CN111228267A

CN111228267A - Pharmaceutical composition with maltose hydrolase inhibition activity and application thereof

Info

Publication number: CN111228267A
Application number: CN202010127939.XA
Authority: CN
Inventors: 董悦生; 余娜; 李夏; 修志龙
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2020-02-28
Filing date: 2020-02-28
Publication date: 2020-06-05
Anticipated expiration: 2040-02-28
Also published as: CN111228267B

Abstract

The invention discloses a pharmaceutical composition with maltose hydrolase inhibition activity and application thereof, wherein the pharmaceutical composition with maltose hydrolase inhibition activity comprises a flavonoid compound and 1-deoxynojirimycin, and the flavonoid compound is selected from at least one of the following monomers, organic salts of the monomers or inorganic salts of the monomers: baicalein, scutellarein, and hexamethylolflavone. The pharmaceutical composition of the present invention can more effectively reduce postprandial blood glucose, can inhibit the activity of maltose hydrolase using maltose as a substrate, and uses less maltose hydrolase inhibitors, so that the drug effect can be improved.

Description

Pharmaceutical composition with maltose hydrolase inhibition activity and application thereof

Technical Field

The invention relates to a pharmaceutical composition with maltose hydrolase inhibitory activity and application thereof, belonging to the field of medical biology.

Background

Diabetes (diabetes) is a chronic hyperglycemic disorder that can be largely classified into type 1 (insulin-dependent diabetes) and type 2 (non-insulin-dependent diabetes), with type 2 diabetes being the most common form, accounting for over 90% of all diabetic patients, and in addition, pre-diabetic conditions when blood glucose is above the normal blood glucose range, but below the diabetic blood glucose range, also known as prediabetes. Prediabetes are a pre-stage of type 2 diabetes, and more than 70% of pre-diabetic patients will develop type 2 diabetes if they do not change their state of life (The Lancet,2012,379: 2279-. Diabetes and pre-diabetes are accompanied by sugar, lipid and protein metabolism disorder caused by insulin secretion or resistance, the causes are many and complex, and the diabetes and pre-diabetes are metabolic diseases and are one of the most important public health problems all over the world. According to the statistics of the international diabetes union, the number of diabetes patients reaches 4.25 hundred million globally by 2017, and the number of diabetes patients rises to 6.29 hundred million in 2045 years. Every year 500 million people die from diabetes, and the medical expenses applied to diabetes are second in the medical expenses for the disease. Diabetes medical costs in china are currently second in the world, second only to the united states (International Diabetes mellitus federation. Epidemiological investigation also shows that the prevalence rate of prediabetes in China is 35.7%. A series of complications caused by diabetes mellitus often involve multiple organs, such as vascular injury, atherosclerosis, increased risk of common infection and cancer, and the like, seriously affect physical and mental health and life quality of patients, increase morbidity and mortality, and become a heavy burden for families and society. The high incidence of diabetes has a significant impact on both the quality of life and the economic cost of the medical system, which is a significant public health problem.

In the hydrolysates of these α -amylases, maltose and maltooligosaccharide linked with α -1,4 glucosidic bonds account for the major part, these maltoses or maltooligosaccharides are hydrolyzed in the small intestine by maltogenic enzymes which specifically hydrolyze α -1,4 glucosidic bonds, can produce glucose, are absorbed into the blood, and are thus classified as α -glucosidic enzymes, but precisely, maltogenic amylases are only 2-glucosidic enzymes having the function of hydrolyzing α -1,4 glucosidic bonds in the small intestine, but have the function of inhibiting the formation of maltogenic amylase linked with maltogenic linked with other enzymes, such as glucoamylase enzymes responsible for the postprandial activity of the glucose-hydrolase, glucose-linked with one another (e.g. maltogenic amylase), glucose-linked with one another), thus the development of a number of glucose-linked glucoamylases, maltogenic linked with the usual glucose-linked amylase-linked glucoamylases (e.g. maltogenic amylase-linked amylase), which has been found to be effective as a carbohydrate-hydrolyzing enzyme inhibitor in mammals, especially for the glucose-linked with the glucose-linked amylase-linked enzyme (e.g. glucose-linked amylase), thus the glucose-linked amylase-linked glucose inhibitors (e.g-linked glucose-linked with the glucose-linked glucose inhibitors of several maltogenic enzyme of mammals, glucose-linked.

