2 days. After 13 consecutive days, the tumor tissues of the mice were collected and weighed.

[0204] Histopathological staining: The tumor tissues of each group of mice were dehydrated and embedded. After the slices were baked, they were placed in hematoxylin solution for staining for 3-5 minutes. After gradient dehydration in 85% ethanol and 95% ethanol, they were placed in eosin solution for staining for 5 minutes, and then dehydrated and sealed.

[0205] Table 4

[0206] Tumor weight (g) <![CDATA[Tumor volume (mm 3 )]]> Control group 0.99±0.25 1213.97±337.77 0.25mg / ml 0.89±0.08 1004.24±139.70 0.50mg / ml 0.57±0.25 546.64±54.93 0.75mg / ml 0.24±0.13 343.33±149.24

[0207] Figure 4.The average volume change of tumors in each group after 15 days of treatment (*P<0.05, **P<0.01); Figure 5 .H&E tissue staining results.

[0208] The experimental results showed that total flavonoids from hibiscus flowers showed good anti-tumor effects in mouse models. After 15 days of treatment, the tumors in the mice were removed and weighed, showing that the tumor weight in the drug-treated group was significantly lower than that in the model group (n=4). Especially in the higher-dose group (0.75 mg / kg, P<0.05), the tumor weight was significantly reduced. The average tumor weight in the hibiscus flower total flavonoids-treated group decreased significantly with the increase in dose, and the 0.5 mg / kg and 0.75 mg / kg dose groups showed stronger anti-tumor effects, indicating that the anti-tumor effect of total flavonoids from hibiscus flowers is closely related to its dose, and higher-dose treatment can more effectively inhibit tumor growth. During the treatment with total flavonoids from hibiscus flowers, the results of histopathological examination showed that there was no significant change in the tumor tissue structure of mice in each dose group.

[0209] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a total flavonoid extract from hibiscus flowers, characterized in that: The steps include: A) crushing hibiscus flowers, extracting with an alcohol solvent, filtering and concentrating to obtain an extract; B) purifying the extract using a macroporous resin to obtain an eluent; C) The eluate is concentrated and dried to obtain the product.

2. The preparation method according to claim 1, characterized in that: The alcohol solvent is 50% to 90% ethanol solution, and the extraction solid-liquid ratio is 1:25 g / mL to 1:75 g / mL.

3. The preparation method according to claim 1, characterized in that: The hibiscus flower is dried hibiscus flower, and the crushing mesh size is 50 meshes.

4. The preparation method according to claim 1, characterized in that: Step A) The extraction is carried out by high-speed homogenization-ultrasonic assisted extraction; the high-speed homogenization parameters are: homogenization speed 5000rpm-7000rpm, homogenization time 60min-90min; The ultrasonic-assisted extraction parameters are: ultrasonic power 300W-700W, temperature 50-70°C, and extraction time 60min-120min.

5. The preparation method according to claim 1, characterized in that: The macroporous resin is an AB-8 type macroporous adsorption resin; The resin purification specifically comprises: The extract was adjusted to pH 4-6 using acetic acid-sodium acetate buffer to obtain a loading solution; After the sample solution is loaded, it is eluted with pure water, then with 5% ethanol, and finally with 40-90% ethanol, and the eluate with 40-90% ethanol is collected.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the sample solution to the resin is 1:1-1:2, and the adsorption rate is 1 BV / h; pure water is 5-6 BV of the resin; 5% ethanol solution is 0.5 BV of the resin; 40-90% ethanol is 2-5 BV of the resin; and the elution rate of 40-90% ethanol is 1-1.5 BV / h.

7. The preparation method according to claim 1, characterized in that: The total flavonoid extract comprises hyperoside, isoquercetin, quercetin-3'-O-β-glucoside and quercetin-3'-O-glucoside.

8. A total flavonoids extract from hibiscus flowers, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the total flavonoid extract prepared by the preparation method according to any one of claims 1 to 7 in the preparation of a drug for treating melanoma.

10. The use according to claim 9, characterized in that: The total flavonoid extract includes hyperoside, isoquercetin, gossypol-3'-O-β-glucoside, and quercetin-3'-O-glucoside; more preferably, the flavonoid components have the following mass ratio: 5.0-24.0% hyperoside, 6.0-23.0% isoquercetin, 23.0-33.0% gossypol-3'-O-β-glucoside, and 8.0-23.0% quercetin-3'-O-glucoside; the treatment includes inhibiting tumor cell growth, reducing the cell viability of tumor cells, reducing the migration ability of melanoma cells, inhibiting the cloning ability of tumor cells, and reducing tumor weight.

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