CN107349244A - Application in the extracting method and treatment diabetic nephropathy of malonyl ginsenoside - Google Patents
Application in the extracting method and treatment diabetic nephropathy of malonyl ginsenoside Download PDFInfo
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- CN107349244A CN107349244A CN201710763361.5A CN201710763361A CN107349244A CN 107349244 A CN107349244 A CN 107349244A CN 201710763361 A CN201710763361 A CN 201710763361A CN 107349244 A CN107349244 A CN 107349244A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention provides the application in a kind of extracting method of malonyl ginsenoside and treatment diabetic nephropathy; using fresh ginseng or American Ginseng as raw material; after cellulase degradation is handled; ultrasonic extraction; solvent is recovered under reduced pressure, concentrates as the aqueous solution, then separated with the polymeric adsorbents of NKA 12; ethanol and phosphate buffered saline solution gradient elution, malonyl ginsenoside is obtained after sephadex desalination.The method efficiently using cellulose degraded ginseng and the cell membrane of American Ginseng, so as to improve the recovery rate of malonyl ginsenoside, has the characteristics that simple to operate, quick, cost is cheap and extraction efficiency is high；Animal experiments prove that malonyl ginsenoside has significant preventive and therapeutic action to diabetes B nephrosis, the preparation of health food or medicine can be applied to.
Description
Technical field
The present invention relates to the preparation method of Chinese medicinal material extract and its application in medicine is prepared, especially discloses one kind third
The extracting method of diacyl ginsenoside, while additionally provide it and preparing the application of prevention and treatment diabetic nephropathy, belong to
Field of traditional Chinese medicine technology.
Background technology
Diabetic nephropathy（Diabetic nephropathy, DN）It is one of diabetes complication the most serious, and
Cause clinically one of diabetic's main causes of death.It is reported that about 20% in I type or patients with NIDDM
~40% can develop into nephrosis；In China, the incidence of disease of diabetic nephropathy is about 40%.Diabetic nephropathy is by more
The coefficient result of factor：The dynamic (dynamical) change of carbohydrate metabolism disturbance, renal blood flow, cytokine profiles and genetic background are equal
Play a very important role.When clinically there is continuous proteinuria, renal lesions has not belonged to early stage, and kidney has occurred not
Reversible lesion, effective measures there is no to prevent it from occurring and develop so far.At present, the remedy measures of diabetic nephropathy mainly wrap
Include：Blood glucose is controlled, blood pressure is controlled, protein intake limitation, controls blood fat, dialysis, kidney transplant etc..But these remedy measures obtain
Effect it is unsatisfactory, it is impossible to effectively suppress diabetic nephropathy development.
Ginseng and American Ginseng are China's Chinese herbal medicines the most rare, have multiple biological activities, there is long medicine in China
With with tonic history.Ginsenoside is the main component of both medicinal plants, can be divided into neutral saponin(e (ginsenoside-
Rb1, -Rb2 , -Rc, -Rd, -Rg1, -Re, -Rg3, -Rg2, -Rh1Deng) and acid saponin(e（Malonyl ginseng soap
Glycosides-Rb1, -Rb2, -Rc, -Rd, -Rg1With-Re etc.）.Isolated at present from ginseng and American Ginseng and identify 100
A variety of ginsenosides.Malonyl ginsenoside（Malonyl- ginsenosides）It is a kind of acid saponin(e, fusing point（mp）One
As be 150~Between 161 DEG C, soluble in water, methanol, ethanol, n-butanol are insoluble in, insoluble in chloroform, ether.Malonyl people
It is very high to join the content of saponin(e, the 50% of general ginsenoside is accounted in ginseng, it is stronger yet with the polarity of this saponins, it is difficult to
Separation identification, it is less to its chemical composition and pharmacology activity research report at present.
