CN115814021A - Rhizoma polygonati total flavone extract and extraction method and application thereof - Google Patents
Rhizoma polygonati total flavone extract and extraction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of polygonatum sibiricum ingredient extraction, and discloses a polygonatum sibiricum total flavone extract and an extraction method and application thereof, wherein the extraction method comprises the steps of cleaning crude polygonatum sibiricum, soaking in a wine-water mixture A till the crude polygonatum sibiricum is completely soaked, steaming for 7-9 h by using a wine-water mixture B, moistening for 8-12 h, and steaming for 7-9 h to obtain wine-siberian solomonseal; drying and crushing polygonatum sibiricum, then uniformly dispersing in an alcohol solution, adjusting the pH to 4.7-4.9, adding cellulase, activating at 38-42 ℃, then performing ultrasonic extraction at 48-52 ℃, inactivating, and filtering, wherein the filtrate contains the polygonatum sibiricum total flavonoids. The extraction method is simple and easy to operate, the extraction effect is stable, the repeatability is high, the extraction method is reliable, the extracted rhizoma polygonati total flavone extract can relieve acute kidney injury of mice caused by cisplatin, a new thought is provided for synergistic attenuation treatment of non-small cell lung cancer by the cisplatin, and the rhizoma polygonati total flavone extract can be used for preparing medicines for relieving acute kidney injury induced by the cisplatin.
Description
Technical Field
The invention relates to the technical field of extraction of polygonatum components, in particular to a polygonatum total flavone extract and an extraction method and application thereof.
Background
Modern pharmacological research shows that the polygonatum has various pharmacological activities of regulating blood fat and blood sugar, enhancing immunity, resisting oxidation, resisting aging and the like due to the existence of various nutritional ingredients such as flavone, polygonatum polysaccharide, saponin and the like, wherein the flavone has various positive effects of resisting oxidation, resisting inflammation, resisting tumors and the like and has a very wide application prospect, so that the provision of an effective extraction method of the polygonatum total flavone is very necessary.
At present, in the prior art, the extraction method of the total flavonoids in polygonatum sibiricum mainly comprises an extraction method, ultrasonic-assisted extraction, microwave-assisted extraction and the like, but the content of the total flavonoids in raw polygonatum sibiricum is low, and the method needs high-pressure or high-temperature treatment to cause loss of effective components of the polygonatum sibiricum, so that the content of the total flavonoids in polygonatum sibiricum obtained by extraction by the method is low, and further application and research of the total flavonoids in polygonatum sibiricum are not facilitated.
Therefore, the invention provides a rhizoma polygonati total flavone extract and an extraction method and application thereof.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a rhizoma polygonati total flavone extract and an extraction method and application thereof.
The invention relates to a rhizoma polygonati total flavone extract, an extraction method and application thereof, which are realized by the following technical scheme:
the first purpose of the invention is to provide an extraction method of rhizoma polygonati total flavone extract, which comprises the following steps:
the first purpose of the invention is to provide an extraction method of rhizoma polygonati total flavone extract, which comprises the following steps:
and 3, uniformly dispersing the polygonatum sibiricum powder in an alcohol solution, adjusting the pH to 4.7-4.9 by using a buffer solution, adding cellulase, carrying out activation treatment at 38-42 ℃ in an ice bath, carrying out ultrasonic extraction at 48-52 ℃, and then inactivating and filtering to obtain filtrate, namely the polygonatum sibiricum total flavone.
Further, the wine-water mixture A and the wine-water mixture B are both a mixture of wine and water, and the mass ratio of the wine to the water is 1:9-11.
Further, in the wine-water mixture A and the wine-water mixture B, the wine is yellow wine, and the alcoholic strength of the yellow wine is 11%.
Further, the feed-liquid ratio of the raw sealwort to the wine-water mixture A is 1-3 mL.
Furthermore, a citric acid-sodium citrate buffer solution is adopted as a buffer solution, and the concentration of the citric acid-sodium citrate buffer solution is 0.08-0.12 mol/L.
Further, the material-liquid ratio of the polygonatum sibiricum powder to the alcoholic solution is 1g.
