CN1081922C - Medicinal composition containing ginsenoside Re, preparation and usage thereof - Google Patents
Medicinal composition containing ginsenoside Re, preparation and usage thereof Download PDFInfo
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- CN1081922C CN1081922C CN98117830A CN98117830A CN1081922C CN 1081922 C CN1081922 C CN 1081922C CN 98117830 A CN98117830 A CN 98117830A CN 98117830 A CN98117830 A CN 98117830A CN 1081922 C CN1081922 C CN 1081922C
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Abstract
The present invention discloses a medical composition which contains ginsenoside-Re components, one kind or multiple kinds of carriers and/or excipient, which can be accepted in medicine, a preparing method thereof and an application thereof in the prevention and the treatment of whole body or partial ischemic damage. A ginsenoside-Re component is basically composed of ginsenoside-Re, ginsenoside-Rg2 and ginsenoside-Rg1 which respectively account for 80%, 10% and 5% of the total ginsenoside in the composition according to an optimal implementation method of the present invention. In addition, the method for preparing the ginsenoside-Re component does not comprise a column chromatography procedure according to an optimal implementation method of the present invention.
Description
The present invention relates to contain the pharmaceutical composition of ginsenoside Re's component, its preparation method and the application in treatment or the damage of prevention tissue ischemia.
Nearly all part of Radix Ginseng (panax gingseng), Radix Panacis Quinquefolii (panax quinguefolium) and Radix Notoginseng araliaceae ginseng plants such as (panax notoginseng), for example root, rhizome, stem and leaf, flower and fruit all contain abundant ginsenoside.
Recent two decades comes, Saponin to panax species has carried out research very in depth, separate in succession and purification nearly 20 kinds of Panax's Saponin monomers, and to these monomers and/or contain these monomeric components and carried out biologic activity and pharmacotoxicological effect research widely.
People typically use the silica gel thin-layer chromatography technology, and are polar different with it according to the charge of each component among total ginsenoside, isolate different monomer components at silica gel on extremely.And, in general, based on the result of silica gel thin-layer chromatography, Radix Ginseng total saponins can be divided into two different components, wherein Rf value is the bigger component I of polarity below the Rf value of panaxoside monomer Re, Rf value at the Re place and above be the less component I I of polarity.Component I is mainly by ginsenoside-R
0,-Rb
1,-Rb
2,-Rb
3,-Rc and-any several or all monomers among the Rd form, component I I then mainly by ginsenoside-Re ,-Rf,--R
F11,-Rg
1,-Rg
2,-Rg
3,-RT
5, and denier-Rh
1With-Rh
2In any several or all monomers form.
Therefore, in ginsenoside's research and production practices, usually with the standard control thing of panaxoside monomer Re as saponin content in the quality of identifying Radix Ginseng and detection ginsenoside or the preparation.
The ginsenoside identifies the earliest and isolating, and is one of the clearest and the most definite panaxoside monomer of character.Know that now ginsenoside-Re structurally is a kind of tetracyclic triterpenoids compound that belongs to dammarane type 20 (s) Protopanaxatriol type.Further structural analysis confirms that it is 20 (s)-Protopanaxatriol-6-[0-α-2 Fructus rhamni (Rhamnus davurica Pall.) pyrans glycosyl (1-2)-β-D-glucose pyranoside]-20-0-β-D glucoside.
Ginsenoside-Re extensively is present among the araliaceae ginseng plant, for example in the root of Radix Ginseng, stem and leaf, alabastrum, the fruit, and the root of Radix Notoginseng, rhizome, Lu Touzhong, and in the root of Radix Panacis Quinquefolii, leaf, fruit.Wherein content is higher in fruit of Radix Ginseng, flower and the alabastrum, is respectively about 6% and about 2.94%.
From ginsenoside's chemistry and pharmaceutical research angle, generally can prepare Radix Ginseng total saponins, and then further therefrom separate and purification panaxoside monomer composition from above-mentioned different natural origins.Yet, production practices angle from medical industry, painstakingly prepare pharmaceutically useful panaxoside monomer composition with it with expensive low yield, it would be better to comparatively easy method and relatively low cost preparation and mainly contain certain monomer, and the compositions that contains a small amount of other monomers or its mixture, condition is said other monomer components or its mixture to the physicochemical property of the principal monomer composition in the said compositions and/or pharmacological activity without any adverse effect, and preferably also has some collaborative or assosting effect between them.
For example, the inventor await the reply No. 98100070, Chinese patent application and the preparation that discloses improvement for No. 98100068 respectively with separate protopanoxadiol group and Protopanaxatriol and organize the method for Saponin, and the pharmaceutical composition and the application thereof that contain protopanoxadiol group and triol group Saponin.Use disclosed preparation method in these patent applications, can improve significantly from Radix Ginseng total saponins and to separate and the product response rate of preparation protopanoxadiol group Saponin and triol group Saponin, simplify production stage greatly and reduce production costs.From the medicinal application angle, though protopanoxadiol group and triol group are formed by the monomer more than six kinds or six kinds separately, but, thereby determined proximity on their each comfortable pharmacologys and the biologic activity because each the monomer member in said two components is at chemical constitution and the qualitative similarity of physics.For example; a large amount of pharmaceutical researches show; protopanoxadiol group Saponin mainly has mutation, free radical resisting, antioxidation and protection biomembrane and cyto-architectural function, and the Protopanaxatriol organizes the effect that Saponin then mainly has radioprotective and promotes anabolism and anti-tissue injury.Therefore, can be under situation about needn't separate with the purification monomeric compound, to organize saponin mixture by the protopanoxadiol group or the Protopanaxatriol of described method preparation, add suitable pharmaceutically acceptable carrier or excipient, and one or more natural or synthetic other active component are made the pharmaceutical composition that is used for particular treatment or prevention purpose.