In order to find new maltohydrolase inhibitors, many attempts have been made to find out that flavonoids exhibit a good inhibitory effect on α -glucosidase, which are widely distributed in nature, are abundant in plants of the family Compositae, Leguminosae, Labiatae, Rutaceae, and often exist in the form of aglycone or glycoside, plants such as Scutellaria baicalensis, Caesalpinia clouded fruit, quercus acutissima, Kalopanax pictus, Ginkgo biloba, honeysuckle, Olea officinalis, and beet all contain flavone components (J Nutr Sci amino, 2006,52,149-153), which exhibit a wide range of biological activities such as antioxidant, anti-inflammatory, and antidiabetic, wherein scutellarin can be isolated from Scutellaria baicalensis and oroxylum indicum, quercetin can be isolated from Kalopanax pictus, ginsenna, luteolin can be isolated from Marigold, luteolin can be isolated from Tagetes, flavonoids can be isolated from Tagetes marigold, and flavonoids have some reports on the inhibitory effect on maltohydrolase, e.g., some of the extracts containing compounds and other plant extracts have a strong inhibitory effect on maltohydrolase, inhibitory effect on animal maltohydrolase, quercetin, and other biological effects on animal glucose oxidase, quercetin.

1-deoxynojirimycin (1-DNJ) belongs to piperidine polyhydroxy alkaloids which are similar to pyranose in structure and are a series of compounds formed by substituting oxygen atoms on pyranose rings with nitrogen atoms. The 1-DNJ is mainly derived from microorganisms such as streptomyces lividans and bacillus, and plants such as mulberry and hyacinth, and has strong maltose hydrolase inhibition activity and blood sugar reducing effect. However, the 1-DNJ content in microorganisms and plants is as low as about 0.1% (100mg/100g dry product) and the bioavailability is low. Although 1-DNJ has good inhibitory activity against maltase hydrolase in vitro, it has a mild efficacy and weak hypoglycemic activity, and cannot be used clinically (Journal of Agricultural foods and chemistry,2007,55,8928, Shenyang pharmaceutical university, 2000,17, 456-460).

Although the drug synergy is the main source of drug discovery, different synergy mechanisms are different, people can choose some methods to evaluate the synergy of drugs, for example, Chou et al propose to evaluate the synergy between drugs by calculating a synergy index (CI), and such a calculation model is applicable to both animal experiments and in vitro enzymology, bacteriostasis and cell experiments, and has no very strict requirements on the dose setting of the drugs used in Combination. The CI calculated for drug interaction can be divided into three intervals based on synergy, stacking, and antagonism among drugs. Wherein the synergistic effect CI is < 0.9; additive effect 0.9< CI < 1.1; antagonism CI > 1.1. When the strength of the drug synergy is evaluated, the CI value can be divided into a plurality of intervals, and strong synergy is obtained when the CI value is less than 0.3; stronger synergy is obtained when the ratio is more than 0.3 and less than 0.7; greater than 0.7 and less than 0.9 are slightly synergistic. Provides an effective evaluation mode for evaluating the strength of the synergistic effect of the medicaments.

For diabetes with more complex mechanisms of action, the development of various drugs with synergistic action has been started, two drugs for combination of type II diabetes mellitus, Glucovance and Avandamet, are approved by FDA to be on the market, and the combination of metformin and glyburide and the combination of metformin and rosiglitazone can reduce postprandial blood glucose through different sugar metabolic pathways, but cannot avoid secondary failure of the diabetes drugs and are easy to cause hypoglycemia for patients.

Disclosure of Invention

In order to solve the problems of large side effect and the like of maltose hydrolase inhibitors in the prior art, the invention provides a pharmaceutical composition comprising 5,6, 7-trihydroxy substituted flavonoid compounds and 1-deoxynojirimycin, the composition can ensure that the combined medicines have synergistic effect to improve the drug effect, and the pharmaceutical composition is natural product active ingredients, has low side effect, can reduce postprandial blood sugar, and can prevent or treat diabetes and obesity.

The technical purpose of the invention is realized by the following technical scheme:

a pharmaceutical composition having maltose hydrolase inhibiting activity comprising a 5,6, 7-trihydroxy substituted flavonoid compound and 1-deoxynojirimycin, the 5,6, 7-trihydroxy substituted flavonoid compound being selected from at least one of the following monomers, organic salts of the monomers or inorganic salts of the monomers: baicalein, scutellarein or hexamethylolflavone.

Further, the mixing ratio of the 5,6, 7-trihydroxy substituted flavonoid compound and the 1-deoxynojirimycin is 5-50,000: 1 in a molar ratio.

