CN101311160A - Method for preparing red sage root salviandic acid A - Google Patents
Method for preparing red sage root salviandic acid A Download PDFInfo
- Publication number
- CN101311160A CN101311160A CNA2007100996188A CN200710099618A CN101311160A CN 101311160 A CN101311160 A CN 101311160A CN A2007100996188 A CNA2007100996188 A CN A2007100996188A CN 200710099618 A CN200710099618 A CN 200710099618A CN 101311160 A CN101311160 A CN 101311160A
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- CN
- China
- Prior art keywords
- salvianolic acid
- danshen root
- root salvianolic
- hpd
- preparation
- Prior art date
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- AJSGWTLZEUBGFV-UHFFFAOYSA-N salviol Natural products CC(C)c1cc2CCC3C(CC(O)CC3(C)C)c2cc1O AJSGWTLZEUBGFV-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
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- 230000002861 ventricular Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method for salvianolic acid A in salvia miltiorrhiza and is characterized in that the method takes salvianolic acid B in the salvia miltiorrhiza as the raw material and converts the salvianolic acid B into the salvianolic acid A in the salvia miltiorrhiza after adjusting pH value to 3.5-6.0 and chemical reaction, thus greatly increasing the extraction yield of the salvianolic acid A; the pharmacological tests prove that a preparation prepared by the salvianolic acid A in the salvia miltiorrhiza has excellent pharmacological action.
Description
Technical field
The present invention relates to medicine, health food technology field, be specifically related to a kind of preparation method of danshen root salvianolic acid A, promptly is raw material with the salvianolic acid B from salvia miltiorrhiza, through overregulate PH to 3.5-6.0, chemical reaction changes into danshen root salvianolic acid A.
The application's chemical reaction method is meant chemical reaction method conventional in the organic chemistry, does not need to add catalyzer, under certain pH value condition, and the conversion reaction that self carries out.
The application's salvianolic acid B from salvia miltiorrhiza content is 10%-100%, and promptly salvianolic acid B from salvia miltiorrhiza can be extract, efficient part, component significant part, effective constituent or pure product; Also can be that content is lower than 10% crude extract.
Background technology
The red sage root is flavour of a drug promoting blood circulation and removing blood stasis classical in the Chinese medicine, it has the main water soluble component of chemical ingredients of function of promoting blood circulation to disperse blood clots, red sage root water soluble ingredient has the phenolic acid structure more, it is salvianolic acid A, B, C, D, effective constituents such as E, the highest in the Radix Salviae Miltiorrhizae total phenolic acids with content of danshinolic acid B, therefore, the emphasis of people's exploitation concentrates on salvianolic acid B, and still, what the red sage root water soluble ingredient activity was best is salviol acid A (Du Guanhua [preclinical medicine and clinical, 2000,20 (5): 10～14], Hu Yiyang [herbal pharmacology journal, 1997,18 (5): 478-480]), but danshen root salvianolic acid A content in the red sage root is very low, is ignored by people because can't carry out suitability for industrialized production.Consult document, we can learn therefore the content of salvianolic acid A in the red sage root is about 5/10000ths,, even it is extracted by a series of method, can only obtain the trace danshen root salvianolic acid A, be applied to clinical in, can not be accepted by the patient because its price is too high; But danshen root salvianolic acid A has good pharmacologically active, can be applied in medicine, the protective foods, and therefore, a lot of medical workers wish salvianolic acid A is carried out suitability for industrialized production, are developed as the patent medicine preparation.
The processing method of danshen root salvianolic acid A extraction purifying is disclosed in existing document and the patent, though can access danshen root salvianolic acid A, but its emphasis all is placed on how with on danshen root salvianolic acid A and the separate impurities, its extraction yield is very low, can't in suitability for industrialized production, realize, also can't obtain the danshen root salvianolic acid A of " patent medicine " level, this is one difficulty of pendulum in face of a lot of researchers.Chinese patent CN1397276A disclosed water or alcohol extracting, resin chromatography in 2002, prepared the method for salvianolic acid A again through the purifying of silica gel column chromatography; Chinese patent CN1513848IA disclosed and adopts water temperature centrifugal, ultrafiltration, acid adjustment, organic solvent extraction to prepare the method for salvianolic acid A again through reversed phase column chromatography in 2003; Chinese patent CN1887849A disclosed the method for adjust pH behind water or alcohol extracting, the column chromatography, organic solvent extraction in 2006; These methods all are directly danshen root salvianolic acid A to be extracted to design a series of purification process again and obtain salvianolic acid A from the red sage root, but because salvianolic acid A content is low in the red sage root, the extraction yield of these methods is very low, can't realize suitability for industrialized production, also can't develop and be applied to clinical pharmaceutical preparation; Chinese patent CN1830947A, disclosed that red sage root water is carried in 2006 or purification process such as alcohol extracting, high-temperature high-voltage reaction, column chromatography, it is 101-140 ℃ that this method adopts high-temperature and high-pressure conditions, pressure is 0.01-0.25MPa, this can't realize in actually operating obviously, because temperature and pressure have certain relation, and this application does not obviously meet this relation, therefore can't realize operation.