The same with well-known many natural drugs, the activity of the monomeric compound that extracts from the natural plants, animal and the mineral that comprise panax species and microbial material usually is the less important or auxiliary element synergism relevant with some and and then unites target tissue or organ are brought into play its biological function.Therefore, in many cases, in order to keep the biologic activity of effective ingredient better, reduce the production cost of natural drug, simplifying production technology, usually is with its some component as the natural drug source, or certain component or its subfraction, obtain having the pharmaceutical composition of identical or better pharmacological activity, can add one or more natural or synthetic other active component or its mixture in the said compositions in addition with monomeric compound.
Chinese patent application No. 87108992 (November 24 1987 applying date) discloses and prepared ginsenoside-Re monomer methods from the ginseng juice, this method reclaims ethanol after comprising concentrated solution with the ethanol precipitation ginseng juice, and with the resultant Re monomer of extracted with diethyl ether semifinished product.Then, with the silica gel G post be further purified and from methanol (1: 1) recrystallization, obtain pure basically ginsenoside-Re monomeric compound.In addition, the flat 7-89863 of Japanese Patent Application Laid-Open number (1993 applyings date JIUYUE 24 days) discloses method for preparing ginsenoside-Re from Radix Ginseng, leaf or alabastrum and panaquilon-Re or the application of its pharmaceutical composition in the treatment arrhythmia that makes by described method.The method of describing in this patent application for preparing the ginsenoside comprises: concentrate aqueous methanol or the aquiferous ethanol or the water extract of described medicinal plants, this extract is crossed post (D101) and with aqueous methanol or ethanol elution.To obtain coarse crystallization behind the gained eluent concentrating under reduced pressure.Obtain the pure product of ginsenoside-Re with 70% methanol aqueous solution recrystallization then.But this patent application does not offer some clarification on degree of purity of production.
An object of the present invention is to provide a kind of pharmaceutical composition of forming by ginsenoside-Re component and pharmaceutically acceptable carrier and/or excipient basically.
This purpose preferred embodiment according to the present invention, said ginsenoside-Re component contains the ginsenoside Re monomer that accounts for total ginsenoside's weight at least 80%, and contains the ginsenoside-Rg that accounts for total ginsenoside's weight 10% in the compositions in the said component in addition
2
This purpose preferred embodiment according to the present invention, wherein said compositions also contain or do not contain the active component that one or more natural or synthetic other and ginsenoside-Re have collaborative or assosting effect.
Another object of the present invention provides the method for the pharmaceutical composition that a kind of preparation limits as mentioned, and this method comprises:
Provide basically by ginsenoside-Re and ginsenoside-Rg
2Ginsenoside-Re component of forming; Then said ginsenoside-Re component is mixed mutually with one or more pharmaceutically acceptable carriers and/or excipient.
This purpose preferred embodiment according to the present invention, the preparation method of wherein said ginsenoside-Re component comprises:
Will be as raw-material panax species or its part in the aqueous solution of lower alkyl alcohol lixiviate 12-72 hour; After filtration and the concentrated said lixiviating solution, remove wherein impurity and pigment with alkali metal hydroxide and suitable adsorbing material, to remove desaccharide and protein, the filtration back is with suitable organic solvent extraction and obtain required ginsenoside-Re component behind recrystallization with high concentration ethanol aqueous solution precipitation.
According to this preferred embodiment of the present invention, wherein said panax species is a Radix Ginseng, and wherein said plant part is a fruit of Radix Ginseng.
According to this preferred embodiment of the present invention, wherein omit the preceding step of recrystallization with organic solvent extraction.
According to this preferred embodiment of the present invention, wherein said alkali metal hydroxide is CaOH
2Or lime water solution (lime cream); And wherein said adsorbing material can be selected from activated carbon, kieselguhr, Kaolin, bleaching earth and activated alumina.
According to this preferred embodiment of the present invention, the wherein said organic solvent that is used for extractive crystallization attitude ginsenoside-Re component can be selected from ethyl acetate, dichloromethane and ether, but ethanol ethyl ester preferably.
According to this preferred embodiment of the present invention, the wherein said lower alkyl alcohol that is used to soak panax species or its part is selected from methanol, ethanol, normal propyl alcohol, isopropyl alcohol and n-butyl alcohol.
A further object of the present invention provides the application of pharmaceutical composition in treatment or prevention whole body or local organization ischemia injury that contains ginsenoside-Re component.
According to a preferred embodiment of this purpose of the present invention, wherein said tissue comprises cerebral tissue, cardiac muscular tissue, nephridial tissue and extremity tissue.
The present invention relates to panax species, particularly Radix Ginseng or its part, root, stem and leaf, alabastrum and fruit as plant are raw material, extract and the method for separating panaxoside-Re component, contain pharmaceutical composition as the said ginsenoside-Re component of primary activity composition, and the application of said pharmaceutical composition in treatment or prevention whole body or local organization ischemia injury.
Term as used herein " ginsenoside Re's component " is meant and contains 50 to 95% ginsenoside Res, and the mixture of the relevant panaxoside monomer of other polarity.According to a preferred embodiment of the invention, wherein ginsenoside Re monomer content is preferably more than 80%, and other monomers that wherein contained are selected from panaxoside Rg
1, Rg
2, Rg
3, RT
5, and Rh
1And Rh
2
As previously mentioned, comprise the araliaceae ginseng plant's of Radix Ginseng, Radix Panacis Quinquefolii and Radix Notoginseng root, rhizome, stem and leaf, flower and alabastrum, and all contain abundant ginsenoside, particularly dammarane type Saponin in the fruit.At present, from these plants or its part, separate and identified nearly 20 kinds of dammarane type four-ring triterpenoid monomeric compounds, and their chemical constitution, physicochemical property and biological function carried out studying extensively and profoundly.According to the transfer behavior (Rf value) of these chemical compounds in silica gel thin-layer chromatography (TLC), after suitable eluant expansion, the monomer separation in the Radix Ginseng general glycoside can be become protopanoxadiol group and Protopanaxatriol to organize two big components.Be used to distinguish the separation ginsenoside-Re residing position after swimming on the silica gel plate just of dividing a word with a hyphen at the end of a line of protopanoxadiol component and three alkoxide components.
