CN105560313B - A kind of compound Jin stork herbal medicine object and preparation method thereof for treating diabetes - Google Patents
A kind of compound Jin stork herbal medicine object and preparation method thereof for treating diabetes Download PDFInfo
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Abstract
The present invention relates to a kind of compound Jin stork herbal medicine objects and preparation method thereof for treating diabetes, this method is to mix Geranium collinum with two kinds of medicinal materials of rough branch Hypericum Chinense, using ethyl alcohol extraction, purifying resin, concentration, dry acquisition extract, extract is mixed with pharmaceutical grade supplementary product starch, dextrin, mannitol, sodium carboxymethyl starch again, particle, capsule or tablet is made.Enzyme activity level can effectively inhibit PTP 1B (PTP1B) and have good antioxidant effect the extract that the method obtains through the invention in vitro, can be used as insulin sensitizer for treating diabetes.The compound medicine is, in conjunction with long-term civil clinical practice, to be enriched with active principle in the compound medicine according to traditional medicine theory using modern extraction and separation technology, and confirm drug effect, provide convenience for patient.
Description
Technical field
The present invention relates to a kind of compound Jin stork herbal medicine objects and preparation method thereof for treating diabetes, belong to national medicine technology
Field.
Background technique
Diabetes are to be distributed wider epidemic disease in the world, and number of patients is in growth trend year by year.Diabetes
Have become the third-largest chronic disease for seriously threatening health of people after tumour, cardiovascular disease, diabetes and its complication
Serious burden is brought to human health and social development.Conventional medication diabetes have " multicomponent, multipath, more
The features such as target spot ", and side effect is low, therefore, finds and develop the drug for the treatment of diabetes from conventional medicament with wide
Prospect.
Type-2 diabetes mellitus is the common chronic disease of China middle-aged and the old.There is 30% to suffer from II type glycosuria in China overweight people
Disease.Since type-2 diabetes mellitus and obesity belong to the multiple-factor inheritance disease closely related with life style, pathogenesis
It is still not very clear.Recent studies have shown that protein tyrosine phosphatase 1B (protein tyrosine phosphatase
1B, PTP1B) insulin (insulin) and leptin (leptin) signal transduction can be weakened, cause Anomalous lipid metablism, is to lead to pancreas
Insulin resistance and fat key link;Meanwhile it is demonstrated experimentally that protein tyrosine phosphatase 1B (PTP1B) gene is knocked out
Mouse do not occur insulin resistance and obesity under the fat eating condition that causes fat.Thus cause people to protein junket ammonia
The broad interest of acid phosphoric acid enzyme 1B (PTP1B) inhibitor, protein tyrosine phosphatase 1B (PTP1B) gene, which becomes, increasingly to be drawn
One of the target gene that people gazes at.Protein tyrosine phosphorylation is the important of cell metabolism, signal transduction and cell cycle regulating
Regulating step.Protein tyrosine phosphatase 1B (PTP1B) belongs to protein tyrosine phosphatase family, more than the family 40 phosphorus
The homologous sequence that all there is sour enzyme about 240 amino acid to form is located at enzymatic activity position, with transmembrane receptor sample albumen
Exist with endocellular enzyme form, the reaction of catalytic proteins tyrosine dephosphorylation.Protein tyrosine phosphatase 1B (PTP1B) passes through
Insulin receptor kinase (insulin receptor kinase, IRK) or the phosphorylated tyrosine residues of its active fragment are gone
Phosphorylation carries out negative regulator to insulin signal transduction.Foreign countries are just in screening protein tyrosine phosphatase 1B (PTP1B)
There are two main classes for inhibitor: one kind is the substrate analogue of protein tyrosine phosphatase 1B (PTP1B) --- phosphotyrosine
Polypeptide;Another kind of is aryl orthophosphate micromolecular substance, but is also being researched and developed, and the clinic of type-2 diabetes mellitus is had not been used in
Treatment.Though sodium vanadate is effective inhibitor of protein-tyrosine enzyme (PTP), it does not have selectivity, is only used for external yet
Research.Therefore, the research of non-peptides, nonphosphate proteinoid tyrosine phosphatase 1B (PTP1B) inhibitor becomes this neck
The hot topic in domain.The most effective strategy of screening protein tyrosine phosphatase 1B (PTP1B) inhibitor is exactly with protein junket at present
The active site of propylhomoserin phosphatase (PTP) is target spot, the catalytic structure of research protein tyrosine phosphatase 1B (PTP1B)
Domain carries out the screening of inhibitor, can be used for clinical, effective, specific inhibitor to obtain.The present invention directly against
Protein tyrosine phosphatase 1B (PTP1B) target spot carries out the pharmacy in vitro screening of compound medicine extract.