Consulting document and patent, is not raw material with the salvianolic acid B from salvia miltiorrhiza, and after chemical reaction obtains the processing method of danshen root salvianolic acid A.
Summary of the invention
For these reasons, we arrive through consulting literature, danshen root salvianolic acid A is that a part Salvianic acidA and two molecule coffic acid condensations form, and being three molecule Salvianic acidAs and the condensation of a part coffic acid, salvianolic acid B from salvia miltiorrhiza forms, the chemical structure of salvianolic acid A and salvianolic acid B has similar place, does the two have onrelevant that salvianolic acid A and salvianolic acid B? be with this query, our scientific research personnel experimental studies have found that by secular, with the salvianolic acid B is raw material, do not need to add any catalyzer, only need change into danshen root salvianolic acid A by regulating PH to 3.5-6.0 and chemical reaction, the extraction yield of the salvianolic acid A that this method obtains can reach 20%-50%, can finely carry out suitability for industrialized production.
The present invention is achieved through the following technical solutions.
A kind of preparation method of danshen root salvianolic acid A is a raw material with the salvianolic acid B from salvia miltiorrhiza, through overregulate PH to 3.5-6.0, chemical reaction changes into danshen root salvianolic acid A.
The application's chemical reaction method is meant chemical reaction method conventional in the organic chemistry, does not need to add catalyzer, under certain PH condition, and the conversion reaction of self.
The application's salvianolic acid B from salvia miltiorrhiza content is 10%-100%, and promptly salvianolic acid B from salvia miltiorrhiza can be that extract, row are imitated position, component significant part, effective constituent or pure product; Also can be that content is lower than 10% crude extract.
Through our preliminary study, the salvianolic acid B from salvia miltiorrhiza of above-mentioned different content is in chemical reaction process, and all the other materials also can change into salvianolic acid B from salvia miltiorrhiza and change into danshen root salvianolic acid A then.
One, processing method
Method 1: get salvianolic acid B from salvia miltiorrhiza, be dissolved in water fully, transfer pH value to 3.5-6.0,110-130 ℃ temperature, heated 1-6 hour, solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract;
Annotate: under " 110-130 ℃ of temperature heated 1-6 hour " condition, carry out chemical reaction, make salvianolic acid B from salvia miltiorrhiza be converted into danshen root salvianolic acid A.
Method 2: get salvianolic acid B from salvia miltiorrhiza, be dissolved in water fully, transfer pH value to 3.5-6.0, put into the microwave extraction device, frequency is that 915MHz-2450MHz, power are 1000-15000 watt microwave extraction 0.5-2 hour, extracting liquid filtering, the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Annotate: under " put into the microwave extraction device, frequency is that 915MHz-2450MHz, power are 1000-15000 watt microwave extraction 0.5-2 hour " condition, carry out chemical reaction, make salvianolic acid B from salvia miltiorrhiza be converted into danshen root salvianolic acid A.
The danshen root salvianolic acid A extract can by nonpolar or the separation of low-pole macroporous resin column chromatography, silica gel column chromatography separation, sephadex lh-20 chromatographic separation, polymeric amide chromatography and separate and the organic solvent extracting access method in one or more methods carry out purifying.
Wherein said macroporous resin column is HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
Silica gel is purification on normal-phase silica gel, reverse phase silica gel.
Wherein organic solvent is selected from a kind of in ethyl acetate, propyl acetate, butylacetate, propyl carbinol and the Virahol.
Contain the pharmaceutical composition that the danshen root salvianolic acid A extract is an activeconstituents.
Formulation preparation: get above-mentioned danshen root salvianolic acid A, be prepared into preparation according to the conventional requirement of the pharmaceutics of tablet, capsule, granule, soft capsule, pellet, pill, oral liquid, powder injection, aqueous injection, infusion solution.
