CN101167816B - preparation method of total glycosides of ranunculus and application thereof - Google Patents
preparation method of total glycosides of ranunculus and application thereof Download PDFInfo
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- CN101167816B CN101167816B CN2007100312287A CN200710031228A CN101167816B CN 101167816 B CN101167816 B CN 101167816B CN 2007100312287 A CN2007100312287 A CN 2007100312287A CN 200710031228 A CN200710031228 A CN 200710031228A CN 101167816 B CN101167816 B CN 101167816B
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Abstract
The invention discloses a ranunculus japonicus total glycoside, which is extracted from ranunculus japonicus whole grass which is used as the raw material, and contains 60%-90% of terpenoid saponin ingredient, and the invention provides a medicament method and the application of the ranunculus japonicus total glycoside, and simultaneously provides the application of the ranunculus japonicus totalglycoside on the aspect of preparing cardiovascular medicine, and applications in preparing medicine which resists myocardium hypertrophy, diastoles blood vessel smooth muscle, inhibits heart action,and eases pain and resists inflammatory. The further research of the ranunculus japonicus total glycoside is to discover novel cardiovascular and a pain-easing antiphlogistic medicine which has high selective performance, strong active performance, small toxic and side effects, which can lay a solid foundation for the extensive application of the ranunculus japonicus total glycoside, and has important potential industrialization developing value and makes significant distribution for human beings in developing novel natural medicine.
Description
Technical field
The present invention relates to medical technical field, be specifically related to a kind of preparation method of Herba Ranunculi Japonici herb extract Herba Ranunculi Japonici total glycosides and in the application in medicine and pharmacology field.
Background technology
Diseases such as cardiovascular disease and pain inflammation are common clinical diseases.Myocardial hypertrophy is a kind of target organ reaction to chronic pressure or volume overload generation, can cause myocardium pliability to descend, and the heart systolic and diastolic function is incomplete, causes serious cardiovascular event, causes mortality rate and disability rate to increase;
Inflammation is to have the defense reaction of the biological tissue of vascular system to the damage factor generation, and its local Clinical symptoms is red and swollen heat pain and dysfunction.
At above-mentioned disease, though many medicines are arranged,, seek the new drug of this respect based on the problem of aspects such as curative effect and untoward reaction, remain the focus of research at present and in the future.
Herba Ranunculi Japonici has another name called fish furuncle grass, flipper, smallage, taberpsychine, tiger claw grass, hair Herba Apii graveolentis, the grass that bubbles, and is grown in hills or limit, low gully, limit, paddy field or wet meadow.Chinese medicine dictionary record Herba Ranunculi Japonici is in the migraine that is used for the treatment of among the people, stomachache, and rheumatic arthritis, multiple inflammatory pains such as toothache show the material that contains certain or certain class anti-inflammatory and antalgic in the Herba Ranunculi Japonici.It is studied as new drug, and be applied to clinically, have broad application prospects and social benefit and economic benefit.
Summary of the invention
(Total glycosides of Ranunculus TGOR), by the present invention, can obtain the effective site of Herba Ranunculi Japonici medicine effect clearly to an object of the present invention is to provide a kind of natural plant extracts Herba Ranunculi Japonici total glycosides.
Another object of the present invention provides the preparation method of the simple possible of described Herba Ranunculi Japonici total glycosides.
A further object of the invention provides the application of Herba Ranunculi Japonici total glycosides in the medicine and pharmacology field, specifically is the application at preparation cardiovascular drugs or antalgic anti-inflammatory agent object space face.
Technical scheme of the present invention provides a kind of Herba Ranunculi Japonici total glycosides (TGOR), is that the raw material extraction obtains with the Herba Ranunculi Japonici herb, contains 60%～80% triterpene saponin constituents.
The invention provides the preparation method of described Herba Ranunculi Japonici total glycosides, may further comprise the steps:
(1) the Herba Ranunculi Japonici herb dries chopping, and water or Different concentrations of alcohol or methanol eddy or Different concentrations of alcohol or methanol percolation extract, filter, and merge extractive liquid,, decompression and solvent recovery concentrates;
(2) in (1) concentrated solution, add equal volume of ethyl acetate; Take off a layer water layer solvent evaporated, get paste;
(3) a certain amount of water dissolution of paste, through macroporous resin adsorption, extremely clear with distillating washing resin earlier, reuse 50～95% ethanol or methanol-eluted fractions concentrate, and solvent evaporated gets paste Herba Ranunculi Japonici total glycosides product.
The ethanol or the methanol of water or 10～95% is adopted in the described extraction of step (1), ethanol or methanol-water liquid according to Herba Ranunculi Japonici herb weight ratio be 7: 1 consumption, extract 2～3 times.