The present invention is according to folk remedy use experience, by Geranium collinum (Geranium collinum Steph.) and rough
Branch Hypericum Chinense (Hypericum Scabrum Lnn.) two kinds of herbal medicine are used in mixed way in specific proportions, and carry out extraction process,
Purifying process, moulding process and Pharmacodynamics in vitro research, so that it is determined that the Preparation method and use of the compound medicine.
Both plant main products are largely distributed in Xinjiang of China in Central Asia, medical usage " Chinese Pharmacopoeia ", " Ah
Wei Senna cures allusion quotation " it is on the books.Geranium wilfordii be China's traditional Chinese medicine, there are three types of the kinds that Chinese Pharmacopoeia records, primary chemical at
It is divided into tannin, flavones, organic acid and volatile oil;With anti-inflammatory and antalgic, antibacterial, antiviral, antidiarrheal, anti-oxidant, uvioresistant
With the pharmacological actions such as antibechic.It is clinically used for rheumatic arthralgia, the card such as numbness contracture, muscles and bones are ached, dysentery.Modern study table
Bright, there are also the effects of antitumor, hypoglycemic for geranium wilfordii.It is increasingly subject to about the chemical component of geranium wilfordii and the research of pharmacological action
The attention of domestic and international researcher.Geranium collinum (Geranium collinum Steph.ex Willd.) is Mang ox
Miao Ke (Geraniaceae) Geranium (Geranium) plant, is distributed in the new of the Central Asia, West Asia, Central Europe and China's Mainland
The ground such as boundary are grown on 2200 meters to 3500 meters of height above sea level of area, are often born in montane grassy marshland, Subalpine region and high mountain, at present
Not yet by artificial introducing and planting.Rough branch Hypericum Chinense (Hypericum scabrum L.) is Guttiferae (Guttiferae) Hypericum Chinense
Belong to (Hypericum L.) plant also known as gland point Hypericum Chinense, the country is only distributed in Altay Mountains, records in " Kazak medicine will "
(first volume), all herbal medicine.It is reported that rough branch Hypericum Chinense is rich in flavone compound, extract has good anti-oxidant work
Property, there is good removing to 1,1- diphenyl -2- trinitrophenyl-hydrazine (DPPH) free radical, hydroxyl radical free radical, superoxide radical
Ability.
Summary of the invention
The object of the present invention is to provide a kind of compound Jin stork herbal medicine object and preparation method thereof for treating diabetes, the party
Method mixes Geranium collinum with two kinds of medicinal materials of rough branch Hypericum Chinense, is mentioned using ethyl alcohol extraction, purifying resin, concentration, drying
Take object, then extract mixed with pharmaceutical grade supplementary product starch, dextrin, mannitol, sodium carboxymethyl starch, be made particle, capsule or
Tablet.Enzyme activity level can effectively inhibit albumen junket ammonia to the compound Jin stork herbal medicine object that the method obtains through the invention in vitro
Acid phosphoric acid enzyme 1B (PTP1B), and there is good antioxidant effect, it can be used as insulin sensitizer for treating diabetes.This is multiple
Side's gold stork herbal medicine object is according to traditional medicine theory, in conjunction with long-term civil clinical practice, using modern extraction and separation technology richness
Collect effective component in the compound medicine, and confirm drug effect, is provided convenience for patient.