The solid preparation of the application's preparation carries out the dissolution rate experiment, and dissolution rate reaches more than 80% in the time of 45 minutes.
Two, detection method
Experimental technique:
Chromatographic column: C18 reverse-phase chromatographic column, NUCLEODUR, 250*4.6mm, ODS;
Chromatographic condition and system suitability experiment: with the octadecylsilane chemically bonded silica is weighting agent; Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 286nm; Number of theoretical plate should be not less than 60000 by salvianolic acid A; With acetonitrile-0.2% aqueous acetic acid is moving phase, carries out gradient elution by following condition of gradient elution, moves 90 minutes;
In the time of 0-15 minute, the ratio of acetonitrile reduces to 80% by 10% ratio that rises to 20%, 0.2% aqueous acetic acid by 90%; In the time of 15-55 minute, the ratio of acetonitrile reduces to 70% by 20% ratio that rises to 30%, 0.2% aqueous acetic acid by 80%; In the time of 55-65 minute, the ratio of acetonitrile reduces to 50% by 30% ratio that rises to 50%, 0.2% aqueous acetic acid by 70%; In the time of 65-72 minute, the ratio of acetonitrile reduces to 20% by 50% ratio that rises to 80%, 0.2% aqueous acetic acid by 50%; In the time of 72-77 minute, the ratio 20% of ratio 80%, 0.2% aqueous acetic acid of acetonitrile; In the time of 77-80 minute, the ratio of acetonitrile rises to 90% by 80% ratio of reducing to 10%, 0.2% aqueous acetic acid by 20%; In the time of 80-90 minute, keep acetonitrile-0.2% aqueous acetic acid to carry out wash-out with 10: 90 ratio;
The preparation of reference substance solution: precision takes by weighing the salvianolic acid A reference substance in volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of sample solution: sample thief adds dissolve with methanol and shakes up; Or precision measures or takes by weighing preparation, adds dissolve with methanol and shakes up, and be diluted to scale;
Assay method: the accurate reference substance solution of drawing, inject liquid chromatograph, the record color atlas; The accurate sample solution of drawing injects liquid chromatograph, calculates peak area ratio.
Get the application's processing method and obtain danshen root salvianolic acid A, through above-mentioned check and analysis experiment, its content is less than 100% more than or equal to 50%.
Three, extraction yield experiment
Experimental technique: get red rooted salvia, according to above-mentioned check and analysis experimental technique, measure danshen root salvianolic acid A content, be divided into equal portions, portion is method (Chinese patent CN1887849A traditionally, scheme 1) obtains the danshen root salvianolic acid A extract, a according to processing method (Chinese patent CN1830947A, scheme 2), scheme 3 obtains the danshen root salvianolic acid A extract according to the application's processing method, according to salvianolic acid A weight in the above-mentioned check and analysis experimental calculation danshen root salvianolic acid A extract, according to the extraction yield of salvianolic acid A weight calculating different process method, experimental result sees Table 1:
The extraction yield of table 1 different process method salvianolic acid A
Experiment conclusion: show by above-mentioned experiment, the extraction yield that the application's processing method obtains salvianolic acid A improves greatly than the extraction yield of salvianolic acid A in the prior art, this can't realize by simple extraction and purification process, must Radix Salviae Miltiorrhizae total phenolic acids be changed into salvianolic acid A by certain conversion, could fundamentally improve the extraction yield of salvianolic acid A.
Four, regulate the importance of pH value
Experimental program:
Scheme 1: get content and be 90% salvianolic acid B from salvia miltiorrhiza 100 grams, be dissolved in water fully, do not regulate pH value, directly carry out chemical reaction, reaction back concentrate drying obtains extract.
Scheme 2: get content and be 90% salvianolic acid B from salvia miltiorrhiza 100 grams, be dissolved in water fully, regulate pH value to 2.0, carry out chemical reaction, reaction back concentrate drying obtains extract.
Scheme 3: get content and be 90% salvianolic acid B from salvia miltiorrhiza 100 grams, be dissolved in water fully, regulate pH value 3.5-6.0, carry out chemical reaction, reaction back concentrate drying obtains extract.
Scheme 4: get content and be 90% salvianolic acid B from salvia miltiorrhiza 100 grams, be dissolved in water fully, regulate pH value 7.5, carry out chemical reaction, reaction back concentrate drying obtains extract.
