CN1839819A - Medicinal composition containing tanshinone and its preparation process and usage - Google Patents

Abstract

Disclosed is a medicinal composition containing tanshinone and its preparing process, wherein the composition comprises tanshinone, oil, emulsifying agent and water. The composition can be prepared into oral administration emulsion or venous emulsion, and can be used for treating or preventing infection, cerebral apoplexy, cerebral ischemia / reperfusion injury, ischemic heart disease, tumor, liver fibrosis, diabetic complication, pulmonary fibrosis, chronic nephritis, chronic kidney functional insufficiency and Alzheimer's disease.

Description

The pharmaceutical composition and preparation method thereof and the purposes that contain TANSHINONES

Technical field

The present invention relates to the TANSHINONES is pharmaceutical composition of main active and preparation method thereof, is specially Emulsion and preparation thereof, and the purposes of this Pharmaceutical composition in pharmacy.

Background technology

Salviamiltiorrhizabung (Salvia miltiorrhiza Bge.) is the dry root and rhizome of profile of tooth section plant, and Radix Salviae Miltiorrhizae is as a kind of Chinese medicine, and it is applied to rare 1500 history, has stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the merit of the relieving restlessness that clears away heart-fire can significantly increase coronary flow.These curative effects of coming out owing to it is shown make it become many important component that are usually used in the cardiovascular and cerebrovascular disease Chinese patent medicine.It is generally acknowledged to have the active effective site of activating blood circulation to dissipate blood stasis in the Radix Salviae Miltiorrhizae two classes are arranged: fat-soluble tanshinone and water solublity salvianolic acid at present.Fat-soluble tanshinone mainly comprises Tanshinone I (tanshinone-I), Tanshinone I I (tanshinone-II), cryptotanshinone (cryptotanshinone) etc., wherein TANSHINONES-II comprise tanshinone-IIA, tanshinone-IIB, 1-hydroxytanshinone-IIA (1-hydroxyl TANSHINONES-IIA), ethytanshinoate (Methyl tanshinoate), 3a-hydroxytanshin one-IIA (and 3a-hydroxyl TANSHINONES-IIA), tanshinol-A etc., TANSHINONES-I comprises tanshinol-A, tanshinone I, dihydrotanshinone I; The main water solublity salvianolic acid of Radix Salviae Miltiorrhizae comprises compositions such as salvianolic acid A-C, rosmarinic acid and formicester thereof, danshensu, protocatechualdehyde and protocatechuic acid.

The biological activity of TANSHINONES has: antibacterial, antiinflammatory, removing lipid free radical, influence pulmonary fibrosis pathological process, estrogen-like effects, the influence to platelet aggregation, protection cardiovascular, the many-sided biological activity of antitumor.

The preparation of TANSHINONES is used clinically tanshinone capsule and TANSHINONES-IIA sulfonate sodium injection.Tanshinone is water-soluble hardly, and the external stripping of preparation is poor.The maximum dissolution of certain side's of reply Radix Salviae Miltiorrhizae Tabellae cryptotanshinone in simulated gastric fluid is 25% after measured, and the tanshinone stripping does not measure; The maximum dissolution of certain batch tanshinone capsule cryptotanshinone in simulated gastric fluid is 1.26%, and the tanshinone stripping does not measure.The pharmacokinetics experiment proves that also the tanshinone oral absorption is poor.Therefore tanshinone belongs to the composition that the oral administration stripping is poor, bioavailability is low.Though TANSHINONES-IIA sodium sulfonate can be water-soluble,, make its content in the central nervous system very low because its " strong polarity " is difficult for therefore can not passing through blood brain barrier through the protein biology film with Lipid Bilayer Structure is arranged.The thick luxuriant grade of sieve studies show that TANSHINONES-IIA is after being prepared into soluble derivative TANSHINONES-IIA sulfonate in addition, its bacteriostatic activity obviously reduces [Luo Houwei, Gao Jiwei, Zheng Jiarun, Deng. the structure activity relationship of tanshinone and allied compound bacteriostasis. China Medicine University's journal, 1988,19 (4): 258～262].

Summary of the invention

The technical problem to be solved in the present invention is prescription of the fat-soluble effective site tanshinone emulsion pharmaceutical composition in the salviamiltiorrhizabung and preparation method thereof and purposes, and wherein Emulsion comprises Orally taken emulsion and vein emulsion.

For addressing the above problem, the invention provides following technical scheme:

A kind of pharmaceutical composition of TANSHINONES, contain cryptotanshinone, tanshinone, Tanshinone I and the dihydrotanshinone I (hereinafter to be referred as refining tanshinone) for the treatment of effective dose, described pharmaceutical composition can contain the adjuvant of pharmaceutically said oral or intravenously administrable emulsion preparations.

Contain the refining tanshinone for the treatment of effective dose in the TANSHINONES, be meant in the TANSHINONES by weight/weight meter refining tanshinone content is not less than 50%, in the preferred TANSHINONES by weight/weight meter refining tanshinone content is 50%-99%.

Pharmaceutical composition of the present invention is more preferably formed: contain by weight/weight meter content is 50%～95% refining tanshinone, pharmaceutically can be used as oil, emulsifying agent and the water of carrier; Its proportioning is refining tanshinone 0.05～0.2g, midchain oil 4g, long-chain oil 1～16g, emulsifying agent 0.5～5.0g, enuatrol 0.02-0.1g, adds water to 100ml;

Preferred compositions proportioning is refining tanshinone 0.05～0.15g, midchain oil 4g, long-chain oil 1～10g, emulsifying agent 1.0～2.0g, and enuatrol 0.02-0.08g adds water to 100ml; Every 100ml Emulsion contain isoosmotic adjusting agent by weight/weight counts 2.0%～3.0%.

Most preferred compositions proportioning is refining tanshinone 0.05～0.10g, midchain oil 4g, long-chain oil 1～6g, emulsifying agent 1.0～1.6g, and enuatrol 0.04-0.07g adds water to 100ml; Every 100ml Emulsion contain isoosmotic adjusting agent by weight/weight meter 2.0～3.0%.

Wherein: long-chain oil is vegetable oil or animal oil or the two mixture.Vegetable oil is selected from: soybean oil, Semen Maydis oil, Oleum Arachidis hypogaeae semen, olive oil, Oleum Ricini, Petiolus Trachycarpi oil, Oleum Cocois, Oleum Brassicae campestris, Oleum sesami, cotton seed wet goods.Animal oil is selected from: derive from Animal fat usually, comprise fish oil, enuatrol, Whale wax oil etc.Midchain oil (middle-chain triglycerides (MCT)) is a median chain triglyceride oil, and promptly hot capric acid glyceride is semisynthetic natural function oils and fats, and carbon chain lengths C8-10 derives from Oleum Cocois.Emulsifying agent is selected from soybean phospholipid, egg yolk lecithin, hydrogenated phospholipid, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, serinephosphatide, lipositol, phosphatidic acid, cephalin or their mixture, and isoosmotic adjusting agent is selected from glycerol.

The present invention screens in the dissolving of oil phase TANSHINONES: TANSHINONES dissolubility in long-chain oil such as soybean oil is very little, does not reach the clinical treatment effective dose; TANSHINONES has dissolubility preferably in midchain oil, because midchain oil (MCT) does not contain the alpha-linolenic acid and the linoleic acid sodium of needed by human, and must have long-chain oil (LCT) to replenish, this just makes midchain oil (MCT) use bad separately, our TANSHINONES of discovering is better than its dissolubility in midchain oil at the miscella dissolubility of long-chains such as soybean oil oil and midchain oil simultaneously, so the present invention adopts the miscella of midchain oil and long-chain oil as the oil phase carrier; The present invention screens the ratio of long-chain oil and midchain oil, has determined the optimal proportion of the oily and midchain oil of long-chain, can make the TANSHINONES dissolubility reach the clinical treatment effective dose, can make steady quality again and meet the Emulsion of clinical drug safety.The present invention screens emulsifier proportion, has determined optimal proportion.

