CN117462531A - Dihydromyricetin composition and application - Google Patents
Dihydromyricetin composition and application Download PDFInfo
- Publication number
- CN117462531A CN117462531A CN202310375677.2A CN202310375677A CN117462531A CN 117462531 A CN117462531 A CN 117462531A CN 202310375677 A CN202310375677 A CN 202310375677A CN 117462531 A CN117462531 A CN 117462531A
- Authority
- CN
- China
- Prior art keywords
- dihydromyricetin
- weight
- group
- carbocisteine
- gluconic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
Disclosed are compositions comprising dihydromyricetin and uses thereof. In one aspect, it relates to the use of a combination comprising 1 part by weight of dihydromyricetin, 0.1 to 0.3 part by weight of carbocisteine, 1 to 3 parts by weight of a divalent metal gluconate salt for the preparation of a medicament for the prevention and/or treatment of tumors. Also relates to a composition comprising the components. The composition of the present invention exhibits excellent biological effects.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a dihydromyricetin composition and application thereof
Background
Ampelopsin, also known as dihydromyricetin, was first isolated from leaves of the ampelopsis plant Fujian tea, meliaefolia, family Vitaceae, genus Ampelopsis, kotake and Kubota at 1940, named ampelopsin, as described in CN112353793A (China patent application No. 202011273451.4). Dihydromyricetin has been proved to have various effects such as anti-tumor, antioxidation, hypolipidemic, anti-inflammatory and antibacterial, but related products of the dihydromyricetin monomer are not clinically available at present.
Phase I metabolism in the liver is dominated by CYP450 enzymes, and CYP450 enzymes are simultaneously expressed in the intestinal tract, in particular CYP3A4 sub-enzymes. CYP450 enzymes can be induced or inhibited by some chemical drugs, and can also be regulated and controlled by some auxiliary materials. The inhibition of the auxiliary materials on the metabolism of the medicine is different according to the physical and chemical properties of the medicine, and is also related to the dosage and physical and chemical properties of the auxiliary materials. That is, the metabolic processes in different medicines are different under the influence of auxiliary materials, and the regularity is not unified. So far, no relevant report is made on the influence and the influence mechanism of auxiliary materials on the in-vivo metabolism of the dihydromyricetin, and the biological influence of the auxiliary materials on the dihydromyricetin is not further researched and verified.
The inventor of the application finds that the metabolism of dihydromyricetin in vivo is mainly dominated by three sub-enzymes CYP3A4, CYP1A2 and CYP2E1 in the research, the metabolism speed is extremely high, the prototype medicine is greatly reduced within about 10min, and Km and Vmax are 737.7 mu M and 158.7 mu M/min/mg protein respectively. The group of inventors of the present application has unexpectedly found in CN112353793a that some adjuvants for increasing the solubility of drugs have also an effect of inhibiting the metabolism of dihydromyricetin. When the dihydromyricetin is mixed with the auxiliary materials to prepare the oral preparation, the dissolution of the dihydromyricetin can be promoted, the absorption can be increased, the metabolism of the dihydromyricetin in the body can be reduced, and the blood concentration of the dihydromyricetin in the body after oral administration can be effectively improved, so that the curative effect of the dihydromyricetin is improved or enhanced.
However, there remains a need in the art for new methods to increase the biological activity of dihydromyricetin, and for dihydromyricetin compositions having superior pharmaceutical properties and biological activity.
Disclosure of Invention
One of the purposes of the invention is to provide a dihydromyricetin pharmaceutical preparation which contains an effective amount of dihydromyricetin serving as a pharmaceutical active ingredient and auxiliary materials with an inhibiting effect on the in-vivo metabolism of the dihydromyricetin. Alternatively, another object of the present invention is to provide a method for improving the biological activity of flavonoid compounds. It has been found that at least one of the objects described herein is achieved by the method of the present invention, and the present invention has been completed based on such findings.
In a first aspect of the present invention, there is provided a method for improving or enhancing the bioavailability of dihydromyricetin by oral administration, characterized in that dihydromyricetin is formulated into an oral formulation by mixing with one or more excipients having metabolic inhibition, which is effective in improving or enhancing the concentration of dihydromyricetin in the plasma in vivo.
In one embodiment of the method of the present invention, the auxiliary material having a metabolic inhibition effect on dihydromyricetin according to the present invention is selected from the group consisting of: a mixture of one or more of poloxamer 188 (also commonly known as pluronic F68), HP-beta-CD (also commonly known as hydroxypropyl-beta-cyclodextrin), PVP K30 (also commonly known as povidone K30), tween-80, polyoxyethylene 40 hydrogenated castor oil (also commonly known as polyoxyethylene hydrogenated castor oil 40). Preferred are polyoxyethylene 40 hydrogenated castor oil, PVP K30.
In one embodiment of the method of the invention, the adjuvant used in the invention having an inhibitory effect on the in vivo metabolism of dihydromyricetin is present in an amount of between 0.1% and 20%, preferably between 1% and 10% by weight of the formulation.
In one embodiment of the method of the invention, the formulation of the invention is an oral formulation, the dosage form may be any suitable oral dosage form, preferably capsules, tablets, granules, solutions, suspensions, emulsions, and may be prepared using conventional techniques of pharmacy such as: mixing adjuvants (poloxamer 188, HP-beta-CD, PVP K30, tween-80, polyoxyethylene 40 hydrogenated castor oil) with dihydromyricetin, and making into oral preparation (including capsule, tablet, granule, solution, and suspension).
In one embodiment of the method of the invention, an oral emulsion is prepared by mixing and emulsifying a certain proportion of auxiliary materials with metabolism inhibition (poloxamer 188, tween-80, polyoxyethylene 40 hydrogenated castor oil) and a certain proportion of dihydromyricetin.