The present invention provides the application of described Herba Ranunculi Japonici total glycosides aspect the preparation cardiovascular drugs simultaneously, the especially application aspect preparation resisting cardiac hypertrophy medicine, and the application aspect preparation vasodilator smooth muscle medicine, aspect preparation inhibition heart drugs with function,
The present invention also provides the application of described Herba Ranunculi Japonici total glycosides at preparation antalgic anti-inflammatory agent object space face, can be used for treatment of diseases such as scapulohumeral periarthritis, rheumatic arthritis, the pharmaceutical composition of Herba Ranunculi Japonici total glycosides of the present invention can be forms such as tablet, granule, capsule, pill, drop pill, effervescent tablet, ointment, syrup, injection, oral liquid, mixture, slow releasing agent, controlled release preparation or targeting preparation.
The invention has the beneficial effects as follows:
(1) the invention provides a kind of new natural plant extracts, and carried out the discriminatory analysis research of feasible quality control aspect.
(2) provide the extraction process of Herba Ranunculi Japonici total glycosides, method is simple, and safety is easily gone, and cost is lower.
(3) the present invention has studied the medical usage of Herba Ranunculi Japonici total glycosides, further research to described Herba Ranunculi Japonici total glycosides is expected to find selectivity height, activity is strong, toxic and side effects is little novel cardiovascular and antalgic and inflammation relieving medicine, lay a solid foundation in the extensive use aspect the medical science for Herba Ranunculi Japonici total glycosides, have that important potential industrialization development is worth and the major contribution of human research's new type natural medicine.
Description of drawings
Fig. 1 Herba Ranunculi Japonici total glycosides thin layer chromatography sketch map
Fig. 2 myocardial cell form (HE dyeing)
Fig. 3 Herba Ranunculi Japonici total glycosides is to the inhibitory action of myocardial hypertrophy
Fig. 4 Herba Ranunculi Japonici total glycosides is to the inhibitory action of isolated frog heart
Fig. 5 Herba Ranunculi Japonici total glycosides antagonism NE causes the effect that vascular ring shrinks
Fig. 6 Herba Ranunculi Japonici total glycosides causes that to NE the suppression ratio of vascular ring contraction compares
The inhibitory action of the inductive mice auricle swelling of Fig. 7 Herba Ranunculi Japonici total glycosides xylol
Fig. 8 Herba Ranunculi Japonici total glycosides is to the influence of the inductive rat paw edema of chondrus ocellatus Holmes
Fig. 9 Herba Ranunculi Japonici total glycosides stimulates the influence of inductive rat pain model to hot plate
Figure 10 Herba Ranunculi Japonici total glycosides Dichlorodiphenyl Acetate stimulates the influence of inductive mice pain model
The specific embodiment
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Embodiment 1
The preparation of Herba Ranunculi Japonici total glycosides, composition are differentiated and assay
One, preparation
(1) Herba Ranunculi Japonici herb 2kg dries chopping, uses 14kg water or 10～95% ethanol or methanol eddy or percolation to extract three times, and 80 mesh sieves filter, merge extractive liquid,, and decompression and solvent recovery is concentrated into 1.05kg.
(2) add in (1) concentrated solution with concentrated solution equal volume of ethyl acetate 4 times to ethyl acetate layer be light green color, take off a layer water layer solvent evaporated, paste 200g;
(3) the 200g paste is through the D101 macroporous resin adsorption, and 50% ethanol to 95% methanol-eluted fractions is washed, the pure consumption of 5～10L, concentrate, adopt conventional method normal pressure or evaporated under reduced pressure solvent, get paste Herba Ranunculi Japonici total glycosides product 100g, Herba Ranunculi Japonici total glycosides effective site dry weight is about 5% in the Herba Ranunculi Japonici herb.
Two, composition is differentiated
A. chemistry is differentiated: the Libermann-Burchard reaction: the above-mentioned paste Herba Ranunculi Japonici total glycosides product that takes a morsel, put on the white plaque, with the dissolving of 1ml glacial acetic acid, add 1ml acetic anhydride-concentrated sulphuric acid reagent (19: 1), mix homogeneously, change color are yellow → brown → sepia, change very fast.Show and contain the triterpene saponin constituents.
B. thin layer is differentiated: sample treatment: take by weighing the above-mentioned paste Herba Ranunculi Japonici total glycosides product of 7.5g, with 200-300 purpose purification on normal-phase silica gel column chromatography, CHCl
3-CH
3OH (3: 1-1: 1) gradient elution, collect pipe 51-58 number, solvent evaporated adds 110 ℃ of hydrolysis 3h of 20% aqueous sulfuric acid, takes out cooling, uses chloroform extraction three times, takes off a layer chloroform layer.
Thin layer chromatography board: silica gel-0.3%CMC-Na (1: 2.5) bed board, room temperature is dried, 105 ℃ of activation 30min;
Developing solvent: petroleum ether-ethyl acetate (1: 1);
Uviol lamp is observed: wavelength adopts 254nm or 365nm;
Developer: concentrated sulphuric acid-vanillin.The results are shown in Figure 1.
