CN108938709A - A kind of cigarette beans water extract and application thereof - Google Patents
A kind of cigarette beans water extract and application thereof Download PDFInfo
- Publication number
- CN108938709A CN108938709A CN201710386666.9A CN201710386666A CN108938709A CN 108938709 A CN108938709 A CN 108938709A CN 201710386666 A CN201710386666 A CN 201710386666A CN 108938709 A CN108938709 A CN 108938709A
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- Prior art keywords
- drug
- cigarette beans
- cigarette
- group
- nephrotic syndrome
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- A—HUMAN NECESSITIES
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Abstract
The present invention provides the purposes of cigarette beans Glycine tabacina (Labill.) Benth and its extract in the drug of preparation treatment nephrotic syndrome.The present invention provides cigarette beans water extracts (Glycine tabacina aqueous extract, GTE) to prepare the application in anti-nephrotic syndrome natural drug, is applied to clinical treatment nephrotic syndrome for cigarette beans and provides foundation.
Description
Technical field
The present invention relates to cigarette beans water extracts and application thereof, specifically in the drug of preparation treatment nephrotic syndrome
Purposes.
Background technique
Nephrotic syndrome (nephrotic syndrome) is the Glomerular lesions as caused by Different types of etiopathogenises and makes glomerulus
Basement membrane permeability increases, and showing as High-grade Proteinuria, Hypoproteinemia, treating serious edema caused, hyperlipidemia and other metabolic disorders is
One group of clinical syndrome of feature.It is divided into primary nephrotic syndrome and secondary nephrotic syndrome according to pathogenic factor, the former
Account for 90%, the disease incidence in China is 2.1%~8.8%, can be complicated by infection, protein and fat metabolic disturbance, embolism, acute kidney
The severe complications such as functional failure.Glucocorticoid, cytotoxic drug and immunosuppressant treatment are clinically commonly used, but long
Phase can cause a series of side effects using these drugs, such as infection, diabetes, hypertension, osteoporosis, artery sclerosis, marrow
Inhibition, hepar damnification, induced tumor etc..Therefore, the new medical instrument of the treatment nephrotic syndrome of new and effective low toxicity is researched and developed
There is important value.
Cigarette beans (Glycine tabacina (Labill.) Benth) are pulse family (Leguminosae) Glycines
(Glycine) perennial grass plant (as shown in the figure), also known as Penghu soybean, one, Penghu root, one root of sharp leaf, come into leaves silver-colored beans
Deng being distributed in Australia, the South Sea Islands and coastal area of southeastern China and Taiwan etc..Cigarette beans contain isoflavones abundant,
Including isoflavone aglycone, genistin, daidzein and genistein.Its rhizome have wind-damp dispelling, strengthen the bone, beneficial spleen kidney the effect of,
It is civil to be used to treat the illness such as treating rheumatic ostealgia, deficiency of vital energy wind-puff, soreness and weakness of waist and knees.But have no the pharmacological activity of reported in literature cigarette beans.
Summary of the invention
The technical solution of the present invention is to provide a kind of cigarette beans and its water extracts；Another technical solution of the invention is to mention
The purposes of the water extract is supplied.
The present invention provides a kind of cigarette beans Glycine tabacina (Labill.) Benth water extracts, it is cigarette beans
80-100 DEG C of water extract.
Wherein, the cigarette beans derive from root, stem, leaf or the complete stool of cigarette beans.
Wherein, the extract is to extract to obtain by following methods: cigarette beans are taken, by 1:10 solid-liquid ratio, add distilled water, in
Heating and refluxing extraction 1h at 80-100 DEG C, filtered on buchner funnel obtains filtrate, after concentrated, is freeze-dried to obtain water extract dry powder.
It is further preferred that the Extracting temperature is 80 DEG C.
The HPLC spectrogram of cigarette beans water extract of the present invention is as shown in Figure 1, chromatographic condition are as follows: chromatographic column Cosmosil
5C18-MS-II (4.6mm I.D. × 250mm) chromatographic column, 25 DEG C of column temperature, Detection wavelength 254nm；Eluent gradient elution requirement
Are as follows: 0-25min, 10-50%MeOH, 90-50%H2O；25-30min, 50-100%MeOH, 50-0%H2O；30-40min,
100%MeOH, 0%H2O。
The present invention provides cigarette beans Glycine tabacina (Labill.) Benth or the cigarette beans water extract to exist
Purposes in the drug of preparation treatment nephrotic syndrome.
