CN108310226B - Composition with effect of preventing and treating diabetes as well as preparation method and application thereof - Google Patents

Composition with effect of preventing and treating diabetes as well as preparation method and application thereof Download PDF

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CN108310226B
CN108310226B CN201810245505.2A CN201810245505A CN108310226B CN 108310226 B CN108310226 B CN 108310226B CN 201810245505 A CN201810245505 A CN 201810245505A CN 108310226 B CN108310226 B CN 108310226B
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CN108310226A (en
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王旭
狄留庆
许惠琴
吴滨
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Shanghai Jianshun Biotechnology Co ltd
Nanjing University of Chinese Medicine
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Nanjing University of Chinese Medicine
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Abstract

The invention discloses a composition with the effect of preventing and treating diabetes, a preparation method and application thereof. According to the traditional Chinese medicine theory, the raw material composition and the weight ratio thereof are screened through a large number of experiments, and the experiment results show that the mulberry leaf extract, the tartary buckwheat extract, the dendrobium officinale and the ganoderma lucidum spore powder are used as the optimal composition, so that the content of active ingredients such as active polysaccharide, rutin, quercetin and naringenin can be improved, the effects of reducing blood sugar and blood fat are good, the green and safe effects are realized, and no toxic or side effect is caused after long-term use. The preparation method provided by the invention has reasonable process design and can realize industrial production.

Description

Composition with effect of preventing and treating diabetes as well as preparation method and application thereof
Technical Field
The invention relates to a composition, in particular to a composition with the effect of preventing and treating diabetes, a preparation method and application thereof.
Background
Diabetes is a common and frequently occurring disease worldwide and has become the third largest disease after tumors and cardiovascular diseases. Diabetes combined with stagnant heat is a main complication of diabetes, and clinically common diabetic heart disease comprises diabetes specific cardiomyopathy, diabetes related cardiac microangiopathy, macroangiopathy, heart vegetative neuropathy and the like, and also commonly accompanies nonspecific coronary atherosclerotic heart disease and (or) hypertensive heart disease, and can be collectively called as diabetic heart disease on the basis of having diabetes. Compared with coronary atherosclerotic heart disease and cardiomyopathy of non-diabetic patients, the diabetic heart disease has the characteristics of serious disease condition, more contradictions, large treatment difficulty, poor prognosis and high fatality rate.
At present, most of the traditional Chinese medicines for treating diabetes and complications thereof are single-prescription and proved-prescription medicines, clinical western medicines have certain curative effect, but most of the traditional Chinese medicines have large adverse reactions, such as rebound after stopping taking the medicines, liver and kidney damage of patients, rhabdomyolysis and other side effects, and the symptoms are not treated at the same time.
Therefore, the active search and the research of the composition which has good safety, low adverse reaction and good effect of preventing and treating diabetes have important significance.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects in the prior art and provide the composition which is scientific and reasonable in proportion, good in safety and good in auxiliary blood sugar reducing effect. The invention also aims to provide a preparation method of the composition, an identification method and application thereof.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
the composition with the effect of preventing and treating diabetes is characterized by comprising the following raw materials:
mulberry leaf, tartary buckwheat, dendrobium officinale and ganoderma lucidum spore powder.
As a preferred scheme, the composition with the effect of preventing and treating diabetes is prepared from the following raw materials in parts by weight:
125-250 parts of mulberry leaf extract, 125-250 parts of tartary buckwheat extract, 250-500 parts of dendrobium officinale and 125-250 parts of ganoderma lucidum spore powder.
As a preferred scheme, the composition with the effect of preventing and treating diabetes is prepared from the following raw materials in parts by weight:
125 parts of mulberry leaf extract, 125 parts of tartary buckwheat extract, 250 parts of dendrobium officinale and 125 parts of ganoderma lucidum spore powder.
The preparation method of the composition with the effect of preventing and treating diabetes comprises the following steps:
the mulberry leaf extract is prepared by the following method: extracting folium Mori with 10 times volume of 80% ethanol under reflux for 2 times (each for 1.5 hr), and filtering to obtain ethanol extractive solution; extracting the residue with 10 times of water for 1 time, each time for 1.5 hr, filtering to obtain water extract, mixing the ethanol extract and the water extract, and concentrating under reduced pressure to relative density of 1.20 at 60 deg.C; vacuum drying or spray drying the concentrated solution, pulverizing, and sieving to obtain folium Mori extract;
the tartary buckwheat extract is prepared by the following method: extracting radix Et rhizoma Fagopyri Tatarici with 10 times volume of 60% ethanol under reflux for 3 times, each time for 1.5 hr, filtering, mixing filtrates, recovering ethanol under reduced pressure, and concentrating to relative density of 1.20 at 60 deg.C; drying the concentrated solution under normal pressure or reduced pressure, pulverizing at low temperature, and sieving to obtain radix Et rhizoma Fagopyri Tatarici extract;
the dendrobium officinale is prepared by the following method: drying the dendrobium officinale at 100 ℃ for 30 minutes, crushing, and sieving with a 100-mesh sieve to obtain the dendrobium officinale powder.
