CN100506962C

CN100506962C - Garlic oil refining process and the Preparation process of its emulsion and solid nanometer liposome particle

Info

Publication number: CN100506962C
Application number: CNB2005100479498A
Authority: CN
Inventors: 郭涛; 何进; 孙学惠; 邓意辉
Original assignee: GUO TAO HE JIN SUN XUEHUI DENG YIHUI; GUO TAO HE JIN SUN XUEHUI DENG
Current assignee: GUO TAO HE JIN SUN XUEHUI DENG YIHUI; GUO TAO HE JIN SUN XUEHUI DENG
Priority date: 2005-12-09
Filing date: 2005-12-09
Publication date: 2009-07-01
Anticipated expiration: 2025-12-09
Also published as: CN1810947A

Abstract

The present invention relates to garlic oil refining process and preparation. The technological process of refining garlic oil includes filling the chromatographic separating column with adsorbent, mixing adsorbent and garlic oil material, decompression filling the mixture to the chromatographic separating column, eluting with proper amount of organic solvent, collecting the effluent, and decompression recovering organic solvent to obtain refined garlic oil. The refined garlic oil is yellowish clear liquid with garlic smell, refractive index over 1.5720 at 20 deg.c, garlicin content over 37.0 % and 15 fingerprint peaks in its fingerprint spectrum. The refined garlic oil may be further prepared into different preparations, including emulsion, nanometer liposome granule, etc, which may be used in intravenous injection to resist deep fungus and bacteria infections to treating and preventing various diseases.

Description

The preparation technology of the process for purification of Oleum Bulbus Allii and emulsion thereof and solid lipid nanoparticle

One, technical field: the present invention relates to a kind of Oleum Bulbus Allii feed purification that will be common and obtain refined garlic oil after handling, and utilize the Oleum Bulbus Allii of purifying to prepare emulsion and solid lipid nanoparticle and method thereof.

Two, background technology: deep fungal infection is one of clinical each section's severe complication.In recent years because extensive application such as Broad spectrum antibiotics, chemotherapeutics and immunosuppressor clinically; Extensively carrying out of deep intravenous hyperalimentation method and vein, trachea cannula, organ transplantation, immunosuppression that chronic wasting disease caused such as diabetes, malignant tumour, acquired immune deficiency syndrome (AIDS) or immune deficiency patient's increase in addition, mycosis especially deep mycosis sickness rate significantly rises.National ward infection detection system data according to U.S. CDC confirms, from 1980 to nineteen ninety, the fungi infestation rate significantly rises in the institute, surgical patients has increased by 124%, medical patient has increased by 73%.And candidiasis becomes pathogenic bacterium in the 6th the modal institute.The invasive fungi disease occurs in the patient of serious underlying diseases mostly, the case fatality rate height.

The high-efficiency low-toxicity medicine of treatment deep mycosis is few at present, still select for use to prolong and used nearly 40 years amphotericin B, but its toxicity is bigger, uses limited.5-flurocytosine (5-FC) narrow antimicrobial spectrum easily produces resistance, does not generally use separately.Synthesize clotrimazole and miconazole the end of the sixties.After the eighties, new antifungal drugs such as KETOKONAZOL, fluconazole are come out one after another, and wherein fluconazole is that present clinical application is the widest, the antifungal drug that effect is best.Yet these medicine majorities only have bacteriostatic action and make the course of treatment longer, need several weeks to the several months usually.Because fungi and human cell are all eukaryotic cell, often the host are had suitable toxicity behind the long-term prescription.Therefore press for the novel anti fungi-medicine of developing efficient, low toxicity, wide spectrum clinically.The Chinese medicine side effect is little, the source is wide, cheap, resistance seldom occurs, be suitable for reaching for a long time these characteristics of prophylactic application, makes the antimycotic Chinese medicine of research and development have good prospect.

Being applied to antimycotic Chinese medicine mainly is some volatile oils, as the Oleum Bulbus Allii that extracts from garlic, the Oleum Cinnamomi that extracts from Chinese cassia tree, aubepine of extracting from fennel etc.Wherein the antifungic action of Oleum Bulbus Allii is leading.Oleum Bulbus Allii be mashed garlic after pulverizing through water distillation or organic solvent extraction and volatile oil.Natural Oleum Bulbus Allii complicated component, kind is a lot, and its main component is to contain the thio-ether type compounds that multiple group such as allyl group, propyl group and methyl is formed.Modern medicine confirms that the Oleum Bulbus Allii pharmacological action is extensive, antitumor, reducing blood-fat, enhance immunity power, the hematoblastic congregation that reduces, aspect resisting pathogenic microbes, various pathogens such as staphylococcus, meningitis, pneumococcus, suis, diphtheria, dysentery, typhoid fever, intestinal bacteria, tubercule bacillus, vibrio cholerae etc. are all had suppress or killing action, enjoy the laudatory title of " natural Broad spectrum antibiotics ".

At present, domestic have ten surplus enterprise of family produce Oleum Bulbus Allii, mainly contain the special pharmaceutical factory of Henan Olympic, Henan Xuchang Yuanhua Biotechnology Co., Ltd., Jinxiang edible oil company limited of Shandong Jin Gui group etc.Natural Oleum Bulbus Allii is mainly used in food and fodder additives, healthcare products etc., and the refined garlic oil of medicinal specification is not arranged so far as yet, purifying (making with extra care) method of the natural Oleum Bulbus Allii raw material that also do not appear in the newspapers, and the preparation of corresponding high quality feedstock production is also failed exploitation.The commercially available garlic similar drug of China only has garlic enteric coated tablet, garlicin enteric capsule and pill, garlicin injection etc. at present.The curative effect fast in order to obtain stable is usually wished direct intravenously administrable clinically.Commercially available garlicin injection is the garlicin synthetics, but garlicin injection is single component preparation, and antimicrobial spectrum is obviously narrow, and bigger to the toxicity of heart, once has the report garlicin injection to have the toxic action and the blood vessel irritation that cause asystole strong.Now still do not have commercially available garlic oils preparation, this blank is demanded urgently filling up.

