CN104161799B - Compound red sage root extract and its preparing method - Google Patents
Compound red sage root extract and its preparing method Download PDFInfo
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Abstract
The invention provides a compound salvia miltiorrhiza extract and a preparation method thereof, wherein the preparation method of the extract comprises the following steps: extracting Saviae Miltiorrhizae radix and Notoginseng radix with water, refining the obtained extractive solution with macroporous resin, concentrating the obtained eluate, hydrolyzing with alkali, and concentrating to obtain soft extract or drying. The preparation method can effectively improve the production efficiency. The compound salvia miltiorrhiza extract removes impurity components such as saccharides, proteins and the like in the prior art, and greatly improves the content of active ingredients. The invention also discloses the application of the compound salvia miltiorrhiza extract in preparing the medicine for treating cardiovascular diseases.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to a compound salvia miltiorrhiza extract and a preparation method thereof.
Background
With the improvement of living standard, the aging of the world population and the younger of the attack population, cardiovascular and cerebrovascular diseases increase year by year and become diseases seriously harming the health and the life quality of human beings. The cardiovascular and cerebrovascular diseases have the characteristics of high morbidity, high disability rate, high mortality rate, high recurrence rate and more complications, namely more than four and one, and at present, the cardiovascular and cerebrovascular disease patients in China exceed 2.7 hundred million people! Nearly 300 million people die of cardiovascular and cerebrovascular diseases in China every year, and the death causes account for 51 percent of the total death causes of China every year. While 75% of surviving patients lose labor capacity to varying degrees and 40% are heavily disabled.
The red sage root is also named red ginseng, purple salvia root, red root, etc. Bitter and slightly cold. It enters heart and liver meridians. Can dilate coronary artery, increase coronary blood flow, improve myocardial ischemia, infarction and heart function, regulate heart rhythm, dilate peripheral blood vessel, and improve microcirculation; can improve the hypoxia tolerance of the organism; has anticoagulant, fibrinolysis promoting, platelet aggregation inhibiting, and thrombosis inhibiting effects; can reduce blood lipid and inhibit the formation of coronary atherosclerosis; can inhibit or relieve hepatocyte degeneration, necrosis and inflammatory reaction, promote hepatocyte regeneration, and has anti-fibrosis effect; can shorten the recovery period of erythrocyte and hemoglobin, increase reticulocyte, promote tissue repair, and accelerate fracture healing; has central nerve inhibiting effect; has anti-tumor effect; can enhance the immune function of the organism; can reduce blood sugar; has inhibitory effect on various bacteria such as tubercle bacillus.
Pseudo-ginseng has the functions of removing blood stasis and stopping bleeding, and is a common medicine for blood syndromes. From Wang catalpa medicinal solution, it is said that it "harmonizes Ying to stop bleeding, unblocks collaterals to remove blood stasis, and removes blood stasis to astringe new blood. All blood stasis is broken after delivery, in menstrual period, traumatic injuries, carbuncle and swelling; it can stop any new blood, such as hematemesis, epistaxis, metrorrhagia, knife wound, and flechage wound. "(New compilation of materia medica)" indicates that: notoginseng root, radix Notoginseng, a hemostatic herb. It is used independently when the blood flows from the upper, middle and lower parts. Can be added into blood-tonifying and qi-invigorating herbs to refresh mind. The medicine can be used for tonifying without boiling, and the medicine can be used for tonifying without causing a quiet body. "
Clinically, the salvia miltiorrhiza and the pseudo-ginseng are usually applied in a combined way to treat cardiovascular diseases, and the marketed medicines comprise compound salvia miltiorrhiza tablets, compound salvia miltiorrhiza injection and the like. However, the production process of the common tablet and the capsule is laggard, the content of active ingredients is low, no quality control index exists, the tablet and the capsule need to be orally taken and absorbed by gastrointestinal tracts, the tablet and the capsule are absorbed into blood after the first pass effect of liver, and the bioavailability is low and the absorption is slow. Can not be suitable for emergency treatment of angina pectoris patients.
Disclosure of Invention
The compound salvia miltiorrhiza dripping pill is a coronary heart disease treatment drug with quick response and good curative effect, and is deeply welcomed by patients clinically. Aiming at the preparation process of the compound salvia miltiorrhiza dropping pill, the invention carries out deep research from the aspect of extraction process, and utilizes macroporous resin to refine and remove components such as saccharides, proteins and the like, thereby improving the content of effective components and reducing the dosage.
The invention aims to provide a compound salvia miltiorrhiza extract and a preparation method thereof.
The invention also aims to provide application of the compound salvia miltiorrhiza extract.
The preparation method of the compound salvia miltiorrhiza extract comprises the following steps: extracting Saviae Miltiorrhizae radix and Notoginseng radix with water, refining the obtained extractive solution with macroporous resin, concentrating the obtained eluate, hydrolyzing with alkali, and concentrating to obtain soft extract or drying.
According to one embodiment of the present invention, the method for preparing the extract solution comprises: reflux-extracting Saviae Miltiorrhizae radix and Notoginseng radix with 2-12 times of water for 1-3 times (each for 1-3 hr), filtering, and mixing the filtrates to obtain the extractive solution.
Preferably, in the preparation method of the extracting solution, the weight percentages of the salvia miltiorrhiza and the panax notoginseng are 20-98% and 2-80% respectively.
According to another embodiment of the present invention, the eluent is prepared by the following method: and (3) putting the extracting solution into a macroporous resin column, and eluting with water and 70-100% ethanol in sequence to obtain ethanol eluent, namely the eluent.
Preferably, the macroporous resin column is pretreated by the following method before use: taking macroporous resin, filling the macroporous resin into a column by using a 70-100% ethanol wet method, washing the column by using the 70-100% ethanol until filtrate is mixed with water and is not white and turbid, and finally washing the column by using distilled water until no alcohol smell exists.
More preferably, the macroporous resin column is pretreated by the following method before use: loading macroporous resin into a column by a 95% ethanol wet method, washing with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white turbid, and finally washing with distilled water until no alcohol smell exists.
Most preferably, the weight ratio of the macroporous resin to the salvia miltiorrhiza and panax notoginseng is 0.5-2.
According to yet another embodiment of the present invention, the hydrolysis method is: and (3) taking the eluent, concentrating under reduced pressure until no alcohol smell exists, then adding alkali liquor, and heating and refluxing to obtain the alcohol-free water-soluble organic solvent.
