CN111956641B - Flavonoid compound composition and preparation method thereof - Google Patents
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Abstract
The invention discloses a flavonoid compound composition and a preparation method thereof. According to the invention, the composition is formed by adding the absorption slow-release substance and the solubilizer into the flavonoid compound, so that the water solubility of the flavonoid compound is improved, and the bioavailability is improved; meanwhile, the cell injury is reduced, and compared with single flavonoid administration, the cell activity is improved by at least 1.17 times, which shows that the composition can effectively reduce the cytotoxicity.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical compositions, and particularly relates to a flavonoid compound composition and a preparation method thereof.
Background
More than 20 kinds of flavonoids in the whole marijuana plant are reported, and apigenin, luteolin, quercetin, orientin, rutin, vitexin and the like are common. In addition, geranylflavone A (Cannflavin A) and geranylflavone B (Cannflavin B) are two specific flavonoids in marijuana plants. Flavonoids in Cannabis have been shown to have a variety of pharmacological activities, as Barrett et al reported that geranylflavone A/B in Cannabis could potentiate anti-inflammatory effects greatly, and that prostaglandin E-2 in rheumatoid synovial cells was inhibited 30 times more than aspirin (Barrett, M.L., A.M.Scutt, et al, "Cannflavin A and B, pretreated flavones from Cannabis sativa". Experientia 42(4): 452 453); amay K et al reported that geranylflavone A/B and derivatives thereof in Cannabis have significant antimicrobial and antiprotozoal activity (Amay K. Ibrahim t, et al, "Microbial metabolism of canflavan A and B isolated from Cannabis sativa". Phytochemistry 71(2010) 1014-. US20190083452a1 discloses the use of cannabinoids and derivatives thereof for the prevention and treatment of neurodegenerative diseases. US patent No. 20180353462a1 discloses the use of cannabixanthone derivatives for the treatment and prevention of cancer.
Thus, the cannabinoids and the derivatives thereof have wide application prospects in the aspect of medicines. However, some flavonoids, such as geranylflavone a, are insoluble in water and only soluble in organic solvents such as ethanol, propylene glycol, ethyl acetate, etc., resulting in low bioavailability. Therefore, large dose administration is necessary to achieve the effectiveness of medication, and the use of large dose inevitably brings compliance problems and increases the economic burden of patients; meanwhile, the burden of the liver and the kidney is also increased by taking the medicine in a large dose, and unpredictable liver and kidney damage is caused. In addition, in some practical experiments, geranylflavone A also shows certain cytotoxicity, so that the compound of plant origin is not effectively developed and applied in the field of medicine.
At present, no solution for the above problems is available at home and abroad. Therefore, the solubilization and attenuation technology of flavonoids is in need of development.
Disclosure of Invention
In order to solve the technical problems, the invention provides a flavonoid compound composition and a preparation method thereof, and the flavonoid compound is added with an absorption sustained-release substance and a solubilizer, so that the water solubility of the flavonoid compound is improved, and the bioavailability of the flavonoid compound is improved; meanwhile, the addition of the absorption slow-release substance delays the metabolic time of the medicine, so that the cytotoxic effect of the medicine is effectively relieved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
on one hand, the invention provides a flavonoid compound composition which comprises a flavonoid compound, an absorption slow-release substance and a solubilizer.
Optionally, the content of each component in the flavonoid compound composition is as follows: 1-20 parts of flavonoid compound, 1-10 parts of absorption slow release substance and 15-80 parts of solubilizer.
Specifically, the flavonoid compound is geranylflavonoid A.
The lower limit of the content of the flavonoid compound can be independently selected from 1 part by weight, 2.5 parts by weight, 5 parts by weight, 10 parts by weight and 15 parts by weight; the upper limit of the content of the flavonoid compound can be independently selected from 8 parts by weight, 12 parts by weight, 16 parts by weight, 18 parts by weight and 20 parts by weight.