In one embodiment of the method of the present invention, dihydromyricetin and the above auxiliary materials can also be prepared into preparation intermediates (inclusion compounds, dispersions, nanoparticles) by adopting special technology, and then further prepared into oral preparations, such as:
mixing adjuvants (poloxamer 188, tween-80, polyoxyethylene 40 hydrogenated castor oil) with certain proportion and dihydromyricetin to obtain dihydromyricetin nano micelle by film dispersion method, lyophilizing or spray drying to obtain preparation intermediate, and further making into oral preparation (including capsule, tablet, granule);
mixing adjuvants (HP-beta-CD) with certain proportion with dihydromyricetin, grinding to obtain dihydromyricetin clathrate, drying to obtain intermediate, and further making into oral preparation (including capsule, tablet, and granule);
mixing adjuvants (PVP K30) with certain proportion and dihydromyricetin with certain proportion, melting to obtain solid dispersion, and making into oral preparation (including capsule, tablet, and granule).
In a second aspect of the present invention, there is provided a method of increasing the biological activity of a flavonoid comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of dihydromyricetin and a pharmaceutical excipient. Such as antitumor activity.
Based on a second aspect of the present invention, the present invention provides a pharmaceutical composition comprising dihydromyricetin, carbocisteine, a divalent metal gluconate salt (e.g. calcium gluconate or zinc gluconate salt).
According to a preferred embodiment of the pharmaceutical composition according to the invention, it comprises 1 part by weight of dihydromyricetin, 0.1 to 0.3 part by weight of carbocisteine, 1 to 3 parts by weight of a divalent metal gluconate salt, for example a calcium salt of gluconic acid or a zinc salt of gluconic acid.
According to a preferred embodiment of the pharmaceutical composition according to the invention, 1 part by weight of dihydromyricetin, 0.15 to 0.25 part by weight of carbocisteine, 1.5 to 2.5 parts by weight of a divalent metal gluconate salt (for example calcium gluconate salt or zinc gluconate salt) are included.
According to a preferred embodiment of the pharmaceutical composition according to the invention, 1 part by weight of dihydromyricetin, 0.2 part by weight of carbocisteine, 2 parts by weight of a divalent metal gluconate salt (for example calcium gluconate or zinc gluconate).
According to a preferred embodiment of the pharmaceutical composition according to the invention, pharmaceutically acceptable excipients are optionally also included. It has been unexpectedly found that the present invention adds at the same timeCarbocisteinePharmaceutical compositions of (also commonly referred to as carbocisteine) and a divalent metal salt of gluconic acid (e.g. a calcium salt of gluconic acid or a zinc salt of gluconic acid) exhibit significantly more excellent biological activity, especially antitumor activity.
According to a preferred embodiment of the pharmaceutical composition according to the invention, it is in the form of a formulation for oral administration.
According to a preferred embodiment of the pharmaceutical composition according to the invention, maltodextrin is also comprised. For example, in an amount of 3 to 6 times the weight of dihydromyricetin, for example 4 to 5 times the weight of dihydromyricetin, for example 4.5 times the weight of dihydromyricetin,
further, a third aspect of the invention provides the use of a combination comprising dihydromyricetin, carbocisteine, a divalent metal gluconate salt (e.g. calcium gluconate or zinc gluconate) for the manufacture of a medicament for the prevention and/or treatment of a tumor.
A preferred embodiment of the use according to the invention, wherein the combination comprises 1 part by weight of dihydromyricetin, 0.1 to 0.3 part by weight of carbocisteine, 1 to 3 parts by weight of a divalent metal gluconate salt, such as a calcium salt of gluconic acid or a zinc salt of gluconic acid.
A preferred embodiment of the use according to the invention, wherein the combination comprises 1 part by weight of dihydromyricetin, 0.15 to 0.25 part by weight of carbocisteine, 1.5 to 2.5 parts by weight of a divalent metal gluconate salt, such as a calcium salt of gluconic acid or a zinc salt of gluconic acid.
According to a preferred embodiment of the use according to the invention, wherein the combination comprises 1 part by weight of dihydromyricetin, 0.2 part by weight of carbocisteine, 2 parts by weight of a divalent metal gluconate salt, such as for example the calcium salt of gluconic acid or the zinc salt of gluconic acid.
According to a preferred embodiment of the use according to the invention, wherein the medicament further optionally comprises pharmaceutically acceptable excipients.
According to a preferred embodiment of the use according to the present invention, wherein said tumor is lung cancer.
It has been unexpectedly found that the pharmaceutical composition of the present invention, to which both carbocisteine and a divalent metal gluconate salt (e.g. calcium salt of gluconic acid or zinc salt of gluconic acid) are added, exhibits significantly more excellent biological activity, in particular antitumor activity.
According to a preferred embodiment of the use according to the invention, wherein the medicament is in the form of an orally administered formulation.
According to a preferred embodiment of the use according to the invention, maltodextrin is further comprised in the medicament. For example, in an amount of 3 to 6 times the weight of dihydromyricetin, for example 4 to 5 times the weight of dihydromyricetin, for example 4.5 times the weight of dihydromyricetin,
it has been found that the present invention can significantly enhance the antitumor activity of dihydromyricetin by combining dihydromyricetin, carbocisteine, a divalent metal gluconate salt (e.g., calcium salt of gluconic acid or zinc salt of gluconic acid).