Wherein, the drug is the drug for treating high protein urine disease.
Wherein, the drug is the drug for reducing nephrotic syndrome glomerular injury.
Wherein, the drug is the drug for improving nephrotic syndrome renal function.
Wherein, the drug is the drug for treating hyperlipidemia.
Wherein, the drug is the drug for improving oxidation resistance or inhibiting nephrotic syndrome response to oxidative stress.
The present invention provides cigarette beans water extracts (Glycine tabacina aqueous extract, GTE) to prepare
Application in anti-nephrotic syndrome natural drug is applied to clinical treatment nephrotic syndrome for cigarette beans and provides foundation.
The experimental results showed that GTE significantly reduces the nephrotic syndrome Balb/c mouse retention total protein and urine of adriamycin induction
The discharge rate of albumin.Glomerulus PAS dyeing observation and appraisal result show GTE treatment group murine glomerular and Renal vascular mesentery
Region PAS positive material substantially reduces, and mesangial matrix * is effectively controlled.Meanwhile the plasma urea nitrogen of GTE treatment group mouse and
Creatinine concentration of plasma is substantially reduced after modeling 2 weeks compared to model group.These the result shows that cigarette beans extract have significantly control
The effect for the treatment of nephrotic syndrome.GTE can also be substantially reduced mRNA IN ADRIAMYCIN NEPHROPATHY syndrome mice plasma cholesterol and triglycerides
It increases, prompts cigarette beans extract that can significantly improve the generation of patients of nephrotic syndrome with hyperlipidemia.Mechanism Study shows that GTE treatment is obvious
The superoxide dismutase (Superoxide Dismutase, SOD) increased in nephrotic syndrome mice plasma and tissue is living
Power and its malonaldehyde (malonaldehyde, MDA) content is reduced, while to the nephrotic syndrome Mouse Kidney of adriamycin induction
Increasing for ROS content is obviously improved in tissue, prompts GTE by improving oxidation resistance and inhibiting response to oxidative stress
And have the function that anti-nephrotic syndrome.Cigarette beans water extract of the present invention, which has, is developed into the latent of anti-nephrotic syndrome drug
Power has wide development and application prospect.
Detailed description of the invention
Extractive HPLC map under Fig. 1 different temperatures extraction conditions is (upper: 90 DEG C of extracts；In: 100 DEG C of extracts；
Under: 80 DEG C of extracts.Eluent gradient elution requirement are as follows: 0-25min, 10-50%MeOH, 90-50%H2O；25-30min,
50-100%MeOH, 50-0%H2O；30-40min, 100%MeOH, 0%H2O.Detection wavelength 254nm.)
Fig. 2 cigarette beans extract significantly mitigate nephrotic syndrome high protein urine disease (the 0th after adriamycin modeling, 4,7,11,18,
It 25 days, collects each group mouse twenty-four-hour urine liquid and eye socket takes a blood sample, isolates blood plasma, detect mouse retention total protein (A) and the white egg of urine
The content of white (B).Experimental result Mean ± SEM expression, n=6.* p < 0.01 p < 0.05, * *, compared with the control group；#p<
0.05, ##P < 0.01, compared with model group.)
Fig. 3 cigarette beans extract substantially reduces nephrotic syndrome glomerular injury, and ((A) each group mouse kidney tissue is sliced
PAS dyeing.(B) result of each group mouse PAS dyeing scoring.Experimental result Mean ± SEM expression, n=6.* p < 0.05, * * p
< 0.01, compared with the control group；#p < 0.05, ##P < 0.01, compared with model group.)
Fig. 4 cigarette beans extract significantly increases nephrotic syndrome renal function (experimental result Mean ± SEM expression, n=6.*
P < 0.01 p < 0.05, * *, compared with the control group；#p < 0.05, ##P < 0.01, compared with model group.)
Fig. 5 cigarette beans extract significantly reduces patients of nephrotic syndrome with hyperlipidemia (experimental result Mean ± SEM expression, n=
6.* p < 0.01 p < 0.05, * *, compared with the control group；#p < 0.05, ##P < 0.01, compared with model group.)
Fig. 6 cigarette beans extract significantly improves oxidation resistance and inhibits nephrotic syndrome response to oxidative stress (modeling 25 days
After put to death mouse, isolate kidney and blood plasma, measure SOD vigor, MDA content and ROS content respectively.Experimental result with Mean ±
SEM expression, n=6.* p < 0.01 p < 0.05, * *, compared with the control group；#p < 0.05, ##P < 0.01, compared with model group.)
