CN110041386A - Preparation method, Specnuezhenide and its application of Specnuezhenide - Google Patents
Preparation method, Specnuezhenide and its application of Specnuezhenide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Specnuezhenide, Specnuezhenide and its application, which obtains fragrans seed extract the following steps are included: extract the Specnuezhenide in fragrans seed by the way of seepage pressure effects;Specnuezhenide in fragrans seed extract is successively extracted using petroleum ether, methylene chloride, n-butanol, obtains n-butanol layer extract;N-butanol layer extract is subjected to two step normal phase column chromatographies, obtains Specnuezhenide.In the present invention, using fragrans seed as raw material, the Specnuezhenide that purity is up to 95% or more is obtained by operation preparations such as seepage pressure effects, fractional extraction and two step normal phase column chromatographies separation, when the Specnuezhenide is used to prepare anti-obstruction of the heart qi drug or health care product, the formation that Rabbit Blood Platelets agglutination can be significantly inhibited, the metabolic rate for improving cardiac muscle cell, anti-inflammatory activity is improved, inhibit thrombus has important very meaning for prevention and treatment obstruction of the heart qi disease and developing new drug.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, are related to preparation method, Specnuezhenide and its application of a kind of Specnuezhenide.
Background technique
The obstruction of the heart qi, the logical heart close two words, the i.e. obstructed justice of impatency.The obstruction of the heart qi refers to heart arteries and veins impatency in "Nei Jing", and qi and blood is obstructed to be caused
With the pained disease for cardinal symptom.The pained rheumatic heart disease illustrated with modern medicine of the obstruction of the heart qi, coronary disease and angina pectoris have
Have correlation, pathogenic factor mainly due to heart coronary artery vascular sclerosis lesion makes its luminal stenosis lead to treating myocardial ischemia damage,
Myocardium requirementing keto quantity and oxygen-supplying amount dysequilibrium.Clinical manifestation is angina pectoris and myocardial infarction.
Coronary disease and angina pectoris is to make its 60% or more luminal stenosis due to coronary sclerosis lesion, and myocardial ischemia is caused to cause the heart
Caused by imbalance between flesh oxygen demand and oxygen-supplying amount.Myocardial infarction is then since the lumen of coronary artery branch is serious narrow or even complete
Fully closed plug, caused by coronary artery perfusion stops.The pathologic basis of the two is all myocardial damage.Studies have shown that causing the machine of myocardial ischemia
It is formed with a variety of, including atheromatous plaque and thrombosis in coronary artery, coronarospasm, parteriole lesion etc. in cardiac muscle.Mesh
Before, the method for improving myocardial ischemia mainly includes that expansion blood vessel improves supply of blood flow, reduces myocardial oxygen consumption, improves cardiac muscle cell's generation
Thank rate and anti-platelet aggregation.And showing the drug centainly to resist myocardial ischemia through animal and clinical trial has following several classes:
Nitrate esters, calcium antagonist, beta-blocker, angiotensin converting enzyme inhibitors, coronary artery dilator, the specific heart
Rate slows down medicine and Chinese herbal medicine extract etc..
When studying myocardial ischemia, other than using whole animal model, tested with the cardiac muscle cell of in vitro culture
Evaluation is also currently used experimental method.By constructing the model of cardiomyocyte injury induced by hypoxia/reoxygenation, it can reflect the heart indirectly
Dirty ischemia/reperfusion injury situation, simulates the pathophysiological process.In addition, thrombosis to the influence in cardiovascular disease
The confirmation on basis and clinical research has been obtained, and platelet activation aggregation and thrombosis and development are closely related: blood platelet
After activation aggregation, thrombosis can lead to, vasopasm influences blood circulation, ischemic or obstruction is generated, to promote disease
Occurrence and development.Therefore research drug is to platelet aggregation, Myocytes Anoxia injury protection, anti-inflammatory effect, antithrombotic
The influence of effect has important very meaning for prevention and treatment obstruction of the heart qi disease and developing new drug.
Sweet osmanthus Osmanthus fragrans (Thunb.) Lour. also known as sweet-scented osmanthus are one of the ten great tradition famous flowers in China,
It is both famous spice berry and excellent Landscape Trees.It is mainly distributed on to the north of Nanling on the south the Huaihe River of the Qinling Mountains, north
To the Shandong Peninsula and other places.Currently, the analysis and exploitation aspect of osmanthus essential oil and medicinal extract ingredient are related to more to the report of Osmanthus Fragrans resources,
Correlative study is concentrated mainly on the traditional fields such as Osmanthus Fragrans resources, kind, cultivation, development, armaticity.And to the other positions of sweet osmanthus
Seldom, information is extremely limited in terms of being related to the chemistry and its pharmacological activity of nonaro-maticity ingredient for research report.
Cassia bud is the ripening fruits of sweet osmanthus.Currently, China five big main sweet osmanthus producing region (Guilin, xianning,hubei, four
River new capital, Law Firm Suzhou Jiangsu, Zhejiang Hangzhou) can be achieved produce per year 1,000,000 kilograms of dried flower, corresponding osmanthus concrete yield be greater than 1000
Kilogram.Therefore under Osmanthus Fragrans resources background abundant, the annual mature period can generate a large amount of fragrans seed resource therewith.And work as
The application of preceding fragrans seed is most of all to regard agriculture waste other than minority carries out sweet osmanthus nursery and cultivation as germ plasm resource
Object processing, causes very big waste.