Furthermore, the dosage of the cellulase is 1.5 to 3.5 percent of the polygonatum kingianum.
Further, the time of the activation treatment is 20 to 40min.
Further, the inactivation treatment time is 5-20 min.
Furthermore, the ultrasonic power of the ultrasonic extraction is 450-500W, the ultrasonic frequency is 30-50 KHz, and the ultrasonic time is 5-45 min.
The second purpose of the invention is to provide a rhizoma polygonati total flavone extract obtained by the extraction method.
The third purpose of the invention is to provide an application of the rhizoma polygonati total flavone extract in preparation of a cisplatin-induced acute kidney injury medicament.
Compared with the prior art, the invention has the following beneficial effects:
the traditional Chinese medicine considers that Huang Jiuqi is bitter, sweet, pungent and hot in taste, has the main efficacy of promoting the circulation of medicine, and has the efficacies of eliminating evil and evil, dredging channels and collaterals, promoting blood circulation, spleen and stomach, nourishing skin, dissipating dampness, strengthening liver, dispelling wind and descending qi, promoting blood circulation, promoting urination and the like. The wine is used as an organic solvent, the polygonatum sibiricum is treated by using a wine-water mixture, and the wine-polygonatum sibiricum is obtained by steaming at normal pressure in the treatment process, so that the dissolution of flavonoids, lipids, volatile oil, alkaloid, salts thereof and the like in the polygonatum sibiricum is facilitated, the irritation of the polygonatum sibiricum can be relieved, the spleen-tonifying and lung-moistening effects of the polygonatum sibiricum are enhanced, the attenuation and the synergism are realized, and the content of flavone in the polygonatum sibiricum after being processed by wine is increased compared with that in biological products and decoction pieces; meanwhile, in the process of preparing the polygonatum sibiricum with the wine, the method adopts a normal pressure steaming mode, can avoid the possible influence of the concentration of negative pressure liquid and the negative pressure time on the appearance properties of the polygonatum sibiricum decoction pieces, also avoids the high requirement of high pressure steaming on equipment and the possible loss of effective substances, and improves the extraction effect of the total flavonoids in the polygonatum sibiricum. Then crushing polygonatum sibiricum and placing the crushed polygonatum sibiricum in an alcohol solution, extracting the total flavonoids in the polygonatum sibiricum by using the ultrasonic synergistic alcohol solution, wherein the extraction rate of the total flavonoids in the polygonatum sibiricum can be obviously improved, and in the process of extracting the total flavonoids in the polygonatum sibiricum by using the ultrasonic synergistic alcohol solution, cellulase is added, so that the wall breaking rate of plant cells can be increased, the flavone yield is improved, and the total flavone yield can reach 1.19 mg.g -1 。
The extraction method provided by the invention is simple and easy to operate, stable in extraction effect, high in repeatability and reliable, and is favorable for researching the pharmacological activity of the rhizoma polygonati total flavonoids.
The sealwort total flavone extracted by the extraction method can remove DPPH, hydroxyl free radical and ABTS, has obvious antioxidant capacity, and especially has IC50 of hydroxyl free radical which is almost 5 times of vitamin C and 11 times of BHT (2,6-di-tert-butyl-4-methylphenol), so the sealwort total flavone extracted by the extraction method is a potential natural free radical strong scavenging agent, can resist A549 cell proliferation in vitro in dose dependence, can relieve acute kidney injury of mice caused by cisplatin, and provides a new idea for synergistic attenuation and toxicity reduction of non-small cell lung cancer.