The quality identification method of the composition with the effect of preventing and treating diabetes comprises the following steps: the method comprises the following steps:
(1) qualitative study
(1.1) microscopic identification of wall-broken spore powder of Ganoderma
Microscopic identification: the ganoderma wall-broken spore powder is a tan powder, has extremely fine and smooth appearance, does not form lump, is lightly rubbed with a mother finger and a forefinger, and has smooth and fine feeling, no adhesion, no sense of foreign matters such as gravel and the like; observing under 1500 times of magnification, not breaking the wall spore to be complete and plump, the double-wall structure is clear, the outer wall is colorless, the inner wall has small thorns, and is light brown; the individual forms are consistent and elliptical; the top end is truncated, and the size is about 8.5-11 multiplied by 5.2-6.9 μm; the wall-broken spore has several forms, and can be divided into mild wall breaking- -complete spore form, small holes on the wall, moderate wall breaking- -incomplete spore form, defect, severe wall breaking- -breaking of spore coat and spore content according to different wall-breaking procedures;
(1.2) thin-layer identification of sitosterol in tartary buckwheat:
weighing tartary buckwheat control medicinal material and composition powder 3g respectively, adding 25mL chloroform solution, performing ultrasonic extraction for 30min, filtering, taking 10mL filtrate, evaporating to dryness on an evaporating dish, dissolving with chloroform, and diluting to 2mL volume; sucking L0 μ L of test solution of the composition and contrast solution of radix Et rhizoma Fagopyri Tatarici, respectively dropping on the same silica gel G plate containing 0.3% sodium carboxymethylcellulose as adhesive, and mixing at a volume ratio of 7: 3, developing by using petroleum ether-diethyl ether as a developing agent, taking out, airing, spraying a 10% sulfuric acid solution, drying at 105 ℃ until spots are developed, and inspecting by sunlight;
(2) quantitative study
(2.1) determination of polysaccharide content in Ganoderma spore powder
Accurately weighing 0.1g of anthrone, adding 100mL of 80% sulfuric acid solution, stirring to dissolve, and shaking up to obtain the final product; performing anthrone-sulfuric acid color reaction, taking anhydrous glucose as a reference substance, and determining at 625nm by a colorimetric method;
preparing a reference substance solution:
accurately weighing anhydrous glucose dried to constant weight, accurately weighing, placing in a measuring flask, adding distilled water to dissolve and dilute to scale, shaking, and making into reference solution;
preparation of test solution
Weighing Ganoderma spore powder, precisely weighing, placing in a round-bottom flask, adding water, standing, heating and refluxing, filtering while hot, washing filter and residue with a small amount of hot water, placing residue and filter paper in the flask, adding water, heating and refluxing, filtering while hot, mixing filtrates, evaporating on water bath to dryness, dissolving residue with water, slowly adding ethanol dropwise while stirring, shaking, centrifuging, discarding supernatant, dissolving precipitate with hot water and transferring to a measuring flask, cooling, adding water to scale, shaking, taking appropriate amount of solution, centrifuging, precisely taking supernatant, placing in the measuring flask, adding water to scale, and shaking to obtain the final product;
drawing of standard curve
Precisely measuring control solution 0.4, 0.6, 0.8, 1.0, and 1.2mL, respectively placing in 10mL test tubes with plugs, respectively adding water to 2.0mL, rapidly and precisely adding sulfuric acid in the form of anthrone solution, shaking, standing, immediately cooling in ice bath, taking corresponding reagent as blank, measuring absorbance at 625nm wavelength by ultraviolet-visible spectrophotometry, taking absorbance as ordinate, and concentration as abscissa, and drawing standard curve;
(2.2) determination of naringenin content in Dendrobium officinale
Measuring the content of naringenin, which is an effective component of dendrobium officinale in the composition, by adopting a high performance liquid chromatography; the chromatographic conditions are as follows: XB 18 chromatographic column with specification of 4.6mm × 250mm and 5 μm; the column temperature is 30 ℃; mobile phase 0.2% phosphoric acid as phase A-methanol as phase B, gradient elution; the detection wavelength is 290 nm; the flow rate is 1.0 mL/min;
precisely weighing appropriate amount of naringenin reference substance, placing in 50mL brown measuring flask, adding methanol to dissolve to scale, shaking, and making into naringenin reference substance solution;
putting dendrobium officinale into a round-bottom flask, precisely adding a mixed solution of methanol and 20% hydrochloric acid, weighing, heating and refluxing in a water bath, cooling, weighing again, complementing with methanol to reduce weight loss, shaking up, standing, filtering supernate with a 0.22 mu m microporous membrane, taking a subsequent filtrate, injecting 10 mu L of the subsequent filtrate, and preparing a sample solution for later use;
precisely sucking the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions by adopting an external standard one-point method;
(2.3) establishment of method for measuring rutin and quercetin content in tartary buckwheat extract
Preparation of control solutions
Precisely weighing a rutin reference substance and a quercetin reference substance, respectively placing in volumetric flasks, dissolving with methanol, fixing the volume to a scale, and shaking up to obtain a rutin and quercetin reference substance solution;
preparation of test solution
Extracting radix Et rhizoma Fagopyri Tatarici with ethanol under reflux, mixing filtrates, concentrating under reduced pressure, dissolving the residue with methanol, transferring to a measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate;
precisely sucking rutin control substance and quercetin control substance solution, diluting with methanol for 6 times, sequentially injecting into liquid chromatograph, measuring standard curve, and sucking Fagopyrum tataricum sample solution and injecting into liquid chromatograph;
the chromatographic conditions are as follows: chromatography column Thermo C18, 4.6mm × 250mm,5 μm; the mobile phase is acetonitrile-0.2% formic acid water solution, and the gradient elution is as follows: 0-18 min, 17-40% of acetonitrile, 18-19 min and 40-17% of acetonitrile; the volume flow is 1 mL/min; detection wavelength: λ 256 nm; sample introduction amount: 10 mu L of the solution;
taking the concentration of each reference substance as an abscissa C, and taking the peak area A of each reference substance as an ordinate to draw a standard curve;
(2.4) content determination of rutin in mulberry leaves:
preparation of control solutions
Taking a proper amount of rutin reference substance, precisely weighing, and preparing a solution containing 0.1mg per 1mL by using methanol;
preparation of test solution
Weighing folium Mori powder, heating and refluxing, filtering, extracting residue with methanol for 2 times, mixing filtrates, recovering solvent under reduced pressure, dissolving residue with methanol, transferring to 25mL measuring flask, adding methanol to scale, shaking, and filtering to obtain filtrate;
preparation of the Standard Curve
Diluting rutin control solution 500 μ L with methanol for 6 times to obtain control solutions with concentrations of 0.0095mg/mL, 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, and 0.3034mg/mL, respectively, injecting into liquid chromatograph, and measuring by sample injection;
the chromatographic conditions are as follows: the chromatographic column is C18 column, 4.6mm × 250mm,5 μm; the mobile phase is methanol-0.5% phosphoric acid water solution, and the gradient elution is as follows: 0-5 min, 35% methanol; 8-16 min, 40% methanol; 19-24 min, 51% methanol; 26-30 min, 70% methanol; 30-35 min, 35% methanol. The volume flow is 1 mL/min; the detection wavelength is 358nm, and the sample injection amount is 10 mu L;
and (4) drawing a standard curve by taking the concentration as an abscissa and the peak area as an ordinate.