A kind of compound Jin stork herbal medicine object for treating diabetes of the present invention, the drug each component hills by weight are old
50-90 parts of stork grass roots, 10-60 parts of rough branch Hypericum Chinense aerial part, auxiliary material are starch, dextrin, mannitol or sodium carboxymethyl starch
10-50 parts, by extraction, purifying, it is dry after the extract that obtains, then extract and auxiliary material prepared according to a conventional method and is made
Grain, capsule or tablet.
The preparation method of the compound Jin stork herbal medicine object of the treatment diabetes, follows these steps to carry out:
A, dry 10-60 parts of 50-90 parts of Geranium collinum root and rough branch Hypericum Chinense aerial part are crushed by weight,
It is extracted 1-3 times with the alcohol reflux that 5-30 times of volumetric concentration is 30%-80%, 30-90 DEG C of temperature, extracts 1-3 hours, close every time
And extracting solution, filtration, filtrate decompression are concentrated into no alcohol taste, obtain extracting solution;
B, the extracting solution obtained step a filters, filtrate with resin column is HP-600, AB-8, D101, HPD-300,
LD601, DM-130 or LX-38 purifying, first with the water elution of 1-4 times of column volume, then the 30-90% ethyl alcohol with 3-8 times of column volume
Ethanol eluate is collected in elution, is concentrated under reduced pressure, is dry, crushing, obtaining extract;
C, the extract for obtaining step b and medical grade auxiliary material are starch, dextrin, mannitol or sodium carboxymethyl starch 10-
50 parts of mixings, routinely particle, capsule or tablet is made in pharmaceutical methods.
A kind of compound Jin stork herbal medicine object and preparation method thereof for treating diabetes of the present invention, by the old stork in hills
Careless (Geranium collinum Steph.) and rough branch Hypericum Chinense (Hypericum Scabrum Lnn.) are mixed by different proportion
It closes, is extracted using different concentration ethanol, purifying resin, utmostly extracted and active constituent-enriched, and is mixed with pharmaceutical grade auxiliary material
It closes, particle, tablet and capsule is made.Through having carried out the research of anti-diabetic Pharmacodynamics in vitro, result to compound Jin stork herbal medicine object
Show: enzyme activity level has effectively inhibition PTP 1B to compound Jin stork herbal medicine object provided by the invention in vitro
(PTP1B) and good antioxidation, it can be used as the purposes of antidiabetic medicine.
The extract that described compound medicine for the treatment of diabetes and preparation method thereof obtains through the invention is in sepia
Powder, color is uniform, and viscosity is low, and hygroscopicity is small, can be easily made particle, capsule or tablet.Pharmacodynamic tests prove that the compound
Golden stork herbal medicine object enzyme activity level significantly effectively can inhibit PTP 1B (PTP1B) and have well in vitro
Antioxidant effect.Carry out glycometabolism and protein level detection in cellular level, there is significant hypoglycemic effect, and there is enhancing pancreas
The effect of island element, the mechanism of action are activation Adenylate cyclase (AMPK) signal path, regulated insulin signaling
The key molecule insulin receptor substrate-1 (IRS-1) of approach, protein kinase B (AKT) overcome insulin resistance, thus in pancreas
Increase glucose uptake in the element resisting cell of island, achievees the purpose that hypoglycemic.
Detailed description of the invention
Fig. 1 is that glucose consumption of the present invention tests (human liver cancer cell, HepG2) figure;
Fig. 2 is that glucose consumption of the present invention tests (rat myoblasts, L6) figure;
Fig. 3 is that glucose uptake of the present invention tests (mouse embryonic fibroblasts, 3T3L1) figure;
Fig. 4 is that the golden stork grass compound extract of the present invention acts on three phosphorus of adenosine after human liver cancer cell (HepG2) cell
Sour (ATP) horizontal variation diagram;
Fig. 5 is that the golden stork grass compound extract of the present invention acts on protein level variation diagram after human liver cancer cell (HepG2) cell.