Specifically, the lower limit of the content of the absorption sustained-release substance may be independently selected from 1 part by weight, 1.5 parts by weight, 3 parts by weight, 5 parts by weight, 7 parts by weight; the upper limit of the content of the absorption slow release substance can be independently selected from 2 parts by weight, 4 parts by weight, 6 parts by weight, 8 parts by weight and 10 parts by weight.
Specifically, the lower limit of the content of the solubilizer may be independently selected from 15 parts by weight, 15.5 parts by weight, 35 parts by weight, 50 parts by weight, 70 parts by weight; the upper limit of the content of the solubilizer can be independently selected from 45 parts by weight, 65 parts by weight, 73 parts by weight and 80 parts by weight.
Optionally, the absorption delaying substance comprises at least one of polysaccharide and edible protein.
Optionally, the polysaccharide is a polysaccharide extract extracted from a plant or microorganism;
the edible protein is protein extract extracted from plant, animal or microorganism.
Specifically, the polysaccharide is at least one of a linear polysaccharide and a branched polysaccharide.
Optionally, the polysaccharide extract is at least one of lycium barbarum polysaccharide, cucurbitan polysaccharide, algal polysaccharide, dextran, and pectin;
the protein extract is at least one of pea protein, cottonseed protein, whey protein, yeast protein, and gelatin.
Preferably, the polysaccharide extract is at least one of water-soluble yeast glucan and pectin.
Preferably, the protein extract is at least one of whey protein, yeast protein and edible gelatin.
Optionally, the solubilizing agent is selected from at least one of a non-ionic surfactant and an ionic cyclodextrin derivative;
the nonionic surfactant is at least one selected from poloxamer, polyethylene glycol, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, and medium chain triglyceride;
the ionic cyclodextrin derivative is at least one selected from hydroxyethyl beta cyclodextrin, hydroxypropyl beta cyclodextrin and sulfobutyl-beta-cyclodextrin.
Specifically, the poloxamer is at least one of poloxamer 188, poloxamer 237 and poloxamer 407; the polyethylene glycol is selected from at least one of polyethylene glycol 4000 and polyethylene glycol 6000.
Preferably, at least poloxamer and ionic cyclodextrin derivatives are added simultaneously as solubilising agents.
Further preferably, at least poloxamer 188 and hydroxypropyl beta cyclodextrin are added simultaneously as solubilizing agents.
Wherein the dosage of the poloxamer is 10-65 parts by weight, and the dosage of the ionic cyclodextrin derivative is 2.5-40 parts by weight.
Specifically, the lower limit of the amount of poloxamer can be independently selected from 10 parts by weight, 12 parts by weight, 20 parts by weight, 25 parts by weight and 30 parts by weight; the upper limit of the amount of poloxamer can be independently selected from 40 parts by weight, 45 parts by weight, 50 parts by weight, 55 parts by weight and 65 parts by weight.
Specifically, the lower limit of the amount of the ionic cyclodextrin derivative may be independently selected from 2.5 parts by weight, 5 parts by weight, 10 parts by weight, 15 parts by weight, and 20 parts by weight; the upper limit of the amount of the ionic cyclodextrin derivative may be independently selected from 18 parts by weight, 25 parts by weight, 30 parts by weight, 35 parts by weight, and 40 parts by weight.
Optionally, the flavonoid composition further comprises a preservative or/and an antioxidant;
the preservative is at least one of potassium sorbate and citric acid;
the antioxidant is at least one of vitamin C and vitamin E.
Optionally, the content of the preservative is 0.01-0.1 weight part;
the content of the antioxidant is 0.2-4 parts by weight.
Specifically, the lower limit of the content of the preservative may be independently selected from 0.01 parts by weight, 0.015 parts by weight, 0.02 parts by weight, 0.03 parts by weight, 0.05 parts by weight; the upper limit of the content of the preservative may be independently selected from 0.06 parts by weight, 0.07 parts by weight, 0.08 parts by weight, 0.09 parts by weight, 0.1 parts by weight.