Preferably, the quality identification method of the composition with the effect of preventing and treating diabetes comprises the following steps: the method comprises the following steps:
(1) qualitative study
(1.1) microscopic identification of wall-broken spore powder of Ganoderma
Microscopic identification: the ganoderma wall-broken spore powder is a tan powder, has extremely fine and smooth appearance, does not form lump, is lightly rubbed with a mother finger and a forefinger, and has smooth and fine feeling, no adhesion, no sense of foreign matters such as gravel and the like; observing under 1500 times of magnification, not breaking the wall spore to be complete and plump, the double-wall structure is clear, the outer wall is colorless, the inner wall has small thorns, and is light brown; the individual forms are consistent and elliptical; the top end is truncated, and the size is about 8.5-11 multiplied by 5.2-6.9 μm; the wall-broken spore has several forms, and can be divided into mild wall breaking- -complete spore form, small holes on the wall, moderate wall breaking- -incomplete spore form, defect, severe wall breaking- -breaking of spore coat and spore content according to different wall-breaking procedures;
(1.2) thin-layer identification of sitosterol in tartary buckwheat:
weighing tartary buckwheat control medicinal material and composition powder 3g respectively, adding 25mL chloroform solution, performing ultrasonic extraction for 30min, filtering, taking 10mL filtrate, evaporating to dryness on an evaporating dish, dissolving with chloroform, and diluting to 2mL volume; sucking L0 μ L of test solution of the composition and contrast solution of radix Et rhizoma Fagopyri Tatarici, respectively dropping on the same silica gel G plate containing 0.3% sodium carboxymethylcellulose as adhesive, and mixing at a volume ratio of 7: 3, developing by using petroleum ether-diethyl ether as a developing agent, taking out, airing, spraying a 10% sulfuric acid solution, drying at 105 ℃ until spots are developed, and inspecting by sunlight;
(2) quantitative study
(2.1) determination of polysaccharide content in Ganoderma spore powder
Accurately weighing 0.1g of anthrone, adding 100mL of 80% sulfuric acid solution, stirring to dissolve, and shaking up to obtain the final product; performing anthrone-sulfuric acid color reaction, taking anhydrous glucose as a reference substance, and determining at 625nm by a colorimetric method;
preparing a reference substance solution:
precisely weighing 1.521mg of anhydrous glucose dried at 105 deg.C to constant weight, precisely weighing, placing in 10mL measuring flask, adding distilled water to dissolve and dilute to scale, shaking, and making into 0.1521 mg/mL-1The control solution of (4);
preparation of test solution
2g of ganoderma spore powder is taken, precisely weighed, placed in a round-bottom flask, added with 60mL of water and kept stand for 1 hour, heated and refluxed for 4 hours, filtered while hot, the filter and filter residue are washed by a small amount of hot water, the filter residue and filter paper are placed in the flask, added with 60mL of water, heated and refluxed for 3 hours, filtered while hot, the filtrates are combined, placed on a water bath and evaporated to dryness, the residue is dissolved by 5mL of water, 75mL of ethanol is slowly dripped while stirring, shaking is uniformly carried out, placed at 4 ℃ for 12 hours, centrifuged, the supernatant is discarded, the precipitate is dissolved by hot water and transferred to a 50mL measuring flask, cooling is carried out, added with water to scale, shaking is carried out, a proper amount of solution is taken, centrifuged, 3mL of the supernatant is precisely measured and placed in a 25mL measuring flask;
drawing of standard curve
Accurately weighing control solution 0.4, 0.6, 0.8, 1.0, and 1.2mL, respectively placing in 10mL test tubes with plugs, respectively adding water to 2.0mL, quickly and accurately adding sulfuric acid (accurately weighing anthraketone 0.lg, adding sulfuric acid 100mL for dissolving, and shaking) 6mL, immediately shaking, standing for 15min, immediately placing in ice bath for cooling for 15min, taking out, taking corresponding reagent as blank, irradiating with ultraviolet-visible spectrophotometry, measuring absorbance at 625nm wavelength, with absorbance as ordinate and concentration as abscissa, drawing standard curve, and y is 0.0051 x-0.0036;
(2.2) determination of naringenin content in Dendrobium officinale
Measuring the content of naringenin, which is an effective component of dendrobium officinale in the composition, by adopting a high performance liquid chromatography; the chromatographic conditions are as follows: XB 18 chromatographic column with specification of 4.6mm × 250mm and 5 μm; the column temperature is 30 ℃; mobile phase 0.2% phosphoric acid as phase A-methanol as phase B, gradient elution; the detection wavelength is 290 nm; the flow rate is 1.0 mL/min;
precisely weighing appropriate amount of naringenin reference substance, placing in 50mL brown measuring flask, adding methanol to dissolve to scale, shaking, and making into reference substance solution containing 26 μ g per 1 mL;
taking 0.5g of the composition, precisely weighing, placing the composition in a 50mL round-bottom flask, precisely adding 25mL of a mixed solution of methanol and 20% hydrochloric acid in a volume ratio of 4: 1, weighing, placing the mixture in a water bath, heating and refluxing, cooling, weighing again, complementing weight loss by using methanol, shaking up, standing, taking supernatant, filtering by using a 0.22 mu m microporous membrane, taking subsequent filtrate, injecting 10 mu L of the sample, and preparing a sample solution for later use;
precisely sucking the reference substance solution and the sample solution, injecting into a liquid chromatograph, and measuring naringenin content by adopting an external standard one-point method;
(2.3) establishment of method for measuring rutin and quercetin content in tartary buckwheat extract
Preparation of control solutions
Precisely weighing 5.952mg of rutin reference substance and 3.075mg of quercetin reference substance, respectively placing in 10mL volumetric flasks, dissolving with methanol, fixing the volume to scale, and shaking to obtain rutin and quercetin reference substance solutions with concentrations of 0.5952mg/mL and 0.3075mg/mL respectively;
preparation of test solution
Reflux-extracting radix Et rhizoma Fagopyri Tatarici with 10 times of 60% ethanol for 3 times (each time for 1 hr), mixing filtrates, concentrating under reduced pressure, dissolving residue with methanol, transferring to 25mL measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate;
precisely absorbing rutin reference substance and quercetin reference substance solution, sequentially diluting in equal gradient for 6 parts, sequentially injecting into liquid chromatograph, measuring standard curve, and absorbing Fagopyrum tataricum sample solution for injection into liquid chromatograph;
the chromatographic conditions are as follows: chromatography column Thermo C18, 4.6mm × 250mm,5 μm; the mobile phase is acetonitrile-0.2% formic acid water solution, and the gradient elution is as follows: 0-18 min, 17-40% of acetonitrile, 18-19 min and 40-17% of acetonitrile; the volume flow is 1 mL/min; detection wavelength: λ 256 nm; sample introduction amount: 10 mu L of the solution;
taking the concentration of each control as abscissa (C, peak area A of each control as ordinate to draw standard curve, and rutin linear regression equation as Y ═ 2 × 107+ 2506.8; the linear regression equation of quercetin is Y ═ 4 × 107－11208；
(2.4) content determination of rutin in mulberry leaves:
preparation of control solutions
Taking a proper amount of rutin reference substance, precisely weighing, and preparing a solution containing 0.1mg per 1mL by using methanol;
preparation of test solution
Weighing folium Mori powder lg, precisely weighing, placing in a round-bottom flask, adding 50mL of methanol, heating and refluxing for 30min, filtering, extracting the residue with 50mL of methanol for 2 times, mixing filtrates, recovering solvent under reduced pressure, dissolving the residue with methanol, transferring to a 25mL measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate;
preparation of the Standard Curve
Diluting rutin control solution 500 μ L with methanol for 6 times to obtain control solutions with concentrations of 0.0095mg/mL, 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, and 0.3034mg/mL, respectively, injecting into liquid chromatograph, and measuring by sample injection;
the chromatographic conditions are as follows: the chromatographic column is C18 column, 4.6mm × 250mm,5 μm; the mobile phase is methanol-0.5% phosphoric acid water solution, and the gradient elution is as follows: 0-5 min, 35% methanol; 8-16 min, 40% methanol; 19-24 min, 51% methanol; 26-30 min, 70% methanol; 30-35 min, 35% methanol. The volume flow is 1 mL/min; the detection wavelength is 358nm, and the sample injection amount is 10 mu L;
plotting the concentration as abscissa to obtain the standard curve y 2 × 107x-10419, linear range of 0.0095-0.3034 mg/mL, correlation coefficient r of 1.
