Method for extracting and purifying quercetin, and preparation method and application of antioxidant
Through microwave-assisted extraction, ultrasonic enhancement and dual resin purification technology, the problems of low quercetin extraction efficiency and insufficient purity were solved, and high-purity quercetin was prepared, which is an antioxidant used for gastric tumor treatment, with significant anti-cancer activity and environmental protection advantages.
Patent Information
- Application Number
- CN202510209511.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, the extraction and purification of quercetin is inefficient and costly, and traditional methods are prone to damage their structure and purity, which is difficult to guarantee, making it difficult to meet the application needs of gastric tumor treatment.
Microwave-assisted extraction combined with ultrasonic strengthening extraction, double purification was performed using tea polyphenol cross-linked sodium alginate resin and polyamide resin, combined with recrystallization technology, high-purity quercetin was prepared, and antioxidants were prepared with tea polyphenols and carrier materials.
It improves the extraction efficiency and purity of quercetin, reduces energy consumption and environmental pollution, enhances antioxidant performance, significantly inhibits tumor cell growth, and has good anti-cancer activity.
Smart Images
Figure CN120271542A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and particularly to the preparation and application of an antioxidant containing quercetin. Background Art
[0002] Quercetin, as an important member of the flavonoid compound family, is widely distributed in various plants in nature. Its unique antioxidant, anti-inflammatory and potential anti-tumor properties have shown great application potential for quercetin in the biomedical field. Quercetin can scavenge free radicals and reduce oxidative stress reactions, thus providing new ideas for the prevention and treatment of some diseases.
[0003] However, despite the many pharmacological activities of quercetin, its efficient extraction and deep purification are still current technical problems. Traditional extraction methods, such as multiple heating reflux, concentration and fine extraction, etc., are not only inefficient, but also easily damage the structure of quercetin, affect its physiological activity, and the purity of the obtained product is often difficult to guarantee, and the accompanying cost is relatively high. In addition, due to the low content of quercetin in plants, the initial work of enriching quercetin is difficult to implement, and the process is often accompanied by extremely high energy consumption, such as long-time heating and multiple refluxes, which not only restricts the production cost, but also burdens the environment.
[0004] In recent years, some progress has been made in the research on quercetin in anti-tumor. Research shows that quercetin can inhibit the growth and proliferation of tumor cells, induce apoptosis of tumor cells, and regulate tumor-related signaling pathways. These research results provide a theoretical basis for the application of quercetin in the treatment of tumors. However, despite certain achievements in the research, the preferred application plan of quercetin in the treatment of gastric tumors still needs to be further explored. Therefore, there is an urgent need for an efficient, safe and environmentally friendly quercetin extraction method, as well as in-depth research on the application of quercetin in the direction of gastric tumors. Summary of the Invention
[0005] In view of the problems existing in the prior art, on the first hand, the present invention provides a method for extracting and purifying quercetin.
[0006] The technical solution of the present invention to solve the above problems is as follows:
[0007] Provide a method for extracting and purifying quercetin, including the following steps:
[0008] S1. Place the sophora flower powder in a microwave-assisted extraction device, perform microwave extraction with a mixed solvent, and then perform ultrasonic enhanced extraction to obtain a crude quercetin extract; in this step, the material-liquid ratio of microwave extraction is 1:(12-18) g / mL; the mixed solvent is an ethanol aqueous solution with a volume fraction of 65-75%.
[0009] S2. Load the crude quercetin extract onto the activated tea polyphenol cross-linked sodium alginate resin column, let it stand for at least 30 min, then pass it through the resin column, and subsequently elute with an ethanol aqueous solution with a volume fraction of 65% - 75% to obtain a preliminarily purified quercetin solution;
[0010] S3. Load the preliminarily purified quercetin solution onto the activated polyamide resin column, let it stand for at least 30 min, wash it with at least 3 times the volume of distilled water, and then elute with an ethanol aqueous solution in an ascending gradient concentration to obtain a quercetin eluate;
[0011] S4. Concentrate the quercetin eluate under vacuum to no less than 3 mg / mL, and then cool it to precipitate crystal deposits;
[0012] S5. Recrystallize the crystal deposits to obtain highly purified quercetin.
