CN101935359A - Liriope muscari baily polysaccharide and preparation method thereof - Google Patents
Liriope muscari baily polysaccharide and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of liriope muscari baily polysaccharide, which comprises the following steps of: taking a liriope muscari baily medicinal material, extracting with ethanol with the concentration of more than 75 percent, and discarding an extracting solution; adding water into the residues of the medicinal material for extraction, adding the ethanol in a gradient mode into the extracting solution until the ethanol concentration reaches 60-95 percent, and taking precipitates after placement to obtain ethanol precipitated polysaccharide; and carrying out cellulose DE-52 column chromatography on the ethanol precipitated polysaccharide, sequentially eluting with water and NaCl, collecting a water washing part, and carrying out freeze drying after concentration to obtain the liriope muscari baily (crude) polysaccharide. The invention also provides liriope muscari baily (refined) polysaccharide obtained by the method; and the liriope muscari baily polysaccharide obtained through extraction and refinement by the method has the definite action of treating cardiovascular diseases.
Description
Technical field
The invention provides a kind of liriope muscari Baily polysaccharide and preparation method thereof, especially contain the liriope muscari Baily polysaccharide of steroidal saponin constituents, and the preparation method of purification of this total saponins, this method is extracted the refining liriope muscari Baily polysaccharide that obtains and is had antitumor action.
Background technology
The beginning tuber of dwarf lilyturf is stated from Shennong's Herbal, is enriching yin Chinese medicine commonly used, has the effect of nourishing Yin and moistening lung, reinforcing stomach reg fluid, the relieving restlessness that clears away heart-fire." the class medicinal material tuber of dwarf lilyturf that records of Chinese pharmacopoeia 2010 version (an one) comprises the tuber of dwarf lilyturf and Radix Liriopes, and wherein the source of the tuber of dwarf lilyturf is the dried root of the Liliaceae Ophiopogon tuber of dwarf lilyturf (Ophiopogon japonicus (Thumb.) Ker-Gawl); The source of Radix Liriopes belongs to the dried root of the Hubei tuber of dwarf lilyturf (Liriope spicata (Thunb.) Lour.Var.prolifera Y.T.Ma) and liriope muscari Baily (L.muscari (Decne.) Bailey) for the Liliaceae Radix Liriopes.The tuber of dwarf lilyturf class medicinal material in state-owned 4 big producing regions, be respectively Zhejiang, Sichuan, Hubei and Fujian.Ground main product RADIX OPHIOPOGONIS from Hangzhou of China such as Zhejiang Cixi, Xiaoshan wherein; Main product river, ground such as Mianyang, Sichuan tuber of dwarf lilyturf; And the main producing region of Hubei tuber of dwarf lilyturf in Xiangyang in river Han basin, Xiangcheng, Gucheng city, Laohekou etc.; Liriope muscari Baily mainly is distributed in counties and cities such as Quanzhou, Xianyou, Yongtai.
On the chemical ingredients, except that the homoisoflavone class did not detect, main chemical ingredients classification was similar to the tuber of dwarf lilyturf, also contains trace elements such as saponin(e, amino acid, polysaccharide and copper, iron, magnesium, cobalt in the Radix Liriopes.Because the effect of Radix Liriopes is equal to traditional medicinal material tuber of dwarf lilyturf, so Chang Zuowei is used as medicine the tuber of dwarf lilyturf on an equal basis in actual applications.
The early stage research of class medicinal material mainly concentrates on saponin(e and flavones position to the tuber of dwarf lilyturf, the polysaccharide in the discovered in recent years class tuber of dwarf lilyturf medicinal material also have resist myocardial ischemia, various pharmacological activities such as hypoglycemic, enhancing immunity, antianaphylaxis.The existing research to polysaccharide component in the tuber of dwarf lilyturf class medicinal material at present relates to aspects such as monose composition, polysaccharide structures, pharmacologically active, quality control, for the development and use of polysaccharide component in the tuber of dwarf lilyturf class medicinal material are laid a good foundation, but also comes with some shortcomings.Such as, existing research concentrates on the tuber of dwarf lilyturf, and the Radix Liriopes correlative study is less, does not still have the report of liriope muscari Baily polysaccharide chemistry research at present.The pharmacology activity research aspect concentrates on Crude polysaccharides, lacks the conclusive evidence to effective constituent.Many in the quality control is means with the colorimetric method for determining total sugar content, the method selectivity is not high, being subjected to impurity easily disturbs, sensitivity is limited, and the difference that measurement result is chosen with standard monose has very big-difference, accuracy that can not method for guaranteeing, simultaneously, because the activity of polyose is relevant with its molecular weight distribution often, measures total sugar content merely and controls quality of medicinal material, can not accomplish real rationally effective.Therefore, existing quality controlling means remains further to improve and improve.
Therefore, a kind of preparation separation method that can isolate the liriope muscari Baily polysaccharide is badly in need of in this area, purpose is to obtain having liriope muscari Baily polysaccharide clear and definite relatively composition, that result of treatment is clear and definite, make polysaccharide can be directly used in the clear and definite various preparations of therapeutic purpose of field of medicaments, the medical usage of clear and definite this plant milk extract of standard reduces the side effect of the product that contains the liriope muscari Baily polysaccharide and reduces its medicinal use risk.
Summary of the invention
The present invention develops a kind of liriope muscari Baily polysaccharide and extraction thereof and purified method through a large amount of experiment, and this method is simply efficient, and the resolution height makes that the activity of polysaccharide is more effective, accurate and with strong points, relatively the purity height.
The object of the invention is to provide the preparation method of polysaccharide in a kind of liriope muscari Baily, and the liriope muscari Baily extract that it obtains in the existing conventional technology further through refining, thereby has been purified activeconstituents, makes its useful active effect clearer and more definite.
The tuber of dwarf lilyturf, the early stage research of class medicinal material mainly concentrated on saponin(e and flavones position, discovered in recent years polysaccharide wherein also have resist myocardial ischemia, various pharmacological activities such as hypoglycemic, enhancing immunity, antianaphylaxis.Existing research to polysaccharide component in the tuber of dwarf lilyturf class medicinal material relates to aspects such as chemical structure, pharmacologically active, quality control.Wherein the pharmacology activity research result show Radix Ophiopogonis polysaccharide its have good resist myocardial ischemia, hypoglycemic, enhancing immunity, antianaphylaxis isoreactivity, be one of important function composition.In the middle of the kind that three kinds of pharmacopeia are recorded, the liriope muscari Baily STUDY ON POLYSACHAROSE is less, has not yet to see the report of its structural research.Existing pharmacology activity research concentrates on Crude polysaccharides, lacks the conclusive evidence of efficient part.Many in quality of medicinal material control is means with the colorimetric method for determining total reducing sugar, because the selectivity of colorimetry own is bad, sensitivity is limited, measurement result is chosen difference with the monose contrast very big-difference is arranged, and the activity of polysaccharide molecular weight often is relevant, the simple total sugar content of measuring is controlled quality of medicinal material, can not be real rationally effectively, existing quality controlling means remains further to be improved with perfect.The object of the invention is the research of polysaccharide component in the tuber of dwarf lilyturf class medicinal material is related to aspects such as monose composition, polysaccharide structures, pharmacologically active, quality control.