Detailed Description
Example 1: influence of different auxiliary materials on metabolism of dihydromyricetin in rat liver microsomes
As described in detail in example 1 of CN112353793A of the present inventors, 6 auxiliary materials (polyoxyethylene 40 hydrogenated castor oil, tween-80, HP-beta-CD, poloxamer 188, PVP K30 and PEG 400) were selected, added into a rat liver microsome (protein concentration: 1.0 mg/ml) incubation system respectively in a certain amount, and after shaking for 15min in a 37 ℃ water bath at 100rpm, dihydromyricetin (80. Mu. Mol/l) and NADPH coenzyme (1 mmol/l) were added respectively to initiate the reaction, after 10min, three times of ice methanol was added to terminate the reaction, vortexing was performed for 1min, and a low temperature high speed centrifuge (4 ℃ C., 13000 rpm) was centrifuged for 8min, and the supernatant was taken and the remaining amount of dihydromyricetin was compared by LC-MS/MS detection. The results show that the polyoxyethylene 40 hydrogenated castor oil, tween-80, HP-beta-CD, poloxamer 188 and PVP K30 with low and high doses (20 and 40 mug/ml) all show remarkable inhibition effect on the metabolism of dihydromyricetin and are dose-dependent. Whereas PEG400 has an inhibitory effect on metabolism, but is not dose-dependent, and when the dose reaches more than 100. Mu.g/ml, instead, there is a tendency for metabolic induction. This also shows that the effect of different adjuvants on dihydromyricetin metabolism is different.
Example 2: IC50 value determination of five auxiliary materials for inhibiting dihydromyricetin metabolism
As described in detail in example 2 of CN112353793A by the inventor team, 5 excipients were mixed in different concentration ranges: polyoxyethylene 40 hydrogenated castor oil (1.0-40.0 mug/ml), tween-80 (10.0-200.0 mug/ml), HP-beta-CD (4.0-100.0 mug/ml), poloxamer 188 (10.0-200.0 mug/ml), PVP K30 (4.0-100.0 mug/ml) are added into a rat liver microsome (protein concentration 1.0 mg/ml) incubation system according to a certain amount, after shaking for 15min at 100rpm in a 37 ℃ water bath, dihydromyricetin (80 mu mol/l) and NADPH coenzyme (1 mmol/l) are added respectively to start the reaction, after 10min, three times of ice methanol is added to stop the reaction, the shaking treatment is carried out for 1min, a low-temperature high-speed centrifuge (4 ℃ and 13000 rpm) is centrifuged for 8min, supernatant is taken, the residual amount of dihydromyricetin is compared through LC-MS/MS detection, and the IC50 value of each auxiliary material is calculated. The results show that the five auxiliary materials have strong and weak inhibiting effect on the metabolism of the dihydromyricetin: polyoxyethylene 40 hydrogenated castor oil > PVP K30> HP-beta-CD > Tween-80> poloxamer 188. The specific results are as follows: polyoxyethylene 40 hydrogenated castor oil had an IC50 value for inhibition of dihydromyricetin metabolism (μg/ml, n=3) of 3.69, tween-80 had an IC50 value for inhibition of dihydromyricetin metabolism (μg/ml, n=3) of 53.96, HP- β -CD had an IC50 value for inhibition of dihydromyricetin metabolism (μg/ml, n=3) of 25.35, poloxamer 188 had an IC50 value for inhibition of dihydromyricetin metabolism (μg/ml, n=3) of 61.76, PVP K30 had an IC50 value for inhibition of dihydromyricetin metabolism (μg/ml, n=3) of 15.25.
Example 3: pharmacokinetic comparison of adjuvant to oral administration of Dihydromyricetin
As described in detail in example 3 of CN112353793A of the inventor team, a solution was prepared by mixing a proportional amount of metabolic inhibition adjuvants (polyoxyethylene 40 hydrogenated castor oil, tween-80, HP-beta-CD, poloxamer 188, PVP K30) with a concentration of dihydromyricetin. The administration of the dihydromyricetin alone was performed by the intragastric administration method using dihydromyricetin as a control, and the orbital blood collection was started to determine the drug concentration in plasma at each time point. The operation is specifically as follows: rats (male, weighing 180-220 g) were randomly divided into 6 groups of 5 animals each. Fasted overnight before administration, and was free to drink water. The same dose of the mixed solution of the auxiliary material and the dihydromyricetin and the single dihydromyricetin solution (the dihydromyricetin is dissolved in the water solution containing dimethyl sulfoxide) (50 mg/kg) are respectively and gastrically administrated, blood is taken from orbital venous plexus respectively at 5, 10, 15, 30, 45, 60, 90, 120, 240, 360, 480 and 720min after administration, and the blood is placed in a centrifuge tube added with heparin sodium, and the blood is centrifugally separated at 4000rpm to obtain blood plasma. 100 μl of plasma was taken, 200 μl of acetate buffer (pH=4.5) was added, 1.2ml of ethyl acetate was added, vortexed for 30s, and centrifuged at 12000rpm for 10min. The upper organic phase was aspirated, dried in a 50℃water bath with nitrogen, and the residue was dissolved in ice acetonitrile, and 20. Mu.l of the sample was taken. Dihydromyricetin was determined by HPLC method and the chromatographic conditions were as follows: chromatographic column: ODS-SP C18 column (4.6 mm. Times.150 mm,5 μm), pre-column: dimma (C18, 10mm×4.6 mm), mobile phase: 2% aqueous acetic acid-acetonitrile (9:1), flow rate: 1ml/min, column temperature: 25 ℃, detection wavelength: 292nm, sample injection amount: 20 μl. The results of the blood concentration time curves of the six experimental groups show that the oral bioavailability of the dihydromyricetin is respectively improved to different degrees by mixing and dosing five different auxiliary materials of polyoxyethylene 40 hydrogenated castor oil, tween-80, HP-beta-CD, poloxamer 188 and PVP K30 with the dihydromyricetin, and the oral bioavailability of the dihydromyricetin is respectively improved to 3.08 times, 1.92 times, 2.26 times, 1.63 times and 2.45 times in sequence compared with the single administration of the dihydromyricetin solution.
Examples 4-8 below are provided with reference to examples 4-8 of CN112353793A of the inventor team.