Three, summary of the invention:

1, goal of the invention: the preparation technology who the invention provides a kind of process for purification and emulsion and solid lipid nanoparticle of Oleum Bulbus Allii, its purpose is to solve the preparation method that the refined garlic oil of medicinal specification is not arranged so far as yet, and the preparation of corresponding high quality Oleum Bulbus Allii feedstock production such as also fails to develop at the problem that the aspect exists.

2, technical scheme: the present invention is achieved through the following technical solutions:

A kind of process for purification of Oleum Bulbus Allii is characterized in that: this method is undertaken by following step:

A, get each raw material: Oleum Bulbus Allii 1: sorbent material I 0.25～1.25: sorbent material II1～5 by following weight ratio;

B, with above-mentioned Oleum Bulbus Allii and sorbent material II mixing;

C, sorbent material I is packed into chromatography column are under reduced pressure packed the decompression of step b gained mixture on it again;

D, get solvent decompression wash-out, collect effluent liquid;

E, reclaim under reduced pressure solvent promptly get refined garlic oil;

Described sorbent material I, sorbent material II are selected from a kind of in silica gel, aluminum oxide, Magnesium Silicate q-agent, polymeric amide, diatomite, the macroporous adsorbent resin; Eluent is selected from C ₁～C ₅Alcohols, C ₃～C ₈Organic acid ester, C ₁～C ₄Organic acid, C ₃～C ₈A kind of in ketone, the ethers.

The solid lipid nanoparticle agent of the refined garlic oil preparation that a kind of usefulness is above-mentioned, it is characterized in that: mainly contain refined garlic oil and pharmaceutical excipient, pharmaceutical excipient is mainly solid lipid, emulsifying agent, assistant for emulsifying agent composition, and the mass ratio of Oleum Bulbus Allii and pharmaceutical excipient is: 5:1～1:50; Each composition consumption of its pharmaceutical excipient is: solid lipid 1%～10% (w/v), emulsifying agent 1%～10% (w/v), assistant for emulsifying agent 0.05～5% (w/v); Other composition is a water.

Described solid lipid is a kind of in glyceride, lipid acid, steroid or the wax class; Glyceride is selected from a kind of in myristin, tripalmitin, the Tristearoylglycerol; Lipid acid is selected from a kind of in stearic acid, palmitinic acid, capric acid, the behenic acid; Steroid is selected from cholesterol; The wax class is selected from a kind of in cetyl palmitate, microcrystalline wax, the whale vinegar wax; Described emulsifying agent is selected from a kind of in phospholipid, HS15, vitamin E polyethylene glycol succinic acid ester, poloxamer 188, cholic acid salt, Tweens, the short-chain alcohols; The phospholipid consumption is 0.5～5% (w/v), HS15, consumption is 0.1～5% (w/v), vitamin E polyethylene glycol succinic acid ester, consumption is 0.1～5% (w/v), poloxamer 188, consumption is 0.05～5% (w/v), cholic acid salt, consumption is 0.1%～5% (w/v), Tweens, consumption is 0.1%～5% (w/v), short-chain alcohols, and consumption is 0.05～5% (w/v); Described assistant for emulsifying agent be selected from oleic acid, sad, certain herbaceous plants with big flowers is sour, a kind of in the lauric acid, palmitinic acid, linolic acid, linolenic acid, stearic acid, docosahexenoic acid, cholic acid.

A kind of preparation technology of above-mentioned refined garlic oil solid lipid nanoparticle agent, it is characterized in that: this technology is undertaken by following step:

A, get each raw material by the prescription of the described refined garlic oil solid lipid nanoparticle of claim 2 agent;

B, in preparing tank, water, emulsifying agent and assistant for emulsifying agent are evenly made water 50～90 ℃ of following dispersed with stirring;

C, in preparing tank, under uniform temp, solid lipid is melted mutually, add Oleum Bulbus Allii again, stir, form the lipid phase;

D, with the disposable quick adding lipid phase of water, high-speed stirring 3 minutes stirs revolution 6000～28000rpm, makes colostrum;

E, cross homogeneous instrument or microjet instrument, regulate homogenize pressure to 40～120MPa, with solution homogenize repeatedly, or colostrum popped one's head under power 400W ultrasonic 5 minutes, keeps 70 ℃, obtain uniform emulsion, millipore filtration is reduced to room temperature, promptly excessively.

3, advantage and effect: by the enforcement of technical solution of the present invention, can solve the refined garlic oil that medicinal specification is not arranged so far as yet well, also the do not appear in the newspapers process for purification of natural Oleum Bulbus Allii raw material, the preparation of corresponding high quality Oleum Bulbus Allii feedstock production such as also fail to develop at the problem that the aspect exists.The present invention makes with extra care existing natural Oleum Bulbus Allii, foundation meets the method for the refining purifying Oleum Bulbus Allii of suitability for industrialized production, formulate medicinal, injection specification Oleum Bulbus Allii quality standard, and adopt purified high quality Oleum Bulbus Allii raw material to study corresponding various high quality preparation.Oleum Bulbus Allii behind the process for purification purifying of the present invention becomes faint yellow by original pale brown look; Measure garlicin content greater than 37.0% with the GC method of being set up, 15 of the total fingerprint peakses of finger printing; Intravenous injection emulsion with the refined garlic oil preparation has external anti-deep fungal effect, and the security raising, for clinical application provides reliable foundation.