The object of the invention also is to provide the therapeutic action of liriope muscari Baily polysaccharide activity against myocardial ischemia aspect.
The invention provides the preparation method of polysaccharide in a kind of liriope muscari Baily, this method comprises:
1). get the liriope muscari Baily medicinal material, the extraction using alcohol that 75% concentration is above obtains extracting solution and medicinal material residue;
2). the material residue of getting it filled adds water extraction, and extracting solution concentrates, and it is 60%～95% that gradient adds ethanol to determining alcohol, for example, gradient adds ethanol to determining alcohol 20%, 40%, 60%, 80% or higher, places, get the precipitation part, get ethanol sedimentation polysaccharide, i.e. Crude polysaccharides medicinal extract;
3). the ethanol sedimentation polysaccharide is crossed Mierocrystalline cellulose DE-52 column chromatography, and water, NaCl wash-out are collected the washing part successively, concentrate postlyophilization, get the liriope muscari Baily polysaccharide.
What obtain above-mentioned steps 1) mainly is the saponin(e part, also be that the present invention mainly wants the part of removing, in order to verify that this part mainly is a saponin(e, can adopt the method for further verification experimental verification, comprise: extracting solution concentrates the back and crosses the AB-8 macroporous resin, water and by the ethanol elution of 10% to 90% concentration is collected 60% ethanol elution position successively, can obtain saponin(e position S; Wash-out is very important with the concentration of ethanolic soln, and the ethanol of the preferred 10-90%wt of the present invention carries out wash-out, collects 60% ethanol elution position.Through conventional saponin(e verification method checking, it is a saponin component.Wash-out is very important with the concentration of ethanolic soln, and the ethanol of the preferred 10-90%wt of the present invention carries out wash-out, collects 60% ethanol elution position.
Above-mentioned step 2) comprise that adding ethanol to determining alcohol is 60%, and to determining alcohol be 80%, place, get the precipitation part, 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide, add up to Crude polysaccharides medicinal extract.
Above-mentioned steps 3) be that 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide are crossed Mierocrystalline cellulose DE-52 column chromatography respectively, water, 0.1mol/lNaCl, 0.2mol/lNaCl wash-out successively, collect the washing part, concentrate postlyophilization, get polysaccharide position T1, T2, add up to the liriope muscari Baily polysaccharide.
Conventional explanation to the quality control tuber of dwarf lilyturf or efficient part all is to concentrate on saponin(e, so the present invention prepares saponin(e and polysaccharide respectively, is not saponin(e but polysaccharide with the clear and definite efficient part of method of pharmacological evaluation.
The present invention preferably provides the preparation method of polysaccharide in a kind of liriope muscari Baily, and this method comprises:
1). remove saponin(e: get the liriope muscari Baily medicinal material, the extraction using alcohol that 75% concentration is above obtains extracting solution;
2). extract Crude polysaccharides: the medicinal material residue adds water extraction, extracting solution concentrates, adding ethanol to determining alcohol is 60%, and to alcohol concn be 80%, place, get 60% and 80% precipitation part, promptly get Crude polysaccharides medicinal extract, this Crude polysaccharides medicinal extract comprises 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide;
Also Crude polysaccharides medicinal extract can be dissolved in water, after the lyophilize, get Crude polysaccharides; This 60% ethanol sedimentation partly is T1, and 80% ethanol sedimentation partly is T2, and T1 and T2 merge into Crude polysaccharides;
The ethanol that above-mentioned said extracted saponin(e is used adopts the extraction using alcohol of 75%～95% concentration, in the preferred embodiment of the present invention, and the ethanol of preferred 95% concentration.
3). primary separation: 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide are crossed Mierocrystalline cellulose DE-52 column chromatography respectively, water, NaCl (0.1mol/lNaCl, 0.2mol/lNaCl) wash-out successively, collect the washing part, concentrate postlyophilization, get polysaccharide position T1, T2.
Liriope muscari Baily polysaccharide crude extract of the present invention preferably makes by following method:
1. remove saponin(e: step is with above-mentioned step 1);
2. extraction Crude polysaccharides: step is with above-mentioned step 2), Crude polysaccharides medicinal extract;
3. fractionation precipitation: get a certain amount of Crude polysaccharides medicinal extract and be dissolved in an amount of distilled water, add ethanol successively to determining alcohol 20%, 40%, 60%, 80% fractionation precipitation, all 4 ℃ of standing over night, centrifugal at every turn; 20%, 40% alcohol precipitation does not all have obvious sediment; Get 60%, 80% ethanol sedimentation, suitable quantity of water dissolving postlyophilization obtains 60%, 80% ethanol sedimentation polysaccharide;
4. Mierocrystalline cellulose DE-52 post decolours and primary separation: with 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide, cross Mierocrystalline cellulose DE-52 column chromatography respectively, water, NaCl (0.1mol/lNaCl, 0.2mol/lNaCl) wash-out successively, collect the washing part, concentrate postlyophilization, get polysaccharide position T1, T2.
" medicinal extract " of the present invention is known in this field, and its relative density is about more than 1.25, for example 1.3.
Raw material of the present invention can be product liriope muscari Baily Crude polysaccharides crude extract in the middle of liriope muscari Baily or its, the latter can by commercially available buy or routinely extracting method obtain.
Prove through pharmacological testing, this pharmacological testing will be described in detail in this paper aftermentioned part, and polysaccharide position T2 has activity against myocardial ischemia significantly, therefore, the present invention does further separation and purification to T2, in the hope of therefrom obtaining the go forward side by side structural research of line correlation of homogeneous polysaccharide.Concrete separation and purification process is as follows:
Instrument Agilent 8453 ultraviolet-visible spectrophotometers, Mettler AE-240 electronic balance, SB3200 Branson ultrasonic apparatus (ultrasonic company limited must be believed in Shanghai, 220V, 50kHz), FD-1 freeze drier (Beijing rich doctor health technology company),
Rotavapar R-205 Rotary Evaporators, U.S. Waters company highly effective liquid phase chromatographic system: Waters 515 high performance liquid chromatographs; 717 automatic sampling instruments; 2410 differential refraction detectors; The Empower2 workstation.