Specific embodiment
The present invention is further elaborated below with reference to specific embodiments and the drawings, these examples are merely to illustrate mesh
, rather than the limitation scope of the invention.
Raw material, equipment used in the specific embodiment of the invention are known product, are obtained by purchase commercial product.
Cigarette beans used root is purchased from FuQing, Fujian Province city in present invention experiment, by DNA bar code plant identification kind, by
It completes in China Medical Sciences Academy Medical Plants Institute doctor Han Jianping laboratory.
The preparation and characterization of the cigarette beans extract of the present invention of embodiment 1
The crushing of cigarette beans root is placed in extraction flask, by 1:10 solid-liquid ratio, is added distilled water, is heated to reflux at 80 DEG C -100 DEG C
1h is extracted, filtered on buchner funnel obtains filtrate, after concentrated, is freeze-dried to get cigarette beans extract dry powder of the present invention.Difference is extracted
Under the conditions of recovery rate be shown in Table 1.
The recovery rate of extract under 1 different temperatures extraction conditions of table
| Extracting temperature | Recovery rate (mg/g dw) |
| 80℃ | 135.0 |
| 90℃ | 137.5 |
| 100℃ | 145.5 |
Above-mentioned 9 extracts are detected using HPLC, HPLC condition: being detected using HPLC, eluent gradient elution requirement are as follows:
0-25min, 10-50%MeOH, 90-50%H2O；25-30min, 50-100%MeOH, 50-0%H2O；30-40min, 100%
MeOH, 0%H2O.Detection wavelength 254nm.As shown in Figure 1, being extracted within the temperature range of the preparation of provided cigarette beans extract
The ingredient height of object is similar, shows in foregoing Extracting temperature, can be prepared into cigarette beans extract of the present invention.
Further, cigarette beans root crushing is placed in extraction flask, by 1:10 solid-liquid ratio, is added distilled water, is heated at 80 DEG C
Refluxing extraction 1h, filtered on buchner funnel obtains filtrate, after concentrated, is freeze-dried to get cigarette beans extract dry powder of the present invention.
Above-mentioned water extract is dispensed, is saved in -80 DEG C of refrigerators.
Beneficial effects of the present invention are proved below by way of effect experiment.
1 cigarette beans extract of experimental example significantly mitigates nephrotic syndrome high protein urine disease
Experimental design
Mouse is randomly divided into 5 groups before experiment: control group, model group, 2.5g/kg GTE group, 5g/kg GTE group and the positive
Medicine benazepil group, 6/group, totally 30.Each group mouse adaptive feeding 3 days.Model group, 2.5g/kg GTE group, 5g/kg
GTE group (being prepared by embodiment 1) and positive drug benazepil group (5mg/kg) mouse through tail vein single injection 12mg/kg Ah
Mycin induced renal disease syndrome.The control group mice physiological saline isometric through tail vein single injection.Mouse is made through adriamycin
0th day beginning gastric infusion after mould, 1 time a day, each every 250 μ l of mouse, until experiment terminates.The 0th after modeling, 4,7,
11,18,25 days collection each group mouse twenty-four-hour urine liquid, detection urine total protein, urinary albumin and concentration of urinary creatinine.
Experimental method
30 female SPF Balb/c mouse (20-25g) are purchased from health science institute of University of Macao Experimental Animal Center
(Animal Experimental Ethical credit number: UMAEC-0211-2015).Zoopery is entrusted in strict accordance with University of Macao's experimental animal ethics
The requirement of member's meeting carries out.Experiment is purchased from FuQing, Fujian Province city with cigarette beanstalk leaf, through DNA bar code plant identification kind, in
It completes in Academy of Medical Sciences Institute of Medical Plants of state doctor Han Jianping laboratory.90 DEG C of hot water extract 30 points after cigarette beanstalk leaf crushes
Clock, Rotary Evaporators concentration, is settled to every milliliter of crude drug containing 2g for water extract, dispenses, and saves in -80 DEG C of refrigerators.Positive drug salt
Benazepril Hydrochloride is purchased from U.S. Sigma-Aldrich, with the benazepil stock solution of 0.9% normal saline 2mg/ml,
It dispenses, is saved in -80 DEG C of refrigerators.Adriamycin is purchased from Aladdin Reagent Company (Chinese Shanghai), with 0.9% normal saline dilution
At the solution of 2mg/ml, packing, -80 DEG C of preservations.BCA protein determination kit is purchased from Thermo company of the U.S..Albumin and flesh
Acid anhydride assay kit is purchased from Shanghai Foxing Changzheng medical science Co., Ltd.