The report for being largely related to ginseng and American Ginseng treatment diabetic nephropathy existing at present.Ginseng aoxidizes to diabetes rat
Stress or AGEs formed caused by renal damage there is protective effect；Ginseng is not only able to reduce the blood glucose and glycosyl of diabetes rat
Change protein level, while kreatinin and urine protein level can also be reduced（Biol.Pharm.Bull.2016,29(8):1678-
1684）.Panax quinquefolium 20 s-protopanaxdiol Saponins can reduce diabetic nephropathy rats blood glucose, reduce kidney cell base
Matter, proliferation of mesangial cells is reduced, protects the ultra microstructure of kidney cell, improve Pathological infringement caused by rat diabetes,
It is relevant that its mechanism may reduce kidney GLUT-1 functions with it（《Jilin University's journal：Medicine》, 2007,33（5）：845-
848.《Chinese Journal of Pathophysiology》, 2008,24（6）：1237-1239）.Monomer ginsenoside Rb1DN Rat renals can be improved
Dirty function, mitigate Pathological infringement, its specific mechanism may be with lowering renal tissues of rats MCP-1 mRNA and protein expression water
It is flat relevant.（《Chinese combination of Chinese tradiational and Western medicine nephrosis magazine》, 2008(7): 578-581）.Ginsenoside Rg1Can reduce TNF-α,
ED-1, TGF-β 1, MCP-1 level, improve the pathological lesion of diabetic nephropathy rats sertoli cell and kidney, sugar can be substantially reduced
Urinate disease rat 24h Urine proteins and serum creatinine, have certain renal protection (《Sichuan University's journal：Medicine》2009,40
（3）：466-471.《Biomedical engineering magazine》, 2010,27（2）：342-347).Panaxoside Rg3Sugar can be significantly reduced
Disease rat blood sugar, serum creatinine, 24h Urine proteins are urinated, glomerular basement membrane thickening degree mitigates, and extracellular matrix accumulation is reduced, energy
Improve the pathological lesion of Renal of Diabetic Rats.（《Modern biomedical is in progress》, 2014,36：7015-7018）.
But up to the present, have no and the research of diabetic nephropathy therapeutic action is reported about malonyl ginsenoside.
In the preparation method of malonyl people's saponin(e, Chinese patent（Application number 200510016845.0）Disclose one kind
The method that malonyl ginsenoside is prepared using fresh ginseng, this method are used the ethanol cold extraction of high concentration, then use water
Saturation n-butanol is extracted, and finally utilizes the isolated malonyl ginsenoside of macroreticular resin.It is but effective in ginseng
Composition is wrapped up by the cell membrane rich in cellulose mostly, causes that the extraction efficiency of ginsenoside is relatively low, and extraction time is longer.Separately
Outside, because malonyl ginsenoside is a kind of acid saponin(e, the ethanol-water system of routine difficult to realize the third two is used
The separation of acyl group ginsenoside and neutral saponin(e, the purity for causing it to prepare is relatively low, and yield is relatively low.
The content of the invention
The present invention discloses a kind of method for extraction and purification of malonyl ginsenoside, solves existing preparation technology recovery rate
It is low, isolate and purify the difficult shortcoming such as low with high-purity malonyl ginsenoside yield.
It is medical in treatment diabetes and its caused nephrosis invention further provides malonyl ginsenoside
Purposes, cellulase degradation ginseng and the cell membrane of American Ginseng, the extraction purification malonyl ginseng of efficient green can be passed through
Saponin(e, the utilization for ginseng and Effective Components of American Ginseng provide technical support.
A kind of method for extraction and purification of malonyl ginsenoside of the present invention, comprises the following steps：
1）Using fresh ginseng or American Ginseng as raw material, chopping, the 1-10 times of distilled water measured is added；
2）Sample quality 1%-20% cellulase is added, in 20-60 DEG C of constant temperature oscillation water-bath 1-12h；
3）Second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 0-80%, ultrasonic extraction 3-5 times, extracts 0.5-4h every time,
The dosage of Extraction solvent is 10-50 times；
4）Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated as the aqueous solution；
5）By step 4）Extract is separated with NKA-12 polymeric adsorbents, and eluent is ethanol and phosphate buffered saline solution gradient
Elution, elution fraction is collected respectively, is detected using thin-layered chromatography, collect, merge and malonyl ginsenoside positive control
Product Rf value identical fraction solution, is recovered under reduced pressure solvent, and concentrate uses sephadex desalination, and freeze-drying obtains the third two
Acyl group ginsenoside powder；The purity of malonyl ginsenoside is more than 70%, and yield is up to 60%.
Malonyl ginsenoside preparation method step of the present invention（1）It is preferred that：After fresh ginseng or American Ginseng chopping, 3- is added
The distilled water of 5 times of amounts；Then the cellulase of sample quality 5% is added.
