CN107513092A - A kind of malonyl ginsenoside Rb1Preparation method and its medical application - Google Patents
A kind of malonyl ginsenoside Rb1Preparation method and its medical application Download PDFInfo
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Abstract
The invention provides a kind of malonyl ginsenoside Rb1Preparation method and medical application, preparation method of the present invention comprise the following steps：Fresh ginseng or American Ginseng are shredded, after laccase enzymolysis processing, solvent is recovered under reduced pressure at 40 DEG C in ultrasonic extraction, extract solution, concentrates as the aqueous solution, then is separated with the polymeric adsorbents of NKA 12, ethanol gradient elution, obtains malonyl ginsenoside extract.ODS pressurized column chromatographies are separated on malonyl ginsenoside extract, and eluent is ethanol or acetonitrile and buffer salt solution gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1Eluent concentration and recovery solvent, concentrate uses sephadex desalination, then through ethyl alcohol recrystallization, obtains malonyl ginsenoside Rb1Sterling.Drug effect and pharmacological research show, malonyl ginsenoside Rb1There is significant therapeutic effect to diabetes B and its Nephropathy, can be applied to the preparation of health food or medicine, securely and reliably, have no toxic side effect, there is good prospect in medicine.
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
Diacyl ginsenoside Rb1Preparation method, the present invention further discloses malonyl ginsenoside Rb1In treatment glycosuria
Medical application in sick and its caused nephrosis, belongs to field of traditional Chinese medicine technology.
Background technology
Diabetes（Diabetes mellitus, DM）It is comprehensive as one group of endocrine metabolism of principal character using blood glucose rise
Simulator sickness, is divided into 1 type and diabetes B, and diabetes B accounts for more than 90%.Modern clinic and pharmaceutical research show that ginseng has
Adjust the effect of glycolipid metabolism, anti-diabetic and obesity, and significant effect, better tolerance.Further investigations have shown that ginseng
Saponin(e is the principle active component that diabetes are treated in ginseng, general ginsenoside and ginseng saponinses（Ginsenoside Rb1、Rb2、
Rc、Re、Rg1、Rh2、Rg3With CK etc.）Hyperglycaemia caused by different diabetes models can be resisted, hence it is evident that improve sugar tolerance, enhancing
Influence of the insulin to glycometabolism.
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；At present, the remedy measures of diabetic nephropathy mainly include：Blood glucose is controlled, controls blood pressure, albumen
Matter intake limitation, controls blood fat, dialysis, kidney transplant etc..Ginseng also has the effect of notable, text in the treatment of diabetic nephropathy
Offer and report that ginseng forms caused renal damage to diabetes rat oxidative stress or AGEs and has protective effect；Ginseng is not only able to
The blood glucose and glycosylation protein level of diabetes rat are reduced, while kreatinin and urine protein level can also be reduced.Folium Panacis Quinquefolii
20s- protopanoxadiols saponins can reduce diabetic nephropathy rats blood glucose, reduce kidney cell matrix, reduce mesangial cell
Hyperplasia, the ultra microstructure of kidney cell is protected, improve Pathological infringement caused by rat diabetes.In addition monomer ginsenoside
Rb1、Rg1And Rg3DN Renal Functions can be improved, mitigate Pathological infringement, diabetes rat 24h urine can be substantially reduced
Albumen and serum creatinine, there is certain renal protection.