Ginsenoside-Re is separated from Radix Ginseng by people such as Sanata at first and carries out structure and character and identify (Chem, Pharma, Bult.22,2407-14 (1974)).Because this panaxoside monomer on silica gel thin-layer chromatography (TLC) flat board through launch with organic solvent and migration after, basically be to be in dull and stereotyped centre position (being determined) by its molecular size and charge, so, total ginsenoside roughly is divided into protopanoxadiol group Saponin and the Protopanaxatriol organizes two big components of Saponin according to the difference of the mobility of the panaxoside monomer composition before and after its migration position.In the Chinese patent application No.98100070.3 that submitted on January 22nd, 1998, describing in detail with the Radix Ginseng total saponins is parent material, prepares the method for protopanoxadiol group and triol group Saponin the inventor.From people such as Sanata at first separate and purification ginsenoside-Re monomeric compound since, people have used various method separation and purification ginsenoside-Re.Particularly in recent years the biologic activity of ginsenoside-Re and clinical value are compared research in depth, for example discovery such as Pei Yu duckweed and reported ginsenoside's arrhythmia effect (the clear 7-89863 of TOHKEMY), thus further excite people to separating and the interest of purification ginsenoside-Re and correlational study.Yet, comprise that disclosed ginsenoside-Re isolation and purification method is the conventional method of commonplace use in the prior art document of the inventor's No. 87108002, Chinese patent application and Japanese Patent Application Laid-Open Zhaoping 7-89863 number.For example, these methods also comprise silicagel column or resin column chromatography ground step mostly except that comprising methanol or ethanol extraction, re-crystallization step.Though resin (for example D101) chromatography can constitute a technological progress significantly with respect to silica gel column chromatography, for example use resin column comparatively easy on handling, and cost is lower etc., but, still there is the problem that to use a large amount of solvents and time to handle chromatographic column from large-scale industrialization production angle.
On the other hand, although claim among the 7-89863 of Japanese Patent Application Laid-Open Zhaoping that available its disclosed method makes highly purified ginsenoside-Re monomeric compound, and has higher yield (account for raw material weight about 0.76%), but our repeated experiments and comparative experiments result show, wherein said method is difficult to obtaining enough repeatabilities and high product yield and purity, and also is difficult to realize large-scale industrial production.Particularly as previously mentioned, from the suitability for industrialized production angle, by laboratory method preparation very high-purity is arranged, for example purity is 95% or the above pure ginsenoside-Re of spectrum analysis that particularly reaches, and might not have the value on the industry of corresponding raising.On the contrary, if with ginsenoside-Re as component in the Radix Ginseng total saponins or subfraction, for example subgroup of organizing Saponin as the Protopanaxatriol preparation of assigning to then may have bigger using value and the feasibility on the suitability for industrialized production.
Therefore, an object of the present invention is to provide ginsenoside-Re component, it is about 80% to it is characterized in that wherein the content of ginsenoside-Re (weight ratio) is no less than, and also contains the ginsenoside-Rg of about 10% (w/w) in the said component
2, and ginsenoside-Rg of about 3-5% (w/w)
1
Our result of study shows, in the ginsenoside-Re component by method preparation of the present invention, although there is a small amount of (for example about 10%) ginsenoside-Rg
2, and the ginsenoside-Rg of less amount
1,-Rh
1With-Rh
2, but they to ginsenoside Re's biologic activity without any adverse effect, on the contrary, these monomers and-also may have certain collaborative or assosting effect (data not shown goes out) between the Re.Therefore, from economy and practical application angle, with relative simple but effective method provided by the invention, preparation said ginsenoside-Re component from panax species or its part, and be the primary activity composition with this Re component, adding one or more pharmaceutically acceptable carrier and/or excipient, make the pharmaceutical composition of the various different dosage forms that are suitable for clinical practice, is fully feasible and valuable.
Therefore, another object of the present invention provides the method for producing above-mentioned ginsenoside-Re component, and this method comprises:
To in the aqueous solution of lower alkyl alcohol, soak 12-72 hour as panax species or its part of raw material; After filtering and concentrating, remove impurity and pigment with alkali metal hydroxide and suitable adsorbing material, precipitate to remove desaccharide and protein with dehydrated alcohol or high concentration ethanol aqueous solution then, filter after, obtain required ginsenoside-Re component with suitable organic solvent extraction and recrystallization.
The raw material that is used to prepare ginsenoside-Re component can be panax species or its part that is selected from Radix Ginseng, Radix Panacis Quinquefolii and Radix Notoginseng etc., for example root of these plants, rhizome, stem and leaf, flower or alabastrum, and fruit.Among the embodiment 1 that provides hereinafter, use fruit of Radix Ginseng, special Radix Ginseng marc is as the raw material of preparation ginsenoside-Re component.Discover that the content of Radix Ginseng total saponins is about 4 times of ginsenoside content in the Radix Ginseng in the fruit of Radix Ginseng.Utilize fruit of Radix Ginseng to prepare in Herba Herminii saponin's the practice at us and find, fruit of Radix Ginseng is squeezed the juice and is still contained relatively large ginsenoside in the residual Radix Ginseng marc in back.Therefore, in order to make full use of for example ginseng resource of panax species, can from the fruit of Radix Ginseng that contains more rich ginsenoside, squeeze the ginseng juice, be used to prepare fruit of Radix Ginseng Saponin or its component, collect the residual fruit of Radix Ginseng residue in back of squeezing the juice then, be used to prepare ginsenoside-Re component.