The preparation method step of malonyl ginsenoside of the present invention（2）It is preferred that：The temperature of cellulase degradation is 40 DEG C,
Constant temperature oscillation water-bath 4h.
The preparation method step of malonyl ginsenoside of the present invention（3）It is preferred that：Second alcohol and water is added after enzymolysis, makes ethanol
Concentration reach 0-20%, the dosage of solvent is 20-30 times, and then ultrasonic extraction 3 times, extract 1h every time.
The preparation method step of malonyl ginsenoside of the present invention（5）It is preferred that：Phosphate buffered saline solution is selected from：
NaH2PO4、 KH2PO4、Na2HPO4、K2HPO4。
Malonyl ginsenoside is preparing the medical application in treating diabetes medicament.
Malonyl ginsenoside is preparing the medical application in treating the nephrosis as caused by diabetes.
Present invention also offers according to a kind of malonyl ginsenoside preparation for having very strong pharmacological activity.
The extract that the medicine of malonyl ginsenoside of the present invention contains therapeutically effective amount is active component, and is contained
One or more pharmaceutically acceptable carriers.
Additionally it should it is emphasized that one or more days can be added in the drug regimen of invention as needed
So or synthesis it is other with this active material with cooperateing with or the composition of booster action, these may be added into natural or synthetic
Auxiliary element be well known by persons skilled in the art and envisioned.Described composition can be configured to oral administration
Formulation, such as tablet, pill, granule, capsule, powder.
In order to prepare tablet, pill, granule, capsule etc. for being suitable to be administered orally, can use sucrose, galactolipin,
Cornstarch, gelatin, microcrystalline cellulose, talcum powder etc. are used as carrier or excipient., can be with the formulation of these oral administrations
Contain suitable other additives, such as disintegrant, lubricant, filler, binder, flavouring, preservative, dispersant, table
Face activating agent, colouring agent etc..
The most preferred method of administration of pharmaceutical composition of the present invention is the formulation of various oral administrations, for example, tablet, granule,
Capsule or pill, the daily dosage of these oral administration formulations typically contain this active material of 10-1000mg.
The positive effect of the present invention is：Malonyl ginsenoside in fresh ginseng and American Ginseng is carried out using new method
Extraction purification.Compared with existing preparation technology, the method extracted using cellulase degradation can make malonyl ginsenoside
Recovery rate improve 1 times；Simultaneously NKA-12 polymeric adsorbents and ethanol-phosphate-buffered salt elution system are used on separation purifying technique
The shortcomings that overcoming malonyl ginsenoside and more difficult neutral saponin separation, improve the purity of malonyl ginsenoside
And yield.The present invention takes full advantage of ginseng resource, extracts to greatest extent, is enriched with malonyl ginsenoside, reach it is simple,
Quickly, environmental protection, the purpose of low cost enrichment, prepare the method that provides for industrialized production, new drug and ensure.
Brief description of the drawings
Fig. 1：Influence of the hydrolysis temperature to malonyl ginsenoside recovery rate；
Fig. 2：Influence of the enzyme concentration to malonyl ginsenoside recovery rate；
Fig. 3：Influence of the enzymolysis time to malonyl ginsenoside recovery rate；
Fig. 4：Influence of the concentration of alcohol to malonyl ginsenoside recovery rate.
Embodiment
By following examples further illustrate description the present invention, do not limit the invention in any way, without departing substantially from
On the premise of the technical solution of the present invention, what those of ordinary skill in the art made for the present invention easily realized any changes
Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1
With fresh ginseng（Fibrous root, supporting root, rhizome, main root etc.）For raw material, chopping, 2 times of distilled water measured are added；Then ginseng is added
The cellulase of sample quality 1%, in 20 DEG C of constant temperature oscillation water-bath 2h.Appropriate distilled water is added after enzymolysis, makes the dosage of solvent
For 10 times, then ultrasonic extraction 3 times, extract 0.5h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated to be water-soluble
Liquid；Then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.01mol/L K2HPO4Gradient elution, receive respectively
Collection elution fraction, detected using thin-layered chromatography, collect, merge it is identical with malonyl ginsenoside positive reference substance Rf values
Fraction solution, be recovered under reduced pressure solvent, concentrate uses sephadex desalination, and freeze-drying obtains malonyl ginsenoside
Powder.