Example 4: precisely weighing 0.8g of dihydromyricetin, 15.5g of poloxamer 188, adding 80ml of purified water, fully standing, stirring for dissolving, adding a proper amount of preservative and sweetener, adding purified water to a volume of 100ml, filtering, quantitatively packaging into small bottles, and thus obtaining the dihydromyricetin oral solution.
Example 5: precisely weighing 1.0g of dihydromyricetin, 6g of polyoxyethylene 40 hydrogenated castor oil, 3.5g of Tween-80, 15ml of olive oil and 50ml of purified water, placing into a high-speed homogenizing machine, treating for 3min at 6000rpm, adding a proper amount of preservative and sweetener, adding purified water to a volume of 100ml, mixing uniformly, and quantitatively packaging into small bottles to obtain the dihydromyricetin oral emulsion.
Example 6: 1.0g of dihydromyricetin, 10g of HP-beta-CD and 5g of PVP K30 are precisely weighed and uniformly mixed. Adding appropriate amount of purified water, making into soft material, and sieving with 18 mesh sieve to obtain wet granule. Drying in oven at 60deg.C for 2 hr, granulating, adding magnesium stearate as lubricant, mixing, and packaging into bags with granule packaging machine according to 3g per bag to obtain dihydromyricetin oral granule.
Example 7: 3.0g of dihydromyricetin, 15g of HP-beta-CD, 65g of microcrystalline cellulose and 15g of compressible starch are precisely weighed and uniformly mixed. The dihydromyricetin oral tablet is prepared by pressing into a tablet of 0.3 g/tablet by a rotary tablet press.
Example 8: accurately weighing 2.0g of dihydromyricetin, 15g of HP-beta-CD and 10g of poloxamer 188, and uniformly mixing. Adding appropriate amount of purified water, making into soft material, and sieving with 18 mesh sieve to obtain wet granule. Drying in oven at 60deg.C for 2 hr, granulating, adding magnesium stearate as lubricant, mixing, and filling into hard capsule shell with full-automatic capsule filling machine at a ratio of 0.2 g/granule to obtain dihydromyricetin oral capsule.
Example 11: dihydromyricetin composition
Example 11a: respectively crushing the calcium salt of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed calcium salt of dihydromyricetin, carbocisteine and gluconic acid by a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.2:2 to obtain the composition of example 11a (E11 a for short), which can be further made into solid preparations such as capsules, granules, tablets, etc.
Example 11b: respectively crushing the calcium salt of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed calcium salt of dihydromyricetin, carbocisteine and gluconic acid by a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.15:2.5 to give a composition of example 11b (abbreviated as E11 b).
Example 11c: respectively crushing the calcium salt of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed calcium salt of dihydromyricetin, carbocisteine and gluconic acid by a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.25:1.5 to give a composition of example 11c (abbreviated as E11 c).
Example 11d: respectively crushing zinc salts of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed zinc salts with a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.2:2 to give a composition of example 11d (abbreviated as E11 d).
Example 11e: respectively crushing zinc salts of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed zinc salts with a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.15:2.5 to give a composition of example 11E (abbreviated as E11E).
Example 11f: respectively crushing zinc salts of dihydromyricetin, carbocisteine and gluconic acid, sieving the crushed zinc salts with a 120-mesh sieve, and then carrying out the following steps according to the weight ratio of 1:0.25:1.5 to give a composition of example 11f (abbreviated as E11 f).
In the present invention, the carbocisteine is S- (carboxymethyl) cysteine. When a drug is added in a test at a cellular level or an animal level, each drug is dissolved in hot water and diluted to an appropriate concentration before use unless otherwise specified.
Example 12: effect of Dihydromyricetin on A549 cell proliferation
This example uses the classical MTT method to test the effect of dihydromyricetin on a549 cell proliferation. The operation process is as follows:
digesting A549 cells in logarithmic phase with pancreatin to obtain cell suspension at a ratio of 5×10 4 Inoculating 100 μl of each well into 96-well culture plate, setting blank control group, normal cell group and dosing group, setting 3 multiple wells in each group, and heating at 37deg.C and 5% CO 2 Is cultured in a sterile incubator for 60 hours.
Dihydromyricetin was added to the dosing groups at final concentrations of 1. Mu.M, 5. Mu.M, 20. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 500. Mu.M, respectively, and the culture medium was used as a blank (0. Mu.M) and incubated in a sterile incubator under the same conditions for 24 hours.
Adding 5g/L MTT solution 20 μl into each well 4 hr before culturing, absorbing supernatant after culturing, adding 100 μl dimethyl sulfoxide into each well, shaking for 10min to dissolve purple crystals, and detecting absorbance of each well by enzyme-labeling instrument 570nm ) Values, cell viability (relative percentage to the blank) was calculated, results: four groups of 1. Mu.M, 5. Mu.M, 20. Mu.M, and 50. Mu.M had cell viability in the range of 96 to 101%, for example, the 5. Mu.M group had cell viability of 98.2%, and the 100. Mu.M, 200. Mu.M, and 500. Mu.M groups had cell viability of 87.5%, 78.2%, and 47.3%, respectively.
The above results show that 0-50. Mu.M of the drug has no effect on the proliferation of A549 cells, the survival rate of A549 cells can be reduced to below 80% when the drug concentration reaches 200. Mu.M, and the survival rate of A549 cells can be reduced to below 50% when the drug concentration reaches 500. Mu.M, which shows that the dihydromyricetin has smaller cytotoxicity on A549 cells in the concentration range of 0-50. Mu.M.
Example 121: effect of Dihydromyricetin on A549 cell proliferation
This example refers to example 12 for testing the effect of dihydromyricetin on a549 cell proliferation using the classical MTT method. The operation process is as follows:
digesting A549 cells in logarithmic phase with pancreatin to obtain cell suspension at a ratio of 5×10 4 Inoculating 100 μl of each well into 96-well culture plate, setting blank control group, normal cell group and dosing group, setting 3 multiple wells in each group, and heating at 37deg.C and 5% CO 2 Is cultured in a sterile incubator for 60 hours.