The composition with the effect of preventing and treating diabetes is applied to preparing hypoglycemic drugs or health care products.
The composition with the effect of preventing and treating diabetes is applied to preparation of blood fat reducing medicines or health care products.
The composition with the effect of preventing and treating diabetes is applied to the preparation of hypoglycemic and hypolipidemic drugs or health care products.
Has the advantages that: the composition with the effect of preventing and treating diabetes provided by the invention has the following advantages:
1. according to the traditional Chinese medicine theory, the raw material compositions and the weight ratio thereof are screened through a large number of experiments, and the experiment results show that when the weight ratio of the mulberry leaf extract to the tartary buckwheat extract to the dendrobium officinale to the ganoderma lucidum spore powder is 1:1:2:1, the traditional Chinese medicine composition has the best effects of reducing blood sugar and blood fat, is green and safe, and has no toxic or side effect after being taken for a long time.
2. The animal experiment results show that: the composition provided by the invention has very good effects of reducing blood sugar, reducing blood fat, improving sugar tolerance, increasing insulin generation and the like.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1
1. Folium mori ethanol reflux extraction process optimization test
Design of factor level
By using L9(34) And (3) an orthogonal design table, wherein the content of rutin and total flavone in the mulberry leaves is used as an index, and the influence of alcohol concentration, material-liquid ratio and extraction frequency on the extraction of index components is inspected.
TABLE 1 orthogonal test factor horizon
Factors of the fact Alcohol concentration (%) Liquid-to-liquid ratio of B Number of C extractions D blank
1 60 1:8 1 1
2 70 1:10 2 2
3 80 1:12 3 3
2. Analysis of results
According to L9(34) Orthogonal table is used for orthogonal test, 10g of mulberry leaf medicinal material is weighed, ethanol is added for reflux, filtration is carried out, and liquid medicine is combined. Each set of experiments was performed in parallel 3 times.
TABLE 2 results of orthogonal experiments
Figure GDA0002782080620000081
Figure GDA0002782080620000091
TABLE 3 analysis of rutin variance
Factors of the fact Sum of squares of deviation Degree of freedom F ratio Critical value of F Significance of
Alcohol concentration 0.041 2 20.500 19.000 *
Ratio of material to liquid 0.011 2 5.500 19.000
Number of times of extraction 0.147 2 73.500 19.000 *
Error of the measurement 0.00 2
TABLE 4 analysis of Total Flavonoids variance
Factors of the fact Sum of squares of deviation Degree of freedom F ratio Critical value of F Significance of
Alcohol concentration 0.273 2 9.750 19.000
Ratio of material to liquid 0.175 2 6.250 19.000
Number of times of extraction 0.469 2 16.750 19.000
Error of the measurement 0.03 2
The result shows that when the rutin is taken as a detection index, the two factors of alcohol concentration and extraction frequency have significant difference, and the sequence of the main factors and the secondary factors is as follows: the extraction times is more than the alcohol concentration and more than the material-liquid ratio, so the best process for selecting the mulberry leaf alcohol extraction is that the material-liquid ratio is 1: 12, the alcohol concentration is 80%, the extraction times are 3 times, but in view of saving cost, the ratio of the materials to the liquid is selected to be 1: 10.
when total flavone is used as an index, all factors have no significant difference, so that the optimal alcohol extraction process is selected as the ratio of material to liquid of 1:10, the alcohol concentration is 80%, and the extraction times are 3 times.
Extracting with 10 times of 80% edible ethanol under reflux for 3 times (1.5 hr each time), filtering, extracting the residue with 10 times of water for 1 time (1.5 hr each time), filtering, and mixing with the ethanol extractive solution. The ethanol was recovered under reduced pressure and concentrated to a relative density of 1.20(60 ℃).
Tartary buckwheat extraction process optimization test
Determination of factor level
Through preliminary experiments, main factors influencing the alcohol extraction process are alcohol concentration, extraction times and material-liquid ratio. L9 (3) was used in this experiment4) And (3) an orthogonal design table, wherein the contents of rutin and quercetin are used as evaluation indexes, and the influence of three factors of alcohol concentration, extraction times and material-liquid ratio on the extraction of index components is inspected.
TABLE 5 factor level table
Factors of the fact Alcohol concentration (%) Number of B extractions C material-to-liquid ratio (g: mL) Blank D
1 60 1 1:6 1
2 70 2 1:8 2
3 80 3 1:10 3
Quadrature test
Weighing 10g of tartary buckwheat medicinal material, arranging experiments according to an orthogonal design table, taking the contents of rutin and quercetin as evaluation indexes, and optimizing the alcohol extraction process, wherein the orthogonal test result is shown in the following table.
Table 6 visual analysis table
Figure GDA0002782080620000101
TABLE 7 analysis of rutin variance
Figure GDA0002782080620000102
Figure GDA0002782080620000111
TABLE 8 Quercetin ANOVA
Factors of the fact Sum of squares of deviation Degree of freedom F ratio Critical value of F Significance of
Alcohol concentrationDegree of rotation 0.015 2 7.500 19.000
Ratio of material to liquid 0.045 2 22.500 19.000 *
Number of times of extraction 0.003 2 1.500 19.000
Error of the measurement 0.00 2
The main and secondary factors influencing the extraction of rutin are as follows: alcohol concentration>Ratio of material to liquid>The extraction times and all factors have no significant difference. The primary and secondary factors influencing the extraction of the quercetin are the material-liquid ratio>Alcohol concentration>The extraction times, wherein the feed-liquid ratio has significant difference, so the optimum process combination is A1B3C3Namely 60 percent ethanol, is extracted for 3 times, and the ratio of material to liquid is 1: 10.
Process verification
On the basis of orthogonal experiments, A is selected1B3C3(60% ethanol, 3 times extraction, 1:10 ratio) for verification test, the results are shown in Table 9.
Table 9 process verification results (n ═ 3)
Serial number Rutin content (%) Quercetin content (%)
1 1.462 0.074
2 1.452 0.073
3 1.474 0.074
Average content 1.463 0.074
RSD(％) 0.72 1.12
The results show that the process is stable and feasible.
Example 2
The composition with the effect of preventing and treating diabetes is prepared from the following raw materials in parts by weight:
125g of mulberry leaf extract, 125g of tartary buckwheat extract, 250g of dendrobium officinale and 125g of ganoderma lucidum spore powder.