Embodiment 2
Using the fibrous root, supporting root and main root of fresh ginseng as raw material, chopping, 3 times of distilled water measured are added；Then samples of Ginseng matter is added
The cellulase of amount 5%, in 40 DEG C of constant temperature oscillation water-bath 4h.Appropriate second alcohol and water is added after enzymolysis, the concentration for making ethanol is
20%, the dosage of solvent is 30 times, and then ultrasonic extraction 4 times, extract 1h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution,
Concentrate as the aqueous solution, then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.02mol/L KH2PO4Gradient is washed
It is de-, elution fraction is collected respectively, is detected using thin-layered chromatography, is collected, is merged and malonyl ginsenoside positive reference substance
Rf value identical fraction solution, solvent is recovered under reduced pressure, concentrate uses sephadex desalination, and freeze-drying obtains malonyl
Base ginsenoside powder.
Embodiment 3
Using fresh American Ginseng as raw material, chopping, 5 times of distilled water measured are added；Then the cellulose of American Ginseng sample quality 10% is added
Enzyme, in 60 DEG C of constant temperature oscillation water-bath 6h.Appropriate second alcohol and water is added after enzymolysis, the concentration for making ethanol is 40%, the use of solvent
Measure as 50 times, then ultrasonic extraction 5 times, extract 2h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated to be water-soluble
Liquid；Then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.05mol/L Na2HPO4Gradient elution, receive respectively
Collection elution fraction, detected using thin-layered chromatography, collect, merge it is identical with malonyl ginsenoside positive reference substance Rf values
Fraction solution, be recovered under reduced pressure solvent, concentrate uses sephadex desalination, and freeze-drying obtains malonyl ginsenoside
Powder.
Embodiment 4
Using fresh American Ginseng rhizome as raw material, chopping, 1 times of distilled water measured is added；Then the fibre of American Ginseng sample quality 20% is added
Plain enzyme is tieed up, in 30 DEG C of constant temperature oscillation water-bath 8h.Appropriate second alcohol and water is added after enzymolysis, the concentration for making ethanol is 60%, solvent
Dosage be 20 times, then ultrasonic extraction 3 times, extract 4h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated as water
Solution；Then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.03mol/L NaH2PO4Gradient elution, respectively
Elution fraction is collected, is detected using thin-layered chromatography, is collected, merges and malonyl ginsenoside positive reference substance Rf value phases
Same fraction solution, solvent is recovered under reduced pressure, concentrate uses sephadex desalination, and freeze-drying obtains malonyl ginseng soap
Glycosides powder.
Embodiment 5
Using the cauline leaf, bud and fruit of ginseng as raw material, chopping, 5 times of distilled water measured are added；Then samples of Ginseng quality 3% is added
Cellulase, in 35 DEG C of constant temperature oscillation water-bath 5h.Appropriate second alcohol and water is added after enzymolysis, the concentration for making ethanol is 5%, molten
The dosage of agent is 40 times, and then ultrasonic extraction 5 times, extract 2h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, concentrates and is
The aqueous solution；Then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.04mol/L KH2PO4Gradient elution, point
Fraction Shou Ji not be eluted, is detected using thin-layered chromatography, collects, merge and malonyl ginsenoside positive reference substance Rf values
Identical fraction solution, solvent is recovered under reduced pressure, concentrate uses sephadex desalination, and freeze-drying obtains malonyl ginseng
Saponin(e powder.
Embodiment 6
Using fresh stem and leaves of American ginseng, bud and fruit as raw material, chopping, 2 times of distilled water measured are added；Then American Ginseng sample matter is added
The cellulase of amount 8%, in 45 DEG C of constant temperature oscillation water-bath 3h.Appropriate second alcohol and water is added after enzymolysis, the concentration for making ethanol is
15%, the dosage of solvent is 30 times, and then ultrasonic extraction 3 times, extract 1h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution,
Concentrate as the aqueous solution；Then separated with NKA-12 polymeric adsorbents, eluent is ethanol and 0.1mol/L NaH2PO4Gradient is washed
It is de-, elution fraction is collected respectively, is detected using thin-layered chromatography, is collected, is merged and malonyl ginsenoside positive reference substance
Rf value identical fraction solution, solvent is recovered under reduced pressure, concentrate uses sephadex desalination, and freeze-drying obtains malonyl
Base ginsenoside powder.