Reagent: DE-52 Mierocrystalline cellulose (Whatman company, Beijing is glad through the packing of Bioisystech Co., Ltd of section); SephadexG-25 (Pharmacia Biotech company); Dehydrated alcohol, sulfuric acid, sodium-chlor and anthrone are homemade AR level reagent.
The separation and purification of T2: get T21.266g, cross the SephadexG-25 post, washing, flow velocity 1ml/min, the 5ml/ bottle is collected.Draw the polysaccharide elution curve after sulfuric acid-anthrone develops the color, press elution curve and merge same composition, concentrate, get white powder polysaccharide LMP (469.3mg) after the vacuum lyophilization.
The present invention only adopts water, can elute different polysaccharide according to asynchronism(-nization), by the molecular sieve principle.
Purity is identified: polysaccharide is a macromolecular substance, and its purity rubric can not be weighed with common micromolecular purity rubric.The purity of polysaccharide is only represented being evenly distributed of similar chain length of a certain polysaccharide.The pure product of usually said polysaccharide come down to the homogeneous component of certain molecular weight scope, and its microcosmic is inhomogenous.The method of measuring purity of polysaccharide has functional group analysis, specific rotatory power method, gel chromatography, efficient gel chromatography, high voltage electrophoresis, ultrafiltration centrifugal analysis method etc.Wherein the efficient gel chromatography adopts the polysaccharide dedicated columns, is equipped with differential detector or laser light scattering instrument, have efficient, fast, characteristics such as favorable reproducibility, be to detect the minimum the most frequently used a kind of method of polysaccharide homogeneity error at present.Do not exceed at molecular weight under the prerequisite of Validity Test scope of chromatographic column, the polysaccharide fraction that presents single symmetrical spike in the HPSEC spectrum can be considered to the homogeneous polysaccharide of purifying.
The trial-product preparation: sample thief 6mg, in the molten 2ml moving phase, promptly through the 0.45um filtering with microporous membrane.
Chromatographic condition: Waters highly effective liquid phase chromatographic system (515 pumps, 717 automatic samplers, 2410 differential refraction detectors); TOSOH TSK gel 2500PWxl (the column gel column of 7.8mm * 300mm); TOSOH TSK PWxl (the column gel pre-column of 6.0mm * 4.0cm); Moving phase: 0.05mol/L sodium sulfate; Detector temperature: 35 ℃; Flow velocity: 0.4ml/min; Working time: 45min.Measurement result shows that separating the water-soluble polysaccharide LMP that obtains from liriope muscari Baily is homogeneous polysaccharide.
The structural analysis of liriope muscari Baily polysaccharide LMP:
The semi-automatic point sample instrument of instrument: CAMAG; The CAMAG electric heating panel; CAMAG laminagraphy system; Agilent 6890N-5975C Gas Chromatography-mass Spectrometer (GCMS) is joined the 7683B automatic sampler, band HP-5 type capillary column;
Rotavapar R-205 Rotary Evaporators; Nicolet5700 type Fourier transform infrared spectrometer (U.S. power ﹠ light company); Centaurus Fourier transform infrared microscope; MALDI-TOF-MS (Autoflex, Bruker); The VNS-500 magnetic nuclear resonance analyzer.
Material: sample LMP; The monose reference substance: glucose, semi-lactosi, rhamnosyl, seminose, pectinose, fructose provide by Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Sorbose and wood sugar are produced for Fluka company.
Reagent: 1-Methylimidazole (sigma company) top grade is pure; Oxammonium hydrochloride, diacetyl oxide, triethyl silicane, boron trifluoride diethyl etherate, trifluoracetic acid, methyl iodide analytical pure; Dimethyl sulfoxide (DMSO), pyridine analytical pure are used before using
Molecular sieve is handled.
The complete acid hydrolysis of polysaccharide acid commonly used has sulfuric acid, hydrochloric acid, nitric acid, trifluoroacetic acid etc.Sulfuric acid or trifluoroacetic acid hydrolysis are adopted in the hydrolysis of general neutral sugar more.Sulfuric acid can be at 0.005mol/l-100 ℃ of following quick hydrolysis Polylevulosan and other five carbon or six carbofuran glycan, hydrolysis five carbon or six carbon pyrans glycan under 1mol/l-100 ℃ of condition; Trifluoroacetic acid hydrolysis how with 2mol/l-100～120 ℃ tube sealing heating a few hours, obtains the mixture of monosaccharides after the hydrolysis.Two kinds of method hydrolysis effects are suitable, but there are differences in aftertreatment.Sulfuric acid adopts the method that adds excessive barium carbonate that acid is neutralized more, and the trifluoroacetic acid boiling point is lower, can blow or the method for evaporated under reduced pressure is removed by nitrogen.Hydrochloric acid hydrolysis is used for the glucosides of more difficult hydrolysis more, and as at 2mol/l～6mol/l, 100～120 ℃ of heating a few hours hydrolysis aminoglycosides can obtain the amino monose of thorough hydrolysis, but neutral sugar and acid sugar are destroyed.Nitric acid hydrolysis glycosidic link requires lower concentration, under rare nitric acid condition of 3%.If there is nitrogen oxide, because it has oxidisability, just the same together with hydrochloric acid, the free monosaccharide that hydrolysis is obtained is destroyed.
Comparatively gentle trifluoroacetic acid hydrolysis method and sulphuric acid hydrolysis method are adopted in experiment of the present invention.Sample after the hydrolysis, detects through thin-layer chromatography and GC-MS method under different condition, and the result is as follows.
Trifluoroacetic acid hydrolysis:
Tlc detects:
Development system: ethyl acetate, alcohol and water (70: 22: 8)
Developer 1: aniline-phthalic acid developer; Developer 2:10% sulfuric acid-ethanol developer;
At first do tentatively groping of hydrolysising condition,, detect with the TLC method then at 2mol/lTFA-120 ℃ of difference hydrolysis 1h, 2h, 4h with total reducing sugar.The result shows that under White R, different hydrolysis time samples have corresponding spot on monose reference substance corresponding position; But under 366nm, different hydrolysis time samples have tangible monose fragment spot in the solvent front.(2mol/lTFA-120 ℃-1h/2h/4h) all be not suitable for the hydrolysis of this product of said hydrolyzed condition is described.
The GC-MS method detects:
Further LMP is pressed above-mentioned condition hydrolysis, GC-MS detects and verifies.Obtain behind the total ion current figure carrying out the preliminary search of monose kind with self-built " carbohydrate " spectrum storehouse, this laboratory, the result shows that first peak may be glucose/semi-lactosi/seminose, second peak may be fructose/sorbose, all the other fragmentogram library searchings do not have corresponding monose, show that they all are the impurity fragments that acid hydrolysis produces.Therefore, 2mol/l TFA-120 ℃ of hydrolysis 1h, 2h all are not suitable for LMP.Use more gentle condition (2mol/l TFA-100 ℃-2h, 0.05mol/l TFA-110 ℃-5h), still have tangible impurity peaks, but the intensity of impurity peaks decreases with the gentleness of acid hydrolysis condition instead.Attempt changing the kind of acid, adopt sulphuric acid hydrolysis, as follows.