Medical usage in relation to sweet osmanthus, modern pharmacology activity aspect only related to sweet osmanthus fragrant ingredient bacteriostasis and
The antioxidant activity of a small amount of sweet osmanthus flavones.Compendium of Material Medica carries: " sweet osmanthus promotes the production of body fluid, wards off smelly, resolving sputum, controls acute toothache." its root,
Flower, fruit can be used as medicine.Fruit of the cassia bud as sweet osmanthus, harvests when mature, after being impregnated with warm water, dries and is used as medicine." plant name
Real figure examines long volume " cassia bud is recorded for treating obstruction of the heart qi pain.However this is only the accumulation of private drug experience, modern pharmacy is to osmanthus
Chemical component and cardiac vascular activity the research report of beggar is extremely limited, therefore fragrans seed is used to treat the material base of the obstruction of the heart qi
With mechanism of action and indefinite.
Currently, Specnuezhenide is mainly acquired from the fruit of glossy privet and fragrans seed.Patents documents are reported from female
The method that Specnuezhenide is prepared in loyal son, prepares Specnuezhenide crude product as patent (CN102372754) is disclosed from the fruit of glossy privet
With the method for high-purity Specnuezhenide, it is pure that this method successively uses macroreticular resin, purification on normal-phase silica gel and reverse phase silica gel technology to be prepared for
Spend the Specnuezhenide up to 98%;Patent (CN101704857A) disclose with extraction, macroreticular resin and reverse phase silica gel technology from
The method of Specnuezhenide is obtained in the fruit of glossy privet;Patent (CN101955504A) is disclosed using high-speed countercurrent chromatography from the fruit of glossy privet
The method of middle separation preparation high-purity Specnuezhenide, glossy privet hardship glycosides and oleanolic acid;Patent (CN106632544A) discloses one
The preparation method of kind of Specnuezhenide reference substance, in the patented technology to Specnuezhenide alcohol extract use in suppress standby column combination pure water
Crystallization technique obtains Specnuezhenide reference substance.Although above-mentioned obtain height from the correlation technique for obtaining Specnuezhenide in the fruit of glossy privet
The Specnuezhenide of purity, but more or less there are operating procedures it is more, preparation flow is long, apparatus for preparation is expensive the problems such as;Together
When, although patent (CN106632544A) is to obtain Specnuezhenide monomer by two steps, preparation step is pressed in its first step
Preparative chromatograph is pressed in middle needs, the instrument price is more expensive, and is not easy to amplification scale up test.In addition, being related to from fragrans seed
Middle separation prepare the method for Specnuezhenide there are the following problems: use be heated to reflux equal extracting modes be easily destroyed effectively at
Dtex Ligustrum lucidum Ait is unfavorable for improving the purity and yield of Specnuezhenide;Extractant includes ethyl acetate in the extraction mode of use,
Wherein the use of ethyl acetate is unfavorable for removing low polar impurity, to be unfavorable for improving the extraction quality of n-butyl alcohol extract;
The isolation technics of use includes three step of MCI column, normal phase silicagel column and reverse phase silica gel column, and complicated operation, is not easy to industrialize
It utilizes, meanwhile, eluant, eluent includes methylene chloride in the isolation technics of use, and wherein methylene chloride is big, at high cost etc. with toxicity
Problem.Therefore, develop a kind of raw material be easy to get, be low in cost, simple process, it is easy to operate, not high to instrument requirements, be easy to big
In the slave fragrans seed of large-scale production obtain high-purity Specnuezhenide method, for utmostly develop and use fragrans seed this
The secondary use value of kind waste resource has a very important significance.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of raw material be easy to get, be low in cost,
Simple process, it is easy to operate, of less demanding to instrument and equipment, be easy to be mass produced, the system of the high Specnuezhenide of product purity
The application of Preparation Method and Specnuezhenide obtained by this method and the Specnuezhenide.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of preparation method of Specnuezhenide, comprising the following steps:
S1, Specnuezhenide in fragrans seed is extracted by the way of seepage pressure effects, obtain fragrans seed extract;
S2, using in fragrans seed extract obtained in petroleum ether, methylene chloride, n-butanol successively extraction step S1
Specnuezhenide obtains n-butanol layer extract;
S3, n-butanol layer extract obtained in step S2 is subjected to two step normal phase column chromatographies, obtains Specnuezhenide.
Above-mentioned preparation method, further improved, the step S1, comprising the following steps:
S1-1, fragrans seed is mixed with solvent, obtains mixture;
S1-2, mixture obtained in step S1-1 is subjected to seepage pressure effects, collects percolate;
Solvent in percolate obtained in S1-3, removal step S1-2, obtains fragrans seed extract.
Above-mentioned preparation method, it is further improved, in the step S1-1, the mass body of the fragrans seed and solvent
Product is than being 1kg: 5L~1kg: 10L;The solvent is methanol solution, ethanol solution, acetone soln, any one in water;Institute
The volumetric concentration for stating methanol solution is 50%~95%;The volumetric concentration of the ethanol solution is 50%~95%;The acetone
The volumetric concentration of solution is 50%~95%;
In the step S1-2, the number of the seepage pressure effects is 2 times~3 times;The time of seepage pressure effects described in single is
6h~for 24 hours.
