CN106552088B - Chinese medicine composition active component and its preparation method and application with treatment diabetic nephropathy - Google Patents
Chinese medicine composition active component and its preparation method and application with treatment diabetic nephropathy Download PDFInfo
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Abstract
The invention discloses a kind of Chinese medicine composition active components and preparation method thereof with treatment diabetic nephropathy, it is made of 10~20 parts of salvianolic acid class, 20~30 parts of total-tanshinone class, 15~25 parts of mulberry leaf total alkaloid, 10~20 parts of total Flavonoids from Mulberry, 20~30 parts of total saponin from leaves of rehmannia.Preparation method provided by the invention includes pretreatment of raw material, water extract-alcohol precipitation, ethyl alcohol extraction, cation exchange resin purifying, macroporous resin purification, concentration, drying and other steps.Drug composition active component provided by the invention has the comprehensive adjustments effect such as apparent hypoglycemic, reducing blood lipid, anti-oxidant, improvement blood circulation; synergistic effect can be played between active component combination, plays the role of significantly protecting injury of kidney and prevention and treatment diabetic nephropathy.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, and in particular to a kind of effective ingredient in Chinese combination for treating diabetic nephropathy
Object and the preparation method and application thereof.
Background technique
Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bge.) is the drying root and rhizome of Lamiaceae Salvia platymiscium, begins to carry
In Shennong's Herbal, it is listed in top grade, bitter, cold nature, converge to heart and liver channels.With stasis-dispelling and pain-killing, blood circulation, blood-nourishing
Tranquilizing the mind, cool blood to disappear carbuncle, relieving restlessness and restlessness and other effects.For chest impediment and cardialgia, stomach duct and abdomen hypochondriac pain, the accumulation of lump in the abdomen lump in the abdomen, hot numbness pain, it is vexed not
It sleeps, irregular menstruation, dysmenorrhea menostasis, sore swell and ache curative.Clinically it is mainly used for cardiovascular and cerebrovascular disease.Main active is in Radix Salviae Miltiorrhizae
Salvianolic acid and tanshinone component.Modern pharmacological studies have shown that salvianolic acid and tanshinone component have anti-artery
Atherosis, improve endothelial function, expansion blood vessel, it is anti-oxidant, improve hemorheology and platelet function, improve microcirculation,
A variety of pharmacological activity such as myocardial oxygen consumption, immunological regulation, reducing blood lipid, hypoglycemic are reduced, separately some researches show that it has kidney
Good protective effect.
Mulberry leaf are the leaf of Moraceae (Moraceae) Mulberry plant mulberry (Morus alba L.).Its cold in nature, sweet-bitter flavor, has
Dispelling wind and heat from the body, clearing away the lungheat and moisturizing, the effect of clearing liver and improving vision.Medicinal history is long, and in Compendium of Material Medica record, " mulberry leaf are brothers Yang Ming
Medicine, juice decocts for tea, can only quench one's thirst ".Therefore Chinese medicine often treats diabetes with mulberry leaf.Modern clinical research shows mulberry leaf or mulberry
Prescription based on leaf can effectively treat diabetes and its complication, and obtain promising result.Chemical constitution study shows in mulberry leaf
Mainly containing ingredients such as alkaloids, flavonoids, phytosterol, microelement kind, vitamins, amino acids.With significant
Hypoglycemic, blood pressure lowering, antibacterial and a variety of physiological activity such as antiviral, wherein with 1-DNJ (1-DNJ) for representative
Alkaloids and flavones ingredient have the function of significantly adjusting blood glucose.
Adhesive rehmannia leaf is the leaf of Scrophulariaceae herbaceos perennial glutinous rehmannia Rehmannia glutinosa Libosch..Begin to carry
In the Tang Dynasty " dietetic materia medica ", adhesive rehmannia leaf slight bitter trembles with fear, enters Liver Channel.Can detoxification sore treatment, control malignant sore, tinea of feet and hands.Currently, adhesive rehmannia leaf
It is not embodied in " Chinese Pharmacopoeia " also, but has been recorded in " Beijing's Chinese medicine standard ".Chemical constitution study shows in adhesive rehmannia leaf
Change containing iridoids and its glycoside, benzyl carbinol and its glycoside, terpene and its glycoside, flavonoids and its glycoside and volatile oil etc.
It studies point, wherein belonging to the acteoside content of benzyl carbinol glycoside much higher than Radix Rehmanniae.Pharmacological research shows that adhesive rehmannia leaf has
It reduces blood glucose, improve the effects of renal function, antibacterial, removing toxic substances, separately have report display total saponin from leaves of rehmannia that there are enriching yin and nourishing kidney, cool blood is living
Blood and other effects is used for the clinical treatment of a variety of kidney troubles such as chronic nephritis mild.
Summary of the invention
Goal of the invention: the purpose of the present invention is to provide a kind of Radix Salviae Miltiorrhizae, mulberry leaf, adhesive rehmannia leaf active component composition, use
In treatment diabetic nephropathy.The preparation method that another object of the present invention is to provide the active component composition is applied with it.
Technical solution: it is achieved by the following technical solution in order to achieve the object of the present invention:
A kind of Chinese medicine composition active component with treatment diabetic nephropathy, it is original by Radix Salviae Miltiorrhizae, mulberry leaf and adhesive rehmannia leaf
Material is prepared.