Tlc detects:
Developping agent: ethyl acetate-pyridine-acetic acid-water (5: 5: 1: 3);
Developer 1: aniline-phthalic acid developer; Developer 2:10% sulfuric acid-ethanol developer;
LMP adds 0.05mol/L H
2SO
4, behind 50 ℃ and 100 ℃ of following hydrolysis 1h, adopt tlc to detect respectively.The result shows that the sample of LMP behind 50 ℃ of hydrolysis 1h has obvious band below the corresponding speckle displacement of monose, illustrate that hydrolysis is incomplete; And LMP is at the sample of 100 ℃ of following hydrolysis 1h, and spot is close with monose reference substance spot, and do not have the fragment spot in the solvent front and occur, and illustrates that this hydrolysising condition is suitable, further with GC-MS method conclusive evidence.
The GC-MS method detects: by the GC-MS detected result as seen, 0.05mol/L desolventize outside the blank peak among the sample total ion current figure of sulfuric acid-100 ℃ hydrolysis 1h, have only peak 1 and peak 2, do not have other impurity fragments, show that the LMP proper hydrolyzing condition is 0.05mol/L sulfuric acid-100 ℃-1h.
Monose compositional analysis: LMP is behind 100 ℃ of hydrolysis 1h of 0.05mol/L sulfuric acid, adopting the GC-MS method to analyze monose forms, after library searching is carried out in total ion current figure that obtains and applicant self-built " carbohydrate " spectrum storehouse, prompting peak 1 may be glucose/semi-lactosi/seminose, and peak 2 may be fructose/sorbose.Get corresponding reference substance derivatize product and carry out the GC-MS analysis, by retention time relatively, determine that peak 1 is glucose, peak 2 is a fructose.Therefore, LMP mainly is made up of fructose, contains minute quantity glucose (ratio of both peak areas is about 32: 1).
The saccharide residue mode of connection is analyzed:
The analysis of general polysaccharides glycosidic link connection site is with behind the polysaccharide exhaustive methylation, after handling by acid hydrolysis, sodium borohydride reduction, acetylize, obtains the ALDI alcohol acetonyl ester of part methylization, carries out GC-MS again and analyzes.But the polysaccharide that contains fructose and glucose during monose is formed simultaneously uses this method to introduce to be obscured.Because fructose is ketose, after reducing, generate corresponding mannitol and sorbitol.If itself contain the glucose of similar connection in the sample, both will obscure.As the fructose and 2 glucose that are connected of 1 connection, after the reduction acetylize, all produced 3,4,6-O-trimethylammonium-1,2,5-O-triacetyl sorbitol so just can't be determined their ratio of components.
And reduce-cracking process is that sample with exhaustive methylation directly reduces with silane, the fracture glycosidic link still keeps the structure that sugar encircles, and the reductive cleavage product of pyranoid ring is still pyranoid ring, the reductive cleavage product of furan nucleus is still furan nucleus, can obviously distinguish on gas-chromatography.As 1,2 glucose that connects behind reductive cleavage, generation be 3,4; 6-O-trimethylammonium-2-O-ethanoyl-1,5-shrink sorbitol, and the generation of the fructose of 1 connection is corresponding 3,4; 6-O-trimethylammonium-1-O-ethanoyl-2,5-shrink sorbitol and mannitol, both can obviously distinguish.
By last figure as seen, 1,2 connect and 6,2 residue of fructose that connect through methylating, behind the reductive cleavage, acetylize, identical product 4 (1-O-Acetyl-2,5-anhydro-3,4,6-tri-O-methyl-D-mannitol) generation being arranged all.In order to determine the area of the compound 4 that two kinds of different mode of connection generate respectively; we (have only 1 with the Root of Medicinal Indian mulberry pentasaccharides; 2 connect) be standard; methylate, behind the reductive cleavage, acetylize; calculate 1,2 and connect the ratio that produces compound 4,5, and then calculate among the LMP 1; 2 connect and 6,2 amounts that connect.
After Root of Medicinal Indian mulberry pentasaccharides and LMP carried out twice methylation reaction, detect, do not have tangible hydroxyl absorption peak through IR, illustrate methylate complete.
GC-MS analyzes: the Root of Medicinal Indian mulberry pentasaccharides of exhaustive methylation and LMP process reductive cleavage and the laggard promoting the circulation of qi of acetylize chromatograph-mass spectrometer coupling analysis mutually again in conjunction with reference, belong to as follows to it successively:
The methylation analysis of LMP (1)
The methylation analysis of LMP (2)
Ratio by calculating Root of Medicinal Indian mulberry pentasaccharides (have only 1,2 connect) methylate-reductive cleavage product 4 and 5 (being mannitol and sorbitol) is 3.45, therefore, the area at the peak 6 of sample LMP multiply by 3.45, add peak 6 areas, the number of promptly corresponding 1,2 connection residue of fructose; After peak 4 deductions 1,2 connect the peak area of residue of fructose generation, add peak 5 areas and promptly connect the residue of fructose numbers corresponding to 6,2.The ratio of various mode of connection is seen above-mentioned two tables (1) and (2).
According to the GC-MS analytical results as can be known, the residue of fructose among the LMP is connected to the master with 2,1, has 2,6 of minority to connect and 1,2,6 connections, and only a few terminal glucose residue is arranged.
According to the classification of Polylevulosan, the Polylevulosan of being made up of 2,1 residue of fructose that connect is called inulin; The Polylevulosan of being made up of 2,6 residue of fructose that connect is called levan; Existing 2, the 6 fructose saccharide residues that connect have the Polylevulosan of 2,1 residue of fructose that connect to be called graminan again.Therefore, LMP is the Polylevulosan of graminan type.
In addition, according to document, might not contain glucose in the Polylevulosan.According to having or not of glucose, Polylevulosan can be divided into Fn type (not containing glucose) and G-Fn type (containing Polylevulosan).The monose compositional analysis of LMP and GC-MS analyze and show that its glucose content is very low, so the fructose chain of the wherein existing Fn type of contriver's deduction, and the fructose chain of G-Fn type is also arranged, and wherein glucosyl residue is connected end.
IR, NMR, the MALDI-TOF-MS spectrometry:
Sample LMP diffuse reflectance infrared spectroscopy absorption band: 3600～3200cm
-1Strong broad peak is intermolecular and the intramolecularly hydroxyl absorbs 3000～2800cm
-1Be c h bond stretching vibration, 1460～1200cm
-1Be the angle vibration of C-H, 1200～1000cm
-1Be C-O stretching vibration among C-O-H and the C-O-C, 931.5cm
-1Be furan nucleus symmetrical stretching vibration, 820.8cm
-1Be the vibration of furan nucleus C-H angle.There is not 1775～1735cm in the collection of illustrative plates
-1And 1250cm
-1So absorption peak is acetyl-containing group not.Find out that by the IR collection of illustrative plates LMP is the furans glycan.
