CN103110680A - Preparation method of total phenolic acid of erigeron breviscapus - Google Patents
Preparation method of total phenolic acid of erigeron breviscapus Download PDFInfo
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Abstract
The invention discloses a medicinal plant extract and a preparation method and application thereof. The medicinal plant extract is powdery total phenolic acid or sodium salt thereof prepared by extraction, concentration, alkali dissolution and acid precipitation, degreasing and the like based on erigeron breviscapus as a raw material. The extract disclosed by the invention enriches various phenolic acid effective substances, the impurities such as pyromeconic acid, carbohydrates, oil and saponin are sufficiently removed, the active ingredients are comprehensive, and moisture absorption is avoided; and the extract can be independently used or combined with other medicinally-allowable substances and is used for preparing medicines and healthcare products for preventing and controlling ischemic cardiovascular and cerebrovascular diseases.
Description
Technical field
The invention belongs to the plant amedica field, be specifically related to total phenolic acid of a kind of Erigeron breviscapus (Vant.) Hand.-Mazz. and preparation method thereof, chemical composition and the application in making control ischemic cardio cerebrovascular diseases Medicines and Health Product.
Background technology
Erigeron breviscapus (Vant.) Hand.-Mazz. is that the Compositae Herba Erigerontis aceris is drafted a document this plant, formal name used at school Erigeron breviscapus (Vant.) Hand.-Mazz, among the people claim again Herba Erigerontis, Herba Erigerontis, Aster dubius (Thunb.) Onno, be exposed to the sun and two sunflower, be Chinese endemic plant, be distributed in the ground such as Guangxi, Yunnan, Guizhou, Hunan, Sichuan.Erigeron breviscapus (Vant.) Hand.-Mazz. has very high medical value, the raw materials for production of the multiple patent medicine such as Herba Erigerontis tablet, herba asari capsule, because the diseases such as apoplexy, coronary heart disease, diabetic nephropathy, the retinal vein occlusion, senile dementia are had good therapeutic effect, are subject to the world of medicine's extensive concern.Tens of kinds of chemical compositions have been isolated from this plant so far, comprise plurality of classes (the pharmacy practice magazines such as flavone, caffeoylquinic acids, ketooctulosonic acid, coumarin, terpenoid, lignanoid, saponin, aromatic acid, gamma-pyrone, cinnamic acid, sterol, fatty acid, aliphatic alcohol, volatile oil, organic acid, 2002,20 (2): 103-107; Chinese herbal medicine, 2005,36 (1): 141-144).The scutellarin that content is higher (scutellarin) is the main effective ingredient of generally acknowledging, breviscapine medicine series take it as raw material production goes on the market in a large number, but medicine is found for research, this composition exists the low problem of oral administration biaavailability (Chinese Clinical pharmacology and therapeutics, 2005,10 (3): 310-313).Coffee mesitoyl quinine acid has antioxidation, anticoagulation isoreactivity, and (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,31 (11): 869-874), but specific examples of such components extensively exists in plant kingdom, is not that Erigeron breviscapus (Vant.) Hand.-Mazz. is peculiar also to be considered to the Erigeron breviscapus (Vant.) Hand.-Mazz. effective ingredient.Pyromeconic acid (pyromeconic acid) is a kind of gamma-pyrone constituents, content is higher in Erigeron breviscapus (Vant.) Hand.-Mazz., this composition is to the cardiovascular and cerebrovascular vessel useful final conclusion that there is no whether, but there is data to show, there is toxicity in it to rat liver, and quilt is as liver poison instrument medicine (Experientia, 1984,40 (8): 894-6; Exp Mol Pathol, 1992,57 (2): 153-66).At present to the not yet limit of understanding of Erigeron breviscapus (Vant.) Hand.-Mazz. chemical composition, new component has report still the time.In a word, the demonstration of prior art data, the composition of Erigeron breviscapus (Vant.) Hand.-Mazz. has multiformity, has both contained plurality of active ingredients, also contains the poisonous or invalid impurity such as pyromeconic acid; Except scutellarin, do not get rid of the probability that also has the stronger effective ingredient of other activity; Also probably there are some unknown pharmacology conspiracy relations between effective ingredient; Overall understanding to Erigeron breviscapus (Vant.) Hand.-Mazz. effective substance and the mechanism of action thereof is still waiting further investigation.
Extraction around the Erigeron breviscapus (Vant.) Hand.-Mazz. active component, existing multinomial patented method is open, its refining means are mainly organic solvent extraction (CN1381236A, CN1709351A, ZL93104701.3), silica gel column chromatography (CN1136434A), macroporous resin adsorption (CN1462620A, CN1327811A, CN1947736A), polycaprolactam (CN1136434A) etc., the extract that these methods obtain is different on component, has to some extent the problems such as effective ingredient is lost, impurity is residual, solvent consumption is large.