Malonyl ginsenoside（Malonyl- ginsenosides）It is the former glycosides of ginseng and American Ginseng, its content is very
Height, the 50% of general ginsenoside is accounted in ginseng.But this saponins polarity is stronger, it is difficult to separation identification, at high temperature under high pressure
The corresponding neutral saponin(e of malonyl generation can be sloughed, therefore studies it that report is less, and pharmacological activity is nearly at sky at present
In vain.In the research of malonyl ginsenoside chemical composition, nineteen eighty-three, Kitagawa etc. is reported first to be produced from Japan
Shine in ginseng and isolate 4 kinds of malonyl ginsenosides（M-Rb1, M-Rb2, M-Rc, M-Rd）；All rain etc. separates from American Ginseng
Malonyl ginsenoside-Rb is gone out1；Liu etc. is checked in pseudo-ginseng and contained using HPLC/ESI-MS analysis saponins
There is malonyl people's saponin(e-Rb1,-Rd and-Rg1.Wang etc. separated from flower of Panax ginseng identify malonyl panax saponin-Re ,-
Rb1、-Rb2,-Rc and-Rd.Sun Guangzhi etc. have studied the extraction and separation identification of malonyl ginsenoside, from ginseng altogether
Separation identifies 6 kinds of malonyl ginsenosides（M-Rb1, M-Rb2, M-Rc, M-Rd, M-Ra3, M-NR4）, wherein M-
NR4And M-Ra3It is the noval chemical compound obtained first.On pharmacological activity, document only reports malonyl ginsenoside total and carried
Take thing to have significant hypoglycemic, adjust the effect of lipid metaboli；Malonyl ginsenoside Rb1Grown with mouse cdontoid process is promoted
Time-histories synergistic effect, and the outer Dorsal ganglion for cultivating chicken embryo of nerve growth factor induction is dashed forward external with humidification.This
Outside, no malonyl ginsenoside pharmacological activity report.Therefore prepared by the separation of research malonyl ginsenoside chemical composition
And pharmacological action, have great importance to the utilization and economic value for improving ginseng.
Learn had so far as described below on malonyl ginsenoside preparation technology through consulting patent document
Patent disclose.
Chinese patent（Application number 200510016845.0）Disclose one kind and prepare malonyl ginseng soap using fresh ginseng
The method of glycosides, after this method is using the ethanol cold extraction of high concentration, is extracted with water-saturated n-butanol, then utilize macropore
The isolated malonyl ginsenoside total extract of resin, finally using the dry post decompression separation of silica gel, chloroform-methanol-washing
De-, ethanol recrystallizes repeatedly, obtains malonyl notoginsenoside-R4.Chinese patent（Application number 201510469832.2）It is open
A kind of malonyl ginsenoside Re and preparation method thereof, by fresh ginseng bud extract solution after macroporous resin column chromatography, warp
Silica gel medium pressure column chromatography is isolated and purified, and is 4 with volume ratio:1:0.1-3:1:0.1-2:1:0.1 dichloromethane:Methanol:
Water is eluted；Eluent is upper after being concentrated under reduced pressure to prepare liquid phase, carries out permanent gradient elution with 20% acetonitrile water, obtains malonyl people
Join saponin monomer Re.But the active ingredient in ginseng is wrapped up by the cell membrane rich in cellulose mostly, causes ginsenoside
Extraction efficiency is relatively low, and extraction time is longer.Further, since malonyl ginsenoside is a kind of acid saponin(e, polarity is stronger,
The above-mentioned silica gel column chromatography on patented technology prepared by the separation of malonyl ginsenoside using routine, technics comparing is complicated,
Yield is relatively low, is difficult to industrialized pharmaceutical manufacturing, and there are no on malonyl ginsenoside Rb1Purification process
Report.
The content of the invention
The present invention discloses a kind of malonyl ginsenoside Rb1Extraction separation method, solve existing preparation technology and carry
Take the shortcomings of rate is low, it is difficult to isolate and purify, yield is low and is not suitable for industrializing pharmaceutical manufacturing.
Invention further provides malonyl ginsenoside Rb1Doctor in treatment diabetes and its caused nephrosis
With purposes, laccase enzymolysis ginseng and the cell membrane of American Ginseng, the extraction purification malonyl ginseng soap of efficient green can be passed through
Glycosides Rb1, the utilization for ginseng and Effective Components of American Ginseng provide technical support.
Malonyl ginsenoside Rb of the present invention1Molecular formula：C57H94O26, molecular weight：1194.