Specific embodiment
Embodiment 1 (prepares granule, to prepare 1000g as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 5 times of volumes are added is 30%
It ethyl alcohol heating and refluxing extraction 2 times, extracts every time 2 hours, 30 DEG C of temperature, combined extract filters, and filtrate decompression is concentrated into no alcohol
Taste obtains extracting solution;
Extracting solution is filtered, filtrate HP-600 resin column purification, first with the water elution of 1 times of column volume, then with 3 times of cylinders
Long-pending concentration is 30% ethanol elution, collects ethanol eluate, is concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, granule is made according to a conventional method, wherein each component is shown in Table 1;
Table 1
Embodiment 2 (prepares granule to prepare 1000g as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 10 times of volumes are added is
50% ethyl alcohol heating and refluxing extraction 2 times is extracted 2 hours every time, and 70 DEG C of Extracting temperature, combined extract filters, and filtrate decompression is dense
It is reduced to no alcohol taste, obtains extracting solution;
Extracting solution is filtered, filtrate AB-8 resin column purification, first with the water elution of 1 times of column volume, then with 3 times of column volumes
Concentration be 70% ethanol elution, collect ethanol eluate, be concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, granule is made according to a conventional method, wherein each component is shown in Table 2;
Table 2
Embodiment 3 (capsule preparation is to prepare 1000 as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 8 times of volumes are added is 50%
It ethyl alcohol heating and refluxing extraction 2 times, extracts every time 2 hours, 90 DEG C of Extracting temperature, combined extract filters, and filtrate decompression is concentrated into
Without alcohol taste, extracting solution is obtained;
Extracting solution is filtered, filtrate D101 resin column purification, first with the water elution of 1 times of column volume, then with 3 times of column volumes
Concentration be 90% ethanol elution, collect ethanol eluate, be concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, capsule is made according to a conventional method, wherein each component is shown in Table 3;
Table 3
Embodiment 4 (prepares capsule to prepare 1000 as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 12 times of volumes are added is
70% ethyl alcohol heating and refluxing extraction 2 times is extracted 2 hours every time, and 80 DEG C of Extracting temperature, combined extract filters, and filtrate decompression is dense
It is reduced to no alcohol taste, obtains extracting solution;
Extracting solution is filtered, filtrate HPD-300 resin column purification, first with the water elution of 2 times of column volumes, then with 6 times of columns
The concentration of volume is 90% ethanol elution, collects ethanol eluate, is concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, capsule is made according to a conventional method, wherein each component is shown in Table 4;
Table 4
Embodiment 5 (prepares tablet to prepare 1000 as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 30 times of volumes are added is
80% ethyl alcohol heating and refluxing extraction 2 times is extracted 2 hours every time, and 80 DEG C of Extracting temperature, combined extract filters, and filtrate decompression is dense
It is reduced to no alcohol taste, obtains extracting solution;
Extracting solution is filtered, filtrate LD601 resin column purification, first with the water elution of 2 times of column volumes, then with 4 times of cylinders
Long-pending concentration is 70% ethanol elution, collects ethanol eluate, is concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, tablet is made according to a conventional method, wherein each component is shown in Table 5;
Table 5
Embodiment 6 (prepares tablet to prepare 1000 as radix)
Dry Geranium collinum root and rough branch Hypericum Chinense aerial part are taken, is crushed, the concentration that 15 times of volumes are added is
70% ethyl alcohol heating and refluxing extraction 2 times is extracted 2 hours every time, and 50 DEG C of Extracting temperature, combined extract filters, and filtrate decompression is dense
It is reduced to no alcohol taste, obtains extracting solution;