Specifically, the lower limit of the content of the antioxidant may be independently selected from 0.2 parts by weight, 0.5 parts by weight, 1 part by weight, 1.5 parts by weight, 2 parts by weight; the upper limit of the content of the antioxidant may be independently selected from 1.8 parts by weight, 2.5 parts by weight, 3 parts by weight, 3.5 parts by weight, and 4 parts by weight.
Optionally, the content of each component in the flavonoid compound composition is as follows: 1-20 parts of flavonoid compound, 1-10 parts of polysaccharide or edible protein, 10-65 parts of poloxamer, 0-30 parts of medium-chain triglyceride, 5-40 parts of hydroxypropyl beta cyclodextrin, 0.01-0.1 part of citric acid, 0.5-2 parts of PEG4000 and 0.01-0.1 part of potassium sorbate.
Specifically, the lower limit of the content of the flavonoid compound can be independently selected from 1 part by weight, 2.5 parts by weight, 5 parts by weight, 10 parts by weight and 15 parts by weight; the upper limit of the content of the flavonoid compound can be independently selected from 8 parts by weight, 12 parts by weight, 16 parts by weight, 18 parts by weight and 20 parts by weight.
Specifically, the lower limit of the content of the polysaccharide or the edible protein may be independently selected from 1 part by weight, 1.5 parts by weight, 3 parts by weight, 5 parts by weight, 7 parts by weight; the upper limit of the amount of the polysaccharide or the edible protein may be independently selected from 2 parts by weight, 4 parts by weight, 6 parts by weight, 8 parts by weight, and 10 parts by weight.
Specifically, the lower limit of the poloxamer content may be independently selected from 10 parts by weight, 12.5 parts by weight, 20 parts by weight, 25 parts by weight, and 30 parts by weight; the upper limit of the poloxamer content can be independently selected from 40 parts by weight, 45 parts by weight, 50 parts by weight, 55 parts by weight and 60 parts by weight.
Specifically, the lower limit of the content of the medium chain triglyceride may be independently selected from 0 part by weight, 1 part by weight, 5 parts by weight, 10 parts by weight, 15 parts by weight; the upper limit of the content of the medium chain triglyceride may be independently selected from 12 parts by weight, 18 parts by weight, 20 parts by weight, 25 parts by weight, and 30 parts by weight.
Specifically, the lower limit of the content of hydroxypropyl beta cyclodextrin can be independently selected from 5 parts by weight, 10 parts by weight, 13 parts by weight, 15 parts by weight, and 20 parts by weight; the upper limit of the content of the hydroxypropyl beta cyclodextrin can be independently selected from 18 parts by weight, 25 parts by weight, 30 parts by weight, 35 parts by weight and 40 parts by weight.
Specifically, the lower limit of the content of citric acid may be independently selected from 0.01 parts by weight, 0.02 parts by weight, 0.03 parts by weight, 0.04 parts by weight, 0.05 parts by weight; the upper limit of the content of citric acid may be independently selected from 0.06 parts by weight, 0.07 parts by weight, 0.08 parts by weight, 0.09 parts by weight, 0.1 parts by weight.
Specifically, the lower limit of the content of PEG4000 may be independently selected from 0.5 parts by weight, 0.7 parts by weight, 0.9 parts by weight, 1.1 parts by weight, 1.3 parts by weight; the upper limit of the content of PEG4000 may be independently selected from 1.2 parts by weight, 1.4 parts by weight, 1.6 parts by weight, 1.8 parts by weight, 2 parts by weight.
Specifically, the lower limit of the content of potassium sorbate may be independently selected from 0.01 parts by weight, 0.02 parts by weight, 0.03 parts by weight, 0.04 parts by weight, 0.05 parts by weight; the upper limit of the content of potassium sorbate can be independently selected from 0.06 part by weight, 0.07 part by weight, 0.08 part by weight, 0.09 part by weight, and 0.1 part by weight.