A kind of malonyl ginsenoside Rb of disclosure of the invention1Method for extraction and purification, comprise 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）Then 10%-80% laccase is added, in 30 DEG C of -70 DEG C of constant temperature oscillation water-bath 2-24h；
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, concentrates as the aqueous solution, is then separated with NKA-12 polymeric adsorbents,
First it is washed with water five column volumes, then with 20%-70% ethanol gradient elutions, obtains malonyl ginsenoside extract；
5）Malonyl ginsenoside Rb1Preparation：
ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluent is ethanol or acetonitrile and buffering
Salting liquid gradient elution；
6）Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1Eluent concentration and recovery solvent, concentrate
Using sephadex desalination, then through ethyl alcohol recrystallization, produce.
A kind of described malonyl ginsenoside Rb1Preparation method step（1）It is preferred that：Fresh ginseng or American Ginseng chopping
Afterwards, the distilled water of 5 times of amounts is added；Then 40% laccase is added.
A kind of described malonyl ginsenoside Rb1Preparation method step（2）It is preferred that：The temperature of laccase enzymolysis is 50
DEG C, constant temperature oscillation water-bath 8h.
A kind of described malonyl ginsenoside Rb1Preparation method step（3）It is preferred that：After enzymolysis add ethanol and
Water, the concentration of ethanol is reached 0-20%, then ultrasonic extraction 3 times, extract 0.5h every time, the dosage of solvent is 30-40 times.
A kind of described malonyl ginsenoside Rb1Preparation method step（5）It is preferred that：ODS pressurized column chromatography methods footpath
Long ratio is more than 1：12, moulding pressure 1-100MPa.
A kind of described malonyl ginsenoside Rb1Preparation method step（5）It is preferred that：Buffer salt solution refers to phosphorus
Phthalate buffer, citrate buffer, acetate buffer, carbonate buffer solution, barbiturates salt buffer, Tris bufferings
Liquid.
A kind of described malonyl ginsenoside Rb1Preparation method step（6）It is preferred that：Malonyl ginsenoside Rb1
Preparation use preparative high performance liquid chromatography, chromatographic condition：ODS posts, using 28% acetonitrile and 72% water as mobile phase, ultraviolet inspection
Survey wavelength is 203nm, flow velocity 120mL/min.
Gained malonyl ginsenoside Rb of the invention1Crystallization through spectrum analysis, there is following physicochemical property：Crystallize and be
Colorless powder（EtOH-BuOH）, mp.150-152 DEG C, methanol, water are soluble in, ethanol, n-butanol are insoluble in, insoluble in chlorine
Imitative and ether.After deploying on the tlc plate, 10%H is used2SO4Aubergine is presented in reagent spray, heating；Liebermann-
Burchard reactions are positive；Molish reactions are positive, and it is triterpene saponin componds to show the compound.Partial hydrolysis
Disaccharide is detected, followed by carries out all-hydrolytic, detects glucose.The sugar that C-20 positions link is prompted as the double of two molecule glucoses composition
Sugar.The sugar connected on aglycon is all glucose.It is consistent with reference substance panoxadiol Rf values that aglycon Rf values are known in TLC inspections.
Crystallization and reference substance malonyl ginsenoside-Rb1With three kinds of different solvents [A of plate: CHCl3-MeOH-H2O (
65：35：10) lower floor；B：CHCl3-MeOH-H2O ( 6：4：1)；C: CHCl3-MeOH- AcOEt -H2O (2：2：4：1)]
Thin layer altogether, Rf values, the color of the two are consistent, and do not decline with reference substance mixed melting point.
In being composed by LCQ-MSm/z 1193[M―H]-, understand its molecular weight be 1194, with malonyl ginsenoside-Rb1
It is consistent；1107 [M -87]+To take off a malonyl, 946 [M -87-162-H] are to lose a malonyl and one
Molecule glucose, 621 [M -87-3 × 162-H] are the fragment peak for losing a malonyl and three molecule glucoses（Data
Ownership is shown in Table 1）.