Above-mentioned preparation method, further improved, the step S2, comprising the following steps:
S2-1, fragrans seed extract obtained in step S1 is dispersed in water, obtains the dispersion of fragrans seed extract
Liquid;
S2-2, using the Specnuezhenide in fragrans seed extract dispersion liquid obtained in petroleum ether extraction step S2-1,
Petroleum ether layer is removed, surplus solution is extract liquor A;
S2-3, using the Specnuezhenide in extract liquor A obtained in methylene chloride extraction step S2-2, remove methylene chloride
Layer, surplus solution are extract liquor B;
S2-4, using the Specnuezhenide in extract liquor B obtained in extracting n-butyl alcohol step S2-3, gained extracting n-butyl alcohol
Liquid is concentrated, and n-butyl alcohol extract is obtained.
Above-mentioned preparation method, it is further improved, in the step S2-1, the matter of the fragrans seed extract and water
Measuring volume ratio is 1kg: 1L;
In the step S2-2, the volume ratio of the fragrans seed extract dispersion liquid and petroleum ether is 1: 1;
In the step S2-3, the volume ratio of the extract liquor A and methylene chloride is 1: 1;
In the step S2-4, the volume ratio of the extract liquor B and n-butanol is 1: 1.
Above-mentioned preparation method, further improved, the step S3, comprising the following steps:
S3-1, n-butanol layer extract is dissolved into solvent, purification on normal-phase silica gel is added, mixing obtains n-butanol layer extraction
Object sample;
S3-2, normal phase column layer is carried out to n-butanol layer extract sample obtained in step S3-1 using normal phase silicagel column
Analysis, after using acetate-methanol-water mixed solution as eluant, eluent carry out gradient elution, collection eluent;
S3-3, using normal phase silicagel column to eluent obtained in step S3-2 carry out normal phase column chromatography, after with second
Acetoacetic ester-methanol-water mixed solution carries out gradient elution as eluant, eluent, collects eluent, obtains Specnuezhenide.
Above-mentioned preparation method, it is further improved, in the step S3-1, the n-butanol layer extract and solvent
Mass volume ratio is 1kg: 1L~1kg: 5L;The solvent is methanol and/or ethyl alcohol;The n-butanol layer extract and positive silicon
The mass ratio of glue is 1: 5~1: 30;The mesh number of the purification on normal-phase silica gel is 100 mesh~200 mesh;
In the step S3-2, the mesh number of purification on normal-phase silica gel is 300 mesh~400 mesh in the normal phase silicagel column;The acetic acid
Ethyl acetate in ethyl ester-methanol-water mixed solution, methanol, water volume ratio be 20: 1: 1~2: 1: 1;
In the step S3-3, the mesh number of purification on normal-phase silica gel is 300 mesh~400 mesh in the normal phase silicagel column;The acetic acid
Ethyl acetate in ethyl ester-methanol-water mixed solution, methanol, water volume ratio be 5: 1: 1~1: 1: 1.
As a general technical idea, the present invention also provides a kind of Specnuezhenides, and the Specnuezhenide is by above-mentioned
Preparation method is made;The purity of the Specnuezhenide is greater than 95%.
As a general technical idea, the present invention also provides a kind of above-mentioned Specnuezhenides to prepare anti-obstruction of the heart qi drug
Or the application in health care product.
Above-mentioned application, it is further improved, tablet, glue is made in the Specnuezhenide and pharmaceutically acceptable carrier
Wafer, granule, pill, pellet, powder, pill, decoction, syrup, mixture, soft extract or extract dosage form drug or
Health care product.
In the present invention, the structure of Specnuezhenide are as follows:
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of preparation methods of Specnuezhenide, using fragrans seed as raw material, by fragrans seed
It carries out seepage pressure effects, fractional extraction and the separation of two step normal phase column chromatographies and high-purity Specnuezhenide is prepared.In the present invention, use
The mode of seepage pressure effects can be to avoid the destruction to effective component Specnuezhenide in fragrans seed;Using petroleum ether, methylene chloride,
N-butanol is successively extracted, and can obtain more n-butyl alcohol extracts on the basis of removing low polar impurity as far as possible;
The loss of Specnuezhenide during the separation process can be reduced using two step normal phase column chromatography isolation technics, to improve yield;Together
When separation during use ethyl acetate, methanol, water system, compared with existing methylene chloride, methanol, water system, have toxicity
Smaller, the characteristics of cost is relatively low.The preparation method of Specnuezhenide of the present invention have raw material be easy to get, be low in cost, simple process, behaviour
Make it is convenient, of less demanding to instrument and equipment, be easy to large-scale production, the advantages that product purity is high, can develop to the greatest extent
It is worth using the secondary use of fragrans seed, there is higher use value and application prospect.
(2) in preparation method of the present invention, in two step normal phase column chromatography isolation technics of use, required silica filler and column layer
Analysing material is the very common equipment of medicine intermediate production field, cheap, is easy to large-scale application;And prepared by the present invention
Gradient elution is carried out to normal phase silicagel column using acetate-methanol-water mixed solution in method, is conducive to rapidly remove miscellaneous
Matter separates Specnuezhenide, saves disengaging time and cost.
(3) the present invention also provides a kind of Specnuezhenides, using fragrans seed as raw material, by carrying out diacolation to fragrans seed
It extracts, the Specnuezhenide that purity is up to 95% or more, and the spy female is prepared in fractional extraction and the separation of two step normal phase column chromatographies
When loyal glycosides is used to prepare anti-obstruction of the heart qi drug or health care product, Rabbit Blood Platelets agglutination can be significantly inhibited, improve cardiac muscle cell
Metabolic rate, improve anti-inflammatory activity, inhibit thrombus formation, for prevention and treatment obstruction of the heart qi disease and developing new drug have it is important very
Meaning.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
Fig. 1 is the preparation technology flow chart of Specnuezhenide in the embodiment of the present invention 1.