Nuclear magnetic resonance map is analyzed: from LMP's
1As seen, proton signal is serious overlapping 3.2 to δ 4.2 of δ in the H-NMR spectrogram, is difficult for resolving.And be the chemical shift of glucose anomeric proton at the proton signal of δ 5.43, illustrate that the glycosidic link of glucose is configured as α-configuration.(the general α-anomeric proton of configuration glucosides is at δ more than 5.0, and the anomeric proton of beta configuration glucosides is at δ below 5.0).
Lot of documents report Polylevulosan is arranged
13Each peak ownership in the C-NMR spectrum consistently thinks that each carbon atom of residue of fructose lays respectively at three zones: be the peak of fructose end group carbon C-2 about 105ppm; It about 75-83ppm the peak of fructose C-3, C-4, C-5; It about 62-65ppm the peak of fructose C-1, C-6.Each peak of glucosyl residue is easier to separate with the peak ratio of fructose, is respectively: be the peak of C-1 about 92-94ppm; It about 70-74ppm the peak of glucose C-2, C-3, C-4, C-5; Less than the 62ppm zone is the peak of glucose C-6.
LMP's
13The C-NMR spectrum presents the feature of the furan type Polylevulosan of tangible β connection.Because the content of glucose seldom, so signal is not obvious in the carbon spectrum.
According to document, the carbon spectrum signal of LMP is belonged to, in being listed in the table below.
13C-NMR spectrum analysis result shows that LMP contains the residue of fructose of various mode of connection, and is consistent with the analytical results of GC-MS.
Chemical shift ownership (the D of the 13C-NMR spectrum of LMP
2O, δ)
aExpression signal is overlapping
MALDI-TOF-MS measures:
MALDI-TOF-MS is a kind of soft ionization mode, and sensitivity is the highest in the various ionization modes, and the molion of generation is stable, is difficult for cracking, and collection of illustrative plates do not have the multi-charge characteristic among the ESI-MS, is easy to resolve.
Mainly be that one group of signal differs 162 [M+Na] by force successively among the MALDI-TOF-MS figure of LMP
+Peak, i.e. 851.239 (pentasaccharides), 1013.316 (six sugar), 1175.372 (seven sugar), 1499.472 (eight sugar), 1661.522 (nine sugar), 1823.574 (ten sugar), 1985.623 (11 sugar), 2147.885 (ten disaccharides), 2309.691 (ten trisaccharides).MALDI-TOF-MS result shows that LMP is made up of 5～13 sugar.As can be seen from the figure, the MALDI-TOF-MS figure of LMP meets the oligosaccharides of single monose composition or the distinct characteristic of polysaccharide collection of illustrative plates, promptly presents the mass spectra peak that one group of adjacent m/z difference equates, and intensity reduces with the m/z increase.This may be because natural oligosaccharides and polysaccharide molecular weight have certain dispersity, and the desorption ionization of the big quality glycan molecule of less ion pair has restraining effect.
LMP mainly is made up of fructose, contains minute quantity glucose (both peak area ratios are 32: 1), and residue of fructose is β-type, and glucosyl residue is α-type; Fructose is connected to the master with 2-1, has minority 2-6 connection and 2-1,2-6 to connect.
The present invention also provides a kind of liriope muscari Baily polysaccharide, it obtains for adopting above-mentioned method, in this liriope muscari Baily polysaccharide, in this liriope muscari Baily polysaccharide, in fructose content after the hydrolysis of HPLC-ELSD method mensuration, the content of described liriope muscari Baily polysaccharide in medicinal material is 0.6%～6% (measured different batches medicinal material polysaccharide content average and be about 3%); In (T1+T2)/Crude polysaccharides medicinal extract quality, yield is 2%.
The present invention also provides the effect of liriope muscari Baily polysaccharide aspect the treatment cardiovascular disorder, and this cardiovascular disorder comprises myocardial ischemia, diabetes, vascular smooth muscle hyperplasia etc.; Especially liriope muscari Baily polysaccharide purposes in the medicine of preparation treatment myocardial ischemia.Its activity against myocardial ischemia research is as follows:
A 1 animal cleaning level SD rat, male, body weight 200～220g has company's (animal conformity certification number: SCXK (capital) 2002-0003) available from Beijing dimension tonneau China laboratory animal technology.
2 medicine Crude polysaccharides, S, T1, T2; Propranolol is white tablet, and it is the 0.8mg/ml suspension that the adding normal saline solution is made into medicament contg; (Isoprenaline Iso) faces with preceding and is made into 8mg/ml solution with physiological saline Racemic isoproterenol.
3 reagent and instrument urethane; ECG-6951E type electrocardiograph; 751 type digital display spectrophotometers (day island proper Tianjin); Biological function signalling system (Chengdu Instruement Factory).
4 methods cause the influence of rat heart muscle ischemic injuries to Racemic isoproterenol
4.1 56 of experiment grouping SD rats are divided into 7 groups at random, 8 every group, are respectively normal control group, model group, Crude polysaccharides group (10mg/kg), S group (10mg/kg), T1 group (10mg/kg), T2 and organize (10mg/kg); Positive control propranolol group (8mg/kg).
4.2 model preparation and administration, detection normal control group and model group are irritated stomach respectively and are given normal saline solution (1ml/100g); Crude polysaccharides group, S group, T1 group, T2 group give to be mixed with physiological saline the solution of 1mg/ml concentration, 1ml/100g respectively; Positive controls gives the propranolol solution 1ml/100g that physiological saline is mixed with 0.8mg/ml.Every day 1 time, successive administration 5 days.All the other each groups gave 8mg/ml Racemic isoproterenol (0.1ml/100g, subcutaneous injection) respectively at the 2nd day and the 3rd day behind the administration 1h except that the normal control group, and the normal control group waits the physiological saline of capacity.
Write down respectively before the administration and the 2nd injection Racemic isoproterenol after the electrocardiogram(ECG (standard I I leads) of 30min, measure the displacement of J point, calculate the absolute value of respectively organizing rat J point displacement change behind the injection Iso.
4.3 the statistical method data are all represented with x ± S, adopt SPSS 11.10 statistical softwares to carry out statistical study, relatively with the t check, a plurality of sample averages are relatively used one-way analysis of variance to mean between group.