Preferably, the above-described Chinese medicine composition active component with treatment diabetic nephropathy, it includes
Salvianolic acid class, total-tanshinone class, mulberry leaf total alkaloid, total flavonoids from mulberry tree leaf and total saponin from leaves of rehmannia.
As more preferred scheme, the above-described Chinese medicine composition active component with treatment diabetic nephropathy, it
Including 10~20 parts of salvianolic acid class, 20~30 parts of total-tanshinone class, 15~25 parts of mulberry leaf total alkaloid, total Flavonoids from Mulberry
10~20 parts and 20~30 parts of total saponin from leaves of rehmannia.
The preparation method of the Chinese medicine composition active component with treatment diabetic nephropathy of the present invention comprising with
Lower step:
(1) Radix Salviae Miltiorrhizae is first taken, coarse granule is ground into, first uses 8~12 times of pure water boiling and extractions, obtains aqueous extract, is concentrated,
95% ethyl alcohol of volumetric concentration is added in concentrate to concentration of alcohol up to 60%~80%, filtering precipitating obtains alcoholic supernatant, dense
Contracting obtains salvianolic acid effective kind part;
Water intaking extract after Radix Salviae Miltiorrhizae residue, using 70%~95% ethyl alcohol heating and refluxing extraction, filtering or centrifugation, filtrate or
Supernatant is concentrated under reduced pressure, and obtains alcohol extract, dry, obtains total-tanshinone effective kind part;
(2) mulberry leaf are taken, with 8~10 times of pure water refluxing extractions, filtering or centrifugation after crushing, filtrate or supernatant decompression are dense
95% ethyl alcohol is added in concentrate to concentration of alcohol up to 50%~80%, stands, filters to take supernatant for contracting, after concentration Shangyang from
Sub-exchange resin is first eluted with the ammonium hydroxide that PH is 7~13, collects eluent, and the effective portion of mulberry leaf total alkaloid is lyophilized to obtain in concentration
Position;
Take mulberry leaf, after crushing, with 50%~80% alcohol reflux of volumetric concentration extract, filtering or centrifugation, take filtrate or on
Clear liquid, upper macroreticular resin after concentration, with 60%~70% ethanol elution, the effective portion of total flavonoids from mulberry tree leaf is lyophilized to obtain in concentrate eluant
Position;
(3) it after taking adhesive rehmannia leaf to crush, is extracted with 60~90% alcohol refluxs, filtering or centrifugation take filtrate or supernatant, dense
Contracting freeze-drying, obtains total saponin from leaves of rehmannia active component.
Preferably, the preparation side of the above-described Chinese medicine composition active component with treatment diabetic nephropathy
Method, 10~20 parts of the salvianolic acid effective kind part for taking step (1) to be prepared, total-tanshinone effective kind part 20~30
Part, 10~20 parts of 15~25 parts of mulberry leaf total alkaloid active component, the total flavonoids from mulberry tree leaf effective kind part that step (2) is prepared
20~30 parts of the total saponin from leaves of rehmannia active component being prepared with step (3) is mixed.
Preferably, the preparation side of the above-described Chinese medicine composition active component with treatment diabetic nephropathy
Method, salvianolic acid percentage composition is 4%~8% in Chinese medicine composition active component;Tanshinone component percentage composition is 4%
~6%;Mulberry leaf total alkaloid percentage composition is 3%~5%;Total flavonoids from mulberry tree leaf percentage composition is 6%~8%;Total saponin from leaves of rehmannia
Percentage composition is 5%~10%.
As more preferred scheme, in Chinese medicine composition active component, salvianolic acid content >=4.5%;Tanshinone
Component content >=4.34%;Mulberry leaf total alkaloid content >=5.91%;Total flavonoids from mulberry tree leaf content >=1.61%;Total saponin from leaves of rehmannia contains
Amount >=5.0%.Salvianolic acid, tanshinone, total flavonoids from mulberry tree leaf and total saponin from leaves of rehmannia content high effective liquid chromatography for measuring,
Mulberry leaf total alkaloid is measured using liquid chromatogram and mass spectrometer.
Various clinical pharmaceutical formulations can be made in present composition active component with pharmaceutical necessities appropriate, such as tablet, glue
Wafer, pill, granule, sustained-release and controlled release preparation, oral solution etc..
The Pharmacological experiment result shows that the traditional chinese medicine composition of the invention active component has significant prevention and treatment diabetic nephropathy and prevents
Control the effect of renal cells fibrosis.