[0013] Compared with the traditional methods of multiple heating reflux and fine extraction, the above technical solution of the present invention adopts the technology of microwave-assisted extraction combined with ultrasonic enhanced extraction, significantly improving the extraction efficiency of quercetin, shortening the extraction time, and reducing the energy consumption. At the same time, through the double purification of the tea polyphenol cross-linked sodium alginate resin column and the polyamide resin column, impurities are effectively removed, the purity of quercetin is improved, and the physiological activity of the product is ensured not to be damaged. This innovative method not only solves the problems of high cost, long time consumption, and low efficiency of traditional extraction methods, but also eliminates the drawback that high-temperature treatment will damage the molecular structure of quercetin, thereby improving the quality and application effect of the final product.
[0014] Compared with the traditional methods of multiple heating reflux and fine extraction, the above technical solution of the present invention that adopts the technology of microwave-assisted extraction combined with ultrasonic enhanced extraction reduces the usage amount of organic solvents and the generation amount of waste liquid, and reduces the environmental pollution.
[0015] As a preference of the above technical solution, the microwave extraction specifically uses a microwave-assisted extraction device with a power of 500 W - 750 W, and the temperature is adjusted to 70 - 80 °C for extraction for 10 - 15 min.
[0016] As a preference of the above technical solution, the ultrasonic enhanced extraction specifically uses an ultrasonic extraction device with a power of 190 - 220 W, and extracts at 40 - 45 °C for 10 - 15 min.
[0017] As a preference of the above technical solution, the mass concentration of the preliminarily purified quercetin solution in step S2 is 4.5 - 5.5 mg / mL; or the preliminarily purified quercetin solution loaded onto the polyamide resin column in step S3 is pre-adjusted to 4.5 - 5.5 mg / mL.
[0018] As an optimization of the above technical solution, in step S2, the sample loading flow rate is 1.8 - 2.5 mL / min, the elution flow rate is 1.8 - 2.5 mL / min, and the volume of the eluent is 1 - 1.5 times that of the sample solution.
[0019] As an optimization of the above technical solution, in step S2, standing for at least 30 min specifically means standing for 2 - 2.5 h.
[0020] As an optimization of the above technical solution, in step S3, the elution flow rate is 1.5 - 1.8 mL / min.
[0021] As an optimization of the above technical solution, in step S3, standing for at least 30 min specifically means standing for 2 - 2.5 h.
[0022] As an optimization of the above technical solution, in step S3, the gradient concentrations are respectively 45% - 55%, 65% - 75%, and 85% - 95% by volume fraction.
[0023] As an optimization of the above technical solution, in step S3, at least 3 times the amount of distilled water specifically means 5 - 6 times the amount of distilled water.
[0024] As an optimization of the above technical solution, in step S4, the crystal precipitate is also dried to obtain quercetin dry powder; in step S5, the recrystallization specifically means first dissolving the quercetin dry powder in a first solvent with relatively high solubility, then adding a second solvent with relatively low solubility until it becomes turbid, and then heating it to clarity, allowing it to cool, and precipitating crystals to obtain purified quercetin.
[0025] As an optimization of the above technical solution, in step S4, drying the crystal precipitate specifically means drying at 65°C - 70°C.
[0026] As an optimization of the above technical solution, in step S4, concentrating to no less than 3 mg / mL under vacuum specifically means concentrating to a quercetin content of 3.5 - 10.0 mg / mL under vacuum.
[0027] As an optimization of the above technical solution, in step S4, the cooling temperature when cooling to precipitate crystals is 0 - 5°C.
[0028] As an optimization of the above technical solution, in step S5, the mass ratio of quercetin dry powder to the mixed volume of the first and second solvents is 1:25 - 1:30 g / mL.
[0029] As an optimization of the above technical solution, in step S5, the combination of the first solvent - second solvent is ethanol - water, methanol - water, or methanol - ethanol; the volume ratio of the first solvent to the second solvent is 1:(0.5 - 2).