Fig. 2 is the purity test HPLC spectrogram of the Specnuezhenide prepared in the embodiment of the present invention 1.
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Material employed in following embodiment and instrument are commercially available.In the embodiment of the present invention, unless otherwise noted,
Used technique is common process, and used equipment is conventional equipment, and the data obtained be test more than three times it is flat
Mean value.
Embodiment 1:
A kind of preparation method of Specnuezhenide, preparation process flow are as shown in Figure 1, comprising the following steps:
S1, fragrans seed extract is prepared
S1-1, fragrans seed 5kg is taken, removed the peel, crushed, the ethanol solution that 50L volumetric concentration is 95% is added, mixing is equal
It is even, obtain mixture.
S1-2, mixture obtained in step S1-1 is subjected to seepage pressure effects, is specially continuously added above percolator
The ethanol solution that fresh volumetric concentration is 95%, while at percolator lower exit port, percolate is collected, collects 25L every time.It should
In step, the number of seepage pressure effects is 3 times, and the time of single seepage pressure effects is 12h.
After the completion of S1-3, each seepage pressure effects, using Rotary Evaporators in 40 DEG C, the ethyl alcohol in extracting solution is recovered under reduced pressure,
Gained concentrate merges, and obtains fragrans seed extract.
S2, n-butyl alcohol extract is prepared
S2-1, according to mass volume ratio be 1kg: 1L, fragrans seed extract obtained in step S1 is dispersed in water,
Obtain fragrans seed extract dispersion liquid.
S2-2, according to volume ratio be 1: 1, petroleum ether is added to sweet osmanthus kind obtained in fragrans seed extract dispersion liquid
In seed extract dispersion liquid, the Specnuezhenide in fragrans seed extract dispersion liquid is extracted, removes petroleum ether after the completion
Layer, surplus solution are extract liquor A.
S2-3, according to volume ratio be 1: 1, methylene chloride is added in extract liquor A obtained in step S2-2, to extraction
Specnuezhenide in liquid A is extracted, and removes dichloromethane layer after the completion, and surplus solution is extract liquor B.
S2-4, according to volume ratio be 1: 1, n-butanol is added in extract liquor B obtained in step S2-3, to extract liquor
Specnuezhenide in B is extracted, and gained butanol extraction liquid, in 40 DEG C, is concentrated under reduced pressure using Rotary Evaporators, recycles positive fourth
Alcohol obtains n-butyl alcohol extract.
S3, Specnuezhenide is prepared
S3-1, according to mass volume ratio be 1kg: 2L, n-butanol layer extract obtained in step S2 is dissolved with methanol,
It is dissolved into methanol;It is 1: 15 according to n-butanol layer extract and the mass ratio of purification on normal-phase silica gel, the positive of 100-200 mesh is added
Silica gel mixed sample obtains n-butanol layer extract sample.
S3-2, n-butanol layer obtained in step S3-1 is extracted using 300-400 mesh (purification on normal-phase silica gel) normal phase silicagel column
Object sample carry out normal phase column chromatography, after using acetate-methanol-water mixed solution as eluant, eluent to normal phase silicagel column
Gradient elution is carried out, eluent is collected, specifically: successively use the volume ratio of ethyl acetate, methanol, water for 20: 1: 1,15: 1:
1,10: 1: 1,5: 1: 1,2: 1: 1 acetate-methanol-water mixed solution carries out gradient elution, and each ratio elutes 5
Column volume is concentrated under reduced pressure using Rotary Evaporators in 40 DEG C, 25 parts of fractions is obtained, according to TLC thin layer point plate to each eluting fraction
It merges, is divided into 10 parts.Show that Specnuezhenide ingredient is most concentrated with TLC lamellae, impure least fraction conduct
The sample of next stage.
S3-3, using 300-400 mesh (purification on normal-phase silica gel) normal phase silicagel column to eluent (step obtained in step S3-2
Containing the fraction that Specnuezhenide is most in S3-2) carry out normal phase column chromatography, after with acetate-methanol-water mixed solution
Gradient elution is carried out to normal phase silicagel column as eluant, eluent, collects eluent, specifically: successively use ethyl acetate, methanol, water
Volume ratio be 5:1:1,2:1:1,1:1:1 acetate-methanol-water mixed solution carry out gradient elution, each ratio
8 column volumes are eluted, using Rotary Evaporators in 40 DEG C, each fraction is concentrated under reduced pressure to obtain.It is tracked, is obtained according to TLC thin layer point plate
Fraction where Specnuezhenide is stood, and a large amount of Specnuezhenides are precipitated, and amounts to 3g.Spy glossy privet obtained in the embodiment of the present invention 1
The purity test result of glycosides is as shown in Figure 2.As shown in Figure 2, the purity of Specnuezhenide obtained is greater than in the embodiment of the present invention 1
95%.
Specnuezhenide obtained is white amorphous powder (methanol), molecular formula C in embodiment 131H42O17Molecular weight is
686.2.Specific NMR data is as shown in table 1 below.