The mulberry leaf extract is prepared by the following method: extracting folium Mori with 10 times volume of 80% ethanol under reflux for 2 times (each for 1.5 hr), and filtering to obtain ethanol extractive solution; extracting the residue with 10 times of water for 1 time, each time for 1.5 hr, filtering to obtain water extract, mixing the ethanol extract and the water extract, and concentrating under reduced pressure to relative density of 1.20 at 60 deg.C; vacuum drying or spray drying the concentrated solution, pulverizing, and sieving to obtain folium Mori extract;
the tartary buckwheat extract is prepared by the following method: extracting radix Et rhizoma Fagopyri Tatarici with 10 times volume of 60% ethanol under reflux for 3 times, each time for 1.5 hr, filtering, mixing filtrates, recovering ethanol under reduced pressure, and concentrating to relative density of 1.20 at 60 deg.C; drying the concentrated solution under normal pressure or reduced pressure, pulverizing at low temperature, and sieving to obtain radix Et rhizoma Fagopyri Tatarici extract;
the dendrobium officinale is prepared by the following method: drying the dendrobium officinale at 100 ℃ for 30 minutes, crushing, and sieving with a 100-mesh sieve to obtain the dendrobium officinale powder.
The ganoderma lucidum spore powder is produced and provided by Jiangsu Hongshou biotechnology limited company.
Example 3
The quality identification method of the composition with the effect of preventing and treating diabetes mellitus described in example 2 comprises the following steps: the method comprises the following steps:
(1) qualitative study
(1.1) microscopic identification of wall-broken spore powder of Ganoderma
Microscopic identification: the ganoderma wall-broken spore powder is a tan powder, has extremely fine and smooth appearance, does not form lump, is lightly rubbed with a mother finger and a forefinger, and has smooth and fine feeling, no adhesion, no sense of foreign matters such as gravel and the like; observing under 1500 times of magnification, not breaking the wall spore to be complete and plump, the double-wall structure is clear, the outer wall is colorless, the inner wall has small thorns, and is light brown; the individual forms are consistent and elliptical; the top end is truncated, and the size is about 8.5-11 multiplied by 5.2-6.9 μm; the wall-broken spore has several forms, and can be divided into mild wall breaking- -complete spore form, small holes on the wall, moderate wall breaking- -incomplete spore form, defect, severe wall breaking- -breaking of spore coat and spore content according to different wall-breaking procedures;
(1.2) thin-layer identification of sitosterol in tartary buckwheat:
weighing tartary buckwheat control medicinal material and composition powder 3g respectively, adding 25mL chloroform solution, performing ultrasonic extraction for 30min, filtering, taking 10mL filtrate, evaporating to dryness on an evaporating dish, dissolving with chloroform, and diluting to 2mL volume; sucking L0 μ L of test solution of the composition and contrast solution of radix Et rhizoma Fagopyri Tatarici, respectively dropping on the same silica gel G plate containing 0.3% sodium carboxymethylcellulose as adhesive, and mixing at a volume ratio of 7: 3, developing by using petroleum ether-diethyl ether as a developing agent, taking out, airing, spraying with a 10% sulfuric acid solution, and drying at 105 ℃ until spots develop color and have the same color with the same Rf value as the reference substance;
(2) quantitative study
(2.1) determination of polysaccharide content in Ganoderma spore powder
Accurately weighing 0.1g of anthrone, adding 100mL of 80% sulfuric acid solution, stirring to dissolve, and shaking up to obtain the final product; performing anthrone-sulfuric acid color reaction, taking anhydrous glucose as a reference substance, and determining at 625nm by a colorimetric method;
preparing a reference substance solution:
precisely weighing 1.521mg of anhydrous glucose dried at 105 deg.C to constant weight, precisely weighing, placing in 10mL measuring flask, adding distilled water to dissolve and dilute to scale, shaking, and making into 0.1521 mg/mL-1The control solution of (4);
preparation of test solution
2g of ganoderma spore powder is taken, precisely weighed, placed in a round-bottom flask, added with 60mL of water and kept stand for 1 hour, heated and refluxed for 4 hours, filtered while hot, the filter and filter residue are washed by a small amount of hot water, the filter residue and filter paper are placed in the flask, added with 60mL of water, heated and refluxed for 3 hours, filtered while hot, the filtrates are combined, placed on a water bath and evaporated to dryness, the residue is dissolved by 5mL of water, 75mL of ethanol is slowly dripped while stirring, shaking is uniformly carried out, placed at 4 ℃ for 12 hours, centrifuged, the supernatant is discarded, the precipitate is dissolved by hot water and transferred to a 50mL measuring flask, cooling is carried out, added with water to scale, shaking is carried out, a proper amount of solution is taken, centrifuged, 3mL of the supernatant is precisely measured and placed in a 25mL measuring flask;
drawing of standard curve
Accurately weighing control solution 0.4, 0.6, 0.8, 1.0, and 1.2mL, respectively placing in 10mL test tubes with plugs, respectively adding water to 2.0mL, quickly and accurately adding sulfuric acid (accurately weighing anthraketone 0.lg, adding sulfuric acid 100mL for dissolving, and shaking) 6mL, immediately shaking, standing for 15min, immediately placing in ice bath for cooling for 15min, taking out, taking corresponding reagent as blank, irradiating with ultraviolet-visible spectrophotometry, measuring absorbance at 625nm wavelength, with absorbance as ordinate and concentration as abscissa, drawing standard curve, and y is 0.0051 x-0.0036;
(2.2) determination of naringenin content in Dendrobium officinale
Measuring the content of naringenin, which is an effective component of dendrobium officinale in the composition, by adopting a high performance liquid chromatography; the chromatographic conditions are as follows: XB 18 chromatographic column with specification of 4.6mm × 250mm and 5 μm; the column temperature is 30 ℃; mobile phase 0.2% phosphoric acid as phase A-methanol as phase B, gradient elution; the detection wavelength is 290 nm; the flow rate is 1.0 mL/min;
precisely weighing appropriate amount of naringenin reference substance, placing in 50mL brown measuring flask, adding methanol to dissolve to scale, shaking, and making into reference substance solution containing 26 μ g per 1 mL;
taking 0.5g of the composition, precisely weighing, placing the composition in a 50mL round-bottom flask, precisely adding 25mL of a mixed solution of methanol and 20% hydrochloric acid in a volume ratio of 4: 1, weighing, placing the mixture in a water bath, heating and refluxing, cooling, weighing again, complementing weight loss by using methanol, shaking up, standing, taking supernatant, filtering by using a 0.22 mu m microporous membrane, taking subsequent filtrate, injecting 10 mu L of the sample, and preparing a sample solution for later use;