[0030] Preferably, as the above technical solution, the tea polyphenol crosslinked sodium alginate resin is prepared by a method comprising the following steps:
[0031] 1), Add sodium alginate to deionized water, heat to 60 - 75 °C, and stir simultaneously until completely dissolved, remove air bubbles, to obtain a sodium alginate solution; Add tea polyphenols to absolute ethanol, and stir simultaneously until completely dissolved, to obtain a tea polyphenol solution;
[0032] 2), Add the tea polyphenol solution to the sodium alginate solution, while adding, stir magnetically, after mixing evenly, perform ultrasonic treatment, to obtain a sodium alginate / tea polyphenol mixture;
[0033] 3), Prepare a 3% - 8% CaCl2 solution as a crosslinking agent; Use a syringe equipped with a needle to drop the sodium alginate / tea polyphenol mixture into the CaCl2 solution drop by drop to form gel beads, and crosslink in the CaCl2 solution for at least 30 min to obtain crosslinked gel beads;
[0034] 4), Take out the crosslinked gel beads, wash with deionized water to remove residual CaCl2 and unreacted tea polyphenols; Subsequently, place the washed gel beads in a petri dish, wipe off the surface moisture, and then perform freeze-drying.
[0035] In the above technical solution of the present invention, a 3% - 8% CaCl2 solution is prepared as a crosslinking agent. Calcium ions can complex with the COO- groups of sodium alginate in water to promote the formation of gel. Tea polyphenols can also complex with calcium ions to further promote the formation of gel. And sodium alginate, as a natural polysaccharide, has the stability and safety required for pharmaceutical excipients. Tea polyphenols, also as natural substances, have stability and safety. Therefore, even if a trace amount of sodium alginate and / or tea polyphenols are introduced during the purification of quercetin, there is no need to specifically remove them. And this solution also avoids the use of traditional chemical crosslinking agents, further improving the environmental friendliness of the entire production process; At the same time, the tea polyphenol crosslinked sodium alginate resin prepared by the freeze-drying method can endow the resin with a porous property, meeting the actual requirements as an adsorption resin.
[0036] In the second aspect, the present invention provides a preparation method of an antioxidant.
[0037] A preparation method of an antioxidant, comprising:
[0038] Mixing quercetin, tea polyphenols, and a flowing carrier material in any order to form a material liquid process; and
[0039] Spray-drying or freeze-drying the material liquid to prepare an antioxidant containing quercetin process.
[0040] In the above technical solution of the present invention, by combining high-purity quercetin and tea polyphenols with a carrier material and then drying to prepare an antioxidant, not only the bioavailability of quercetin is improved, but also the product is endowed with stronger antioxidant performance. The application research of this antioxidant in gastric tumors shows that it can effectively inhibit the growth and proliferation of tumor cells and exhibits good anti-cancer activity. This discovery provides a new idea for the application of quercetin in the prevention and treatment of gastric tumors and has important clinical significance and potential market value.
[0041] As a preference of the above technical solution, the mass ratio of quercetin, tea polyphenols, and the carrier material is 1:(0.1 - 0.3):(6 - 8).
[0042] As a preference of the above technical solution, quercetin, tea polyphenols, and the flowing carrier material are mixed evenly in any order. Specifically, quercetin and the carrier material are first mixed evenly to obtain a basic solution, and then tea polyphenols are mixed with the basic solution.
[0043] As a preference of the above technical solution, the flowing carrier material is an aqueous solution of pregelatinized starch and / or maltodextrin.
[0044] As a preference of the above technical solution, the concentration of the aqueous solution of pregelatinized starch and / or maltodextrin is 10% - 30% (w / v).
[0045] As a preference of the above technical solution, the specific process parameters of the spray drying are: inlet air temperature 160 - 180 °C, feeding speed 30 - 40 r / min, and liquid concentration 10 - 15%.
[0046] As a further preference of the above technical solution, the specific process parameters of the spray drying are: inlet air temperature 170 °C, feeding speed 35 r / min, and liquid concentration 12%.
[0047] In the third aspect, the present invention provides the application of the above antioxidant.
[0048] The application of the antioxidant prepared according to the foregoing method in the preparation of drugs for treating gastric tumors. Description of the Drawings
[0049] Figure 1 It is the chromatogram of quercetin extracted and purified in Example 1 of the present invention;
[0050] Figure 2 It is the result graph of the DPPH free radical scavenging ability of the antioxidant prepared from the quercetin extracted and purified in Example 1;
[0051] Figure 3 It is the result graph of the ·OH free radical scavenging ability of the antioxidant prepared from the quercetin extracted and purified in Example 1;
[0052] Figure 4 The result graph of the therapeutic effect of the quercetin-containing antioxidant prepared for the present invention on gastric (cancer) cells;
[0053] Figure 5 The tumor growth curve graph of the therapeutic effect of the quercetin-containing antioxidant prepared for the present invention on mice;
[0054] Figure 6 The detection result graph of biochemical indexes of the therapeutic effect of the quercetin-containing antioxidant prepared for the present invention on mice;
[0055] Figure 7 The pathological staining graph of the therapeutic effect of the quercetin-containing antioxidant prepared for the present invention on mice. Detailed implementation manners
[0056] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. The embodiments are only explanations of the present invention and do not limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims.