Three, assay
The content of spectrophotometry Herba Ranunculi Japonici total saponins is adopted in this experiment.With the oleanolic acid is reference substance.Precision takes by weighing extract and places 10ml tool plug test tube in right amount, adds 5% vanillin glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of new preparation, is incubated 15min in 70 ℃ of water-baths, place frozen water to cool off 5min immediately, add glacial acetic acid 5ml, shake up, place 10min.Blank pipe is not except that adding oleanolic acid or Herba Ranunculi Japonici total glycosides, and all the other reagent are identical.According to spectrophotography [one one of Chinese Pharmacopoeia (version in 2005)] experiment, absorbance A is measured at the place immediately at the 560nm wavelength.To the concentration C mapping, getting regression equation is A=4.5245 * 10 with A (absorbance)
-2C+2.032 * 10
-3, r=0.9975.Linear range is 6.0mg/L～18.0mg/L.Methodological study is up to specification.This paper adopts the content of colorimetric method for determining saponin, and method is simple, quick, cheap.Measure Herba Ranunculi Japonici total glycosides content between 60～80% by this law.
Embodiment 2
Resisting cardiac hypertrophy effect experiment of the present invention
1. medicine
Angiotensin II, Sigma company, lot number: 125K51011
DMEM dry powder, Gibco company, lot number: 1324945
Hyclone, Hyclone company, lot number: 20060404
Trypsin, Beijing ancient cooking vessel state, lot number: DH355-2
II Collagen Type VI enzyme, Invitrogen, lot number: DH071
Penicillin, Sigma, lot number: DH231-1
Streptomycin, Sigma, lot number: DH325
5-BrdU, Roche, lot number: 280879
The sulfo group rhodamine B, Sigma-Aldrich, lot number: 3520-42-1
Trichloroacetic acid, Genebase, lot number: G2365
Other reagent are homemade analytical pure
2. instrument
CO
2Incubator, company of Haier;
Microplate reader 850 types, Biotech laboratories;
Biological inverted microscope XSP-15C, the rectangular optical instrument company limited in Shanghai;
Low speed centrifuge LD4-2A type, Beijing Medical Centrifugal Machine Factory;
Vertical pressure steam autoclave YXQ-LS-30S II, Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd..
3. the separation of myocardial cell and cultivation
Give 1～4 day SD neonatal rat of birth sterilization just with 75% ethanol, in aseptic workbench, get its heart, put into the D-Hanks liquid of pre-cooling, lung, trunk and sheath around the rejecting heart.Then heart is changed in another D-Hanks plate that pre-cooling is arranged, the extruding heart is washed hemocyte off.Then all hearts move in the 25ml beaker, and beaker has a homemade magnetic stir bar, shreds heart to grains of sand shape.Add 0.125% pancreatin: 0.08%=1: 1 collagenase digesting liquid, room temperature digestion 6 minutes stops digestion with calf serum, leaves standstill a moment, inhales and removes supernatant.Add isopyknic above-mentioned collagenase digesting liquid again and continue digestion, collect supernatant.Digestion is repeatedly collected all supernatants, the centrifugal 8min of 1000rpm so continuously.Supernatant discarded is with D-Hanks liquid resuspending cell, the centrifugal 8min of 1000rpm.Supernatant discarded.Collecting precipitation is used the 20%FBS suspendible, crosses 120 order cells sieve, be seeded to cultivate 2h in the culture bottle after, the sucking-off supernatant is adjusted cell density to 1 * 10
5～4 * 10
5Individual/ml, add the BrdU that final concentration is 0.1mmol/l.Change 5%FBS cultivation 24h behind the cell culture 72h into and can carry out following experiment.
4. the structure of myocardial hypertrophy model
The structure condition
4.1 examine or check cell density with orthogonal experimental design method, these three factors of angiotensin Ang II concentration and dosing intervention time are determined the optimum condition that the myocardial cell hypertrophy model makes up to the influence of hypertrophic cardiomyopathy cell, experiment repeats 2 times.
Table 1 factor level table
4.2 cell grouping
In cell inoculation to 96 hole, be divided into 9 groups, handle according to the dosing of table 2 method.
Table 2 L
9(3
3)
4.3 index detects
Myocardial cell is a kind of well differentiated terminal cell, and contractile function is arranged, and can not breed generally speaking, with the increase of volume or length, and increases without quantity.The myocardial hypertrophy responsing reaction that to be myocardial cell and Interstitial cell done neuro humor factor or mechanically stretching, these neuro humor factors comprise Angiotensin II (angiotensin II, Ang II), endothelin-1 (endothelin-1, ET-1), catecholamines mediator, insulin-like growth factor-i (insulin-like grow factor-1), tumor necrosis factor-alpha.These factors all are the positivity regulation and control factors that development takes place myocardial hypertrophy.Their corresponding receptors then become the main target molecule of present myocardial hypertrophy control.Bibliographical information Angiotensin II and its I receptor (AT
1) in conjunction with after can directly induce the myocardial cell plumpness.Show as the increase of volume after the myocardial cell plumpness on form, the inherence shows as intracellular total protein content, albumen synthetic quantity and DNA synthetic quantity to be increased.