According to the present invention, in order to obtain ginsenoside-Re component, at first panax species such as Radix Ginseng or its part RUGEN, rhizome, stem and leaf, flower or fruit are carried out necessary pretreatment as after cleaning and cutting into suitable fragment or fragment, immerse the aqueous solution of lower alkyl alcohol, the lower alkyl alcohol that for example contains the 1-5 carbon atom, in the aqueous solution as methanol, ethanol, normal propyl alcohol, isopropyl alcohol, n-butyl alcohol, isobutanol or amylalcohol, but better be in methanol or alcoholic acid aqueous solution, soaking at room temperature 12-72 hour.Collect the pure lixiviating solution of said plant or its part then, use conventional filtration device filtering and collecting filter liquid, and add an amount of lime cream (being the cream that calcium hydroxide forms) in water the pH value of filtrate is transferred to about 10-13.Static about 30 minutes to remove the impurity in the extract.In the filtrate of this alkalization, add one or more adsorbents that are selected from activated carbon, Kaolin, kieselguhr, bleaching earth and activated alumina that weight ratio is about 0.3-1.0% then, abundant stir about 30 minutes was also placed 12-48 hour, to remove the pigment in the said lixiviating solution.For the ease of separating, be used for preferably activated carbon of adsorbent of the present invention.
Filter then above-mentioned through with the lixiviating solution of sorbent treatment, collect filtrate after, excessive to wherein slowly adding about 4 times of volumes under strong agitation, the ethanol of concentration 〉=95% is separated out sugar, nucleic acid and protein substance in the said lixiviating solution with precipitation.Static and separate out post precipitation, to filter and/or centrifugal collection supernatant, decompression suction concentrated filtrate is down also reclaimed ethanol.Then the gained concentrated solution is moved in the separatory funnel, to wherein adding the organic solution that is selected from ethanol ethyl ester, dichloromethane and ether, difference according to the partition coefficient of required ginsenoside-Re component between water and these organic solvents, after placing about 12-48 hour, can in organic facies, separate out the crystallization of required ginsenoside-Re component.In case of necessity, can further this crystal be dissolved in the methanol (1: 1) again, obtain said ginsenoside-Re component behind the recrystallization.
Be astoundingly, the inventor finds in the long term studies practice, in order further to simplify the separation and the purification step of ginsenoside Re's component, in pure lixiviate of process and adsorbents adsorb, and high concentration ethanol post precipitation, recrystallization and needn't make organic solvent extraction such as ethyl acetate and ether further repeatedly to the gained supernatant, the ginsenoside Re's component of the present invention that promptly can sufficiently high yield be defined as mentioned.
Therefore, according to one embodiment of the invention, wherein can omit the organic solvent extraction step.Promptly in initial step, use the soak with ethanol according to method of the present invention, continue after step do not need any organic solvent.
The TCL analysis result shows, Zhi Bei ginsenoside of the present invention-Re component contains weight ratio and is about 80% ginsenoside-Re monomer and weight ratio and is about ginsenoside-Rg of 10% as stated above
2All the other 10% mainly by a small amount of ginsenoside-Rg
1, micro-ginsenoside-Rh
1With-Rh
2, and other impurity are formed.
Compare with the conventional method of generally using in the past, major advantage of separating the method for preparing ginsenoside-Re from panax species or its part of the present invention is, save the step of column chromatography in this method, and replace lixiviating solution with lime cream (the emulsified water suspension of calcium hydroxide) and charcoal treatment panax species material, need to consume than multi-solvent and time step thereby saved with the processing chromatographic column.In addition, the method according to this invention, required ginsenoside prepares with pure fully only monomer form, but obtain with the form that comprises the monomeric mixture of 80% (W/W) ginsenoside-Re at least.Said mixture except that the ginsenoside-Re that contains at least 80% (W/W), also contain about 10% (W/W)-Rg
2, and other about 10% (W/W)-Rg
1,-Rh
1,-Rh
2And trace impurity.Comparative experiments is the result prove, use myocardial ischemia one reperfusion injury of ginsenoside of the present invention-Re component treatment laboratory animal (mice), have almost completely identical effect with the ginsenoside-Re monomeric compound of purity about 98%, and in indivedual matched groups even better therapeutic effect (referring to EXPERIMENTAL EXAMPLE 1-3) as seen arranged.
The present invention further provides with said ginsenoside-Re component is the primary activity composition, and contains the pharmaceutical composition of one or more pharmaceutically acceptable carriers and/or excipient.
Can ginsenoside of the present invention-Re component be mixed by proper proportion with one or more pharmaceutically acceptable carriers or excipient according to known method in the pharmaceuticals industry, make the pharmaceutical composition of the different dosage form that is suitable for using clinically.For example said compositions can be mixed with the injection that can supply intravenous, intramuscular, intraperitoneal, subcutaneous, marrowbrain intracavity and intraocular injection administration, perhaps make the tablet, powder agent, pill, capsule and the suspending agent that are suitable for oral administration, and the spray, cream, ointment, elixir and the suppository that are suitable for topical.
In order to prepare the solution that is suitable for the outer administration of gastrointestinal tract, for example can use distilled water, water for injection, isotonic sodium chlorrde solution or glucose solution, perhaps low concentration (for example 1-100mM) phosphate buffered saline (PBS) (PBS) is as carrier or excipient.Can in the preparation of these gastrointestinal tract external administrations, add one or more other auxiliary elements or additives, for example can use ascorbic acid as antioxidant, use sodium benzoate or methyl hydroxybenzoate as antiseptic, use dimethyl sulfoxide as absorption enhancer etc.
In order to prepare tablet, powder agent, suspending agent or the suppository that is suitable for oral administration, can use sucrose, galactose, corn starch, gelatin, lipid, microcrystalline Cellulose, Pulvis Talci etc. as carrier or excipient.Be suitable for also can containing other proper additive in the preparation of oral administration, for example solubilizing agent, disintegrating agent, lubricant, absorption enhancer, dispersant, surfactant, flavouring agent or coloring agent etc. at these.