[0057] Example 1
[0058] S1 Raw material treatment
[0059] Use an ultrafine pulverizer to pulverize fully dried sophora flower buds to 500 meshes, ensure that the powder is fine and uniform, and set aside;
[0060] S11 Extraction
[0061] Add sophora flower bud powder to an ethanol-water mixed solvent with an ethanol volume fraction of 70%; the solid-liquid ratio is 1:15 g / mL; place this mixture in a microwave-assisted extraction device, set the microwave power to 750 W, adjust the extraction temperature to 75 °C, and set the microwave extraction time to 12 minutes; subsequently, use an ultrasonic processor to process it at 42 °C and 220 W for 18 minutes to promote the full release of the active ingredients in sophora flower buds; after extraction, filter the extract to remove impurities to obtain a crude quercetin extract with a concentration of 5.5 mg / mL, and set aside;
[0062] S12 Purification
[0063] First, take 70 mL of the crude quercetin extract (concentration 5.5 mg / mL) and load it onto an activated tea polyphenol cross-linked sodium alginate resin column (2.5 g), and let it stand for 2.5 hours for adsorption; then, pass it through the resin column at a flow rate of 2.5 mL / min; afterwards, elute it with 70 mL of ethanol (75 vol%), and set the elution speed to 2.5 mL / min as well, collect the eluate to obtain a preliminarily purified quercetin solution;
[0064] Then, the preliminary purified quercetin solution was loaded onto a polyamide resin column and statically adsorbed for 2.5 hours to ensure complete adsorption. Subsequently, the polyamide column was rinsed with 6 times the volume of distilled water to remove impurities. Then, ethanol-aqueous solutions with gradient concentrations of 55%, 75%, and 95% by volume were used as eluents for ascending elution. Each fraction was 12 mL, the elution flow rate was 1.7 mL / min, and the eluate was collected. The eluate was concentrated under vacuum to a quercetin content of 5.0 ± 0.1 mg / mL. It was left to stand at 4°C to precipitate crystals, the precipitated crystals were filtered, and dried at 70°C to obtain crude quercetin.
[0065] Finally, according to the total material-liquid ratio of 1:30 g / mL, the crude quercetin was recrystallized using a methanol-ethanol mixed solvent. Specifically, the crude quercetin was first dissolved in methanol, then an equal amount of ethanol was added, resulting in turbidity. Subsequently, it was slowly heated to clarity, allowed to cool, and crystals precipitated to obtain purified quercetin.
[0066] S2 Preparation of antioxidant
[0067] The purified quercetin and maltodextrin were mixed evenly at a mass ratio of 1:7, and then 20% of the mass of quercetin of tea polyphenols was added as an antioxidant synergist and mixed evenly again. It was spray-dried to obtain an antioxidant. The specific process parameters of spray drying were: the inlet air temperature was set at 170°C, the feeding speed was adjusted to 35 r / min, and the feed liquid concentration was set at 12% to ensure the stability of the spray drying process and the uniformity of product quality.
[0068] In the above S12 purification step, the tea polyphenol cross-linked sodium alginate resin was prepared by a method comprising the following steps:
[0069] 1) Dissolve 2 g of sodium alginate powder in 100 mL of deionized water, heat and stir in a water bath at 65°C for 1 hour until the sodium alginate is completely dissolved, and remove the bubbles to obtain a sodium alginate solution. Dissolve 1 g of tea polyphenol powder in 50 mL of absolute ethanol, and magnetically stir at room temperature for 30 minutes until the tea polyphenol is completely dissolved to obtain a tea polyphenol solution.
[0070] 2) Slowly drip the tea polyphenol solution into the sodium alginate solution while magnetically stirring. After stirring and mixing evenly, perform ultrasonic treatment for 30 minutes using an ultrasonic cell disruptor. The ultrasonic interval is 5 seconds of work and 5 seconds of pause, and the amplitude is set at 60%.