Extracting solution is filtered, filtrate LX-38 resin column purification, first with the water elution of 3 times of column volumes, then with 5 times of cylinders
Long-pending concentration is 80% ethanol elution, collects ethanol eluate, is concentrated under reduced pressure, dry, pulverize, obtain extract;
Extract and auxiliary material are mixed well again, tablet is made according to a conventional method, wherein each component is shown in Table 6;
Table 6
Embodiment 7
The anti-diabetic drug effect evaluation of the compound Jin stork herbal medicine object for the treatment of diabetes of the present invention:
PTP 1B (PTP1B) Inhibition test:
Experimental material:
Test medicine: golden stork grass compound extract 50#, preparation method is the same as embodiment 2;Golden stork grass compound extract 70#, system
Preparation Method is the same as embodiment 4;Precise, which is dissolved in again in dimethyl sulfoxide (DMSO), is made test mother liquor, extract concentrations 10mg/
mL;
P- nitrophenylphosphate disodium (pNPP) is used to utilize enzyme mark as substrate to be control in positive drug sodium vanadate
Instrument carries out the high flux screening of PTP 1B (PTP1B) enzyme inhibitor, according to PTP 1B
(PTP1B) it hydrolyzes the phosphate group of p- nitrophenylphosphate disodium (pNPP) and generates color reaction to measure protein-tyrosine
The activity of phosphatase 1 B (PTP1B);Enzyme reaction system composition is as follows: buffer (50mM hydroxyethyl piperazine second thiosulfonic acid, pH7.3,
100mM sodium chloride, 0.1% bovine serum albumin(BSA) and 1mM dithiothreitol dithio), PTP 1B (PTP1B) fusion
Albumen, p- nitrophenylphosphate disodium (pNPP), PTP 1B (PTP1B) specific inhibitor sodium vanadate (100 μ
g/mL);In 37 DEG C of placement 30min of temperature after reaction system mixing, 1M sodium hydroxide is added and terminates reaction, sets and is measured on colour comparatour
Absorption value (A) under 405 wavelength conditions, measurement result calculate enzymatic activity after subtracting background values;
Measurement result of the golden stork grass compound extract to PTP 1B activity suppression
Sample | IC50 |
Compound Jin stork grass extract 50# | *0.12±0.02 |
Golden stork grass compound extract 70# | *0.14±0.03 |
From the results showed that golden stork grass compound extract has effects that protein-tyrosine phosphatase 1B inhibitor.
Glycometabolism experiment:
Experimental material
Test medicine: golden stork grass compound extract 30#, the preparation method is the same as that of Example 1;Golden stork grass compound extract 50#, system
Preparation Method is the same as embodiment 2;Golden stork grass compound extract 70#, preparation method is the same as embodiment 4;Golden stork grass compound extract 80#, system
Preparation Method is with embodiment 5, and precise, which is dissolved in again in dimethyl sulfoxide, is made test mother liquor, extract concentrations 100mg/mL;
It is thin in main sugar metabolizing cells strain human liver cancer cell (HepG2), rat myoblasts (L6), mouse embryo fibroblast
Sugared utilization, Sugar intake experiment, golden stork grass compound extract 30#, 50#, 70#, 80# the best use concentration are carried out on born of the same parents (3T3L1)
Respectively 25 μ g/mL, 25 μ g/mL, 10 μ g/mL, 10 μ g/mL, with negative control and positive control jamaicin (Berberine),
Melbine (Metformin) comparison, golden stork grass compound extract has significant hypoglycemic effect, and 50#, 70#, 80# have
Enhance the effect of insulin.
The variation of adenosine triphyosphate (ATP) level:
Adenylate cyclase (AMPK) participates in a variety of metabolic processes, activity as a kind of important protein kinase
By Adenosine acid/regulation of adenosine triphyosphate (AMP/ATP) ratio, referred to as " metaboreceptor " of cell
Or " fuel cock " of regulating cell adenosine triphyosphate (ATP) and sour (AMP) level of Adenosine;Work as cell
By it is any cause adenosine triphyosphate (ATP) generate reduce with consume increased stress stimulation when, adenine ribose core
Thuja acid/adenosine triphyosphate (AMP/ATP) ratio increases, and Adenylate cyclase (AMPK) is then activated.
In human liver cancer cell (HepG2), rat myoblasts (L6) cell strain, pass through gland after detection active constituent effect
Purine nucleosides triphosphoric acid (ATP) yield, discovery cell is under the effect of golden stork grass compound extract, adenosine triphyosphate
(ATP) yield reduces, thus it is speculated that extract activates Adenylate cyclase signal path.