The MR data of Specnuezhenide obtained in 1 embodiment 1 of table
Embodiment 2
The anti-obstruction of the heart qi activity rating of fragrans seed extract, n-butyl alcohol extract, Specnuezhenide monomer
Fragrans seed extract obtained, n-butyl alcohol extract, Specnuezhenide are investigated as tested material using in embodiment 1
Following experiment:
Influence to ADP induction Platelet Aggregation in Rabbits function
Weight 2.5~3.0kg health Japan large ear rabbit is taken, auricular vein takes blood 3mL (anti-coagulants: blood=1: 9), at once
It mixes, sealing, 500 revs/min are centrifuged 10 minutes, preparation enrichment thrombocyte plasma (PRP).After isolating PRP, then it is centrifuged
(3000rpm/min, 10min) prepares platelet poor plasma (PPP).PRP is taken to be put into ratio test glass of the 300 μ L with magnetic bead, it is remaining
Blood mixes, and 3000 revs/min, is centrifuged 10 minutes.It takes PPP to be put into the ratio test glass of 300 μ L, after being returned to zero with PRP, takes 200 μ L PRP
Tested material DMSO (dimethyl sulfoxide) solution of 5 μ L equimolar concentrations is added, solvent control group adds isometric(al) solvent DMSO, incubates
After 2min, using 20 μ L5mmol/L ADP as inducer, measurement platelet aggregation reaction.(preparation of ADP: use disodium hydrogen phosphate phosphorus
Acid dihydride sodium prepares buffer (pH=7.4,2mg/mL).T examines the conspicuousness of each group difference.
Influence to AA induction Platelet Aggregation in Rabbits function
Weight 2.5~3.0kg health Japan large ear rabbit is taken, auricular vein takes blood 3mL (anti-coagulants: blood=1: 9), at once
It mixes, sealing, 500 revs/min are centrifuged 10 minutes, preparation enrichment thrombocyte plasma (PRP).After isolating PRP, then it is centrifuged
(3000rpm/min, 10min) prepares platelet poor plasma (PPP).PRP is taken to be put into ratio test glass of the 300 μ L with magnetic bead, it is remaining
Blood mixes, and 3000 revs/min, is centrifuged 10 minutes.It takes PPP to be put into the ratio test glass of 300 μ L, after being returned to zero with PRP, takes 200 μ L PRP
The tested material DMSO solution of 5 μ L equimolar concentrations is added, solvent control group adds isometric(al) solvent DMSO, after incubating 2min, with
20umol/L AA is inducer, and measurement platelet aggregation reacts, and t examines the conspicuousness of each group difference.
Influence to collagen-induced Platelet Aggregation in Rabbits function
Weight 2.5~3.0kg health Japan large ear rabbit is taken, auricular vein takes blood 3mL (anti-coagulants: blood=1: 9), at once
It mixes, sealing, 500 revs/min are centrifuged 10 minutes, preparation enrichment thrombocyte plasma (PRP).After isolating PRP, then it is centrifuged
(3000rpm/min, 10min) prepares platelet poor plasma (PPP).PRP is taken to be put into ratio test glass of the 300 μ L with magnetic bead, it is remaining
Blood mixes, and 3000 revs/min, is centrifuged 10 minutes.It takes PPP to be put into the ratio test glass of 300 μ L, after being returned to zero with PRP, takes 200 μ L PRP
The tested material DMSO solution of 5 μ L equimolar concentrations is added, solvent control group adds isometric(al) solvent DMSO, after incubating 2min, with
1mg/mL collagen is inducer, and measurement platelet aggregation reacts, and t examines the conspicuousness of each group difference.
Under three kinds of guidance models, the experimental results are shown inthe following table for tested material:
Influence of the different tested materials of table 2 to ADP induced platelet aggregation
* is P < 0.05 compared to the blank group, and * * is P < 0.01.
The influence that the different tested materials of table 3 assemble collagen-induced platelet
Concentration (mg/mL) | Maximum aggregation rate (%) | Inhibiting rate (%) | |
Blank | 26.02±10.15 | ||
Specnuezhenide | 2.8 | 18.13±15.49* | 30.32 |
Specnuezhenide | 1.4 | 19.47±9.32 | 25.18 |
Specnuezhenide | 0.7 | 20.71±11.95 | 20.38 |
Fragrans seed extract | 2.8 | 10.75±0.07** | 58.68 |
Fragrans seed extract | 1.4 | 14.57±5.00* | 44.02 |
Fragrans seed extract | 0.7 | 18.95±11.10 | 27.17 |
N-butyl alcohol extract | 2.8 | 14.57±3.08* | 44.02 |
N-butyl alcohol extract | 1.4 | 15.65±0.49* | 39.85 |
N-butyl alcohol extract | 0.7 | 19.55±47.72 | 24.86 |
Danshen injections | 1.4 | 20.2±6.98* | 22.36 |
* is P < 0.05 compared to the blank group, and * * is P < 0.01.
Influence of the different tested materials of table 4 to AA induced platelet aggregation
* is P < 0.05 compared to the blank group, and * * is P < 0.01.
It can be seen from the above results in the model ofthrombocytic aggregation of ADP induction, compared to the blank group, middle and high concentration
Lower fragrans seed extract and n-butanol layer extract show significant inhibiting effect.Inhibiting rate and sun both under high concentration
Property comparison medicine danshen injections it is close, Specnuezhenide has significant inhibiting effect in higher concentrations;Under collagen-induced model,
Compared to the blank group, fragrans seed extract, n-butyl alcohol extract show significant inhibition Rabbit Blood Platelets agglutination.It is high
Their blood platelet inhibiting rate is twice of danshen injections under concentration.In addition, Specnuezhenide is also shown under middle and high concentration
The result higher than positive drug inhibiting rate;In arachidonic acid-induction model, fragrans seed extract and n-butanol under high concentration
Layer extract shows the inhibiting rate higher than danshen injections.The inhibitory effect close to danshen injections is then shown under middle concentration.