Aminoacid such as leucine, phenylalanine, lysine, valine are the essential amino acids of human body synthetic protein, therefore mix in the myocardial cell of In vitro culture
3The H-leucine or
14The C-phenylalanine can be investigated the synthetic situation of myocardial cell albumen.In general the incorporation size becomes positive correlation with myocardial cell albumen synthetic quantity.Right with liquid scintillation counter
3H,
14Low energy beta rays such as C carry out radiocounting to be measured, with the albumen synthetic quantity of indirect reaction myocardial cell.
Comprise adenine, guanine, thymus pyrimidine and 4 kinds of bases of cytosine in the dna structure, thymus pyrimidine is the peculiar base of DNA, also is the synthetic essential material of DNA.Thymus pyrimidine consumption prompting DNA is synthetic active, with
3The incorporation of H-TDR can carry out quantitatively the DNA of myocardial cell is synthetic.
Traditional stainings such as Coomassie brilliant blue method, biuret method and lorry-phenol reagent process can be measured the protein content of cell, can carry out quantitatively the intracellular total protein of hypertrophic cardiomyopathy with these methods.Sulfo group rhodamine B (SRB) is a kind of protein binding dyestuff, can combine with the basic amino acid in the cell protein after TCA is fixing and develop the color, present a kind of bright pink, measure absorbance, the numerical value of the cell protein content of a sensitivity can be provided with enzyme-linked immunosorbent assay instrument.Srb assay avoids using radioelement, and experimental technique and mtt assay relatively have its outstanding advantage: it is shorter 1. to operate the required time, and the TCA fixed cell needs 1h, and SRB dyeing only needs 10～30min, and cell adds after the MTT in the mtt assay, also needs 4h; 2. srb assay might not be finished whole operations in one period set time, cell with the fixing back of TCA or with all can deposit after SRB combines, detect again to the suitable time, its OD value is still very stable, and mtt assay must once be finished whole operations; 3. srb assay is sensitiveer more than mtt assay, if in the hole 150 cells are only arranged, can detect with srb assay, and mtt assay can not be measured.With traditional Coomassie brilliant blue method, biuret method and lorry-phenol reagent process, operational approach is simpler, the sensitivity height.
In view of the above, this experiment is dyeed to myocardial cell with SRB, to measure intracellular total protein content.
Cell culture is after the some time, take out 96 orifice plates, siphon away culture medium, every hole adds 50% trichloroacetic acid of 50ul, and fixing 30min in 4 ℃ outwells trichloroacetic acid, deionization washing 5 times, placed under the room temperature 24 hours, and allowed the moisture of culture hole volatilize, then with the 70ul 0.4%SRB 20min that at room temperature dyes, outwell SRB, wash 5 times with 1% acetic acid, placed under the room temperature 24 hours, after the moisture in the culture hole volatilizes, every hole adds 200ul 10mmol/LUnbuffered Tris-base, jolting 10min on oscillator plate detects absorbance in each hole with microplate reader then under 492nm, see Table 3.
The absorptance numerical value of 9 test groups of table 3
| Tested number | AngII concentration (A) | Cell density (B) | Incubation time (C) | The absorbance average |
| ?1?2?3?4?5?6?7?8?9 | 1 1 1 2 2 2 3 3 3 | 123123123? | 1 2 3 2 3 1 3 1 2 | ?0.2695?0.4726?0.3702?0.2715?0.3760?0.3942?0.2307?0.5203?0.5023 |
| ?T1?T2?T3?R×3 | 1.1123 1.0417 1.2763 0.2346 | 0.77171.39191.26670.6206? | 1.1840 1.2464 0.9769 0.2695 | ? |
Above-mentioned result of the test is shown do intuitive analysis: judge secondary factors with R value size, cell density is a principal element, is dosing incubation time and Ang II concentration successively.Optimum condition is A
3B
2C
2, promptly Ang II concentration is 10
-6Mol/L, cell density are 2 * 10
5/ ml, it is 48h that incubation time is intervened in dosing.This is combined in the orthogonal table and does not occur, and in order to prove its reliability, presses A
3B
2C
2Condition is tested, and the absorbance that result of the test obtains is: 0.5433, and the absorbance that obtains than any battery of tests in the table 3 is all big, proves that resulting optimum condition is reliable.
4.4 HE dyeing morphology relatively
The cell grouping:
The diameter that cell culture is equipped with 2 * 2cm wave carrier piece in the bottom is in the culture dish of 3.5cm, and each culture dish adds 1ml cell suspension, is divided into three groups: (1) normal group: do not add medicine and handle; (2) model group: add Ang II and hatch, final concentration is 10
-6Mol/L; (3) medicine group: add Ang II and losatan simultaneously and handle, final concentration is 10
-6Mol/l adds simultaneously for reagent and intervened 48 hours.
The capable HE dyeing of pair cell, Fig. 2 is seen in microphotograph, SD neonatal rat myocardial cell HE coloration result (* 100) shown in Figure 2 1～3 day, A normal myocardium cell, the inductive hypertrophic cardiomyopathy cell of B Angiotensin II, C hypertrophic cardiomyopathy cell was intervened 48 hours through losatan.