Said ginsenoside-Re component and the application of the pharmaceutical composition that contains said component in treatment or prevention part or general histanoxia or ischemic injury have been the present invention further provides.
In the mice general anoxia animal test model that element causes on using subcutaneous injection isopropyl kidney, give injection various dose (5 in the mouse peritoneal in advance, 10 and the 15mg/kg body weight) ginsenoside of the present invention-Re component, after about 15 minutes, each experimental mice subcutaneous injection vasoconstrictor isoproterenol (20mg/kg body weight).The animal of using an injecting normal saline is as negative control group, and the animal of ginsenoside-Re monomeric compound of using beta blocker administration Propranolol and purity about 98% respectively is as positive controls.After the injection, animal is placed under the airtight anaerobic environment, observe and the record animals survived time, so as to inferring the ability of ginsenoside of the present invention-inductive general tissue ischemia-anoxia-induced apoptosis of Re component antagonism vasoconstrictor.Experimental result shows, the ginsenoside of intraperitoneal injection in advance-Re component can strengthen the general tissue ischemia anoxia due to the animal antagonism isoproterenol significantly, prolongs the time-to-live (P＜0.01) of animal under the environment that suffocates.And under the situation of using more heavy dose of ginsenoside-Re component, the matched group of anoxybiotic ability of this anti-tissue ischemia and beta blocker administration Propranolol similar (P＜0.01).
Making us interested especially is, high dose (20mg/kg body weight) ginsenoside of the present invention-Re component is compared with the single chemical compound of ginsenoside-Re of our the former purity about 98% for preparing, as if the performance of ginsenoside in this experimental system-Re component have stronger anti-general histanoxia ability (referring to EXPERIMENTAL EXAMPLE 1, table 1).
In addition, we also in the experiment of using the experimental rat acute myocardial infarction model to carry out, have observed ginsenoside of the present invention-Re component to experimental rat acute myocardial infarction scope and animal serum creatine phosphokinase (CPK) and the active influence of lactic acid dehydrogenase (LDH).Briefly, rat is caused acute myocardial infarction model with the coronary artery ligation method down in anesthesia, give the ginsenoside of the present invention-Re component of animal intravenous injection various dose (5,10, the 20mg/kg body weight) then immediately.As negative control group, and be about the animal of 98% ginsenoside-Re monomeric compound and FUFANG DANSHEN ZHUSHEYE as positive controls with the animal pattern of an injecting normal saline to inject the purity of preparation before us.Behind the ligation coronary artery 24 hours in the animal body blood sampling measure CPK and LDH activity, and after handling animal preparation animal cardiac muscle tissue slice (thick about 3mm), the myocardial infarction area of each treated animal is calculated and compares in the dyeing back with weight method.The result shows, ginsenoside of the present invention-Re component can reduce the area of the experimental myocardial infarction of rat acute significantly, and observe after the acute myocardial infarction that the activity (these enzymatic activitys are important zymetology indications of acute myocardial necrosis) of CPK and LKH enzyme is starkly lower than untreated control animals (referring to EXPERIMENTAL EXAMPLE 2 in 24 hours living animal serum, and the blood plasma prostaglandin E table 2),
2(PGE
2) level then obviously raise.The result is similar to previous experiments, in this experimental system, and ginsenoside of the present invention-Re component, as if ginsenoside-Re the monomer than purity about 98% has the effect of better inhibition Experimental Acute Myocardial Infarction.
At last, also observed of the influence of ginsenoside of the present invention-Re component to the ECG change of the inductive acute myocardial ischemia rat of pituitrin.Ginsenoside-the Re of animal intravenous injection various dose (5,10, the 20mg/kg body weight), the about 5 minutes interior injection of pituitrin of posterior veins are also monitored animal ECG change in 20 minutes after this vasoconstrictor of injection.Found that, the animal of injecting various dose ginsenoside of the present invention-Re component in advance is behind injection vasoconstrictor pituitrin, the typical cardiac electrical figure of myocardial ischemia changes (the ST section raises and the T wave height is alarmmed) and is subjected to obvious inhibition (referring to EXPERIMENTAL EXAMPLE 3, table 3).
Although relevant ginsenoside-Re component suppresses body tissue; particularly the mechanism of action of cardiac muscle and cerebral tissue ischemic injury is still not exclusively clear at present; but we in the past in addition-studies show that; in the cerebral tissue ischemic animal model that ligation rat subclavian artery causes; the serum superoxide dismutases activity of the experimental group animal of the ginsenoside-Re component that comes into operation obviously raises than matched group; the level of lipid peroxidation metabolite malonaldehyde then significantly reduces, so infer that ginsenoside-Re component may be relevant with lipid peroxidation with the inhibition oxygen-derived free radicals to the protective effect of the courageous and upright damage of rat cerebral tissue's anoxia.In addition, in the rat heart muscle ischemia model, observe untreated fish group animal blood slurry prostaglandin E
2(PGE
2) level obviously raises, the matched group of injecting ginsenoside-Re component in advance is then than PGE
2Level raises and is subjected to suppressing significantly, shows that ginsenoside-Re component may be by suppressing PGE
2Inductive hat blood vessel hypovolemia, myocardial cell membrane structural deterioration and increase myocardial cell Ca
2+Load and quicken pathologic damage effect such as myocardial cell internal protein degraded and bring into play its function.