Embodiment 7
The malonyl total saposins 1kg for taking scheme 1, scheme 2 or scheme 5 to prepare, the medicinal cyclodextrin 10kg of excipient is added, dried
Appropriate starch, appropriate carboxymethyl cellulose, appropriate talcum powder, appropriate sucrose.Sucrose is used for sugar coating with talcum powder, mixes, system
Grain tabletting, tablet weight 0.50g, the every 100mg of total saposins containing malonyl is made.
Embodiment 8
The malonyl total saposins 1kg for taking scheme 3, scheme 4 or scheme 6 to prepare, addition cornstarch 20kg, appropriate cyclodextrin,
Mix, load capsule, the every 100mg of total saposins containing malonyl.
In order to further illustrate the extraction and purification process of malonyl ginsenoside and its treatment glycosuria in the inventive method
The medicinal usage of sick nephrosis, following experiments further demonstrate preparation technology and the pharmacological action of medicine of the present invention：
Test example 1
1 experiment material and reagent
Fresh ginseng（Panax ginseng C.A.Meyer）5 years raw roots, buy in ten thousand good market of Fusong；Cellulase is purchased from
Shanghai Bai Ao Bioisystech Co., Ltd；STZ is Sigma products, and blood sugar test kit is purchased from Beijing Li Deman biochemical technologies
Co., Ltd；Microalbuminuria enzyme exempts from detection kit and is purchased from Nanjing Xin Fan bio tech ltd.Ginsenoside Rb1、
Rb2, Rc, Rd, Re and Rg1Buy in National Institute for Food and Drugs Control；Malonyl ginsenoside-Rb1、-Rb2,-Rc and-
Rd is that self-control through nominal purities such as HPLC, NMR, MS is 99%.Chromatographic grade acetonitrile（Fisher companies of the U.S.）, other reagents are point
Analyse pure.
2 experimental animals
Healthy SD rat 100, male, body weight 200-220g, is provided by Jilin University's Experimental Animal Center.
Experimental method
Processing of 2.1 cellulases to samples of Ginseng
After the chopping of fresh ginseng root, precision weighs 5g sample several pieces, is placed in 250ml triangular flasks, investigates temperature respectively（20 DEG C,
30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C）, cellulase enzyme concentration（0,1%, 5%, 10%, 20%）, enzymolysis time（1h, 2h, 4h, 6h, 8h）
And concentration of alcohol（0,20%, 40%, 60%, 80%）Influence to the recovery rate of malonyl ginsenoside.
The configuration of 2.2 standard solutions
It is each appropriate that precision weighs each monomer ginsenoside standard items, is placed in same volumetric flask, adds the dissolving of 80% ethanol and constant volume,
The concentration for being configured to each ginsenoside reference substance is 1 mg/ml, is shaken up, and obtains hybrid standard product storing solution.Precision draws 0.2
Ml deposit liquors are placed in 1ml volumetric flasks, are diluted to scale with 80% ethanol water, are obtained mixed reference substance solution, 4 DEG C are deposited
Storage, it is standby.
The preparation of 2.3 test samples to be measured
Ultrasonic extraction 3 times after cellulase degradation is handled of fresh ginseng sample, 0.5h is extracted every time, is concentrated under reduced pressure at 40 DEG C, so
Afterwards with 80% methanol constant volume in 25 mL volumetric flasks;Shake up, 0.45 μm of filter membrane is crossed, for HPLC quantitative analyses.
2.4 chromatographic condition
The C of chromatographic column Cosmosil 518-MS（250 mm × 4.6 mm, 5 μm）；Mobile phase：Acetonitrile（A）- 0.05 mol/l phosphorus
Acid dihydride aqueous solutions of potassium（B）；Gradient：0-20 min, 22%A；20-25 min, 22%-29%A；25-45 min, 29%A；
45-55 min, 29%-35%A；55-60 min, 35%-50%A；Column temperature：25 ℃；Detection wavelength：203 nm；Flow velocity：1 ml/
min；Sample size：20μl；Analysis time：60 min.
The drafting of 2.5 standard curves
Precision absorption ginseng reference substance storing solution is appropriate, is diluted to the mixed reference substance solution of 7 various concentrations respectively, accurate to inhale
20 μ l are taken, are analyzed according to above-mentioned condition sample introduction, the peak area at each peak are recorded, with peak areaYTo mass concentrationX (mg/ml）
Linear regression is carried out, each monomer saponin linear relationship in the range of 0.010 mg/ml-0.640 mg/ml is good, obtains the side of recurrence
Journey, the range of linearity and coefficient correlation.