The LCQ-MS attribution datas that table 1 crystallizes
| m/z | Fragment belongs to |
| 1193 | [M -H]- |
| 1149 | [M- CO2 -H]- |
| 1107 | [M - COCH2COOH] |
| 946 | [M - COCH2COOH -glc -H]- |
| 783 | [M - COCH2COOH -2glc -H]- |
| 621 | [M - COCH2COOH -3glc -H]- |
Conclusion：Data above shows that crystallization is malonyl ginsenoside-Rb1。
Malonyl ginsenoside Rb of the present invention1According to there is very strong pharmacological activity, treatment diabetes can be prepared
Medicine, treatment nephrosis as caused by diabetes can also be prepared.
Malonyl ginsenoside Rb of the present invention1Medicine contain one or more pharmaceutically acceptable carriers.Should
It is emphasized that one or more natural or synthetic other and sheets can be added in the drug regimen of invention as needed
Active material has the composition of collaboration or booster action, and these natural or synthetic auxiliary elements that may be added into are this areas
Technical staff is known and envisioned.Described composition can be configured to the formulation of oral administration, such as tablet, ball
Agent, granule, capsule, powder etc..
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 5-1000mg.
The positive effect of the present invention is：
Using new method to malonyl ginsenoside Rb in fresh ginseng and American Ginseng1Carry out extraction purification.With existing preparation work
Skill is compared, and can make malonyl ginsenoside Rb using the method for laccase enzymolysis and extraction1Recovery rate improve nearly 80%；Simultaneously
Eluted, overcome using silica gel column chromatography malonyl using ODS pressurized column chromatographies and buffer salt solution on separation purifying technique
Ginsenoside Rb1The shortcomings that separation is more difficult, improve the Rb of malonyl ginsenoside1Purity and yield.Fully profit of the invention
With ginseng resource, extract to greatest extent, be enriched with malonyl ginsenoside Rb1, reach simple, quick, environmentally friendly, inexpensive
The purpose of enrichment, prepare the method that provides for industrialized production, new drug and ensure.
Brief description of the drawings
Fig. 1：Enzyme concentration is to malonyl ginsenoside-Rb1The influence of recovery rate；
Fig. 2：Hydrolysis temperature is to malonyl ginsenoside-Rb1The influence of recovery rate；
Fig. 3：Enzymolysis time is to malonyl ginsenoside-Rb1The influence of recovery rate；
Fig. 4：Concentration of alcohol is to malonyl ginsenoside-Rb1The influence of recovery rate；
Fig. 5：Malonyl ginsenoside-Rb1Influence to blood glucose in diabetic rats；
Fig. 6：Malonyl ginsenoside-Rb1Influence to diabetes rat urinary albumin；
Fig. 7：Malonyl ginsenoside-Rb1Influence to diabetes rat HbAlC；
Fig. 8：Malonyl ginsenoside-Rb1Influence to diabetes rat creatinine；
Fig. 9：Malonyl ginsenoside-Rb1Influence to diabetes rat BUN.
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 sample is added
The laccase of quality 10%, in 30 DEG C of constant temperature oscillation water-bath 2h.Appropriate second alcohol and water is added after enzymolysis, reaches the concentration of ethanol
To 20%, then ultrasonic extraction 3 times, 0.5h is extracted every time, the dosage of solvent is 10 times.Extract solution is recovered under reduced pressure at 40 DEG C
Solvent, concentrate as the aqueous solution, separated with NKA-12 polymeric adsorbents, be first washed with water five column volumes, then with 20%-70% ethanol
Gradient elution, obtain malonyl ginsenoside extract.ODS pressurized column chromatographies on malonyl ginsenoside extract are entered
Row separation, eluent is ethanol and phosphate buffer gradient elution.Detected using thin layer plate chromatography, malonyl people will be contained
Join saponin(e Rb1Eluent concentration and recovery solvent, concentrate uses sephadex desalination, then through ethyl alcohol recrystallization, obtains
Malonyl ginsenoside Rb1Sterling.