Preferably, the alkali liquor is selected from a solution of sodium bicarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and the pH value is 7-10.
More preferably, the lye is a 10% sodium bicarbonate solution.
According to yet another embodiment of the present invention, the hydrolysis time is 0.5 to 8 hours.
Most preferably, the preparation method of the compound salvia miltiorrhiza extract comprises the following steps:
(1) respectively taking radix salviae miltiorrhizae and pseudo-ginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, adding 4 times of water for reflux extraction for 1 hour, filtering, removing dregs, combining filtrates, and keeping the obtained extract for later use;
(2) macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: crude drug quantity (the amount of the salvia miltiorrhiza and panax notoginseng in the step (1) = 1: 1, filling the column with 95% ethanol by a wet method, washing the column with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column with a large amount of distilled water until no alcohol smell exists for later use;
(3) taking the extracting solution in the step (1), loading the extracting solution into a column at the flow rate of 1BV/hr, washing with 1BV water at the flow rate of 1BV/hr to remove impurities, finally eluting with 1BV/hr by using 2.5BV 95% ethanol as an eluent, and collecting the eluent for later use;
(4) concentrating the eluate of step (3) at 60 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (1/4 of the medicinal material amount) is present to obtain refined compound Saviae Miltiorrhizae radix concentrated solution; adding 10% sodium bicarbonate solution, heating and refluxing for 2 hours to obtain compound salvia miltiorrhiza hydrolysate, and concentrating under reduced pressure at 70 ℃ until the compound salvia miltiorrhiza hydrolysate is dried to obtain the compound salvia miltiorrhiza extract.
The compound salvia miltiorrhiza extract is used as a pharmaceutical active substance, and a pharmaceutically acceptable carrier is added as required to prepare a required preparation according to the conventional technology of pharmaceutics. The pharmaceutically active substance in the preparation can be 0.1-99.9%, and the rest is pharmaceutically acceptable carrier. The pharmaceutical preparation of the present invention is in a unit dosage form, which means a unit of preparation, such as each tablet of a tablet, each capsule of a capsule, each bottle of an oral liquid, each bag of a granule, and the like.
The formulation of the invention may be in any pharmaceutically acceptable dosage form including: tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, dripping pill, capsule, oral liquid, buccal agent, granule, pill, powder, unguent, pellet, suspension, powder, injection, suppository, ointment, plaster, cream, spray, drop, and patch.
Preferably, the formulation of the invention is an oral dosage form, such as: dripping pill, capsule, granule, pill, powder, pellet, and unguent.
Most preferably, the formulation of the present invention is a drop pill.
According to the invention, the dripping pill is prepared by the following method: dissolving the compound red sage root extract in a proper amount of water, and mixing the dissolved extract with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1: 2-5, adding a proper amount of borneol, and melting at 60-100 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Preferably, the auxiliary material is PEG 6000.
Preferably, the drug-adjuvant ratio is 1: 3.
Preferably, the melting temperature is 80 ℃.
Most preferably, the drop pill is prepared by the following method: dissolving the compound red sage root extract in a proper amount of water, and mixing the dissolved extract with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1:3, uniformly mixing, adding a proper amount of borneol, and melting at the temperature of 80 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
According to the invention, the preparation method of the compound salvia miltiorrhiza extract comprises the following steps: extracting Saviae Miltiorrhizae radix and Notoginseng radix with water, refining the obtained extractive solution with macroporous resin, concentrating the obtained eluate, hydrolyzing with alkali, and concentrating to obtain soft extract or drying.
According to one embodiment of the present invention, the method for preparing the extract solution comprises: reflux-extracting Saviae Miltiorrhizae radix and Notoginseng radix with 2-12 times of water for 1-3 times (each for 1-3 hr), filtering, and mixing the filtrates to obtain the extractive solution.
Preferably, in the preparation method of the extracting solution, the weight percentages of the salvia miltiorrhiza and the panax notoginseng are 20-98% and 2-80% respectively.
According to another embodiment of the present invention, the eluent is prepared by the following method: and (3) putting the extracting solution into a macroporous resin column, and eluting with water and 70-100% ethanol in sequence to obtain ethanol eluent, namely the eluent.
Preferably, the macroporous resin column is pretreated by the following method before use: taking macroporous resin, filling the macroporous resin into a column by using a 70-100% ethanol wet method, washing the column by using the 70-100% ethanol until filtrate is mixed with water and is not white and turbid, and finally washing the column by using distilled water until no alcohol smell exists.
More preferably, the macroporous resin column is pretreated by the following method before use: loading macroporous resin into a column by a 95% ethanol wet method, washing with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white turbid, and finally washing with distilled water until no alcohol smell exists.
Most preferably, the weight ratio of the macroporous resin to the salvia miltiorrhiza and panax notoginseng is 0.5-2.
According to yet another embodiment of the present invention, the hydrolysis method is: and (3) taking the eluent, concentrating under reduced pressure until no alcohol smell exists, then adding alkali liquor, and heating and refluxing to obtain the alcohol-free water-soluble organic solvent.
Preferably, the alkali liquor is selected from a solution of sodium bicarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and the pH value is 7-10.
More preferably, the lye is a 10% sodium bicarbonate solution.
According to yet another embodiment of the present invention, the hydrolysis time is 0.5 to 8 hours.
Most preferably, the preparation method of the compound salvia miltiorrhiza extract comprises the following steps:
(1) respectively taking radix salviae miltiorrhizae and pseudo-ginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, adding 4 times of water for reflux extraction for 1 hour, filtering, removing dregs, combining filtrates, and keeping the obtained extract for later use;
(2) macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: crude drug quantity (the amount of the salvia miltiorrhiza and panax notoginseng in the step (1) = 1: 1, filling the column with 95% ethanol by a wet method, washing the column with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column with a large amount of distilled water until no alcohol smell exists for later use;
(3) taking the extracting solution in the step (1), loading the extracting solution into a column at the flow rate of 1BV/hr, washing with 1BV water at the flow rate of 1BV/hr to remove impurities, finally eluting with 1BV/hr by using 2.5BV 95% ethanol as an eluent, and collecting the eluent for later use;
(4) concentrating the eluate of step (3) at 60 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (1/4 of the medicinal material amount) is present to obtain refined compound Saviae Miltiorrhizae radix concentrated solution; adding 10% sodium bicarbonate solution, heating and refluxing for 2 hours to obtain compound salvia miltiorrhiza hydrolysate, and concentrating under reduced pressure at 70 ℃ until the compound salvia miltiorrhiza hydrolysate is dried to obtain the compound salvia miltiorrhiza extract.