Further, it is preferable that the mixing ratio of the baicalein, the organic salt thereof or the inorganic salt thereof and 1-deoxynojirimycin is 10 to 50,000:1 in terms of a molar ratio, wherein it is more preferable that the mixing ratio of the baicalein, the organic salt thereof or the inorganic salt thereof and 1-DNJ is 40 to 40,000:1 in terms of a molar ratio, most preferably 101 to 25740: 1; preferably the mixing ratio of scutellarein, organic salt or inorganic salt thereof and 1-deoxynojirimycin is 5-30,000: 1 in a molar ratio, more preferably the mixing ratio of scutellarein, organic salt or inorganic salt thereof and 1-DNJ is 20-20,000: 1 in a molar ratio, most preferably 58: 1-14947: 1; preferably, the mixing ratio of the hexahydroxyflavone, the organic salt or the inorganic salt thereof to the 1-deoxynojirimycin is 10-20,000 in a molar ratio of: 1, wherein the mixing ratio of the hexahydroxyflavone, the organic salt or the inorganic salt thereof to the 1-DNJ is more preferably 40-12,000: 1 in a molar ratio, and is most preferably 136-8695: 1.

Any technical scheme of the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient. The pharmaceutically acceptable carrier and/or excipient is one or more of common fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants or carriers of flavoring agents. The filler can be selected from starch, sucrose, lactose or microcrystalline cellulose; the binder is selected from cellulose derivatives, alginate, gelatin or polyvinylpyrrolidone; the disintegrating agent is selected from sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate or sodium bicarbonate; the surfactant may be cetyl alcohol or sodium lauryl sulfate; the lubricant is selected from pulvis Talci, calcium stearate, magnesium, silica gel micropowder or polyethylene glycol.

The invention also aims to provide a pharmaceutical dosage form containing any one of the pharmaceutical compositions, which comprises tablets, capsules, dripping pills or granules.

Various pharmaceutical dosage forms of the pharmaceutical composition of the invention can be prepared into the required preparation according to the conventional production method in the pharmaceutical field. For example, the tablet can be a common tablet, a film tablet, an enteric tablet, etc., and can be prepared by adding an appropriate amount of diluent selected from starch, dextrin, mannitol and microcrystalline cellulose, an appropriate amount of binder selected from water, ethanol, cellulose, starch and gelatin, an appropriate amount of disintegrating agent selected from sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and sodium alginate, and an appropriate amount of lubricant selected from magnesium stearate, talcum powder and polyethylene glycol, adding sweetener selected from D-xylose, xylitol, maltitol, steviosin and aspartame, granulating by a conventional wet method, granulating by a drying method, granulating by a whole granule method or a dry method, tabletting, such as a film-coated tablet, coating with a film-forming material selected from celluloses and polyethylene glycols by a conventional method, and subpackaging in a closed bottle or an aluminum plastic plate. The capsule can be common capsule, enteric capsule, etc., and can be prepared by adding appropriate adjuvant selected from calcium carbonate, mannitol, magnesium oxide, silica gel micropowder, etc., appropriate lubricant selected from pulvis Talci, magnesium stearate, glycol ester, and silicone, appropriate binder selected from mineral oil and edible oil, and appropriate sweetener selected from D-xylose, xylitol, maltitol, steviosin, and radix asparagi, mixing to obtain dry powder or making into granule, filling into capsule, and packaging in sealed bottle or aluminum plastic plate.

The pharmaceutical composition with maltose hydrolase inhibition activity can be used for preparing a medicament for treating diabetes, wherein the diabetes is type I diabetes, type II diabetes and pre-diabetes, and the α -glycosidase is maltose as a substrate.

The pharmaceutical compositions of the present invention may be administered orally to a patient in need of such treatment.

The invention has the beneficial effects that:

the invention provides a pharmaceutical composition comprising 5,6, 7-trihydroxy-substituted flavonoid and 1-deoxynojirimycin, which has a good hypoglycemic effect, can effectively improve the drug effect under a synergistic effect, and has low side effect.

Therefore, the pharmaceutical composition of the invention can more effectively reduce postprandial blood sugar, can inhibit the activity of maltose hydrolase which takes maltose and maltose as a substrate, and completely uses natural pharmaceutical active ingredients, thereby improving the drug effect, having low side effect, and effectively solving the problems of easy hypoglycemia and the like caused by drug combination.

Drawings

FIG. 1A to FIG. 1E. the chemical structures of flavone and 1-deoxynojirimycin; FIG. 1A is baicalein; FIG. 1B is scutellarein; FIG. 1C is hexahydroxyflavone; FIG. 1D is quercetin; FIG. 1E is 1-DNJ.