[0071] 3) Prepare a 5% CaCl2 solution as a cross-linking agent. Pour the sodium alginate / tea polyphenol mixture into a syringe with a needle diameter of 1 mm. Fix the syringe on a titration device, and drop the liquid droplets into the CaCl2 solution one by one to form gel beads, and cross-link in the CaCl2 solution for 2 hours.
[0072] 4) Take out the crosslinked gel beads, wash them 3 times with deionized water to remove residual CaCl2 and unreacted tea polyphenols; then put the washed gel beads into a petri dish and dry the surface moisture with absorbent paper; put the petri dish into a freeze dryer, pre-freeze it to below -40 °C, then evacuate and freeze-dry for 48 hours.
[0073] Example 2
[0074] Use high performance liquid chromatography to determine the purity of the quercetin extracted and purified in Example 1
[0075] Inject a standard quercetin solution with a known concentration, record the corresponding mV value, and plot the relationship curve between concentration and mV value. The concentration X is the abscissa; the mV value is the ordinate, represented by Y. Plot the regression equation of the quercetin standard curve, Y = 3.29×107X + 63778.2, R2 = 0.9995; R2 is the value obtained by software calculation and fitting.
[0076] Then, by measuring the mV value of quercetin in the test sample, from Figure 1 it can be seen that it is about 58 mV to calculate the quercetin concentration in the product.
[0077] Finally, calculate the quercetin purity with (quercetin concentration / product concentration) × 100%, and the product concentration can be directly measured. The output result of the quercetin purity computer is 98.78%. In fact, qualitative observation can be roughly seen from Figure 1 the ratio of the peak area to the area under the curve in
[0078] This is because in the present invention, sophora flower buds are pulverized to 400 - 500 meshes by using an ultrafine pulverizer, which greatly increases the contact area between the raw material and the solvent, thereby improving the extraction efficiency of quercetin; at the same time, combined with microwave-assisted extraction and ultrasonic enhanced extraction technologies, it further accelerates the release of active ingredients and ensures the efficient extraction of quercetin;
[0079] Secondly, in the purification process, the present invention ingeniously uses tea polyphenol crosslinked sodium alginate resin and polyamide resin for two-step purification; the tea polyphenol crosslinked sodium alginate resin, relying on its unique adsorption performance, can efficiently adsorb quercetin from the extract and remove most of the impurities at the same time; while the polyamide resin further purifies the adsorbed quercetin, and through impurity removal with distilled water and elution with ethanol solutions of gradient concentrations, effectively removes the remaining trace impurities, thereby obtaining quercetin with extremely high purity.
[0080] Example 3
[0081] Performance evaluation: For the antioxidant prepared from the quercetin extracted and purified in Example 1, the following indexes are measured.
[0082] Determination of antioxidant property: Quercetin (Qu) purified by extraction in Example 1, the prepared antioxidant containing quercetin (Qu+GTP), and VC were formulated into different mass concentrations (0.125, 0.250, 0.375, 0.500, 0.725, 1.000, 2.000, 3.000, 4.000 mg / mL). Using VC as a control, the antioxidant property was determined.
[0083] ① Using the DPPH free radical scavenging ability detection method, the scavenging ability of the antioxidant on DPPH free radicals was detected: The Qu antioxidant solution, Qu+GTP antioxidant solution were mixed with the newly prepared 0.1 mmol / L DPPH solution in equal volume and mixed evenly. After reacting in the dark for 30 min, the absorbance value at 517 nm was measured. Using the same method with VC as a control, the scavenging rate was calculated according to the formula: DPPH· scavenging rate = (1 - (A1 - A2) / A0) × 100%; where, in the formula, A1, A2, and A0 are the absorbance values of the blank group, the sample group, and the absorbance value of absolute ethanol replacing the DPPH· solution, respectively.
[0084] ② ·OH free radical scavenging ability: 2 mL of the Qu antioxidant solution and Qu+GTP antioxidant solution were added successively, 1 mL of the newly prepared FeSO4 solution and H2O2 solution with the same concentration (6 mmol / L) were added, and finally 1 mL of the salicylic acid-ethanol solution was added. After shaking well, it was placed in a constant temperature water bath at 37°C for 30 min. The absorbance value was measured at 510 nm. Using VC as a control at the same time, the scavenging rate was calculated according to the formula: ·OH free radical scavenging rate = (1 - (A1 - A2) / A0) × 100%; where, in the formula, A1, A2, and A0 are the absorbance values of the blank group, the absorbance value after adding the sample solution, and the absorbance value without adding the H2O2 solution, respectively.