Described preparation of drug combination method is: with TANSHINONES or contain treatment effective dose refining tanshinone and mixes with oil, be heated to 60～80 ℃, stir, make to be dissolved as clear and bright solution, add enuatrol and emulsifying agent, stirring makes to be dissolved as clear and bright solution, gets oil phase; With isotonic agent formation water soluble in water, be heated to 60-80 ℃; Under high-speed stirred, oil phase is added to aqueous phase and forms colostrum, after the gained colostrum adds the water standardize solution,, obtain Emulsion through high pressure homogenizer emulsifying.Preferred manufacturing procedure is: will contain treatment effective dose refining tanshinone, soybean phospholipid and/or egg yolk lecithin, midchain oil mixes with long-chain oil, is heated to 70～80 ℃, the adding enuatrol makes to be dissolved as clear and bright solution, must oil phase; With glycerol formation water soluble in water, be heated to 70-80 ℃; The oil phase that contains TANSHINONES is added to aqueous phase forms colostrum, after the gained colostrum adds the water standardize solution,, make Emulsion through high pressure homogenizer emulsifying under the condition of pressure 100Mpa with high-speed stirred.

TANSHINONES of the present invention is extracted by lixiviate of red rooted salvia ethanol or CO2 supercritical fluid extraction and is obtained, and through column chromatography purification, its main component is cryptotanshinone, tanshinone, Tanshinone I and dihydrotanshinone I.

Cryptotanshinone, tanshinone, Tanshinone I and dihydrotanshinone I are referred to as " refining tanshinone ", are the effective site of Radix Salviae Miltiorrhizae, are the active ingredient in the pharmaceutical composition of the present invention.

According to new drug registration management way, effective site content is not less than 50% in the crude drug.For pharmaceutical composition of the present invention, the crude drug refining tanshinone by weight/weight ratio meter content must not be lower than 50%.

Described oil is meant biological acceptable material, refers generally to vegetable oil, animal oil, artificial oil or its mixture.Vegetable oil can be among a kind of or several in soybean oil, Semen Maydis oil, Oleum Arachidis hypogaeae semen, olive oil, Oleum Ricini, Petiolus Trachycarpi oil, Oleum Cocois, Oleum Brassicae campestris, midchain oil, Oleum sesami or the Oleum Gossypii semen; Be preferably the oil that soybean oil, Semen Sesami wet goods are rich in triglyceride.Also can be selected from Fructus Schisandrae oil, Oleum Hippophae, peach kernel oil, almond oil, safflower oil, Oleum Camelliae, Radix Oenotherae erythrosepalae oil, Semen Tritici aestivi germ oil, perilla oil, litsea cubeba oil, Oleum Fructus Bruceae or the Oleum Curcumae one or more; Perhaps be selected from linoleic acid sodium, conjugated linoleic acid sodium, enuatrol ethyl ester, castor oil hydrogenated, hydrogenated groundnut, hydrogenated cottonseed oil, linoleic acid sodium ethyl ester, conjugated linoleic acid sodium ethyl ester, self-emulsifying monostearate, suffering/caprin or the glycerol triethyl one or more.Animal oil is selected from: derive from Animal fat usually, comprise fish oil, enuatrol, Whale wax oil etc.

Emulsifying agent of the present invention can be one or more in anion surfactant, cationic surface agent, amphoteric surfactant or the non-ionic surface active agent.Wherein anion surfactant is selected from one or more in salt, sulfated oil and the high fatty alcohol sulfuric acid ester of higher fatty acids, as enuatrol sodium, enuatrol potassium, 12 (14,16,18) alkyl sodium sulfate etc.Cationic surfactant is selected from azochlorosulfonate acid compound, as in octyl sulphuric acid (sulfonic acid) sodium, dioctyl sodium sulfosuccinate, the dodecylbenzene sodium sulfonate one or more; Amphoteric surfactant is selected from one or more in fabaceous lecithin, egg yolk lecithin, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, serinephosphatide, lipositol, phosphatidic acid, cephalin or the hydrogenated phospholipid; Non-ionic surface active agent is selected from one or more in sucrose-fatty esters (as sucrose ester), spans, Tweens, polyoxyethylene fatty acid ester class (as Myrij Myrij), polyoxyethylene aliphatic alcohol ether class (as Bian Ze Brij), Cetomacrogol class, Cremophore EL class or the poloxamer class (as poloxamer).In the preferred soybean phospholipid of emulsifying agent of the present invention, egg yolk lecithin, the hydrogenated phospholipid etc. one or more, more preferably soybean phospholipid and/or egg yolk lecithin.

The isoosmotic adjusting agent of tanshinone emulsion of the present invention is conventional adjuvant, conventional amount used, oozes purpose as long as reach etc., and isoosmotic adjusting agent is a glycerol as described.

Because TANSHINONES is difficult to be dissolved in the water, and does not still have the use of injection aspect so far.Tanshinone emulsion provided by the invention was placed 12 months through room temperature, placed 6 months study on the stability results for 30 ℃ and showed that laser particle size analyzer is measured its mean diameter and changed in prescribed limit, and epigranular, meets the requirement of national drug registration management way; High performance liquid chromatogram (HPLC) method is measured refining tanshinone content and is kept stable.Other every indexs all meet the injection requirement, show that the gained quality of the pharmaceutical preparations is stable.

Tanshinone emulsion provided by the invention can change TANSHINONES in the intravital distribution of people, improve bioavailability of medicament, TANSHINONES is easily absorbed by reticuloendothelial system after making Emulsion, has significant targeting in vivo, thereby reach treatment concentration at cardia and central nervous system's enrichment, improve of the effect of this medicine, than the heart targeting of aqueous injection under the same dose and the raising of central nervous system's targeting more than 300% at treatment cardiovascular disease and central nervous system disease.And can keep the TANSHINONES original activity.This injection is stablized, can be tolerated autoclaving, and the adjuvant of use has biocompatibility, and shows less side effect.

The present invention also provides the described application of compositions in preparing treatment or prophylaxis against infection diseases, ischemia apoplexy, Cerebral Ischemia damage, ischemic heart desease, tumor, hepatic fibrosis, diabetic complication, pulmonary fibrosis, chronic nephritis, chronic renal insufficiency, Alzheimer medicine that contains TANSHINONES.

The specific embodiment

TANSHINONES of the present invention can adopt commercially available product (producer with corresponding extraction equipment or commercial scale supercritical equipment all can produce, as the various content products of its product purification TANSHINONES 10-80% of Tianfeng Biological Science ﹠ Technology Co., Ltd., Xi'an).The commercially available product that Oleum Camelliae, midchain oil, refined soybean oil, refining Oleum sesami, fish oil, Semen Maydis oil, Oleum Arachidis hypogaeae semen all adopt Tieling Beiya Medical Oil Co., Ltd. to produce among the embodiment.The commercially available product that fabaceous lecithin, egg yolk lecithin, cephalin, hydrogen flower phospholipid, enuatrol, glycerol all adopt Shanghai Dong Shang Industrial Co., Ltd. to produce.

Preparation example 1: preparation refining tanshinone

Get and work as red rooted salvia, pulverize, 95% ethanol lixiviate is reclaimed ethanol and is got extractum, purification by silica gel column chromatography, and petroleum ether-ether gradient elution is collected and is rich in the refining tanshinone position, obtains refining tanshinone.High performance liquid chromatogram (HPLC) method is measured, and contains refining tanshinone 56% (w/w).

Preparation example 2: preparation refining tanshinone

The red rooted salvia crushing screening places 5L supercritical extraction equipment, CO ₂The supercritical fluid extraction thing (extraction kettle 35MPa, 60 ℃, time 40min, 95% ethanol is entrainer, circulating load is 675mlh ^-1, promptly consumption is 30% of a medical material total amount, pressure 5MPa, and 45 ℃ of temperature, the Radix Salviae Miltiorrhizae extract filters, and filtrate is left standstill, and gets precipitation, purification by silica gel column chromatography, petroleum ether-ether gradient elution is collected and is rich in the refining tanshinone position, obtains refining tanshinone.High performance liquid chromatogram (HPLC) method is measured, and contains refining tanshinone 89% (w/w).

Embodiment 1: the preparation of refining tanshinone Emulsion

With refining tanshinone 50mg, fabaceous lecithin 0.5g, join in the miscella of Oleum Camelliae 1.0g and midchain oil 4.0g, add enuatrol 0.02g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 20ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 166nm.

Embodiment 2: the preparation of refining tanshinone Emulsion

With refining tanshinone 200mg, egg yolk lecithin 5.0g, join in the miscella of refining Oleum sesami 16g and midchain oil 4g, add enuatrol 0.1g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.25g is dissolved in the 60ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 168nm.