5. result: with the normal control group relatively, the model group rat is behind subcutaneous injection Iso, electrocardiogram J point displacement significantly changes (P＜0.01), the active of LDH obviously raises (P＜0.05) in the blood plasma, myocardial ischemia takes place in prompting, modeling is successful.Electrocardiogram J point displacement behind the model group subcutaneous injection Iso obviously raises, and with normal control group comparing difference statistical significance (P＜0.01) is arranged; Positive drug propranolol group can obviously suppress rat electrocardiogram J point displacement due to the Iso, with model group relatively, difference has statistical significance (P＜0.05); Displacement has obvious restraining effect (P＜0.05) to the T2 group to electrocardiogram J point; The T1 group also has restraining effect to the displacement of electrocardiogram J point, compares not statistically significant (P＜0.05) with model group; Crude polysaccharides group, S group do not have tangible effect to the electrocardiogram J point displacement that Iso causes.
Table 2-1 is to rat electrocardiogram J point Influence of Displacement due to the Iso
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Above presentation of results: T2 group has function of resisting myocardial ischemia, and the T1 group also has trend from mean data, but statistics does not have significance, and other 2 kinds of compositions do not show function of resisting myocardial ischemia.
Detect through HPLC-RI, T1 comprises A, B, C three parts, and T2 mainly contains B, C two portions, and very a spot of A is only arranged.In conjunction with pharmacological activity experiment results, can draw, the position that has activity against myocardial ischemia in the liriope muscari Baily is a polysaccharide.
The tuber of dwarf lilyturf is to the antagonistic action of inducibility vascular smooth muscle cell proliferation
1.1 reagent D-Hanks damping fluid: RPMI-1640 substratum (GIBCO, USA) (every liter contains 20% calf serum, L one glutamine 0.33mg, penicillin 100 μ, Streptomycin sulphate 100 μ, pH:7.4); Digestive pharmaceutical is 0.25 trypsinase; Tetramethyl-nitrogen azoles salt (MTT); Dimethyl sulfoxide (DMSO) (DMSO).
1.2 cell cultures VSMC takes from livid purple blue rabbit aorta middle level childhood.After the carotid artery bloodletting, rapidly aorta being taken out in body under aseptic technique places D-Hanks (to include penicillin 100IU/ml, Streptomycin sulphate lO0 μ g/ml) in the liquid, remove intra-arterial blood and clot, peel off the fiber lipid layer of tunica adventitia vasorum, vertically cut off blood vessel, remove endotheliocyte, washing for several times in D-Hansk liquid, little of the middle level of laterally tearing is cut into the fritter of 1～2mm, is inoculated in a sidewall of culturing bottle, after slowly adding contains the RPMI-1640 of 20% calf serum along the sidewall that does not have the tissue block culturing bottle, culturing bottle is stood on 37 ℃, after 3～4 hours, keep flat culturing bottle and make tissue block be soaked in the nutrient solution fully in the incubator of 5%CO2, after treating cell growth and converging into sheet, the cultivation (nutrient solution contains 10 calf serum) of going down to posterity of the tryptic digestion with 0.25.Observation of cell is logarithmic growth under the light microscopic.Test with 3～5 subtituted culturing cells.
1.3 4x10 is got in the experiment grouping
4The cell of density is in Tissue Culture Plate (Nunc Denmark), and when treating that the cell growth reaches 50% fusion, control group adds 3% calf serum, the blank substratum of 100 μ l; Model group adds the blank substratum of 3% calf serum, 5%HLS, Ins1000 μ and 100 μ l; Tuber of dwarf lilyturf group on the basis of model group, add again 5% the tuber of dwarf lilyturf solution 100 μ l.
1.4MTT colorimetric estimation: in the 24th hour absorption 100 μ l supernatant liquors from 96 well culture plates of former generation VSMC cultivation, the dimethyl sulfoxide (DMSO) that adds 100 μ l to each hole, vibration 5min, after formed formazan fully dissolves behind the cellular metabolism MTT, measure 490nm place light absorption value with enzyme-linked immunosorbent assay instrument.
1.5 statistical procedures statistics adopts the t check, with P＜0.05 expression difference the significance meaning is arranged.
2 cell proliferation control groups, model group and after the tuber of dwarf lilyturf, group was hatched 24 hours jointly with vascular smooth muscle cell as a result, through the MTT colorimetric method for determining, control group is 0.142 ± 0.017; Model group is 0.191 ± 0.004; The tuber of dwarf lilyturf, group was 0.1774 ± 0.004.The result shows: VSMC cell had significant proliferation after hyperinsulinism, the clear modeling of hyperlipemia, compare with control group, and difference has the significance meaning, P＜0.05.On the modeling basis, add the medicine tuber of dwarf lilyturf after, can obviously suppress hyperinsulinism, the hyperlipemia VSMC propagation due to clear, with model group relatively, difference has the significance meaning, P＜0.05.
The result shows: the tuber of dwarf lilyturf, group had significantly resisting vascular smooth muscle cell proliferation effect, and the cellular form change that the growth-stimulating factor is caused has certain mitigation.
Influence to rat diabetes model blood sugar
1.1 animal: male Wistar rat (180～200g), the SPF level, available from Beijing Vital River Experimental Animals Technology Co., Ltd., animal conformity certification number: SCXK (capital) 2002-0003.
1.2 reagent and medicine: acarbose, Bayer HealthCare Co, (lot number: 108579); Streptozotocin is available from Sigma company (lot number: 024k1220); Sucrose test kit (lot number: 060631); Available from Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd..
1.3 instrument: Bio-RAD, the model-680 microplate reader; SHZ82 type gas bath vibrator, big instrument plant produces in the Jintan, Jiangsu; The LDZ0.8 whizzer, Beijing Medical Centrifugal Machine Factory's product; The AT250 electronic balance, Switzerland METTLER company product.
50 of extracting male Wistar rats, body weight 180-200g, (ice bath pH4), brings out rat diabetes with tail vein injection streptozotocin 50mg/kg.The injection streptozotocin is after 7 days, and fasting 10h cuts the tail blood sampling and measures blood sugar.Select the rat of fasting blood sugar more than 11.1mmol/L, be defined as the diabetes model rat, be divided into 5 groups at random, be respectively 20 μ gml-1,50 μ gml-1,100 μ gml-1 organize the tuber of dwarf lilyturf; Model group, 8 every group.Every morning timing ig administration 1 time, administration volume 10mlkg-1, successive administration 7d.Shift to an earlier date fasting 10h, 0.5h measures with method and respectively organizes rat fasting blood-glucose after the last administration.
1.5 the tuber of dwarf lilyturf is to the influence of rat sugar tolerance: medication and dosage are the same, successive administration 7d, fasting 10h before the last administration, rapid ig sucrose solution (4gkg-1) after the last administration, cut tail blood sampling, measure behind the rat oral gavage 0,0.5,1.0 and the blood glucose value of 2.0h with sucrose oxydase-peroxidase method.