Embodiment 2
Using the fibrous root, supporting root and main root of fresh ginseng as raw material, chopping, 4 times of distilled water measured are added；Then sample quality is added
20% laccase, in 40 DEG C of constant temperature oscillation water-bath 4h.Appropriate distilled water is added after enzymolysis, the dosage for making solvent is 20 times, so
Ultrasonic extraction 4 times afterwards, extract 1h every time.Solvent, concentrate NKA-12 polymeric adsorbents is recovered under reduced pressure in extract solution at 40 DEG C
Separated, be first washed with water five column volumes, then with 20%-70% ethanol gradient elutions, obtain the extraction of malonyl ginsenoside
Thing.ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluent is acetonitrile and Citrate buffer
Liquid gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1Eluent concentration and recovery solvent,
Concentrate uses sephadex desalination, then through ethyl alcohol recrystallization, obtains malonyl ginsenoside Rb1Sterling.
Embodiment 3
Using the cauline leaf, bud and fruit of ginseng as raw material, chopping, 6 times of distilled water measured are added；Then sample quality 30% is added
Laccase, in 50 DEG C of constant temperature oscillation water-bath 6h.Appropriate second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 30%, solvent
Dosage is 30 times, and 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, is then separated with NKA-12 polymeric adsorbents, is first washed with water five column volumes, then with 20%-70% ethanol gradient elutions, is obtained
To malonyl ginsenoside extract.ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluted
Liquid is ethanol and acetate buffer gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1's
Eluent concentration and recovery solvent, concentrate use sephadex desalination, then through ethyl alcohol recrystallization, obtain malonyl ginseng
Saponin(e Rb1Sterling.
Embodiment 4
Using fresh American Ginseng rhizome as raw material, chopping, 8 times of distilled water measured are added；Then the laccase of sample quality 40% is added, in
60 DEG C of constant temperature oscillation water-bath 8h.Appropriate second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 50%, the dosage of solvent is
40 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 the aqueous solution, so
Separated afterwards with NKA-12 polymeric adsorbents, be first washed with water five column volumes, then with 20%-70% ethanol gradient elutions, obtain third
Diacyl ginsenoside extract.ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluent is
Acetonitrile and carbonate buffer solution gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1Elution
Liquid concentration and recovery solvent, concentrate use sephadex desalination, then through ethyl alcohol recrystallization, obtain malonyl ginsenoside
Rb1Sterling.
Embodiment 5
Using fresh American Ginseng as raw material, chopping, 10 times of distilled water measured are added；Then the laccase of sample quality 50% is added, in 70 DEG C
Constant temperature oscillation water-bath 12h.Appropriate second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 70%, the dosage of solvent is 50
Times, then ultrasonic extraction 4 times, extract 0.5h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, is concentrated as the aqueous solution, so
Separated afterwards with NKA-12 polymeric adsorbents, be first washed with water five column volumes, then with 20%-70% ethanol gradient elutions, obtain third
Diacyl ginsenoside extract.ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluent is
Ethanol and barbiturate buffer solution for gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1's
Eluent concentration and recovery solvent, concentrate use sephadex desalination, then through ethyl alcohol recrystallization, obtain malonyl ginseng
Saponin(e Rb1Sterling.
Embodiment 6
Using fresh stem and leaves of American ginseng, bud and fruit as raw material, chopping, 5 times of distilled water measured are added；Then sample quality 60% is added
Laccase, in 45 DEG C of constant temperature oscillation water-bath 24h.Appropriate second alcohol and water is added after enzymolysis, the concentration of ethanol is reached 80%, it is molten
The dosage of agent is 35 times, and then ultrasonic extraction 5 times, extract 1h every time.Solvent is recovered under reduced pressure at 40 DEG C in extract solution, concentrates and is
The aqueous solution, is then separated with NKA-12 polymeric adsorbents, five column volumes is first washed with water, then washed with 20%-70% ethanol gradients
It is de-, obtain malonyl ginsenoside extract.ODS pressurized column chromatographies on malonyl ginsenoside extract are divided
From eluent is acetonitrile and Tris buffer solution for gradient elution.Detected using thin layer plate chromatography, malonyl ginsenoside will be contained
Rb1Eluent concentration and recovery solvent, concentrate uses sephadex desalination, then through ethyl alcohol recrystallization, obtains malonyl
Base ginsenoside Rb1Sterling.