The invention also aims to provide a compound salvia miltiorrhiza preparation.
The compound salvia miltiorrhiza preparation is prepared by taking the compound salvia miltiorrhiza extract as a medicinal active substance, adding other active ingredients and/or pharmaceutically acceptable carriers as required and adopting the conventional technology of pharmaceutics. The formulation of the invention may be in any pharmaceutically acceptable dosage form including: tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, dripping pill, capsule, oral liquid, buccal agent, granule, pill, powder, unguent, pellet, suspension, powder, injection, suppository, ointment, plaster, cream, spray, drop, and patch.
Preferably, the formulation of the invention is an oral dosage form, such as: dripping pill, capsule, granule, pill, powder, pellet, and unguent.
Most preferably, the formulation of the present invention is a drop pill.
According to the invention, the dripping pill is prepared by the following method: dissolving the compound red sage root extract in a proper amount of water, and mixing the dissolved extract with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1: 2-5, adding a proper amount of borneol, and melting at 60-100 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Preferably, the auxiliary material is PEG 6000.
Preferably, the drug-adjuvant ratio is 1: 3.
Preferably, the melting temperature is 80 ℃.
Most preferably, the drop pill is prepared by the following method: dissolving the compound red sage root extract in a proper amount of water, and mixing the dissolved extract with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1:3, uniformly mixing, adding a proper amount of borneol, and melting at the temperature of 80 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Compared with the traditional water extraction and alcohol precipitation technology without macroporous resin refining, the method has the following advantages:
in the aspect of product quality, the compound salvia miltiorrhiza extract removes non-drug-effect components such as saccharides, proteins and the like, and greatly improves the content of the drug-effect components. Compared with the compound red sage root extract prepared by the traditional water extraction and alcohol precipitation process, the content of danshensu is improved by 198 percent, the content of notoginsenoside is improved by 113 percent, and the content of monosaccharide and disaccharide is reduced from 14.4 percent to 0 percent, thereby reflecting the characteristics of the modern traditional Chinese medicine and being beneficial to the control of the preparation process.
In the aspect of taking dosage, the refined compound salvia miltiorrhiza dripping pill prepared by taking the compound salvia miltiorrhiza extract as the raw material has 46.7 percent of daily dosage reduction on the premise of ensuring the drug effect, is more convenient to take, and thus improves the compliance of patients.
The extract disclosed by the invention is applied to preparation of a medicine for treating cardiovascular diseases.
Preferably, the extract is applied to the preparation of medicines for treating diseases caused by myocardial ischemia.
Preferably, the extract is applied to preparation of a medicament for inhibiting myocardial damage.
Experiments show that the compound salvia extract can remarkably relieve the main symptom indexes of acute myocardial infarction caused by rat coronary artery ligation, and can be used for treating various diseases caused by myocardial ischemia, including myocardial infarction, acute coronary syndrome, atrioventricular block, arrhythmia, coronary heart disease and angina pectoris.
Experiments show that the compound salvia extract can obviously improve the main symptom index of toxic myocarditis. Can be used for treating myocardial cell injury caused by myocarditis and myocardial infarction, and inhibiting myocardial ischemia caused by myocarditis.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1: the compound salvia miltiorrhiza extract has a phenolic acid component fingerprint spectrum.
FIG. 2: the saponin component fingerprint spectrum of the compound salvia miltiorrhiza extract is disclosed.
FIG. 3: the sugar component fingerprint spectrum of the compound salvia miltiorrhiza extract is disclosed.
FIG. 4: influence of compound red sage root extract on acute myocardial infarction caused by rat coronary artery ligation tests cardiac slice digital photos of different administration groups.
FIG. 5: in the test (toxic myocarditis model in vivo) for the effect of compound Saviae Miltiorrhizae radix extract on toxic myocarditis, the electron microscope images of myocardial cell ultrastructure of different administration groups are respectively blank control group, model group, captopril group, extract low group, extract middle group and extract high group.
Detailed Description
The embodiments of the present invention will be described in detail below, and the embodiments described with reference to the drawings are exemplary only for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Experimental example one macroporous resin refining process flow of compound salvia miltiorrhiza extract:
1. extracting medicinal materials: respectively taking 450g of salvia miltiorrhiza and 88g of panax notoginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, adding 4 times of water for reflux extraction for 1 hour, filtering, and discarding residues. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, AB-8 macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: crude drug quantity = 1: 1, filling the column with 95% ethanol by a wet method, washing the column with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column with a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 1BV/hr, washing with 1BV water at flow rate of 1BV/hr to remove impurities, eluting with 2.5BV 95% ethanol as eluent at flow rate of 1BV/hr, and collecting the eluent.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 60 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (1/4 of medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 16.1ml of 10% sodium bicarbonate solution, heating and refluxing for 2 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate.
3.2, concentrating and drying: concentrating compound Saviae Miltiorrhizae radix hydrolysate at 70 deg.C under reduced pressure to dry to obtain compound Saviae Miltiorrhizae radix extract.
4. Preparing refined compound salvia miltiorrhiza extract dropping pills:
taking about 5g of refined compound salvia extract, adding a proper amount of water to dissolve, and mixing with pre-dissolved PEG6000 according to the medicine-assistant ratio of 1:3, mixing uniformly, adding 1g of borneol, and melting at the temperature of 80 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Experimental example II detection data and analysis of refined compound radix Salviae Miltiorrhizae extract and unrefined sample
The contents of tanshinol, notoginsenoside, monosaccharide and disaccharide in the refined compound red sage root extract are respectively measured and compared with the compound red sage root extract prepared by the traditional water extraction and alcohol precipitation process. And (3) displaying data: the content of tanshinol in the compound Saviae Miltiorrhizae radix extract refined by macroporous resin is increased by 198%, the content of notoginsenoside is increased by 113%, and the content of monosaccharide and disaccharide is reduced from 14.4% to 0% (see figure 1, figure 2 and figure 3). The dosage is reduced by 46.7% in daily administration.