[0085] Experimental conclusion
[0086] As Figure 2 shown, the detection results of the DPPH free radical scavenging ability indicate that the antioxidants prepared from the quercetin purified by extraction in Example 1 all have a significant DPPH scavenging effect, which is better than the single quercetin solution, and the scavenging rate is positively correlated with the concentration. Through the ·OH free radical scavenging ability ( Figure 3 ), it was further found that the antioxidants prepared in Example 1 all showed good ·OH free radical scavenging ability (also better than the single quercetin solution), and with the increase of the concentration, the scavenging rate gradually increased.
[0087] Compared with Figure 2 the corresponding data is shown in Table 1.
[0088] Table 1 Antioxidant property determination table for each group (DPPH· scavenging rate)
[0089] Mass concentration (mg / mL) Vc Qu Qu + GTP 0.1250 80.10 57.39 67.543 0.2500 80.20 61.45 69.876 0.3750 80.34 65.27 72.456 0.5000 80.36 69.12 75.345 0.7250 81.00 70.23 77.278 1.0000 81.35 73.67 78.567 2.0000 81.50 77.78 82.123 3.0000 81.80 81.22 86.005 4.0000 82.00 83.55 90.576
[0090] The data corresponding to Figure 3 is shown in Table 2.
[0091] Table 2 Antioxidant Activity Determination Table for Each Group (·OH Scavenging Rate)
[0092] Mass concentration (mg / mL) Vc Qu Qu + GTP 0.1250 82.100 57.22 68.423 0.2500 83.123 61.34 70.756 0.3750 83.145 65.28 73.098 0.5000 83.667 69.12 75.342 0.7250 83.982 71.29 77.876 1.0000 84.234 72.89 79.657 2.0000 84.265 77.84 82.413 3.0000 84.298 80.23 85.315 4.0000 84.483 83.23 88.901
[0093] Example 4
[0094] Antitumor Effect in Vitro
[0095] Suspend gastric cancer cells HGC-27 and normal gastric cells GES-1 in the culture medium, adjust the cell density to 1×10 4 cells / mL, inoculate 100 μL into a 96-well plate. After culturing at 37 °C for 24 h, discard the original culture medium in the wells. Divide the HGC-27 cells and GES-1 cells into 3 groups respectively: Control, Qu, and Qu+GTP group (20 μM). Add 100 μL of the culture medium containing the above groups, continue to incubate for 24 h, then add 10 μL of CCK8 solution into the wells, incubate at 37 °C in the dark for 1.5 h, measure the absorbance at 450 nm, and calculate the cell viability.
[0096] The results are shown in Table 3.
[0097] Table 3 Cell Viability Determination of Gastric Cancer Cells HGC-27 and Normal Gastric Cells GES-1 (Absorbance at 450 nm)
[0098] Qu Qu + GTP Control Qu Qu + GTP GES-1 99.3 98.0 99.2 99.3 - - - HGC-27 - - 99 98 78 80 61 63
[0099] By measuring the viability of HGC-27 / GES-1 cells using the CCK8 method, we found that there were significant differences in the effects of different treatment groups on cell viability. As Figure 4 shown, compared with the Control group, the cell viability of the normal gastric cell Qu (quercetin) treatment group remained almost unchanged, and there was no significant difference in the absorbance value, which fully demonstrated that the quercetin extracted and purified in the present invention had good biocompatibility and low toxicity in normal gastric cells. The Qu+GTP (quercetin + tea polyphenols, that is, the new antioxidant prepared in the present invention) treatment group also showed cell viability similar to that of the Control group, further verifying the safety and biocompatibility of the antioxidant of the present invention in normal gastric cells.
[0100] For the gastric cancer cell HGC-27 group, the Qu treatment group significantly inhibited the growth of gastric cancer cells, indicating that quercetin has a significant inhibitory effect on gastric cancer cells. More importantly, the inhibitory effect of the Qu+GTP treatment group on gastric cancer cells was more significant, which fully demonstrated that the antioxidant containing quercetin prepared in the present invention was superior to the treatment with quercetin alone in inhibiting the growth of gastric cancer cells and synergistically inhibited the growth of gastric cancer cells HGC-27.