Urinate total protein content measurement: each group mouse twenty-four-hour urine total protein concentration is detected using bicinchoninic acid (BCA) method.
Peptide bond in protein molecule under alkaline condition can be with Cu2+Complexing, and by Cu2+It is reduced into Cu+.BCA and its sodium salt are a kind of
Water soluble compound, under alkaline condition can be with Cu+In conjunction with generation darkviolet compound, the depth and protein of compound colors
Concentration it is proportional, the compound at 562nm wavelength have strong absworption peak, the content of protein can be determined with colorimetric method.This
Total protein content measurement result is urinated in experiment to be indicated with the ratio for urinating total protein concentration (mg/ml) and concentration of urinary creatinine (mg/ml).
Urinary albumin content measurement: mouse twenty-four-hour urine albumin concentration is detected using Bromocresol green.Due in acidity
Under the conditions of, albumin forms blue-green complex compound in conjunction with bromocresol green.React albumin in the complex compound formed and urine sample
Concentration is directly proportional.The complex compound has strong absworption peak at 630nm, can be used for detecting the concentration of albumin in urine sample.This experiment
Standard curve is done for standard items with bovine serum albumin(BSA) (2mg/ml) and is quantified.Urinary albumin content measurement result is with urinary albumin
The ratio of concentration (mg/ml) and concentration of urinary creatinine (mg/ml) indicate.
Concentration of urinary creatinine measurement: mouse twenty-four-hour urine creatine concentration is detected using picric acid method.Since creatinine can be with alkalinity
Picric acid reaction generate red Janovski compound.Creatinine is dense in the red complex and sample formed in reaction process
It spends directly proportional.The compound has strong absworption peak at 510nm wavelength, can be used for detecting the concentration of creatinine in urine.This experiment
Standard curve is done with creatinine standard items (2mg/ml) and is quantified.
Experimental result
High protein urine is one of notable feature of nephrotic syndrome, wherein mainly albumin.Adriamycin model group and not
With the discharge rate of concentration GTE treatment group mouse retention total protein (Fig. 2A) and urinary albumin (Fig. 2 B) after modeling 1 week progressive liter
Height, and control group significant difference (p<0.05), and positive drug group and control group ratio are without significant difference (p>0.05).It is controlling
After treating 11 days, compared with model group, the discharge rate of mouse retention total protein and urinary albumin is equal for GTE various concentration group and positive drug group
There is different degrees of reduction, difference is statistically significant (p < 0.05).These as the result is shown cigarette beans extract can be relieved Ah
The high protein of the Balb/c mouse nephrotic syndrome of mycin induction urinates symptom.
2 cigarette beans extract of experimental example substantially reduces nephrotic syndrome glomerular injury
Experimental design
Mouse is randomly divided into 5 groups before experiment: control group, model group, 2.5g/kg GTE group, 5g/kg GTE group are (by reality
Apply the preparation of example 1) and positive drug benazepil group, 6/group, totally 30.Each group mouse adaptive feeding 3 days.Model group, 2.5g/
Kg GTE group, 5g/kg GTE group and positive drug benazepil group (5mg/kg) mouse through tail vein single injection 12mg/kg Ah
Mycin induced renal disease syndrome.The control group mice physiological saline isometric through tail vein single injection.Mouse is made through adriamycin
0th day beginning gastric infusion after mould, 1 time a day, each every 250 μ l of mouse, until experiment terminates.The 25th day after modeling, place
Dead mouse simultaneously isolates kidney, is fixed and is stayed overnight with Fu Ermalin, does paraffin section and PAS dyeing, observes between glomerulus and kidney
The structure of matter changes and scores.