Summary of the invention
One of purpose of the present invention is to provide the Erigeron breviscapus (Vant.) Hand.-Mazz. extract that a kind of effective ingredient is comprehensive, structure is clear and definite, quality controllable, in order to natural many target spots pharmacology synergism that the performance medical material exists originally, obtains better medical health effect.The inventor has carried out the basic research of system to Erigeron breviscapus (Vant.) Hand.-Mazz., based on to its chemical composition and bioactive understanding, effective site is locked as total phenolic acid, illustrate more up hill and dale the chemical constitution at this position, wherein comprised the structure types such as flavone (comprising flavanone, flavonol), caffeic acid ester, coumarin, aromatic acid, cinnamic acid, chromone, ketooctulosonic acid.
Two of purpose of the present invention is to provide the preparation method of the total phenolic acid of a kind of above-mentioned Erigeron breviscapus (Vant.) Hand.-Mazz. and sodium salt thereof, the method can be extracted multifarious phenolic acids effective ingredient in medical material, fully utilize expeditiously herb resource, can fully remove the impurity such as pyromeconic acid, erigeroside, saccharide, oils and fats, volatile oil, saponin, inorganic salt again, to reduce dose, improve drug safety, can also improve yield, reduce production costs.Accompany simultaneously the difference of all kinds of impurity on physicochemical property of depositing in inventor's phenolic acid total according to Erigeron breviscapus (Vant.) Hand.-Mazz. and plant, grope to sum up the preparation were established of an original creation, through repetition test and optimization, reached the extraction purification purpose of the present invention's expection.
Three of purpose of the present invention is to provide the total phenolic acid of above-mentioned Erigeron breviscapus (Vant.) Hand.-Mazz. or its sodium salt is prevented and treated the medicine of ischemic cardio cerebrovascular diseases or the purposes in health product in preparation.
The preparation method of the total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. of the present invention and sodium salt thereof comprises following in-sequence operational step:
A. medical material is with one of aqueous alcohol, methanol or water, and preferred concentration is 60%～95% ethanol, reflux, extract, 2～5 times, and merge extractive liquid,, concentrating under reduced pressure gets extractum;
B. extractum with 2～15 times of water gaging suspendibles, slowly adds dilute sodium hydroxide, sodium carbonate or sodium bicarbonate solution under stirring, regulates pH value 7.0～8.5, and is centrifugal, filters;
C. filtrate is with dilute hydrochloric acid or dilute sulfuric acid adjust pH 1～3, and is standing, minute gets precipitation, is washed to pH closely neutral, and drying is pulverized;
D. precipitation is used the lipotropy organic solvent degreasing, volatilizes, and gets total phenolic acid;
E. total phenolic acid further with dilute sodium hydroxide, sodium carbonate or sodium bicarbonate solution dissolving, transfers pH closely neutral, filters, and concentrated, drying gets the total phenols acid sodium-salt.
Preparation method of the present invention is characterised in that to have the link of alkali extraction and acid precipitation in its process route, and namely extractum is first used dilute sodium hydroxide, sodium carbonate or sodium bicarbonate solution hydrotropy, and the clear liquid after filtration with dilute hydrochloric acid or dilute sulfuric acid acidify, makes the active component Precipitation again.
The feature of preparation method of the present invention also is, has the defat link in its process route, namely use one of normal hexane, cyclohexane extraction, petroleum ether or gasoline, preferred boiling range is the petroleum ether of 60～90 ℃, under room temperature or heating, the total phenols acid crude is flooded or reflux, extract, 2～10 times, be no less than 1 hour at every turn.The oils and fats that removes is the brown oily, and the intense stimulus abnormal smells from the patient is arranged; The acute toxicity test in mice demonstration, this oils and fats gastric infusion was observed 7 days, recorded its LD
50Be 2.8g/kg; The poisoning symptoms such as that animal subject has all occurred is restless, tic of the limbs and convulsions; The toxicity of oils and fats is significantly higher than the total phenolic acid after defat.This defat link can be removed and be accounted for 4% oily impurity in crude product, to reducing total phenolic acid toxicity, eliminate bad smell, improving powder flowbility and brought into play pivotal role.
Total phenolic acid of the present invention, one of its feature are to contain scutellarin 4%～20% (weight).
Total phenolic acid of the present invention is further characterized in that to contain 3,5-, two-O-caffeoylquinic acids (1, see accompanying drawing 1) 0.1%～5% (weight) and 4,5-, two-O-caffeoylquinic acids (2) 0.5%～8% (weight).
