Anti-tumor crude extract containing total flavonoids and solution

Abstract

A preparation method of the anti-tumor crude extract comprises the following steps: step 1, taking plant source powder containing the total flavonoids, adding the plant source powder containing the total flavonoids into an extracting solution according to a solid-liquid ratio of 1: (10-50), and performing constant-temperature treatment under a constant-temperature treatment condition of 40-80 DEG C for 10-50 minutes to obtain the extracting solution containing the total flavonoids; and step 2, drying the extracting solution containing the total flavonoids to obtain an extract solid matter containing the total flavonoids, and the extract solid matter is an anti-tumor crude extract. By improving the extraction process of the general flavone, the crude extract with anti-tumor activity can be obtained, and the method has potential commercial application value.

Description

Technical Field

[0001] This invention relates to the field of natural products, and in particular to an antitumor crude extract and solution containing total flavonoids. Background Technology

[0002] Total flavonoids, as an important class of bioactive substances, are essential nutrients for maintaining normal human life activities. They exhibit diverse and significant biological activities, playing a vital role in antioxidation, anti-inflammation, anti-tumor, antibacterial, and antiviral activity, as well as in the prevention and improvement of diseases such as hypertension and liver fibrosis. They are an important dietary supplement for maintaining health.

[0003] Natural medicines, with their unique biological activity and low side effects, have become a key research focus. There are also studies on the application of natural products in anti-tumor research. However, traditional natural product extraction techniques have the problem of reducing the activity of natural products. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide a crude extract of a natural product with anti-tumor activity.

[0005] This invention discloses an antitumor crude extract containing total flavonoids, the preparation method of which includes:

[0006] Step 1. Take the plant-derived powder containing total flavonoids, add the extract at a material-to-liquid ratio of 1:(10-50), and treat at a constant temperature of 40-80℃ for 10-50 minutes to obtain the extract containing total flavonoids.

[0007] Step 2. The extract containing total flavonoids is dried to obtain a solid extract containing total flavonoids, wherein the solid extract is an antitumor crude extract.

[0008] Furthermore, the preparation method of the plant-derived powder containing total flavonoids is as follows: the plant containing total flavonoids is dried, pulverized, and passed through a 50-200 mesh sieve to obtain the plant-derived powder containing total flavonoids.

[0009] Furthermore, the plant containing total flavonoids is *Hosta lanceolata*.

[0010] Furthermore, the extract is a solution with an ethanol content of 50%-100%.

[0011] Furthermore, the extract comprises a solution with an ethanol content of 70%-90%.

[0012] Furthermore, the constant temperature treatment conditions are 50-70℃.

[0013] Furthermore, the material-to-liquid ratio is 1:(30-40).

[0014] Furthermore, the isothermal treatment time is 20-40 minutes.

[0015] Furthermore, the isothermal treatment is specifically ultrasonic isothermal treatment.

[0016] Another aspect of the present invention discloses a solution containing the above-mentioned crude antitumor extract, wherein the solution preparation method includes:

[0017] Take the crude antitumor extract, add solvent, and filter through a 0.22 μm microporous membrane to obtain the solution.

[0018] Compared with the prior art, the present invention can obtain a crude extract with anti-tumor activity by improving the extraction process of total flavonoids. Attached Figure Description

[0019] Figure 1 This is a graph showing the experimental results of the effect of a single factor on the extraction of total flavonoids in Example 1 of the present invention;

[0020] Figure 2 This is a graph showing the inhibitory effect of different concentrations of total flavonoids on BGC-823 cells in an embodiment of the present invention.