The final concentration of dihydromyricetin, carbocisteine and calcium salt of gluconic acid are respectively 1 mu M,5 mu M, 20 mu M, 50 mu M, 100 mu M, 200 mu M and 500 mu M, the concentration of carbocisteine is 0.2 times of that of dihydromyricetin, the concentration of calcium salt of gluconic acid is 2 times of that of dihydromyricetin, and in addition, the culture medium is used as a blank control group (0 mu M), and the culture is carried out for 24 hours in a sterile incubator under the same conditions.
Adding 5g/L MTT solution 20 μl into each well 4 hr before culturing, absorbing supernatant after culturing, adding 100 μl dimethyl sulfoxide into each well, shaking for 10min to dissolve purple crystals, and detecting absorbance of each well by enzyme-labeling instrument 570nm ) Values, cell viability (relative percentage to the blank) was calculated, results: the cell viability of four groups of 1. Mu.M, 5. Mu.M, 20. Mu.M and 50. Mu.M of dihydromyricetin is in the range of 96-103%, for example, the cell viability of 5. Mu.M group is 97.6%, and the cell viability of 100. Mu.M, 200. Mu.M and 500. Mu.M groups is 84.1%, 77.5% and 49.6%, respectively.
The above results show that 0-50 mu M dihydromyricetin and the calcium salt of carbocisteine and gluconic acid have no effect on the proliferation of A549 cells, when the drug concentration reaches 200 mu M, the survival rate of A549 cells can be reduced to below 80%, and when the drug concentration reaches 500 mu M, the survival rate of A549 cells can be reduced to below 50%, which shows that the cytotoxicity of 0-50 mu M dihydromyricetin added with the calcium salt of carbocisteine and gluconic acid on A549 cells is smaller.
Example 121a: referring to the method of example 121, except that only carbocisteine was added and calcium salt of gluconic acid was not added, the results of the test were substantially identical to those of example 121, and the survival rate of dihydromyricetin 1. Mu.M to 50. Mu.M was in the range of 97 to 102%, for example, the survival rate of 5. Mu.M group was 98.5%, and the survival rates of 100. Mu.M, 200. Mu.M, 500. Mu.M group were 81.7%, 74.3%, 42.7%, respectively.
Example 121b: referring to the method of example 121, except that only calcium salt of gluconic acid was added without carbocisteine, the results of the test were substantially identical to those of example 121, and the viability of dihydromyricetin 1. Mu.M to 50. Mu.M was in the range of 96 to 101%, for example, the cell viability of 5. Mu.M group was 97.3%, and the cell viability of 100. Mu.M, 200. Mu.M, 500. Mu.M group was 83.5%, 76.4%, 45.8%, respectively.
Example 121c: referring to the method of example 121, except that the calcium salt of gluconic acid was changed to zinc salt of gluconic acid in the same amount, the results of the test were substantially identical to those of example 121, and the cell viability of four groups of dihydromyricetin 1. Mu.M to 50. Mu.M was in the range of 98 to 102%, for example, the cell viability of 5. Mu.M group was 99.7%, and the cell viability of 100. Mu.M, 200. Mu.M, 500. Mu.M groups was 86.3%, 76.8%, 47.3%, respectively.
Example 121d: referring to the method of example 121b, except that the calcium salt of gluconic acid was changed to zinc salt of gluconic acid in the same amount, the results of the test were substantially identical to those of example 121b, and the cell viability of four groups of dihydromyricetin 1. Mu.M to 50. Mu.M was 98.7% in the range of 98 to 100%, for example, the cell viability of groups of 5. Mu.M, 84.8%, 79.3% and 48.5% in the groups of 100. Mu.M, 200. Mu.M and 500. Mu.M, respectively.
Example 13: effect of dihydromyricetin on immune cells
In this example, the activity of dihydromyricetin in killing immune cells was examined by a conventional method. The operation process is as follows:
digesting A549 cells in logarithmic phase with pancreatin to obtain cell suspension at a ratio of 5×10 4 The concentration of/mL was inoculated into 96-well plates, 100. Mu.L per well, a blank group, a normal cell group, a maximum LDH release group, an IFN-. Gamma.group and a dosing group were set, 3 duplicate wells were set per group, and cultured in a sterile incubator at 37℃with 5% CO2 for 24 hours.
Adding dihydromyricetin or positive control 1-methyl-tryptophan (1-MT) with final concentration of 10 μm and 20 μm into the dosing group of A549 cells respectively for 2h, and then adding IFN-gamma; each group was cultured in a sterile incubator at 37℃with 5% CO2 for 24 hours.
A549 medium was changed with 100 μl of NK cell complete medium, and NK cells were then plated at 5×10 5 A96-well plate plated with A549 cells was inoculated at a concentration of/mL, 100. Mu.L per well, and cultured in a sterile incubator at 37℃with 5% CO2 for 4 hours.
To the maximum Lactate Dehydrogenase (LDH) release group, 20. Mu.L of 1.5% NP40 lysate was added and the mixture was treated in a sterile incubator at 37℃and 5% CO2 for 60 minutes to complete the lysis.
The 96-well plate was centrifuged at 1500rpm for 5 minutes, 50. Mu.L of the supernatant was placed in a fresh 96-well plate, 50. Mu.L of the substrate was added, the reaction was carried out in the dark for 0.5 hour, and then 50. Mu.L of a stop solution was added to each well, and the absorbance at 492nm was measured.