Emulsion that Oleum Bulbus Allii after utilizing the present invention refining makes and solid lipid nanoparticle agent be intravenously administrable directly, and every index is all qualified after after testing, and particle size range is 10nm～5 μ m; 17 of the total fingerprint peakses of finger printing.Preparation of the present invention has anti-deep fungal and infectation of bacteria, anti-treating acute and chronic bacillary dysentery and enteritis, Whooping cough, lung and gastral fungi infestation, Candida albicans microbemia, cryptococcal meningitis, pulmonary tuberculosis etc.

Four, description of drawings:

Accompanying drawing 1 is refined garlic oil stability influence factorial experiments result curve figure of the present invention;

Accompanying drawing 2 is the stability test result curve figure under the different storage requirements of refined garlic oil of the present invention;

Accompanying drawing 3 is refined garlic oil GC finger printing of the present invention;

Accompanying drawing 4 is Oleum Bulbus Allii intravenous injection emulsion finger printing of the present invention;

Accompanying drawing 5 is Oleum Bulbus Allii intravenous injection emulsion standard finger-print of the present invention;

Accompanying drawing 6 is Oleum Bulbus Allii solid lipid nanoparticle fingerprint spectrogram of the present invention;

Accompanying drawing 7 gives rabbit ear edge vein pathological section photo after the glucose injection for the present invention;

Accompanying drawing 8 gives the rabbit ear edge vein pathological section photo of 72h behind the Oleum Bulbus Allii intravenous injection emulsion for the present invention;

Accompanying drawing 9 gives behind the Oleum Bulbus Allii intravenous injection emulsion 14 days rabbit ear edge vein pathological section photo for the present invention.

Five, embodiment:

Oleum Bulbus Allii feed purification method of the present invention is got each raw material by following weight ratio: Oleum Bulbus Allii 1: sorbent material I0.25～1.25: sorbent material II1～5; Can choose its proportionlity arbitrarily in this scope as required, concrete operations are: 1, Oleum Bulbus Allii and sorbent material II mixing; 2, the chromatography column of under reduced pressure sorbent material I being packed into is packed the decompression of step 1 gained mixture on it again; 3, get solvent decompression wash-out, collect effluent liquid; 4, the reclaim under reduced pressure solvent promptly gets refined garlic oil.Sorbent material I and II are one or more in silica gel, aluminum oxide, Magnesium Silicate q-agent, polymeric amide, diatomite, the macroporous adsorbent resin; Eluent is C ₁～C ₅Alcohols is as ethanol, methyl alcohol; C ₃～C ₈Organic acid ester, C ₁～C ₄Organic acid, C ₃～C ₈Ketone, ethers.The refined garlic oil of gained, its quality standard is as follows: 1, proterties: this product is faint yellow or yellow clarified liq, the tool garlic odour; 2, refractive index: during 20 ℃ of this product refractive index is more than 1.5720; 3, garlicin assay and content limit: adopt the GC method, tetrahydrofuran (THF) is that interior mapping is decided garlicin content in the Oleum Bulbus Allii, and garlicin content should be greater than 37.0%; 4, finger printing is: chromatographic condition: 6890N GC, and it is that U.S. Agilent company produces, chromatographic column: HP-5, its specification is 30m * 320 μ m * 0.25 μ m, 250 ℃ of injector temperatures, 60～220 ℃ of column temperatures, temperature programming, temperature rise rate: 8 ℃/min, N ₂Do carrier gas, flow velocity 1.0mlmin ^-1, fid detector, 300 ℃ of sensing chamber's temperature, tetrahydrofuran (THF) is interior mark; The total fingerprint peaks retention time (min) of Oleum Bulbus Allii GC finger printing is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00.

The Oleum Bulbus Allii of above-mentioned preparation is made intravenous injection emulsion, and it contains Oleum Bulbus Allii behind the purifying of effective dosage as medicine, and contains pharmaceutical excipient, and the mass ratio of Oleum Bulbus Allii and pharmaceutical excipient is 1:1～1:300; Pharmaceutical excipient is mainly oil, emulsifying agent, isotonic regulator, pH regulator agent, oxidation inhibitor composition, and each composition consumption of its pharmaceutical excipient is: oil 1～30% (w/v); Emulsifying agent 0.05%～5% (w/v); Isotonic regulator 0.5～5% (w/v); Oxidation inhibitor 0.1～5.0% (w/v); Other composition is a water; The PH optimum range is 4～9.

Described grease separation is from C ₆～C ₂₈Vegetables oil or animal oil, it comprises through vegetables oil, triglyceride level, Oleum Cocois, sweet oil, soybean oil, Thistle oil, seal oil, hot certain herbaceous plants with big flowers acid glyceride after structure of modification and the hydrolysis; And the derivative of lipid acid, it comprises ethyl oleate, glyceryl linoleate, polyoxyethylene glycol laurin, ethyl linoleate, oleic acid sorbitol ester, olein, Oleum Cocois C ₈～C ₁₀Monoglyceride or dibasic acid esters, Oleum Cocois C ₈～C ₁₀Propylene glycol dibasic acid esters, Oleum Cocois C ₈～C ₁₀One or more mixtures in triglyceride level, the acetylizad direactive glyceride of purifying, the purifying sunflower oil direactive glyceride, consumption are 1～30% (w/v); Described emulsifying agent comprises phosphatidase 10 .5%～5% (w/v), polyoxyethylene glycol 12-hydroxy stearic acid ester (polyethylene glycol 660 hydroxystearate, HS15), consumption is that the consumption of 0.1%～5% (w/v), vitamin E polyethylene glycol succinic acid ester 0.1%～5% (w/v), poloxamer 188 is one or more mixtures in 0.05%～5% (w/v), Tweens 0.1%～5% (w/v), the spans 0.1%～5% (w/v); Orally taken emulsion can also adopt gelatin, gum arabic, western Huang to have a liking for glue, human serum albumin as emulsifying agent 0.05%～5% (w/v); Add assistant for emulsifying agent or sanitas as required, assistant for emulsifying agent be selected from oleic acid, sad, certain herbaceous plants with big flowers is sour, a kind of 0～5% (w/v) in the lauric acid, palmitinic acid, linolic acid, linolenic acid, stearic acid, docosahexenoic acid, cholic acid; Sanitas is selected from parabens, benzoic acids 0～5% (w/v).Described isotonic regulator be selected from be in glycerine, glucose, N.F,USP MANNITOL, the Xylitol a kind of (0.5～5%) (w/v); The pH regulator agent is selected from a kind of in hydrochloric acid, sodium hydroxide, acetic acid, sodium-acetate, phosphoric acid, sodium phosphate, citric acid, the Trisodium Citrate etc., and pH value scope is 4～9; Oxidation inhibitor is rare gas element, ethylenediamine tetraacetic acid (EDTA) and its esters, alpha-tocopherol, α-Vitamin E-acetate, alpha-lipoic acid, vitamins C and derivative thereof, and one or more mixtures (0.1～5.0%) in the Ascorbyl Palmitate (w/v).