Be worth further should be mentioned that, compare with pure basically ginsenoside-Re monomeric compound and have better anti-part or general tissue ischemia-anoxia activity (EXPERIMENTAL EXAMPLE that vide infra 1 and 2) in view of the present invention tentatively proves ginsenoside-Re component, so be expected to use ginsenoside-Re component, rather than pure basically ginsenoside-Re monomeric compound is as the primary activity composition, be used for preparation treatment and prevention of brain, heart, the pharmaceutical composition of tissue local such as kidney and limbs ischemia injury, and be expected with of the present invention with ginsenoside-Re component present some natural drug composition that generally uses clinically of pharmaceutical composition replacement, for example FUFANG DANSHEN ZHUSHEYE as the primary activity composition.Although be not limited to theory, the above-mentioned advantage of inferring ginsenoside-Re component may be owing to a small amount of-Rg in ginsenoside-Re and the said ginsenoside-Re component
2With-Rg
1Deng existing between the monomer component due to some collaborative and/or assosting effect.
Following examples are intended to further describe for example the present invention, rather than limit the await the reply scope of claim of the present invention by any way.
Preparation embodiment 1: with the Radix Ginseng marc is feedstock production ginsenoside-Re component
It is the method for feedstock production ginsenoside-Re component that present embodiment is described with the Herba Herminii.At first during being used for fruit of Radix Ginseng residue (about 1000g) residual behind fruit of Radix Ginseng preparation Herba Herminii saponin, add appropriate amount 80% (V/V) ethanol, under room temperature, soaked about 24 hours behind the mixing.With conventional filtration device suction filtration gained soak to remove insoluble matter.In the clear filtrate of gained, add a small amount of lime cream (CaOH
2Aqueous suspension) its PH is transferred to about 12, adds activated carbon (8.0g) then, and fully stirred 10 minutes.This mixture room temperature placement was used the buchner funnel filtration under diminished pressure after 24 hours.Collection filtrate and adding are therein seldom measured 6N hydrochloric acid filtrate are transferred to PH=7, reduce pressure then to aspirate down to reclaim also concentrated this filtrate of ethanol.Continue to stir down, in the gained concentrated solution, add 95% ethanol of about 4 times of volumes, up to separating out visible precipitate, and filter (G4 funnel) once more and remove these precipitate.Collect filtrate and after decompression concentrates down, the gained concentrated solution is added in the separatory funnel, and adds ethanol ethyl ester (20ml) therein.Vibrate repeatedly gained mixture and placing about 10 minutes, as seen the adularescent crystallization is separated out gradually.Filtration is collected this crystal and it is dissolved in the methanol (1: 1), obtains 3.2g ginsenoside-Re component behind the recrystallization.
The silica gel thin-layer chromatography result shows, it is about 80% to contain ginsenoside-Re monomer in the ginsenoside who makes as stated above-Re component ,-Rg
2Monomer is about 10% ,-Rg
1Monomer 5%, all the other are 2% for seldom measuring ginsenoside-Rh
1,-Rh
2And trace impurity.
Preparation embodiment 2: with the Stem and leaf of Radix Ginseng is feedstock production ginsenoside-Re component
The stem and leaf that present embodiment is described with panax ginseng plant is a raw material, prepares ginsenoside-Re component by the method described in the embodiment 1 basically.Briefly, fresh Stem and leaf of Radix Ginseng cleaned and air-dry after cut into pieces (about 1000g), to wherein adding 90% (V/V) ethanol and immersion 48 hours under room temperature.Filter this soak to remove plant residue and other insoluble matters with the conventional filtration device.In clarifying filtrate, add the 1N sodium hydroxide solution, transfer to PH11.In filtrate, add about 7.5g active carbon then and stirred 15 minutes.Gained mixture room temperature is placed the back of spending the night G3 funnel filtration under diminished pressure.Collect filtrate adding 50% (V/V) aqueous hydrochloric acid solution filtrate is transferred to PH7.5.Aspirate recovery ethanol down in decompression then and obtain concentrated solution.Continue to stir 95% ethanol that in the gained concentrated solution, adds about 4 times of volumes down, with precipitation sugar and protein wherein.After removing by filter precipitate, collect filtrate and concentrating under reduced pressure.This spissated solution is added in the separatory funnel, and adds about 20ml ether therein, after strong agitation, separate out crystallization gradually.The gained crystal is dissolved in the methanol (1: 1), and recrystallization obtains about 3.0g white crystals shape ginsenoside-Re component.
The silica gel thin-layer chromatography analysis result shows, the ginsenoside who makes as stated above-Re component contains 82% the ginsenoside-Re of having an appointment, ginsenoside-Rg of about 10%
2, ginsenoside-Rg of about 5%
1, and ginsenoside-Rh of about 3%
1,-Rh
2And trace impurity.
Preparation embodiment 3: from the Radix Ginseng marc, prepare ginsenoside Re's component with the non-solvent extraction
Basically according to the method described in the embodiment 1, press in the fruit of Radix Ginseng residue (1000g) residual behind the leaf with fruit of Radix Ginseng and to extract the ginsenoside Re, but different be the step of wherein omitting before the recrystallization with ethyl acetate or extracted with diethyl ether.
The silica gel thin-layer chromatography result shows, it is about 81% that the ginsenoside Re's component that makes by the described method of present embodiment contains ginsenoside Re monomer ,-Rg
2Monomer is about 11% ,-Rg
1Monomer is about 6% ,-Rh
1,-Rh
2And other impurity are about 2%.
EXPERIMENTAL EXAMPLE 1: the ginsenoside-inductive mice whole body of Re component p-isopropyl epinephrine
The influence of property anoxia tolerance
The male mice of 70 about 19-21 grams of body weight is divided into 7 groups at random, every group each 10.Three experimental group difference intraperitoneal injection ginsenoside of the present invention-Re component 5,10 and 20mg/kg body weight.Blank group injection equal-volume (2ml) normal saline; Treat matched group the panaxoside monomer chemical compound 20mg/kg body weight and beta-blocker Propranolol (propranolol) the 10mg/kg body weight of intraperitoneal injection purity about 98% respectively for two simultaneously.After the administration 15 minutes, except that the blank group, the equal subcutaneous injection isoproterenol of animal 20mg/kg body weight of all the other each groups.After 15 minutes, each treated animal is almost put into the airtight wide mouthed bottle of 250ml that sodica calx (10g) is housed respectively in the same time again, observe and write down the time-to-live of animal at once.Shown in the following tabulation 1 of result.The result is with myocardial infarction area (mm
2) or the mean+SD of sero-enzyme concentration (units per liter) represent (n=8-10).