Embodiment 7
Malonyl ginsenoside-the Rb for taking scheme 1, scheme 2 or scheme 3 to prepare1 1g, the medicinal cyclodextrin 5g of excipient is added,
It is appropriate to dry starch, appropriate carboxymethyl cellulose, appropriate talcum powder, appropriate sucrose.Sucrose is used for sugar coating with talcum powder, mixes
It is even, pelletizing press sheet, tablet weight 0.25g, the every-Rb of ginsenoside containing malonyl is made1 20mg。
Embodiment 8
Malonyl ginsenoside-the Rb for taking scheme 4, scheme 5 or scheme 6 to prepare1 1g, adds cornstarch 5g, and cyclodextrin is fitted
Amount, mix, load capsule, the every-Rb of ginsenoside containing malonyl1 20mg。
In order to further illustrate malonyl ginsenoside Rb in the inventive method1Extraction and purification process and its preparing
Treat the application in diabetes and its Nephropathy medicine, following experiments further demonstrate medicine of the present invention preparation technology and
Pharmacological action.
1 experiment material and reagent
Fresh ginseng（Panax ginseng C.A.Meyer）5 years raw roots, buy in ten thousand good market of Fusong；Laccase is purchased from Shanghai
Bai Ao Bioisystech Co., Ltd；STZ is Sigma products, and it is limited that blood sugar test kit is purchased from Beijing Li Deman biochemical technologies
Company；Microalbuminuria enzyme exempts from detection kit and is purchased from Nanjing Xin Fan bio tech ltd.Malonyl ginsenoside-
Rb1It is 99% through nominal purities such as HPLC, NMR, MS for self-control.Chromatographic grade acetonitrile（Fisher companies of the U.S.）, other reagents are
Analyze pure.
2 experimental animals
Healthy SD rat 100, male, body weight 200-220g, is provided by Jilin University's Experimental Animal Center.
3 experimental methods
Processing of 2.1 laccases to samples of Ginseng
After the chopping of fresh ginseng root, precision weighs 5g sample several pieces, is placed in 250ml triangular flasks, investigates laccase enzyme concentration respectively
（0,10%, 20%, 40%, 80%）, temperature（30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C）, enzymolysis time（2h, 4h, 8h, 12h,
24h）And concentration of alcohol（0,20%, 40%, 60%, 80%）To malonyl ginsenoside-Rb1The influence of recovery rate.
The configuration of 2.2 standard solutions
Precision weighs malonyl ginsenoside-Rb1Appropriate standard items, it is placed in volumetric flask, dissolves simultaneously constant volume with 30% acetonitrile,
The concentration for being configured to reference substance is 1 mg/ml, shakes up, obtains hybrid standard product storing solution.Precision is drawn 0.2ml deposit liquors and put
In 1ml volumetric flasks, with 30% dilution in acetonitrile to scale, mixed reference substance solution is obtained, 4 DEG C of storages are standby.
The preparation of 2.3 test samples to be measured
Ultrasonic extraction 3 times after laccase enzymolysis processing of fresh ginseng sample, 30min is extracted every time, is concentrated under reduced pressure at 40 DEG C, Ran Houyong
30% acetonitrile constant volume is 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 draws malonyl ginsenoside-Rb1Reference substance storing solution is appropriate, is diluted to the mixing control of 7 various concentrations
Product solution, it is accurate to draw 20 μ l, analyzed according to above-mentioned condition sample introduction, the peak area at each peak is recorded, with peak areaYIt is dense to quality
DegreeXCarry out linear regression, malonyl ginsenoside-Rb1Linear relationship is good in the range of 0.010 mg/ml-0.640 mg/ml
It is good, obtain regression equation, the range of linearity and coefficient correlation.