Drawings
FIG. 1 shows the total flavone extraction rates of examples 1 to 5;
FIG. 2 shows the total flavone extraction yield of example 1, and example 6 to example 13;
FIG. 3 shows the total flavone extraction yield of example 1, and example 14-example 17;
FIG. 4 shows the total flavone extraction rates of example 1, and of examples 19 to 26;
FIG. 5 shows the ability of total flavonoids from Polygonatum sibiricum Red to scavenge DPPH free radicals;
FIG. 6 shows the ability of total flavonoids of Polygonatum sibiricum Red to scavenge hydroxyl radicals;
FIG. 7 shows the ability of total flavonoids of Polygonatum sibiricum Red to scavenge ABTS free radicals;
FIG. 8 shows the effect of total flavonoids of polygonatum sibiricum extracted according to the present invention at different dosages on the proliferation activity of A549 cells;
FIG. 9 is a photograph of mouse urine;
FIG. 10 is a photograph of a mouse kidney;
FIG. 11 shows the effect of total flavonoids extracted from Polygonatum sibiricum Red of the present invention on cisplatin-induced acute kidney injury;
FIG. 12 shows the MDA content in serum of mice after each experiment, wherein group 1 is blank; group 2 is the acute kidney injury model group; group 3 is the polygonatum sibiricum flavone extract low-dose group; group 4 is the medium dosage group of the polygonatum sibiricum flavone extract; group 5 is a high-dose group of flavone extract of polygonatum sibiricum; # indicates P <0.01, compared to blank; * P <0.01, P <0.05, compared to the acute kidney injury model group;
FIG. 13 is the MDA content in the kidney tissue of mice after each experiment, wherein group 1 is blank; group 2 is the acute kidney injury model group; group 3 is a low dose group of polygonatum sibiricum flavone extract; group 4 is the medium dosage group of the polygonatum sibiricum flavone extract; group 5 is a high-dose group of flavone extract of polygonatum sibiricum; # indicates P <0.01, compared to blank; * P <0.01, P <0.05, compared to the acute kidney injury model group.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
Example 1
The embodiment provides an extraction method of a rhizoma polygonati total flavone extract, which comprises the following steps:
The present invention is not limited to the specific amount of the alcoholic beverage mixture a, as long as the raw materials are completely soaked. Optionally, in this embodiment, the usage ratio of the wine-water mixture a to the raw rhizoma polygonati is 2ml. In the present embodiment, a mixture of wine and water at a mass ratio of 1.
it should be noted that the specific process of the drying treatment is not limited in the present invention, as long as the excess wine-water mixture of polygonatum sibiricum can be removed, which facilitates the subsequent pulverization treatment, and optionally, the drying temperature in this embodiment is-80 ℃ for freeze drying, and the drying time is 72 hours.
The invention does not limit the concrete process of the crushing treatment, as long as the polygonatum sibiricum can be crushed to be a No. 6 sieve, namely a 100-mesh sieve.
it should be noted that the present invention does not limit the specific amount of the alcoholic solution, but in consideration of the effect of extracting total flavonoids from polygonatum sibiricum powder, alternatively, this example uses 45% ethanol as the alcoholic solution, and the ratio of polygonatum sibiricum powder to the alcoholic solution is 1g.
The invention does not limit the concrete type of the buffer solution, as long as the pH of the solution system can be adjusted to about 4.8, and the invention can be selected from citric acid-sodium citrate buffer solution with the concentration of 0.1mol/L as the buffer solution.
In this embodiment, the amount of cellulase is 1.5%, and the activation time can be flexibly adjusted according to the actual activation temperature, for example, the activation treatment is performed at 40 ℃ for 30min. In the embodiment, the ultrasonic extraction temperature is 50 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, and the ultrasonic time is 5min; and the inactivation time is 10min.
Example 2
The embodiment provides an extraction method of a rhizoma polygonati total flavone extract, which comprises the following steps:
In this example, the ratio of the wine-water mixture a to the raw polygonatum sibiricum is 1ml. And the mixture with the mass ratio of wine to water of 1:9 is preferably used as the wine-water mixture A in the invention.
the drying temperature in this example was-70 ℃ for 80 hours.
in this example, ethanol with a concentration of 55% was used as an alcoholic solution, and the stock-to-solution ratio of polygonatum sibiricum powder to the alcoholic solution was 1g.
In this example, the concentration was set to 0.08 mol. L -1 The citric acid-sodium citrate buffer solution of (4) is used as a buffer solution.
In this example, the amount of cellulase used was 2.5%, and the activation treatment time was 40min.
In the embodiment, the ultrasonic extraction temperature is 48 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 30KHz, and the ultrasonic time is 20min; and the inactivation time is 5min.