Experimental method
Periodic acid-Xi Fushi (Periodic acid-Schiff stain, PAS) decoration method can sufficiently be shown in tissue
Glycoprotein components are usually used in specific observations glomerular capillary basement membrane and extracellular matrix.PAS dyeing after, glomerulus and
Renal tubular basement membrane, collagenous fibres, protein cast are in aubergine.Experiment is Nanchang rain with 10% neutral formalin fixer
Reveal the production of experiment equipment Co., Ltd.Solid paraffin is purchased from Shanghai Yi Yang Instrument Ltd..Glycogen PAS staining kit is purchased from
Beijing Regen Biotech Inc..After modeling 25 days, puts to death mouse and isolate kidney and be placed in 10% formalin solution
Middle fixation is stayed overnight, and then, takes 0.3cm3The lipid peroxicition of size, step by step dehydration of alcohol, dimethylbenzene permeabilization, paraffin embedding.Make
Nephridial tissue is cut into 4 μm of slabs with paraffin slicing machine, is then dewaxed, PAS dyeing is done, random selection at least 50 kidneys are small
Ball, the structure for observing glomerulus and renal interstitial change and score.Glomerular epithelial cell scores (0-3), and 0 point: normal；1 point: slightly weak to arrive
Weak performance shows the expansion of faint proliferation of glomerular mesangial cells and Glomerular mesangium component *；2 points: heavier performance,
The expansion of apparent proliferation of glomerular mesangial cells and extracellular matrix；3 points: there is obvious leaflet structure in glomerulus and glomerulus is glutinous
In Bowman's capsule inner surface.
Experimental result
In order to further look at whether cigarette beans water extract mitigates damage of the nephrotic syndrome of adriamycin induction to glomerulus,
PAS dyeing has been carried out to each group mouse kidney.As the result is shown (Fig. 3 A): Normal group Renal Cortex area's glomerulus and renal tubule
Clear in structure, glomerulus and renal cells marshalling, basement membrane structure are complete.Model group murine glomerular cortical area
Interstitial blood vessel is largely expanded, congested, and capillary loops opening is bad, it is seen that apparent glomerulus sectional type leaflet structure, kidney are small
, there are a large amount of PAS stained positive substances in ball capillary and epithelial cell basilar memebrane irregular thickening.GTE and positive drug shellfish that
Puli acts on the pathological change that mRNA IN ADRIAMYCIN NEPHROPATHY syndrome mouse has been relieved kidney cortical area, after mouse gives GTE treatment,
Glomerular mesangium expansion is reduced, and glomerulus capillary epithelium cell Proliferation is reduced, and glomerular basement membrane substantially returns to normal water
Flat, glomerulus and tubule PAS stained positive substance significantly reduce.The treatment of positive drug benazepil alleviates the expansion of glomerular mesangium area
It opens and reduces capillary epithelial cell proliferation, however glomerulus blister cavities basilar memebrane shows irregular thickening, Bowman's capsule
The visible slight protein sample deposition of chamber, glomerulus and renal tubule mesangial region PAS stained positive substance significantly reduce.
3 cigarette beans extract of experimental example significantly improves nephrotic syndrome renal function
Experimental design
Mouse is randomly divided into 5 groups before experiment: control group, model group, 2.5g/kg GTE group, 5g/kg GTE group are (by reality
Apply the preparation of example 1) and positive drug benazepil group, 6/group, totally 30.Each group mouse adaptive feeding 3 days.Model group, 2.5g/
Kg GTE group, 5g/kg GTE group and positive drug benazepil group (5mg/kg) mouse are through tail vein single injection adriamycin
(12mg/kg) induced renal disease syndrome.The control group mice physiological saline isometric through tail vein single injection.Mouse is from modeling
Start within the 0th day daily continuous gavage administration, each every 250 μ l of mouse, until experiment terminates afterwards.The 0th after modeling, 7,14,
25 days, each group mouse isolated blood plasma through eye socket blood sampling, while detecting plasma urea nitrogen and serum creatinine concentration.
Experimental method
Urea and creatinine assay kit are purchased from Shanghai Foxing Changzheng medical science Co., Ltd.Mouse the 0th after modeling,
Blood plasma is isolated through eye socket blood sampling within 7,14,25 days, detect the concentration of plasma urea nitrogen and plasma creatinine.
Plasma urea nitrogen concentration mensuration: mice plasma urea nitrogen concentration is detected using urease Source of UV Rate Method.Urea warp
The effect of urease generates glutamic acid and NAD+, NAD+Generating rate it is directly proportional to the concentration of urea nitrogen in sample.By
The fall off rate that the brightness of Fixed Time Interval interior suction is measured at 340nm, measures the concentration of urea nitrogen in sample.In this experiment use
The urea nitrogen that state's food and medicine examines and determine research institute's production is urea nitrogen standard items.Plasma urea nitrogen concentration calculation formula are as follows:
Serum creatinine concentration mensuration: mouse serum creatinine method for measurement of concentration is measured with concentration of urinary creatinine.