2.6 modelings and experimental therapy
SD rats, male, after adapting to sexual balance raising 5d, for testing.Rat Fast can't help water 24h, disposable celiac injection
Streptozotocin（STZ）60mg/kg（With 0.1 mol/L, pH4.4 citric acid-sodium citrate buffer solutions are configured to 0.1mol/
L）, rat freely ingests water after injecting medicine.Glycosuria is used as using the non-empty stomach tail vein sugar level >=16.7mmol/L of modeling 7d
The sick successful selection standard of rat model.4 groups are randomly divided into mould rat, blank control group, model group, malonyl ginseng soap
The high low dose group of glycosides；Every group 12, each group is gastric infusion.The body weight, feed water, urine of each group rat are observed in experiment
Amount, hair etc., successive administration is after 8 weeks, and rat is placed in metabolic cage and 12h urine volume is inscribed, with 10% chloraldurate intraperitoneal injection of anesthesia,
Abdominal aortic blood.Fasting plasma glucose uses glucose oxidase method, and urinary albumin is determined using method of exempting from is put, BUN and creatinine
Determined using chemical colorimetry.
4 results are with discussing
4.1 single factor experiments investigate laccase to malonyl ginsenoside-Rb1The influence of recovery rate
4.1.1 enzyme concentration is to malonyl ginsenoside-Rb1The influence of 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 0,10%, 20%, 40%, 80% laccase, at 40 DEG C, digest 4h；After enzymolysis processing, appropriate ethanol and distillation are added
Water, the concentration of ethanol is reached 80%, ultrasonic extraction 3 times, extract 0.5h every time.Sample after extraction is used for HPLC quantitative analyses.
As a result Fig. 1 is seen, with the increase of enzyme concentration, recovery rate is in increased trend, but when enzyme concentration is 40% and 80%, malonyl people
Join saponin(e-Rb1Recovery rate is 40% to reduce cost to select enzyme concentration without obvious increase.
4.1.2 hydrolysis temperature is to malonyl ginsenoside-Rb1The influence of 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 laccase of sample quality 40% is added, respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C and 70 DEG C, digests 4h；After enzymolysis processing, add
Enter ethanol in proper amount and distilled water, the concentration of ethanol is reached 80%, ultrasonic extraction 3 times, extract 0.5h every time.Sample is determined after extraction
It is dissolved in 25 mL volumetric flasks, for HPLC quantitative analyses.Hydrolysis temperature is to malonyl ginsenoside-Rb1The influence of recovery rate
See Fig. 2.From Fig. 2, at 50 DEG C, malonyl ginsenoside-Rb1Extraction effect it is optimal, as medium temperature raises,
Malonyl ginsenoside-Rb1Recovery rate is on a declining curve on the contrary.
4.1.3 enzymolysis time is to malonyl ginsenoside-Rb1The influence of 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 laccase of quality 40%, at 50 DEG C, 2h, 4h, 8h, 12h, 24h are digested respectively；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 malonyl ginsenoside-Rb is illustrated1With the extension of enzymolysis time, start a period of time recovery rate and be continuously increased,
When enzymolysis time is after 8h, with the extension of extraction time, recovery rate amplification unobvious, therefore optimal enzymolysis time are 8h
（Fig. 3）.
4.1.4 concentration of alcohol is to malonyl ginsenoside-Rb1The influence of 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
Quality is 40% laccase, at 50 DEG C, digests 8h；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
Ginsenoside-Rb1Extraction；Therefore optimal Extraction solvent is 0-20% ethanol（Fig. 4）.
4.2 malonyl ginsenoside-Rb1Influence to blood glucose in diabetic rats
Before modeling, each group Hair of Rats is pure white and glossy, movable agility, and food ration, amount of drinking water, urine volume are normal.After modeling
In addition to blank control group rat, remaining each group rat aggregate performance is that hair is withered and yellow, matt, One's spirits are drooping, slow in reacting, arch
The back of the body is curled up sleeping, and food ration, amount of drinking water and urine volume increase.Malonyl ginsenoside-Rb1After treating 3 weeks, the state of mind is good,
Hair is relatively moist, and diabetic symptom substantially mitigates.
As shown in Figure 5, with blank control group ratio, model group rats blood glucose is significantly raised, and reagent malonyl ginseng soap
Glycosides-Rb1 10th, two dosage of 20mg/kg, which have, reduces the effect of rat blood sugar content, after being administered 3 weeks, malonyl ginseng soap
Glycosides-Rb1 20mg/kg group blood sugar reducing functions are notable（P ＜ 0.01）, illustrate malonyl ginsenoside-Rb1To diabetes B model
Animal has blood sugar reducing function.