[0101] Example 5
[0102] Antitumor effect in vivo
[0103] Twenty healthy male C57BL / 6 mice (20 - 22 g) aged 6 - 8 weeks were selected and raised in an animal house. They were fed with basal diet for one week to adapt to the environment, and then randomly divided into four groups: normal group (ND), model group (MD), quercetin group (Qu), and antioxidant group (Qu+GTP), with 5 mice in each group and 5 mice in each cage. The mice in the ND and MD groups were continuously fed with ordinary feed and gavaged with a fixed amount of pure water. The mice in the Qu group were fed with ordinary feed and gavaged with 50 mg / kg quercetin extract. The mice in the Qu+GTP group were continuously fed with ordinary feed and gavaged with 50 mg / kg antioxidant (Qu+GTP, calculated by the active ingredient) for 14 days. On the 8th day, except for the normal group (ND), the other three groups (MD, Qu, Qu+GTP) of mice were subcutaneously injected with MFC gastric cancer cell suspension to establish a tumor model. After injection, the mental state, activity ability, and tumor growth of the mice were observed daily. The body weights of all mice were measured every 2 days, and the long diameter (L) and short diameter (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the formula V = 0.5×(L×W 2 )). At the end of the 14-day experiment, blood samples of the mice were collected by orbital blood sampling, and the serum was separated for detecting gastric cancer-related tumor markers (CEA), antioxidant enzyme activity (SOD), and oxidative stress index (MDA), etc., to evaluate the physiological state and antioxidant capacity of the mice in each group. After the mice were sacrificed, the tumor tissues were quickly dissected and fixed in formalin for histological examination (HE staining).
[0104] (1) Tumor growth inhibition: As Figure 5 shown, compared with the model group (MD), the tumor volumes of the mice in the quercetin group (Qu) and the antioxidant group (Qu+GTP) were significantly reduced (P<0.05), and the tumor inhibitory effect of the Qu+GTP group was more significant, indicating that Qu+GTP has stronger activity in inhibiting the growth of gastric cancer.
[0105] Compared with Figure 5 the corresponding data are shown in Table 4.
[0106] Table 4 Calculation results of tumor volume (mm3 )
[0107]
[0108]
[0109] (2) Biochemical indexes: As Figure 6 shown, compared with the model group, the CEA level in the Qu group decreased, while the CEA level, a gastric cancer-related tumor marker in the serum of mice in the Qu+GTP group, further decreased compared with the Qu group, and the expression of tumor-related antigens in vivo was further inhibited. During the growth and metastasis of gastric cancer cells, a large amount of oxygen free radicals and reactive oxygen species are produced, leading to an increase in the level of oxidative stress in the body, while the activity of SOD enzyme is inhibited. Compared with the model group, the activity of SOD oxidase in the Qu group increased, and the SOD level in the Qu+GTP group further increased, alleviating the inhibition of SOD enzyme activity. MDA is one of the products of lipid peroxidation, and the level of MDA can reflect the degree of lipid peroxidation and oxidative stress damage in the body. Compared with the model group, the MDA level in the Qu group decreased, and the MDA level in the Qu+GTP group further decreased, indicating that Qu+GTP can effectively reduce oxidative stress and may inhibit the development of gastric cancer by regulating tumor-related inflammation and oxidative stress pathways.
[0110] Compared with Figure 6 the corresponding data are shown in Table 5.
[0111] Table 5 Detection results of gastric cancer-related tumor markers (CEA), antioxidant enzyme activity (SOD), and oxidative stress index (MDA)
[0112] CEA SOD MDA ND 1.00 1.00 1.00 MD 2.78 0.62 1.89 Qu 1.60 0.78 1.56 Qu + GTP 1.20 0.88 1.23
[0113] (3) Pathological examination: As Figure 7 shown by HE staining, compared with the normal group, the tissue structure in the MD group was disordered and the cell arrangement was disordered; compared with the model group, the HE staining sections of the Qu group showed less signs of tissue necrosis or fibrosis; in the Qu+GTP group, the cellular atypia in the tumor tissue was reduced, the mitotic figures were reduced, and the degree of inflammation and damage in the gastric tissue was reduced, which was the group with the most obvious pathological improvement among the three groups.