Experimental result
Urea nitrogen and creatinine are the metabolites of protein and itrogenous organic substance.In normal renal function, these are small
Molecular substance is filtered out from glomerulus and is excluded in vitro, therefore can be used as the diagnosis pointer of detection of glomeruli filtration function.Work as glomerular filtration
When function lowers, serum creatinine and blood urea nitrogen can increase because of retention.Compared with the control group, model group mice plasma urea nitrogen
And the concentration of plasma creatinine significantly increases (p < 0.05), and the 14th day after modeling reaches peak value, then begin to reduce (Fig. 4 A and
Fig. 4 B).2.5g/kg and 5g/kg GTE and positive drug benazepil significantly reduce adriamycin induction nephrosis after treating 7 days is comprehensive
Simulator sickness mice plasma urea nitrogen and serum creatinine concentration (p < 0.05).These significantly improve Ah mould the result shows that cigarette beans extract has
The effect of plain nephrotic syndrome Balb/c mouse renal function.
4 cigarette beans extract of experimental example significantly reduces patients of nephrotic syndrome with hyperlipidemia
Experimental design
Mouse is randomly divided into 5 groups before experiment: control group, model group, 2.5g/kg GTE group, 5g/kg GTE group are (by reality
Apply the preparation of example 1) and positive drug benazepil group, 6/group, totally 30.Model group, 2.5g/kg GTE group, 5g/kg GTE group and
Positive drug benazepil group (5mg/kg) mouse is through tail vein single injection adriamycin (12mg/kg) induced renal disease syndrome.
The control group mice physiological saline isometric through tail vein single injection.Mouse daily continuous gavage the 0th day after modeling
Administration, each every 250 μ l of mouse, until experiment terminates.The 0th, 7,14,25 day after modeling, mouse takes a blood sample and divides through eye socket
Blood plasma is separated out, the concentration of cholesterol and triglycerides in blood plasma is detected.
Experimental method
Cholesterol and triglyceride determination kit are purchased from Shanghai Foxing Changzheng medical science Co., Ltd.Each group mouse exists
Blood plasma is isolated through eye socket blood sampling within the 0th, 7,14,25 day after modeling, detect the concentration of plasma cholesterol and triglycerides.
Plasma cholesterol concentration measurement: mice plasma cholesterol concentration is detected using cholesterol oxidation enzyme process.Cholesterol can
Hydrogen peroxide is generated by cholesterol oxidation oxydasis, it is sub- that the latter reacts the red quinone of generation with P-hydroxybenzoic acid and amino-antipyrine
Aminochrome.The production quantity of imines pigment is directly proportional to the concentration of cholesterol in sample, inhales brightness by measuring at 500nm wavelength
The concentration of cholesterol in sample can be obtained in value.This experiment is done standard curve with cholesterol standards (2mg/ml) and is quantified.
Plasma triglyceride concentration measurement: mice plasma triglyceride concentration is detected using enzyme process.Triglycerides can be sweet
Oleophosphoric acid oxydasis is phosphoric acid dihydroxyacetone (DHA) and hydrogen peroxide.The latter reacts with phenolsulfonate and amino-antipyrine
Generate red quinone imines pigment.The production quantity of imines pigment is directly proportional to the concentration of triglycerides in sample, by 520nm
Brightness value is inhaled in measurement can be obtained the concentration of triglycerides in sample.This experiment does standard song with triglycerides standard items (2mg/ml)
Line is simultaneously quantitative.
Experimental result
Hyperlipidemia is that the raising of another notable feature of nephrotic syndrome, wherein cholesterol and triglycerides is most obvious.Such as
Shown in Fig. 5 A, compared with the control group, model group mice plasma triglycerides and cholesterol concentration are significant after modeling 7 days and 14 days
It increases (p < 0.05).Compared with model group, GTE and positive drug treatment group plasma triglyceride and cholesterol concentration are significantly reduced
(p<0.05).These are the result shows that cigarette beans extract can be obviously improved mRNA IN ADRIAMYCIN NEPHROPATHY syndrome induced Hyperlipidemia in Mice.
5 cigarette beans extract of experimental example significantly improves oxidation resistance and inhibits nephrotic syndrome response to oxidative stress
Experimental design
Mouse is randomly divided into 5 groups before experiment: control group, model group, 2.5g/kg GTE group, 5g/kg GTE group are (by reality
Apply the preparation of example 1) and positive drug benazepil group, 6/group, totally 30.Model group, 2.5g/kg GTE group, 5g/kg GTE group and
Positive drug benazepil group (5mg/kg) mouse is through tail vein single injection adriamycin (12mg/kg) induced renal disease syndrome.