Giving the traditional chinese medicine composition of the invention active component stomach-filling to high glucose and high fat adds streptozotocin to cause diabetic nephropathy model
Rat 2 weeks, it was observed to fasting blood-glucose, high-density lipoprotein, low-density lipoprotein, total cholesterol, triglycerides, saccharification serum
The influence of the indexs such as albumen, serum creatinine, urea nitrogen, rat cystatin C, blood β2-microglobulin, Kidney coefficients, Urine proteins, knot
Fruit finds that the Chinese medicine composition active component has following pharmacological action: diabetic nephropathy rats fasting blood-glucose is decreased obviously, with
Model group, which is compared, significant difference (p < 0.05).Diabetic nephropathy rats high-density lipoprotein, low-density lipoprotein, total gallbladder are solid
Alcohol, triglycerides, glycated serum protein, serum creatinine, urea nitrogen, rat cystatin C, blood β2-microglobulin, Kidney coefficients, urine
The content of albumen is decreased obviously, and has significant difference (p < 0.05) compared with model group.Diabetic nephropathy model group rat kidney
Renal tubular epithelial vacuolar degeneration is obvious, and cell infiltration is obvious in official jargon, and the micro- expansion of glomerulus blister cavities, administration group pathological state connects
It is normal group nearly.The traditional chinese medicine composition of the invention active component can significantly be adjusted in people's renal cells of high glucose induction in vitro
E-cadherin,α-SMA,FN,p-ERK,NOX1,NOX2,NOX4,TGF-β,TGF-βRI,TGF-βRII,Smad2,Smad3,
The relative amount of Smad7 albumen has effects that prevent and treat renal cells fibrosis well.
The utility model has the advantages that the Chinese medicine composition active component provided by the invention with treatment diabetic nephropathy is with following excellent
Point:
The Chinese medicine composition active component with treatment diabetic nephropathy provided by the invention, screens medicine by many experiments
Object composition, and by preferred method prepare optimum amount match obtain active component, the experimental results showed that, it is provided by the invention
Chinese medicine composition active component has effects that reduce diabetic nephropathy rats fasting blood-glucose well, and can significantly reduce glycosuria
Sick nephrotic rats high-density lipoprotein, low-density lipoprotein, total cholesterol, triglycerides, glycated serum protein, serum creatinine,
The content of urea nitrogen, rat cystatin C, blood β2-microglobulin, Kidney coefficients and Urine proteins can be played with multiple target point, multipath
The effect for the treatment of diabetic nephropathy.And the experimental results showed that the present invention also has prevention and treatment renal cells fibre well
The effect of dimensionization.In experimentation, obvious toxic-side effects are had no, safety is good.
The preparation method of the Chinese medicine composition active component with treatment diabetic nephropathy provided by the invention, preparation process
Design is reasonable, strong operability, and in the active component being prepared, active component content is high.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies specific material proportion and its result described in example and is merely to illustrate the present invention, without claim should will not be limited
The present invention described in detail in book.The techniques implemented on the basis of the foregoing are all within the scope of the present invention.
Embodiment 1
A kind of preparation method of the Chinese medicine composition active component with treatment diabetic nephropathy comprising following steps:
(1) preparation of salvianolic acid effective kind part: taking red rooted salvia to be ground into coarse granule, is first decocted using 10 times of pure water
Extraction 2 times, each 1.5h, filtering or centrifugation are boiled, aqueous extract is obtained, is concentrated into certain volume, 95% ethyl alcohol is added to ethyl alcohol
Up to 80%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part concentration;
(2) preparation of total-tanshinone effective kind part: Radix Salviae Miltiorrhizae residue of the step (1) after water extracts is taken, is filtered dry, then use
95% ethyl alcohol heating and refluxing extraction 2 times, each 1.5h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry,
Obtain total-tanshinone effective kind part.
(3) with 8 times of pure water refluxing extractions after taking mulberry leaf to crush, 2 the preparation of mulberry leaf total alkaloid effective kind part: are extracted
Secondary, it is quiet to concentration of alcohol up to 80%, 4 DEG C that 95% ethyl alcohol is added in each 1h~2h, filtering or centrifugation, filtrate or supernatant, concentration
It sets for 24 hours, filters to take supernatant, concentrate is slowly added to after concentration with certain flow velocity to the cation exchange resin column installed
In, first with pure water rinsing to neutrality, partial impurities are removed, then washed with the flow velocity of 1.5ml/min with the ammonium hydroxide of 0.5mol/L
It is de-, it is then eluted with the ammonium hydroxide of pH=7~13, collects the eluent of PH=7~10.5, mulberry leaf total alkaloid is lyophilized to obtain in concentration
Active component.
(4) preparation of total flavonoids from mulberry tree leaf effective kind part: taking mulberry leaf to crush, with 80% alcohol reflux extract 2 times, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 2BV/h
The macroporous resin column installed is added, 60%~70% alcohol is used to be eluted with the flow velocity of 3ml/min, collects eluent, it is dense
Contracting, is lyophilized to obtain total flavonoids from mulberry tree leaf effective kind part.
(5) preparation of Radix Rehmanniae total glucosides effective kind part: adhesive rehmannia leaf crush after, with 70% alcohol reflux extract 2 times, every time
1h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia effective kind part is lyophilized to obtain.
Take 15 parts of obtained salvianolic acid effective kind part made above, 25 parts of total-tanshinone effective kind part, mulberry leaf
25 parts of 20 parts of total alkaloid active component, 15 parts of total flavonoids from mulberry tree leaf effective kind part, total saponin from leaves of rehmannia active component combinations obtain.
Through detecting, salvianolic acid content is 6.75% in composition;Total-tanshinone constituents content is 5.42%;Mulberry leaf total alkaloid
Content is 7.88%;Total flavonoids from mulberry tree leaf content is 2.42%;Total saponin from leaves of rehmannia content is 6.25%.