precisely sucking the reference substance solution and the sample solution, injecting into a liquid chromatograph, and measuring naringenin content by adopting an external standard one-point method;
(2.3) establishment of method for measuring rutin and quercetin content in tartary buckwheat extract
Preparation of control solutions
Precisely weighing 5.952mg of rutin reference substance and 3.075mg of quercetin reference substance, respectively placing in 10mL volumetric flasks, dissolving with methanol, fixing the volume to scale, and shaking to obtain rutin and quercetin reference substance solutions with concentrations of 0.5952mg/mL and 0.3075mg/mL respectively;
preparation of test solution
Reflux-extracting radix Et rhizoma Fagopyri Tatarici with 10 times of 60% ethanol for 3 times (each time for 1 hr), mixing filtrates, concentrating under reduced pressure, dissolving residue with methanol, transferring to 25mL measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate;
precisely absorbing rutin reference substance and quercetin reference substance solution, sequentially diluting in equal gradient for 6 parts, sequentially injecting into liquid chromatograph, measuring standard curve, and absorbing Fagopyrum tataricum sample solution for injection into liquid chromatograph;
the chromatographic conditions are as follows: chromatography column Thermo C18, 4.6mm × 250mm,5 μm; the mobile phase is acetonitrile-0.2% formic acid water solution, and the gradient elution is as follows: 0-18 min, 17-40% of acetonitrile, 18-19 min and 40-17% of acetonitrile; the volume flow is 1 mL/min; detection wavelength: λ 256 nm; sample introduction amount: 10 mu L of the solution;
taking the concentration of each control as abscissa (C, peak area A of each control as ordinate to draw standard curve, and rutin linear regression equation as Y ═ 2 × 107+ 2506.8; the linear regression equation of quercetin is Y ═ 4 × 107－11208；
(2.4) content determination of rutin in mulberry leaves:
preparation of control solutions
Taking a proper amount of rutin reference substance, precisely weighing, and preparing a solution containing 0.1mg per 1mL by using methanol;
preparation of test solution
Weighing folium Mori powder lg, precisely weighing, placing in a round-bottom flask, adding 50mL of methanol, heating and refluxing for 30min, filtering, extracting the residue with 50mL of methanol for 2 times, mixing filtrates, recovering solvent under reduced pressure, dissolving the residue with methanol, transferring to a 25mL measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate;
preparation of the Standard Curve
Diluting rutin control solution 500 μ L with methanol for 6 times to obtain control solutions with concentrations of 0.0095mg/mL, 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, and 0.3034mg/mL, respectively, injecting into liquid chromatograph, and measuring by sample injection;
the chromatographic conditions are as follows: the chromatographic column is C18 column, 4.6mm × 250mm,5 μm; the mobile phase is methanol-0.5% phosphoric acid water solution, and the gradient elution is as follows: 0-5 min, 35% methanol; 8-16 min, 40% methanol; 19-24 min, 51% methanol; 26-30 min, 70% methanol; 30-35 min, 35% methanol. The volume flow is 1 mL/min; the detection wavelength is 358nm, and the sample injection amount is 10 mu L;
plotting the concentration as abscissa to obtain the standard curve y 2 × 107x-10419, linear range of 0.0095-0.3034 mg/mL, correlation coefficient r of 1.
Through qualitative identification, the content of active polysaccharide, rutin, quercetin and naringenin in the composition is higher than the sum of the content of polysaccharide, rutin, quercetin and naringenin in the composition of 125g of single mulberry leaf extract, 125g of tartary buckwheat extract, 250g of dendrobium officinale and 125g of ganoderma lucidum spore powder. The dissolution content of active ingredients of polysaccharide, rutin, quercetin and naringenin can be obviously improved after the mulberry leaf extract 125g, the tartary buckwheat extract 125g, the dendrobium officinale 250g and the ganoderma lucidum spore powder 125g are combined.
Example 4 efficacy experiment for lowering blood sugar and blood lipid
First, experimental material
1. Medicine, reagent and its preparing method
1.1 drugs and reagents
The tested drugs are: the composition prepared in the embodiment 2 of the invention (125 g of mulberry leaf extract, 125g of tartary buckwheat extract, 250g of dendrobium officinale and 125g of ganoderma lucidum spore powder) has a clinical planned dosage of 5g (preparation dosage)/60 kg, and a mouse design dosage in an experiment is 2.5g/kg (respectively 30 times of the clinical dosage); the designed dose of rat is 0.21g/kg, 0.42g/kg, 0.84g/kg, 1.26g/kg (2.5, 5, 10, 15 times of clinical dose respectively).
Metformin, saint-economy pharmaceutical limited, guizhou, lot number: 20160505. the designed dose for rats in the experiment was 0.125 g/kg.
Alloxan, Sigma company, batch number: batch number: BCBW 1234V.
Normal saline, am double crane pharmaceutical industry, llc, lot number: 1603012D.
Citric acid, national drug group chemical reagents ltd, lot number: 20130801.
sodium citrate, metropolis chemical reagent plant, batch number: t20100913.
Glucose, Amresco corporation, usa, lot number: 0188.
triglyceride (TG) assay kit (single reagent GPO-PAP method), tokyo, bio-engineering institute, lot number: 20161126.
total Cholesterol (TC) determination kit (single reagent GPO-PAP method), tokyo institute of biotechnology, lot number: 20161126.
rat serum insulin assay kit (Elisa method), tokyo institute of bioengineering, lot number: 20161201.
glycated Serum Protein (GSP) kit (NBT method), tokyo institute for bioengineering, lot no: 20161128.
rat glycated hemoglobin assay kit (Elisa method), Nanjing institute of bioengineering, lot number: 20161201.
1.2 methods of formulating drugs and reagents
1.2.1 blood glucose test in Normal mice
High dose group of the composition of the invention (2.5g formulation/kg): weighing 50g of extract powder, and preparing into 400ml with distilled water, wherein the concentration is 0.125g/ml, and the administration volume is 20 ml/kg. The dose administered is calculated as the formulation dose.
1.2.2 diabetic rat blood sugar test
Inventive composition I (0.21g formulation/kg): weighing extract powder 4.2g, preparing into 400ml with distilled water, the concentration is 0.0105g/ml, and the administration volume is 20 ml/kg. The dose administered is calculated as the formulation dose.
Composition II according to the invention (0.42g formulation/kg): 8.4g of extract powder is weighed and prepared into 400ml by distilled water, the concentration is 0.021g/ml, and the administration volume is 20 ml/kg. The dose administered is calculated as the formulation dose.
Composition III according to the invention (0.84g formulation/kg): weighing 16.8g of extract powder, preparing into 400ml with distilled water, wherein the concentration is 0.042g/ml, and the administration volume is 20 ml/kg. The dose administered is calculated as the formulation dose.
Inventive composition IV (1.26g formulation/kg): weighing 25.2g of extract powder, preparing into 400ml with distilled water, the concentration is 0.063g/ml, and the administration volume is 20 ml/kg. The dose administered is calculated as the formulation dose.