Embodiment 3: the preparation of refining tanshinone Emulsion

With refining tanshinone 150mg, egg yolk lecithin 2.0g, join among the midchain oil 4g, refined soybean oil 10g adds enuatrol 0.08g, is heated to 60～80 ℃, stir to make to be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 30ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 165nm.

Embodiment 4: the preparation of refining tanshinone Emulsion

With refining tanshinone 50mg, egg yolk lecithin 1.0g, join in the miscella of fish oil 1.0g and midchain oil 4.0g, add enuatrol 0.02g, be heated to 70～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 40ml water forms water, be heated to 70-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 174nm.

Embodiment 5: the preparation of refining tanshinone Emulsion

With refining tanshinone 100mg, egg yolk lecithin 1.6g, join in the miscella of fish oil 6.0g and midchain oil 4.0g, add enuatrol 0.07g, be heated to 70～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 30ml water forms water, be heated to 70-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 174nm.

Embodiment 6: the preparation of refining tanshinone Emulsion

With refining tanshinone 50mg, hydrogenated phospholipid 1.0g, join in the miscella of midchain oil 4.0g and Semen Maydis oil 1.0g, add enuatrol 0.04g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 50ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 164nm.

Embodiment 7: the preparation of refining tanshinone Emulsion

With refining tanshinone 80mg, cephalin 1.4g, join in the miscella of midchain oil 4.0g and Oleum Arachidis hypogaeae semen 4.0g, add enuatrol 0.06g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 40ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 155nm.

Embodiment 8: the preparation of refining tanshinone Emulsion

With refining tanshinone 120mg, egg yolk lecithin 1.2g, join in the miscella of midchain oil 6g and soybean oil 2g, add enuatrol 0.05g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 30ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 158nm.

Embodiment 9: the preparation of refining tanshinone Emulsion

With refining tanshinone 150mg, egg yolk lecithin 1.2g, join in the miscella of midchain oil 6.0g and Semen Maydis oil 4.0g, add enuatrol 0.07g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 40ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 158nm.

Embodiment 10: the preparation of refining tanshinone Emulsion

With refining tanshinone 200mg, egg yolk lecithin 1.6g, join in the miscella of midchain oil 10g and soybean oil 5g, add enuatrol 0.05g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 40ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform greenish orange yellow emulsion, mean diameter 158nm.

Comparative example 1: the preparation of refining tanshinone oil phase

Refining tanshinone 20mg is joined in the 10g Semen Maydis oil, be heated to 60～80 ℃, stir, refining tanshinone is insoluble to Semen Maydis oil, can't make oil phase.

Comparative example 2: the preparation of refining tanshinone oil phase

Refining tanshinone 20mg is joined in the 20g soybean oil, be heated to 60～80 ℃, stir, refining tanshinone is insoluble to soybean oil, can't make oil phase.

Comparative example 3: the preparation of refining tanshinone oil phase

Refining tanshinone 200mg is joined in the 4.0g midchain oil, be heated to 60～80 ℃, stir, refining tanshinone can not be dissolved in midchain oil entirely, can't make oil phase.

Comparative example 4: the preparation of refining tanshinone oil phase

Refining tanshinone 200mg is joined in 4.0g midchain oil and the 6.0g soybean oil, be heated to 60～80 ℃, stir, refining tanshinone is dissolved as clear and bright solution, can make oil phase.

Comparative example 5: the preparation of refining tanshinone Emulsion

With refining tanshinone 150mg, egg yolk lecithin 1.6g, join among the midchain oil 10g, add enuatrol 0.025g, be heated to 60～80 ℃, stir to make and be dissolved as clear and bright solution, oil phase; Glycerol 2.5g is dissolved in the 100ml water forms water, be heated to 60-80 ℃; The oil phase that contains refining tanshinone is added to aqueous phase is emulsified into colostrum with high-speed stirred, after the gained colostrum added water and is settled to 100ml, after emulsifying under the condition of pressure 100Mpa, 121 ℃ of sterilizations were 15 minutes after the packing, make Emulsion through high pressure homogenizer.Gained Emulsion is uniform pale red emulsion, mean diameter 168nm.

Below the tanshinone emulsion of test example 1 employing is pressed embodiment 1,2,5 preparations.The tanshinone emulsion of all the other test examples is pressed embodiment 10 preparations.The commercially available product that tanshinone sulfonate injection described in the test example is produced available from Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd.Various antibacterials are available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Various laboratory animals are all available from unming Medical College's Experimental Animal Center.

Test is given an example: tanshinone emulsion is characteristic distributions (medicine is in the intravital tissue distribution research of rat) in vivo

48 of Sprague Dawley (being called for short SD) rats, male and female half and half, be divided into 8 groups at random, every group 6, press 20mg/kg body weight tail vein injection medicine, the contrast medicine is a tanshinone sulfonate injection, all the other four groups is tanshinone emulsion (according to embodiment 5 preparations), after the administration respectively at 15min, 30min, 60min, 120min sacrificed by exsanguination (6 animals of every time point, male and female half and half), take out the heart, liver, spleen, lung, kidney, gastrointestinal, brain rapidly, with normal saline flushing surface blood stains, suck dry moisture ,-25 ℃ of preservations are for test; Make the normal saline homogenate use of 400mg/ml before the test with Potter-Elvehjem Tissue Grinders.Tanshinone content in test blood plasma and each tissue adopts the high performance liquid chromatography quantitative analysis with calculated by peak area, the results are shown in Table 1.

Medicine each tissue distribution concentration data table (tanshinone μ g/g tissue) in vivo behind the table 1 TANSHINONES breast drug administration by injection

The administration group		Brain	Lung	The heart	Liver	Spleen	Kidney
								Tanshinone emulsion	15min	85±28.6	165±24.1	219.8±32.2	48.29±1.01	18.5±3.9	0.16±0.07
30min	93±34.4	198±35.9	219.±24.83	38.01±2.15	26.4±5.1	0.12±0.10
							60min		58.9±18.6	141±19.6	68.4±13.4	26.58±4.56	14.5±5.3	0.23±0.23
120min	40.4±15.3	134±27.4	58.5±8.4	16.99±3.98	13.45±3.9	0.26±0.18
							Tanshinone sulfonate injection		15min	0.0	160±24	110±30.21	62.1±1.31	10.3±0.98	0.28±0.01
30min	0.0	200±36	130.±12.1	92.2±10.3	20.2±1.3	0.32±0.02
								60min	0.0	140±15	100±10.21	82.15±5.24	14.2±2.5	0.22±0.01
120min	0.0	80±23	70±9.21	46.3±6.78	8.3±0.98	0.18±0.01

The result shows:

1, Emulsion can significantly improve the targeting of medicine at heart;

2, the result shows after this medicine is made Emulsion and can see through blood brain barrier, and the abundance in brain obviously improves than injection.

Test example 1: the external bacteriostasis test of tanshinone emulsion

1 method

Adopt dull and stereotyped punch method to detect tanshinone emulsion group (0.5mg/ml, 1mg/ml, 2mg/ml), tanshinone sulfonate injection respectively to staphylococcus aureus, escherichia coli, bacillus pyocyaneus, Hemolytic streptococcus, Bacillus tuberculosis's fungistatic effect.

2 results

The result shows: each dosage group of tanshinone emulsion all has significant fungistatic effect to staphylococcus aureus, escherichia coli, bacillus pyocyaneus, Hemolytic streptococcus, Bacillus tuberculosis.With the contrast of tanshinone sulfonate injection significant difference is arranged.

Table 2 tanshinone emulsion bacteriostasis

Strain	Inhibition zone diameter meansigma methods (d/cm)
					Tanshinone emulsion			Tanshinone sulfonate injection
	0.5mg/ml	1mg/ml	2mg/ml	2mg/ml
					Staphylococcus aureus	1.04 *	1.21 **	1.32 **	0.53
Escherichia coli	0.94 *	1.06 **	1.14 **	0.23
					Bacillus pyocyaneus	0.67 **	0.88 **	1.08 **	0
Hemolytic streptococcus	0.45 **	0.64 *	0.89 **	0
					The people tulase	0.79 *	1.02 **	1.13 **	0.11

Annotate: compare * P＜0.05, * * P＜0.01 with tanshinone sulfonate injection.

Below the animals administer method is all intravenous administration in the test example.The tanshinone emulsion that adopts is all pressed embodiment 10 preparations.