[0021] Figure 3 The cell survival rates after incubation at different concentrations for 24, 48, and 72 hours in the embodiments of the present invention are shown. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0023] The instruments and reagents used in the embodiments of this invention are as follows:

[0024] Test instruments: UV-1800 UV-Vis spectrophotometer, Shanghai Meipuda Instrument Co., Ltd.; ultrasonic cleaner, Shanghai Bilang Instrument Manufacturing Co., Ltd.; micropipette, Prandtl GmbH, Germany; Neo1600R high-speed refrigerated centrifuge, Shanghai Lishen Scientific Instrument Co., Ltd.; GT224C electronic balance, Shanghai Youke Instrument Co., Ltd.; BXH-280S precision programmable oven, Shanghai Boxun Medical Biological Instrument Co., Ltd.; 78-1 electric thermostatic incubator, Huangshi Medical Equipment Factory, Hubei Province; BSC-1000ⅡB2 biosafety cabinet, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; HD-TMXZ McElligott turbidimeter, Shandong Holder Electronic Technology Co., Ltd.

[0025] Test material: Hosta scabra was collected from the campus and identified by Associate Professor Ma Haiying of the College of Ecology and Environment, Yunnan University.

[0026] Test reagents: anhydrous sodium acetate, Tianjin Ruijinte Chemical Co., Ltd.; anhydrous aluminum chloride (AlCl3), Tianjin Yongda Chemical Reagent Co., Ltd.; 95% ethanol and anhydrous ethanol, Liaoning Quanrui Reagent Co., Ltd.

[0027] This invention discloses an antitumor crude extract containing total flavonoids, the preparation method of which includes:

[0028] Step 1. Take the plant-derived powder containing total flavonoids, add the extract at a material-to-liquid ratio of 1:(10-50), and treat at a constant temperature of 40-80℃ for 10-50 minutes to obtain the extract containing total flavonoids.

[0029] Step 2. The extract containing total flavonoids is dried to obtain a solid extract containing total flavonoids, wherein the solid extract is an antitumor crude extract.

[0030] In some optional embodiments, the method for preparing the total flavonoid-containing plant source powder is as follows: the total flavonoid-containing plant is dried, pulverized, and passed through a 50-200 mesh sieve to obtain the total flavonoid-containing plant source powder.

[0031] In some optional embodiments, the plant containing total flavonoids is *Hosta lanceolata*.

[0032] In some optional embodiments, the extract is a solution with an ethanol content of 50%-100%.

[0033] In some optional embodiments, the extract comprises a solution with an ethanol content of 70%-90%.

[0034] In some optional embodiments, the isothermal treatment conditions are 50-70°C.

[0035] In some optional embodiments, the feed-to-liquid ratio is 1:(30-40).

[0036] In some optional embodiments, the isothermal treatment time is 20-40 minutes.

[0037] In some alternative embodiments, the isothermal treatment is specifically ultrasonic isothermal treatment.

[0038] Another aspect of this invention discloses a solution containing the above-mentioned crude antitumor extract, wherein the solution preparation method includes:

[0039] Take the crude antitumor extract, add solvent, and filter through a 0.22 μm microporous membrane to obtain the solution.

[0040] Example 1

[0041] The total flavonoid extraction process of this invention includes:

[0042] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0043] Take 0.5g of powdered hosta folia containing total flavonoids, add it to a solution with 60%-100% ethanol at a material-to-liquid ratio of 1:(10-50), and sonicate at 40-80℃ for 10-50 minutes to obtain an extract containing total flavonoids.

[0044] The embodiments of this invention employ a single-factor experimental design, as shown in the table below. Four factors are selected, with five levels for each factor, to investigate their effects on the extraction of total flavonoids from Hosta salsa.

[0045] Factor A is the effect of ethanol concentration on the extraction of total flavonoids. The specific treatment method is as follows:

[0046] Five bottles of dried powder of Hosta salsa were weighed, 0.5 g per bottle, and 10 mL of 60%, 70%, 80%, 90%, and 100% ethanol solutions were added respectively. Extraction was carried out under the conditions of ultrasonic temperature of 60℃, material-liquid ratio of 1:20, power of 100 W, and ultrasonic time of 30 min.

[0047] Factor B is the effect of ultrasound time on the extraction of total flavonoids. The specific treatment method is as follows:

[0048] Weigh out 5 bottles of dried powder of Hosta salsa flowers, 0.5 g per bottle, add 10 mL of 80% ethanol, with a material-to-liquid ratio of 1:20, and extract at 60℃ for 10 min, 20 min, 30 min, 40 min and 50 min respectively.