Embodiment 2
A kind of preparation method of the Chinese medicine composition active component with treatment diabetic nephropathy comprising following steps:
(1) preparation of salvianolic acid effective kind part: taking red rooted salvia to be ground into coarse granule, is first decocted using 8 times of pure water
Extraction 2 times, each 1.5h, filtering or centrifugation are boiled, aqueous extract is obtained, is concentrated into certain volume, 95% ethyl alcohol is added to pure and strong
Up to 70%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part degree;
(2) preparation of total-tanshinone effective kind part: the Radix Salviae Miltiorrhizae residue after water of learning from else's experience extraction is filtered dry, then uses 80% second
Alcohol heating and refluxing extraction, 2 times, each 1.5h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry, get Zong Dan
Join ketone active component.
(3) with 10 times of pure water refluxing extractions after taking mulberry leaf to crush, 2 the preparation of mulberry leaf total alkaloid effective kind part: are extracted
Secondary, each 1.5h, filtering or centrifugation, filtrate or supernatant, concentration, 95% ethyl alcohol of addition to determining alcohol are stood up to 60%, 4 DEG C
For 24 hours, supernatant is filtered to take, concentrate is slowly added to after concentration with certain flow velocity to the cation exchange resin column installed
In, first with pure water rinsing to neutrality, partial impurities are removed, then washed with the flow velocity of 1.5ml/min with the ammonium hydroxide of 0.5mol/L
It is de-, eluted with the ammonium hydroxide of pH=7~13, collect the eluent of PH=7~10.5, concentration, be lyophilized mulberry leaf total alkaloid is effective
Position.
(4) preparation of total flavonoids from mulberry tree leaf effective kind part: taking mulberry leaf to crush, with 60% alcohol reflux extract 2 times, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 2BV/h
The macroporous resin column installed is added, 60% ethyl alcohol is used to be eluted with the flow velocity of 2ml/min, collects eluent, is concentrated, freezes
Do to obtain total flavonoids from mulberry tree leaf effective kind part.
(5) preparation of Radix Rehmanniae total glucosides effective kind part: adhesive rehmannia leaf crush after, with 80% alcohol reflux extract 2 times, every time
2h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia effective kind part is lyophilized to obtain.
Take 10 parts of obtained root of red-rooted salvia phenolic acid effective kind part made above, 30 parts of tanshinone active component, mulberry leaf biology
20 parts of alkali active component, 10 parts of flavones in mulberry leaves effective kind part, 30 parts of total saponin from leaves of rehmannia active component.Through detecting, Radix Salviae Miltiorrhizae total phenol
Acid content is 4.5%;Total-tanshinone constituents content is 6.51%;Mulberry leaf total alkaloid content is 5.88%;Total flavonoids from mulberry tree leaf
Content is 1.61%;Total saponin from leaves of rehmannia content is 7.5%.
Embodiment 3
A kind of preparation method of the Chinese medicine composition active component with treatment diabetic nephropathy comprising following steps:
(1) preparation of salvianolic acid effective kind part: taking red rooted salvia to be ground into coarse granule, is first decocted using 12 times of pure water
Extraction 2 times, each 1.5h, filtering or centrifugation are boiled, aqueous extract is obtained, is concentrated into certain volume, 95% ethyl alcohol is added to pure and strong
Up to 80%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part degree.
(2) preparation of total-tanshinone effective kind part: the Radix Salviae Miltiorrhizae residue after water of learning from else's experience extraction is filtered dry, then uses 70% second
Alcohol heating and refluxing extraction, 2 times, each 2h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry, obtains total Radix Salviae Miltiorrhizae
Ketone active component.
(3) with 10 times of pure water refluxing extractions after taking mulberry leaf to crush, 2 the preparation of mulberry leaf total alkaloid effective kind part: are extracted
Secondary, each 1.5h, filtering or centrifugation, filtrate or supernatant, concentration, 95% ethyl alcohol of addition to determining alcohol are stood up to 50%, 4 DEG C
For 24 hours, supernatant is filtered to take, concentrate is slowly added to after concentration with certain flow velocity to the cation exchange resin column installed
In, first with pure water rinsing to neutrality, partial impurities are removed, then washed with the flow velocity of 1.5ml/min with the ammonium hydroxide of 0.5mol/L
It is de-, eluted with the ammonium hydroxide of pH=7~13, collect the eluent of PH=7~10.5, concentration, be lyophilized mulberry leaf total alkaloid is effective
Position.
(4) preparation of total flavonoids from mulberry tree leaf effective kind part: taking mulberry leaf to crush, with 70% alcohol reflux extract 2 times, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 3BV/h
The macroporous resin column installed is added, 70% ethyl alcohol is used to be eluted with the flow velocity of 3ml/min, collects eluent, is concentrated, freezes
Do to obtain total flavonoids from mulberry tree leaf effective kind part.
(5) preparation of Radix Rehmanniae total glucosides effective kind part: adhesive rehmannia leaf crush after, with 60% alcohol reflux extract 2 times, every time
1h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia effective kind part is lyophilized to obtain.