[0114] As described above, the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be thought of without creative work should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Claims
1. Method for extracting and purifying quercetin, comprising the following steps: S1. Place the sophora japonica flour in a microwave-assisted extraction device, perform microwave extraction with a mixed solvent, and then perform ultrasonic enhanced extraction to obtain a crude quercetin extract. In this step, the material-liquid ratio for microwave extraction is 1:(12 - 18) g / mL; the mixed solvent is an ethanol aqueous solution with a volume fraction of 65 - 75%. S2. Load the crude quercetin extract onto an activated tea polyphenol cross-linked sodium alginate resin column, let it stand for at least 30 min, then pass it through the resin column, and then elute with an ethanol aqueous solution with a volume fraction of 65% - 75% to obtain a preliminarily purified quercetin solution. S3. Load the preliminarily purified quercetin solution onto an activated polyamide resin column, let it stand for at least 30 min, wash it with at least 3 times the volume of distilled water, and then perform ascending gradient concentration elution with an ethanol aqueous solution to obtain a quercetin eluate. S4. Concentrate the quercetin eluate under vacuum to not less than 3 mg / mL, and then cool it to precipitate crystal precipitate. S5. Recrystallize the crystal precipitate to obtain highly purified quercetin.
2. The method for extracting and purifying quercetin according to claim 1, characterized in that: The specific microwave extraction is to use a microwave-assisted extraction device with 500W - 750W, adjust the temperature range to 70 - 80°C, and extract for 10 - 15 min.
3. The method for extracting and purifying quercetin according to claim 1, wherein: The mass concentration of the preliminarily purified quercetin solution in step S2 is 4.5 - 5.5 mg / mL; or the preliminarily purified quercetin solution loaded onto the polyamide resin column in step S3 is pre-adjusted to 4.5 - 5.5 mg / mL.
4. The method for extracting and purifying quercetin according to claim 1, wherein: In step S2, the loading flow rate is 1.8 - 2.5 mL / min, the elution flow rate is 1.8 - 2.5 mL / min, and the volume of the eluent is 1 - 1.5 times the volume of the sample solution.
5. The method for extracting and purifying quercetin according to claim 1, characterized in that: In step S3, the elution flow rate is 1.5 - 1.8 mL / min; the gradient concentrations are respectively volume fractions of 45% - 55%, 65% - 75%, and 85% - 95%.
6. The method for extracting and purifying quercetin according to claim 1, characterized in that: In step S4, the crystal precipitate is also dried to obtain crude quercetin; in step S5, the recrystallization specifically is to first dissolve the crude quercetin in a first solvent with relatively high solubility, then add a second solvent with relatively low solubility until it becomes turbid, and then heat it to make it clear, let it stand and cool to precipitate crystals to obtain purified quercetin.
7. The method for extracting and purifying quercetin according to claim 1, characterized in that: In step S5, the combination of the first solvent - the second solvent is ethanol - water, methanol - water, or methanol - ethanol; the volume ratio of the first solvent to the second solvent is 1:(0.5 - 2).
8. The method for extracting and purifying quercetin according to claim 1, characterized in that, The tea polyphenol cross-linked sodium alginate resin is prepared by a method comprising the following steps: 1). Add sodium alginate to deionized water, heat it to 60 - 75°C, and stir simultaneously until it is completely dissolved, remove the bubbles to obtain a sodium alginate solution; add tea polyphenols to absolute ethanol, and stir simultaneously until it is completely dissolved to obtain a tea polyphenol solution. 2). Add the tea polyphenol solution to the sodium alginate solution, add it while magnetically stirring, after mixing evenly, perform ultrasonic treatment to obtain a sodium alginate / tea polyphenol mixture. 3), Prepare a 3% - 8% CaCl₂ solution as a crosslinking agent; use a syringe equipped with a needle to drop the sodium alginate / tea polyphenol mixture into the CaCl₂ solution drop by drop to form gel beads, and crosslink in the CaCl₂ solution for at least 30 minutes to obtain crosslinked gel beads; 4), Take out the crosslinked gel beads, wash them with deionized water to remove residual CaCl₂ and unreacted tea polyphenols; then put the washed gel beads into a petri dish, wipe off the surface moisture, and then freeze-dry.
9. A method for preparing an antioxidant, comprising: The step of mixing quercetin, tea polyphenols, and a flowing carrier material in any order to prepare a feed liquid; and The step of spray-drying or freeze-drying the feed liquid to prepare an antioxidant containing quercetin; The mass ratio of quercetin, tea polyphenols, and the carrier material is 1: (0.1 - 0.3): (6 - 8).
10. Use of the antioxidant prepared by the method according to claim 9 in the preparation of a medicament for treating gastric tumors.