The rat body anoxic that table 1 panaxoside-Re component is induced isoprel affect group dosage (mg/kg) time-to-live (minute) survival rate negative control-48.5 ± 2.6*, 6/10 isoprel processed group 20 29.3 ± 4.6 0/10 ginsenoside Re components 5 34.1 ± 6.8 3/10 ginsenoside Re components, 10 44.6 ± 7.7*, 5/10 ginsenoside Re component, 20 47.5 ± 5.4*, 6/10 sterling ginsenoside Re monomer, 20 48.0 ± 3.8*, 6/10 Propranolol, 10 46.8 ± 7.2* 6/10* P＜0.01 of comparing with the isoprel group
As can be seen, the subcutaneous injection isoproterenol can significantly shorten the time-to-live of mice under the normobaric hypoxia environment from last tabulation 1, compares with the negative control group of an injecting normal saline, and difference is (P＜0.01) especially significantly.In advance in the mice body injection ginsenoside of the present invention-Re component 10 or 20mg/kg body weight can resist effectively continue after the animal general anaerobic condition that causes of subcutaneous injection isoproterenol, thereby prolong the time-to-live of animal under the weary oxygen environment of normal pressure greatly, compare with the contrast of beta blocker administration Propranolol (10mg/kg body weight) in advance, its effect is very close, but the therapeutical effect that experimental group showed of injection low dosage ginsenoside-Re component and not obvious.In addition, it can also be seen that as if under similarity condition, the effect of ginsenoside of the present invention-Re component is better than pure basically (purity about 98%) ginsenoside Re monomer chemical compound from the result shown in the table 1.
EXPERIMENTAL EXAMPLE 2: ginsenoside-Re component is to the protection of experimental rat acute myocardial infarction
Effect
70 of the male rats of the about 250-300g of body weight are divided into 7 groups at random, every group each 10.Under etherization face upward the position by known method and be fixed on the operating-table, the 3-4 intercostal is opened breast from the left side, exposes heart, with No. 0 line ligation arteria coronaria left anterior descending branch immediately.Send heart back to thoracic cavity rapidly then, extrude thoracic cavity inner blood and gas, and close the thoracic cavity.Whole surgery was finished in 1 minute.Sublingual vein injection ginsenoside Re monomer 5,10,20mg/kg and ginsenoside of the present invention-Re component (20mg/kg) and FUFANG DANSHEN ZHUSHEYE (2g/kg) respectively after ligation, administration 1 time again after 12 hours.Treat the ligation arteria coronaria after 24 hours, with pentobarbital sodium 30mg/kg (ip) anesthesia, the ventral aorta blood sampling, and measure serum CK and LDH.Take out heart simultaneously, with left chamber flesh crosscut 3-4 thin slice, section is dyeed with chlorination nitro tetrazole orchid (N-BT) behind the normal saline flushing, and the calculating necrotic myocardium accounts for ventricle weight in wet base percentage rate, calculating infarct size (%).Shown in the following tabulation 2 of result.
Table 2 panaxoside-Re component is to protective effect group infarct size (%) CK (U/L) LDH (U/L) the blank group-294.5 of rat acute miocardial infarction ± 44.1 3123.6 ± 451.1 model group 32.5 ± 5.6 366.3 ± 60.2**, 4132.5 ± 786.6** ginsenoside Re monomer 5mg/kg, 25.1 ± 6.9*, 308.4 ± 39.9*, 3510.5 ± 298.6**10mg/kg, 24.3 ± 5.8**, 302.6 ± 30.0**, 3230.3 ± 417.5**20mg/kg, 22.5 ± 7.7**, 301.4 ± 40.2**, 3156.2 ± 428.7** ginsenoside Re component 20mg/kg, 21.4 ± 6.5**, 285.4 ± 45.2**, 3061.5 ± 405.5** danshen injections 20mg/kg, 24.5 ± 4.2**, 307.5 ± 40.2**, 3278.8 ± 315.3**
* compare * P＜0.05 with model group, P＜0.01 * * and blank group be * * P＜0.01 relatively
From result shown in the last table as can be seen, ginsenoside-Re is to the myocardial infarction area of Experimental Acute Myocardial Infarction animal model, and all showed inhibitory action significantly as the CK and the LDH enzymatic activity of the downright bad biochemistry indication of myocardium acute ischemic.In addition, it can also be seen that no matter the ginsenoside Re be single chemical compound or contain ginsenoside-Re and the compositions of other monomeric compounds from result shown in the table 2, to the protective effect of acute myocardial infarction all significantly better than traditional Chinese medicine compound Radix Salviae Miltiorrhizae Injection.Also be worth should be mentioned that further as if the above-mentioned effect of ginsenoside of the present invention-Re compositions be better than the ginsenoside-Re monomeric compound of pure substantially (98% purity).
EXPERIMENTAL EXAMPLE 3: ginsenoside-Re component to the acute core ischemia of rat experimental after electrocardio
The influence that figure changes
Each 25 of the male and female Wistar rats of the about 105g of average weight are divided into 5 groups at random, every group each 10.Three experimental grouies are injected ginsenoside of the present invention-Re component (5,10, the 20mg/kg body weight) through sublingual vein respectively.Negative control group is injected isopyknic normal saline (1ml).Positive controls injection anti-arrhythmic verapamil (Verapamil) (10mg/kg).After 5 minutes, each treated animal is intravenous injection pituitrin (biochemical-pharmaceutical factory, Shenyang) (1 unit/kg), and inject in 10 seconds with constant speed and to finish respectively.Behind injection of pituitrin 15 seconds, 30 seconds and traced the electrocardiogram of animal in 1,2,5,20 minute.Under tabulate and shown that the 30 Electrocardiographic ST sections of animal in second and T ripple change behind the intravenous injection pituitrin in 3.The result represents (n=10) with the mean+SD of electrocardio millivolt (mV) number.