In addition, inhibiting rate of the Specnuezhenide under basic, normal, high three kinds of concentration is all higher than positive drug.
Embodiment 3
Fragrans seed extract obtained, n-butyl alcohol extract, Specnuezhenide are investigated as tested material using in embodiment 1
They influence rats in vitro Myocytes Anoxia injury protection
The culture of neonatal rat myocardial cell
The Wistar suckling mouse in 1-3d age, using 75% (unit is v/v) ethyl alcohol soaking disinfection, coring tip tissue is shredded,
It is placed in centrifuge tube, with 10mL, 0.25% (unit is v/v) trypsin solution, is digested in 37 DEG C of water-baths of constant temperature oscillator
10min, while making its natural sedimentation with suction pipe piping and druming tissue 2min.It is to be precipitated completely after, take supernatant into another pipe, be added
The cold culture medium of 2mL terminates digestion, is then centrifuged for 10min, discards supernatant liquid, and 8mL D-Hanks liquid is added into precipitating, finally uses
Cardiac muscle cell is precipitated and is mixed, cell suspension is made and is placed in culture by the DMEM culture medium containing 20% (unit is v/v) fetal calf serum
In bottle.By culture bottle, 37 DEG C of cultures in carbon dioxide incubator, change liquid afterwards for 24 hours and the bromo- 2 '-deoxidation of 0.1mmol/L5- are added
Uridine inhibits fibroblast growth.
Mtt assay measures metabolism of myocardium rate
Coring myocyte's suspension is added in 96 orifice plates with 100 holes μ L/, after culture for 24 hours, is rinsed 3 times with D-hanks liquid,
It is separately added into the DMEM culture medium containing 20% (v/v) fetal calf serum and normal group and model is made in the DMEM culture medium without serum
Group.N is filled with into closed container2, after 2h by culture plate after cultivating 1h under normal circumstances, 5% (v/v) MTT (thiazole is added
It is blue) liquid is discarded supernatant after 20 holes μ L/ culture 4h, the DMSO in 100 holes μ L/ is added, measures 570nm under microplate reader after vibrating 3min
Absorbance.
Influence of the different tested materials of table 5 to metabolism of myocardium rate
Concentration (mol/L) | Light absorption value (%) | |
Normal group | -- | 0.51±0.015 |
Model group | -- | 0.29±0.042 |
Specnuezhenide | 0.1 | 0.49±0.063## |
Specnuezhenide | 0.01 | 0.43±0.038# |
Specnuezhenide | 0.001 | 0.31±0.054 |
Fragrans seed extract | 0.1 | 0.62±0.016## |
Fragrans seed extract | 0.01 | 0.53±0.044## |
Fragrans seed extract | 0.001 | 0.46±0.091# |
N-butyl alcohol extract | 0.1 | 0.54±0.098## |
N-butyl alcohol extract | 0.01 | 0.45±0.026# |
N-butyl alcohol extract | 0.001 | 0.40±0.063 |
Danshen injections | 0.001 | 0.46±0.051 |
*P < 0.05 and normal group ratio;#P < 0.05,##P < 0.01 and model group ratio.
As can be seen from the above table, each group test drug can significantly improve the metabolic rate of cardiac muscle cell.Wherein, fragrans seed mentions
Take object in high, middle concentration, it is better than positive drug danshen injections that n-butanol layer extract and Specnuezhenide are shown in higher concentrations
Metabolic rate.
Embodiment 4
It is anti-to zebra fish to investigate them as tested material for fragrans seed extract obtained, Specnuezhenide using in embodiment 1
Scorching activity rating
Specific embodiment:
Method: using 3dpf (days post fertilization) healthy macrophage fluorescence transgenic zebrafish Tg
(zlyz:EGFP) study sample fragrans seed extract (5 μ g/mL, 10 μ g/mL and 50 μ g/ are distinguished as experimental animal model
) and influence of the Specnuezhenide (1 μ g/mL, 5 μ g/mL and 10 μ g/mL) to zebra fish inflammatory reaction mL.Two kinds of samples respectively with spot
After horse fish is incubated for for 24 hours altogether, each group zebra fish is handled respectively with 20 μM of copper-baths.CuSO4After being protected from light processing zebra fish 1h,
Each group zebra fish is fixed using 4% (v/v) PFA (paraformaldehyde) at room temperature.After fixed 1h, 4% (v/v) is removed
PFA simultaneously cleans zebra fish using PBST (phosphate Tween buffer).The reaction of fluorescence microscopy microscopic observation Macrophage Inflamatory, meter
Number neuromast peripheral macrophage number.Whether judgement sample has anti-inflammatory activity.
Zebrafish embryo obtains
Male and female zebra fish is separately fed, illumination 14h/ dark 10h alternately, timing feed with artificial grain's shape bait and just
The artemia nauplii (Artemia nauplii) hatched.Take the sexually matured zebra fish of health in the ratio of male and female 1: 1 when adopting ovum
It is put into mating cylinder, when next day 9-10 obtains fertilized eggs.Zebrafish embryo culture is moved into after fertilized eggs are carried out disinfection and washed
With water (NaCl containing 5.0mM, 0.17mM KCl, 0.4mM CaCl2, 0.16mM MgSO4) in, optical culture is controlled at 28 DEG C.