Enlarged markedly by the surface area ratio normal group of the inductive hypertrophic cardiomyopathy cell of Angiotensin II (B figure) as can be seen from the HE coloration result, the hypertrophic cardiomyopathy cell was intervened 48 hours through losatan, and the myocardial cell surface area returns to normal level (C figure) substantially.
5. carry out the plump drug study of anti-myocardial according to above-mentioned experiment condition
5.1 grouping:
In myocardial cell inoculated and cultured to 96 orifice plate, after 72 hours, culture fluid changes 5%FBS into and cultivates 24h, and the pair cell grouping is established 8 multiple holes for every group, and the dosing intervention: (1) normal group adds isopyknic DMEM; (2) hypertrophy model group: Ang II final concentration is 10
-6Mol/l; (3) losatan group: add Ang II and losatan simultaneously, final concentration is 10
-6Mol/l; (4) Herba Ranunculi Japonici total glycosides group of the present invention: Ang II final concentration is 10
-6Mol/l, Herba Ranunculi Japonici total glycosides final concentration are 50 μ g/ml, and dosing was intervened 48 hours.
5.2 index detects:
This experiment is examined or check the content of respectively organizing total protein in the myocardial cell with srb assay, and concrete grammar is with method described in the structure of 4. myocardial hypertrophy models.Observe the influence of Herba Ranunculi Japonici total glycosides to the hypertrophic cardiomyopathy cell.Detect the optimum condition of determining that hypertrophy model makes up, i.e. cell concentration according to orthogonal experiment design and HE dyeing: 2 * 10
5/ ml, Ang II final concentration: 10
-6Mol/L, dosing intervention time: 48 hours.As condition, experiment is divided into the normal myocardium groups of cells, hypertrophy model group, Losartan treatment group, pharmaceutical intervention group, result show that Herba Ranunculi Japonici total glycosides has significant resisting cardiac hypertrophy effect, effect is suitable with the Losartan group.
5.3 experimental result:
Each organizes cell through 10
-6After the Ang II of mol/l was hatched 48 hours, it is obviously big than normal group that srb assay obtains the OD value, shows that the total protein of cell content after Ang II induces increases, and promptly myocardial cell is through 10
-6After the Ang II of mol/l was hatched 48 hours, plumpness had taken place in cell.Result of experiment draws, and the Herba Ranunculi Japonici total glycosides of 50 μ g/ml has the effect of remarkable inhibition myocardial hypertrophy, compares p＜0.01 with model group.Experimental result is seen Fig. 3.
8 multiple holes are established in every group of experiment, and experiment repeats to be illustrated as a representational result more than 3 times.Data represent with meansigma methods ± standard deviation,
*P＜0.01, expression is compared with Ang II group, and significant difference is arranged.
Embodiment 3
Herba Ranunculi Japonici total glycosides suppresses heart effect experiment
1 instrument, medicine and animal
1.1 instrument
Bio signal acquisition system (MS4000U), Guangzhou dragon fly to reach the 10g of Science and Technology Ltd. tonotransducer, Beijing industrial scientific ﹠ trading Co., Ltd. of newly navigating
1.2 medicine
The general naphthalene Nore of hydrochloric acid sheet, Yongshou, Shaanxi Pharmaceutical Co, lot number: 20051028 isoproterenol hydrochloride injs, Shanghai Hefeng Pharmaceutical Co., Ltd., lot number: 5E20004
1.3 animal
Rana nigromaculata, about 60～90g, ♀ ♂ dual-purpose, is provided by Guangzhou fringe north animal center by 30;
2 experimental techniques
2.1 frog heart cannula:
Make Si Shi isolated heart specimen with reference to existing experimental technique, clamp the apex of the heart with frog heart clip then, connect the bio signal acquisition system through tonotransducer, each sleeve pipe adds the ringer solution of 1ml, retouches one section normal heart beating curve of meter, and tests by following grouping.
2.2 experiment grouping
2.2.1 Herba Ranunculi Japonici total glycosides is to the tensile influence in isolated frog heart basis:
Add 10 μ g/ml Propranolol respectively in sleeve pipe, the Herba Ranunculi Japonici total glycosides of 1 μ g/ml and 10 μ g/ml is retouched the meter effect curve, calculates dosing front and back shrinkage amplitude ratio, and dosing after-contraction amplitude/basic shrinkage amplitude is represented with D/F, sees Table 4.
2.2.2 Herba Ranunculi Japonici total glycosides is to the inhibitory action of isolated frog heart
Add 10 μ g/ml isoproterenol (Iso) solution in the ringer solution, after treating effect obviously, the Herba Ranunculi Japonici total glycosides that adds 10 μ g/ml Propranolol, 1 μ g/ml or 10 μ g/ml more respectively, retouch the meter effect curve, calculate dosing front and back shrinkage amplitude ratio, dosing after-contraction amplitude/add Iso after-contraction amplitude is represented with D/I, sees Table 4.