According to salvianolic acid A weight in the above-mentioned check and analysis experimental calculation danshen root salvianolic acid A extract, according to the extraction yield of salvianolic acid A weight calculating different process method, experimental result sees Table 2:
The extraction yield of table 2 different process method salvianolic acid A
Annotate: with the content of the salvianolic acid B of above-mentioned raw materials medicine, the difference of its extraction yield is close with above-mentioned experimental result.
Experiment conclusion: show that by above-mentioned experiment before carrying out chemical reaction, must regulate the pH value of salvianolic acid B, though not so carry out certain chemical reaction, its extraction yield can not satisfy industrial production requirement
Five, pharmacological evaluation
Experiment 1
1. to the provide protection of anesthetized rat myocardial ischemia-reperfusion injury
Experimental technique:
Get the healthy SD rat, body weight 240-260g, random packet: blank group, FUFANG DANSHEN PIAN group, the application's solid preparation of salvianolic acid A of red sage root group.Place the pre-raising of equivalent environment 2 days, free diet.After pre-raising finishes, test, animal is weighed, and 20% urethane is pressed the 0.6ml/100g abdominal injection, after treating that anesthesia is satisfied, lie on the back and be fixed on the mouse plate, trachea cannula connects respirator, by 10～12ml Tidal volume, 70 times/minute frequency is exhaled, and continuous positive pressure breathing is inhaled: exhale than being 1: 1.Adjust respiration parameter according to the respiratory rate and the degree of depth.Connect electrocardiograph subsequently, survey normal ECG.Cut off front field of operation hair, iodine disinfection, cut off skin, subcutis, front muscle and manadesma 3～4cm, it is long to separate intercostal muscle 3cm with the 18# vascular clamp along the 3rd intercostal passivity, open thoracic cavity and pericardium, recording ecg, strut 3,4 ribs, refer to hold thoracic cavity, rat right side with left hand four, the assistant upwards pushes away thymus gland with the ophthalmology tweezer, finds ligation sign blood vessel great cardiac vein between left auricle of heart and pulmonary conus, the 2mm place is with there not being wound roundlet pin band 6-0 silk thread threading below left auricle of heart, depth of needle is 1～1.5mm, wide 2～3mm, recording ecg behind the threading, give corresponding soup through the tail vein, recording ecg behind the administration 10min, and with one the band groove little plastics pipe pad at the ligation position, the ligation thereon of two rear line heads.At once recording ecg after the ligation is cyanosis or the II S-T section back of a bow that leads with left chamber antetheca and upwards raises greater than 0.1mv and be that ligation successfully indicates (it is superseded that the S-T section does not have the changer) more than the lasting 0.5h.10min recording ecg is once more cut off ligature behind the ligation 30min after the ligation, realizes perfusion again, and record pours into electrocardiogram(ECG at once again, removes in the thoracic cavity layer-by-layer suture wall of the chest behind the hematocele, removes respirator, animal recovery autonomous respiration, and incision of trachea does not process.Irritate again at once, 10min, 20min, 40min, 1h, 2h, 3h recording ecg respectively.With heart after refrigerator and cooled is frozen 10min, from the apex of the heart entad the parallel coronary sulcus direction in the end 5 of equal thickness are cut in left chamber, put into the 1%TTC dye liquor, 37 ℃ of dyeing 10min, the necrotic area is not a garnet, the necrotic area is canescence.Digital camera is taken pictures.Weighed respectively in necrotic area and non-necrotic area, calculate the per-cent that the necrotic area accounts for left ventricular mass, i.e. infarction size.