Experimental example three pharmacodynamic evaluation tests of compound red sage root extract
1. Drugs and reagents:
1.1 Compound Danshen extract (prepared according to the first embodiment of the present invention, divided into 3 dose groups, low extract, medium extract and high extract, respectively). The preparation method comprises the following steps: weighing the sample, and dissolving the sample with distilled water to prepare the liquid medicine with the required concentration.
1.2 captopril tablets (positive drug): shanghai Shi Guibao pharmaceutical Co., Ltd, Zhongmei, lot number: 1203032, respectively; the preparation method comprises the following steps:
the captopril tablets are ground into powder and dissolved by distilled water to prepare the liquid medicine with the required concentration.
1.30.9% sodium chloride injection: nanjing Xiaoying pharmaceutical industry group Limited, lot number: 2012011301.
1.4 Doxorubicin hydrochloride for injection: zhejiang Haizhen pharmaceutical industry, batch number: 120101.
1.5 chloral hydrate: national pharmaceutical group chemical reagents limited, batch number: 20100111.
1.6 Creatine Kinase (CK) assay kit, lot No.: 20120713, respectively; a kit for measuring Lactate Dehydrogenase (LDH),
batch number: 20120712, respectively; malondialdehyde (MDA) assay kit, lot No.: 20120717, respectively; superoxide dismutase (SOD) assay kit, lot number: 20120709, respectively; creatine kinase isoenzyme (CK-MB) assay kit, lot No.: 20120720, respectively; lactate Dehydrogenase (LDH) assay kit, lot No.: 20120709, respectively; aspartate Aminotransferase (AST) kit, lot number: 20120713. are provided by Nanjing institute of biological engineering.
1.7 red tetrazolium (TTC), national group chemical agents corporation, lot number: f20120308; the preparation method comprises the following steps: the TTC powder was weighed and dissolved in PBS buffer to prepare a 1% TTC solution.
1.8 high-glucose DMEM: product of GIBCO BRL company, USA.
1.9 calf serum: supplied by Hangzhou Sijiqing Co., Ltd. — 20 ℃.
1.10 Trypsin: provided by SUNSHINE technologies, Inc. of Nanjing.
1.11 dimethyl sulfoxide: shanghai Linfeng Chemicals, ltd., batch number: 051123.
1.12 penicillin: shandong anti-medicine, Inc., batch number: B041103.
1.13 streptomycin: shandong anti-medicine, Inc., batch number: 040601.
2. preparing main reagents:
2.1 preparation of high-glucose DMEM: adding NaHCO into 1L DMEM culture solution33.7G of penicillin G sodium, 0.05G of streptomycin sulfate, and 7.4% NaHCO3Or 1mol/L HCl is adjusted to pH 7.2-7.4. Calf serum (20% concentration) was added for cell culture.
2.2PBS buffer saline solution (g/L): NaCl8.0g, KCl0.20g, Na2HPO4·12H2O1.56g，KH2PO40.20 g. With 7.4% NaHCO3The pH of the solution was adjusted to 7.4 by 1mol/L HCl.
2.3 digestive juice: pancreatin, a product of AMRESCO corporation; EDTA-2Na, product of national drug group chemical reagents Co. Preparing pancreatin to 0.25% by PBS, filtering and sterilizing; EDTA was made up to 0.02% with PBS and autoclaved. Pancreatin, EDTA 1: 1 mixing and storing in a refrigerator at 4 ℃.
3. Experimental animals:
2 batches of clean grade SD rats, 230 ~ 250g, the first batch of rats are provided by the Suzhou industrial park Elmmet science and technology Limited company, animal certification number: SCXK (su) 2009-: SCXK (Zhe) 2008 + 0033; newborn 1-3 days SD rat suckling mice, male and female are not limited, and are provided by Qinglong mountain animal farms in Jiangning district of Nanjing city.
4. An experimental instrument:
4.1HX-300 ventilator: chengtai Union technologies, Inc.
4.2ECG-6511 electrocardiograph: shanghai optoelectronic medical electronics, Inc.
4.3HH-2 digital display constant temperature water bath: products of Guohua electric appliances Limited.
4.4BS224s model electronic balance: product of Beijing Sidolis Instrument systems, Inc.
4.5 electronic balance model BS110 s: product of Beijing Sidolis Instrument systems, Inc.
4.6DHG-9053A type electric heating constant temperature air-blast drying oven: shanghai sperm macro laboratory facilities, Inc.
4.7LDZ5-2 centrifuge: beijing medical centrifuge works.
4.8 superclean bench: suzhou clean plant.
4.9 inverted microscope: OLYMPUS, Inc. is a product of model CKX 41.
4.10 Water jacketed CO2Sterile incubator: thermo Electron Corporation, made in USA, FORMA3111, S/N: 304481-7185.
4.11 bench low speed centrifuge: shanghai medical instruments (group) Co., Ltd surgical instruments factory, type: CH 80-2.
4.12 constant temperature magnetic stirrer: shanghai selec instruments ltd, model: 85-2.
4.13 electrothermal blowing dry box: shanghai fuma experimental facilities ltd, model: DGX-9143B-1.
4.14 vertical self-control electric heating pressure steam sterilizer: shanghai Boxun practice Co., Ltd, type: YXQ-LS-50 SII.
4.15 electronic balance: beijing sydolis instruments systems ltd, model: BS 224S.
4.16BIO-RAD Model680 microplate reader: BIO-RAD Laboratories Ltd, UK.
5. The influence of the compound salvia miltiorrhiza extract on acute myocardial infarction caused by coronary artery ligation of rats:
cleaning male SD rats were randomly divided into 9 groups by weight, namely a pseudo-surgery group (distilled water), a model group (distilled water), low extract (crude drug dose of 81 mg/kg), medium extract (crude drug dose of 162 mg/kg), high extract (crude drug dose of 324 mg/kg), and 10 rats per group. Each group of rats was administered by gavage once a day for 7 consecutive days. The volume of administration was 0.5ml/100g body weight. 1h after the last administration, 300mg/kg of chloral hydrate is injected into the abdominal cavity for anesthesia, the abdominal cavity is fixed in a supine position, the incision part of the operation is disinfected by iodine tincture and alcohol in sequence, then the chest is opened at the third rib and the fourth rib, artificial respiration is carried out, the heart is exposed, the left anterior descending branch of the coronary artery is ligated by a medical non-invasive suture needle 5/0, the thoracic cavity is closed rapidly and disinfected in a conventional way, the operation process is not more than 30s, the artificial respiration is continued for 1-2 min after the operation, and 20 ten thousand units of penicillin is injected into the muscle (i.m) to prevent. Recording standard II-lead electrocardiogram 5min before operation, 0s, 1min, 5min, 15min, 1h and 4h after ligation, and inspecting the change of J point of electrocardiogram.