The ratio of the absorbance of each well to the absorbance of the maximum LDH release group was calculated, and the LDH killing rate (%) was calculated as follows:
results of LDH killing rate: 12.6% of IFN-gamma group, 36.4% of IFN-gamma+10. Mu.M dihydromyricetin group, 49.7% of IFN-gamma+20. Mu.M dihydromyricetin group and 22.3% of IFN-gamma+1-MT group. From the results, when IFN-gamma exists, the A549 cells and the NK cells are co-cultured, and after the A549 cells are pretreated by the dihydromyricetin, the killing activity of the NK cells to the A549 cells can be obviously improved, which shows that the dihydromyricetin can enhance the killing activity of immune cells.
Example 131: effect of dihydromyricetin on immune cells
This example refers to example 13, which uses conventional methods to examine the killing immune cell activity of dihydromyricetin. The operation process is as follows:
digesting A549 cells in logarithmic phase with pancreatin to obtain cell suspension at a ratio of 5×10 4 Inoculating 100 μl of the culture medium into 96-well culture plate, and setting blank control group, normal cell group, maximum LDH release group, IFN-gamma group and drug administration group, each group3 duplicate wells were incubated in a sterile incubator at 37℃with 5% CO2 for 24h.
Adding dihydromyricetin with a final concentration of 6.5 μm (wherein carbocisteine with a concentration of 0.2 times that of dihydromyricetin and calcium salt of gluconic acid with a concentration of 2 times that of dihydromyricetin) and dihydromyricetin with a final concentration of 13 μm (wherein carbocisteine with a concentration of 0.2 times that of dihydromyricetin and calcium salt of gluconic acid with a concentration of 2 times that of dihydromyricetin) respectively to the dosing group of A549 cells, treating for 2 hours, or positive control 1-methyl-tryptophan (1-MT), and then adding IFN-gamma; each group was incubated at 37℃in a 5% CO2 sterile incubator for 24h.
A549 medium was changed with 100 μl of NK cell complete medium, and NK cells were then plated at 5×10 5 A96-well plate plated with A549 cells was inoculated at a concentration of 100. Mu.L per well at 37℃with 5% CO 2 Is cultured in a sterile incubator for 4 hours.
To the maximum Lactate Dehydrogenase (LDH) release group, 20. Mu.L of 1.5% NP40 lysate was added and the mixture was treated in a sterile incubator at 37℃and 5% CO2 for 60 minutes to complete the lysis.
The 96-well plate was centrifuged at 1500rpm for 5 minutes, 50. Mu.L of the supernatant was placed in a fresh 96-well plate, 50. Mu.L of the substrate was added, the reaction was carried out in the dark for 0.5 hour, and then 50. Mu.L of a stop solution was added to each well, and the absorbance at 492nm was measured.
The ratio of the absorbance of each well to the absorbance of the maximum LDH release group was calculated, and the LDH killing rate (%) was calculated as follows:
results of LDH killing rate: IFN-gamma group 11.3%, IFN-gamma+6.5mu.M dihydromyricetin (supplemented with carbocisteine and calcium salt of gluconic acid) group 38.7%, IFN-gamma+13. Mu.M dihydromyricetin (supplemented with carbocisteine and calcium salt of gluconic acid) group 52.2%, IFN-gamma+1-MT group 23.8%. From the results, when IFN-gamma exists, the A549 cells and the NK cells are co-cultured, and after the A549 cells are pretreated by the dihydromyricetin, the killing activity of the NK cells to the A549 cells can be obviously improved, which shows that the dihydromyricetin can enhance the killing activity of immune cells. In addition, comparable killing rates were also obtained with lower concentrations of drug compared to example 13, indicating that the killing activity of dihydromyricetin on a549 cells by immune NK cells was enhanced by the addition of carbocisteine and the calcium salt of gluconic acid.
Example 131a: referring to the method of example 131, only a549 cells were dosed separately: adding dihydromyricetin with final concentration of 10 μm (wherein 0.2 times of carboxymethyl and no calcium salt of gluconic acid are added), adding dihydromyricetin with final concentration of 20 μm (wherein 0.2 times of carboxymethyl and no calcium salt of gluconic acid are added), or treating positive control 1-MT for 2 hr, adding IFN-gamma, standing at 37deg.C and 5% CO 2 Culturing for 24 hours in a sterile incubator; the test results were substantially the same as in example 131, with a value of 36.4% for the 10. Mu.M dihydromyricetin (carbocisteine-containing) group and 51.4% for the 20. Mu.M dihydromyricetin (carbocisteine-containing) group.
Example 131b: referring to the method of example 131, only a549 cells were dosed separately: adding dihydromyricetin with final concentration of 10 μm (wherein calcium salt of gluconic acid with 2 times of dihydromyricetin concentration is also added but carboxymethyl span is not added), adding dihydromyricetin with final concentration of 20 μm (wherein calcium salt of gluconic acid with 2 times of dihydromyricetin concentration is also added but carboxymethyl span is not added), or positive control 1-MT is treated for 2h, adding IFN-gamma, standing at 37deg.C, and 5% CO 2 Culturing for 24 hours in a sterile incubator; the test results were substantially the same as in example 131, with a value of 34.7% for the 10. Mu.M dihydromyricetin (calcium salt containing gluconic acid) group and 48.4% for the 20. Mu.M dihydromyricetin (calcium salt containing gluconic acid) group. The results of examples 131a and 131b show that the effect of dihydromyricetin in this test cannot be enhanced without the simultaneous addition of both carbocisteine and the calcium salt of gluconic acid.
Example 131c: referring to the method of example 131, except that the calcium salt of gluconic acid therein was changed to zinc salt of gluconic acid in an equivalent amount, the results of the test were substantially identical to those of example 131, and the killing rate was found to be 40.3% for IFN-. Gamma. + 6.5. Mu.M dihydromyricetin (supplemented with carboxymethyl and zinc salt of gluconic acid) and 55.6% for IFN-. Gamma. + 13. Mu.M dihydromyricetin (supplemented with zinc salt of carboxymethyl and gluconic acid).