Annotate: compare with the blank group,
*P＜0.01; Compare #P＜0.05 with positive FUFANG DANSHEN PIAN group
Experiment 2
Research to the provide protection of intraluminal middle cerebral artery occlusion in rats ischemical reperfusion injury
Experimental technique:
Animal random packet: blank group, Radix Salviae Miltiorrhizae Injection group, the application's red sage root A magnesium injection preparation group.The continuous tail intravenously administrable of each dosage group 3 days was made middle cerebral artery occlusion (MCAO) model with improvement line bolt method in 20 minutes behind the 4th day medicine.Behind the rat anesthesia, it is fixing that it is lain on the back.Separate right carotid (CCA), internal carotid artery (ICA) and external carotid artery (ECA), ligation ECA and CCA, after closing the ICA distal end with bulldog clamp folder, make a kerf in ECA and ICA crotch rapidly, insert the nylon wire (diameter is 0.25mm, marks apart from pommel 18mm place, is stained with heparin solution before the insertion) that an end is heated into smooth, spherical and has been coated with 0.1% poly-lysine, depth of penetration is 18mm, realizes that middle cerebral artery occlusion causes cerebral ischemia.Ligation ingress, nylon wire are stayed about 1cm, skin suture outward.Lift extremely slightly resistance of institute's the end of a thread that stays after 2 hours gently, realize that arteria cerebri media pours into again, modeling is finished.At ischemic 2h with pour into the body temperature of keeping rat in the 1h with electric blanket again, body temperature maintains 36.5～37.5 ℃ of anus temperature.The animal inclusion criteria is pressed Longa Pyatyi point system, gets the neural function behavior scoring and be 1,2,3,4 minute animal, (0 minute: the impassivity defective symptom; 1 minute: the offside forelimb can not stretch fully; 2 minutes: to sideway swivel; 3 minutes: topple over to offside; 4 minutes: can not oneself walk or stupor).The cerebral infarction scope is measured, rat model pours into 24h again, after the study of behaviour scoring, broken end is got brain, removes olfactory bulb, cerebellum and low brain stem, and remainder is at-20 ℃ of freezing 10min of refrigerator, crownly on ice pan be cut into 6, rapidly the brain sheet is placed the TTC dye liquor, 37 ℃ of lucifuge temperature are incubated 1h, take out to be placed on the 24h that keeps in Dark Place in 10% formalin.The non-ischemic region in dyed back is a rose, and infarct is a white.White organized carefully to dig down weigh, account for full brain weight per-cent as the cerebral infarction scope with blocking tissue's weight.
Brain water content is measured: after TTC dyeing is weighed, brain placed oven dry 12h claims dry weight in 120 ℃ of vacuum driers.Brain water content=(brain weight in wet base-brain stem is heavy)/brain weight in wet base * 100%.Experimental result sees table 4 for details.
The influence of table 4 pair intraluminal middle cerebral artery occlusion in rats ischemical reperfusion injury rat cerebral infarction scope and brain water content (X ± S)
Annotate: compare with the blank group,
*P＜0.01; Compare #P＜0.05 with positive FUFANG DANSHEN PIAN group
Experiment 3
To CCl
4Cause the influence of rat liver fibrosis
Experimental technique: select 60 of male and healthy Wistar rats, body weight 180-200g, all divide normal group, colchicine group (0.17mg/kg), the application's danshen root salvianolic acid A preparation group at random with rat, except that the normal control group, all the other respectively organize rat first in subcutaneous injection CCl
45ml/kg, 2 back subcutaneous injection 40%CCl4 sweet oil 3ml/kg, totally 6 weeks weekly later on.Except that normal group, the 1st～2 week, each group all gave the Semen Maydis powder feed that 20% lard adds 0.5% cholesterol at experimental session, and the 3rd～6 week raised with normal diet.Each administration group is irritated the soup that stomach gives corresponding dosage simultaneously when modeling begins, administration volume 1ml/100g, and administration time is totally 12 weeks.Each is organized after the medication cycle finishes, and cuts open the belly under the etherization, through the lower chamber dooor venous blood collection, detects Serum ALT, AST, Alb respectively, and gets a part of hepatic tissue and make liver homogenate, is used to detect oxyproline (Hyp) content.Other gets hepatic tissue and does HE dyeing and be used for pathological study.Experimental result sees Table 5:
The influence of Hyp in the table 5 pair Liver Fibrosis Model liver tissues of rats (X ± S)
Annotate: compare with the blank group,
*P＜0.01;
Experiment 4
Influence to the mouse pulmonary fibrosis model
Experimental technique:
Age in animal: 8-1 week, male, Kunming mouse, body weight 18-22g.Reagent: bleomycin for inj A5, Hebei, Tianjin pharmaceutical factory, prednisone acetate tablets: fairy house pharmaceutical Co. Ltd produces, and the time spent grinding powder is made into 50% suspension with dissolved in distilled water.PBS liquid, oneself is prepared.Adopt bleomycin A5 to duplicate animal diffuse interstitial pulmonary fibrosis model.Mouse is lain on the back on experiment table after with etherization, and fixing head and four limbs cut skin of neck, by the disposable injection bleomycin A5 of tracheae solution 0.05ml (pastille 0.1mg, 5mg/kg).Skin suture after injection finishes, mouse is upright, rotation make soup uniform distribution in lung as far as possible.The strict aseptic technique of surgical procedure.The blank group is in kind injected equivalent physiologic saline for substitute bleomycin A5, the clear-headed back of animal ad libitum access.The mouse modeling is divided into 6 groups at random after 24 hours, be respectively blank group, Prednisone acetate group (6.5mg/kg), the application's solid preparation group.The blank group is irritated stomach and is given distilled water 0.2ml/10g, every day three times; Positive controls is irritated stomach and is given Prednisone acetate, 6.5mg/kg, 0.2ml/10g, every day three times after the modeling; The administration group is irritated the soup that stomach gives each corresponding dosage by 0.2ml/10g after the modeling.More than each the group, successive administration 28 days.The result sees table 6 for details.