Example 3
The embodiment provides an extraction method of a rhizoma polygonati total flavone extract, which comprises the following steps:
In this example, the ratio of the wine-water mixture a to the raw polygonatum sibiricum is 1ml. And the preferred mixture of the invention is the mixture A with the mass ratio of wine to water of 1.
the drying temperature in this example was-90 ℃ for 48h of lyophilization.
in the present embodiment, 85% ethanol is used as an alcoholic solution, and the ratio of polygonatum sibiricum powder to the alcoholic solution is 1g.
In this example, a citric acid-sodium citrate buffer solution having a concentration of 0.12mol/L was used as the buffer solution.
In this example, the cellulase was used in an amount of 3.5% and the activation treatment time was 40min.
In the embodiment, the ultrasonic extraction temperature is 52 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 50KHz, and the ultrasonic extraction time is 45min; and the inactivation time is 20min.
Example 4
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 1 only in that:
in the embodiment, the material-liquid ratio of polygonatum sibiricum powder to an alcohol solution is 1g, 15ml, the alcohol solution is 70% ethanol, the cellulase dosage is 1.5%, and the ultrasonic time is 15min.
Example 5
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 4 only in that:
in this example, the amount of cellulase used was 2%.
Example 6
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 4 only in that:
in this example, the amount of cellulase used was 2.5%.
Example 7
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 4 only in that:
in this example, the amount of cellulase used was 3%.
Example 8
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 4 only in that:
in this example, the cellulase amount was 3.5%.
Example 9
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 1 only in that:
in the embodiment, the feed-liquid ratio of polygonatum sibiricum powder to an alcohol solution is 1g, 15ml, the dosage of cellulase is 2.5%, the ultrasonic time is 15min, and the concentration of ethanol is 45%.
Example 10
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
the concentration of ethanol was 50%.
Example 11
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 55%.
Example 12
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 60%.
Example 13
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 65%.
Example 14
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 70%.
Example 15
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 75%.
Example 16
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 80%.
Example 17
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 9 only in that:
in this example, the concentration of ethanol was 85%.
Example 18
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 1 only in that:
in the embodiment, the dosage of the cellulase is 2.5%, the concentration of the ethanol is 70%, the ultrasonic time is 15min, and the feed-liquid ratio of polygonatum sibiricum powder to the alcoholic solution is 1g.
Example 19
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 18 only in that:
in this example, the ratio of polygonatum sibiricum powder to alcoholic solution was 1g, 10ml.
Example 20
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 18 only in that:
in the embodiment, the ratio of the polygonatum sibiricum powder to the alcoholic solution is 1g.
Example 21
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 18 only in that:
in the present example, the ratio of polygonatum sibiricum powder to alcoholic solution was 1g.
Example 22
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 18 only in that:
in the embodiment, the material-liquid ratio of polygonatum sibiricum powder to the alcoholic solution is 1g.
Example 23
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 18 only in that:
in this example, the material-to-liquid ratio of polygonatum sibiricum powder to the alcohol solution is 1g.
Example 24
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 1 only in that:
the dosage of the cellulase is 2.5%, the concentration of the ethanol is 55%, the feed-liquid ratio of polygonatum sibiricum powder to the alcoholic solution is 1g.
Example 25
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
the ultrasonic extraction time is 10min.
Example 26
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasonic extraction time was 15min.
Example 27
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 20min.
Example 28
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 25min.
Example 29
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 30min.
Example 30
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 35min.
Example 31
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 40min.
Example 32
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 24 only in that:
in this example, the ultrasound extraction time was 45min.
Example 33
This example provides a method for extracting total flavonoids from polygonatum sibiricum, which is different from example 1 only in that:
in the embodiment, the cellulase dosage is 1.5%, the ethanol concentration is 45%, the ultrasonic time is 16min, and the feed-liquid ratio is 1g.
Test section
1. Influence of different cellulose dosage on total flavone content
In the present invention, the sealwort total flavone extract solutions prepared in examples 4 to 8 are taken as examples, and the total flavone content thereof is tested to investigate the influence of different cellulose dosages on the total flavone content, and the test results are shown in fig. 1, which shows that the total flavone extraction rate is also improved along with the increase of the cellulase content, but the total flavone extraction rate is decreased when the cellulase content is increased to a certain degree, so the preferable cellulose dosage of the present invention is 2% to 3%.