Take 20 parts of obtained root of red-rooted salvia phenolic acid effective kind part made above, 20 parts of tanshinone active component, mulberry leaf biology
20 parts of alkali active component, 20 parts of flavones in mulberry leaves effective kind part, 20 parts of total saponin from leaves of rehmannia active component.Through detecting, Radix Salviae Miltiorrhizae total phenol
Acid content is 9%;Total-tanshinone constituents content is 4.34%;Mulberry leaf total alkaloid content is 7.88%;Total flavonoids from mulberry tree leaf contains
Amount is 3.22%;Total saponin from leaves of rehmannia content is 5%.
Embodiment 4
Reference composition 1: 15 parts of salvianolic acid effective kind part, the total-tanshinone that 1 method of Example is prepared
25 parts of 25 parts of effective kind part, total saponin from leaves of rehmannia active component combinations obtain.
Reference composition 2: 15 parts of salvianolic acid effective kind part, 25 parts of total-tanshinone effective kind part, mulberry leaf are always given birth to
15 parts of 20 parts of alkaloids active component, total flavonoids from mulberry tree leaf effective kind part combinations obtain.
Reference composition 3: 25 parts of total saponin from leaves of rehmannia active component, 20 parts of mulberry leaf total alkaloid active component, mulberry leaf are always yellow
The 15 parts of combinations of ketone active component obtain.
Embodiment 5 is hypoglycemic, reducing blood lipid and protection injury of kidney experiment
1, SD rat, weight (220 ± 20) g are taken, male barrier environment rat (SPF grades) adaptive feeding 5 days, is tested
10 rats of period blank group feed always conventional feed, remaining rat feeds always high glucose and high fat and 0.5% glucose water, and 3
Zhou Hou, by 30mg/kg, intraperitoneal injection STZ, high glucose and high fat and glucose water continue after feeding 4 days for three days on end, and Rat Fast is not
Prohibit water, 12h rear molding venous blood sampling measures fasting blood-glucose, is higher than 13.1mmol/L as modeling success using blood glucose value.Model success rate
It is 95.7%.The successful diabetes rat of modeling is randomly divided into 8 groups according to fasting blood glucose level, every group 10, difference is continuous
15 days stomach-filling Huang Kui capsules (0.75g/kgd), Irbesartan (13.5mg/kgd), 1 sample of embodiment (0.5g/kgd),
2 sample of embodiment (0.5g/kgd), 3 sample of embodiment (0.5g/kgd), 1 sample of reference composition (0.5g/kgd),
2 sample of reference composition (0.5g/kgd) and 3 sample of reference composition (0.5g/kgd).During administration, normal group and mould
0.5% sodium carboxymethylcellulose of type group rat oral gavage same dose.Animal feeding is in 21~23 DEG C of room temperature, opposite during experiment
Humidity 60%, 12h periodicity of illumination SPF grade barrier environment in.After being administered, Rat Fast be can't help into water and be placed in metabolic cage,
Collect urine for 24 hours, for 24 hours when claim rat weight, subsequent tail vein takes hematometry each group rat fasting blood-glucose value, last abdomen master
Arterial blood extracting collects every rat blood serum, and removes rat or so nephridial tissue rapidly and claim its quality, then cuts left kidney portion rapidly
Cortex portion is divided to be located in formalin for HE dyeing, Masson dyeing, PAS dyeing.It is detected with Coomassie brilliant G-250 method
The content of albumen in urinating for 24 hours, it is solid that corresponding kit measures serum middle-high density lipoprotein, low-density lipoprotein, total gallbladder respectively
Alcohol, triglycerides, glycated serum protein, serum creatinine, urea nitrogen, rat cystatin C, blood β2-microglobulin, and calculate kidney
Index (renal index=(left kidney quality+right kidney quality)/(rat weight * 100)).
1. the influence of pair fasting blood-glucose
By tail vein take the pharmaceutical composition in hematometry blank group, model group, positive drug group and embodiment 1,2,3 to
The fasting blood-glucose of medicine group rat, as a result, it has been found that, the sky of 1,2,3 sample administration group rat of positive drug administration group and the embodiment of the present invention
Abdomen blood glucose is significantly lower than model group (p < 0.01) and reference composition 1, reference composition 2 and reference composition 3.As a result such as table
Shown in 1.
Influence of 1 drug of table to diabetic model rats fasting blood-glucose
2. the influence pair glycolipid metabolism related biochemical indicator
Pass through the pharmaceutical composition administration group in kit measurement blank group, model group, positive drug group and embodiment 1,2,3
With rat blood serum middle-high density lipoprotein, low-density lipoprotein, total cholesterol, the triglycerides, saccharification of reference composition 1 to 3
The content of haemocyanin, as a result, it has been found that, the pharmaceutical composition administration group rat blood serum in positive drug administration group and embodiment 1,2,3
Middle-high density lipoprotein, low-density lipoprotein, total cholesterol, triglycerides, glycated serum protein content be significantly lower than mould
Type group (p < 0.01) and reference composition 1 to 3.As shown in table 2.