Example 131d: referring to the method of example 131b, except that the calcium salt of gluconic acid was changed to a zinc salt of the same amount of gluconic acid, the results of the test were substantially identical to those of example 131b, the killing rate was found to be 33.3% in the 10. Mu.M dihydromyricetin (zinc salt containing gluconic acid) group, and 51.2% in the 20. Mu.M dihydromyricetin (zinc salt containing gluconic acid) group.
Example 14: in vivo antitumor Activity
In this example, the antitumor activity of dihydromyricetin in animals was examined using the typical tumor inhibitor 1-methyl-tryptophan as a positive drug. The operation process is as follows:
dihydromyricetin (crushed and sieved by a 120-mesh sieve) is suspended in 1% sodium carboxymethylcellulose to prepare a suspension with proper concentration.
40C 57 BL/6 mice (Male, 6w old, 18-22g, SPF grade, SYXK (Min) 2021-0005, megaku Co.) were divided into 4 groups (10/each group), each with 2X 10 inoculation of the left underarm 6 And (3) establishing a lung cancer transplantation tumor model by using Lewis lung cancer cells of the mice. 72 hours after inoculation, the control group was given 1% sodium carboxymethylcellulose, the positive group was given 20mg/kg of 1-methyl-tryptophan, and the 2 drug groups were given 10mg/kg of dihydromyricetin or 25mg/kg of body weight orally. The positive group was administered once daily to the abdominal cavity, the remaining groups were administered once daily to the stomach by gavage, and animals were free to drink water for 14 consecutive days. The weight of the mice was measured every two days, and the weight of the mice and the tumor weight were weighed after the last administration.
For weight change, the weight change percentage is obtained by dividing the daily average weight of each group of animals by the daily average weight of the blank group of animals and multiplying the daily average weight of each group of animals by 100 percent, and the weight change percentages of the positive group and the 2 drug groups in the test period are in the range of 96-103 percent, so that no obvious difference exists between the weight of each group of animals and the weight of the blank group.
Average tumor inhibition was calculated from the average tumor weights of the animals in each group, as a result: the positive group 37.6%, the 10mg/kg dose group 44.2% and the 25mg/kg dose group 62.7% show that the dihydromyricetin can obviously inhibit the growth of the Lewis lung cancer transplants of mice.
Example 141: in vivo antitumor Activity
This example refers to example 14, which uses the typical tumor suppressor 1-methyl-tryptophan as a positive drug, to examine the antitumor activity of dihydromyricetin in animals. The operation process is as follows:
dihydromyricetin (crushed and sieved by a 120-mesh sieve) is suspended in 1% sodium carboxymethylcellulose to prepare a suspension with proper concentration.
40C 57 BL/6 mice (Male, 6w old, 18-22g, SPF grade, SYXK (Min) 2021-0005, megaku Co.) were divided into 4 groups (10/each group), each with 2X 10 inoculation of the left underarm 6 And (3) establishing a lung cancer transplantation tumor model by using Lewis lung cancer cells of the mice. 72h after inoculation, the control group was given 1% sodium carboxymethylcellulose, the positive group was given 20mg/kg 1-methyl-tryptophan, the first group was given 6mg/kg dihydromyricetin orally (to which was also added carbocisteine at a dose of 0.2 times the dose of dihydromyricetin, the calcium salt of gluconic acid) and the second group was given 15mg/kg dihydromyricetin orally (to which was also added carbocisteine at a dose of 0.2 times the dose of dihydromyricetin, the calcium salt of gluconic acid) respectively. The positive group was administered once daily to the abdominal cavity, the remaining groups were administered once daily to the stomach by gavage, and animals were free to drink water for 14 consecutive days. The weight of the mice was measured every two days, and the weight of the mice and the tumor weight were weighed after the last administration.
For weight changes, the percentage of weight change in the positive group and the 2 drug groups in the test period were in the range of 97-103%, indicating that there was no significant difference in weight between each group of animals and the blank group.
Average tumor inhibition was calculated from the average tumor weights of the animals in each group, as a result: positive group 37.6%, (supplemented with carbocisteine and calcium salt of gluconic acid) 6mg/kg dose group 42.8%, (supplemented with carbocisteine and calcium salt of gluconic acid) 15mg/kg dose group 64.3%, and the results show that dihydromyricetin can obviously inhibit the growth of Lewis lung cancer transplantation tumor of mice.
Example 141a: referring to the method of example 141, only two drug group settings were varied: the first group was given 10mg/kg dihydromyricetin (to which was also added carbocisteine at a dose of 0.2 times but no calcium salt of gluconic acid), and the second group was given 25mg/kg dihydromyricetin (to which was also added carbocisteine at a dose of 0.2 times but no calcium salt of gluconic acid), and as a result, the average tumor suppression rates of the two groups were 44.6% and 61.3%, respectively, and the weight change of each group of animals was substantially identical to example 141.
Example 141b: referring to the method of example 141, only two drug group settings were varied: the first group was given 10mg/kg dihydromyricetin (with 2 times the dose of dihydromyricetin calcium salt but no carbocisteine added) and the second group was given 25mg/kg dihydromyricetin (with 2 times the dose of dihydromyricetin calcium salt but no carbocisteine added) and as a result, the average tumor inhibition rates of the two groups were 41.3% and 59.7%, respectively, and the weight changes of the animals of each group were substantially identical to those of example 141.
Example 141c: with reference to the procedure of example 141, except that the calcium salt of gluconic acid therein was changed to a zinc salt of gluconic acid in an equivalent amount, the results of the test were substantially identical to those of example 141, with a dose of 40.4% in the 6mg/kg dose group (supplemented with the zinc salts of carbocisteine and gluconic acid) and a dose of 65.8% in the 15mg/kg dose group (supplemented with the zinc salts of carbocisteine and gluconic acid).