Total phenolic acid of the present invention is further characterized in that to contain 3-O-caffeoyl-γ-quinide (3) 0.01%～1% (weight).This composition is very rare at nature, only found from roasting coffee beans so far, known this Compound Phase is for common di-coffee mesitoyl quinine acid, have that molecular weight is little, lipotropy is strong, be easy to penetrate the advantage such as blood brain barrier, to much higher (the Z Lebensm Unters Forsch of cerebrovascular activity, 1994,199 (1): 17～21).Infer thus, this composition has significant contribution to the drug effect of the total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz., classifies it as key index composition.Accompanying drawing 3 has shown the existence of this compound in total phenolic acid.The inventor uses chromatographic process and has obtained this compound monomer from the Erigeron breviscapus (Vant.) Hand.-Mazz. separation, and has measured molecular structure, and its spectral data is as follows:
UVλmax(nm)：221，240，305(sh)，330。
ESI-MS?m/z：335[M-H]
-。
1H-NMR(DMSO-d
6，400MHz)δppm：1.93(1H，t，J＝12Hz，H-2a)，2.04(1H，m，H-2b)，2.21(1H，m，H-6a)，2.37(1H，d，J＝12Hz，H-6b)，4.14(1H，q，J＝4.8Hz，H-4)，4.69(1H，m，H-5)，4.72(1H，m，H-3)，5.67(1H，d，J＝4.8Hz，4-OH)，6.14(1H，s，1-OH)，6.24(1H，d，J＝16Hz，H-8’)，6.76(1H，d，J＝8.OHz，H-5’)，7.00(1H，dd，J＝8.0，1.6Hz，H-6’)，7.03(1H，d，J＝1.6Hz，H-2’)，7.52(1H，d，J＝16Hz，H-7’)，9.16(1H，s，Ar-OH)，9.60(1H，s，Ar-OH)。
13C-NMR(DMSO-d
6，100MHz)δppm：36.1(C-6)，36.9(C-2)，63.2(C-4)，69.0(C-3)，71.9(C-1)，76.1(C-5)，114.2(C-8’)，115.2(C-2’)，116.2(C-5’)，121.9(C-6’)，125.9(C-1’)，146.0(C-7’，4’)，149.0(C-3’)，166.1(C-9’)，177.6(C-7)。
Total phenolic acid of the present invention is further characterized in that to contain 1,3-, two-O-caffeoylquinic acids (4) 0.01～2% (weight).This compound claims again Lay silibin (cynarin), has the effect that suppresses Biosynthesis of cholesterol, is used for clinical (Arzneimittelforschung.1975,25 (8): 1311-4 as blood lipid-lowering medicine; Z Gastroenterol.1973,11 (3): 183-6).Infer thus, this composition also has significant contribution to the drug effect of the total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz..Accompanying drawing 3 has shown the existence of this compound in total phenolic acid.The inventor uses chromatographic process and separates from Erigeron breviscapus (Vant.) Hand.-Mazz. and obtained this compound monomer, and has measured molecular structure, and its spectral data is as follows:
UVλmax(nm)：219，243，309(sh)，324。
1H-NMR(CD
3COCD
3，400MHz)δ：1.78(1H，dd，J＝13.6，11.3Hz，H-6
a)，2.26(1H，dd，J＝16.0，3.0Hz，H-2
a)，2.42(1H，dt，J＝13.6，3.0Hz，H-6
b)，2.72(1H，dt，.J＝16.0，3.0Hz，H-2
b)，3.64(1H，dd，J＝9.5，3.0Hz，H-4)，4.21(1H，ddd，Ｊ＝11.3，9.5，3.0Hz，H-5)，5.32(1H，q，Ｊ＝3.0Hz，H-3)，6.09(1H，d，J＝15.9Hz，H-8’)，6.20(1H，d，J＝15.9Hz，H-8”)，6.53(1H，d，J＝8.2Hz，H-5’)，6.59(1H，dd，J＝8.2，1.8Hz，H-6’)，6.66(1H，d，J＝8.2Hz，H-5”)，6.77(1H，dd，J＝8.2，1.8Hz，H-6”)，6.88(1H，d，J＝1.8Hz，H-2’)，7.00(1H，d，Ｊ＝1.8Hz，H-2”)，7.39(1H，d，J＝15.9Hz，H-7’)，7.42(1H，d，J＝15.9Hz，H-7”)。
13C-NMR(DMSO-d
6，100MHz)δppm：81.1(C-1)，32.9(C-2)，73.0(C-3)，75.3(C-4)，67.8(C-5)，41.3(C-6)，174.6(C-7)，127.4(C-1’)，115.1(C-2’)，146.5(C-3’)，149.3(C-4’)，116.1(C-5’)，122.0(C-6’)，147.2(C-7’)，115.5(C-8’)，167.8(C-9’)，127.5(C-1”)，115.4(C-2”)，146.7(C-3”)，149.7(C-4”)，116.6(C-5”)，122.9(C-6”)，147.7(C-7”)，115.4(C-8”)，168.9(C-9”)。
total phenolic acid of the present invention, be further characterized in that and further contain 1, 5-two-O-caffeoylquinic acids (5), 3, 4-two-O-caffeoylquinic acids (6), chlorogenic acid (7), 3-O-Cafeoylquinic acid (8), 4-O-caffeoylquinic acids (9), caffeic acid (10), syringic acid (11), protocatechuic acid (12), P-hydroxybenzoic acid (13), vanillic acid (14), coumaric acid (15), 5, 7-dihydroxy chromone (16), scopoletin (17), umbelliferone (18), aesculetin (19), multiradiate fleabane glycosides (20), high baicalin (21), apigenin (22), Quercetin (23), eriodictyol (24), apigenin-7-O-glucuronide (25), apigenin-7-O-glucoside (26, see accompanying drawing 2), apigenin-7-O-galacturonic acid glycosides (27), naringenin-7-O-glucuronide (28), eriodictyol-7-O-glucuronide (29), Quercetin-3-O-galacturonic acid glycosides (30), the above composition of at least one in Quercetin-compositions such as 7-O-glucuronide (31).These monomer components separate from Erigeron breviscapus (Vant.) Hand.-Mazz. by the inventor and obtain, and have identified molecular structure through Spectrum Analysis.Available data shows, these liposoluble ingredients all have the activity that antioxidation etc. is of value to cardiovascular and cerebrovascular vessel, are effective ingredient.