[0049] Factor C represents the effect of the solid-liquid ratio on the extraction of total flavonoids. The specific treatment method is as follows:

[0050] Weigh out 5 bottles of dried powder of Hosta salsa, 0.5 g per bottle, add 10 mL of 80% ethanol solution, extract at 60℃, sonicate for 20 min, and set the material-to-liquid ratios to be 1:10, 1:15, 1:20, 1:25, and 1:30 respectively.

[0051] Factor D is the effect of ultrasonic temperature on the yield of total flavonoids. The specific treatment method is as follows:

[0052] Five bottles of dried powder of Hosta fasciata flowers were weighed, 0.5g per bottle, and 10 mL of 80% ethanol was added. The material-to-liquid ratio was 1:30. The samples were extracted by ultrasonication for 20 min at temperatures of 40℃, 50℃, 60℃, 70℃ and 80℃, respectively.

[0053] In each group of single-factor experiments, after each experiment, solid-liquid separation was performed by centrifugation at 12000 r / min for 20 min. The supernatant was collected, diluted to volume, and shaken well. The absorbance value was then measured. Figure 1 As shown.

[0054] Single-factor level table of total flavonoid extraction

[0055]

[0056] Based on the results of the single-factor experiment, select the factors with significant effects and their levels.

[0057] from Figure 1 A indicates that the extraction efficiency of total flavonoids from *Hosta davidii* is best when the ethanol concentration is 80%. When the ethanol volume fraction is between 60% and 80%, the total flavonoid yield increases continuously with increasing ethanol volume fraction, reaching its maximum at 80%. Beyond 80%, the yield decreases. This may be because at low ethanol concentrations, flavonoids are not fully dissolved. At 80% ethanol volume fraction, the solvent polarity is similar to that of the flavonoids in *Hosta davidii*, leading to greater flavonoid dissolution according to the principle of "like dissolves like." Further increases in ethanol volume fraction result in the dissolution of large amounts of fat-soluble substances and plant pigments, competing with flavonoids for dissolution and causing a decrease in the total flavonoid yield. Therefore, orthogonal experiments were conducted using ethanol concentrations of 70%, 80%, and 90%.

[0058] from Figure 1 As shown in Figure B, the total flavonoid yield initially increased and then decreased with prolonged extraction time, reaching its highest value at 20 min. Further increases in extraction time led to a decreasing yield, which may be due to oxidation of the flavonoid extract. Therefore, orthogonal experiments were conducted with extraction times of 10 min, 20 min, and 30 min.

[0059] from Figure 1 As shown in Figure C, the extraction yield of total flavonoids initially increases and then decreases as the solid-liquid ratio decreases. The yield reaches its highest point at a solid-liquid ratio of 1:30. Further increasing the amount of extraction solvent leads to a decrease in the extraction rate. This is because the solubility of total flavonoids in the solvent increases with the amount of solvent, resulting in a higher extraction rate. However, excessive solvent dilution leads to a decrease in the extraction rate. Therefore, orthogonal experiments were conducted with solid-liquid ratios of 1:20, 1:30, and 1:40.

[0060] from Figure 1As shown in D, the total flavonoid yield initially increases and then decreases with increasing water extraction temperature, reaching its highest at 60℃. This may be because a certain range of temperature increases accelerates molecular motion, which is beneficial for the dissolution of flavonoids and cell wall rupture, leading to an increase in the total flavonoid dissolution rate. However, excessively high temperatures may cause flavonoid instability or competition for dissolution with other substances, resulting in a decrease in the total flavonoid yield. Therefore, orthogonal experiments were conducted using extraction temperatures of 50℃, 60℃, and 70℃.

[0061] Example 2

[0062] The total flavonoid extraction process of this invention includes:

[0063] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0064] 0.5g of powdered *Hosta lanceolata* containing total flavonoids was added to a 60% ethanol aqueous solution at a material-to-liquid ratio of 1:10. The mixture was then sonicated at 40℃ for 10 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was 4.778 mg / g. (Yield calculation formula: Yield (mg / g) = c × V / m, where C is the concentration of the crude total flavonoid extract (mg / mL); V is the final volume (mL); and m is the pollen mass (g).)