The influence of table 6 pair pulmonary fibrosis model mouse lung coefficient (X ± S)
Annotate: compare with the blank group,
*P＜0.01
Experiment 5
The MTT reduction method detects the preparation anti-tumor activity test
Experiment material:
MTT: with phosphate buffered saline buffer (PBS) the dissolving MTT final concentration 5mg/ml of 0.01mol/L, filtration sterilization, 4 ℃ keep in Dark Place after the packing.
The preparation of MTT lysate: the 80g sodium laurylsulfonate is dissolved in the N-N-dimethyl methyl phthalein amine of 200ml, and the heating in water bath hydrotropy adds 200ml distilled water, mixes with 1N hydrochloric acid (1: 1) with 80% acetate and transfers pH to 4.7.
Cell strain is selected for use: human normal cell line strain, human hepatoma cell strain and National People's Congress's sclc cell line.
Experimental technique: single cell suspension is inoculated in 96 orifice plates (with the RPM-1640 basic medium with cell dilution to 30000/ml, every hole adds 200 μ l and dilutes good cell), cultivates 24 hours under 37 ℃, 5% carbonic acid gas saturated humidity; Every group of four parallel samples; Remove substratum, get new preparation substratum and prepare cancer therapy drug (the application's preparation) solution by series concentration, every hole 20 μ l cultivated 48 hours; Every hole adds the MTT20 μ l of 2mg/ml, hatches 4 hours; Nutrient solution in the sucking-off hole (as far as possible fully) adds DMSO liquid (150 μ l/ hole), vibrates 10 minutes, and crystallisate is fully dissolved; Microplate reader detects each hole OD value, (detecting wavelength 560nm); Draw the cell viability graphic representation, obtain the IC50 value.Experimental result is as shown in table 7:
Table 7 cytotoxicity experiment result
Brief summary: the application's solid preparation has stronger cell toxicant, and its toxicity has certain selectivity to normal cell and cancer cells, and the application's solid preparation can be used for making each antitumor drug.
Experiment 6
Comparison to the rats'liver tumor suppression
Laboratory animal: rat, 150g-180g, male and female are regardless of.
Experiment medicine: physiological saline; The present invention respectively organizes preparation; Commercially available sodium cantharidinate tablet.
Experimental technique: get rat and be divided into physiological saline group, sodium cantharidinate tablet group, preparation group of the present invention, make W
256Liver in inoculation, inoculate after 7 days, press the dosage intraperitoneal injection of anesthesia of 35mg/kg with vetanarcol, fixing, cutting open the belly exposes liver, tumor surface maximum diameter (a) and path (b) are pressed (a*b on the measurement liver
2)/2=V (gross tumor volume).Separate stomach, arteria duodenalis, arteria hepatica communis and proper hepatic artery, ligation stomach, arteria duodenalis far-end, with silver brain clip blocking-up arteria hepatica communis, in sending into proper hepatic artery again at the gastroduodenal artery upper cut and after inserting external diameter 0.3mm conduit under the operating loupe, inject respectively by the experiment grouping then and be subjected to the reagent thing, postoperative tube drawing ligation gastroduodenal artery, decontrol the arteria hepatica communis silver brain clip, sew up the incision again, place animal housing to wait to revive rat, continue breeding observing, performed the operation back 8 days, detect gross tumor volume by last method, experimental result sees Table 8:
Table 8 is respectively organized preparation to the rejection ratio of tumour
Annotate: compare with the physiological saline group
*P＜0.01;
Experiment 7
Anti-ageing experiment
Experimental technique:
With aged Shanghai is the mouse random packet, 14/group, and male and female half and half, the administration group is irritated stomach preparation 15mg/kg of the present invention, 3 weeks of administration altogether every day.Blood 50 μ l are got in the mouse docking, measure the activity of SOD according to the autoxidizable method of pyrogallol.With the mouse sacrificed by decapitation, take out liver, inhale with filter paper and remove residual blood, shred and weigh, add physiological saline, be prepared into 1% homogenate, adopt the thiobarbituricacid method to measure the content of LPO in the hepatic tissue.Experimental result sees Table 9:
Table 9 solid preparation is to the influence of SOD, LPO
Annotate: compare with control group
*P＜0.01,
*P＜0.05; Compare #P＜0.05 with positive controls
Six, preparation embodiment
Embodiment 1
Salvianolic acid B from salvia miltiorrhiza is a raw material, through overregulate PH to 3.5-6.0, chemical reaction changes into danshen root salvianolic acid A.