In another aspect of the present invention, a method for preparing a flavonoid composition is provided, which at least comprises:
crushing solid components in the flavonoid compound composition to obtain solid powder;
mixing the solid powder with other components to obtain a mixture;
adding purified water into the mixture, and uniformly mixing to obtain a liquid flavonoid compound composition;
the amount of the purified water is 50 to 85 parts by weight.
In the preparation method, pectin or edible gelatin is preferably adopted as the absorption slow-release substance, wherein the dosage of the pectin is 1-5 parts by weight, and the dosage of the edible gelatin is 1-10 parts by weight.
When preparing, all solid components are mixed and crushed in advance, and then are mixed with pectin (such as containing pectin) and purified water uniformly, otherwise, the flavonoid compound can not be completely dissolved.
Specifically, the lower limit of the amount of pectin may be independently selected from 1 part by weight, 1.5 parts by weight, 2 parts by weight, 2.5 parts by weight, and 3 parts by weight; the upper limit of the amount of pectin can be independently selected from 2.7 parts by weight, 3.5 parts by weight, 4 parts by weight, 4.5 parts by weight and 5 parts by weight.
Specifically, the lower limit of the amount of the edible gelatin may be independently selected from 1 part by weight, 2 parts by weight, 3 parts by weight, 4 parts by weight, and 5 parts by weight; the upper limit of the amount of the edible gelatin can be independently selected from 6 parts by weight, 7 parts by weight, 8 parts by weight, 9 parts by weight and 10 parts by weight.
In a third aspect of the present invention, a method for preparing a flavonoid compound composition is provided, which at least comprises:
weighing each component of the composition of the flavonoid compounds, mixing, and preparing into solid powder;
the method for preparing solid powder comprises at least one of physical grinding, heating and melting, spray drying, dry granulation, wet granulation and freeze drying.
In particular, the amount of the solvent to be used,
a) physical grinding: weighing the components according to the prescription amount, placing the components in a mortar with proper size or special grinding machine equipment, fully grinding the components until the components in the prescription are uniformly mixed, and then crushing the uniformly mixed materials in a physical or airflow crushing or pharmacopeia sieving mode;
b) heating and melting: placing the components of the prescription amount into a container with a proper size, heating to above 65 ℃ to enable the components of the prescription to be in a molten state, fully and uniformly stirring at the temperature, cooling to room temperature, and crushing the uniform mixture by adopting the method in the a);
c) spray drying: weighing the components according to the prescription amount, dispersing the components into purified water according to the solid content of 1-5 wt%, after uniform dispersion, performing spray drying by a spray dryer to obtain a powdery material, and then crushing the material after the spray drying by adopting the method in a);
d) and (3) dry granulation: putting the components in the prescription amount into a dry granulating machine for dry granulation, and crushing the granules obtained by the method in a);
e) and (3) wet granulation: placing the components of the prescription amount in a wet granulator, adding ethanol as a wetting agent, performing wet granulation, drying the prepared granules at the temperature of below 50 ℃ to remove ethanol, and finally crushing the granules by adopting the method in a).
f) And (3) freeze drying: weighing the components according to the prescription amount, dispersing the components in purified water according to the solid content of 5-20 wt%, and preparing the components into fluffy powdery materials through a freeze dryer after uniform dispersion.
The invention has the beneficial effects that:
1. according to the invention, the absorption slow-release substance and the solubilizer are added into the flavonoid compound to form the composition, so that the cell injury is reduced, and compared with the administration of the flavonoid compound alone, the cell activity is improved by at least 1.17 times, which shows that the composition can effectively reduce the cytotoxicity; meanwhile, the water solubility of the flavonoid compounds in the composition is obviously improved, and the bioavailability of the flavonoid compounds is improved.
2. The combination rate of the flavonoid compound composition and the plasma protein is obviously reduced and can be as low as 78% of the combination rate of the flavonoid compound and the plasma protein, which shows that the flavonoid compound composition is helpful for solving the potential accumulation problem in the metabolism of the flavonoid compound.