Table 4 Herba Ranunculi Japonici total glycosides to the inhibitory action of isolated frog heart (n=8,
)
| Group | Final concentration (μ g/ml) | D/F? | D/I? |
| Propranolol Herba Ranunculi Japonici total glycosides Herba Ranunculi Japonici total glycosides | 10110? | 0.506±0.071 0.898±0.058 ** 0.836±0.115 ** | 0.712±0.098 0.919±0.058 ** 0.885±0.056 ** |
Annotate:
*, compare p＜0.01 with the Propranolol group
2.2.3 date processing
Experimental data is so that (x ± s) expression adopts the t check to carry out statistical procedures, and P＜0.05 is as the dividing value of significant difference.
3 experimental result experimental results are seen Fig. 4.
4 conclusions
This experiment shows that 1 μ g/ml and 10 μ g/ml Herba Ranunculi Japonici total glycosides can reduce the basic tension force of isolated frog heart, and can resist the heart contractility reinforcement that isoproterenol causes.
Embodiment 4
Herba Ranunculi Japonici total glycosides of the present invention is tested the vascular ring effect
1 instrument, medicine and animal
1.1 instrument
The bio signal acquisition system, Medlab-U/4C501 type, the Nanjing Mei Yi 10g of scientific ﹠ technical corporation tonotransducer, Beijing industrial scientific ﹠ trading Co., Ltd. of newly navigating
Ultrathermostat 501 types, Shanghai City experimental apparatus factory
1.2 medicine
Noradrenaline bitartrate injection (NE), Tianjin gold credit aminoacid company limited, lot number: 0503221
Acecoline, Shanghai San'aisi Reagent Co., Ltd., lot number: 20030620 phentolamine mesylate injection (Phe), the rising sun East Sea is general, lot number: 060201
1.3 animal
The SD rat, the SPF level, about 200g, the male and female dual-purpose, is provided credit number by 30 by Guangdong Medical Lab Animal Center: SCXK (Guangdong) the word 2006A015 that checks and affirm in 2003-0002 Guangdong
2. experimental technique
2.1 experiment grouping:
2.1.1 Herba Ranunculi Japonici total glycosides causes the influence that vascular ring shrinks to NE
Under logical oxygen to vascular ring, apply preload 1.5g, dripping final concentration behind the balance 1.5h is 10
-6The mol/l norepinephrine causes vasoconstriction to plateau, and experiment is divided into four groups: (1) blank group adds normal saline 0.1ml; (2) Herba Ranunculi Japonici total glycosides low dose group, its final concentration are 1mg/ml; (3) Herba Ranunculi Japonici total glycosides high dose group, its final concentration are 2mg/ml; (4) phentolamine group, its final concentration are 5 * 10
-6Mol/L.The results are shown in Figure 5.
Always resist the effect that NE causes that vascular ring shrinks for the quantitative expedition Herba Ranunculi Japonici, be calculated as follows for reagent antagonism NE and cause the suppression ratio that vascular ring shrinks, vascular ring tension force behind suppression ratio (%)=(vascular ring tension force-Jia is for antiotasis after the reagent after adding NE)/the add NE.The results are shown in Figure 6, Fig. 6 shows that final concentration is that the Herba Ranunculi Japonici total glycosides of 1mg/ml and 2mg/ml obviously suppresses by the inductive vasoconstriction effect of norepinephrine, and its suppression ratio is tangible dose dependent.
The experiment of Herba Ranunculi Japonici total glycosides anti-inflammatory and analgesic effect of the present invention
It all is classical inflammatory model that chondrus ocellatus Holmes is induced rat paw edema model and mice caused by dimethylbenzene xylene auricle edema model, with telangiectasis, capillary permeability is hyperfunction is cardinal symptom.It is to produce due to the acute inflammation by the abdominal cavity indirectly that glacial acetic acid causes mouse writhing reaction, and the PGE in the peritoneal fluid is raise with the PGF level, reacts thereby produce peripheral pain; And the animal pain model that thermostimulation causes is the central pain model.
1 medicine, instrument and animal
1.1 medicine
Dexamethasone (DEX), Zhejiang Province XianJu Pharmacy stock Co., Ltd, lot number: 060105 rotundine sulfate injection (Rotundine), Guangdong Boluo Pioneer Medicinal Group Co., Ltd., lot number: 060104
Other reagent: dimethylbenzene (analytical pure), the 90662nd factory of the Chinese People's Liberation Army produces.
1.2 instrument
The FA2104S electronic analytical balance, Shanghai balance equipment factory
Electronic platform scale, the new and basic scientific and technological development company limited in Zhongshan city, Guangdong is produced ultrathermostat (501 type), Shanghai City experimental apparatus factory
1.3 animal
SPF level SD rat is provided credit number by Guangdong Medical Lab Animal Center: SCXK (Guangdong) 2003-002, and word 2005A010 checks and affirm in Guangdong; Body weight 150g-160g.SPF level NIH mice is provided credit number by Guangdong Medical Lab Animal Center: SCXK (Guangdong) 2003-002, and word 2004A018 checks and affirm in Guangdong; Body weight 18-22g, male and female half and half.SPF level NIH mice is provided credit number by Guangdong Medical Lab Animal Center: SCXK (Guangdong) 2003-002, and word 2005A012 checks and affirm in Guangdong; Body weight 18-22g, male and female half and half.