The Electrocardiographic group ST section that influences of acute myocardial ischemia rat that table 3 ginsenoside-Re component is brought out pituitrin, (mV) T ripple, (mV) negative control group 0.19 ± 0.04 0.28 ± 0.08 verapamil processed group, 0.03 ± 0.02***, 0.05 ± 0.02*** experimental group 5mg/kg, 0.11 ± 0.10*, 0.14 ± 0.03*10mg/kg, 0.07 ± 0.03**, 0.10 ± 0.04**20mg/kg, 0.04 ± 0.01***, 0.11 ± 0.02**
Compare with matched group: * p＜0.05, * * p＜0 01, * * * p＜0.001
From the result shown in the table 3 as can be seen, to animal come into operation ginsenoside of the present invention-Re component can obviously suppress continue after the pituitrin Acute Myocardial Ischemia in Rats ECG change of bringing out (the ST section is raised with the T wave height and alarmmed).After promptly injecting ginsenoside-Re component in advance, can suppress the acute hat vasospasm that vasoconstrictor causes effectively, and then alleviate acute myocardial ischemia.
The acute hat vasospasm that gua pipe contracting agent causes, and then alleviate acute myocardial ischemia.
Claims (6)
1, the pharmaceutical composition of forming by ginsenoside-Re component and pharmaceutically acceptable carrier or excipient, wherein said ginsenoside-Re component contains the ginsenoside-Re monomer that accounts for ginsenoside's gross weight about 80 to 95% in the compositions, and is selected from panaxoside Rg
1, Rg
2, Rg
3, RT
5, Rh
1And Rh
2One or more other panaxoside monomers.
2, the method for the pharmaceutical composition of preparation claim 1 qualification, this method comprises: provide basically by about 80% ginsenoside-Re and about 10% ginsenoside-Rg
2And-Rg
1Ginsenoside-Re component of forming, and said ginsenoside-Re component mixed mutually with one or more pharmaceutically acceptable carriers or excipient.
3, according to the method for claim 2, the preparation method of wherein said ginsenoside-Re component comprises: will be as raw-material panax species or its part in the aqueous solution of lower alkyl alcohol lixiviate 12-72 hour; After filtering and concentrating said lixiviating solution, remove wherein impurity and pigment with lime cream and suitable adsorbing material, with high concentration ethanol aqueous solution precipitation with except that desaccharide and protein; The filtration back is with suitable organic solvent extraction and obtain required ginsenoside-Re component behind recrystallization.
4, according to the method for claim 3, wherein said panax species is that Radix Ginseng and wherein said plant part are fruit of Radix Ginseng.
5, according to the method for claim 3, the organic solvent that wherein is used for extractive crystallization attitude ginsenoside-Re component is selected from ethyl acetate, dichloromethane and ether.
6, according to the method for claim 3, wherein said method can be omitted the preceding step with organic solvent extraction of recrystallization.
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| CN98117830A CN1081922C (en) | 1998-08-28 | 1998-08-28 | Medicinal composition containing ginsenoside Re, preparation and usage thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98117830A CN1081922C (en) | 1998-08-28 | 1998-08-28 | Medicinal composition containing ginsenoside Re, preparation and usage thereof |
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| CN1081922C true CN1081922C (en) | 2002-04-03 |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386085C (en) * | 2003-12-17 | 2008-05-07 | 昆明紫健生物技术有限公司 | Fat Emulsion transfusion liquid for preventing and curing tumour and production method thereof |
| CN100443497C (en) * | 2004-04-27 | 2008-12-17 | 吉林省宏久生物科技股份有限公司 | Method for extraction of ginseng fruit glucoside |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310649C (en) * | 2002-11-07 | 2007-04-18 | 北京鑫利恒医药科技发展有限公司 | Application of ginsenoside Re for preparing medicine for curing dysmnesia |
| CN102464693B (en) * | 2010-11-05 | 2015-05-13 | 李慧 | Ginsenoside Re extraction and separation method |
| CN116370367A (en) * | 2023-01-31 | 2023-07-04 | 西安绿天生物技术有限公司 | Plant extract for cosmetics and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033278A (en) * | 1987-11-24 | 1989-06-07 | 白求恩医科大学 | Technology for producing ginsenoside re monomer |
-
1998
- 1998-08-28 CN CN98117830A patent/CN1081922C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033278A (en) * | 1987-11-24 | 1989-06-07 | 白求恩医科大学 | Technology for producing ginsenoside re monomer |
Non-Patent Citations (3)
| Title |
|---|
| 沈阳药科大学学报，12(2) 1995.4.1 刘崇铭等人"人参总皂甙与单体皂甙 Re对动物心功能和血液动力学的影响" * |
| 白求恩医科大学学报，21(3) 1995.1.1 张伟等人"人参皂甙Re对人红细胞膜脂流动性的影响";沈阳药科大学学报，12(2) 1995.4.1 刘崇铭等人"人参总皂甙与单体皂甙 Re对动物心功能和血液动力学的影响" * |
| 白求恩医科大学学报，21(3) 1995.1.1 张伟等人"人参皂甙Re对人红细胞膜脂流动性的影响" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386085C (en) * | 2003-12-17 | 2008-05-07 | 昆明紫健生物技术有限公司 | Fat Emulsion transfusion liquid for preventing and curing tumour and production method thereof |
| CN100443497C (en) * | 2004-04-27 | 2008-12-17 | 吉林省宏久生物科技股份有限公司 | Method for extraction of ginseng fruit glucoside |
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