The application's chemical reaction method is meant chemical reaction method conventional in the organic chemistry, does not need to add catalyzer, under certain pH value condition, and the conversion reaction that self carries out.
The application's salvianolic acid B from salvia miltiorrhiza content is 10%-100%, and promptly salvianolic acid B from salvia miltiorrhiza can be extract, efficient part, component significant part, effective constituent or pure product; Also can be that content is lower than 10% crude extract.
Embodiment 2
Getting content is that 10% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 3.5,110 ℃ of temperature, heats 1 hour, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 3
Getting content is that 10% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 3.5, puts into the microwave extraction device, and frequency is that 915MHz, power are 1000 watts microwave extraction 0.5 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 4
Getting content is that 50% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5,120 ℃ of temperature, heats 3 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 5
Getting content is that 50% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5, puts into the microwave extraction device, and frequency is that 2450MHz, power are 10000 watts microwave extraction 1 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 6
Getting content is that 99.9% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 6.0,130 ℃ of temperature, heats 6 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 7
Getting content is that 99.8% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 6.0, puts into the microwave extraction device, and frequency is that 2450MHz, power are 15000 watts microwave extraction 2 hours, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 8
Getting content is that 20% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.0,115 ℃ of temperature, heats 2 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 9
Getting content is that 20% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.0, puts into the microwave extraction device, and frequency is that 915MHz, power are 2000 watts microwave extraction 0.8 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 10
Getting content is that 75% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 5.5,125 ℃ of temperature, heats 5.5 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 11
Getting content is that 85% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 5.0, puts into the microwave extraction device, and frequency is that 2450MHz, power are 14500 watts microwave extraction 1.8 hours, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 12
Getting content is that 95% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5,120 ℃ of temperature, heats 4.5 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 13
Getting content is that 85% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5, puts into the microwave extraction device, and frequency is that 2450MHz, power are 10000 watts microwave extraction 1 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 14
Getting content is that 5% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 3.5,110 ℃ of temperature, heats 4.5 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 15
Getting content is that 8% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 3.5, puts into the microwave extraction device, and frequency is that 2450MHz, power are 15000 watts microwave extraction 1 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 16
Getting content is that 4% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 6.0,130 ℃ of temperature, heats 4.5 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 17
Getting content is that 2% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 6.0, puts into the microwave extraction device, and frequency is that 915MHz, power are 1000 watts microwave extraction 1 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 18
Getting content is that 9% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5,120 ℃ of temperature, heats 4.5 hours, and solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 19
Getting content is that 4% salvianolic acid B from salvia miltiorrhiza is dissolved in water fully, transfers pH value to 4.5, puts into the microwave extraction device, and frequency is that 2450MHz, power are 10000 watts microwave extraction 1 hour, extracting liquid filtering, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
Embodiment 20
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate is separated with a kind of macroporous resin column chromatography among the AB-8 through HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400, obtains danshen root salvianolic acid A.
Embodiment 21
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate through HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 with AB-8 in a kind of macroporous resin column chromatography separate and silica gel column chromatography, obtain danshen root salvianolic acid A.
Embodiment 22
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate through HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 separate with a kind of macroporous resin column chromatography among the AB-8 and ethyl acetate, propyl acetate, butylacetate, propyl carbinol and Virahol in a kind of organic solvent extraction, obtain danshen root salvianolic acid A.
Embodiment 23
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate is separated through silica gel column chromatography, obtains danshen root salvianolic acid A.
Embodiment 24
Get above-mentioned any embodiment danshen root salvianolic acid A extract, be dissolved in water, filter, filtrate obtains danshen root salvianolic acid A through the sephadex lh-20 chromatographic separation.
Embodiment 25
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate is separated through the polymeric amide chromatography, obtains danshen root salvianolic acid A.
Embodiment 26
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate is separated with the polymeric amide chromatography with a kind of macroporous resin among the AB-8 through HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 and is separated, organic solvent extraction obtains salvianolic acid A.