Metformin (0.125g/kg), 20 capsules (0.25 g/capsule) were prepared into 400ml with distilled water, concentration of 0.0125g/ml, and administration volume of 10 ml/kg. The dose administered is calculated as the formulation dose.
Tetraoxypyrimidine (100mg/kg), 2g of tetraoxypyrimidine powder is dissolved in 200ml of citric acid-sodium citrate buffer solution with the concentration of 0.01g/ml, and the volume of intraperitoneal injection is 10 ml/kg.
2. Experimental animal and feed
Clean grade ICR male mice, weighing 18-22g, 30, provided by the university of Yangzhou comparative medical center, quality eligibility license number: SCXK (su) 2012-.
The SPF male SD rat with the weight of 150-: SCXK (zhe) 2014-0001 for use in the experiment of "impact on hyperglycemic model animals".
The experimental mouse pellet feed and the high-heat energy feed are provided by a Qinglongshan animal breeding farm in Jiangning district of Nanjing city.
3. Laboratory apparatus
TD6001 electronic balance, tianma instruments factory, tianjin.
Electronic analytical balance (FA2104), shanghai precision instruments ltd.
Blood glucose test paper (FAD glucose dehydrogenase method), bayer medicine health care limited.
Bai an kang contourr TS glucometer, bayer medicine health ltd.
DHG-9140A type electric heating constant temperature forced air drying oven, Shanghai sperm macro experimental facilities Co.
Direct-Q ultrapure water instrument, MILLIPORE, USA.
Mix-100 Mix elfin, Hangzhou Osseo instruments ltd.
Synergy HT microplate reader, Bio-Tek, USA.
Second, experimental conditions and statistical method
The mice are fed with common pellet feed, the mice are fed with the common pellet feed in the adaptation period and the 1 st week of formal experiments, and the high-heat energy feed is replaced in the later 3 weeks. Temperature: 20-24 ℃; humidity: 50-60%. SPSS 22.0 statistical software is adopted, single-factor variance analysis is adopted for the comparison among groups, t test is adopted for the comparison between every two groups, and data are calculated according to the statistical analysis result
Figure GDA0002782080620000171
And (4) showing.
Third, experimental method and result (I) blood sugar reduction experiment for normal animal
1. Grouping, molding and administration method
Taking 30 clean ICR male mice, 18-22g, after adapting to feed for 3-5 days, fasting for 5h, and measuring fasting blood glucose value before administration. Then randomly divided into two groups of 15 each, which were (1) blank control groups: 20ml/kg of physiological saline; (2) the composition of the invention in the high dose group: 2.5 g/kg. Gavage is carried out for 1 time every day, the gavage volume is 20ml/kg, fasting is carried out for 5 hours after continuous administration for 35 days, and the fasting blood glucose value after administration is measured.
2. Observation index
2.1 general case
Mice were observed daily for diet, water intake, and urination.
2.2 body weight
The body weight before (week 0), in the middle of (week 2), and after (week 5) the administration was measured.
2.3 blood sugar level
Fasting is carried out for 5h after 35 days of continuous administration, fasting blood glucose values are measured, and blood glucose values of the two groups of animals are compared.
3. Results
3.1 general case observations
The mice in the blank control group and the high-dose group of the composition have normal diet and drinking water conditions and normal urination conditions, and the weight of the mice in the blank control group before, in the middle and after administration and the high-dose group of the composition have no significant difference (see table 10).
TABLE 10 Effect of the compositions of the present invention on body weight of Normal mice: (
Figure GDA0002782080620000181
n＝15)
Figure GDA0002782080620000182
3.2 Effect on fasting plasma glucose in Normal mice
The experimental results show that: the high dose group of the composition of the invention has no obvious influence on the fasting blood glucose of normal mice, and has no significant difference compared with a blank control group (see table 11).
TABLE 11 Effect of the compositions of the present invention on fasting plasma glucose (mmol/L) in Normal mice
Figure GDA0002782080620000183
Figure GDA0002782080620000184
(II) Effect on hyperglycemic model animals
1. Basal blood glucose
120 male SD rats with 150-170g weight are taken, after the normal maintenance material is adapted to be fed for 3-5 days, fasting is carried out for 4 hours, and the basic blood sugar value is measured (namely, the blood sugar value before (namely 0h) glucose is measured, and the blood sugar value after 2.5g/kg glucose is measured for 0.5h and 2 h).
2. Grouping, administering and moulding
Selecting rats with fasting blood glucose of 4-7mmol/L, and randomly grouping (no significant difference exists between fasting blood glucose groups), wherein each group comprises 15 rats, namely (1) a blank control group, without treatment; (2) model control group: NS 10 ml/kg; (3) metformin group: 0.125 g/kg; (4) compositions of the invention group i: 0.21 g/kg; (5) composition of the invention group II: 0.42 g/kg; (6) composition of the invention group III: 0.84 g/kg; (7) composition VII of the invention: 1.26 g/kg. The administration was continued for 33 days in each of the groups except group (1). The first 1 week is fed with maintenance feed, and the last 3 weeks is fed with high-heat feed. After 4 weeks of administration, fasting is carried out for 24h (without water prohibition), alloxan is given for 100mg/kg intraperitoneal injection, and after injection, high-calorie feed is continuously given for 5 days.
3. Observation index
3.1 general case
Rats were observed daily for diet, water intake, and urination.
3.2 body weight
Body weights before (week 0), during (week 3), and after (week 5) administration were measured.
3.3 fasting blood glucose and glucose tolerance
After 33 days of administration, animals of each group were fasted for 4 hours, fasting blood glucose and glucose tolerance were measured (after 15min, glucose was orally administered to each group at 2.5g/kg, and blood glucose levels of rats of each group after administration of glucose were measured at 0.5 and 2 hours, respectively), and the change in area under the blood glucose curve (AUC) was calculated.
The area under the blood glucose curve is 0.25 × (0h blood glucose level +4 × 0.5h blood glucose level +3 × 2h blood glucose level).
3.4 serum insulin, Cholesterol, triglyceride and glycated serum protein, glycated hemoglobin
After the blood sugar measurement is finished, 4ml of blood is taken from the orbit of the rat, the orbit is kept stand for 2h at room temperature and then centrifuged (3000rpm,10min) to take serum, the levels of the insulin, cholesterol, triglyceride and glycosylated serum of the serum to be measured are measured, and simultaneously 1ml of anticoagulated whole blood is taken to measure the level of the glycosylated hemoglobin.
4. Results
4.1 general case observations
The food intake, water intake and weight of the model group rats are increased, and more urination is realized; the metformin group and the composition groups I, II, III and IV of the invention have reduced dietary water intake and reduced urine output compared with the model group. After injecting alloxan into the abdominal cavity, the rats in the model group died 6, the rats in the metformin group died 5, the rats in the composition I of the invention died 3, the rats in the composition II of the invention died 2, the rats in the composition III of the invention died 5, and the rats in the composition IV of the invention died 5.