FIG. 2 is a graph showing the CI values of combinations of baicalein and 1-deoxynojirimycin for synergistically inhibiting mouse intestinal maltose hydrolase.

FIG. 3 shows the synergistic effect of scutellarein and 1-deoxynojirimycin on the inhibition of mouse intestinal maltose hydrolase, where the values are the CI values for the combination.

FIG. 4 shows that hexamethyloxyflavone and 1-deoxynojirimycin synergistically inhibit mouse intestinal maltose hydrolase, and the numerical values in the figure are CI values of the combined drug.

FIGS. 5A and 5B, C/N-terminal electrophoretic analysis plots of purified maltose-glucoamylase.

FIG. 6 shows that quercetin and 1-deoxynojirimycin synergistically inhibit mouse intestinal maltose hydrolase, and the values in the figure are CI values of the combined drug.

FIG. 7. inhibition of mouse intestinal maltose hydrolase by acarbose.

FIG. 8 experiment of baicalein for reducing postprandial blood glucose elevation in maltose-burdened mice (^*p<0.05，^#p<0.01)。

FIG. 9.1-DNJ postprandial blood glucose elevation experiments in maltose-burdened mice (II)^*p<0.05，^#p<0.01)。

FIG. 10 combination of baicalein and 1-deoxynojirimycin and experiment on lowering maltose load by acarbose for postprandial increase in blood glucose in mice (^*p<0.05，^#p<0.01)。

FIG. 11 area analysis under the curve of the experiment of postprandial blood glucose elevation in mice with baicalein in combination with 1-deoxynojirimycin and carbopol to reduce maltose load (. # p <0.05, # p < 0.01).

FIG. 12. baicalein and 1-deoxynojirimycin alone and in combination reduce maltose load in miceArea under the curve analysis of postprandial blood glucose rise experiment. (^*p<0.05，)。

FIG. 13. combination of baicalein and 1-deoxynojirimycin with glucose-loaded mice postprandial blood glucose elevation test: (^*p<0.05)。

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Materials and methods used in the present invention:

materials:

the 1-DNJ used in the invention is purchased from Kyormant Biotechnology limited company, and the purity is more than or equal to 98 percent; baicalein and quercetin are purchased from Goldmaste Biotechnology limited, and the purity is more than or equal to 98 percent; scutellarein and hexamethylolflavone are purchased from Shanghai-sourced leaf Biotech, Inc., the purity is more than or equal to 98%, and acarbose is purchased from Bayer medicine health promotion, Inc.; maltose is purchased from shanghai solibao biotechnology limited.

The method comprises the following steps:

the calculation method of the inhibition rate comprises the following steps:

the mouse small intestine α -glucosidase is used as an enzyme source, maltose is used as a substrate, the concentration of generated glucose is measured by a glucose oxidase method, and the inhibition rate of the inhibitor on the enzyme by single or mixed use is calculated according to the following formula:

wherein:

sample set (a): absorbance after addition of inhibitor

Control group (a): addition of control buffer and absorbance of enzyme alone without inhibitor

The inhibition rates of the single drugs and the combined drugs are plotted along with the change of the dosage, and the difference of the inhibition rates between the single drugs and the combined drugs is compared.

Calculation of synergy index:

according to Chou et al, CompuSyn software was used to calculate synergy index (CI) for the combination at each inhibition point.

Wherein:

(D)₁: inhibitor 1 is used singly in the dosage required for achieving a specific drug effect

(D)₂: inhibitor 2 is used singly in the dosage required for achieving a specific drug effect

(D_x)₁: combination of inhibitors to achieve a particular potency required the dose of inhibitor 1

(D_x)₂: combination of inhibitors to achieve a particular potency required the dose of inhibitor 2

If CI is less than 0.9, then synergy is obtained; if the CI is more than 0.9 and less than 1.1, the superposition effect is obtained; if CI is greater than 1.1, antagonism is obtained. Within the scope of synergy, the smaller the CI value, the stronger the synergy.

The following examples serve to illustrate the invention. These examples are not intended to limit the invention or its scope in any way. These examples illustrate the synergistic relationship achieved with the compositions of the present invention.

Example 1 synergistic inhibition of mouse intestinal maltose hydrolase by baicalein and 1-DNJ

Fasting Kunming mice (20-23 g) for 16h, taking small intestines after neck breaking and death, splitting, washing with precooled PBS (0.1M, pH7.0) buffer solution, adding the PBS buffer solution according to the ratio of 1:10(W/V), cutting the small intestines into fragments, homogenizing, centrifuging at 4 ℃, 10000r/min for 15min, and taking supernatant as α -glucosidase mother liquor for test.