Test example 2: tanshinone emulsion is to the influence of rat local cerebral ischemia damage

1. divide into groups and experimental technique

The Wistar rat is divided into (1) sham operated rats (waiting the capacity solvent) at random, (2) model control group (waiting the capacity solvent), (3) Tanshinone I I-A sodium sulfonate injection group (6mg/kg), (4) tanshinone emulsion low dose group (3mg/kg); (5) dosage group (6mg/kg) in the tanshinone emulsion; (6) tanshinone emulsion high dose group (12mg/kg).Every group 10.After the fasting 12 hours, and chloral hydrate (350mg/kg, i.p.) anesthesia separates right carotid, and folder closes in the neck, common carotid artery, external carotid artery proximal part and distal end ligation, cut off the centre.The external carotid artery free-end is pulled to internal carotid artery in alignment, bolt line (selecting diameter 0.24mm nylon wire for use, length 5.0cm) is inserted into intracranial by external carotid artery, stop when meeting slight resistance, insertion depth is about 2cm.Ligation external carotid artery opening, and open the common carotid artery bulldog clamp, the disinfection and stitching wound causes left side middle cerebral artery ischemia model; Sham operated rats is only carried out the separation (above experiment is all carried out at 23 ℃～25 ℃) of right carotid, internal carotid artery, external carotid artery.Each treated animal intravenous injection relative medicine of postoperative.Press document [Liu Xiaoguang, Xu Lina, a kind of rat brain medium-sized artery model that can estimate thrombolytic and anti-thrombolytic after 24 hours, Acta Pharmaceutica Sinica, 1995,30:662] described method and standard is observed and the behavior disorder of record rat: (A) carry the Mus tail and observe forelimb flexing situation, stretch to ground as two forelimb symmetries, count 0 fen, the wrist flexing occurs as operation offside forelimb and count 1 fen, the elbow flexing is counted 2 fens, the shoulder inward turning is counted 3 fens, existing wrist flexing and/or elbow flexing have shoulder inward turning person again, count 4 fens.(B) animal is placed on the plane earth, push away both shoulders respectively, check resistance to side shifting.As bilateral resistance equity and strong, count 0 fen, as resistance descender when the operation offside promotes, according to decline degree difference be divided into gently, in, weigh three degree, count 1,2 and 3 fen respectively.(C) the two forelimbs of animal are put on the wire netting, observed the muscular tension of two forelimbs.Two muscle of anterior limb tension force equities and strong person count 0 fen.Count 1,2 and 3 fen according to operation offside muscular tension decline degree difference equally.(D) animal has ceaselessly to a side person of turn-taking, and counts 1 fen.According to the standard scoring, full marks are 11 minutes, and mark is high more, and expression animal behavior obstacle is serious more.

Put to death rat behind the behavior scoring, get brain, remove olfactory bulb, cerebellum and low brain stem, crownly be cut into 5, the brain sheet takes on a red color after normal structure is dyed with red tetrazolium (TTC) dyeing, and blocking tissue is white in color, taking a picture in dyeing back, asks the infarct size ratio with Chinese Aero-Space university pathological image analysis software.Data are represented with X ± s, carry out statistical procedures with t check between group.

2. result

As shown in table 3, ischemia is after 24 hours, and rat shows tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain; Give the tanshinone emulsion of various dose, the animal behavior obstacle has alleviating in various degree, and the rat cerebral ischemia district also takes an evident turn for the better, and is dose dependent.Compared significant difference with Tanshinone I I-A sodium sulfonate injection group.

The influence that table 3 tanshinone emulsion damages the rat local cerebral ischemia (n=10, X ± s)

Group		Behavior disorder	Ischemic areas (%)
				Sham operated rats		0	0
Model control group		9.12±1.35	24.09±4.13
				Tanshinone I I-A sodium sulfonate injection group		8.90±1.24	23.34±2.73
The tanshinone emulsion group	3mg/kg	7.48±3.04 *	20.52±2.66 *
				6mg/kg	5.83±1.27 **#	16.09±3.59 **#
	12mg/kg	4.44±1.26 **##	10.09±3.69 **##

Annotate: compare * P＜0.05 with model control group, #P＜0.05, ##P＜0.01 are compared with Tanshinone I I-A sodium sulfonate injection group in * * P＜0.01.

Test example 3 tanshinone emulsions are to the influence of rat cerebral ischemia/reperfusion injury

1. divide into groups and experimental technique

Grouping is the same.After the fasting 12 hours, cause left side middle cerebral artery ischemia by experimental example 2 described methods; Each treated animal intravenous injection relative medicine of postoperative 10min.Behind the ischemia 2 hours, extract nylon wire out, cause rat cerebral ischemia/reperfusion injury; After irritating 22 hours again, by carrying out behavior scoring, the dyeing of brain sheet by test example 2 described methods.Data are represented with X ± s, carry out statistical procedures with t check between group.

2. result

As shown in table 4, after ischemia was irritated 22 hours in 2 hours again, rat showed tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain; Give the tanshinone emulsion of various dose, the animal behavior obstacle has alleviating in various degree, and the rat cerebral ischemia district also takes an evident turn for the better, and is dose dependent.Compared significant difference with Tanshinone I I-A sodium sulfonate injection group.

Table 4 tanshinone emulsion is to the influence of rat cerebral ischemia/reperfusion injury (n=10, X ± s)

Group		Behavior disorder	Ischemic areas (%)
				Sham operated rats		0	0
Model control group		10.19±1.25	25.37±4.78
				Tanshinone I I-A sodium sulfonate injection group		9.98±2.34	24.56±7.09
The tanshinone emulsion group	3mg/kg	7.87±1.21 *#	18.38±2.97 *#
				6mg/kg	7.54±1.36 **#	13.53±3.54 **##
	12mg/kg	5.34±1.01 **##	10.54±3.12 **##

Annotate: compare * P＜0.05 with model control group, #P＜0.05, ##P＜0.01 are compared with Tanshinone I I-A sodium sulfonate injection group in * * P＜0.01.

Test example 4: tanshinone emulsion is to the influence of rat heart muscle ischemia

1. divide into groups and experimental technique

Grouping is the same.After the fasting 12 hours, the limb lead electrocardiogram is surveyed in ip urethane (1.2g/kg) anesthesia.Cut off left front fur, iodine tincture and alcohol disinfecting, along left border of sternum 1cm place, cut off thoracic wall muscle and two ribs, open the thoracic cavity rapidly, expose heart, the ligation left coronary artery is put back to heart immediately between arterial cone and left auricle, squeezes the thoracic cavity air, use the mosquito forceps closed-chest, cause Model Rats with Acute Myocardial Ischemia.Each treated animal intravenous drip (injecting) relative medicine (administration volume 3ml, 30min drips off) of postoperative with infusion pump.Record postoperative 0,2, the 6h electrocardiogram takes out heart subsequently, after cleaning with cold saline ,-20 ℃ of refrigerator freeze overnight.Next day, refrigerated heart is cut into 5 by ligation place to apex uniform thickness, immerse in the freshly prepared 0.5%N-BT phosphate buffer (pH 7.4).37 ℃ of water-bath jolting 10～15min.Blot the dyeing liquor of slice surface with filter paper, separate the coloured portions and the part of being unstained, weigh the compute infarct size.Myocardial infarction percentage ratio (%)=infarction part weight/(non-infarction part weight+infarction part weight) * 100%.

3. result: as shown in table 5, the tanshinone emulsion of various dose all obviously reduces the myocardial infarction area that Acute Myocardial Ischemia in Rats causes due to the ligation coronary artery, and can reduce the rising of the limb lead electrocardiogram J point that is caused by myocardial ischemia.Tanshinone emulsion has protective effect to cardiac muscle, can reduce the myocardial damage that ischemia causes.Middle and high dosage group has been compared significant difference with Tanshinone I I-A sodium sulfonate injection group.

Table 5 tanshinone emulsion is to the influence of rat heart muscle ischemic injuries (n=10, X ± s)

Grouping		Myocardial infarction percentage ratio (%)	The J point raises (10 * mV)
							Normally	0h	2h	6h
			Model control group		25±3	0.8±0.6					6.8±2.2	6.3±2.0	5.8±1.5
Tanshinone I I-A sodium sulfonate injection group		19±6 *					0.8±0.6	6.0±1.4 *	5.6±1.7 *	5.6±1.7 *
			Tanshinone emulsion	3mg/kg	18±6 *△	0.7±0.6					5.3±1.1 *△	5.8±1.3 *△	5.1±1.5 *△
6mg/kg	14±6 **#	0.7±0.6					4.5±1.5 *#	4.1±1.4 *	3.5±1.0 *#
				12mg/kg	13±6 **#	0.7±0.5				4.4±1.2 *#	4.0±1.6 *#	3.0±1.0 *##

Annotate: compare * P＜0.05 with model control group, #P＜0.05, ##P＜0.01 are compared with Tanshinone I I-A sodium sulfonate injection group in * * P＜0.01.