The Oleum Bulbus Allii intravenous injection emulsion of gained, the GC finger printing is: chromatographic condition: 6890N GC (U.S. Agilent company), chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m), 250 ℃ of injector temperatures, 60～220 ℃ of column temperatures, temperature programming, temperature rise rate: 8 ℃/min, N ₂Do carrier gas, flow velocity 1.0mlmin ^-1, fid detector, 300 ℃ of sensing chamber's temperature, tetrahydrofuran (THF) is interior mark; The total fingerprint peaks retention time (min) of GC finger printing is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00,20.0～21.0,23.0～25.

Solid lipid nanoparticle with the preparation of the refined garlic oil of gained of the present invention, the Oleum Bulbus Allii that contains effective dosage is as medicine, and contain pharmaceutical excipient, and pharmaceutical excipient comprises oil, solid lipid, emulsifying agent, assistant for emulsifying agent, the mass ratio of Oleum Bulbus Allii and pharmaceutical excipient is: 5:1～1:50; Each composition consumption of its pharmaceutical excipient is: solid lipid 1%～10% (w/v), emulsifying agent 1%～10% (w/v), assistant for emulsifying agent 0.05～5% (w/v); Other composition is a water.Its solid lipid mainly includes glyceride, lipid acid, steroid and wax class; Glyceride is selected from a kind of in myristin, tripalmitin, the Tristearoylglycerol, during wherein Tristearoylglycerol comprises, the glyceryl ester of longer chain fatty acid, glyceryl behenate (Compritol888ATO), partial glycerol such as Imwitor, glyceryl monostearate contain one or more mixtures in list, two, the Witepsol W-S 55; Lipid acid is selected from a kind of in stearic acid, palmitinic acid, capric acid, the behenic acid; Steroid is selected from cholesterol; The wax class is selected from a kind of in cetyl palmitate, microcrystalline wax, the whale vinegar wax; It is 0.5～5% (w/v), HS15 that described emulsifying agent is selected from the phospholipid consumption, consumption is 0.1～5% (w/v), vitamin E polyethylene glycol succinic acid ester (TPGS), consumption is 0.1～5% (w/v), poloxamer 188, consumption is 0.05～5% (w/v), cholic acid salt, consumption is 0.1%～5% (w/v), Tweens, consumption is 0.1%～5% (w/v), short-chain alcohols, and consumption is 0.05～5% (w/v); Described phospholipid is selected from soybean phospholipid, egg phospholipids, Yelkin TTS; HS15 is selected from polyoxyethylene glycol 12-hydroxy stearic acid ester, polyethylene glycol hydroxystearate 660; The cholic acid salt is selected from Sodium cholic acid, Sodium glycocholate, Taurocholic acid sodium salt, deoxidation Taurocholic acid sodium salt; Short-chain alcohols is selected from butanols, butyric acid; Described assistant for emulsifying agent be selected from oleic acid, sad, certain herbaceous plants with big flowers is sour, a kind of in the lauric acid, palmitinic acid, linolic acid, linolenic acid, stearic acid, docosahexenoic acid, cholic acid.

The solid lipid nanoparticle agent of gained, the GC finger printing is: chromatographic condition: 6890N GC (U.S. Agilent company), chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m), 250 ℃ of injector temperatures, 60～220 ℃ of column temperatures, temperature programming, temperature rise rate: 8 ℃/min, N ₂Do carrier gas, flow velocity 1.0mlmin ^-1, fid detector, 300 ℃ of sensing chamber's temperature, tetrahydrofuran (THF) is interior mark; The total fingerprint peaks retention time (min) of Oleum Bulbus Allii solid lipid nanoparticle GC finger printing is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00,20.0～21.0,23.0～25.

Can also prepare tablet, hard, soft capsule (comprising self-emulsifying), granule, suspensoid with the Oleum Bulbus Allii raw material behind the purifying.Refined garlic oil can be used for anti-deep fungal infection, prevention and treatment thrombosis, hyperlipidemia, atherosclerosis control.

Embodiment 1:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 40g, and elder generation is in ratio and the 80g silica gel mixing of Oleum Bulbus Allii and silica gel weight ratio 1:2; Get silica gel 10g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and silica gel weight ratio 1:0.25 in addition; Again the decompression of above-mentioned Oleum Bulbus Allii and silica-gel mixture is packed on it; With the sherwood oil wash-out that reduces pressure, collect effluent liquid; The reclaim under reduced pressure sherwood oil obtains refined garlic oil.Yield 87%, outward appearance are little yellow clear liquid, and refractive index is 1.5820, garlicin content 37.83%.

Embodiment 2:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 60g, earlier in Oleum Bulbus Allii and ratio and the 60g aluminum oxide mixing of alumina weight than 1:1; Get silica gel 30g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and silica gel weight ratio 1:0.5 in addition; Again the decompression of above-mentioned Oleum Bulbus Allii and alumina mixture is packed on it; With petroleum ether-ethyl acetate (95:5) decompression wash-out, collect effluent liquid; Decompression and solvent recovery obtains refined garlic oil, yield 86%, and outward appearance is little yellow clear liquid, refractive index is 1.5730, garlicin content 37.79%.