[0065] Example 3

[0066] The total flavonoid extraction process of this invention includes:

[0067] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0068] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to a 60% ethanol aqueous solution at a material-to-liquid ratio of 1:20, and the mixture was sonicated at 50℃ for 20 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.808 mg / g.

[0069] Example 4

[0070] The total flavonoid extraction process of this invention includes:

[0071] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0072] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to a 60% ethanol aqueous solution at a material-to-liquid ratio of 1:30, and the mixture was sonicated at 60℃ for 30 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.780 mg / g.

[0073] Example 5

[0074] The total flavonoid extraction process of this invention includes:

[0075] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0076] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to a 70% ethanol aqueous solution at a material-to-liquid ratio of 1:30, and the mixture was sonicated at 40℃ for 20 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.724 mg / g.

[0077] Example 6

[0078] The total flavonoid extraction process of this invention includes:

[0079] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0080] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to a 70% ethanol aqueous solution at a material-to-liquid ratio of 1:10, and the mixture was sonicated at 50℃ for 30 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was 5.391 mg / g.

[0081] Example 7

[0082] The total flavonoid extraction process of this invention includes:

[0083] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0084] 0.5g of powdered *Hosta lanceolata* containing total flavonoids was added to a 70% ethanol aqueous solution at a material-to-liquid ratio of 1:20, and the mixture was sonicated at 60℃ for 10 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.530 mg / g.

[0085] Example 8

[0086] The total flavonoid extraction process of this invention includes:

[0087] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0088] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to an 80% ethanol aqueous solution at a material-to-liquid ratio of 1:20, and the mixture was sonicated at 40℃ for 30 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.780 mg / g.

[0089] Example 9

[0090] The total flavonoid extraction process of this invention includes:

[0091] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0092] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to an 80% ethanol aqueous solution at a material-to-liquid ratio of 1:30, and the mixture was sonicated at 50℃ for 10 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.947 mg / g.

[0093] Example 10

[0094] The total flavonoid extraction process of this invention includes:

[0095] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0096] 0.5g of powdered *Hosta davidii* containing total flavonoids was added to an 80% ethanol aqueous solution at a material-to-liquid ratio of 1:10, and the mixture was sonicated at 60℃ for 20 min to obtain an extract containing total flavonoids. This process was repeated 5 times. The average yield of total flavonoids was determined to be 5.641 mg / g.

[0097] Example 11

[0098] The total flavonoid extraction process of this invention includes:

[0099] After drying, the flowers of the sword-leaf hosta are pulverized and passed through a 200-mesh sieve to obtain sword-leaf hosta powder, which is a plant-derived powder containing total flavonoids.

[0100] Take 0.5g of powdered hosta folia containing total flavonoids, add it to an aqueous solution with 90% ethanol at a material-to-liquid ratio of 1:40, and sonicate at 60℃ for 20min to obtain an extract containing total flavonoids.

[0101] The methods of this invention are listed in 3 groups, with each group being repeated 3 times. The total flavonoid yield is measured as shown in the table below.

[0102]

[0103] It can be seen that, compared with Examples 2-10, Example 11 of the present invention has a significantly improved yield, and can achieve efficient extraction of total flavonoids.

[0104] Example 12

[0105] The total flavonoid extract obtained in Example 11 was evaporated to obtain an extract paste (water content approximately 5%-10%), with a total flavonoid content of approximately 60% or more. 100 mg of the extract paste was dissolved in 10 mL of 1% DMSO aqueous solution to achieve a total flavonoid concentration of 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, and 45 mg / mL, respectively, and their antitumor activity was tested.