4.2 Effect on body weight of diabetic rats
There were no significant differences between the body weight groups of the animals before (week 0), during (week 3) and at the end of (week 5) dosing (see table 12).
TABLE 12 Effect of the compositions of the invention on rat body weight
Figure GDA0002782080620000191
Figure GDA0002782080620000201
Note: the number of rats per group n is 15 before and during administration##P<0.01 in comparison with blank control
4.3 Effect on fasting plasma glucose in diabetic rats
The experimental results show that: compared with the rats in the blank control group, the fasting blood glucose of the rats in the model group is obviously increased, and compared with the rats in the metformin group, the fasting blood glucose of the rats in the compositions II and III of the invention is obviously reduced (see table 13).
TABLE 13 Effect of the compositions of the present invention on fasting plasma glucose (mmol/L) in hyperglycemic rats
Figure GDA0002782080620000202
Figure GDA0002782080620000203
##P<0.01 vs. placebo P<0.01，*P<0.05 comparison with model group
4.4 Effect on glucose tolerance in diabetic rats
The experimental results show that: the area under the blood glucose curve of the rats in the model group is obviously increased compared with that of the rats in the blank control group, and the area under the blood glucose curve of the rats in the metformin group, namely the rats in the compositions I, II, III and IV is obviously reduced compared with that of the rats in the model group (see table 14).
TABLE 14 Effect of the compositions of the present invention on the area under the blood glucose curve of rats
Figure GDA0002782080620000204
Figure GDA0002782080620000205
##P<0.01 vs. placebo P<0.01 comparison with model group
4.5 Effect on glycated serum proteins and hemoglobin in diabetic rats
The experimental results show that: there were no significant differences (P >0.05) between the placebo, model, and each of the administered groups between glycated serum protein and hemoglobin groups (see Table 15).
TABLE 15 Effect of the compositions of the present invention on glycated serum protein and glycated hemoglobin in rats
Figure GDA0002782080620000211
Figure GDA0002782080620000212
4.6 Effect on fasting serum insulin in diabetic rats
The experimental results show that: the fasting serum insulin of the rats in the model group is obviously reduced compared with that in the blank control group, and the serum insulin level can be increased to different degrees in each administration group, wherein the fasting serum insulin level of the compositions II and III in the metformin group is obviously increased compared with that in the model group (see table 16).
TABLE 16 Effect of the compositions of the present invention on fasting serum insulin in rats
Figure GDA0002782080620000213
Figure GDA0002782080620000214
##P<0.01 vs. placebo P<0.01，*P<0.05 comparison with model group
4.7 Effect on blood lipids in diabetic rats
The experimental results show that: the serum triglyceride and total cholesterol levels of the rats in the model group are obviously increased compared with those in the blank control group, the serum triglyceride levels of the rats in the metformin group and the compositions II and III in the invention are reduced, and the serum total cholesterol levels of the rats in the metformin group and the compositions I, II and III in the invention are obviously different from those in the model group (see table 17).
TABLE 17 Effect of the compositions of the present invention on rat serum triglycerides
Figure GDA0002782080620000221
Figure GDA0002782080620000222
##P<0.01 vs. placebo P<0.01，*P<0.05 compared with the model group, the experimental results show that:
1. the high-dose group (2.5g/kg, 30 times) of the composition has no obvious influence on the fasting blood glucose value of normal mice.
2. The composition I (0.21g/kg, 2.5 times) of the invention can reduce the area under the blood sugar curve of diabetic rats (P <0.01) and serum cholesterol (P <0.05) by preventive administration;
the composition II (0.42g/kg, 5 times) can obviously reduce fasting blood glucose (P <0.05), area under the blood glucose curve (P <0.01), increase serum insulin (P <0.05), reduce serum triglyceride (P <0.01) and cholesterol (P <0.01) of diabetic rats by preventive administration;
the composition III (0.84g/kg, 10 times) of the invention can obviously reduce fasting blood sugar (P <0.01) of diabetic rats, area under the blood sugar curve (P <0.01), increase serum insulin (P <0.01), reduce serum triglyceride (P <0.01) and cholesterol (P <0.01) by preventive administration;
the composition IV (1.26g/kg, 15 times) can obviously reduce the area under the blood sugar curve of a diabetic rat (P <0.01) by preventive administration.
According to the blood sugar reducing experiment, other weight ratios of the mulberry leaf extract, the tartary buckwheat extract, the dendrobium candidum and the ganoderma lucidum spore powder are screened, and comprise raw materials in different weight ratios of 1:1:1:1, 1:2:1:1, 2:1:1:1, 1:1:1:2, 3:1:1:1, 1:3:1, 1:1:3:1 and 1:1:3, and the comparison result shows that the weight ratios of the mulberry leaf extract, the tartary buckwheat extract, the dendrobium candidum and the ganoderma lucidum spore powder are as follows: the ratio of 1:1:2:1 has the best effect of reducing blood sugar and blood fat. The weight ratio shows that the 4 raw materials play a synergistic effect and are the optimal dosage ratio of the invention.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (3)

1. A composition with the effect of preventing and treating diabetes is characterized by being prepared from the following raw materials in parts by weight:
125 parts of mulberry leaf extract, 125 parts of tartary buckwheat extract, 250 parts of dendrobium officinale and 125 parts of ganoderma lucidum spore powder;
the mulberry leaf extract is prepared by the following method: extracting folium Mori with 10 times volume of 80% ethanol under reflux for 2 times (each for 1.5 hr), and filtering to obtain ethanol extractive solution; extracting the residue with 10 times of water for 1 time, each time for 1.5 hr, filtering to obtain water extractive solution, mixing the ethanol extractive solution and the water extractive solution, and concentrating under reduced pressure to relative density of 1.20 at 60 deg.C; vacuum drying or spray drying the concentrated solution, pulverizing, and sieving to obtain folium Mori extract;
the tartary buckwheat extract is prepared by the following method: extracting radix Et rhizoma Fagopyri Tatarici with 10 times volume of 60% ethanol under reflux for 3 times, each time for 1.5 hr, filtering, mixing filtrates, recovering ethanol under reduced pressure, and concentrating to relative density of 1.20 at 60 deg.C; drying the concentrated solution under normal pressure or reduced pressure, pulverizing at low temperature, and sieving to obtain radix Et rhizoma Fagopyri Tatarici extract;
the dendrobium officinale is prepared by the following method: drying the dendrobium officinale at 100 ℃ for 30 minutes, crushing, and sieving with a 100-mesh sieve to obtain the dendrobium officinale powder.
2. The use of the composition with the effect of preventing and treating diabetes mellitus of claim 1 in the preparation of hypoglycemic drugs.
3. The use of the composition with the effect of preventing and treating diabetes mellitus of claim 1 in the preparation of a medicament for reducing blood fat.
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