Embodiment 3:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 80g, and elder generation is in ratio and the 240g Magnesium Silicate q-agent mixing of Oleum Bulbus Allii and Magnesium Silicate q-agent weight ratio 1:3; Get Magnesium Silicate q-agent 60g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and Magnesium Silicate q-agent weight ratio 1:0.75 in addition; Again the decompression of above-mentioned Oleum Bulbus Allii and Magnesium Silicate q-agent mixture is packed on it; With the chloroform wash-out that reduces pressure, collect effluent liquid; The reclaim under reduced pressure chloroform obtains refined garlic oil.Yield 94%, outward appearance are little yellow clear liquid, and refractive index is 1.5750, garlicin content 38.71%.

Embodiment 4:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 20g, in ratio and the 80g diatomite mixing of Oleum Bulbus Allii and diatomite weight ratio 1:4, gets silica gel 20g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and silica gel weight ratio 1:1 in addition; Again the decompression of above-mentioned Oleum Bulbus Allii and diatomite mixture is packed on it; With the methyl alcohol wash-out that reduces pressure, collect effluent liquid; Decompression and solvent recovery obtains refined garlic oil.Yield 87%, outward appearance are little yellow clear liquid, and refractive index is 1.5810, garlicin content 38.83%.

Embodiment 5:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 40g, in ratio elder generation and the 200g polymeric amide mixing of Oleum Bulbus Allii and polymeric amide weight ratio 1:5; Get polymeric amide 200g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and polymeric amide weight ratio 1:1.25 in addition; Again the decompression of above-mentioned Oleum Bulbus Allii and polyamide compound is packed on it; With the sherwood oil wash-out that reduces pressure, collect effluent liquid; The reclaim under reduced pressure sherwood oil obtains refined garlic oil.Yield 85%, outward appearance are little yellow clear liquid, and refractive index is 1.5760, garlicin content 37.23%.

Embodiment 6:

The method of refined garlic oil of the present invention is got Oleum Bulbus Allii 100g, in ratio and the macroporous adsorbent resin 400g mixing of Oleum Bulbus Allii and macroporous adsorbent resin weight ratio 1:4; Get macroporous adsorbent resin 50g, the chromatography column of under reduced pressure packing in the ratio of Oleum Bulbus Allii and macroporous adsorbent resin weight ratio 1:0.5 in addition; With the sherwood oil wash-out that reduces pressure, collect effluent liquid; The reclaim under reduced pressure sherwood oil obtains refined garlic oil.Yield 80%, outward appearance are little yellow clear liquid, and refractive index is 1.5790, garlicin content 38.01%.

To the above-mentioned refined garlic oil stability influence factorial experiments that makes:

Get embodiment 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5, the refined garlic oil that embodiment 6 makes, (4500Lx ± 500Lx) placed 10 days under the condition is respectively sampling in the 5th, 10 day 40 ℃, 60 ℃ and illumination respectively, measure garlicin content, and compare with 0 day result.With embodiment 1 is example, the results are shown in Figure 1, and the y axle is represented the garlicin percentage composition among Fig. 1, and the x axle is represented the time, and unit is day, and wherein trilateral, rhombus, square are represented result curve under 40 ℃, illumination, 60 ℃ of conditions respectively.The result shows that Oleum Bulbus Allii of the present invention is all responsive to light, heat, and temperature is high more, and stability is poor more.

To the stability test under the different storage requirements of the above-mentioned refined garlic oil that makes:

Get the refined garlic oil that embodiment 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5, embodiment 6 make, deposit respectively at airtight shading under room temperature, refrigeration, the freezing conditions, respectively at sampling in 0,0.5,1,3,6,9,12 month, measure garlicin content, with embodiment 1 is example, the results are shown in Figure 2, the y axle is represented the garlicin percentage composition among Fig. 2, the x axle is represented the time, unit is minute, and wherein trilateral, rhombus, square are represented result curve under freezing, room temperature, the refrigerated condition respectively.The result shows that Oleum Bulbus Allii of the present invention was under freezing conditions stored one month, and content tends towards stability, and all descends and store content under room temperature lucifuge and the refrigerated condition.

To the above-mentioned Oleum Bulbus Allii GC determining fingerprint pattern that makes:

Chromatographic condition: 6890N GC (U.S. Agilent company), chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m), 250 ℃ of injector temperatures, 60～220 ℃ of column temperatures, temperature programming, temperature rise rate: 8 ℃/min, N ₂Do carrier gas, flow velocity 1.0mlmin ^-1, fid detector, 300 ℃ of sensing chamber's temperature, tetrahydrofuran (THF) is interior mark.

Under above-mentioned chromatographic condition, garlicin is at 0.005～2.4mgml ^-1In the scope, linear relationship good (r=0.9998), average recovery rate 102.52% (RSD=0.41%).RSD＜2% in the daytime in a few days, repeated RSD＜2%, the sample solution room temperature is placed 48h and is had good stability.As shown in Figure 3,15 at the total peak of Oleum Bulbus Allii finger printing of the present invention, retention time (min) is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00.

The precision of finger printing, stability, repeated up to specification.

Embodiment 7:

The emulsion of gained refined garlic oil preparation of the present invention, its composition is the refined garlic oil 3.0g of effective dose, soya-bean oil 3.0g, phosphatidase 10 .8g, alpha-tocopherol 0.53g, glycerine 1.6g, oleic acid 0.13g, sodium hydroxide is an amount of, and all the other are water for injection, altogether 100ml.