Example 141d: with reference to the procedure of example 141b, except that the calcium salt of gluconic acid was changed to a zinc salt of the same amount of gluconic acid, the results of the test were substantially identical to those of example 141b, and the average tumor suppression rates of the two dose groups of 10mg/kg and 25mg/kg were 39.6% and 58.2%, respectively.
Example 141e: referring to the method of example 141, only the drug group was set as two groups: group A given 5mg/kg of carbocisteine and 50mg/kg of calcium salt of gluconic acid, group B given 5mg/kg of carbocisteine and 50mg/kg of zinc salt of gluconic acid, as a result of A, B, the average tumor suppression rates of the two groups were-3.6% and 2.2%, respectively, and the weight change of animals was substantially identical to that of example 141.
The experiments described above by way of reference to example 121a, example 131a, example 141a, etc. are performed in parallel in the experiments to which reference is made respectively, and the description of the present invention is merely for convenience of explanation.
The results of examples 141, 141a, 141b, 141c, 141d and 141e show that dihydromyricetin can significantly inhibit the growth of mouse Lewis transplanted tumors, and the effect of dihydromyricetin on tumor growth inhibition can be enhanced after the addition of the calcium salt/zinc of carboxymethyl and gluconic acid, and the effect of dihydromyricetin in the test cannot be enhanced without the simultaneous addition of the calcium salt/zinc of carboxymethyl and gluconic acid.
Example 15: dihydromyricetin composition
Example 15a: respectively crushing dihydromyricetin, carbocisteine, calcium salt of gluconic acid and maltodextrin, sieving with a 120-mesh sieve, and then mixing the materials according to the weight ratio of 1:0.2:2:4.5 to obtain the composition of example 15a (E15 a for short), which can be further made into solid preparations such as capsules, granules, tablets, etc.
Example 15b: crushing dihydromyricetin, carbocisteine, zinc salt of gluconic acid and maltodextrin respectively, sieving with a 120-mesh sieve, and then mixing the materials according to the weight ratio of 1:0.2:2:4.5 to obtain the composition of example 15b (E15 b for short), which can be further made into solid preparations such as capsules, granules, tablets, etc.
Example 16: stability investigation of the composition
Dihydromyricetin is known to be unstable in the presence of certain divalent metal ions (He Guixia, et al, stability study of Dihydromyricetin, J.New China, 2007, 16 (22): 1888), this example uses the HPLC method described in the He Guixia literature to determine the content of Dihydromyricetin in each solid composition at 0 month and 6 months after the solid compositions of examples 11 a-11 f, 15 a-15 b are left to stand for 6 months at a temperature of 40 ℃, and the percent (%) of the residue of Dihydromyricetin in that sample is obtained by dividing the content of Dihydromyricetin by the content of Dihydromyricetin at 6 months and multiplying the product by 100%. Results: the dihydromyricetin residual rates of examples 11a to 11f are in the range of 72 to 77%, for example, the dihydromyricetin residual rates of examples 11a and 11d are 75.7% and 73.2%, respectively, and the dihydromyricetin residual rates of examples 15a and 15b are 95.8% and 97.3%, respectively. In addition, referring to example 11a and example 11d, solid compositions were prepared without adding a divalent metal gluconate salt, and the residual dihydromyricetin rates at 6 months were 96.4% and 95.3%, respectively, as determined by the above method. In addition, referring to example 11a and example 11d, but substituting the divalent metal gluconate salt added thereto with an equivalent amount of gluconic acid to prepare a solid composition, the residual dihydromyricetin rates at 6 months were 98.2% and 96.8%, respectively, as determined by the above method. It has surprisingly been found that the effect of divalent metal gluconate on the instability properties of dihydromyricetin can be overcome when a defined amount of maltodextrin is added to the composition of the present invention.
Claims (10)
1. The use of a combination comprising 1 part by weight of dihydromyricetin, 0.1 to 0.3 part by weight of carbocisteine and 1 to 3 parts by weight of a divalent metal gluconate salt for the preparation of a medicament for the prevention and/or treatment of tumors.
2. Use according to claim 1, wherein the combination comprises 1 part by weight of dihydromyricetin, 0.15 to 0.25 part by weight of carbocisteine, 1.5 to 2.5 parts by weight of a divalent metal gluconate.
3. Use according to claim 1, wherein the combination comprises 1 part by weight of dihydromyricetin, 0.2 part by weight of carbocisteine and 2 parts by weight of a divalent metal gluconate.
4. The use according to claim 1, wherein the medicament further comprises maltodextrin in an amount of 3 to 6 times the weight of dihydromyricetin.
5. Use according to claim 1, wherein the divalent metal gluconate is a calcium or zinc gluconate.
6. The use according to claims 1-5, wherein the medicament further comprises pharmaceutically acceptable excipients; the medicament is in the form of an orally administered preparation; the tumor is lung cancer; alternatively, the maltodextrin is 4 to 5 times the weight of dihydromyricetin.
7. A pharmaceutical composition comprises 1 part by weight of dihydromyricetin, 0.1-0.3 part by weight of carbocisteine and 1-3 parts by weight of bivalent metal gluconate.
8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition comprises 1 part by weight of dihydromyricetin, 0.15 to 0.25 part by weight of carbocisteine, and 1.5 to 2.5 parts by weight of a divalent metal gluconate; or comprises 1 part by weight of dihydromyricetin, 0.2 part by weight of carbocisteine and 2 parts by weight of bivalent metal gluconate.
9. The pharmaceutical composition according to claim 7, which optionally further comprises pharmaceutically acceptable excipients; alternatively, it is in the form of an orally administered formulation.
10. The pharmaceutical composition according to claim 7, further comprising maltodextrin in an amount of 3 to 6 times the weight of dihydromyricetin; alternatively, the divalent metal gluconate is a calcium or zinc gluconate.
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