Detailed Description
The present invention will be described in further detail with reference to examples, but the method of carrying out the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified; the equipment used in the following examples, unless otherwise specified, was subject to the manufacturer's recommended use parameters.
The geranylflavone A adopted in the embodiment of the invention is produced by pharmaceutical limited companies of Yunnan Han Union, the content is more than or equal to 95 percent, and the structural formula of the geranylflavone A is as follows:
the pectin adopted in the embodiment of the invention is Wanguo YN-1, and the manufacturer is Anhui Yuning Biotechnology Limited; the edible gelatin is food grade, and the manufacturer is Xian Yuhua biotechnology limited company; the medium chain triglyceride is MCT powder with the specification of 70 percent, and the manufacturer is Jiangsu Yibeineng medicine technology company Limited; poloxamers of the type KolliphorRP188, manufactured by Pasteur USA; hydroxypropyl beta cyclodextrin, model No. MB1904 (see>98%) of Dalian, Melam Biotechnology, Inc.; the potassium sorbate is food grade, and the manufacturer is Hebei Runsheng Biotechnology Co. PEG4000 is of pharmaceutical grade, and the manufacturer is used for Xian Tianzheng medicineLimited adjuvants.
Example 1
Weighing 2.5 parts of geranylflavone A, 1.5 parts of pectin, 12 parts of poloxamer 188, 2.5 parts of hydroxypropyl beta cyclodextrin, 0.5 part of PEG4000, 0.1 part of potassium sorbate and 80.4 parts of purified water according to parts by weight.
Mixing geranylflavone A, poloxamer 188, hydroxypropyl beta cyclodextrin, PEG4000 and potassium sorbate, grinding into mixed powder, adding pectin and purified water into the mixed powder, stirring, and uniformly mixing to obtain a liquid composition, which is recorded as a formula 1.
Example 2
Weighing 20 parts of geranylflavone A, 5 parts of edible gelatin, 65 parts of poloxamer 188, 15 parts of hydroxypropyl beta cyclodextrin and 0.1 part of citric acid according to parts by weight. Grinding into 100-200 mesh powder in a mortar, and marking as formula 2.
Example 3
Weighing 20 parts of geranylflavone A, 5 parts of edible gelatin, 5 parts of glucan, 25 parts of medium-chain fatty acid powder, 40 parts of hydroxypropyl beta cyclodextrin, 5 parts of PEG4000, 0.01 part of potassium sorbate and 0.2 part of vitamin C according to parts by weight. Dispersing the materials in purified water according to the solid content of 10 wt%, uniformly dispersing, and performing freeze drying for 24 hours by using a freeze dryer (model FD-1A-50) to obtain fluffy fine powder of 80-100 meshes, wherein the fluffy fine powder is recorded as a formula 3.
Example 4
Weighing 10 parts of geranylflavone A, 5 parts of pectin, 30 parts of poloxamer 188, 5 parts of hydroxypropyl beta cyclodextrin, 0.1 part of citric acid and 4 parts of vitamin E according to parts by weight. Grinding the mixture into fine powder of 100 to 200 meshes in a mortar.
Solubility test of composition in water
100ml of deionized water is respectively placed in 4 beakers, geranylflavonoid A and a formula of 1-3 are respectively added into the beakers, the dosage of geranylflavonoid A is increased by 0.1% each time, a glass rod is broken up, the mixture is uniformly stirred, and heating and ultrasonic assistant dissolution at 50 ℃ are assisted. When the dissolution is obviously stopped, adding the solution again to supersaturate the solution, continuously performing ultrasonic treatment and heating at 50 ℃ to assist the dissolution for 30min, standing the solution to room temperature, filtering the solution by using a 0.45-micrometer needle filter, taking the continuous filtrate, introducing the continuous filtrate into a liquid phase (instrument model Agilent 1100) to detect the concentration of geranylflavone A, and converting the solubility of geranylflavone A in 100ml of water to obtain the result shown in Table 1.