The feature of the total phenolic acid of the present invention also is substantially not contain pyromeconic acid (32).May be to the toxic and side effects of liver generation in order to eliminate pyromeconic acid, preparation method of the present invention is to pyromeconic acid and glucoside thereof, and namely erigeroside (33), carried out directed removal, and its residual quantity is controlled at below 10ppm.Pyromeconic acid is met ferric chloride, and the aobvious redness of complex reaction can occur, and this reacts rapid sensitive, can be used for the qualitative and limit detection of pyromeconic acid.
In addition, the total phenolic acid of the present invention also contains the esters composition that some ketooctulosonic acids and 1～2 molecule caffeic acid, coumaric acid or benzoic acid condensation form.Some carboxylic compositions in total phenolic acid, with the Ethanol Exposure process in condensation reaction can occur, form the ethyl ester product, can be converted into apigenin-7-O-glucuronide-6 as apigenin-7-O-glucuronide "-ethyl ester; 3,5-, two-O-caffeoylquinic acids can be converted into 3,5-, two-O-caffeoylquinic acids ethyl ester; Cause the composition number to increase.The conversion ratio of esterification is relevant with factors such as the time of alcohol exposure, temperature, pH, concentration, usually secondary metabolite seldom, so substantially can produce obviously impact to drug effect and the safety of total phenolic acid.
The total phenolic acid of the present invention or its sodium salt yield are up to 5% left and right, outward appearance is chocolate brown powder, nonhygroscopic, easily flow, stable chemical nature, can be separately or with the combinations of substances of other medicinal licenses, make easily various oral administration solid pharmaceutical dosage forms, as capsule, tablet, granule, drop pill, soft capsule.Total phenols acid sodium-salt water soluble of the present invention easily absorbs, and also is fit to make liquid preparation, as oral liquid, spray.
The present invention has significant difference compared with the prior art, outstanding behaviours exists: 1. preparation method of the present invention uses alkali extraction and acid precipitation and organic solvent degreasing as main refining means, rather than the means such as prior art silica gel column chromatography used, macroporous resin adsorption, polycaprolactam, organic solvent liquid-liquid extraction, operate simpler, solvent consumption still less, be more suitable for large-scale industrial production, the oral formulations raw material production that particularly dosage is larger; 2. the total phenolic acid of the present invention not only contains known scutellarin, 3,5-two-O-caffeoylquinic acids, 4, the compositions such as 5-two-O-caffeoylquinic acids, also contain the various active composition that prior art had not related to, as 3-O-caffeoyl-γ-quinide, naringenin-7-O-glucuronide, eriodictyol-7-O-glucuronide etc., its liposoluble ingredient is more complete, and structure is clearer and more definite; 3. the total phenolic acid of the present invention has been removed pyromeconic acid and hydrolyzable is the erigeroside of pyromeconic acid, has therefore eliminated the major hidden danger that Long-term taking medicine may cause the liver toxic and side effects; 4. the total phenolic acid of the present invention has been removed the lipotropy impurity such as a large amount of oils and fatss, volatile oil, pigment, has improved drug safety; 5. the total phenolic acid of the present invention has been removed the water-solubility impurities such as a large amount of saccharides, inorganic salt, saponin, and its outward appearance is pulverulent solids, and stable chemical nature is nonhygroscopic, can carry out easily quality control, storage, allocates and feed intake as intermediate.