2 experimental techniques
2.1 the influence of Herba Ranunculi Japonici total glycosides xylol induced mice auricle edema
Get 50 of NIH mices, male and female half and half are divided equally 5 groups at random, 10 every group.Be basic, normal, high three the dosage groups of normal saline group, Herba Ranunculi Japonici total glycosides, i.e. 200mg, 400mg, 800mg/kg, Dexamethasone group, the positive, everyone accumulated dose of being grown up is 30mg, becomes body weight for humans to press 60kg and calculates, mice is 20 times of administrations, i.e. 30mg * 20/60=10mg/kg of people's dosage proportionately.Be administered once every day, for three days on end, after the last administration 1 hour, each is organized mouse right ear and only is coated with dimethylbenzene 0.05ml/, left side ear compares, and puts to death mice after 15 minutes, uses the card punch of diameter 7mm that ears are downcut with the position homalographic, weigh respectively with electronic balance, be calculated as follows auris dextra swelling rate: auris dextra swelling rate (%)=(auris dextra sheet weight-left auricle is heavy)/left auricle heavy * 100%.The results are shown in Figure 7.
2.2 Herba Ranunculi Japonici total glycosides is to the influence of rat paw edema due to the chondrus ocellatus Holmes
Get 50 of SD rats, male and female half and half are divided equally 5 groups at random, i.e. normal saline group, basic, normal, high three the dosage groups of Herba Ranunculi Japonici total glycosides, be 400mg, 200mg, 100mg/kg, Dexamethasone group, the positive, everyone accumulated dose of being grown up is 30mg, becoming body weight for humans to press 60kg calculates, rat is 10 times of administrations of people's dosage proportionately, i.e. 30mg * 10/60=5mg/kg, and be administered once every day.For three days on end, after the last administration 2 hours, measure the normal ankle joint girth of every Mus right hind earlier with the moccasin chi, at the right back sufficient plantar subcutaneous injection 1% carrageenin suspension 0.1ml/ of rat only, cause inflammation respectively, subsequently respectively at 1 hour, 2 hours, 4 hours, respectively measured one time the ankle joint girth in 6 hours, the record result is calculated as follows sufficient perimeter change
Ankle joint girth before the scorching back of foot perimeter change=cause ankle joint girth-cause is scorching
Calculate each Mus foot perimeter change.The results are shown in Figure 8, Fig. 8 shows that 200mg/kg and 400mg/kg Herba Ranunculi Japonici total glycosides can significantly suppress rat paw edema, and the prompting Herba Ranunculi Japonici total glycosides has good antiinflammatory action.
2.3 Herba Ranunculi Japonici total glycosides stimulates the influence that causes the mice pain reaction to hot plate
The temperature of regulating thermostatic water-circulator bath is at 55 ℃ ± 0.5 ℃, with hot plate preheating 10min.Get 18～22g female mice, each 1 is placed on the hot plate, and mice is from being placed on the hot plate to the pain threshold of metapedes required time number second as this Mus occurring licking.Allly lick the metapedes time, give it up less than 5 seconds or greater than 30 seconds or leaper.50 qualified mices are divided into 5 groups at random, be basic, normal, high three the dosage groups of normal saline group, Herba Ranunculi Japonici total glycosides, be 200mg, 400mg, 800mg/kg, the rotundine group, the positive, everyone accumulated dose of being grown up is 90mg, becomes body weight for humans to press 60kg and calculates, mice is 20 times of administrations, i.e. 90mg * 20/60=30mg/kg of people's dosage proportionately.Each experimental mice is administered once every day, and matched group is given the normal saline of equivalent, continuous three times, measured the mice pain threshold after the last administration in 60 minutes, as 60 seconds still reactionless, mice is taken out, in order to avoid scald, its pain threshold calculated by 60 seconds.The results are shown in Figure 9, Fig. 9 shows: 200mg/kg, 400mg/kg and 800mg/kg Herba Ranunculi Japonici total glycosides can significantly suppress to stimulate inductive pain reaction by 55 ℃ of hot plates, and this analgesic effect and rotundine are suitable.
2.4 the Herba Ranunculi Japonici total glycosides Dichlorodiphenyl Acetate stimulates the influence that causes the mouse writhing reaction
Get 50 of NIH mices, male and female half and half are divided equally 5 groups at random, i.e. normal saline group, basic, normal, high three the dosage groups of Herba Ranunculi Japonici total glycosides, i.e. 200mg, 400mg, 800mg/kg, rotundine group, 30mg/kg.Each experimental mice is administered once every day, and matched group is given the normal saline of equivalent, continuous three times, after the last administration 1 hour, each Mus lumbar injection 0.6% acetic acid 0.2ml/ only, that observes each Mus appearance in 15 minutes turns round the body number of times, and calculates the analgesia percentage rate of each medicine:
Calculate each Mus analgesia percentage rate.The results are shown in table 7 and Figure 10.