2. result
2.1 the tuber of dwarf lilyturf is to the influence of rat diabetes model blood sugar: rat is 7d behind the injection streptozotocin, and fasting blood sugar significantly raises, the modeling success.After 1 week of medication, different dosing group rat blood sugar value and model group significantly descend (P＜0.05), administration group blood sugar reducing function and dosage be proportionate (r=0.995); The results are shown in Table 1.
Table 1 tuber of dwarf lilyturf is to the rat diabetes model
The influence of sugar (x ± S, n=8)
Compare * P＜0.05 with model group; * P＜0.01
2.2 the tuber of dwarf lilyturf is to the influence of rat sugar tolerance
Blood glucose value peaking behind each treated animal ig sucrose 0.5h, 0.5h begins to 2.0h after ig sucrose, three dosed administration groups reduce (P＜0.05) with significance is arranged with time point model group comparison blood glucose value, illustrate that this experiment condition can improve the sucrose tolerance of diabetes rat the following tuber of dwarf lilyturf, the results are shown in Table 2.
Table 2 tuber of dwarf lilyturf is to greatly
The influence of sugar tolerance (χ ± S, n=8)
Compare * P＜0.05 with model group; * P＜0.01
The result shows, 1 week of successive administration back 3 dosage the tuber of dwarf lilyturf of 0.5h after load sucrose, evenly can significance improve rat sucrose tolerance (P＜0.05), reduce the blood glucose value of body after the load sucrose; Simultaneously, can also bring out the blood glucose value (P＜0.05) of diabetes model rat by significance reduction streptozotocin the tuber of dwarf lilyturf, and this effect is dose-dependently.This shows to have tangible blood sugar reducing function the tuber of dwarf lilyturf.
Description of drawings
Fig. 1: the color atlas of the liriope muscari Baily polysaccharide that the present invention obtains, X-coordinate be the time (minute), ordinate zou is an optical density.
Embodiment
Embodiment 1:
Get liriope muscari Baily medicinal material 10kg, 10 times of 95% alcohol immersion spent the night, and extracts 2h, with 8 times of 95% extraction using alcohol 1h, extracting solution discards, and residue adds 10 times of water gagings, extracts 2h, extract 1h with 8 times of water gagings again, merge, concentrate, adding an amount of ethanol to determining alcohol is 60%, alcohol precipitation 24h gets Crude polysaccharides total reducing sugar medicinal extract.
Get Crude polysaccharides medicinal extract (relative density 1.34) 192g, be dissolved in the 640ml distilled water, to add ethanol centrifugal to 20%, 40%, 60%, 80%, 4 ℃ of standing over night of determining alcohol for gradient successively.Get 60%, 80% ethanol sedimentation vacuum-freeze-dry, get 60% alcohol precipitation polysaccharide 1.065g (yield 0.5546%), 80% alcohol precipitation polysaccharide 2.9774g (yield 1.55%).
Get 60% ethanol sedimentation 1.0505g, be dissolved in the 13.5ml distilled water, centrifugal, supernatant liquor DE-52 cellulose column (2.6*32cm) is used the NaCl eluant solution of distilled water, 0.1mol/l, 0.2mol/l respectively.Merge the water elution part, concentrate.Lyophilize promptly obtains T10.9333g (yield 87.63%).
Get 80% ethanol sedimentation 2.9774g, be dissolved in the 10.5ml distilled water, centrifugal, supernatant liquor DE-52 cellulose column (2.6*32cm) is used the NaCl eluant solution of distilled water, 0.1mol/l, 0.2mol/l respectively.Merge the water elution part, concentrate.Lyophilize promptly obtains T22.4955g (yield 83.81%).
Embodiment 2:
Get Crude polysaccharides medicinal extract (relative density 1.34) 1000g that embodiment 1 obtains, be dissolved in the 2000ml distilled water, add ethanol successively to 20%, 40%, 60%, 80%, 4 ℃ of standing over night of determining alcohol, centrifugal.Get 60% and 80% ethanol sedimentation, vacuum lyophilization obtains 60% alcohol precipitation polysaccharide 3.319g (yield 0.33%), 80% alcohol precipitation polysaccharide 204.206g (yield 20.4%).It is the liriope muscari Baily polysaccharide, and color atlas is referring to Fig. 1
Get 60% ethanol sedimentation 2g, be dissolved in the 10ml distilled water, centrifugal, supernatant liquor is crossed DE-52 cellulose column (1.6*47cm), uses the NaCl eluant solution of distilled water, 0.1mol/l, 0.2mol/l respectively.Merge the water elution part, concentrate, lyophilize promptly obtains T11.624g (yield 81.2%).
Get 80% ethanol sedimentation 1.5g, be dissolved in the 10ml distilled water, centrifugal, supernatant liquor is crossed DE-52 cellulose column (1.6*47cm), uses the NaCl eluant solution of distilled water, 0.1mol/l, 0.2mol/l respectively.Merge the water elution part, concentrate, lyophilize promptly obtains T21.3328g (yield 88.85%).
Embodiment 3: liriope muscari Baily polyoses oral hard capsule
Liriope muscari Baily polysaccharide 100 grams
Dry starch 100 grams
Lactose 50 grams
Sodium starch glycolate 5 grams
Cross 80 mesh sieves after said components is mixed respectively, mix, 70% ethanolic soln of the polyvinylpyrrolidone with an amount of 1.5% is made 20～30 order particles, 50 ℃ of dryings, and the whole grain of 20 orders, filled capsules is promptly.
Embodiment 4: the liriope muscari Baily polysaccharide tablet
Liriope muscari Baily polysaccharide 100 grams
Microcrystalline Cellulose 200 grams
Lactose 150 grams
Sodium starch glycolate 15 grams+15 grams
Magnesium Stearate 1.5 grams; Make 1000.
Liriope muscari Baily polysaccharide, Microcrystalline Cellulose, lactose, 1/2 sodium starch glycolate are crossed 80 mesh sieve mixings respectively, make 20 order particles with 70% ethanolic soln of 3% polyvinylpyrrolidone, 50 ℃ of dryings, the whole grain of 18 orders, added the Magnesium Stearate of 120 mesh sieves, 1/2 sodium starch glycolate in addition of 80 mesh sieves, mixing, compressing tablet, dressing.
Embodiment 5: the liriope muscari Baily polyoses oral liquid
Liriope muscari Baily polysaccharide 100 grams
Sodium Benzoate 15 grams
Stevioside 10 grams
Water adds to 10000ml;
Prepare 1000 altogether.
The water of liriope muscari Baily polysaccharide with dosing amount 60% is dissolved fully, Sodium Benzoate and the stevioside water with dosing amount 20% is dissolved fully, merge above-mentioned two solution, benefit adds water to full dose, cross the filter membrane of 0.8um, can according to a conventional method, seal, step such as sterilization makes the liriope muscari Baily polyoses oral liquid.