The manufacture method of emulsion (aseptic method):

A, prepare oil phase in preparing tank: in preparing tank, soya-bean oil, oleic acid, alpha-tocopherol are heated to 60 ℃, the violent stirring mixing adds medicine, the violent stirring mixing;

B, prepare water in preparing tank: water and glycerine were stirred 5 minutes down at 60 ℃, add phosphatide, high-speed stirring makes its complete miscibility;

C, in the time of 60 ℃, oil phase is added water, the violent stirring mixing forms colostrum;

D, use an amount of sodium hydroxide solution, regulate pH value to 7.5;

E, cross the homogeneous instrument by aseptic method, regulate homogenize pressure to 70Mpa, with solution homogenize repeatedly, obtain uniform emulsion, cross millipore filtration, encapsulation promptly gets this product.The median size of emulsion is 180nm, does not contain the breast grain greater than 5 μ m.Gained emulsion can intravenous injection also can be oral.

Embodiment 8:

The emulsion of gained refined garlic oil preparation of the present invention, composition is the Oleum Bulbus Allii 3g of effective dose, Thistle oil 5g, polyoxyethylene glycol 12-hydroxy stearic acid ester (HS15) 2g, alpha-tocopherol 1.0g, N.F,USP MANNITOL 2.0g, sodium phosphate is an amount of, and all the other are water for injection, altogether 100ml.

The manufacture method of emulsion:

A, prepare oil phase in preparing tank: in preparing tank, Thistle oil, alpha-tocopherol are heated to 70 ℃, the violent stirring mixing adds medicine, the violent stirring mixing;

B, prepare water in preparing tank: water, N.F,USP MANNITOL are stirred 5min down at 60 ℃, add HS15, poloxamer 188, high-speed stirring makes its complete miscibility;

C, by aseptic method in the time of 70 ℃, oil phase is added water, the violent stirring mixing forms colostrum;

D, use an amount of sodium radio-phosphate,P-32 solution, regulate pH value to 7

E, cross the microjet instrument, the first step is regulated homogenize pressure to 40Mpa, and the second step re-adjustment with solution homogenize repeatedly, obtains uniform emulsion to 100Mpa, crosses millipore filtration, fills nitrogen, encapsulation, and sterilization, emulsion gets product.The median size of emulsion is 183nm, does not contain the breast grain greater than 5 μ m.

Embodiment 9:

The emulsion of gained refined garlic oil preparation of the present invention, its composition is the Oleum Bulbus Allii 3.3g of effective dose, hot certain herbaceous plants with big flowers acid glyceride and soybean oil (1:1) 5g, phosphatidase 12 .0g, poloxamer 188 0.5g, stearic acid 1.0g, Ascorbyl Palmitate 1.2g, glucose 1.6g, sodium-acetate is an amount of, all the other are water for injection, altogether 100ml.

The manufacture method of emulsion:

A, prepare oil phase in preparing tank: in preparing tank, hot certain herbaceous plants with big flowers acid glyceride and soybean oil mixed solution, stearic acid, Ascorbyl Palmitate are heated to 80 ℃, the violent stirring mixing adds medicine, the violent stirring mixing;

B, prepare water in preparing tank: water and glucose are stirred 5min down at 80 ℃, add phosphatide and poloxamer 188, high-speed stirring makes its complete miscibility;

C, in the time of 80 ℃, oil phase is added water, the violent stirring mixing forms colostrum;

D, use an amount of sodium acetate soln, regulate pH value to 6;

E, cross the homogeneous instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration solution homogenize repeatedly to 80Mpa, encapsulation, sterilization promptly gets this product.The median size of emulsion is 170nm, does not contain the breast grain greater than 5 μ m.Gained emulsion can intravenous injection also can be oral.

Embodiment 10:

The emulsion of gained refined garlic oil preparation of the present invention, its composition is the refined garlic oil 3.3g of effective dose, seal oil 10g, phosphatidase 11 .5g, lauric acid 0.05g, disodium ethylene diamine tetraacetate 0.2g, alpha-tocopherol 0.5g, Xylitol 1.6g, Trisodium Citrate is an amount of, and all the other are water for injection, altogether 100ml.

The preparation method of emulsion is as follows:

A, prepare oil phase in preparing tank: in preparing tank, seal oil, lauric acid, alpha-tocopherol are heated to 50 ℃, the violent stirring mixing adds Oleum Bulbus Allii, the violent stirring mixing;

B, prepare water in preparing tank: water, disodium ethylene diamine tetraacetate, Xylitol were stirred 5 minutes down at 50 ℃, add phosphatide, high-speed stirring makes its complete miscibility;

C, in the time of 50 ℃, oil phase is added water, the violent stirring mixing forms colostrum;

D, use an amount of sodium citrate solution, regulate pH value to 8;

E, cross the homogeneous instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration solution homogenize repeatedly to 90Mpa, encapsulation, sterilization, emulsion gets product.The emulsion median size is 166nm, does not contain the breast grain greater than 5 μ m, gained emulsion can intravenous injection also can be oral.

Embodiment 11:

The emulsion of gained refined garlic oil preparation of the present invention, composition is the Oleum Bulbus Allii 3.3g of effective dose, glyceryl linoleate and soya-bean oil (1:1) 5.0g, 2.5g, glycerine 1.0g, α-Vitamin E-acetate 0.5g, hydrochloric acid is an amount of, and all the other are water for injection, altogether 100ml.

The manufacture method of emulsion:

A, prepare oil phase by aseptic method in preparing tank: in preparing tank, glyceryl linoleate and soya-bean oil mixed solution, α-Vitamin E-acetate are heated to 90 ℃, the violent stirring mixing adds medicine, the violent stirring mixing;

B, prepare water by aseptic method in preparing tank: water and glycerine are stirred 5min down at 90 ℃, add the alpha-tocopherol polyethanediol succinate, high-speed stirring makes its complete miscibility;

C, by aseptic method in the time of 90 ℃, oil phase is added water, the violent stirring mixing forms colostrum.