TABLE 1 solubility results in water for different formulations
As can be seen from the table, the solubility of geranylflavone A in the composition of formulas 1-3 is obviously increased, which shows that the composition can improve the water solubility and further improve the bioavailability of administration.
In vitro cell experiment (composition attenuation effect) of geranylflavone A
(1) The experimental method comprises the following steps:
collecting L-02 normal liver cells (YS-XB 1681) with good cell morphology in logarithmic growth phase, digesting with 0.25% trypsin containing EDTA to obtain single cell suspension, and regulating cell concentration to about 1 × 105One/ml, inoculated into 96-well culture plates, each well inoculated with 100. mu.l of single cell suspension. Place the plates in CO2Incubator (A)
5%) at 37 ℃ for 24h, and the culture medium was discarded.
Setting a blank control (physiological saline); 0.2mmol/L geranylflavone A aqueous solution (adding dimethyl sulfoxide DMSO (DMSO/water, V/V) with the volume fraction of 2% to assist dissolution to obtain geranylflavone A aqueous solution with the concentration of 0.2 mmol/L); three concentration gradients of high (2mmol/L), medium (0.2mmol/L) and low (0.02mmol/L) are respectively set in the prescriptions 1 to 3, and above 11 samples are respectively provided with 5 identical repeated samples.
Under the aseptic condition, respectively adding 100 mul of blank control, 100 mul of geranyl flavonoid A water solution and 100 mul of three concentration gradient samples with the concentration of 1-3 in the prescription into the cultured cells, and placing the culture plate in CO again2Culture box(
5%) at 37 ℃ for 24 h.
Under the aseptic condition, respectively adding 50 μ l of 1mg/ml MTT solution into each well, continuously culturing for 2h under the same condition, and sucking out supernatant; then 100 mul of isopropanol is added into each hole respectively, the mixture is shaken for 15min until the crystals are completely dissolved, and the absorbance values are measured under the conditions of 570nm and 650nm by an enzyme-labeling instrument.
(2) The experimental results are as follows:
the percent cell viability was calculated according to the following formula:
the cell activity is less than or equal to 70 percent, which indicates that the test article has potential cytotoxicity.
The results of the experimental measurements are shown in table 2:
TABLE 2 cell viability assay results for compositions of different formulations and different concentrations
As can be seen from Table 2, the geranylflavone A composition in the examples of the present invention has significantly reduced cell damage and improved cell viability by at least 1.17 times compared to the flavonoid compounds alone, indicating that the composition of the present invention can effectively reduce cytotoxicity.
Experiment for evaluating plasma protein binding rate by equilibrium dialysis method
(1) The experimental method comprises the following steps:
1) preparing a test solution: taking the composition samples of the formulas 1-3 obtained in the embodiments 1-3 and geranylflavone A raw materials respectively, and adding phosphate buffer solution (PBS solution) with pH7.4 into the formulas 1-3 to prepare 3 gradients of high (2mmol/L), medium (0.2mmol/L) and low (0.02 mmol/L); adding phosphate buffer solution (PBS solution) with pH value of 7.4 into geranylflavone A raw material, and using dimethyl sulfoxide DMSO (DMSO/water, V/V) with volume fraction of 2% to assist dissolution to prepare the PBS solution of geranylflavone A with final concentration of 0.2 mmol/L.
2) Preparing a blank dialysate: accurately weighing 14.110g K2HPO4,2.592g KH2PO41.991g of NaCl was dissolved in 950ml of deionized water, adjusted to pH7.4 with 0.1mol/L of HCl, and then made up to 1000 ml.
3) Dialyzing the internal liquid: taking 200 mu L of fresh rabbit plasma by a liquid transfer gun, and adding 600 mu L of a test solution (geranylflavone A or a composition with a formula of 1-3) to obtain a test solution in a dialysis membrane. The molecular weight cut-off of the dialysis membrane is 5000D (model: G-RC-18-5K), and the dialysis cell is an ampoule bottle containing 20ml of blank dialysate.