Table 7 Herba Ranunculi Japonici total glycosides Dichlorodiphenyl Acetate stimulates the influence of inductive pain model
| Group | n? | Dosage (mg/kg) | 15 ' interior body the number of times of turning round | Analgesia percentage rate (%) |
| Normal saline rotundine Herba Ranunculi Japonici total glycosides Herba Ranunculi Japonici total glycosides Herba Ranunculi Japonici |
10 10 10 10 10 | Equal- |
28.5±10.9 9.0±10.26 *** 15.5±5.4 *** 6.4±4.6 *** 8.8±6.7 *** | 68.4?45.6?47.7?76.8 |
Compare with the normal saline group: * * *, p＜0.01; The T check
Table 7 explanation, the mouse writhing reaction that the Herba Ranunculi Japonici total glycosides Dichlorodiphenyl Acetate causes all has significant inhibition to do, and Figure 10 shows: 200mg/kg, 400mg/kg and 800mg/kg Herba Ranunculi Japonici total glycosides significantly suppress to stimulate inductive pain reaction by acetic acid, and have dose dependent.
2.5 statistical analysis
Experimental data all adopts
Expression, and adopt the T check to carry out statistical procedures
3 experimental results
The result shows: the mice auricle swelling due to the 200mg/kg of Herba Ranunculi Japonici total glycosides, 400mg/kg and three dosage groups of the 800mg/kg xylol all has significant inhibitory effect, and dose dependent is arranged.Two dosage groups of the 200mg/kg of Herba Ranunculi Japonici total glycosides and 400mg/kg have significant inhibitory effect to rat paw edema due to the chondrus ocellatus Holmes; The prompting Herba Ranunculi Japonici total glycosides has good antiinflammatory action.Three obvious antagonisms of dosage group of Herba Ranunculi Japonici total glycosides 200mg/kg, 400mg/kg and 800mg/kg are by the pain reaction of thermostimulation (dose dependent is arranged) and acetic-acid induced, and the prompting Herba Ranunculi Japonici total glycosides may all have antagonism to periphery and central pain.
Claims (7)
1. the preparation method of a Herba Ranunculi Japonici total glycosides is characterized in that may further comprise the steps:
(1) the Herba Ranunculi Japonici herb dries chopping, and water or 10～95% ethanol or methanol eddy or percolation extract, filter, and merge extractive liquid,, decompression and solvent recovery concentrates;
(2) in (1) concentrated solution, add equal volume of ethyl acetate, take off a layer water layer solvent evaporated, get paste;
(3) a certain amount of water dissolution of paste, through macroporous resin adsorption, extremely clear with distillating washing resin earlier, reuse 50～95% ethanol or methanol-eluted fractions concentrate, and solvent evaporated gets paste Herba Ranunculi Japonici total glycosides product.
2. the preparation method of Herba Ranunculi Japonici total glycosides according to claim 1 is characterized in that described extraction employing water of step (1) or 10～95% ethanol or methanol eddy or percolation extract, and solvent and Herba Ranunculi Japonici herb weight ratio are 7: 1 consumption, extract 2～3 times.
3. application of adopting the Herba Ranunculi Japonici total glycosides that claim 1 or 2 described preparation methoies prepare is characterized in that the application aspect the preparation cardiovascular drugs.
4. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 3, it is characterized in that the application aspect preparation resisting cardiac hypertrophy medicine.
5. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 3, it is characterized in that the application aspect preparation vasodilator smooth muscle medicine.
6. according to the application of the described Herba Ranunculi Japonici total glycosides of claim 3, it is characterized in that the application aspect preparation inhibition heart drugs with function.
7. application of adopting the Herba Ranunculi Japonici total glycosides that claim 1 or 2 described preparation methoies prepare is characterized in that the application at preparation antalgic anti-inflammatory agent object space face.
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Non-Patent Citations (7)
| Title |
|---|
| 杨世昆.毛茛叶外敷治愈2例慢性膝关节炎的疗效观察.中国民间疗法 4.1994,(4),37. |
| 杨世昆.毛茛叶外敷治愈2例慢性膝关节炎的疗效观察.中国民间疗法 4.1994,(4),37. * |
| 蔡飒等.毛茛提取液含药血清对家兔子宫平滑肌细胞的影响.中草药36 4.2005,36(4),566-568. |
| 蔡飒等.毛茛提取液含药血清对家兔子宫平滑肌细胞的影响.中草药36 4.2005,36(4),566-568. * |
| 郑威.毛茛化学成分的研究及几种中药新药的开发研究.中国优秀硕士学位论文全文数据库.2006,论文全文. * |
| 郑威等.毛茛化学成分的研究.中国中药杂志31 11.2006,31(11),892-894. |
| 郑威等.毛茛化学成分的研究.中国中药杂志31 11.2006,31(11),892-894. * |
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