Influence of 2 drug of table to glycolipid metabolism index in Serum of Diabetes Rats
3. the influence pair renal impairment index of correlation
Pass through the pharmaceutical composition and control in kit measurement blank group, model group, positive drug group and embodiment 1,2,3
The content of serum creatinine, urea nitrogen, rat cystatin C, blood β2-microglobulin in 1 to 3 administration group rat blood serum of composition, with examining
Mas bright blue G-250 method detects the content of albumen in urine for 24 hours, and calculates renal index (renal index=(left kidney quality+right kidney
Quality)/(rat weight * 100)).As a result, it has been found that the pharmaceutical composition in positive drug administration group and embodiment 1,2,3 is administered
Group rat blood serum creatinine, urea nitrogen, rat cystatin C, blood β2-microglobulin, for 24 hours content of Urine proteins and renal index value are equal
Significantly lower than model group (p < 0.01) and reference composition 1 to 3.As shown in table 3.
Influence of 3 drug of table to diabetic model rats renal function index of correlation
4. the influence pair renal pathology state
After administration, each group renal tissues of rats is taken to carry out HE dyeing, PAS dyeing, Masson dyeing, under light microscopic seen in just
Often group rat renal tubule and glomerulus are normal, and model group renal tubular epithelial vacuolar degeneration, a large amount of cell infiltrations of renal interstitial, kidney are small
Balloon cavity is expanded, and the sample administration group rat kidney structure in positive drug administration group and embodiment 1,2,3 is close to normal group.
Embodiment 6 improves the functional experiment of renal tubule fibrosis and to high saccharide ring border servant's renal cells NADPH
Oxidase/ROS/ERK and TGF-β/Smad signal path regulation experiment
1. experimental cell
Renal cells strain HK-2 is provided by Nanjing KaiJi Biology Science Development Co., Ltd, and cell culture is in interior
Contain, cell culture is in the DMEM high glucose medium for+100 μ g/mL streptomysin of fetal calf serum+100U/mL penicillin for including 10%
In, 37 DEG C are placed in, 5%CO2In incubator.
2. drug
Positive drug DPI (nadph oxidase inhibitor), U0126 (ERK inhibitor) and siRNA (NOX1, NOX2, NOX4 suppression
Preparation) it is purchased from Calbiochem Corp. (La Jolla, CA).
3. the grouping and processing of cell
With 96 orifice plate bed board cells, pre-processed with DMEM high glucose medium, 37 DEG C, 5%CO2Middle culture 6h, is at
Synchronized state.It is every to change liquid for 24 hours.Experiment is divided into: blank control is added without intervention factor);High sugar processing group;Component is administered
Not Wei DPI (10 μM), siRNA (10 μM), U0126 (10 μM) and 1,2,3 sample of embodiment (50 μM) administration group and control group 1,
2,3 samples (50 μM).
4. experimental analysis measures E-cadherin, α-SMA, FN, p-ERK, NOX1 using western blot analysis method,
NOX2, NOX4, TGF-β, TGF-β RI, the relative amount of TGF-β RII, Smad2, Smad3, Smad7.
5. experimental result
(1) to the influence of the HK-2 cellular morphology of high glucose induction
Each group HK-2 cell after intervening 48h, the analysis of biological inverted microscope the result shows that, normal HK-2 cell of organizing is presented
There is obvious fibrosis, 1,2,3 sample administration group of positive drug administration group and embodiment in apparent paving stone state, model group cell
Cell and normal group are close.
(2) to HK-2 cell E-cadherin, α-SMA, FN, p-ERK, NOX1, NOX2, NOX4 the albumen table of high glucose induction
Up to the influence of amount
Western blotting measures E-cadherin, α-SMA, FN, p- in each group HK-2 cell of high glucose induction
The expression quantity of ERK, NOX1, NOX2, NOX4 albumen, using β-actin as internal reference, experiment is repeated 3 times.The results are shown in Table 4, positive
α-SMA, FN, p-ERK, NOX1, NOX2, NOX4 albumen is opposite in 1,2,3 pharmaceutical composition administration group cell of medicine and embodiment
Expression quantity is obviously low compared with model group and control group 1,2 and 3, and the relative expression quantity of E-cadherin albumen is obviously compared with model group and right
It is high according to group 1,2 and 3.
4 each group of table is to E-cadherin, the influence of α-SMA, FN, p-ERK, NOX1, NOX2, NOX4 expressing quantity
(3) to the HK-2 cell TGF-β of high glucose induction, TGF-β RI, TGF-β RII, Smad2, Smad3, Smad7 albumen table
Up to the influence of amount
TGF-β in each group HK-2 cell of Western blotting measurement high glucose induction, TGF-β RI, TGF-β RII,
The expression quantity of Smad2, Smad3, Smad7 albumen, using GADPH as internal reference, experiment is repeated 3 times.The results are shown in Table 5, embodiment
1, TGF-β in 2,3 pharmaceutical composition administration group cells, TGF-β RI, the relative expression quantity of TGF-β RII, Smad2, Smad3 albumen
Obviously low compared with model group and control group 1,2 and 3, the relative expression quantity of Smad7 albumen is obviously compared with model group and control group 1,2 and 3
It is high.