4.3 malonyl ginsenoside-Rb1Research to diabetes rat nephrosis
Experimental result is referring to Fig. 6-9, urinary albumin, HbAlC, creatinine and the BUN and Normal group of diabetic model group rat
Compared to significantly raised, and there is significant difference（P ＜ 0.01）；Compared with model group, malonyl ginsenoside of the invention-
Rb1High low dose group is handled 8 weeks, can substantially reduce the total discharge rate (p of rat urinary albumin<0.01).In addition malonyl people
Join saponin(e-Rb1High dose group significantly reduces the amount of rat HbAlC and creatinine, also there is the trend of reduction to BUN.These knots
Fruit illustrates malonyl ginsenoside-Rb1There is preferable therapeutic action to diabetic nephropathy.
Claims (9)
- A kind of 1. malonyl ginsenoside Rb1Method for extraction and purification, comprise 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）Then 10%-80% laccase is added, in 30 DEG C of -70 DEG C of constant temperature oscillation water-bath 2-24h；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, concentrates as the aqueous solution, is then separated with NKA-12 polymeric adsorbents, First it is washed with water five column volumes, then with 20%-70% ethanol gradient elutions, obtains malonyl ginsenoside extract；5）Malonyl ginsenoside Rb1Preparation：ODS pressurized column chromatographies on malonyl ginsenoside extract are separated, eluent is ethanol or acetonitrile and buffering Salting liquid gradient elution；6）Detected using thin layer plate chromatography, malonyl ginsenoside Rb will be contained1Eluent concentration and recovery solvent, concentrate Using sephadex desalination, then through ethyl alcohol recrystallization, produce.
- A kind of 2. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（1） After the fresh ginseng or American Ginseng chopping, the distilled water of 5 times of amounts is added；Then 40% laccase is added.
- A kind of 3. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（2） The temperature of the laccase enzymolysis is 50 DEG C, constant temperature oscillation water-bath 8h.
- A kind of 4. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（3） Second alcohol and water is added after the enzymolysis, the concentration of ethanol is reached 0-20%, then ultrasonic extraction 3 times, extracts 0.5h every time, it is molten The dosage of agent is 30-40 times.
- A kind of 5. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（5） The ODS pressurized column chromatographies method fineness ratio is more than 1：12, moulding pressure 1-100MPa.
- A kind of 6. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（5） Described in buffer salt solution be selected from：Phosphate buffer, citrate buffer, acetate buffer, carbonate buffer solution, Barbiturates salt buffer, Tris buffer solutions.
- A kind of 7. malonyl ginsenoside Rb according to claim 11Preparation method, it is characterised in that：Step（6） The malonyl ginsenoside Rb1Preparation use preparative high performance liquid chromatography, chromatographic condition：ODS posts, with 28% second Nitrile and 72% water are mobile phase, and ultraviolet detection wavelength is 203nm, flow velocity 120mL/min.
- 8. malonyl ginsenoside Rb1Preparing the medical application in treating diabetes medicament.
- 9. malonyl ginsenoside Rb1Preparing the medical application in treating the nephrosis as caused by diabetes.
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| CN110982657A (en) * | 2019-12-29 | 2020-04-10 | 通化市赫思恩科技有限公司 | Method for rapidly preparing red ginseng wine by gradient ultrasonic extraction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103463153A (en) * | 2013-08-26 | 2013-12-25 | 威海金颐阳药业有限公司 | Method for extracting ginsenoside from American ginseng |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103463153A (en) * | 2013-08-26 | 2013-12-25 | 威海金颐阳药业有限公司 | Method for extracting ginsenoside from American ginseng |
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| 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》 * |
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| CN110982657A (en) * | 2019-12-29 | 2020-04-10 | 通化市赫思恩科技有限公司 | Method for rapidly preparing red ginseng wine by gradient ultrasonic extraction method |
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