The control group mice physiological saline isometric through tail vein single injection.Mouse daily continuous gavage the 0th day after modeling
Administration, each every 250 μ l of mouse, until experiment terminates.The 25th day execution mouse and blood plasma and kidney group are isolated after modeling
It knits, builds up total number born (SOD) kit of company using Nanjing and malonaldehyde (MDA) assay kit measures respectively
Group mice plasma and SOD vigor and MDA content in nephridial tissue.Meanwhile startability oxygen is answered using the oxidation of GENMED animal tissue
Luminol chemiluminescence standard measure detection kit (GENMED Scientifics, USA) measures the active oxygen in nephridial tissue certainly
By base (ROS) content.
Experimental method
Total number born (SOD) and malonaldehyde (MDA) assay kit are purchased from Nanjing and build up company.GENMED is dynamic
Object tissue oxidizing stress active oxygen luminol chemiluminescence standard measure detection kit be purchased from U.S. GENMED Scientifics
Company.Experiment last day puts to death mouse and isolates blood plasma and nephridial tissue, and measures the SOD vigor and MDA in blood plasma and tissue
Content.The ROS content in mouse tissue is detected simultaneously.
SOD vigor is detected using xanthine oxidase in SOD vitality test each group mice plasma and nephridial tissue.Xanthine
And xanthine oxidase reaction system generates ultra-oxygen anion free radical, the latter aoxidizes azanol and forms nitrite, in color developing agent
Effect is lower to be presented aubergine, and the production quantity of nitrite is directly proportional to SOD vigor in sample, has strong inhale at 550nm wavelength
Receive peak.SOD vigor calculation formula in mice plasma and tissue:
MDA content is detected using malonaldehyde method in MDA assay mice plasma and tissue.Lipid peroxide catabolite
In malonaldehyde can be condensed with thiobarbituricacidα-, form red product, the content of MDA in the production quantity and sample of red product
It is directly proportional, there is maximum absorption band at 532nm wavelength.MDA content calculation formula in mice plasma and tissue are as follows:
ROS assay kidney of mouse ROS content is organized to examine using luminol chemiluminescence method and by product description
It surveys.Luminol is also known as amino phthalyl hydrazine, is a kind of fat-soluble luminous agent, after oxidation generate luminol free radical and it is chemical
It shines, the band-like wide spectrum with 460nm or so peak value is visually observed distinct hyacinthine.Luminol aoxidizes shape with unit price
Formula is reacted with Superoxide radicalanion.It as probe can detect histocyte in hydrogen peroxide, superoxide anion and hydroxyl from
By base.Kidney of mouse ROS calculation formula are as follows:
Experimental result
SOD plays a crucial role the oxidation of body and anti-oxidant balance, can remove ultra-oxygen anion free radical,
Protect cells from damage.And the MDA severity that then indirect reaction body cell is attacked by oxygen radical.Such as Fig. 6 A and 6C
Shown, after modeling 25 days, the SOD vigor in model group mice plasma and tissue is substantially less than control group (p < 0.05), and its MDA
Content (Fig. 6 B and 6D) is then higher than control group (p < 0.05).Give the GTE treatment apparent increase of 2.5g/kg and 5g/kg nephrosis
Syndrome mice plasma and tissue in SOD vigor (p < 0.05) and reduce its MDA content (p < 0.05), alleviate adriamycin
Kidney oxidative damage during the nephrotic syndrome of induction.Contain in addition, GTE treatment group is substantially reduced ROS in kidney of mouse
It measures (Fig. 5 E).These are the result shows that GTE significantly enhances the oxidation resistance of renal tissue, it is suppressed that mRNA IN ADRIAMYCIN NEPHROPATHY syndrome
Oxidative stress, prompt cigarette beans extract by enhancing kidney oxidation resistance achieve the purpose that treatment nephrotic syndrome.
Statistical analysis
Experimental data is indicated with Mean ± SEM.Data analysis is carried out using SPSS13.0 statistical software, wherein mouse retention egg
The data such as white discharge rate, plasma creatinine and urea nitrogen concentration, plasma cholesterol and triglyceride concentration carry out Two-way ANOVA
Variance analysis, comparison among groups use Bonferroni method.Kidney PAS dyeing scoring, blood plasma and nephridial tissue SOD vigor and MDA content,
And the One-way ANOVA variance analysis of the data such as nephridial tissue ROS content, comparison among groups use Tukey method.P < 0.05 is difference
It is statistically significant.