2. Effect of different alcohol concentrations on Total Flavonoids content
In the present invention, the total flavone content of the polygonatum sibiricum extract solutions prepared in examples 9 to 17 is taken as an example, and the total flavone content is tested to investigate the influence of different alcohol concentrations on the total flavone content, and the test results are shown in fig. 2, it can be seen that the total flavone extraction rate is also improved along with the increase of the alcohol concentration, but when the alcohol concentration is increased to a certain degree, the total flavone extraction rate is rather reduced, so the preferable alcohol concentration range of the present invention is 50% to 60%.
3. Influence of different feed liquid ratios on total flavone content during extraction
In the present invention, the total flavone content of the polygonatum sibiricum extract solutions prepared in examples 18 to 22 are taken as examples, and the total flavone content is tested to investigate the influence of different feed liquid ratios on the total flavone content during extraction, and the test results are shown in fig. 3, which shows that the total flavone extraction rate is also increased along with the increase of the feed liquid ratio during extraction, but the total flavone extraction rate is decreased instead when the feed liquid ratio during extraction is increased to a certain degree, so that the preferable solid-liquid ratio of polygonatum sibiricum powder to alcohol solution of 1g.
4. Influence of different ultrasonic time on total flavone content during extraction
Taking the sealwort total flavone extracting solutions prepared in the embodiments 24 to 32 as examples, the invention respectively tests the total flavone content of the sealwort total flavone extracting solutions to investigate the influence of different ultrasonic time on the total flavone content during extraction, and the test results are shown in fig. 3, which shows that the total flavone extraction rate is improved along with the increase of the ultrasonic time during extraction, but the total flavone extraction rate is reduced after the ultrasonic time is increased to a certain degree, so the invention preferably takes the ultrasonic time for extraction of 5 to 25min.
5. In vitro antioxidant capacity
The present invention is exemplified by the total flavone extract obtained in the method of example 33, which was freeze-dried (-80 ℃ C. For 48 hours), prepared into solutions of various concentrations (0, 12.5, 25, 50, 100. Mu.g/mL), and subjected to the following tests, respectively, to determine the antioxidant ability.
Preparing ABTS working solution: the 7mmol/LABTS solution and 140mmol/L potassium persulfate aqueous solution are mixed according to 50.
Sample set for each experiment (A) s The DPPH absolute ethanol solution of the sample was added), and the sample control group (A) bs Adding extractive solution of ethanol (distilled water), and blank control group (A) bc Equal volume of absolute ethanol (distilled water) was substituted for DPPH (hydroxyl radical working solution, ABST) solution of the sample), positive control group (vitamin C (VC) or 2,6-di-tert-butyl-4-methylphenol (BHT)). Each set of experiments was repeated 3 times in parallel.
5.1 measurement of DPPH radical scavenging ability
Sucking 100 μ L of the above prepared rhizoma Polygonati total flavone solutions with different concentrations, adding 100 μ L of 0.1mmol/L DPPH anhydrous ethanol solution, mixing, keeping out of the sun at room temperature for 30min, and measuring A value at 517nm. DPPH radical clearance = [1- (A) s -A bs )/A bc ]X 100%, the test results are shown in FIG. 5.
5.2 measurement of hydroxyl radical scavenging ability
Sucking 50 μ L of the above prepared rhizoma Polygonati total flavone solution with different concentrations, sequentially adding 50 μ L of 6mmol/LFeSO 4 、100μL6mmol/LH 2 O 2 Shaking thoroughly, standing at room temperature for 10min; then 50. Mu.L of salicylic acid with the concentration of 6mmol/L is added, the mixture is fully shaken up and placed for 30min at room temperature, and the A value is measured at 510nm. Hydroxyl radical clearance rate = (a) bs -A s )/A bs 100%, the test results are shown in FIG. 6.
5.3 determination of ABTS Total antioxidant Capacity
Sucking 50 μ L of the above prepared total flavone solution of rhizoma Polygonati Odorati with different concentrations, adding 100 μ L of LABTS, keeping away from light for 10min, and measuring A value at 734nm. Total antioxidant ability = [1- (A) s -A bs )/A bc ]100%, the test results are shown in FIG. 7.