Influence of 2 kinds of samples to zebra fish inflammatory reaction
At development of fertilized ova 3dpf (days post fertilization), selected under stereomicroscope normal
Zebrafish embryo moves into 24 well culture plates, 8 pieces of every hole, and each every group of two repeating holes, experiment is repeated twice.It is separately added into
The fragrans seed extract (5 μ g/mL, 10 μ g/mL and 50 μ g/mL) and Specnuezhenide (1 μ g/mL, 5 μ g/mL and 10 of various concentration
μ g/mL), add culture water to 2.0mL.The Indomethacin of 5 μ g/mL is added in positive controls.Then cover, respectively by tested group with
Positive controls zebra fish, which is placed in illumination box (28 DEG C), allows embryo to continue to develop.After 2h, with 20 μM of CuSO4Solution difference
Handle the above each group zebra fish.It separately sets blank control group zebra fish and is placed in illumination box when this group of zebra fish develops 3dpf
(28 DEG C) allow embryo to continue to develop.This group of zebra fish does not add 20 μM of CuSO after 2h4Solution, continuation are protected from light under the conditions of 28 DEG C
Culture.Observation: the reaction of fluorescence microscopy microscopic observation Macrophage Inflamatory counts neuromast peripheral macrophage number.It utilizes
Influence of the Image Pro Plus software statistics sample to inflammatory reaction.Experimental data is indicated with mean ± SD, soft using SPSS
Part carries out variance analysis between each group, and P < 0.05 is to have significant difference, and P < 0.01 is to have extremely significant sex differernce.
Experimental result
For model group compared with blank control group, macrophage migration digital display work increases (P < 0.05).Positive drug group, osmanthus
Under 5 μ g/mL group of seeds of flowering plants seed extract and 1 μ g/mL group zebra fish macrophage migration number of Specnuezhenide are significant compared with model group
It drops (P < 0.05).
Influence (mean ± SD) of the different samples of table 6 to zebra fish macrophage migration number
* indicate that there is significant difference (P < 0.05) compared with blank control, # indicates there is conspicuousness compared with model group
Difference (P < 0.05).
Experimental result discovery tested material fragrans seed extract is 5 μ g/mL dosage, Specnuezhenide in 1 μ g/mL agent in concentration
Under amount, there is anti-inflammatory effect to zebra fish juvenile fish.
Embodiment 5
It is anti-to zebra fish to investigate them as tested material for fragrans seed extract obtained, Specnuezhenide using in embodiment 1
Thrombus activity rating
Specific embodiment
Method: using 3dpf (days post fertilization) health AB system zebra fish as experimental animal model
To zebra when distinguishing study sample 1 (5 μ g/mL, 10 μ g/mL and 50 μ g/mL) and sample B (1 μ g/mL, 5 μ g/mL and 10 μ g/mL)
The influence of fish thrombosis.After two kinds of samples are incubated for 48h altogether with zebra fish respectively, handled respectively with the ferric trichloride of 60 μ g/mL
Each group zebra fish.Under the microscope since being added ferric trichloride, the time of subintestinal vein vascularization thrombus is recorded, judges sample
Whether product have inhibition thrombosis active.
Zebrafish embryo obtains
Male and female zebra fish is separately fed, illumination 14h/ dark 10h alternately, timing feed with artificial grain's shape bait and just
The artemia nauplii (Artemia nauplii) hatched.Take the sexually matured zebra fish of health in the ratio of male and female 1: 1 when adopting ovum
It is put into mating cylinder, when next day 9-10 obtains fertilized eggs.Zebrafish embryo culture is moved into after fertilized eggs are carried out disinfection and washed
With water (NaCl containing 5.0mM, 0.17mM KCl, 0.4mM CaCl2, 0.16mM MgSO4) in, optical culture is controlled at 28 DEG C.
Influence of 2 kinds of samples to zebra fish thrombosis
In development of fertilized ova 3dpf, normal zebrafish embryo is selected under stereomicroscope, moves into 24 well culture plates
In, 10 pieces of every hole, every group of two repeating holes, experiment are repeated twice every time.Add culture water to 2.0mL, as positive controls.Point
Not Jia Ru various concentration sample 1 (5 μ g/mL, 10 μ g/mL and 50 μ g/mL) and sample B (1 μ g/mL, 5 μ g/mL and 10 μ g/
ML), add culture water to 2.0mL.Being placed in illumination box (28 DEG C) allows embryo to continue to develop.In 48hpf, with 60 μ g/mL's
Ferric trichloride handles each group zebra fish respectively.Under the microscope since being added ferric trichloride, subintestinal vein vascularization is recorded
Whether the time of thrombus, judgement sample have the activity of inhibition thrombosis, and observe embryonic death or orthodontic condition.
Experimental result
Influence of the different samples of table 7 to zebra fish thrombosis time
* indicate that there is significant difference (P < 0.05) compared with the control.
Find that there is inhibition thrombosis activity when sample B concentration is 10 μ g/mL by table 7;1 concentration of sample is 50 μ g/mL
When, thrombus generate the time shorten, illustrate sample 1 under the concentration, promote thrombosis, this may under high concentration caused by the heart
The toxicity phenomenas such as packet oedema are related.