In human liver cancer cell (HepG2), rat myoblasts (L6) cell strain, pass through albumen water after detection drug effect
Flat variation, discovery cell is under the effect of golden stork grass compound extract, phosphorylated adenosine acid activation protein kinase (p-AMPK) albumen
Express key protein phosphorylated insulin receptor substrate (p-IRS-1), the phosphorylated protein kinase of raising, insulin signaling pathway
(p-AKT) expression increases, and illustrates drug activation Adenylate cyclase (AMPK) signal path, regulated insulin signaling
The key molecule insulin receptor substrate-1 (IRS-1) of approach, protein kinase B (AKT) overcome insulin resistance, thus in pancreas
Increase glucose uptake in the element resisting cell of island, achievees the purpose that hypoglycemic.
Carry out in glycometabolism and protein level detection in cellular level, golden stork grass compound extract is imitated with significant hypoglycemic
Fruit, and 50#, 70#, 80# have the function of enhancing insulin, and the mechanism of action is by activating phosphatase adenosine acid activation egg
White kinases (AMPK) signal path, the key molecule insulin receptor substrate-1 (IRS-1) of regulated insulin signaling pathway, egg
White kinase b (AKT) overcomes insulin resistance, to increase glucose uptake in insulin resisting cell, reaches hypoglycemic
Purpose.
Claims (1)
1. a kind of compound Jin stork herbal medicine object for treating diabetes, it is characterised in that the old stork in each component of drug hills by weight
50-90 parts of grass roots, 10-60 parts of rough branch Hypericum Chinense aerial part, auxiliary material are starch, dextrin, mannitol or sodium carboxymethyl starch 10-
50 parts, by extraction, purifying, it is dry after the extract that obtains, then extract and auxiliary material prepared according to a conventional method be made particle,
Capsule or tablet, concrete operations follow these steps to carry out:
A, 10-60 parts of 50-90 parts of Geranium collinum root and rough branch Hypericum Chinense aerial part dry crushing are used by weight
The alcohol reflux that 5-30 times of volumetric concentration is 30-80% extracts 1-3 times, 30-90 DEG C of temperature, extracts 1-3 hours every time, merging mentions
Liquid is taken, is filtered, filtrate decompression is concentrated into no alcohol taste, obtains extracting solution;
B, the extracting solution obtained step a filters, filtrate with resin column is HP-600, AB-8, D101, HPD-300, LD601,
DM-130 or LX-38 purifying is received first with the water elution of 1-4 times of column volume, then with the 30-90% ethanol elution of 3-8 times of column volume
Collect ethanol eluate, is concentrated under reduced pressure, is dry, crushing, obtaining extract;
C, the extract for obtaining step b and medical grade auxiliary material are starch, dextrin, mannitol or 10-50 parts of sodium carboxymethyl starch
It mixes, routinely particle, capsule or tablet is made in pharmaceutical methods.
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CN101757012A (en) * | 2008-12-09 | 2010-06-30 | 凌沛学 | Application of hyperin in preparation of medicament for treating diabetes |
CN101912565A (en) * | 2010-05-25 | 2010-12-15 | 河南中医学院 | Turmeric and astragalus capsules for treating diabetes and incipient diabetic nephropathy |
CN103893361A (en) * | 2012-12-28 | 2014-07-02 | 天津药物研究院 | Pharmaceutical composition for treating diabetes and application thereof |
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CN101757012A (en) * | 2008-12-09 | 2010-06-30 | 凌沛学 | Application of hyperin in preparation of medicament for treating diabetes |
CN101912565A (en) * | 2010-05-25 | 2010-12-15 | 河南中医学院 | Turmeric and astragalus capsules for treating diabetes and incipient diabetic nephropathy |
CN103893361A (en) * | 2012-12-28 | 2014-07-02 | 天津药物研究院 | Pharmaceutical composition for treating diabetes and application thereof |
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