The total volume of the enzyme reaction system is 250 mu L, and the enzyme reaction system comprises 50 mu L of each enzyme solution, baicalein, 1-DNJ, maltose and buffer solution, wherein the baicalein is dissolved in 50% DMSO. The enzyme was first mixed with the two inhibitors, incubated at 37 ℃ for 30min and the reaction was started by adding 50. mu.L of maltose solution. Incubation at 37 ℃ for 20 min. The sample was placed in boiling water for 5min to terminate the reaction. The concentration of the produced glucose was measured using a glucose oxidase kit, and 5. mu.L of each sample was taken after cooling, and 200. mu.L of glucose oxidase working solution was added to a 96-well plate. After mixing well, the mixture reacts for 15min at 37 ℃. And (3) measuring the light absorption value of the sample under 505nm of the microplate reader, and calculating the inhibition rate and the CI value.

When 1-DNJ is used alone, the inhibition rate of maltose hydrolase is increased from 28.7% to 72.1% in the range of 0.023 to 0.368 mu M. When the concentration of baicalein used alone is increased from 37 to 592 mu M, the inhibition rate of maltose hydrolase is increased from 10.2% to 68.1%, and the inhibition activity is lower. However, 37, 74, 148, 296 and 592 μ M baicalein were respectively combined with 0.023, 0.046, 0.092, 0.184 and 0.368 μ M1-DNJ, the combined inhibition rate was significantly improved in each dose point compared with the single inhibition rate, the CI values were all less than 0.7 (table 1), and the partial representative synergistic inhibition CI values are shown in fig. 2, which indicates that the combination of baicalein and 1-DNJ has a stronger synergistic inhibition effect on the process of hydrolyzing maltose by maltose hydrolyzing enzyme.

TABLE 1 Co-ordinated inhibition of CI index of mouse intestinal maltose hydrolase by baicalein and 1-DNJ

Example 2 synergistic inhibition of mouse intestinal maltose hydrolase by scutellarein and 1-DNJ

α -glucosidase was prepared and its inhibition rate was determined by the same method as in example 1, except that the flavonoid was changed to scutellarein, 21.5, 43, 86, 172, 344 μ M scutellarein was combined with 0.023, 0.046, 0.092, 0.184, 0.368 μ M1-DNJ, respectively, the combined inhibition rate was significantly improved at each dose point compared to the inhibition rate alone (table 2), the CI values were all less than 0.6, and the partially representative co-inhibition CI values are shown in fig. 3, which indicates that the combination of scutellarein and 1-DNJ has a strong co-inhibition effect on the hydrolysis of maltose by maltose hydrolase.

TABLE 2 Coincident inhibition of mouse intestinal maltose hydrolase by scutellarein and 1-DNJ CI

Example 3 synergistic inhibition of mouse intestinal maltose hydrolase by hexahydroxyflavone and 1-DNJ

α -glucosidase preparation and inhibition rate determination method is similar to example 1, except that the flavonoid compound is changed into hexahydroxyflavone, the combination of 25, 50, 100 and 200 μ M hexahydroxyflavone with 0.023, 0.046, 0.092 and 0.184 μ M1-DNJ respectively has significantly improved combined inhibition rate (Table 3) compared with single inhibition rate at each dosage point, the CI value is less than 0.8, and partial representative synergistic inhibition CI value is shown in figure 4, and the result shows that the combination of hexahydroxyflavone and 1-DNJ has stronger synergistic inhibition effect on the process of hydrolyzing maltose by maltose hydrolase.

TABLE 3 CI index for synergistic inhibition of mouse intestinal maltose hydrolase by hexahydroxyflavone and 1-DNJ

Example 4 comparison of inhibitory Activity of different flavonoids on mouse maltose hydrolase and recombinant MGAM C-and N-terminal

From examples 1 to 3, it is clear that baicalein, scutellarein and hexamethylolflavone are IC's for maltose hydrolase alone₅₀225.5 + -12.0, 80.1 + -3.8 and 260.0 + -12.9 μ M, respectively, and the quercetin concentration was not 50% inhibited, and the IC of the quercetin concentration was found to be higher than that of the quercetin concentration₅₀322.6 + -5.0 μ M, which is weaker than baicalein, scutellarein and hexamethylolflavone.