After the experiment, the heart is taken out immediately, blood is washed by normal saline, blood vessels at the atria and the bottom of the heart are cut off, the weight of the ventricle is weighed, the ventricle is cut into 5 pieces along the atrioventricular groove on average and is placed into 1 percent TTC solution, the solution is dyed in constant temperature water bath at 37 ℃ for 5min, after the heart is taken out, a digital picture is taken firstly, then the undyed part (namely the infarcted part) is separated and weighed to calculate the percentage of the undyed part accounting for the weight of the whole ventricle (the percentage of myocardial infarction), and the t test between groups is carried out with an ischemia model. The formula for calculating the percent infarct is as follows:
percent infarct (%) = pale zone weight/(ventricular weight) × 100%
Before taking the heart, carrying out orbital venous plexus blood taking on the rat, taking 1-2 ml, centrifuging at the speed of 2000rpm for 25min, separating serum, and determining the content or activity of serum Creatine Kinase (CK), Lactate Dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), Malondialdehyde (MDA) and superoxide dismutase (SOD) according to a kit method. The results are shown in tables 1, 2, 3 and 4.
TABLE 1 Effect on J-Point Change (mv) in rats with myocardial ischemia due to coronary ligation
（n=7）
Note:#P<0.05，##P<0.01, the front-to-back ratio of each time point after the molding and before the molding is carried outComparing;▲P<0.05，▲▲P<0.01, each group was compared to the model group at the same time point.
As can be seen from Table 1, the J point of the electrocardiogram after the coronary ligation operation of the rats in each operation group is significantly higher than that before the operation (p <0.01), which indicates that the modeling is successful. Compared with the model group, 5min after operation, the extract high group can obviously inhibit the increase of J point (p is less than 0.05), 1h after operation, the extract middle and high group can obviously inhibit the increase of J point (p is less than 0.05), and 4h after operation, the extract middle and high group can obviously inhibit the increase of J point (p is less than 0.05). There was no significant difference in comparison among the other groups.
TABLE 2 Effect on myocardial infarction caused by coronary artery ligation in rats
▲P<0.05，▲▲P<0.01, compared to a model set.
As can be seen from Table 2, the myocardial infarction rates of the model group and the administration groups are significantly higher than those of the pseudo-operation group, indicating that the modeling is successful. The high extract group was able to significantly reduce the myocardial infarction rate (p <0.05) compared to the model control group.
TABLE 3 Effect on Biochemical indices of blood in rats with myocardial ischemia due to coronary artery ligation
*P<0.05，**P<0.01, compared to the sham surgical group;▲P<0.05，▲▲P<0.01, compared to a model set.
As can be seen from Table 3, the levels of MDA, LDH, CK and CK-MB in the serum of the model group are significantly higher than that of the pseudo-operation group (P <0.01), and the level of SOD is significantly lower than that of the pseudo-operation group (P <0.01), indicating successful modeling. Compared with a model control group, the high-extract group can obviously reduce the MDA content (P <0.05) of the serum of the myocardial ischemia rat, the high-extract group in the extract can obviously reduce the LDH (P <0.05) of the serum of the myocardial ischemia rat, the high-extract group in the extract can obviously improve the SOD activity (P <0.05) of the serum of the myocardial ischemia rat, the high-extract group in the extract can obviously reduce the CK activity (P <0.05) of the serum of the myocardial ischemia rat, and the high-extract group can obviously reduce the CK-MB (P <0.05) activity of the serum of the myocardial ischemia rat.
6. Effect of compound red sage extract on toxic myocarditis:
6.1 in vitro toxic myocarditis model:
newborn SD rats are killed by dislocation for 1-3 days, the skin of the chest and abdomen is disinfected by 2% iodine tincture and 75% ethanol respectively, the skin of the chest is cut by iris scissors, and the subcutaneous tissue is exposed. The subcutaneous tissue was sterilized with iodine tincture and ethanol, cored by thoracotomy, and washed away with residual hematocele in a dish containing PBS. Separating from the centrifugal chamber, and cutting into pieces of 1mm3The pieces are ready for digestion.
According to the experimental requirements, a fractional digestion method is selected to prepare the myocardial cell suspension. Standing the myocardial cell suspension in a triangular flask or a deep plate at 37 deg.C for 180min to quickly adhere epithelial cells to the wall, carefully sucking out the cell suspension, transplanting the cell suspension in other culture dishes, and culturing at 5 × 105The cells were inoculated in 96-well plates at a density of one ml at 37 ℃ and 5% CO2Under the condition, the culture is carried out in a static way by using a DMEM medium containing 20% calf serum. And replacing the culture medium once every 2-3 days according to the adherent growth of the cells and the change condition of the pH value of the culture medium.
Primarily culturing the myocardial cells for 72h, changing the liquid, adding the medicine, grouping, performing model group, captopril group (40 mu g/ml), low extract (crude drug dose of 600 mu g/ml), medium extract (crude drug dose of 1200 mu g/ml) and high extract (crude drug dose of 2400 mu g/ml), incubating for 24h, adding adriamycin (1 mu g/ml) for acting for 1h, and adding the same amount of culture medium into a blank control group.
The pulse frequency and the percentage of change of the cardiomyocytes before and after molding were observed under a microscope, and the experimental results are shown in table 4.
TABLE 4 beating frequency and percent change of cardiomyocytes before and after molding
*P<0.05，**P<0.01, compared to a blank control group;▲P<0.05，▲▲P<0.01, compared to a model set.
As can be seen from Table 4, the percent change before and after molding of each doxorubicin group compared with the blank control group is very significant, indicating that molding is successful (P < 0.01). Compared with the model group, the positive medicine group and the high-extract group remarkably inhibit the reduction of the beating frequency of the myocardial cells in vitro caused by the adriamycin (P is less than 0.05). There was no significant difference in comparison among the other groups (P > 0.05).