The total phenolic acid of the present invention has antiplatelet aggregation, blood fat reducing, improves hemorheological property, suppresses thrombosis, alleviate free radical increase due to the pharmacological action such as blood vessel injury and tissue necrosis, and have no obvious toxic-side effects, therefore can be used for preparing medicine and the health product of preventing and treating ischemic cardio cerebrovascular diseases, market prospect is wide.
Description of drawings
Fig. 1: the structural formula of Erigeron breviscapus (Vant.) Hand.-Mazz. compound 1～25
Fig. 2: the structural formula of Erigeron breviscapus (Vant.) Hand.-Mazz. compound 26～33
Fig. 3: the HPLC collection of illustrative plates of the total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. [chromatographic condition: chromatographic column VP-ODS, 150L * 4.6; Mobile phase methanol-0.1%TFA gradient (time 0 → 40 → 50 → 60min, methanol 20% → 60% → 100% → 100%, 0.1%TFA80% → 40% → 0% → 0%); Flow velocity 1ml/min; Wavelength 254nm; 25 ℃ of column temperatures]
Fig. 4: 3-O-caffeoyl-γ-quinide
1H-NMR and UV spectrogram
Fig. 5: 1,3-, two-O-caffeoylquinic acids
1H-NMR and UV spectrogram
The below provides specific embodiment that the present invention further is illustrated, but these embodiment are only examples, the present invention are not consisted of any restriction.
The specific embodiment
Preparation and the check of the embodiment 1 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. and sodium salt thereof
The total phenols processed with acid is standby: get the dry herb 10kg of Erigeron breviscapus (Vant.) Hand.-Mazz., cutting with 80% alcohol extraction 3 times (120L/2h/ time), merge extractive liquid,, concentrating under reduced pressure, gets pure extractum 1.7kg (proportion 1.2).Add entry 8kg in the extractum and make suspendible, slowly add 10% sodium hydroxide to make abundant dissolving under stirring, regulating pH value is 8, centrifugal, filter, filtrate is 2 with 5% sulfuric acid acidation to pH value, standing 5 hours, filter collection precipitation, closely neutral with being washed on a small quantity effluent, drain 40 ℃ of vacuum dryings, porphyrize, extract 4 times (2L/10h/ time) with petroleum ether (60～90 ℃ of boiling ranges) dipping, volatilize, get chocolate brown powder 565g, be total phenolic acid.
Total phenols acid sodium-salt preparation: get above-mentioned total phenolic acid 100g, add purified water 400ml to make suspendible, slowly add 10% sodium hydroxide solution under stirring, make abundant dissolving, regulate pH value 7.5 ± 0.5, filter concentrating under reduced pressure, spray drying gets chocolate brown powder 95g, is the total phenols acid sodium-salt.
Pyromeconic acid detects: get above-mentioned total phenolic acid or its sodium salt 1g, add the chloroform supersound extraction 3 times (10ml/10 minute/time), merging filtrate, put evaporate to dryness in water-bath, residue adds the 1ml water dissolution, drips 1 of ferric chloride test solution, not aobvious red, show that total phenolic acid sample does not contain pyromeconic acid substantially.
Assay: with the content of 5 kinds of compositions such as HPLC external standard method Simultaneous Determination scutellarin.Chromatographic condition: chromatographic column VP-ODS150L * 4.6; Mobile phase acetonitrile-0.1%TFA gradient elution (time 0 → 40min, acetonitrile 10% → 50%, 0.1%TFA90% → 50%) is mobile phase; The detection wavelength is 325nm; Flow velocity 1ml/min; 30 ℃ of column temperatures.Sample solution: 8mg/1ml methanol solution; Reference substance: each 50 μ g/1ml methanol solutions.Sampling volume: 10 μ l.Measurement result: scutellarin 13.5%; 4,5-, two-O-caffeoylquinic acids 2.6%; 3,5-, two-O-caffeoylquinic acids 1.1%; 3-O-caffeoyl-γ-quinide 0.08%; 1,3-, two-O-caffeoylquinic acids 0.15%.
The preparation of embodiment 2 Erigeron breviscapus (Vant.) Hand.-Mazz. total phenolic acid tablet agent
Get the embodiment 1 total phenolic acid 60g of gained, add starch 80g, dextrin 5g, mix homogeneously, add 10% starch slurry soft material processed, granulate with 14 order nylon screens, 60～70 ℃ of aeration-dryings, 16 mesh sieve granulate, add magnesium stearate 1.5g, sodium carboxymethyl cellulose 5g mixing, be pressed into 1000, coating and get final product.Every contains total phenolic acid 60mg.