In order to verify the activity of flavone maltose hydrolase, a C end and an N end of MGAM with maltose hydrolase activity are constructed and expressed, and are purified, the MGAM-C/N target genes of humanized MGAM 87-956 amino acids and 960-1853 amino acids are respectively amplified by PCR, are connected with a pPIC9K vector after enzyme digestion, are transformed into escherichia coli E.coli DH5 α by heat shock, are cultured and sequenced, a positive strain with correct sequencing is selected, a recombinant plasmid is extracted by overnight culture, a pPIC9k-MGAM-C/N recombinant plasmid is linearized by SalI digestion and is transformed into pichia pastoris GS115 competent cells, a dot blot method is used for high expression strain screening, protein expression is carried out, and then the recombinant protein is purified by a nickel ion metal chelate chromatography method to obtain the high-purity MGAM-C/N protein (fig. 5A and fig. 5B).

The activities of four flavones on the C-terminal and N-terminal of MGAM were determined in the same manner as in example 1 except that the enzyme solution was changed to the C-terminal and N-terminal activities of MGAM expressed recombinantly, and IC's of baicalein, scutellarein, hexamethyloxyflavone and quercetin on the C-terminal of MGAM₅₀281.9 + -13.1, 17.1 + -0.5, 298.6 + -4.6 and 289.8 + -19.1 μ M, respectively. IC for N terminal of MGAM₅₀Respectively 98.2 +/-8.1, 63.5 +/-0.7, 76.0 +/-14.3 and more than 400 mu M, which shows that the four flavones have different activities on the C end and the N end of the MGAM, scutellarein has better activities on the C end and the N end of the MGAM, scutellarein and hexamethyloxyflavone have better activities on the N end of the MGAM, and quercetin has activity on the C end of the MGAM but is weak and has no activity on the N end.

Comparative example 1 inhibitory Effect of Quercetin and 1-DNJ on mouse intestinal maltose hydrolase

α -glucosidase preparation and inhibition rate determination method is similar to example 1, except that the flavonoid compound is changed to quercetin, 45, 90, 180 μ M quercetin is combined with 0.023, 0.046, 0.092 μ M1-DNJ respectively, the combined inhibition rate is improved compared with the single inhibition rate at each dosage point, but the CI values are all close to 0.9, the CI values of partial synergistic inhibition are shown in figure 6, and the result shows that the combination of quercetin and 1-DNJ has weak synergistic effect on the process of hydrolyzing maltose by maltose hydrolase.

Comparative example 2 inhibitory Effect of baicalin and 1-DNJ on mouse Small intestine maltose hydrolase

α -glucosidase was prepared and the inhibition was determined by a method similar to that of example 1 except that the flavonoid was replaced with baicalin 400. mu.M baicalin was combined with 0.092. mu.M 1-DNJ, and the CI value was greater than 0.9, indicating that the combination of baicalin and 1-DNJ did not have a synergistic effect on the hydrolysis of maltose by maltose hydrolase.

Comparative example 3 inhibitory Effect of Mulberry leaf extract and 1-DNJ on mouse Small intestine maltose hydrolase

Taking 40g folium Mori material of Jinzhou area of Liaoning province, adding 400L 60% ethanol water solution, reflux extracting at 80 deg.C for 3 times, each time for 30min, mixing the three extractive solutions, filtering, removing ethanol, extracting with n-hexane to remove oil component, extracting with ethyl acetate, and evaporating to dryness to obtain extract. Rutin is used as a standard substance, the content of flavone in the extract is 35% by ultraviolet method (490nM), different concentrations (50-500 mug/ml) of flavone and 1-DNJ (0.023-0.184 muM) are combined, and the CI value of the synergy index is calculated, wherein the CI values are all larger than 0.9, and no synergistic inhibition effect is shown.

Example 5 comparison of synergistic inhibition of mouse intestinal maltose hydrolase by various flavonoids and 1-DNJ

As can be seen from examples 1-4, the synergistic effect of flavones with different structures in combination with 1-DNJ on mouse intestinal maltose hydrolase is different, for example, the inhibition ratio of 0.092. mu.M 1-DNJ and 148. mu.M baicalein on maltose hydrolase is 72%, and the CI value is 0.43; 0.092. mu.M of 1-DNJ and 86. mu.M of scutellarein were used in combination, and the inhibition ratio of maltose hydrolase was 68%, and the CI value was 0.65; 0.092. mu.M 1-DNJ in combination with 100. mu.M hexamethylolflavone showed 65% inhibition of maltose hydrolase with a CI value of 0.39; when 0.092. mu.M 1-DNJ and 233. mu.M quercetin were used in combination, the inhibition ratio against maltose hydrolase was 63%, and the CI value was 0.74. That is, when baicalein, scutellarein and hexamethylolflavone are combined with 1-DNJ, the synergistic effect is strong, and when quercetin is combined with 1-DNJ, the synergistic effect is weak, so that the combination of 5,6, 7-trihydroxy substituted flavone and 1-DNJ has better effect of inhibiting mouse small intestine maltose hydrolase.