Brief summary
Present invention firstly discovers that cigarette beans extract has the function of significant anti-nephrotic syndrome, show: (1) significant to mitigate
Glomerular injury；(2) urine total protein and urinary albumin content are reduced；(3) plasma creatinine and urea nitrogen content are reduced；(4) it reduces
Plasma cholesterol and content of triglyceride.Also, cigarette beans extract is by improving oxidation resistance and inhibiting response to oxidative stress
And play the effect of anti-nephrotic syndrome.It is the height of anti-nephrotic syndrome that the present invention, which prompts cigarette beans extract to have potential exploitation,
Less toxic drug is imitated, there is extensive clinical value.
Claims (11)
1. a kind of cigarette beans Glycine tabacina (Labill.) Benth water extract, it is characterised in that: it is the 80- of cigarette beans
100 DEG C of water extracts.
2. cigarette beans water extract according to claim 1, it is characterised in that: the cigarette beans derive from cigarette beans Glycine
Root, stem, leaf or the complete stool of tabacina (Labill.) Benth.
3. cigarette beans water extract according to claim 1 or 2, it is characterised in that: the extract is by following methods
Extraction obtains: cigarette beans taken, by 1:10 solid-liquid ratio, add distilled water, heating and refluxing extraction 1h, filtered on buchner funnel at 80-100 DEG C
Filtrate is obtained, after concentrated, is freeze-dried to obtain water extract dry powder.
4. cigarette beans water extract according to claim 3, it is characterised in that: the Extracting temperature is 80 DEG C.
5. cigarette beans water extract according to any one of claims 1-4, it is characterised in that: the HPLC spectrogram is such as
Shown in Fig. 1, chromatographic condition are as follows:
Chromatographic column be Cosmosil 5C18-MS-II (4.6mm I.D. × 250mm) chromatographic column, 25 DEG C of column temperature, Detection wavelength
254nm；Eluent gradient elution requirement are as follows: 0-25min, 10-50%MeOH, 90-50%H2O；25-30min, 50-100%
MeOH, 50-0%H2O；30-40min, 100%MeOH, 0%H2O。
6. cigarette beans water described in cigarette beans Glycine tabacina (Labill.) Benth or claim 1-5 any one mentions
Take purposes of the object in the drug of preparation treatment nephrotic syndrome.
7. purposes according to claim 6, it is characterised in that: the drug is the drug for treating high protein urine disease.
8. purposes according to claim 6, it is characterised in that: the drug is to reduce nephrotic syndrome glomerular injury
Drug.
9. purposes according to claim 6, it is characterised in that: the drug is the medicine for improving nephrotic syndrome renal function
Object.
10. purposes according to claim 6, it is characterised in that: the drug is the drug for treating hyperlipidemia.
11. purposes according to claim 6, it is characterised in that: the drug is to improve oxidation resistance or inhibition kidney
The drug of sick syndrome response to oxidative stress.
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|---|---|---|---|
| CN201710386666.9A CN108938709B (en) | 2017-05-26 | 2017-05-26 | Water extract of tobacco bean and application thereof |
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| CN201710386666.9A CN108938709B (en) | 2017-05-26 | 2017-05-26 | Water extract of tobacco bean and application thereof |
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| CN108938709A true CN108938709A (en) | 2018-12-07 |
| CN108938709B CN108938709B (en) | 2021-10-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117538461A (en) * | 2024-01-10 | 2024-02-09 | 地奥集团成都药业股份有限公司 | Detection method of related substances of benazepril hydrochloride tablets |
-
2017
- 2017-05-26 CN CN201710386666.9A patent/CN108938709B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| SHYH-SHYUN HUANG等: "Antioxidants, anti-inflammatory,and antidiabetic effects of the aqueousextracts from Glycine species and its bioactive compounds", 《BOTANICAL STUDIES》 * |
| 李燕等: "大豆异黄酮对实验性肾病综合征疗效观察", 《中华肾脏病杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117538461A (en) * | 2024-01-10 | 2024-02-09 | 地奥集团成都药业股份有限公司 | Detection method of related substances of benazepril hydrochloride tablets |
| CN117538461B (en) * | 2024-01-10 | 2024-03-26 | 地奥集团成都药业股份有限公司 | Detection method of related substances of benazepril hydrochloride tablets |
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