The preparation of the embodiment 3 total phenolic acid capsules of Erigeron breviscapus (Vant.) Hand.-Mazz.
Get the embodiment 1 total phenolic acid 110g of gained, with starch 68g, magnesium stearate 2g mixing, cross 20 mesh sieves, 1000 of filled capsules, polishing, and get final product.Every contains total phenolic acid 110mg.
The preparation of the embodiment 4 total phenolic acid drop pill of Erigeron breviscapus (Vant.) Hand.-Mazz.
Get the embodiment 1 total phenolic acid 10g of gained, drop in the polyethylene glycol 6000 of 32g heating and melting, be stirred to dissolving, be transferred in reservoir, airtight and insulation is regulated pill dripping machine drop quantitative valve at 80～90 ℃, splash into from top to bottom in the liquid Paraffin of 10～15 ℃, make altogether 1000, the drop pill that forms is drained and wipe liquid Paraffin, be drying to obtain.Every contains total phenolic acid 10mg.
The preparation of the embodiment 5 aching and limp capsules of Erigeron breviscapus (Vant.) Hand.-Mazz. total phenols
Get PEG400280g, put in appropriate vessel, add PEG600010g, heating in water bath dissolves to PEG6000, stirs evenly, and adds glycerol 18g, add the embodiment 1 total phenolic acid 110g of gained, grind to form even mastic with colloid mill, be transferred to the material storage barrel of encapsulating machine, be pressed into 1000 of soft capsules with the mould of 0.3ml, typing, wash away surface lubricant, dry under 20～30% relative humiditys, 28 ℃ of conditions, and get final product.Every contains total phenolic acid 110mg.
The preparation of the compound capsule of the embodiment 6 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. and arasaponin
Get the embodiment 1 total phenolic acid 100g of gained and the commercially available Radix Notoginseng total arasaponins 20g that meets the Chinese Pharmacopoeia standard, with medical starch 80g, magnesium stearate 2g mixing, cross 20 mesh sieves, 1000 of filled capsules, polishing, and get final product.Every contains total phenolic acid 100mg, contains Radix Notoginseng total arasaponins 20mg.
The embodiment 7 total phenolic acid acute toxicity tests of Erigeron breviscapus (Vant.) Hand.-Mazz.
Select body weight at the healthy mice of 18～22g, adopt the gastric infusion mode, disposablely give total phenolic acid 14g/kg, Continuous Observation 7 days, result has no animal and obvious toxicity and death occur.This dosage is more than 400 times of dosage of clinical plan.
The embodiment 8 total phenolic acid long term toxicity tests of Erigeron breviscapus (Vant.) Hand.-Mazz.
Get 180 of rats, body weight 70～90g, male and female half and half are divided into four groups at random: 50 of matched groups, 50 of heavy dose of groups, 40 of middle dosage groups, 40 of low dose group, every group of male and female half and half.The dosage of three dosage groups is respectively 3g/kg, 1.8g/kg, 0.6g/kg, and medicine adds the twen-80 suspendible before use, gastric infusion, once a day, continuous six months.Matched group gavages the equivalent twen-80.Experimental session observed and recorded every day animal general state, the weighing the weight of animals once weekly.When experiment finishes, each treated animal is carried out electrocardiogram, blood, biochemistry and pathological examination.When proceeding to three month, experiment puts to death each 10 of heavy dose of group and control animals; Each group is put to death 30 in the time of six month, keeps 10, and the presumable toxic effect of medicine and recovery situation were observed in drug withdrawal in one month; All put to death in the time of seven month.Result shows, the total phenolic acid of rats gavaged Erigeron breviscapus (Vant.) Hand.-Mazz. is after six months, and there are no significant changes for the activity of three dosage treated animals, appetite, defecation and growth promoter; The hemogram of each administration treated animal, hepatic and renal function, electrocardiogram and main organs index and matched group relatively there are no significant difference (through t assay statistics, p＞0.05), and all in the normal physiological scope.Pathological examination shows, matched group and three each organs and tissues structures of administration treated animal are showed no obvious pathologic and change.
The impact of the embodiment 9 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. on the Experimental Hyperlipoidemia Rabbits blood fat
Get normal feedstuff and stablize the rabbit in 2 weeks, be divided at random 4 groups (normal saline group, total phenolic acid I group, total phenolic acid II group, zhibituo groups) by body weight, each treated animal is when giving normal feedstuff, and gavage gives cholesterol 0.2g/kg, once-a-day; The administration group is administered once every day; Matched group gives normal saline; In continuous 8 weeks, 2,4,6,8 weeks were got blood by ear vein after modeling, and 3000rpm is centrifugal, and separation of serum is standby to be surveyed.The result demonstration, total phenolic acid can significantly reduce triglyceride and the serum cholesterol level (table 1,2) of Hyperlipidemia Rabbits.