The purifying of 2.6 malonyl ginsenosides
The samples of Ginseng of cellulase processing is concentrated under reduced pressure after ultrasonic method is extracted at 40 DEG C, NKA-12 absorption tree on concentrate
Fat is separated, and investigates different solvents system（Alcohol-water, ethanol-potassium dihydrogen phosphate aqueous solution）Malonyl saponin(e is divided
From purifying.
Influence of the 2.7 malonyl ginsenosides to diabetes B nephrotic rats
SD rats, male, after adapting to sexual balance raising 5d, for testing.After experimental rat fasting 12h, with 10% chloraldurate
The descending left kidney ligation of intraperitoneal anesthesia, ligating methodses are：Formed along kidney while ligature kidney arteriovenous and ureter.Postoperative 2
Week, Rat Fast can't help water 24h, disposable celiac injection Streptozotocin（STZ）50mg/kg（With 0.1 mol/L, pH4.4
Citric acid-sodium citrate buffer solution is configured to 0.1mol/L）, rat freely ingests water after injecting medicine.With the non-empty stomaches of modeling 72h
Tail vein sugar level >=13.8mmol/L is as the successful selection standard of Diabetic nephropathy animal model.Into mould DN rats with
Machine is divided into 4 groups, blank control group, model group, the high low dose group of malonyl ginsenoside（Embodiment 1 prepares the dry of purifying
Powder, 50,100mg/kg/d）；Every group 12, each group is gastric infusion.The body weight of each group rat is observed in experiment, enters drinking water
Amount, urine volume, hair etc., successive administration is after 8 weeks, and rat is placed in metabolic cage and 12h urine volume is inscribed, and rat is with 10% chloraldurate abdominal cavity
Injecting anesthetic, abdominal aortic blood, and take left kidney to remove coating, weigh to calculate organ coefficient (kidney weight/body weight).
As a result with discussion
4.1 single factor experiments investigate influence of the cellulase to malonyl ginsenoside recovery rate
4.1.1 influence of the hydrolysis temperature to malonyl ginsenoside recovery rate
Precision weighs 15 parts of fresh ginseng root samples, every part of 5g, is respectively placed in 250ml triangular flasks, after adding 25ml distilled water,
The cellulase of sample quality 5% is added, respectively at 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C and 60 DEG C, digests 1h；After enzymolysis processing,
Appropriate ethanol and distilled water are added, the concentration of ethanol is reached 80%, ultrasonic extraction 3 times, extracts 0.5h every time.Sample after extraction
Product constant volume is in 25 mL volumetric flasks, for HPLC quantitative analyses.Influence of the hydrolysis temperature to malonyl ginsenoside recovery rate
See Fig. 1.From Fig. 1, at 40 DEG C, the extraction effect of malonyl ginsenoside is optimal, as medium temperature raises, the third two
Acyl group ginsenoside recovery rate is on a declining curve on the contrary.
4.1.2 influence of the enzyme concentration to malonyl ginsenoside recovery rate
15 parts of 5g fresh ginseng roots sample is weighed, is placed in 250ml triangular flasks, after adding 25ml distilled water, is separately added into sample
Quality is 0,1%, 5%, 10%, 20% cellulase, at 40 DEG C, digests 1h；After enzymolysis processing, appropriate ethanol and steaming are added
Distilled water, the concentration of ethanol is reached 80%, ultrasonic extraction 3 times, extract 0.5h every time.Sample after extraction quantitatively divides for HPLC
Analysis.As a result Fig. 2 is seen, with the increase of enzyme concentration, recovery rate is in increased trend, but after enzyme concentration reaches 10%, recovery rate
It is the 5% of ginseng sample quality to reduce cost to select enzyme concentration without obvious increase.
4.1.3 influence of the enzymolysis time to malonyl ginsenoside recovery rate
15 parts of fresh ginseng root samples are weighed, every part of 5g, are placed in 250ml triangular flasks, after adding 25ml distilled water, add sample
The cellulase of quality 5%, at 40 DEG C, 1h, 2h, 4h, 6h, 8h are digested respectively；After enzymolysis processing, add appropriate ethanol and
Distilled water, the concentration of ethanol is reached 80%, ultrasonic extraction 3 times, extract 0.5h every time.Sample after extraction quantifies for HPLC
Analysis.As a result extension of the malonyl ginsenoside with enzymolysis time is illustrated, starts a period of time recovery rate and is continuously increased, when
Enzymolysis time is after 4 h, and with the extension of extraction time, recovery rate amplification unobvious, therefore optimal enzymolysis time are 4h
（Fig. 3）.