6.2 in vivo models of toxic myocarditis:
clean male SD rats were randomly divided into 10 groups by weight, including blank control group (distilled water), model group (distilled water), captopril group (15 mg/kg), low extract (81 mg/kg), high extract (162 mg/kg), and 10 rats per group. Each group of rats was administered by gavage once a day for 7 consecutive days. The volume of administration was 0.5ml/100g body weight. At 1h after the last administration, the weight of the rat was recorded (BW 1), 24h after molding with caudal vein doxorubicin (5 mg/kg), weighed (BW 2), killed by breaking the neck, immediately removed the heart by opening the chest, washed out the blood in cold physiological saline, excised the tissues and blood vessels around the heart, blotted with filter paper, weighed the total heart weight (CW) with an electronic balance, and calculated as the heart weight index (CW/BW, mg/g), with the results shown in Table 5. A conventional myocardial tissue block is rapidly taken and fixed by 2.5 percent glutaraldehyde, and the ultramicro structure of the myocardial cells is observed after the conventional myocardial tissue block is prepared into an electron microscope slice, as shown in figure 5. Another myocardial tissue was ground into a homogenate and the activities of myocardial Lactate Dehydrogenase (LDH), serum Creatine Kinase (CK) and aspartate Aminotransferase (AST) were determined according to the kit protocol, with the results shown in Table 6.
TABLE 5 Effect on body weight and Heart weight index of rats in an in vivo myocarditis model
*P<0.05，**P<0.01, compared to a blank control group;▲P<0.05，▲▲P<0.01, compared to a model set.
As can be seen from Table 5, the percent weight change of each administration group was not significantly different (P >0.05) compared with the blank control group and the model group; for the heart weight index, compared with a blank control group, the heart weight index of the model group is obviously increased, which indicates that the molding is successful. The captopril group and the extract high group had significantly reduced heart weight indices (P <0.05) compared to the model group. There was no significant difference in comparison among the other groups (P > 0.05).
As can be seen from FIG. 5, the cardiomyocytes in the blank control group had a regular arrangement of myofilaments, a clear Z-line structure, abundant mitochondria, and parallel and dense ridges. The arrangement of myofilaments and mitochondria of the model group and the extract low group is disordered, the cells are swelled, vacuole-like degeneration is generated, and the nuclear edema is serious. The positive drug captopril group has obviously reduced injury, clear Z line, rich mitochondria and no vacuole-like degeneration. The damage of the myocardial cells of the middle and high groups of the extract is lighter than that of the model group, the arrangement of the myofilaments and the mitochondria is more regular, the vacuole-like degeneration is obviously reduced, only the mild swelling of the nucleus is seen, and the edema is reduced.
TABLE 6 Effect on in vivo myocardial enzymes in rats with toxic myocarditis
*P<0.05，**P<0.01, compared to a blank control group;▲P<0.05，▲▲P<0.01, comparing with a model group;○P<0.05，○○P<0.01, compared to the captopril group.
As can be seen from Table 6, the myocardial enzymes in the doxorubicin-administered groups were significantly increased compared with the blank control group, indicating successful molding (P <0.01, P < 0.05). Compared with the model group, the positive drug captopril group can remarkably inhibit the activity of the adriamycin myocardial-induced enzyme LDH (P <0.01), and the extract high group can remarkably inhibit the activity of the adriamycin myocardial-induced enzyme LDH (P < 0.05); compared with the model group, the captopril group and the high group in the extracts can remarkably inhibit the increase of the activity of myocardial CK caused by adriamycin (P < 0.05); compared with the model group, the positive drug can obviously inhibit the increase of myocardial AST activity caused by adriamycin (P < 0.05). Compared with the positive medicine group, the activity of the myocardial enzyme CK in the low-extract group and the high-extract group is remarkably improved (P <0.05), and the activity of the myocardial enzyme CK in the high-extract group is remarkably improved (P < 0.01). There was no significant difference in comparison among the other groups (P > 0.05).
7. Conclusion of the experiment
Compared with the model group, 15min after operation, the extract high group can obviously inhibit the increase of J point (p is less than 0.05), 1h after operation, the extract medium and high dose group can obviously inhibit the increase of J point (p is less than 0.05), and 4h after operation, the extract medium and high dose group can obviously inhibit the increase of J point (p is less than 0.05). The high dose group of the extract was able to significantly reduce the myocardial infarction rate (p <0.05) compared to the model control group. Compared with a model control group, the extract high-dose group can obviously reduce the MDA content (P <0.05) of the serum of the myocardial ischemia rat, the extract medium-dose group and the extract high-dose group can obviously reduce the LDH (P <0.05) of the serum of the myocardial ischemia rat, the extract medium-dose group and the extract high-dose group can obviously improve the SOD activity (P <0.05) of the serum of the myocardial ischemia rat, the extract medium-dose group and the extract high-dose group can obviously reduce the CK activity (P <0.05) of the serum of the myocardial ischemia rat, and the extract high-dose group can improve the CK-MB (P <0.05) activity of the serum of the myocardial ischemia rat. Experiments show that the compound salvia extract can effectively treat various diseases caused by myocardial ischemia, including myocardial infarction, acute coronary syndrome, atrioventricular block, arrhythmia, coronary heart disease and angina pectoris.
Compared with the model group, the captopril group and the extract high-dose group are improved, so that the reduction of the beating frequency of the myocardial cells of toxic myocarditis in vitro can be remarkably inhibited (P < 0.05). The extract high dose group had significantly reduced cardiac weight index (P <0.05) compared to the model group. Compared with the model group, the extract high-dose group can remarkably inhibit the activity increase of the in vivo toxic myocarditis myocardial enzyme LDH (P < 0.05); compared with the model group, the medium and high dose groups of the extract obviously reduce the ultrastructural damage of the myocardial cells and can obviously inhibit the increase of the activity of the myocardial CK of the toxic myocarditis in vivo (P < 0.05). Compared with the positive medicine group, the activity of the myocardial enzyme CK in the low and high dose groups of the extract is remarkably improved (P <0.05), and the activity of the myocardial enzyme CK in the dose groups of the extract is remarkably improved (P < 0.01). The tests show that the compound salvia extract can inhibit the myocardial cell damage caused by diseases such as myocarditis, myocardial infarction and the like and inhibit the myocardial ischemia caused by diseases such as myocarditis and the like.