D, use appropriate hydrochloric acid solution, regulate pH value to 6;

E, cross the homogeneous instrument by aseptic method, regulate homogenize pressure to 90Mpa, with solution homogenize repeatedly, obtain uniform emulsion, cross millipore filtration, fill nitrogen, encapsulation promptly gets this product.The emulsion median size is 150nm, does not contain the breast grain greater than 5 μ m.Gained emulsion can intravenous injection also can be oral.Above-mentioned each parameter of the emulsion that makes is as follows:

Outward appearance: outward appearance is the milky white emulsion body, no oil droplet, not layering, good fluidity;

PH value: 7.0～7.5;

Particle diameter: median size is 140～180nm;

Garlicin content: GC conditions chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m); Carrier gas: N ₂, flow velocity is 1.0mlmin-1; Injector temperature: 250 ℃; 60～220 ℃ of column temperatures, temperature programming, temperature rise rate is 8 ℃/min; Detector: FID, sensing chamber's temperature: 300 ℃, tetrahydrofuran (THF) is interior mark.Under this chromatographic condition, garlicin separates fully, and peak shape is good, in 0.005～2.4mgml-1 scope, and linear relationship good (r=0.9998), average recovery rate 102.52% (RSD=0.41%).RSD＜2% in the daytime in a few days, repeated RSD＜2%, the sample solution room temperature is placed 48h and is had good stability.The garlicin retention time is 11.7min, and pharmaceutical adjunct is noiseless to measuring.Get embodiment 7, embodiment 8, embodiment 9, embodiment 10, embodiment 11, measure garlicin content, the results are shown in Table 1 by this law.

Table 1 samples contg measurement result (n=3)

The Oleum Bulbus Allii intravenous injection emulsion GC determining fingerprint pattern that the foregoing description makes:

GC conditions: chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m); Carrier gas: N2, flow velocity are 1.0mlmin-1; Injector temperature: 250 ℃; 60～220 ℃ of column temperatures, temperature programming, 8 ℃/min of temperature rise rate; Detector: FID, sensing chamber's temperature: 300 ℃, tetrahydrofuran (THF) is interior mark.With this understanding, the precision of determining fingerprint pattern, stability, repeated up to specification.Get 10 batches of emulsions of the present invention, measure its finger printing, the results are shown in Figure 4.

Through traditional Chinese medicine fingerprint similarity evaluation system handles, set up Oleum Bulbus Allii intravenous injection emulsion GC finger printing common pattern, as shown in Figure 5, i.e. standard finger-print, wherein S represents interior mark; 11 expression garlicins.In standard finger-print, determine 17 of total fingerprint peakses, retention time (min) is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00,20.0～21.0,23.0～25.0.

Wherein 3,5,6,7,8,11, No. 15 peaks have constituted the principal character of fingerprint, though other total peak is less relatively to the finger printing contribution, at the equal stable existence of different sample rooms, have constituted the accidental quality of fingerprint.

Between the 10 batch sample finger printings that the present invention made according to the included angle cosine method are criticized and with the similarity evaluation of standard finger-print, the result shows, the relation conefficient of finger printing is between 0.9593～0.9999, higher and the non-total peak area of degree of agreement accounts for total peak area below 5% each other, and is up to specification.

The Oleum Bulbus Allii intravenous injection emulsion quality evalution that the present invention is made:

(1), the comparison of finger printing before and after the sterilization of Oleum Bulbus Allii intravenous injection emulsion: the finger printing degree of agreement is higher before and after the sterilization, and relation conefficient sees Table 2, shows that the overall diagram looks do not change

Sample finger printing similarity evaluation (n=5) before and after table 2 sterilization

(2), the comparison of the different shelf lives finger printings of Oleum Bulbus Allii intravenous injection emulsion: get Oleum Bulbus Allii intravenous injection emulsion of the present invention, the room temperature lucifuge stores, and measures finger printing every first quarter moon, and carries out similarity evaluation, the results are shown in Table 3.Relation conefficient is 0.9997～0.9999, show the Oleum Bulbus Allii intravenous injection emulsion store 2 months after its oeverall quality considerable change does not take place.

The different shelf lives sample finger printing similarity evaluations (n=3) of table 3

(3), the different condition of storage finger printings of Oleum Bulbus Allii intravenous injection emulsion relatively: get Oleum Bulbus Allii intravenous injection emulsion of the present invention, store 2 months respectively under room temperature lucifuge and refrigerated condition, and carry out determining fingerprint pattern and similarity evaluation, the results are shown in Table 4.Relation conefficient shows that 0.9992～0.9998 considerable change does not take place Oleum Bulbus Allii intravenous injection emulsion its finger printing overall diagram looks under two kinds of conditions of storage.

The different condition of storage sample of table 4 finger printing similarity evaluation (n=5)

As follows to the Oleum Bulbus Allii intravenous injection emulsion dilution test that the present invention makes:

Get Oleum Bulbus Allii intravenous injection emulsion of the present invention respectively with sterilized water for injection, 5% glucose injection, 0.9% sodium chloride injection, Intralipid fat emulsion injection (C ₁₄～ ₂₄) be diluted to 250ml, press the variation that table 5 is investigated Oleum Bulbus Allii intravenous injection emulsion dilution front and back, for clinical application provides foundation.

The result shows that various thinners did not have the significance influence to every inspection index of emulsion in 10 hours.After emulsion is diluted by water, glucose, sodium-chlor, still can smell the distinctive stink of Oleum Bulbus Allii, but can be covered its stink fully after the high-fat emulsion dilution, and high-fat emulsion and garlic oil emulsion are formed close, do not regard sb. as an outsider after both mix and see the change of change and particle diameter, illustrate that garlic oil emulsion can be dispersed in the high-fat emulsion, does not change its stability.Bigger with the particle diameter after the high-fat emulsion dilution, be since the particle diameter of high-fat emulsion own big due to.