As can be seen from FIGS. 5-7, the total flavone extract of Polygonatum sibiricum Red obtained by the present invention has strong antioxidant ability, can dose-dependently remove DPPH, hydroxy radicals and ABST radicals, and has IC50 of (19.43 + -0.96) μ g/mL, (4.46 + -0.14) μ g/mL and (31.57 + -0.14) μ g/mL, especially has stronger removing ability for hydroxy radicals than vitamin C (IC 50=22.23 + -0.99 μ g/mL) and BHT (IC 50=54.83 + -0.95 μ g/mL).
6. In vitro anti-A549 proliferation capacity
The invention takes the total flavone extract obtained by the method of example 33 as an example, the total flavone extract is prepared into solutions with different concentrations (0,0.06X 10) after being frozen and dried (frozen and dried for 48 hours at minus 80 ℃) 3 ，0.12×10 3 ，0.24×10 3 ，0.48×10 3 ，0.96×10 3 ，1.92×10 3 microgram/mL) to obtain rhizoma Polygonati Odorati flavone extracts with different concentrations, and respectively removing CO at 37 deg.C and 5% 2 Next, A549 cells were treated for 24h (48, 72 h), 10. Mu.L CCK-8 solution was added to each well, incubated for 3.5h, and absorbance (OD) was measured at 600nm, and cell survival% = [ (ODs-OD) b )/(OD c -OD b )]X 100%. The assay was repeated 5 times for each group and the results are shown in FIG. 8.
As can be seen from FIG. 8, after the flavone extract from Polygonatum sibiricum Red was applied to A549 cells, the cells were shriveled and necrosed to different degrees. It can inhibit A549 cells dose-dependently, and the IC50 of the A549 cells is 18.97 +/-3.48 (24 h), 2.77 +/-0.99 (48 h) and 2.01 +/-0.43 (72 h) mu g/mL in sequence.
7. Protection effect on cisplatin-induced acute kidney injury of mice
7.1 preparation of cisplatin-induced acute Kidney injury model in mice
According to the method in the literature (Pan Xiaoyue. Different doses of cisplatin-induced mouse acute kidney injury model observation and biological index detection [ D ]. New county medical college, 2015), cisplatin (20 mg/kg) is injected into the abdominal cavity in a single day in a model group and a polygonatum sibiricum administration group to form a mouse acute kidney injury model.
7.2 grouping and administration
24 male Kunming mice were randomly divided into blank group, model group, polygonatum sibiricum (low, medium, high), and 8 mice per group. The blank group and the model group are subjected to continuous physiological saline intragastric administration for 7d, and the rhizoma polygonati group (equivalent mouse dose is calculated by referring to a method of 'Chinese pharmacopoeia' 2020 edition, and low, medium and high doses are 3.08,9.25 and 27.74 mg/kg) is subjected to continuous extract intragastric administration for 7d. Except for the blank group, each group of mice was injected with cisplatin at a dose of 0.02mL/g in the abdominal cavity. The blank group was injected with 0.9% sodium chloride injection in equal amount.
7.3 mouse urine and Kidney contrast
As shown in figure 9, the urine changes of the mice in each group are observed, and the urine of the blank group is light yellow, clear and transparent, and is neither turbid nor precipitate. The model group was most yellow in color and somewhat turbid compared to the blank group, indicating the most severe damage to the kidneys. The color of urine of the group administered with the polygonatum kingianum extract is not much different from that of the blank group.
7.4 Kidney morphological differences in groups
As shown in fig. 10, the kidneys of mice after each experiment were fixed with 4% paraformaldehyde, dehydrated and cleared, embedded in paraffin, and kidney sections were taken for HE staining, and the renal tubules and peripheral cell changes were observed under a microscope.
As shown in figure 11, by HE staining of kidney tissues of the acute kidney injury model group and the polygonatum sibiricum extract group, the acute kidney injury tissue renal tubular epithelial cell particles or vacuole-like deformation, the lumen expansion, brush-like edge shedding, basement membrane rupture, transparency, particle or cell tube type, and epithelial apoptosis phenomenon in the lumen are observed. And the polygonatum sibiricum extract group has renal tubular vacuole-like deformation, epithelial cell particles are relieved compared with a model group, renal tubular epithelial shedding is relieved, and nucleus-fixing and shrinking epithelial cells shed in a lumen are obviously reduced.