Test example 5. tanshinone emulsions are to the effect of cultured tumor cells in vitro inhibition of proliferation

1. material and test method:

Medicine faces with preceding and is made into respective concentration with complete culture solution.The RPMI1640 culture medium: GIBCO company produces.New-born calf serum (super), Hangzhou Sijiqing Biological Engineering Material Co., Ltd., product batch number: 000528.Trypsin, Sigma company produces.MTT, Sigma company.The injection streptomycin, Shangdong Ruiyang Pharmaceutical Co., Ltd, 1g (1,000,000 unit)/, lot number: 200105123.Benzylpenicillin sodium for injection, Lukang Medical Co., Ltd., Shandong, 800,000 units/, lot number: L020312.Other chemical reagent commonly used is homemade analytical pure.

Cell strain: human hepatoma cell strain SMMC-7721, Bel-7402, human oophoroma cell line H08910, human prostata cancer born of the same parents PC-3M, human lung adenocarcinoma cell line A549, human colon cancer cell strain HCT-8, human esophagus cancer cell strain CaEs-17, human glioma cell line U251, human stomach cancer cell line BGC-823, people's adenocarcinoma of stomach cell strain SGC-7901, human promyelocytic leukemia cell strain HL60, all available from institute of Materia Medica,Chinese Academy of Medical Sciences pharmacological room.

Instrument: the CO2 incubator, superclean bench, microplate reader, inverted microscope, Tissue Culture Flask (Costar, USA), 96 porocyte culture plates (Costar, USA).

Software: Microsoft Excel 7.0 statistical analysis softwares; Microcal Origin 6.0 data processing softwares.

Experimental technique: the RPMI1640 culture medium adds one liter in water for one bag, adds the 2g sodium bicarbonate, and 100,000 unit penicillins and 100mg streptomycin are regulated pH value to 7.4, with the degerming of 0.22um degerming membrane filtration.The 90ml culture medium adds deactivation new-born calf serum 10ml and is complete culture solution.Trypsin dissolves the after-filtration degerming with the D-hanks buffer.

All cells all is incubated at and contains in the 5ml complete culture solution Tissue Culture Flask 37 ℃, 5%CO ₂, full closing under the humidity.Attached cell is gone down to posterity after with 0.25% trypsinization, abandons original fluid behind the centrifugal 10min of suspension cell 1000rpm and adds fresh medium and go down to posterity.

Behind the adhere-wall culture cell tryptase protease digestion, after suspension cell is centrifugal, add complete culture fluid suspension cell, trypan blue dyeing counting living cells number is adjusted cell number to 1 * 10 with complete culture solution ⁵Individual cell/ml.Cell is joined 96 well culture plates, every hole 100 μ l.The culture plate that adds cell is in CO ₂Cultivate after 12 hours in the incubator and outwell original fluid, add the complete culture solution of respectively organizing medicine in 96 orifice plates, drug level is up to 20 μ g/ml, by 0.5 times of doubling dilution, establish 3 Concentraton gradient altogether, other establishes not that the pastille group is a matched group, and four parallel holes are established in each processing.Put into incubator behind the vibration mixing and continue to cultivate 48h.After cultivating end, take out culture plate, outwell original fluid, add 100 μ l and contain the RPMI1640 culture fluid of 0.5mg/ml MTT, continue in incubator, to place 4h, take out culture plate, the careful suction removes to cultivate supernatant in the plate hole, adds 150 μ l DMSO, and vibration is fully dissolved the crystallization of bluish violet first Za.On Bio-TEK Synergy HT microplate reader, measure the light absorption of every hole, measure wavelength 570nm, reference wavelength 630nm.Calculate the suppression ratio of medicine on cell proliferation according to each hole absorbance value.

According to drug level logarithm and corresponding suppression ratio, utilize Micro Origin software to make linear regression, obtain the 503nhibiting concentration (IC of medicine pair cell according to linear equation ₅₀).

2. result: see Table 6.

Table 6 tanshinone emulsion is to the tumor cell proliferation inhibition effect (IC of In vitro culture ₅₀, μ g/ml)

Cell strain	The tanshinone emulsion group	Tanshinone I I-A sodium sulfonate injection group
			SMMC-7721	6.68	4.56
Bel 7402	5.66	3.45
			H08910	4.50	4.04
CaEs-17	6.78	4.33
			A549	5.55	4.12
PC-3M	4.51	4.45
			HCT-8	6.45	4.08
U251	4.15	3.34
			BGC	4.46	3.78
HL-60	3.24	2.34

The result shows that each dosage group of tanshinone emulsion all can obviously suppress the in-vitro multiplication of various human source tumor cell line, its 503nhibiting concentration IC ₅₀All little 10 μ g/ml.And be better than Tanshinone I I-A sodium sulfonate injection group.

Test example 6. tanshinone emulsions suppress the inhibitory action of growth of xenografted in the mice body

1. materials and methods

Animal and tumor strain: Kunming kind white mice, body weight 18-22g, male.C ₅₇The BL/6 mouse inbred lines, body weight 18-22g, male.Mice S180 sarcoma, H22 hepatocarcinoma, Lewis lung cancer, U14 cervical cancer are introduced by institute of Materia Medica,Chinese Academy of Medical Sciences pharmacological room.

Experimental technique: S180, H22 and U14 tumor all go down to posterity in the Kunming mouse intraperitoneal, generally go down to posterity to grow ascites in back 7 days.Asepsis injector takes out after the ascites with after 3 times of the injection normal saline dilutions, and it is subcutaneous to be inoculated in the Kunming mouse oxter, and 0.2ml/ only.Lewis lung cancer goes down to posterity in C57BL/6 mice oxter, connects after the tumor about 10 days, gets the good tumor tissue of growth conditions, grinds to form homogenate after adding 5 times of amount normal saline, crosses 100 mesh sieves and gets tumor cell suspension, is inoculated in C57BL/6 mice oxter, and 0.2ml/ only.Connect administration in second day after the tumor.Successive administration 10 days, after the last administration 24 hours, take off cervical vertebra and put to death mice, strip tumor tissue and weigh, calculate tumour inhibiting rate.

2. result: each dosage group of tanshinone emulsion all can obviously suppress the growth of transplanted tumor in the mice body.And be better than TANSHINONES A sodium sulfonate injection group.See table 7 for details.

Table 7 tanshinone emulsion is to the inhibitory action of growth of xenografted in the mice body

Group	S180	H22	U14	Lewis
					Tanshinone emulsion 24mg/kg group	75.2 **##	85.2 **##	76.7 **##	72.2 **##
Tanshinone emulsion 12mg/kg group	60.4 **#	61.4 **#	60.8 **#	54.2 **#
					Tanshinone emulsion 6mg/kg group	32.8 **	43.3 **	35.0 **	38.3 **
Tanshinone I I-A sodium sulfonate injection group	43.7 **	55.3 **	46.1 **	40.8 **

Annotate: each is organized, and tumor is heavy to be compared with model group: ^*P＜0.01.Compare #P＜0.05, ##P＜0.01 with Tanshinone I I-A sodium sulfonate injection group.

Test example 7 tanshinone emulsions are to the influence of hepatic fibrosis

1. laboratory animal and grouping, male SD rat, body weight 170-230g is divided into 5 groups at random with laboratory animal: normal control group, model group, tanshinone emulsion various dose group, 10 every group.