Embodiment 27
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate through HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 separate with a kind of macroporous resin among the AB-8 and, the sephadex lh-20 chromatographic separation, organic solvent extraction obtains salvianolic acid A.
Embodiment 28
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, the filtrate organic solvent extraction obtains salvianolic acid A.
Embodiment 29
Get any danshen root salvianolic acid A extract of the foregoing description 1-19, be dissolved in water, filter, filtrate obtains danshen root salvianolic acid A through silica gel column chromatography separation, sephadex lh-20 chromatographic separation.
Embodiment 30
Get above-mentioned any embodiment danshen root salvianolic acid A, be prepared into tablet, capsule, granule, soft capsule, pellet, pill, oral liquid, powder injection, aqueous injection, infusion solution according to the conventional requirement of pharmaceutics.
Annotate: the present invention's concrete technical scheme required for protection is not limited to the concrete combination of the expressed technical scheme of the foregoing description.
Claims (8)
1, a kind of preparation method of danshen root salvianolic acid A is characterized in that with the salvianolic acid B from salvia miltiorrhiza being raw material, through overregulate PH to 3.5-6.0, chemical reaction changes into danshen root salvianolic acid A.
2, the preparation method of a kind of danshen root salvianolic acid A according to claim 1, wherein chemical reaction method is:
Method 1: get salvianolic acid B from salvia miltiorrhiza, be dissolved in water fully, transfer pH value to 3.5-6.0,110-130 ℃ temperature, heated 1-6 hour, solution filters, and the filtrate concentrate drying obtains the danshen root salvianolic acid A extract;
Method 2: get salvianolic acid B from salvia miltiorrhiza, be dissolved in water fully, transfer pH value to 3.5-6.0, put into the microwave extraction device, frequency is that 915MHz-2450MHz, power are 1000-15000 watt microwave extraction 0.5-2 hour, extracting liquid filtering, the filtrate concentrate drying obtains the danshen root salvianolic acid A extract.
3, each method of the system of a kind of danshen root salvianolic acid A according to claim 1 and 2, wherein the content of salvianolic acid B from salvia miltiorrhiza is 10%-100%.
4, the preparation method of a kind of danshen root salvianolic acid A according to claim 1 and 2, wherein the danshen root salvianolic acid A extract can carry out purifying by one or more methods in nonpolar or the separation of low-pole macroporous resin column chromatography, silica gel column chromatography separation, sephadex lh-20 chromatographic separation, the separation of polymeric amide chromatography and the organic solvent extracting access method.
5, the preparation method of a kind of danshen root salvianolic acid A according to claim 4, wherein in the purification process nonpolar or low-pole macroporous resin be HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 and AB-8 in a kind of.
6, the preparation method of a kind of danshen root salvianolic acid A according to claim 4, wherein organic solvent is selected from a kind of in ethyl acetate, propyl acetate, butylacetate, propyl carbinol and the Virahol in the purification process.
7, the danshen root salvianolic acid A of the preparation method of a kind of danshen root salvianolic acid A according to claim 1 and 2 preparation.
8, be the pharmaceutical composition of activeconstituents according to the described danshen root salvianolic acid A of claim 7.
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| CN2007100996188A CN101311160B (en) | 2007-05-25 | 2007-05-25 | Method for preparing red sage root salviandic acid A |
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| CN2007100996188A CN101311160B (en) | 2007-05-25 | 2007-05-25 | Method for preparing red sage root salviandic acid A |
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| CN101991565A (en) * | 2010-12-13 | 2011-03-30 | 南京中医药大学 | Application of salvianolic acid A in preparing medicament for inducing apoptosis of tumor cells and/or inhibiting propagation of tumor cells |
| CN102028678A (en) * | 2010-12-13 | 2011-04-27 | 南京中医药大学 | Application of salvianolic acid A in preparing anti-tumor metastasis medicine |
| CN102212005A (en) * | 2010-04-06 | 2011-10-12 | 山东靶点药物研究有限公司 | Method for purifying salvianolic acid A by adopting normal phase chromatography |
| CN102212004A (en) * | 2010-04-06 | 2011-10-12 | 山东靶点药物研究有限公司 | Method for preparing salvianolic acid A by catalytically converting salvianolic acid B |
| CN102219685A (en) * | 2010-04-16 | 2011-10-19 | 北京本草天源药物研究院 | Method for preparing salvianolic acid A of salvia miltiorrhiza |
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