Comparative example 4 combination of flavone and 1-DNJ vs. acarbose alone for mouse intestinal maltose hydrolase inhibitory Activity

The inhibitory activity of acarbose, a clinically used drug, on mouse intestinal maltose hydrolase was measured according to the method of example 1, and the IC was shown₅₀1.7. mu.M (see FIG. 7), and comparative examples 1 to 3 (see FIGS. 2 to 4), when 1-DNJ was used in combination with baicalein, scutellarin and luteolin to achieve 50% inhibition, the dose of 1-DNJ was 0.023 to 0.046. mu.M, which was not only lower than 0.184. mu.M when 1-DNJ was used alone, but also far lower than that when 1-DNJ was used aloneLower than the acarbose dose. Furthermore, from the inhibition curve of acarbose, the IC of acarbose can be seen₅₀Although also lower, indicating that it has better hypoglycemic effect. However, as the concentration of acarbose increases, the inhibition rate increases more slowly, e.g., at a dose of up to 16. mu.M (which is the IC)₅₀9.4 times the dose), does not inhibit the mouse intestinal maltose hydrolase by more than 80%, i.e. even if higher concentrations of acarbose are taken, some maltose is still hydrolyzed into glucose, which causes the blood sugar to rise after entering the blood. After combination of baicalein, scutellarein, hexamethylolflavone and 1-DNJ, the combination can remarkably improve the inhibition effect on mouse small intestine maltose hydrolase, and when the inhibition rate reaches 80 percent, the concentration of 1-DNJ is 0.092-0.184 mu M and is more than the IC of 1-DNJ₅₀The improvement is only 3-4 times, and when 0.184-0.368 mu M of 1-DNJ is used (namely the concentration of 1-DNJ is the IC of the 1-DNJ)₅₀8 times of the total amount of the. Therefore, the above results indicate that the combination of flavone and 1-DNJ has better drug effect than acarbose.

Example 6 combination of baicalein and 1-DNJ to synergistically reduce postprandial blood glucose elevations in maltose-burdened mice

18-23g C57BL/6 mice (male) were bred for 5 days to stabilize their physiological indices. 5, 10, 20 and 40mg/mL of baicalein alone was evaluated for its effect in reducing postprandial blood glucose in maltose-loaded mice, including 5 negative controls, with 8 mice per group, without fasting for 16h prior to administration. The negative control group only contains gavage maltose, the concentration is 2g/kg, and the volume of the gavage solution is 0.2mL/20 g. The administration group was a mixture of maltose and a drug at different concentrations, and was also administered by gavage, and the maltose concentration was the same as that of the negative control group. Maltose load and blood sampling after administration, and blood glucose meter for measuring blood glucose values at 0, 30, 60, 90 and 120min after administration. As shown in FIG. 8, although baicalein of 10, 20 and 40mg/mL was able to significantly reduce the postprandial 30min blood glucose level (p <0.05) when used alone, it was weak in blood glucose lowering ability, had no significant difference in 60min blood glucose level, and had no effect of delaying the postprandial blood glucose peak.

In the same manner, the effect of reducing postprandial blood glucose in maltose-loaded mice was evaluated for 0.4, 0.6, 0.8 and 1mg/mL of 1-DNJ alone, and as a result, as shown in FIG. 9, 0.4, 0.6, 0.8 and 1mg/mL of 1-DNJ alone significantly reduced postprandial 30min blood glucose values (p <0.01) compared with the negative control group, but the effect was weaker in the group of 0.4mg/mL1-DNJ in combination with the other groups.

In the same manner, the effect of reducing postprandial blood glucose in maltose-loaded mice was evaluated by combining 0.4mg/mL of 1-DNJ with 10mg/mL of baicalein, and the difference in the effect of each combination with 0.4mg/mL of 1-DNJ and 10mg/mL of baicalein was compared, and the experiment of this group was divided into 5 groups consisting of a negative control, a positive control, a combined group, 1-DNJ and baicalein, each group consisting of 8C 57BL/6 mice, each group using 1.2mg/mL of acarbose as a positive control. As shown in FIG. 10, the blood glucose levels at 30min after meal were significantly reduced (p <0.05) in the combination of 0.4mg/mL of 1-DNJ and 10mg/mL of baicalein when compared with those of baicalein and 1-DNJ alone (p <0.05), and the area under the curve (AUC) was also shown to be significantly reduced (p <0.05) when compared with that of baicalein and 1-DNJ alone (FIG. 11), indicating that the combination group was able to maintain the stability of blood glucose after meal, and that the blood glucose levels before and after maltose loading in the combination group were higher than 4, i.e., no hypoglycemia occurred.