2. experimental technique

The rat liver fibrosis model copy reaches and respectively organizes method of disposal: with reference to the superfine method [Wu Mengchao that duplicates the rat liver fibrosis model of Wu Meng, Yang Guangshun. the research of rats'liver cirrhosis model copy. Chinese experimental surgery magazine, 1984,1 (4): 145-147], the every 100g body weight of every 4d subcutaneous injection 60% carbon tetrachloride oil solution 0.3ml, with 5% ethanol water as solvent.The every 4d subcutaneous injection of rats in normal control group oil solution 0.3ml/100g body weight, after 1 week, the next day gavage tap water 0.3ml/100g body weight, lumbar injection aquesterilisa 0.1ml/100g body weight every day (6 times weekly), with tap water as beverage; Hepatic fibrosis group rat is as stated above after 1 week of modeling, the next day gavage tap water 0.3ml/100g body weight, lumbar injection aquesterilisa 0.1ml/100g body weight every day (6 times weekly); Tanshinone emulsion group rat is as stated above after 1 week of modeling, the next day gavage tap water 0.3ml/100g body weight, lumbar injection tanshinone emulsion 2mg/100g body weight every day (6 times weekly).4th, 8 weekends, each group is randomly drawed 6 animals, presses 150mg/100g body weight intraperitoneal injection of anesthesia with 25% urethane solution, dissects, and hepatic tissue is left and taken in the portal vein blood sampling, and part is fixed with 10% neutral formalin solution, system paraffin mass in the 24-48h.

Observation index and assay method: HE dyeing is adopted in the liver histopathology inspection, the fibroplasia degree be divided into the 0-4 level [Li Kun, Zhao Yuzhen, Zhu Qiushuan etc. ligustrazine is to the influence of the aged mouse heart, liver superoxide dismutase activity. Heilungkiang medical science, 1998; 21:4-5].Measure Serum hyaluronic acid content with radioimmunoassay, hyaluronic acid radioimmunoassay determination box grinds the medical biotechnology center available from Shang Haihai, and used instrument is that SN-695A type intelligence is put and exempted from the γ measuring instrument.Hydroxyproline determination adopts oxidizing process in the hepatic tissue, and detection kit is built up bio-engineering research institute available from Nanjing.

3. experimental result

Pathological examination: the rats in normal control group liver structure is normal; Tangible fibrosis all appears at 4 weekends of model group rat liver and 8 weekends; Fibrosis is all light than model group in each dosage group of tanshinone emulsion.

Hyaluronic acid contents in the rat blood serum: 4 weekends and 8 weekends, except that heavy dose was organized 8 time-of-week points, tanshinone emulsion other times point rat blood serum hyaluronic acid contents was all low than model group, significant difference (table 8).

Table 8 tanshinone emulsion is to the influence of hepatic fibrosis rats hematohyaloid acid content (μ g/L)

Group	4 weeks	8 weeks
			The normal control group	153±25	140±26
Model group	396±45 △	592±87 △
			Tanshinone emulsion 3mg/kg	262±25 ▲	289±46 ▲
Tanshinone emulsion 6mg/kg	180±26 ▲	265±43 ▲
			Tanshinone emulsion 12mg/kg	135±29 ▲	210±47 ▲

Annotate: △, compare P＜0.05 with the normal control group; ▲ compare P＜0.05 with model group; ▲ ▲ P＞0.05.

Collagen content in the liver tissues of rats: at 4 weekends, collagen content is lower than hepatic fibrosis group, significant difference in each dosage group liver tissues of rats of tanshinone emulsion; During 8 weekends, also low than model group, but heavy dose of difference not remarkable (seeing Table 9) of organizing.

Table 9 tanshinone emulsion is to the influence of hepatic fibrosis rats hepatic tissue collagen content (mg/g hepar siccatum)

Group	4 weeks	8 weeks
			The normal control group	6.20±0.78	5.98±0.88
Model group	13.02±1.69 △	22.03±3.76 △
			Tanshinone emulsion 3mg/kg	10.11±2.16 ▲	14.98±3.34 ▲
Tanshinone emulsion 6mg/kg	9.78±2.10 ▲	12.88±1.98 ▲
			Tanshinone emulsion 12mg/kg	9.23±2.01 ▲	14.13±3.09 ▲

Annotate: △, compare P＜0.05 with the normal control group; ▲, compare P＜0.05 with model group; ▲ ▲, compares P＞0.05 with model group.

Test example 8 tanshinone emulsions are to the active influence of aldose reductase (AR)

1. test method

Grouping is the same.Modeling method adopts alloxan (100mg/kg) to bring out diabetes.With the rat sacrificed by decapitation, get kidney and reject attachment when testing for the 16th week, smash to pieces and get homogenate, AR is active to be used for measuring.

AR activity determination method: promptly write down NADPH and represent enzymatic activity at 340nm place absorption light value decline 10U.The reaction system that this research is used: the Na of 67mmol/L, pH6.2 ⁺-K ⁺NADPH and the enzyme of the DL-glyceraldehyde of-phosphate buffer, 10mmol/L, the 2 mercapto ethanol of 5mmol/L, 0.1mmol/L are an amount of; The experiment cup comprises whole reagent, and the contrast cup replaces substrate DL-glyceraldehyde with distilled water.

Measure the tissue homogenate enzymatic activity, measure protein content simultaneously, a unit enzymatic activity is defined as 1min and produces 1 μ molNADP ⁺The enzyme amount, each histaminase activity compares with specific activity, specific activity=enzyme unit/mg albumen.

2. result

Its AR activity of diabetic mice model group is significantly higher than normal group (P＜0.01), and AR is active behind the use tanshinone emulsion obviously descends, and compares with model group, and the P value all＜0.01; Though each treatment group AR activity still is higher than normal group, the there was no significant difference of comparing with normal group (P＞0.05).See Table 10.

Table 10 experiment is respectively organized the AR activity change relatively

Group	The AR activity
		The normal control group	2.14±0.82
Model group	10.40±3.19 **
		Tanshinone emulsion 3mg/kg	3.61±1.58 ##
Tanshinone emulsion 6mg/kg	3.28±0.88 ##
		Tanshinone emulsion 12mg/kg	3.22±0.69 ##

Annotate: compare * * P＜0.01 with the normal control group; Compare #P＜0.05 with model group; ##P＜0.01.

Test example 9 tanshinone emulsions are to the influence of lung fibrosis in rats model

1. modeling method

Grouping is the same.1.5% sodium pentobarbital with aseptic configuration carries out intraperitoneal anesthesia (2.5ml/kg body weight) to rat, with sterile working's row cervical region median incision, adopt blunt capable partition method to expose trachea, the concentration of slowly injecting the physiological saline solution configuration to pulmonary through trachea puncture is the bleomycin A of 4mg/ml ₅(BLMA ₅) solution 0.2～0.3ml (BLMA ₅Dosage is the 5mg/kg body weight), uprightly rotate animal immediately, medicinal liquid is evenly distributed in lung.The normal control group is injected the equivalent physiological saline solution.

2. observation index: gather abdomen cardinal vein blood sample, use the EDTANa2 anticoagulant, separated plasma ,-20 ℃ are frozen, to be checked.Measure Plasma TNF-α, NO, the content of MDA and the activity of SOD, all strict with the operation of reagent corresponding box description.

3. result

Each dosage of tanshinone emulsion all can reduce experimental pulmonary fibrosis rat plasma TNF-α, NO, MDA content, SOD activity improving, and the injury of lung that bleomycin is caused rat has certain protective role.See Table 11.

The content of TNF-α, NO, MDA is respectively organized in table 11 experiment and the activity change of SOD compares

Group		TNF-α (pg/ml)	NO(μmol/L	MDA(nmol/ml)	SOD(U/ml)
						The normal control group		497.8±52.6	28.5±2.8	5.9±1.3	114.8±19.3
Model group		753.5±82.4 **	35.8±5.5 **	13.8±2.7 **	73.2±15.8 **
						Tanshinone emulsion	3mg/kg	690.9±68.3 **#	32.1±8.2 *#	12.5±2.6 **#	88.7±17.9 **#
6mg/kg	567.7±68.3 **#	31.2±8.0 *#	9.6±2.8 **#	99.9±19.9 **#
					12mg/kg		490.4±68.3 ##	29.3±8.3 ##	8.8±2.4 *##	105.7±15.7 *##

Annotate: compare * P＜0.05, * * P＜0.01 with the normal control group; Compare #P＜0.05 with model group; ##P＜0.01.

Test example 10 tanshinone emulsions are to the influence of rat chronic nephritis model

1. modeling method

It is the same to divide into groups.Adopt the anti-Thy-1 Antibody Preparation rat mesangial proliferative nephritis model (preparation reference literature [Chen Guangping of the anti-Mus Thy-1 of rabbit antiserum (ATS), Guo Muyi, Zhang Yuee, Deng. the foundation of rat Thy-1 Antiserum Preparation and mesangial proliferative nephritis model. clinical and experimental pathology magazine, 1996,3:241], with reference to above-mentioned literature method, normal group is respectively organized rat outward by tail vein injection ATS1ml/100g, 1 time weekly, continuous 4 weeks.