In the experiment, compared with a blank group, a mouse kidney picture shows that the kidney of the model group is most affected by the cisplatin and has the lightest color; the physiological saline given to the blank group has no damage to the kidney of the mouse, and the color is the darkest, which indicates that the molding is successful. The sealwort extract administration group is analyzed from kidney pictures, and the high dose color is closest to that of the blank group. In addition, the picture shows that compared with the blank group, the kidney of the model group mouse is the most swollen, and the kidneys of the low, medium and high polygonatum sibiricum extract administration group are sequentially closer to the blank group, which indicates that cisplatin has a protective effect on the kidneys.
7.5 serum and Kidney tissue Malondialdehyde (MDA) assay
Blood is taken from eyeballs of mice after each group of experiments, and serum is obtained by centrifuging at the rotating speed of 3000r/min for 20min. Preparing 10% kidney tissue homogenate by using kidney tissue and physiological saline according to the proportion of 1:9, centrifuging the homogenate for 10min in a centrifuge 12000r, taking supernatant, and determining the concentration of the kidney tissue protein by using a BCA protein concentration kit. The serum and kidney tissues are sequentially detected on an enzyme labeling instrument by colorimetry at 532nm according to the steps of the kit specification, and the MDA content is calculated, and the test results are respectively shown in fig. 12 and fig. 13, wherein fig. 12 shows the MDA content in the serum of the mouse after each group of experiments, and fig. 13 shows the MDA content in the kidney tissues of the mouse after each group of experiments.
As can be seen from fig. 12 and 13, the oxidative stress and antioxidant imbalance of the model mice, compared to the blank group, is represented by 1.03-fold increase of serum MDA and 6.71-fold increase of kidney MDA. Compared with the model group, the serum MDA of the polygonatum kingianum group is reduced by 41.3 percent (low dose group), 39.9 percent (medium dose group) and 29.9 percent (high dose group), and all the administration groups have obvious difference (P is less than 0.01). Compared with the model group, the kidney tissue MDA of polygonatum sibiricum is reduced by 35.7 percent (low dose group), 43.3 percent (middle dose group) and 19.2 percent (high dose group), and the middle and low dose groups have obvious difference (P is less than 0.05), namely, the total flavone extract of polygonatum sibiricum at different doses can reduce the oxidative stress level of cisplatin-induced acute kidney injury of mice.
The content of the total flavonoids is measured by the following method:
the invention adopts NaNO 2 -Al(NO 3 ) 3 Measuring the content of total flavonoids by NaOH colorimetry, respectively sucking 1.0mL of the rhizoma polygonati total flavonoid extract obtained in each embodiment, respectively measuring the absorbance of the rhizoma polygonati total flavonoid extract of each embodiment, substituting the absorbance into a standard curve of the total flavonoids to measure the concentration, and calculating the extraction rate of the total flavonoids.
The extraction rate of the total flavone is% = (the concentration of the total flavone is multiplied by the volume of the liquid medicine)/the mass of the medicinal material is multiplied by 100%.
Wherein the standard curve of total flavonoids is prepared by dissolving rutin standard substance with chromatographic methanol to obtain 0.2mg/mL reference substance stock solution. Precisely sucking rutin control stock solution 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0mL, placing in 25mL volumetric flask with rutin concentration as horizontal lineCoordinate, absorbance A is ordinate, plotted as standard curve y =10.239x +0.0272 (R) 2 = 0.9976), linear range 0.0080-0.0480 mg/mL.
The invention has stronger capability of removing hydroxyl free radicals compared with water-soluble antioxidant vitamin C and artificially synthesized fat-soluble antioxidant BHT by measuring the antioxidant capability of the obtained polygonatum sibiricum total flavonoids. And the vitamin C has the same capacity of eliminating DPPH free radicals and ABTS, and has stronger capacity of eliminating the DPPH free radicals and the ABTS than BHT. The total flavone of polygonatum sibiricum in the invention is a strong natural antioxidant.