5 drug of table is to E-cadherin, the influence of α-SMA, FN, p-ERK, NOX1, NOX2, NOX4 albumen relative expression quantity
The experimental results showed that, the present invention is red with Chinese medicine by the Chinese medicinal effective-part composition that experiment preferably obtains above
Ginseng, mulberry leaf, adhesive rehmannia leaf are raw material, by salvianolic acid class, total-tanshinone class, mulberry leaf total alkaloid, total flavonoids from mulberry tree leaf, adhesive rehmannia leaf
Total glycosides is formed according to certain weight ratio compatibility, the experimental results showed that have significant hypoglycemic, reducing blood lipid, it is anti-oxidant, change
It is apt to blood circle treatment effect, synergistic effect can be played between active component, plays good protection injury of kidney, improves kidney
The effect of tubule fibrosis and prevention and treatment diabetic nephropathy.It can be used as the short-term of diabetes or diabetic complication patient or long-term auxiliary
Help the raw material or product of therapeutic agent.
Claims (3)
1. a kind of active component of the Chinese medicine composition treat with diabetic nephropathy, which is characterized in that it is total by Radix Salviae Miltiorrhizae
15 parts of phenolic acid effective kind part, 25 parts of total-tanshinone effective kind part, 20 parts of mulberry leaf total alkaloid active component, total flavonoids from mulberry tree leaf
15 parts and 25 parts of total saponin from leaves of rehmannia active component of effective kind part are made;
(1) salvianolic acid effective kind part the preparation method comprises the following steps: red rooted salvia is taken to be ground into coarse granule, first use 10 times of pure water
It decocts and extracts 2 times, each 1.5h, filtering or centrifugation obtain aqueous extract, be concentrated into certain volume, and 95% ethyl alcohol is added to ethyl alcohol
Up to 80%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part concentration;
(2) total-tanshinone effective kind part the preparation method comprises the following steps: take Radix Salviae Miltiorrhizae residue of the step (1) after water extracts, be filtered dry, then adopt
With 95% ethyl alcohol heating and refluxing extraction 2 times, each 1.5h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry,
Obtain total-tanshinone effective kind part;
(3) mulberry leaf total alkaloid active component the preparation method comprises the following steps: with 8 times of pure water refluxing extractions after taking mulberry leaf to crush, extraction 2
Secondary, each 1h ~ 2h, filtering or centrifugation, filtrate or supernatant, concentration, 95% ethyl alcohol of addition to concentration of alcohol are stood up to 80%, 4 DEG C
For 24 hours, supernatant is filtered to take, concentrate is slowly added to after concentration with certain flow velocity to the cation exchange resin column installed
In, first with pure water rinsing to neutrality, partial impurities are removed, then washed with the flow velocity of 1.5ml/min with the ammonium hydroxide of 0.5mol/L
It is de-, then eluted with the ammonium hydroxide of pH=7 ~ 13, collect the eluent of pH=7 ~ 10.5, concentration, be lyophilized mulberry leaf total alkaloid is effective
Position;
(4) total flavonoids from mulberry tree leaf effective kind part the preparation method comprises the following steps: mulberry leaf is taken to crush, extract 2 times with 80% alcohol reflux, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 2BV/h
The macroporous resin column installed is added, 60% ~ 70% alcohol is used to be eluted with the flow velocity of 3ml/min, collects eluent, is concentrated,
Total flavonoids from mulberry tree leaf effective kind part is lyophilized to obtain;
(5) total saponin from leaves of rehmannia active component the preparation method comprises the following steps: take adhesive rehmannia leaf crush after, with 70% alcohol reflux extract 2 times, often
Secondary 1h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia active component is lyophilized to obtain.
2. a kind of active component of the Chinese medicine composition treat with diabetic nephropathy, which is characterized in that it is total by Radix Salviae Miltiorrhizae
10 parts of phenolic acid effective kind part, 30 parts of total-tanshinone effective kind part, 20 parts of Folium Mori alkaloid active component, flavones in mulberry leaves class have
10 parts and 30 parts of total saponin from leaves of rehmannia active component of effect position is made;
(1) salvianolic acid effective kind part the preparation method comprises the following steps: red rooted salvia is taken to be ground into coarse granule, first use 8 times of pure water
It decocts and extracts 2 times, each 1.5h, filtering or centrifugation obtain aqueous extract, be concentrated into certain volume, and 95% ethyl alcohol is added to pure and strong
Up to 70%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part degree;
(2) total-tanshinone effective kind part the preparation method comprises the following steps: take Radix Salviae Miltiorrhizae residue of the step (1) after water extracts, be filtered dry, then adopt
With 80% ethyl alcohol heating and refluxing extraction, 2 times, each 1.5h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry
It is dry, obtain total-tanshinone effective kind part;
(3) Folium Mori alkaloid active component the preparation method comprises the following steps: with 10 times of pure water refluxing extractions after taking mulberry leaf to crush, extraction 2 times,
Each 1.5h, filtering or centrifugation, filtrate or supernatant, concentration are added 95% ethyl alcohol and stand for 24 hours to determining alcohol up to 60%, 4 DEG C, mistake
Concentrate is slowly added in the cation exchange resin column installed with certain flow velocity after concentration, is first used by leaching supernatant
Pure water rinsing removes partial impurities to neutrality, then is eluted with the ammonium hydroxide of 0.5mol/L with the flow velocity of 1.5ml/min, with pH=
7 ~ 13 ammonium hydroxide elution, collects the eluent of pH=7 ~ 10.5, and Folium Mori alkaloid active component is lyophilized to obtain in concentration;
(4) flavones in mulberry leaves effective kind part the preparation method comprises the following steps: mulberry leaf is taken to crush, extract 2 times with 60% alcohol reflux, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 2BV/h
The macroporous resin column installed is added, 60% ethyl alcohol is used to be eluted with the flow velocity of 2ml/min, collects eluent, is concentrated, freezes
Do to obtain flavones in mulberry leaves effective kind part;
(5) total saponin from leaves of rehmannia active component the preparation method comprises the following steps: take adhesive rehmannia leaf crush after, with 80% alcohol reflux extract 2 times, often
Secondary 2h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia active component is lyophilized to obtain.