2. medication

Beginning administration in the 2nd day after injecting ATS first.

3. observation index: after the 4th all footline eyeball excise blood samplings, cut open the belly at once and get kidney.

3.1 ordinary circumstance

The mental status of rat, body weight, chaeta, forage volume, amount of drinking water, two just reach active situation etc.

3.2 immunofluorescence is checked

Get fritter renal cortex tissue and make frozen section, use the Mus IgG of the rabbit Chinese People's Anti-Japanese Military and Political College (Sigma company, 1:64 tires) of Fluorescein isothiocyanate (FITC) labelling to make direct fluorescence staining, AO one-twenty fluorescence microscope.Fluorescence intensity grade scale reference literature [Zou Wanzhong. kidney pathology and clinical. Changsha: Hunan science tech publishing house, 1993.9-10,26-27].

3.3 observation by light microscope

Get the part nephridial tissue and fix with 10% neutral formalin, the ladder ethanol dehydration, paraffin embedding, haematoxylin Yihong dyeing (HE) and periodic acid Schiff reaction, PAS dyeing (PAS) is done in section, observes, takes pictures under the optical microscope.To optional 10 glomerule of every routine specimen, count the cell number of each glomerule, measure the major diameter of each glomerule, and record mesentery hyperplasia degree, its grade scale reference literature [Zou Wanzhong. kidney pathology and clinical. Changsha: Hunan science tech publishing house, 1993.9-10,26-27].

3.4 electron microscope observation

Get 3 rat kidney 1mm for every group ²3 of sizes are made electron microscopic observation, and are conventional with fixing before 2% glutaraldehyde, fixing behind 1% osmic acid, the ladder ethanol dehydration, and epoxy resin ON812 embedding, ultrathin section behind parallel uranium and the plumbous double staining, is put under the JEM-1200EX transmission electron microscope and is observed, Taking Pictures recording.

4. experimental result

4.1 ordinary circumstance

Model group has 1 routine rats death in experimentation, and phenomenons such as lassitude in various degree, the minimizing of ingesting, growth retardation, health are become thin, rolled up, chaeta tarnish, The stool is completely mashed all appear in the rat of respectively organizing of modeling, and are the most remarkable with model group.

4.2 light microscopy checking

The result shows: the normal control group has 1 example to see the glomerular capillary mild hyperaemia, and is surplus no abnormal.The model group intraglomerular mesangial cell all has hypertrophy, the broadening of mesentery substrate, and visible a small amount of neutrophil infiltration.Basement membrane is not seen obviously and is thickened.Most of glomerule increases kidney sacculus obturation.Proximal tubular epithelial cells is moderate or severe intracellular edema, and part renal tubules intracavity is seen albumen.Each treatment group mesentery hypertrophy phenomenon has and alleviates, and edema is clearly better in the proximal tubular epithelial cells, and albumen disappears in the renal tubules.See table 12-14 for details.

Table 12 is respectively organized rat immunity fluoroscopy result relatively (n=10)

Group	Fluorescence intensity
						-	+	++	+++	++++
	The normal control group	10	0	0	0						0 **
Model group						0	1	1	3	4
	Tanshinone emulsion 3mg/kg	2	1	4	2						1
Tanshinone emulsion 6mg/kg						3	2	4	1	0
	Tanshinone emulsion 12mg/kg	5	1	3	1						0 *

Annotate: compare * P＜0.05, * * P＜0.01 with model group.

Table 13 is respectively organized the comparison (x ± s) of glomerule inner cell number, glomerule size

Group	Cell number (individual)	Glomerule diameter (μ m)
			The normal control group	42.11±7.96 **	78.41±7.98 **
Model group	76.66±14.09	130.37±12.35
			Tanshinone emulsion 3mg/kg	58.65±9.33 **	109.57±10.84 **
Tanshinone emulsion 6mg/kg	55.10±9.46 **	96.74±10.34 **
			Tanshinone emulsion 12mg/kg	49.04±10.02 **	87.41±9.68 **

Annotate: compare * P＜0.05, * * P＜0.01 with model group.

Table 14 is respectively organized rat mesentery hyperplasia degree relatively (n=10)

Group	The classification of mesentery hypertrophy
					Do not have	Slightly	Moderate	Severe
	The normal control group	10	0	0					0 **
Model group					0	1	2	6
	Tanshinone emulsion 3mg/kg	3	2	4					1
Tanshinone emulsion 6mg/kg					2	2	5	l
	Tanshinone emulsion 12mg/kg	4	4	2					0

Annotate: compare * P＜0.05, * * P＜0.01 with model group.

4.3 electron microscopic examination

The result shows: the normal group cellularity is complete.The remarkable broadening of model group mesangial region, mesangial cell and mesentery substrate hypertrophy have cloud electron-dense thing to merge at mesangial region deposition part podocytic process, and capillary basement membrane is normal substantially.Each treatment group pathological change is lighter than model group, and is the lightest with tanshinone emulsion high dose group pathological changes, in, low dose group takes second place.

5. conclusion

Tanshinone emulsion has therapeutical effect to the kidney pathological lesion of rat chronic nephritis model.

Test example 11 tanshinone emulsions are to the influence of rat chronic renal insufficiency model

1. modeling method

Except that the normal control group, all the other are respectively organized rat and adopt the Plant method, the most of excision of row 5/6 kidney is made the chronic kidney hypofunction rat model: after pressing 30mg/kg intraperitoneal injection of anesthesia rat with 1% pentobarbital sodium solution, be fixed on the Mus platform, lower back is shaved hair, behind the alcohol disinfecting, make otch in left of spine, open the abdominal cavity, expose left kidney, peel off kidney peplos, with the aseptic suture ligation left side kidney base of a fruit, excise left the kidney utmost point and lateral border up and down, stop blooding with gelfoam in the part, wash the abdominal cavity to protect from infection with metronidazole injection, then layer-by-layer suture muscle and skin.The right kidney of excision behind the 1 week back ligation right renal artery.The nephridial tissue of excision is weighed, and estimates left residual kidney weight indirectly, and requiring the average resection rate of nephridial tissue is about 80%.

2. grouping and administration

Male SD rat; body weight 170-230g; laboratory animal is divided into 5 groups at random: sham operated rats (is performed the operation; but do not excise kidney), model group, protection group (twice postoperative promptly began to give tanshinone emulsion 6mg/kg on the 2nd day), tanshinone emulsion various dose group (twice 1 week of postoperative begins to give tanshinone emulsion 3,6,12mg/kg), 10 every group.8 weeks of the course of treatment.Per 2 weeks weigh 1 time, to adjust the rat dosage.

3. observation index

3.1 ordinary circumstance: mainly observe the mental status, mobility, hair luster degree, diet, body weight etc.

3.2 renal function detects: medication was weighed after 8 weeks, femoral vein is got blood 8ml and is collected in and leaves standstill 1h in the test tube, and separation of serum is measured serum creatinine (Cr) and blood urea nitrogen (BUN) level with automatic biochemical analyzer.Put to death animal, win remaining kidney (rats in normal control group is got 1/6 of two kidney full weights and compared) fast, kidneys such as removal fascia are organized outward, filter paper suck dry moisture behind the normal saline washing, and scales/electronic balance weighing calculates kidney weight/weight ratio.

4. result

4.1 ordinary circumstance: the performance of normal control treated animal is alert, and reaction is fast, the fur densification, and neat and glossy, diet is normal; Each treated animal lethargy after the modeling, movable slow, fur is fluffy, withered tarnish, inappetence.Wherein model control group is the most obvious, and each dosage group of tanshinone emulsion and protection group are better than model control group.

4.2 renal function detects: after the modeling, model group rat blood serum creatinine, blood urea nitrogen are apparently higher than normal control group (P＜001), and after the treatment, each dosage group of tanshinone emulsion and protection group change of serum C r, BUN obviously descend.Although with normal group more variant (P＜001); but significantly be lower than model control group; especially with the heavy dose of group of tanshinone emulsion and protection group to the improvement of BUN comparatively obviously (P＜005), the reduction of the heavy dose of group of tanshinone emulsion Cr is very obviously (P＜001) also.