Cisplatin is still the first-line first choice drug for treating non-small cell lung cancer at present. But inevitably, acute kidney injury caused by cisplatin limits the benefit of the patient. At present, the method for preventing and treating cis-damage by cisplatin mainly comprises hydration, diuresis, electrolyte supplement, word segmentation administration, administration time control, administration of angiotensin converting enzyme inhibitor (the medicine has the effects of increasing blood potassium and causing teratogenesis, so the medicine is forbidden for patients with renal artery stenosis at both sides and pregnant women), trace elements supplement, and glutathione and other antioxidants are combined (the glutathione injection part sometimes has mild pain, and the medicine has the side effects of local irritation, pruritus, conjunctival congestion, blurred vision and the like when being used for eye dropping). The polygonatum sibiricum flavone extract extracted by the method can obviously reduce cisplatin-induced acute kidney injury of mice, and has sweet taste and no irritation. The total flavonoids extracted from the sealwort still show excellent in-vitro anti-proliferation effect on the non-small cell lung cancer, so that a novel synergistic attenuation substance is provided for clinical application of cisplatin to treatment of the non-small cell lung cancer, and the total flavonoids extracted from the sealwort can be used for preparing a medicine for relieving cisplatin-induced acute kidney injury.
In conclusion, the sealwort total flavone extract extracted by the method has high total flavone content, strong free radical scavenging capacity and capability of reducing the oxidative damage of cisplatin to kidney tissues by resisting the peroxidation rate and strength of body lipid. The ability of scavenging free radicals is stronger than that of the polygonatum polysaccharide component which is researched more at present and has kidney protection function.
It is to be understood that the above-described embodiments are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A method for extracting rhizoma polygonati total flavone extract is characterized by comprising the following steps:
step 1, cleaning raw rhizoma polygonati, soaking the cleaned raw rhizoma polygonati in a wine-water mixture A until the rhizoma polygonati is completely soaked, and filtering to obtain soaked raw rhizoma polygonati; steaming the soaked crude polygonatum for 7-9 h by using a wine-water mixture B, then moistening for 8-12 h, and steaming for 7-9 h again to obtain polygonatum;
step 2, drying rhizoma polygonati preparata and then crushing to obtain rhizoma polygonati preparata powder;
and 3, uniformly dispersing the polygonatum sibiricum powder in an alcohol solution, adjusting the pH to 4.7-4.9 by using a buffer solution, adding cellulase, carrying out activation treatment at 38-42 ℃ in an ice bath, carrying out ultrasonic extraction at 48-52 ℃, and then inactivating and filtering to obtain filtrate, namely the polygonatum sibiricum total flavone.
2. The extraction process according to claim 1, wherein the wine-water mixture A and the wine-water mixture B are both a mixture of wine and water, and the mass ratio of wine to water is 1:9-11.
3. The extraction method according to claim 1, wherein the feed-liquid ratio of the raw polygonatum sibiricum to the wine-water mixture A is 1g to 3mL.
4. The extraction method according to claim 1, wherein a citric acid-sodium citrate buffer solution is used as the buffer solution, and the concentration of the citric acid-sodium citrate buffer solution is 0.08-0.12 mol/L.
5. The extraction method according to claim 1, wherein the material-to-liquid ratio of the polygonatum sibiricum powder to the alcoholic solution is 1g;
the mass concentration of the alcoholic solution is 45-85%.
6. The extraction method according to claim 1, wherein the cellulase is 1.5 to 3.5 percent of the polygonatum sibiricum powder by mass.
7. The extraction method according to claim 1, wherein the time of the activation treatment is 20 to 40min;
the inactivation treatment time is 5-20 min.
8. The extraction method according to claim 1, wherein the ultrasonic power of the ultrasonic extraction is 450-550W, the ultrasonic frequency is 30-50 KHz, and the ultrasonic time is 5-45 min.
9. A polygonatum sibiricum total flavone extract obtained by the extraction method according to any one of claims 1 to 8.
10. An application of the rhizoma polygonati total flavone extract according to claim 9 in preparing a medicament for relieving cisplatin-induced acute kidney injury.
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