Meanwhile, when the combined group and the 1-DNJ with different concentrations in the graph 8 are compared, the area under the curve is known (figure 12), when the effect of reducing the postprandial blood sugar is achieved (p is more than 0.05) which is the same as that of the combined group, the single dosage of the 1-DNJ is 0.8mg/mL which is 2 times of that of the 1-DNJ of the combined group, and the effect of reducing the postprandial blood sugar (p is less than 0.05) of the combined group cannot be achieved even if the highest test concentration of 40mg/mL is used by baicalein, and the results show that the combination of the 5,6, 7-trihydroxy substituted flavone 1-DNJ can effectively reduce the usage amount of the medicine and further reduce the risk of side effects of the medicine.

After fasting, the mice were glucose loaded and dosed, and neither 0.4mg/mL1-DNJ nor 10mg/mL baicalein showed significant postprandial glucose lowering effects, as shown in FIG. 13. This result confirmed that the mechanism of lowering postprandial blood glucose by baicalein and 1-DNJ alone or in combination was an inhibitory effect on maltogenic hydrolase carbohydrate.

Comparative example 5 combination of baicalein and 1-DNJ compared with the Activity of acarbose alone to reduce postprandial blood glucose elevation in maltose-loaded mice

In example 6, comparing the effect of 10mg/mL baicalein and 0.4mg/mL1-DNJ in combination with acarbose, a clinically used drug, on reducing postprandial blood glucose elevation in maltose-loaded mice, the results show that blood glucose values at all blood glucose concentration check points in the combination are significantly lower than that in the acarbose group of 1.2mg/mL (FIG. 10), blood glucose is maintained more stably, and the area AUC under the curve of the synergistic group is also significantly lower than that in the acarbose group (p <0.05, FIG. 12), which indicates that the combination of 5,6, 7-trihydroxy-substituted flavone and 1-DNJ has better activity of reducing postprandial blood glucose compared with acarbose.

Claims

1. A pharmaceutical composition having maltase inhibitory activity, characterized in that: comprises 5,6, 7-trihydroxy substituted flavonoid and 1-deoxynojirimycin.

2. The pharmaceutical composition having maltase inhibitory activity according to claim 1, said 5,6, 7-trihydroxy substituted flavonoid is selected from at least one of the following monomers, organic salts of the monomers or inorganic salts of the monomers: baicalein, scutellarein or hexamethylolflavone.

3. The pharmaceutical composition having maltase inhibitory activity according to claim 1, characterized in that: the mixing ratio of the 5,6, 7-trihydroxy substituted flavonoid compound to the 1-deoxynojirimycin is 5-50,000: 1 in a molar ratio.

4. The pharmaceutical composition having maltase inhibitory activity according to claim 3, characterized in that: the mixing ratio of the baicalein, the organic salt or the inorganic salt of the baicalein and the 1-deoxynojirimycin is 10-50,000: 1 in a molar ratio, and the mixing ratio of the scutellarin, the organic salt or the inorganic salt of the baicalein and the 1-deoxynojirimycin is 5-30,000: 1 in a molar ratio; the mixing ratio of the hexahydroxyflavone, the organic salt or the inorganic salt of the hexahydroxyflavone and the 1-deoxynojirimycin is 10-20,000 in molar ratio: 1.

5. the pharmaceutical composition having a maltase inhibitory activity according to any one of claims 1 to 4, characterized in that: also comprises a pharmaceutically acceptable carrier and/or excipient.

6. A pharmaceutical dosage form comprising a pharmaceutical composition with maltose suppressing activity as claimed in any of claims 1 to 5 characterized by: including tablets, capsules, pills or granules.

7. Use of the pharmaceutical composition with maltose suppressing activity as claimed in any one of claims 1 to 5 in the preparation of a medicament for treating diabetes.

8. The use of a pharmaceutical composition with maltose suppressing activity as claimed in claim 7 in the preparation of a medicament for the treatment of diabetes characterized in that: the diabetes is type I diabetes, type II diabetes or pre-diabetes.

9. The use of a pharmaceutical composition with maltose suppressing activity as claimed in claim 7 in the preparation of a medicament for the treatment of diabetes characterized in that: the pharmaceutical composition treats diabetes by inhibiting maltase activity.