Example one
1. Extracting medicinal materials: respectively taking 450g of salvia miltiorrhiza and 88g of panax notoginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, adding 4 times of water for reflux extraction for 1 hour, filtering, and discarding residues. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, AB-8 macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: crude drug quantity = 1: 1, filling the column with 95% ethanol by a wet method, washing the column with 95% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column with a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 1BV/hr, washing with 1BV water at flow rate of 1BV/hr to remove impurities, eluting with 2.5BV 95% ethanol as eluent at flow rate of 1BV/hr, and collecting the eluent.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 60 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (1/4 of medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 16.1ml of 10% sodium bicarbonate solution, heating and refluxing for 2 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate.
3.2, concentrating and drying: concentrating compound Saviae Miltiorrhizae radix hydrolysate at 70 deg.C under reduced pressure to dry to obtain compound Saviae Miltiorrhizae radix extract.
Example two
1. Extracting medicinal materials: respectively taking 490g of radix Salviae Miltiorrhizae and 10g of radix Notoginseng, extracting with 10 times of water under reflux for 1 hr, filtering, extracting with 6 times of water under reflux for 0.5 hr, filtering, and discarding residue. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, D-101 type macroporous resin pretreatment: taking the amount of the dry D-101 type macroporous resin: crude drug = 0.5: 1, filling the column with 85% ethanol by a wet method, washing the column with 85% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column with a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 1.5BV/hr, washing with 2BV water at flow rate of 1.5BV/hr to remove impurities, eluting with 5BV 70% ethanol as eluent at flow rate of 1.5BV/hr, and collecting the eluent.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 80 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (about 1/4 of medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 10ml of 5% sodium carbonate solution, heating and refluxing for 8 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate.
3.2, concentrating and drying: concentrating compound Saviae Miltiorrhizae radix hydrolysate at 60 deg.C under reduced pressure to obtain compound Saviae Miltiorrhizae radix extract.
EXAMPLE III
1. Extracting medicinal materials: respectively taking 100g of salvia miltiorrhiza medicinal material and 400g of panax notoginseng medicinal material, adding 2 times of water for reflux extraction for 3 hours, filtering, then adding 6 times of water for reflux extraction for 2 hours, filtering, and discarding dregs. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, HPD-400 macroporous resin pretreatment: taking the amount of the dry HPD-400 type macroporous resin: crude drug quantity = 1: and 2, filling the mixture into a column by using a 70% ethanol wet method, washing the column by using the 70% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column by using a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 0.5BV/hr, washing with 1.5BV water at flow rate of 1.2BV/hr to remove impurities, eluting with 1BV 100% ethanol as eluent at flow rate of 0.5BV/hr, and collecting the eluent.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 40 deg.C under reduced pressure, and recovering ethanol until no ethanol smell (about 1/4 of medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 1% sodium hydroxide solution 5ml, heating and refluxing for 0.5 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate for use.
3.2, concentrating and drying: concentrating the compound Saviae Miltiorrhizae radix hydrolysate at 40 deg.C under reduced pressure to dry to obtain compound Saviae Miltiorrhizae radix extract.
Example four
1. Extracting medicinal materials: respectively taking 400g of salvia miltiorrhiza and 100g of panax notoginseng, adding 6 times of water for reflux extraction for 2.5 hours, filtering, adding 5 times of water for reflux extraction for 1.5 hours, filtering, and discarding medicine residues. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, pretreating NKA-9 macroporous resin: taking the amount of the dried NKA-9 type macroporous resin: crude drug quantity = 1: 1.5, filling the mixture into a column by a 90% ethanol wet method, washing the column by 90% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing the column by a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 0.8BV/hr, washing with 0.8BV water at flow rate of 0.8BV/hr to remove impurities, eluting with 2BV 90% ethanol as eluent at flow rate of 0.8BV/hr, and collecting the eluent for use.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 65 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (1/4 of the medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 5ml of 1% potassium hydroxide solution, heating and refluxing for 1 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate.
3.2, concentrating and drying: concentrating the compound Saviae Miltiorrhizae radix hydrolysate at 65 deg.C under reduced pressure to dry to obtain compound Saviae Miltiorrhizae radix extract.
EXAMPLE five
1. Extracting medicinal materials: respectively taking 450g of salvia miltiorrhiza and 50g of panax notoginseng, adding 4 times of water for reflux extraction for 2.5 hours, filtering, then adding 3 times of water for reflux extraction for 2 hours, filtering, and discarding medicine residues. Mixing the extractive solutions.
2. Refining macroporous resin:
2.1, DM-301-8 macroporous resin pretreatment: taking the amount of the dry DM-301 type macroporous resin: crude drug quantity = 1: 1.5, filling the column with 80% ethanol by a wet method, washing with 80% ethanol until the filtrate is mixed with water (1: 3) and is not white and turbid, and washing with a large amount of distilled water until no alcohol smell exists for later use.
2.2, loading, eluting and purifying processes: loading the mixed extractive solution into column at flow rate of 1.2BV/hr, washing with 1.2BV water at flow rate of 1.2BV/hr to remove impurities, eluting with 4BV 80% ethanol as eluent at flow rate of 1.2BV/hr, and collecting the eluent.
3. Preparing a compound red sage root extract:
3.1, hydrolysis: concentrating the eluate at 55 deg.C under reduced pressure, and recovering ethanol until no alcohol smell (about 1/4 of medicinal material amount) is obtained to obtain refined compound Saviae Miltiorrhizae radix concentrated solution. Adding 10% sodium bicarbonate solution 20ml, heating and refluxing for 4 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate.
3.2, concentrating and drying: concentrating the compound Saviae Miltiorrhizae radix hydrolysate at 55 deg.C under reduced pressure to obtain compound Saviae Miltiorrhizae radix extract.