It follows that a kind of method that Specnuezhenide is obtained from fragrans seed is provided in the present invention, it is thus obtained
High-purity Specnuezhenide can be used for developing new anti-obstruction of the heart qi drug or health care product, specially by Specnuezhenide and pharmaceutically acceptable
Carrier be made tablet, capsule, granule, pill, pellet, powder, pill, decoction, syrup, mixture, soft extract or
The drug or health care product of extract dosage form.
Above embodiments are only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned reality
Apply example.All technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It is noted that being led for this technology
For the those of ordinary skill in domain, improvements and modifications without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of Specnuezhenide, which comprises the following steps:
S1, Specnuezhenide in fragrans seed is extracted by the way of seepage pressure effects, obtain fragrans seed extract;
S2, using the special female in fragrans seed extract obtained in petroleum ether, methylene chloride, n-butanol successively extraction step S1
Loyal glycosides obtains n-butanol layer extract;
S3, n-butanol layer extract obtained in step S2 is subjected to two step normal phase column chromatographies, obtains Specnuezhenide.
2. preparation method according to claim 1, which is characterized in that the step S1, comprising the following steps:
S1-1, fragrans seed is mixed with solvent, obtains mixture;
S1-2, mixture obtained in step S1-1 is subjected to seepage pressure effects, collects percolate;
Solvent in percolate obtained in S1-3, removal step S1-2, obtains fragrans seed extract.
3. preparation method according to claim 2, which is characterized in that in the step S1-1, the fragrans seed with it is molten
The mass volume ratio of agent is 1kg: 5L~1kg: 10L;The solvent is methanol solution, ethanol solution, acetone soln, appointing in water
It anticipates one kind;The volumetric concentration of the methanol solution is 50%~95%;The volumetric concentration of the ethanol solution is 50%~95%;
The volumetric concentration of the acetone soln is 50%~95%;
In the step S1-2, the number of the seepage pressure effects is 2 times~3 times;The time of seepage pressure effects described in single be 6h~
24h。
4. preparation method according to claim 1, which is characterized in that the step S2, comprising the following steps:
S2-1, fragrans seed extract obtained in step S1 is dispersed in water, obtains fragrans seed extract dispersion liquid;
S2-2, using the Specnuezhenide in fragrans seed extract dispersion liquid obtained in petroleum ether extraction step S2-1, removal
Petroleum ether layer, surplus solution are extract liquor A;
S2-3, using the Specnuezhenide in extract liquor A obtained in methylene chloride extraction step S2-2, remove dichloromethane layer,
Surplus solution is extract liquor B;
S2-4, using the Specnuezhenide in extract liquor B obtained in extracting n-butyl alcohol step S2-3, gained butanol extraction liquid into
Row concentration, obtains n-butyl alcohol extract.
5. the preparation method according to claim 4, which is characterized in that in the step S2-1, the fragrans seed is extracted
The mass volume ratio of object and water is 1kg: 1L;
In the step S2-2, the volume ratio of the fragrans seed extract dispersion liquid and petroleum ether is 1: 1;
In the step S2-3, the volume ratio of the extract liquor A and methylene chloride is 1: 1;
In the step S2-4, the volume ratio of the extract liquor B and n-butanol is 1: 1.
6. preparation method according to any one of claims 1 to 5, which is characterized in that the step S3, including following step
It is rapid:
S3-1, n-butanol layer extract is dissolved into solvent, purification on normal-phase silica gel is added, mixing obtains n-butanol layer extract sample
Product;
S3-2, normal phase column chromatography, knot are carried out to n-butanol layer extract sample obtained in step S3-1 using normal phase silicagel column
Gradient elution is carried out after beam using acetate-methanol-water mixed solution as eluant, eluent, collects eluent;
S3-3, using normal phase silicagel column to eluent obtained in step S3-2 carry out normal phase column chromatography, after with acetic acid second
Ester-methanol-water mixed solution carries out gradient elution as eluant, eluent, collects eluent, obtains Specnuezhenide.
7. preparation method according to claim 6, which is characterized in that in the step S3-1, the n-butanol layer extraction
The mass volume ratio of object and solvent is 1kg: 1L~1kg: 5L;The solvent is methanol and/or ethyl alcohol;The n-butanol layer extraction
Object and the mass ratio of purification on normal-phase silica gel are 1: 5~1: 30;The mesh number of the purification on normal-phase silica gel is 100 mesh~200 mesh;
In the step S3-2, the mesh number of purification on normal-phase silica gel is 300 mesh~400 mesh in the normal phase silicagel column;The ethyl acetate-
Ethyl acetate in the mixed solution of methanol-water, methanol, water volume ratio be 20: 1: 1~2: 1: 1;
In the step S3-3, the mesh number of purification on normal-phase silica gel is 300 mesh~400 mesh in the normal phase silicagel column;The ethyl acetate-
Ethyl acetate in the mixed solution of methanol-water, methanol, water volume ratio be 5: 1: 1~1: 1: 1.
8. a kind of Specnuezhenide, which is characterized in that the Specnuezhenide is by preparation side according to any one of claims 1 to 7
Method is made;The purity of the Specnuezhenide is greater than 95%.
9. a kind of Specnuezhenide as claimed in claim 8 is preparing the application in anti-obstruction of the heart qi drug or health care product.
10. application according to claim 9, which is characterized in that by the Specnuezhenide and pharmaceutically acceptable carrier
Tablet, capsule, granule, pill, pellet, powder, pill, decoction, syrup, mixture, soft extract or extract is made
The drug or health care product of dosage form.
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