Table 1: the affect experimental data of total phenolic acid on triglyceride
Annotate: compare * p＜0.05 with the normal saline group; * p＜0.01
Table 2: the affect experimental data of total phenolic acid on serum cholesterol
Annotate: compare * p＜0.05 with the normal saline group; * p＜0.01
The impact that the embodiment 10 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. form rat suppository
Get the healthy SD rat, male, body weight 200～300g is divided into administration group and matched group at random.Weigh rear successive administration 3 days; Matched group gives normal saline.Weigh during experiment, lumbar injection pentobarbital sodium 0.05g/kg, insert polyethylene tube in trachea in order to remove the trachea endocrine, separate right common carotid artery and left external jugular vein, put into four long trumpeter's art silk threads of a 5cm in the stage casing of three sections polyethylene tubes, polyethylene tube is filled with heparin-saline solution (50u/ml) first through silicidation in pipe, right common carotid artery and left external jugular vein are inserted respectively in two ends, consist of the artery-vein bypass circuit.Recover to use bulldog clamp pinch off blood flow after blood flow 15min, take out rapidly silk thread and weigh, it is wet weight of thrombus that gross weight deducts silk thread weight, calculates suppression ratio.Result demonstration, total phenolic acid can effectively suppress the rat thrombus in vivo and form (table 3).
Table 3: the affect experimental data of total phenolic acid on the formation of rat thrombus in vivo
| Group | Dosage (mg/kg) | Thrombus weight (mg) | Suppression ratio (%) |
| The normal saline group | Deng capacity | 23.69±5.43 | 0 |
| Total phenolic |
30 | 17.55±4.27 ** | 25.9 |
| Total phenolic acid II group | 60 | 17.05±3.38 ** | 28.0 |
Annotate: compare with normal saline
*P＜0.01
The impact of the embodiment 11 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. on stasis syndrome rat model hemorheological property
Get identical raising condition lower body and focus on the Wistar rat of about 300g, male and female half and half are divided into 5 groups (blank group, model group, total phenolic acid I group, total phenolic acid II group, SHUXUENING groups) at random, 1 week of administration group successive administration, once a day; Model group and blank group all give isometric normal saline.After last administration in the 7th day, except the blank group, equal adrenaline subcutaneous injection 0.8ml/kg, totally twice, 5min in frozen water between double injection Adr, is immersed with rat in interval 4 hours, stops eating after disposal, gets blood examination inferior morning and surveys.The result demonstration, total phenolic acid group and model group relatively can significantly reduce the level of whole blood contrast viscosity and serum specific viscosity, improve RBC electrophoresis index, reduce the RBC hematocrit, point out this product to have the effect (table 4) that improves hemorheological property.
Table 4: the affect experimental data of total phenolic acid on the rat blood rheological characteristic
Annotate: compare #p＜0.05, ##p＜0.01 with the normal saline group; Compare * p＜0.05, * * p＜0.01 with model group
The impact of the embodiment 12 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. on rat platelet aggregation
Get male SD rat, gavage gives total phenolic acid 14 days continuously, get blood was administered once in front 1 hour, pentobarbital sodium anesthesia, abdominal aortic blood, 3.8% sodium citrate anticoagulant (1: 9), centrifugal preparation rich platelet (PRP) and anemia platelet (PPP) are with the platelet aggregation of MPG3E type platelet aggregation instrument mensuration ADP (1.25mmol/L) and thrombin induction.Result shows, total phenolic acid can significantly reduce platelet aggregation rate (table 5) due to ADP and thrombin.
The affect experimental data of the total phenolic acid of table 5 on rat platelet aggregation
Annotate: compare * p＜0.05 with reference substance
The embodiment 13 total phenolic acid of Erigeron breviscapus (Vant.) Hand.-Mazz. cause the impact of local organization ischemic necrosis mouse model on radical damage
Give the total phenolic acid of mouse gavaging the present invention, modeling after 15 days, with the rose-red injection mouse vein of photoactive substance, cold light source irradiation mice cervical region with the 560nm wavelength, injected the Evans blue dyestuff through the tongue vein in 3 hours after modeling, put to death animal after 1 hour, get cerebral tissue and make homogenate, with the optical density of the average cerebral tissue of colorimetric method for determining.The infraction severe patient, its dyeing is dark, and optical density is large, and ischemia half dark space area is large.Experimental result shows, total phenolic acid can reduce the optical density of model mice cerebral tissue Evans blue, and reduce the area (table 6) of ischemia half dark space, due to oxygen-derived free radicals is increased, blood vessel injury and thrombosis have inhibitory action, are conducive to prevent and treat blood vessel injury and the tissue necrosis of ischemic vascular disease.