3. a kind of active component of the Chinese medicine composition treat with diabetic nephropathy, which is characterized in that it is total by Radix Salviae Miltiorrhizae
20 parts of phenolic acid effective kind part, 20 parts of total-tanshinone effective kind part, 20 parts of Folium Mori alkaloid active component, flavones in mulberry leaves class have
20 parts and 20 parts of total saponin from leaves of rehmannia active component of effect position is made;
(1) preparation of salvianolic acid effective kind part: taking red rooted salvia to be ground into coarse granule, is first mentioned using 12 times of pure water decoctions
It takes 2 times, each 1.5h, filtering or centrifugation obtain aqueous extract, be concentrated into certain volume, and 95% ethyl alcohol to determining alcohol is added and reaches
80%, filtering precipitating obtains alcoholic supernatant, is concentrated, obtains salvianolic acid effective kind part;
(2) total-tanshinone effective kind part the preparation method comprises the following steps: take Radix Salviae Miltiorrhizae residue of the step (1) after water extracts, be filtered dry, then adopt
With 70% ethyl alcohol heating and refluxing extraction, 2 times, each 2h, filtering or centrifugation, filtrate or supernatant are concentrated under reduced pressure, and alcohol extract is dry,
Obtain total-tanshinone effective kind part;
(3) Folium Mori alkaloid active component the preparation method comprises the following steps: with 10 times of pure water refluxing extractions after taking mulberry leaf to crush, extraction 2 times,
Each 1.5h, filtering or centrifugation, filtrate or supernatant, concentration are added 95% ethyl alcohol and stand for 24 hours to determining alcohol up to 50%, 4 °C, mistake
Concentrate is slowly added in the cation exchange resin column installed with certain flow velocity after concentration, is first used by leaching supernatant
Pure water rinsing removes partial impurities to neutrality, then is eluted with the ammonium hydroxide of 0.5mol/L with the flow velocity of 1.5ml/min, with pH=
7 ~ 13 ammonium hydroxide elution, collects the eluent of pH=7 ~ 10.5, and Folium Mori alkaloid active component is lyophilized to obtain in concentration;
(4) flavones in mulberry leaves effective kind part the preparation method comprises the following steps: mulberry leaf is taken to crush, extract 2 times with 70% alcohol reflux, every time
45min, twice extracting solution filtering or centrifugation, take filtrate or supernatant, are concentrated into no alcohol taste, by concentrate with the flow velocity of 3BV/h
The macroporous resin column installed is added, 70% ethyl alcohol is used to be eluted with the flow velocity of 3ml/min, collects eluent, is concentrated, freezes
Do to obtain flavones in mulberry leaves effective kind part;
(5) total saponin from leaves of rehmannia active component the preparation method comprises the following steps: take adhesive rehmannia leaf crush after, with 60% alcohol reflux extract 2 times, often
Secondary 1h, filtering or centrifugation, take filtrate or supernatant, are concentrated, total saponin from leaves of rehmannia active component is lyophilized to obtain.
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CN1541687A (en) * | 2003-11-07 | 2004-11-03 | 天津中医学院 | Prepared traditional Chinese medicine for treating diabetes |
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CN104666585A (en) * | 2015-03-16 | 2015-06-03 | 河南中医学院 | Application of radix rehmanniae leaf extract in preparation of blood glucose-reducing medicines |
CN105106578A (en) * | 2015-10-10 | 2015-12-02 | 临沂草之美医药科技有限公司 | Traditional Chinese medicine composition for treating diabetic nephropathy and preparation method of traditional Chinese medicine composition |
CN105213776A (en) * | 2015-11-09 | 2016-01-06 | 广东聚智诚科技有限公司 | A kind of Chinese medicine composition for the treatment of diabetic nephropathy and preparation method thereof |
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Patent Citations (5)
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CN1541687A (en) * | 2003-11-07 | 2004-11-03 | 天津中医学院 | Prepared traditional Chinese medicine for treating diabetes |
CN102630790A (en) * | 2012-05-08 | 2012-08-15 | 四川九通慧医药信息科技有限公司 | Tartary buckwheat tea prepared by Ginkgo biloba leaf and preparation process thereof |
CN104666585A (en) * | 2015-03-16 | 2015-06-03 | 河南中医学院 | Application of radix rehmanniae leaf extract in preparation of blood glucose-reducing medicines |
CN105106578A (en) * | 2015-10-10 | 2015-12-02 | 临沂草之美医药科技有限公司 | Traditional Chinese medicine composition for treating diabetic nephropathy and preparation method of traditional Chinese medicine composition |
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