4) And (3) special investigation: and (3) observing the peak appearance condition of the internal and external liquids after the dialysate, the mixed sample of the dialysate and the sample are subjected to dialysis balance in a dialysis tank, and observing whether the interference exists on the measurement result.
5) Preparation of a standard curve: taking 400 μ L of each sample solution, adding 200 μ L of dialysate, mixing, adding 400 μ L of methanol, centrifuging, collecting supernatant, and subjecting to HPLC (Agilent 1100) to test, and preparing standard curve of dialysis external liquid (or internal liquid) with sample concentration as abscissa and peak area as ordinate.
6) And (3) sample recovery rate investigation: taking 200 mu L of fresh rabbit plasma by a pipette, adding high, medium and low concentration samples respectively, and determining the recovery rate of geranylflavonoid A according to the steps 5).
7) Plasma protein binding rate assay: washing dialysis bag with deionized water, ligating one end, adding high, medium and low concentration blood plasma (200 μ L +600 μ L), balancing, performing reference to standard medium post-treatment method, sampling, and determining by HPLC.
HPLC determination conditions:
octadecylsilane chemically bonded silica gel as filler (C18, 4.6X 150mm, 3um chromatographic column), acetonitrile-water (acetonitrile-water volume ratio 70:30) as mobile phase, detection wavelength of 220nm, flow rate of 1ml/min, and column temperature of 25 deg.C. The number of theoretical plates is not less than 1500 calculated according to geranylflavone A peak.
(2) The experimental results are as follows:
the results of the experiment are shown in table 3:
TABLE 3 plasma protein binding Rate assay results for compositions of different formulations and different concentrations
The results show that the plasma protein binding rate of each formula is obviously reduced compared with geranylflavone A. It is shown that the compositions of the present invention help to address the potential accumulation problem in geranylflavonoid a metabolism.
Fourthly, researching pharmacokinetic properties of geranylflavone A and geranylflavone A composition in rat body for 3 prescriptions
After a geranylflavone A sample to be detected is fed to a rat by gavage, whole blood samples at different time are collected, plasma is separated, and the concentration of a drug in the plasma is determined by a liquid chromatography-tandem mass spectrometry method.
(1) Dosing regimens
Healthy SPF-rated rats 40 were purchased from Wittisley center (laboratory animals, Charles River), weighed 150-. Each group was treated by gavage, fasted for 12h before the test, and freely drunk water. The diets were uniformly fed 2h after dosing.
Geranyl flavone A and the composition of the geranyl flavone A of each formula are dissolved in normal saline to be 2mmol/L (calculated by geranyl flavone A pure substance) solution or suspension for experiment.
The liquid was administered intragastrically at a dosing volume of 10ml/kg for each group tested. 1) The blank control group is infused with normal saline; 2) geranylflavone A suspension is directly administrated by intragastric administration: 3) the composition in the formula 1-3 is dissolved in normal saline to be 2mmol/L and is administered by intragastric administration.
(2) Blood sampling time points and sample treatment:
sampling was carried out 10 time points 10min, 20min, 40min, 1.0h, 80min, 2.0h, 4.0h, 8.0h, 12.0h and 24h after administration.
At the time points set above, 0.3ml of venous blood was taken through the retrobulbar venous plexus of rats, placed in heparinized tubes, centrifuged at 11000rpm for 5min, plasma separated, and frozen in a-20 ℃ refrigerator.
(3) Sample testing and data analysis
1) The concentration of geranylflavonoid A in rat plasma was determined by LC/MS/MS.
Pharmacokinetic parameters after drug administration were calculated using non-compartmental analysis with WinNonlin software.
2) Mass spectrum conditions:
the instrument comprises the following steps: AB SCIEX Triple quad 5500 system; ESI: a positive ion; and (3) monitoring mode: SRM; geranylflavone a: [ M + Na ]]+m/z 459.9/152.0; euglycemia (internal standard): [ M + H ]]+ m/z 494.2/169.1.