EXAMPLE six
Taking the compound salvia miltiorrhiza extract in the first embodiment, adding a proper amount of water to dissolve, and mixing the compound salvia miltiorrhiza extract with pre-dissolved PEG6000 according to the medicine-auxiliary ratio of 1:3, uniformly mixing, adding a proper amount of borneol, and melting at the temperature of 80 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
EXAMPLE seven
Taking the compound salvia miltiorrhiza extract in the first embodiment, adding a proper amount of water to dissolve, and mixing with pre-dissolved PEG4000 according to a medicine-auxiliary ratio of 1: 2, uniformly mixing, adding a proper amount of borneol, and melting at the temperature of 70 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Example eight
Taking the compound salvia miltiorrhiza extract in the first embodiment, adding a proper amount of water to dissolve, and mixing with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1: 5, uniformly mixing, adding a proper amount of borneol, and melting at 90 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Example nine
Taking the compound salvia miltiorrhiza extract in the first embodiment, adding a proper amount of water to dissolve, and mixing with pre-dissolved PEG4000 according to a medicine-auxiliary ratio of 1: 2.5, mixing evenly, adding a proper amount of borneol, and melting at 85 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product.
Example ten
Taking the compound salvia miltiorrhiza extract in the first embodiment, adding a proper amount of water to dissolve, and mixing with pre-dissolved auxiliary materials according to a medicine-auxiliary ratio of 1: 3.5, mixing evenly, adding a proper amount of borneol, and melting at 75 ℃. After the materials are evenly dissolved, transferring the materials into a drop pill machine for drip irrigation; dripping the medicinal liquid into liquid paraffin of 0-10 deg.C, taking out dripping pill, removing oil, and sieving to obtain the final product. EXAMPLE eleven
Tablet formulation
[ prescription ] 100g of the extract obtained in example one, microcrystalline cellulose 50g of aerosil 3g of magnesium stearate 1.5g
[ PREPARATION METHOD ] sieving raw materials and adjuvants with 100 mesh sieve respectively; mixing the extract and microcrystalline cellulose, making soft mass with 60% ethanol as binder, sieving with 20 mesh sieve, granulating, drying at 60 deg.C, taking out, sieving with 30 mesh sieve, grading, adding silica gel micropowder and magnesium stearate, mixing, and tabletting to obtain 1000 tablets.
Example twelve
[ prescription ] 75g of microcrystalline cellulose 37g of aerosil 2.3g of magnesium stearate 1.1g of extract obtained in example II
[ PREPARATION METHOD ] sieving raw materials and adjuvants with 100 mesh sieve respectively; mixing the extract and microcrystalline cellulose, making soft mass with 60% ethanol as binder, sieving with 20 mesh sieve, granulating, drying at 60 deg.C, taking out, sieving with 30 mesh sieve, grading, adding appropriate amount of silica gel micropowder and magnesium stearate, mixing, and tabletting to obtain 1000 tablets.
EXAMPLE thirteen
And taking 5.0g of the extract obtained in the third embodiment, 10g of xylitol, 0.8g of isomaltose hypgather, 10g of erythritol, 0.05 of aspartame and the balance of water to obtain the compound salvia miltiorrhiza oral liquid.
Example fourteen
Taking 25% of the extract obtained in the first step, 48% of xylitol, 6% of maltose, 14% of cyclodextrin and 7% of calcium lactate, and preparing the compound red sage root soft capsule according to a conventional soft capsule process.
Claims (4)
1. The compound salvia miltiorrhiza extract is characterized by being prepared by the following method:
(1) respectively taking salvia miltiorrhiza and panax notoginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, then adding 4 times of water for reflux extraction for 1 hour, filtering, removing dregs of a decoction, combining filtrates, and reserving an obtained extracting solution for later use, wherein the weight percentages of the salvia miltiorrhiza and panax notoginseng are 20-98% and 2-80% respectively;
(2) macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: the crude drug amount is 1: 1, filling the column with 95% ethanol by a wet method, and then washing the column with 95% ethanol until the volume ratio of the filtrate to water is 1:3, mixing the materials until the materials are not white and turbid, and washing the materials by using a large amount of distilled water until no alcohol smell exists for later use;
(3) taking the extracting solution in the step (1), loading the extracting solution into a column at the flow rate of 1BV/hr, washing with 1BV water at the flow rate of 1BV/hr to remove impurities, finally eluting with 1BV/hr by using 2.5BV 95% ethanol as an eluent, and collecting the eluent for later use;
(4) taking the eluent in the step (3), concentrating under reduced pressure at 60 ℃, and recovering ethanol until no alcohol smell exists, thereby obtaining a refined compound salvia miltiorrhiza concentrated solution; adding 10% sodium bicarbonate solution, heating and refluxing for 2 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate, and concentrating under reduced pressure at 70 deg.C to dry.
2. The method of preparing an extract of claim 1, comprising the steps of:
(1) respectively taking radix salviae miltiorrhizae and pseudo-ginseng, adding 5 times of water for reflux extraction for 2 hours, filtering, adding 4 times of water for reflux extraction for 1 hour, filtering, removing dregs, combining filtrates, and keeping the obtained extract for later use;
(2) macroporous resin pretreatment: taking the amount of the dry AB-8 type macroporous resin: the crude drug amount is 1: 1, filling the column with 95% ethanol by a wet method, and then washing the column with 95% ethanol until the volume ratio of the filtrate to water is 1:3, mixing the materials until the materials are not white and turbid, and washing the materials by using a large amount of distilled water until no alcohol smell exists for later use;
(3) taking the extracting solution in the step (1), loading the extracting solution into a column at the flow rate of 1BV/hr, washing with 1BV water at the flow rate of 1BV/hr to remove impurities, finally eluting with 1BV/hr by using 2.5BV 95% ethanol as an eluent, and collecting the eluent for later use;
(4) taking the eluent in the step (3), concentrating under reduced pressure at 60 ℃, and recovering ethanol until no alcohol smell exists, thereby obtaining a refined compound salvia miltiorrhiza concentrated solution; adding 10% sodium bicarbonate solution, heating and refluxing for 2 hr to obtain compound Saviae Miltiorrhizae radix hydrolysate, and concentrating under reduced pressure at 70 deg.C to dry.
3. A compound Danshen preparation, characterized in that, the compound Danshen extract of claim 1 is used as the active pharmaceutical ingredient, and pharmaceutically acceptable carriers are added as required, and the preparation is prepared according to the conventional technology of pharmaceutics.
4. Use of the extract of claim 1 for the preparation of a medicament for the treatment of cardiovascular disease.
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