Claims (10)
1. the preparation method of polysaccharide in the liriope muscari Baily, this method comprises:
1). get the liriope muscari Baily medicinal material, the extraction using alcohol that 75% concentration is above obtains extracting solution and medicinal material residue;
2). the material residue of getting it filled adds water extraction, and extracting solution concentrates, and it is 60%～95% that gradient adds ethanol to determining alcohol, places, and gets the precipitation part, gets ethanol sedimentation polysaccharide, i.e. Crude polysaccharides medicinal extract;
3). the ethanol sedimentation polysaccharide is crossed Mierocrystalline cellulose DE-52 column chromatography, and water, NaCl wash-out are collected the washing part successively, concentrate postlyophilization, get the liriope muscari Baily polysaccharide.
2. the described preparation method of claim 1, wherein, described step 2) comprise that adding ethanol to determining alcohol is 60%, and to determining alcohol be 80%, place, get the precipitation part, 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide, add up to Crude polysaccharides medicinal extract.
3. be 80% the described preparation method of claim 1, wherein, described step 2), place, get the precipitation part for adding ethanol to determining alcohol, 80% ethanol sedimentation polysaccharide, it is a Crude polysaccharides medicinal extract.
4. the described preparation method of claim 1, wherein, step 2) adds water extraction for the medicinal material residue, extracting solution concentrates, and adding ethanol to determining alcohol is 60%, and to alcohol concn be 80%, place, get 60% and 80% precipitation part, promptly get Crude polysaccharides medicinal extract, this Crude polysaccharides medicinal extract comprises 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide.
5. the described preparation method of claim 1, wherein, step 3) is that 60% ethanol sedimentation polysaccharide and 80% ethanol sedimentation polysaccharide are crossed Mierocrystalline cellulose DE-52 column chromatography respectively, water, 0.1mol/lNaCl, 0.2mol/lNaCl wash-out successively, collect the washing part, concentrate postlyophilization, get polysaccharide position T1, T2, add up to the liriope muscari Baily polysaccharide.
6. the described preparation method of claim 1, wherein, this preparation method comprises:
1) get the liriope muscari Baily medicinal material, the extraction using alcohol that 75% concentration is above obtains extracting solution and medicinal material residue;
2) the material residue of getting it filled adds water extraction, and extracting solution concentrates, and adding ethanol to determining alcohol is 80%, places, and gets the precipitation part, 80% ethanol sedimentation polysaccharide, it is a Crude polysaccharides medicinal extract;
3) 80% ethanol sedimentation polysaccharide is crossed Mierocrystalline cellulose DE-52 column chromatography, water, 0.1mol/lNaCl, 0.2mol/lNaCl wash-out are collected the washing part successively, concentrate postlyophilization, get the liriope muscari Baily polysaccharide.
7. liriope muscari Baily polysaccharide, it obtains for adopting each described method of claim 1～6.
8. the described liriope muscari Baily polysaccharide of claim 7, in this liriope muscari Baily polysaccharide, in fructose content after the hydrolysis, the content of liriope muscari Baily polysaccharide in medicinal material that described method obtains is 0.6%～6%.
9. the purposes of the described liriope muscari Baily polysaccharide of claim 7 in the medicine of preparation treatment cardiovascular disorder.
10. the described purposes of claim 9, wherein, described cardiovascular disorder comprises myocardial ischemia, diabetes, vascular smooth muscle hyperplasia.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391388A (en) * | 2011-12-13 | 2012-03-28 | 云南瑞升烟草技术（集团）有限公司 | Method for extracting polysaccharides of ophiopogon japonicus by subcritical water and application in cigarette |
| CN102875689A (en) * | 2012-10-09 | 2013-01-16 | 山东省医药工业研究所 | Preparation method and application of ophiopogon japonicus polysaccharide |
| CN104371037A (en) * | 2014-11-11 | 2015-02-25 | 常州大学 | Method for extracting ophiopogonpolysaccharide from radix ophiopogonis |
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| CN103665175A (en) * | 2012-09-07 | 2014-03-26 | 上海张江中药现代制剂技术工程研究中心 | Fructan extract, and preparation method and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1445244A (en) * | 2003-03-11 | 2003-10-01 | 上海中医药大学附属曙光医院 | Lilyturf root polysaccharide MDG-1 and its separation method and application |
| CN1504486A (en) * | 2002-11-29 | 2004-06-16 | 湖北大学 | Extracting technology for mountain ophiopogon root polysaccharide |
| CN101050241A (en) * | 2007-05-11 | 2007-10-10 | 上海中医药大学 | Method for preparing MDG-1 polysaccharide of lilyturf root |
-
2010
- 2010-09-10 CN CN2010102779303A patent/CN101935359B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1504486A (en) * | 2002-11-29 | 2004-06-16 | 湖北大学 | Extracting technology for mountain ophiopogon root polysaccharide |
| CN1445244A (en) * | 2003-03-11 | 2003-10-01 | 上海中医药大学附属曙光医院 | Lilyturf root polysaccharide MDG-1 and its separation method and application |
| CN101050241A (en) * | 2007-05-11 | 2007-10-10 | 上海中医药大学 | Method for preparing MDG-1 polysaccharide of lilyturf root |
Non-Patent Citations (1)
| Title |
|---|
| 《药物分析杂志》 20091231 张萍等， 短葶山麦冬药材定性与定量分析方法研究 35-38 第29卷, 第1期 2 * |
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|---|---|---|---|---|
| CN102391388A (en) * | 2011-12-13 | 2012-03-28 | 云南瑞升烟草技术（集团）有限公司 | Method for extracting polysaccharides of ophiopogon japonicus by subcritical water and application in cigarette |
| CN102391388B (en) * | 2011-12-13 | 2014-04-02 | 云南瑞升烟草技术（集团）有限公司 | Method for extracting polysaccharides of ophiopogon japonicus by subcritical water and application in cigarette |
| CN102875689A (en) * | 2012-10-09 | 2013-01-16 | 山东省医药工业研究所 | Preparation method and application of ophiopogon japonicus polysaccharide |
| CN104371037A (en) * | 2014-11-11 | 2015-02-25 | 常州大学 | Method for extracting ophiopogonpolysaccharide from radix ophiopogonis |
| CN110117332A (en) * | 2018-02-05 | 2019-08-13 | 上海绿谷制药有限公司 | A kind of isolated sposknikovan and application thereof |
| CN110117332B (en) * | 2018-02-05 | 2022-03-04 | 上海绿谷制药有限公司 | Separated radix sileris polysaccharide and application thereof |
| CN109603196A (en) * | 2018-12-25 | 2019-04-12 | 云南民族大学 | A method of the directional separation target compound from plant |
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