3) Ultra Performance Liquid Chromatography (UPLC) conditions:
mobile phase A0.1% by volume aqueous formic acid-acetonitrile solution (v: v, 95:5)
Mobile phase B0.1% by volume formic acid acetonitrile-water solution (v: v, 95:5)
Elution procedure, as shown in table 4:
TABLE 4 elution conditions
4) Chromatographic conditions are as follows:
a chromatographic column: ACQUITY UPLC Protein BEH C18 column, column temperature: 60 ℃, flow rate: 1.2ml/min, retention time: geranylflavone A:6.6min, YOUJIANGTANG (internal standard): 0.87 min;
5) sample preparation: precisely taking 5 mu L of blood sample, adding 100 mu L of internal standard, performing vortex oscillation, performing centrifugation at 1300rpm at 4 ℃ for 10min, and taking 10 mu L of supernatant for sample injection analysis.
(4) Detecting the index
1) Detecting the concentration of geranylflavonoid A in plasma after the rat is subjected to intragastric administration with 10 ml/kg;
2) detection of the pharmacokinetic parameters (Peak concentration C) of the rats after gastric administration of 10ml/kgmaxTime to peak TmaxAUC under the curve of time of administration, and phase using geranylflavone A suspension as reference preparationF%) bioavailability, the results are shown in table 5.
TABLE 5 pharmacokinetic parameters of different geranylflavonoid A formulations after gastric gavage administration to mice
(5) Results of the study
Geranylflavone A has C of formula 1-3 compared with geranylflavone AmaxHigher, TmaxLonger, and can play a long-acting role after oral administration. In addition, the AUC and the relative bioavailability of the formula 1-3 are obviously superior to those of the geranylflavone A raw material, and the geranylflavone A composition has a better absorption effect in vivo and can reduce the dosage.
In conclusion, it is probably because the addition of straight-chain (or branched) polysaccharide such as pectin, edible gelatin and the like in the composition greatly delays the time when geranylflavonoid A reaches the maximum absorption concentration in the digestive tract, and a similar mechanism for delaying transmembrane transport exists in an extracellular experiment, so that the cytotoxic effect of geranylflavonoid A is effectively relieved.
Although the present invention has been described with reference to a few preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. The composition of the flavonoid compound is characterized by comprising the flavonoid compound, an absorption slow-release substance and a solubilizer;
the flavonoid compound is geranylflavone A;
the absorption slow-release substance comprises at least one of polysaccharide and edible protein;
the polysaccharide is at least one of pectin and glucan;
the edible protein is selected from gelatin;
the solubilizer is selected from at least one of poloxamer, polyethylene glycol, medium chain triglyceride and hydroxypropyl beta cyclodextrin;
the content of each component in the composition of the flavonoid compound is as follows: 1-20 parts of flavonoid compound, 1-10 parts of absorption slow release substance and 15-80 parts of solubilizer.
2. The flavonoid composition according to claim 1,
the composition of the flavonoid compound also comprises a preservative or/and an antioxidant;
the preservative is at least one of potassium sorbate and citric acid;
the antioxidant is at least one of vitamin C and vitamin E.
3. The flavonoid composition according to claim 2,
the content of the preservative is 0.01-0.1 part by weight;
the content of the antioxidant is 0.2-4 parts by weight.
4. A method for preparing a flavonoid composition according to any one of claims 1 to 3, characterized in that it comprises at least:
crushing solid components in the flavonoid compound composition to obtain solid powder;
mixing the solid powder with other components to obtain a mixture;
adding purified water into the mixture, and uniformly mixing to obtain a liquid flavonoid compound composition;
the amount of the purified water is 50-85 parts by weight.
5. A method for preparing a flavonoid composition according to any one of claims 1 to 3, characterized in that it comprises at least:
weighing each component of the composition of the flavonoid compounds, mixing, and preparing into solid powder;
the method for preparing the solid powder is at least one of physical grinding, heating and melting, spray drying, dry granulation, wet granulation and freeze drying.
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