[0106] Antitumor activity assay: Following the CCK-8 assay kit procedure, the inhibitory effect of total flavonoids from *Hosta davidii* on the growth of human gastric adenocarcinoma cells BGC-823 was determined. Cell suspensions were seeded in 96-well plates and cultured overnight. Then, samples of various concentrations were added, and the plates were incubated for a certain period. 10 μL of CCK-8 solution was added to each well, and after 1 h of incubation, the absorbance of each well was measured at 450 nm using a microplate reader. The cell inhibition rate was calculated as [(ODc-ODs) / (ODc-ODb)]×100%; the survival rate was calculated as (ODs–ODb) / (ODc–ODb)×100%. ODs: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); ODc: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drug); ODb: Absorbance of blank wells (containing culture medium, CCK-8 solution, and drug, but excluding cells). The half-maximal inhibitory concentration (IC50) was calculated using curves constructed with Origin 2021 software.

[0107] Curves were constructed using Origin 2021 software, and IC50 values ​​were calculated. The results are shown below. Figure 2 The IC50 value of total flavonoids was 23.84 μg / mL, indicating that total flavonoids from the flower had high inhibitory activity against BGC-823 cells. The concentration-dependent inhibition of cell viability was significant, as shown in the experimental results. Figure 3As shown, total flavonoids from *Hosta* exhibited a significant concentration-dependent inhibitory effect on cell viability. At a concentration of 20 μg / mL, cells maintained high viability for 24 h, indicating that total flavonoids at this concentration had low cytotoxicity and cells could maintain normal metabolic function. However, cell viability decreased significantly at 48 h and 72 h. When the concentration was increased to 30 μg / mL, cell viability significantly decreased at 24 h. Further increasing the concentration to 40 μg / mL did not result in a significant difference in cell viability compared to 30 μg / mL. This suggests that 30 μg / mL may be the critical concentration at which total flavonoids from *Hosta* exert cytotoxicity. This phenomenon indicates that total flavonoids from *Hosta* exhibit a threshold effect on cells; lower concentrations may have mild biological activity, while higher concentrations can induce cell damage.

[0108] The inhibitory effect of total flavonoids from Hosta salsa on gastric cancer BGC823 cells in this invention showed that the IC50 of total flavonoids was lower than that of many natural products, and the inhibitory effect increased over time, suggesting that it may exert its effect by inducing apoptosis or cell cycle arrest, indicating its strong anti-tumor potential.

[0109] In summary, the total flavonoids from *Hosta salsa* exhibit antitumor activity in the embodiments of this invention, providing strong scientific evidence for the development of natural drugs and demonstrating enormous development potential and broad application prospects.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.

Claims

1. A crude extract containing total flavonoids for antitumor activity, characterized in that, The method for preparing the antitumor crude extract includes: Step 1. Take the plant-derived powder containing total flavonoids, add the extract at a material-to-liquid ratio of 1:(10-50), and treat it at a constant temperature of 40-80℃ to obtain an extract containing total flavonoids. Step 2. The extract containing total flavonoids is dried to obtain a solid extract containing total flavonoids, wherein the solid extract is an antitumor crude extract.

2. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The preparation method of the plant-derived powder containing total flavonoids is as follows: the plant containing total flavonoids is dried, pulverized, and passed through a 50-200 mesh sieve to obtain the plant-derived powder containing total flavonoids.

3. The antitumor crude extract containing total flavonoids according to claim 2, characterized in that, The plant containing total flavonoids is *Hosta lanceolata*.

4. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The extract is a solution with an ethanol content of 50%-100%.

5. The antitumor crude extract containing total flavonoids according to claim 4, characterized in that, The extract comprises a solution with an ethanol content of 70%-90%.

6. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The constant temperature treatment conditions are 50-70℃.

7. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The material-to-liquid ratio is 1:(30-40).

8. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The constant temperature treatment time is 20-40 minutes.

9. The antitumor crude extract containing total flavonoids according to claim 1, characterized in that, The constant temperature treatment is specifically ultrasonic constant temperature treatment.

10. A solution containing the antitumor crude extract according to any one of claims 1-9, characterized in that, The solution preparation method includes: Take the crude antitumor extract, add solvent, and filter through a 0.22 μm microporous membrane to obtain the solution.

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