The affect experimental data of the total phenolic acid of table 6 Erigeron breviscapus (Vant.) Hand.-Mazz. on the damage of radical damage induction of vascular and cerebral infarction mouse model
| Group | Number of animals | Evans blue (OD value) | Ischemia half dark space area |
| Model group | 6 | 0.1334±0.0769 | ++++ |
| Dihydroergotoxine (positive control) | 7 | 0.1122±0.0451 | + |
| Total phenolic acid I (30mg/kg) | 7 | 0.1098±0.0359 | ++ |
| Total phenolic acid II (60mg/kg) | 8 | 0.1073±0.0313 * | + |
Annotate: compare * p＜0.05 with model group; Ischemia half blanking bar ,+, ++, +++, ++ ++ represent that area is ascending.
Claims (6)
1. the preparation method of the total phenolic acid of an Erigeron breviscapus (Vant.) Hand.-Mazz. [Erigeron breviscapus (Vant) Hand-Mazz] and sodium salt thereof is characterized in that comprising following operating procedure:
A. medical material is with one of aqueous alcohol, methanol or water, and preferred concentration is 60%～95% ethanol, reflux, extract, 2～5 times, and merge extractive liquid,, concentrating under reduced pressure gets extractum;
B. extractum with 2～15 times of water gaging suspendibles, slowly adds dilute sodium hydroxide, sodium carbonate or sodium bicarbonate solution under stirring, regulates pH value 7.0～8.5, and is centrifugal, filters;
C. filtrate is with dilute hydrochloric acid or dilute sulfuric acid adjust pH 1～3, and is standing, minute gets precipitation, is washed to pH closely neutral, and drying is pulverized;
D. precipitation is used the lipotropy organic solvent degreasing, volatilizes, and gets total phenolic acid;
E. total phenolic acid further with dilute sodium hydroxide, sodium carbonate or sodium bicarbonate solution dissolving, transfers pH closely neutral, filters, and concentrated, drying gets the total phenols acid sodium-salt.
2. the defat in the described preparation method of claim 1 is characterized in that with one of normal hexane, cyclohexane extraction, petroleum ether or gasoline preferred boiling range is the petroleum ether of 60～90 ℃, under room temperature or heating, the total phenols acid crude is flooded or reflux, extract, 2～10 times.
3. the described total phenolic acid of claim 1, is characterized in that containing scutellarin 4%～20% (weight), 3,5-two-O-caffeoylquinic acids 0.1%～5% (weight) and 4,5-, two-O-caffeoylquinic acids 0.5%～8% (weight).
4. the described total phenolic acid of claim 1, is characterized in that containing 3-O-caffeoyl-γ-quinide 0.01%～1% (weight) and 1,3-, two-O-caffeoylquinic acids 0.01～2% (weight).
5. the described total phenolic acid of claim 1, is characterized in that the content of pyromeconic acid is lower than 10ppm.
6. the described total phenolic acid of claim 1 or its sodium salt, separately or with the combinations of substances of other medicinal license, in the medicine of preparation control ischemic cardio cerebrovascular diseases or the application in health product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103951721A (en) * | 2014-05-05 | 2014-07-30 | 南京瑞菁医药科技有限责任公司 | Application of quercetin-O-glucoside derivative to treatment of lipid metabolism disorders |
| CN104415046A (en) * | 2013-08-30 | 2015-03-18 | 河北以岭医药研究院有限公司 | Application of apigenin-7-o-beta-D-glucuronide |
| CN113896750A (en) * | 2021-11-08 | 2022-01-07 | 陈磊 | Grading extraction process of effective components of erigeron breviscapus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104415046A (en) * | 2013-08-30 | 2015-03-18 | 河北以岭医药研究院有限公司 | Application of apigenin-7-o-beta-D-glucuronide |
| CN104415046B (en) * | 2013-08-30 | 2019-03-05 | 河北以岭医药研究院有限公司 | A kind of application of apiolin -7-o- β-D-Glucose aldehydic acid glycosides |
| CN103951721A (en) * | 2014-05-05 | 2014-07-30 | 南京瑞菁医药科技有限责任公司 | Application of quercetin-O-glucoside derivative to treatment of lipid metabolism disorders |
| CN103951721B (en) * | 2014-05-05 | 2016-08-24 | 南京瑞菁医药科技有限责任公司 | The application in treatment lipid metabolism disorder of the Quercetin-O-glycosides derivative |
| CN113896750A (en) * | 2021-11-08 | 2022-01-07 | 陈磊 | Grading extraction process of effective components of erigeron breviscapus |
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