4.1.4 influence of the concentration of alcohol to malonyl ginsenoside recovery rate
15 parts of fresh ginseng root samples are weighed, every part of 5g, are placed in 250ml triangular flasks, after adding 25ml distilled water, add sample
The cellulase of quality 5%, at 40 DEG C, digest 4h；After enzymolysis processing, appropriate ethanol and distilled water are added, makes the dense of ethanol
Degree respectively reaches 0,20%, 40%, 60%, 80%, ultrasonic extraction 3 times, extracts 0.5h every time.Sample after extraction quantifies for HPLC
Analysis.As a result the ethanol for illustrating high concentration is advantageous to the extraction of neutral saponin(e, and the ethanol of low concentration is advantageous to malonyl
The extraction of ginsenoside；Therefore optimal Extraction solvent is 0-20% ethanol（Fig. 4）.
The influence that 4.2 different solvents systems purify to malonyl saponin separation
Different solvents system is investigated using NKA-12 polymeric adsorbents（Alcohol-water, ethanol-potassium dihydrogen phosphate aqueous solution）To malonyl
The influence isolated and purified of basis soap glycosides.As a result illustrating the mmol/l potassium dihydrogen phosphate aqueous solutions of ethanol -100 gradient elution can be with
By malonyl ginsenoside and neutral saponin separation, the purity of malonyl ginsenoside is up to 78.6%, yield 62.4%,
And ethanol-water system gradient elution is difficult by malonyl ginsenoside and neutral saponin separation.
Therapeutic action of the 4.3 malonyl ginsenosides to diabetic nephropathy rats
Experimental result is referring to table 1, blood glucose, urinary albumin, HbAlC, creatinine, BUN and the kidney weight/body weight of DN model group rats etc.
Index is significantly raised compared with Normal group, and has significant difference（P ＜ 0.01）；It is of the invention compared with model group
After the high low dose group of malonyl ginsenoside is handled 8 weeks, rat fasting blood-glucose (p ＜ 0.01 or p ＜ can be substantially reduced
0.05), also conspicuousness reduces (p ＜ 0.01) to the total discharge rate of urinary albumin.Other malonyl ginsenoside high dose group is obvious
The amount for reducing rat HbAlC and creatinine, also have the trend of reduction to BUN and kidney weight/body weight.These results show that third
Diacyl ginsenoside has preferable preventive and therapeutic action to diabetic nephropathy.
Improvement result (± s) of the malonyl ginsenoside of table 1 to Diabetic Nephropathy
With Normal group ratio##P ＜ 0.01,#P ＜ 0.05;With model control group ratio * * p ＜ 0.01, * p ＜ 0.05.
Claims (7)
1. a kind of method for extraction and purification of malonyl ginsenoside, comprises the following steps：
1）Using fresh ginseng or American Ginseng as raw material, chopping, the 1-10 times of distilled water measured is added；
2）Sample quality 1%-20% cellulase is added, in 20-60 DEG C of constant temperature oscillation water-bath 1-12h；
3）Second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 0-80%, ultrasonic extraction 3-5 times, extracts 0.5-4h every time,
The dosage of Extraction solvent is 10-50 times；
4）Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated as the aqueous solution；
5）By step 4）Extract is separated with NKA-12 polymeric adsorbents, and eluent is ethanol and phosphate buffered saline solution gradient
Elution, elution fraction is collected respectively, is detected using thin-layered chromatography, collect, merge and malonyl ginsenoside positive control
Product Rf value identical fraction solution, is recovered under reduced pressure solvent, and concentrate uses sephadex desalination, and freeze-drying obtains the third two
Acyl group ginsenoside powder.
A kind of 2. preparation method of malonyl ginsenoside according to claim 1, it is characterised in that：Step（1）Institute
After stating fresh ginseng or American Ginseng chopping, the 3-5 times of distilled water measured is added；Then the cellulase of sample quality 5% is added.