It is obvious respectively to organize remaining kidney compensatory hypertrophy after the modeling, with the normal group comparing difference highly significant (P＜001) is arranged, rat body weight then descends to some extent, especially obviously (compare P＜005 with normal group) with the model group weight loss, each dosage group of tanshinone emulsion and protection group then relatively do not have significant difference with the normal control group; In addition; respectively organizing kidney of rats weight/kidney weight ratio after the modeling obviously increases; with normal control group comparing difference highly significant (P＜001) is arranged; the protection group then decreases and (compares with model group; P＜005); the prompting tanshinone emulsion can improve the chronic kidney hypofunction rat body weight and descend, and has the effect of preventative prevention kidney compensatory hypertrophy.

See table 15 for details.

Table 15 tanshinone emulsion is to the influence of chronic kidney hypofunction kidney of rats function (x ± s)

Group	n	BUN(mmol/L)	Cr(μmol/L)	Kidney heavy (g)	Body weight (g)	Kidney weight/body weight (* 10 -3)
							The normal control group	10	7.54±0.96	43.19±6.09	0.33± 0.08	286.63± 40.95	1.16±0.27
Model group	7	14.45±2.56 ##	145.83± 9.89 ##	0.98± 0.25 ##	236.71± 31.78 #	4.14±1.10 ##
							Tanshinone emulsion 3mg/kg	8	11.89±1.46 ##	103.32± 11.43 ##	1.27± 0.17 ##	284.68± 34.77	4.46±0.78 ##
Tanshinone emulsion 6mg/kg	7	11.23±0.97 ##	94.49± 24.57 ##	0.97± 0.13 ##	290.43± 24.23	3.34±0.69 ##
							Tanshinone emulsion 12mg/kg	9	10.99±2.67 *##	74.57± 13.86 **##	0.87± 0.33 ##	278.99± 41.13 *	3.09±1.14 ##
The protection group	8	11.04±0.99 *##	96.08± 13.33 ##	0.82± 0.32 ##	288.28± 34.77 *	2.84±0.45 ##

Annotate: with the model group ratio, * * P＜001; With normal control group, #P＜005, ##P＜001.

5. conclusion

Tanshinone emulsion 3,6,12mg/kg have the not effect of progress entirely of postponing chronic kidney function.

Test example 12: tanshinone emulsion is to the influence of AD rat model learning memory

1.A _{β 1-40}Induce rat AD sample pathological changes Preparation of model and grouping:

Use condensed state beta-amyloyd peptide 1～40 fragment (A _{β 1-40}) inject the animal model of setting up AD sample pathological changes in the rat hippocampus.Specific as follows:

Totally 50 of SD male rats clean 2 grades, body weight 250～300g.The SD rat after anaesthetizing under stereotaxic instrument with A _{β 1-40}Injection in the row bilateral Hippocampus, each 1 μ l of every side is (in advance with A _{β 1-40}Be dissolved in physiological saline solution 10 μ g/ μ l, under preceding 37 ℃, hatched for 1 week).The injection site is: 3.5mm behind the bregma, and the brain median line is other opens 2.0mm, and its degree of depth is 2.7mm.

Rat is divided into 5 groups at random: (1) A _{β 1-40}The transaction module group; (2) tanshinone emulsion low dose therapy group (3mg/kg); (3) dosage treatment group (6mg/kg) in the tanshinone emulsion; (4) tanshinone emulsion high-dose therapy group (12mg/kg); (5) Sham-operated control group: promptly inject the equivalent normal saline in the Hippocampus.

Medication: each treated animal begins intravenous administration, every day 1 time, continuous 3 weeks in modeling the last week.The labyrinth training period, 0.5h administration before training.

2. observation index and method

Space learning memory test: adopt Morris water maze method.Platform is located at Northeast Quadrant center, labyrinth, and horizontal plane exceeds platform 1.5cm, and water temperature remains on 19 ℃～20 ℃, rat is trained 5d continuously, and every day 2 times, setting the longest walk time is 70s, clock with stopwatch, the record rat find the platform required time (incubation period, EL).

3. result:

Influence to AD rat space learning memory: increase with the training natural law, each is organized the average EL of rat and shortens gradually.Compare with sham operated rats, model group rat EL obviously prolongs, and the 4th day beginning significant difference (P＜0.05) illustrates the modeling success.With the model group ratio, tanshinone emulsion 3mg/kg group rat obviously shortens in the 4th day beginning EL, and the 6mg/kg group then obviously shortens in the 3rd day beginning EL, and the 12mg/kg group then promptly began EL in the 2nd day and obviously shortens, all significant difference; 12mg/kg organizes the 5th day then utmost point significant difference.Show that each dosage group of tanshinone emulsion all can significantly improve A _{β 1-40}Mutually inductive rat space learning dysmnesia, and be dose dependent.See table 16 for details.

Table 16 tanshinone emulsion is to the influence of experimental AD rat model spatial discrimination ability of learning and memory

Group	n	Incubation period/s
							1d	2d	3d	4d	5d
		Sham-operated control group	10	58.88±6.86	47.13±8.79	43.18±11.30						32.56±8.42 *	23.34±4.96 *
Model group	10						60.18±6.69	58.43±4.86	54.87±9.29	52.23±7.15	51.37±10.67
		Tanshinone emulsion 3mg/kg group	10	59.97±7.30	55.44±5.26	50.56±6.12						38.23±7.13 *	30.48±3.99 *
Tanshinone emulsion 6mg/kg group	10						59.44±8.30	53.68±6.89	39.87±6.06 *	33.95±7.09 *	24.31±3.89 *
		Tanshinone emulsion 12mg/kg group	10	62.09±8.38	43.54±6.04 *	35.96±6.03 *						30.14±7.67 *	21.22±3.77 **

Annotate: compare * P＜0.05:**P＜0.01 with the AD model group.

Claims (10)

1, a kind of pharmaceutical composition that contains TANSHINONES, it consists of, and contains in per 100 gram compositionss:

The water of TANSHINONES 0.05～0.2g, oil 5～20g, emulsifying agent 0.5～5.0g, surplus.

2, pharmaceutical composition as claimed in claim 1 is characterized in that, also contains the 0.02-0.1g enuatrol in per 100 gram compositionss.

3, pharmaceutical composition as claimed in claim 1 is characterized in that, in the described TANSHINONES cryptotanshinone, tanshinone, Tanshinone I and dihydrotanshinone I total content by weight/the weight meter is not less than 50%.

4, pharmaceutical composition as claimed in claim 1 is characterized in that, described oil is pharmaceutically as the oil of carrier, is that 1 to 0.25～4 ratio form with midchain oil than long-chain oil by midchain oil and long-chain oil.

5, pharmaceutical composition as claimed in claim 3, it is characterized in that, described long-chain oil is vegetable oil or animal oil or both mixture, described vegetable oil is soybean oil, Semen Maydis oil, Oleum Arachidis hypogaeae semen, olive oil, Oleum Ricini, Petiolus Trachycarpi oil, Oleum Cocois, Oleum Brassicae campestris, Oleum sesami, Oleum Gossypii semen or their mixture, and described animal oil is fish oil, enuatrol, sperm oil or their mixture.

6, pharmaceutical composition as claimed in claim 1, it is characterized in that described emulsifying agent is soybean phospholipid, egg yolk lecithin, hydrogenated phospholipid, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, serinephosphatide, lipositol, phosphatidic acid, cephalin or their mixture.

7, pharmaceutical composition as claimed in claim 1, it is characterized in that, per 100 grams contain in the compositionss TANSHINONES 0.05～0.10g, midchain oil 4g, long-chain oil 1～6g, emulsifying agent 1.0～1.6g, enuatrol 0.04-0.07g, isoosmotic adjusting agent by weight/weight counts 2.0～3.0% and the water of surplus.

8, pharmaceutical composition according to claim 1 is characterized in that described compositions is Orally taken emulsion or vein emulsion.

9, the described preparation of compositions method of claim 1-9 is characterized in that, TANSHINONES and emulsifying agent are mixed with oil, and heating is stirred, and makes to be dissolved as clear and bright solution, gets oil phase; With isotonic agent formation water soluble in water; Under high-speed stirred, oil phase is added to aqueous phase and forms colostrum, after the gained colostrum adds the water standardize solution,, obtain Emulsion through high pressure homogenizer emulsifying.

10, the application of the compositions of claim 1-8 in preparing treatment or prophylaxis against infection diseases, ischemia apoplexy, Cerebral Ischemia damage, ischemic heart desease, tumor, hepatic fibrosis, diabetic complication, pulmonary fibrosis, chronic nephritis, chronic renal insufficiency, Alzheimer medicine.