A kind of 3. preparation method of malonyl ginsenoside according to claim 1, it is characterised in that：Step（2）Institute
The temperature for stating cellulase degradation is 40 DEG C, constant temperature oscillation water-bath 4h.
A kind of 4. preparation method of malonyl ginsenoside according to claim 1, it is characterised in that：Step（3）Institute
Second alcohol and water is added after stating enzymolysis, the concentration of ethanol is reached 0-20%, the dosage of solvent is 20-30 times, then ultrasonic extraction 3
It is secondary, 1h is extracted every time.
A kind of 5. preparation method of malonyl ginsenoside according to claim 1, it is characterised in that：Step（5）In
Described phosphate buffered saline solution is selected from： NaH2PO4、 KH2PO4、Na2HPO4、K2HPO4。
6. malonyl ginsenoside is preparing the medical application in treating diabetes medicament.
7. malonyl ginsenoside is preparing the medical application in treating the nephrosis as caused by diabetes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110025623A (en) * | 2019-03-21 | 2019-07-19 | 李和伟 | A kind of lyophilized preparation and its preparation method and application |
| WO2020056735A1 (en) * | 2018-09-21 | 2020-03-26 | 吉林加一健康产业股份有限公司 | Method for improving content of malonyl-ginsenoside in product |
| CN112755060A (en) * | 2021-01-22 | 2021-05-07 | 北京本草方源药业集团有限公司 | Single-flavor low-temperature vacuum drying extraction powder and extraction method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513869A (en) * | 2003-03-13 | 2004-07-21 | 李向高 | Malonyl ginseng saponin structure modification, enriched ginseng saponin Rb1 technology |
| CN1696144A (en) * | 2005-06-03 | 2005-11-16 | 吉林农业大学 | Technique for preparing malonyl ginsenoside, and application of medication in treating diabetes |
| CN103127192A (en) * | 2011-11-30 | 2013-06-05 | 郑毅男 | Preparation method for fresh ginseng extractive and application thereof in II-type diabetics |
| CN103142669A (en) * | 2013-03-07 | 2013-06-12 | 安徽丰润生物技术有限公司 | Enzyme method for extracting ginsenoside and obtained ginsenoside extractive |
-
2017
- 2017-08-30 CN CN201710763361.5A patent/CN107349244B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513869A (en) * | 2003-03-13 | 2004-07-21 | 李向高 | Malonyl ginseng saponin structure modification, enriched ginseng saponin Rb1 technology |
| CN1696144A (en) * | 2005-06-03 | 2005-11-16 | 吉林农业大学 | Technique for preparing malonyl ginsenoside, and application of medication in treating diabetes |
| CN103127192A (en) * | 2011-11-30 | 2013-06-05 | 郑毅男 | Preparation method for fresh ginseng extractive and application thereof in II-type diabetics |
| CN103142669A (en) * | 2013-03-07 | 2013-06-12 | 安徽丰润生物技术有限公司 | Enzyme method for extracting ginsenoside and obtained ginsenoside extractive |
Non-Patent Citations (6)
| Title |
|---|
| 孙光芝，等: "鲜人参中2种丙二酰基人参皂苷的分离鉴定", 《分析化学》 * |
| 张学凯，等: "Rg1、Rb1对糖尿病肾病大鼠肾脏保护作用及其对肾组织MCP-1 mRNA与蛋白表达的影响", 《中国中西医结合肾病杂志》 * |
| 王铁生，主编: "《中国人参》", 31 December 2001, 辽宁科学技术出版社 * |
| 罗永明，主编: "《中药化学成分提取分离技术与方法》", 31 January 2016, 上海科学技术出版社 * |
| 赵啸虎，等: "正交试验优选人参中人参皂苷Rg1的酶解工艺研究", 《中草药》 * |
| 阮长春: "丙二酰基人参皂苷化学成分及其治疗2型糖尿病大鼠的药效学研究", 《吉林农业大学博士学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020056735A1 (en) * | 2018-09-21 | 2020-03-26 | 吉林加一健康产业股份有限公司 | Method for improving content of malonyl-ginsenoside in product |
| CN110025623A (en) * | 2019-03-21 | 2019-07-19 | 李和伟 | A kind of lyophilized preparation and its preparation method and application |
| CN112755060A (en) * | 2021-01-22 | 2021-05-07 | 北京本草方源药业集团有限公司 | Single-flavor low-temperature vacuum drying extraction powder and extraction method thereof |
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