With after the sodium-chlor dilution, as seen particle diameter increases a little, though the milk sap that also is white in color, color is darker, and As time goes on the trend of reinforcement arranged, and once be placed on refrigerator cold-storage and spent the night, muddy stain, but also do not have the phenomenon of breakdown of emulsion.So the diabetic subject can dilute administration with sodium-chlor, but for guaranteeing that safety should finish in 12 hours innerlich anwendens.

Table 5. Oleum Bulbus Allii intravenous injection emulsion dilution test result

+: white milk sap, evenly, do not see oil droplet, nothing is separated

As follows to the Oleum Bulbus Allii intravenous injection emulsion stability test that makes:

(1), influence factor test

With reference to pharmacopeia (two ones) new governing principle and relevant document that preparation stability is investigated in 2005, carried out the investigation of high temperature, illumination, freezing factor.Experiment shows that Oleum Bulbus Allii intravenous injection emulsion of the present invention was placed 10 days under various conditions, and the outward appearance emulsion that still is white in color is not seen layering, gone out wet goods, and good fluidity takes place obviously to change; Under illumination and the 40 ℃ of conditions, content descends, and particle diameter does not have considerable change, and exposure experiments to light has the greatest impact to pH, may be because illumination can promote due to the oxygenolysis of phosphatide; Under the freezing conditions, the content no change, and particle diameter enlarges markedly.Therefore, this product is all responsive to heat, light, should lucifuge store under optimal temperature, must not be freezing.

Investigated the stability of Oleum Bulbus Allii injection liquid under illumination, high temperature, freezing conditions according to above test method contrast, the result shows, Oleum Bulbus Allii injection liquid pH value under illumination, hot conditions descends slightly, but content descends obviously, compare with the Oleum Bulbus Allii intravenous injection emulsion, under the equal conditions, the rate of descent of garlicin in injection liquid almost is the twice in emulsion, illustrate that emulsion wraps up Oleum Bulbus Allii into interior phase, really can improve its stability.

(2), the freezing test with commercially available VITAMIN high-fat emulsion compares:

The influence factor test-results shows under the freezing conditions, and the content of dispersion of emulsion does not change, but particle diameter obviously increases, and commercially available Vitalipid N Adult liposoluble vitamin injection liquid is done freezing accelerated test equally, and the two particle diameter is compared.

In fact, the freezing front and back of Oleum Bulbus Allii intravenous injection emulsion that Nicomp-380 laser particle diameter instrument is measured, all recommend to use Gaussian distribution, and vitamin injection is after freezing 10 days, particle diameter generation considerable change, can not represent, but be more convenient that the two Nikon is distributed to be compared with Gaussian distribution.Compare with self, the particle diameter of the two all increases, but the particle diameter of freezing back vitamin injection is across 100～500nm, and the particle diameter of Oleum Bulbus Allii intravenous injection emulsion illustrates that at 100～250nm its stability is than the commercial like product excellence.

(3), accelerated test

From the influence factor test-results as can be seen, the Oleum Bulbus Allii intravenous injection emulsion is placed under 40 ℃ of conditions, garlicin content obviously descends, and to the medicine stability test governing principle, accelerated test is carried out under 25 ℃ ± 2 ℃ according to pharmacopeia, and the time is 6 months.The Oleum Bulbus Allii intravenous injection emulsion was placed six months 25 ℃ ± 2 ℃ following lucifuges, in the 1st, 2,3, the sampling in June, check by requirement under the investigation project, compare with 0 day result, the result shows, after 6 months, the basic no change of every inspection index has good stability the Oleum Bulbus Allii intravenous injection emulsion in accelerated test.

(4), the test that keeps sample for a long time:

From the influence factor test-results as can be seen, the Oleum Bulbus Allii intravenous injection emulsion is placed under 40 ℃ of conditions, garlicin content obviously descends, and to the medicine stability test governing principle, test of long duration is carried out under 6 ℃ ± 2 ℃ according to pharmacopeia.Oleum Bulbus Allii intravenous injection emulsion lucifuge under 6 ℃ ± 2 ℃ conditions was placed 6 months, in the 3rd, the sampling in June, check by requirement under the investigation project, comparing with 0 day result. the result shows, the Oleum Bulbus Allii intravenous injection emulsion is keeping sample test after 6 months for a long time, the basic no change of every inspection index has good stability.

Embodiment 12:

Refined garlic oil solid lipid nanoparticle of the present invention, the purified Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, glyceryl monostearate 2.0g, Taurocholic acid sodium salt 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

The preparation method:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are stirred down 30min at 75 ℃, be uniformly dispersed and make water;

B, in preparing tank, under uniform temp, the solid-state lipid of recipe quantity is melted mutually, again the recipe quantity Oleum Bulbus Allii is added, stir, form the lipid phase;

C, the disposable quick adding lipid phase of water, high-speed stirring 3min makes colostrum under the rotating speed of 6000rpm;

D, cross the homogeneous instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 106nm, does not contain the breast grain greater than 5 μ m, and the gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 13:

Refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, glyceryl monostearate 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

The preparation method:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are evenly made water 60 ℃ of following dispersed with stirring;

C, the disposable quick adding lipid phase of water, high-speed stirring (6000rpm) 3min makes colostrum;

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 136nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 14:

Refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, glyceryl monostearate 2.0g, Taurocholic acid sodium salt 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are evenly made water 80 ℃ of following dispersed with stirring;

C, the disposable quick adding lipid phase of water, stirred during high speed 6000rpm 3 minutes, make colostrum;

D, with the colostrum ultrasonic 5min that under power 400W, pops one's head in, keep 70 ℃, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product.Median size is 146nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 15:

Above-mentioned solid lipid nanoparticle preparation method is as follows:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are evenly made water 75 ℃ of following dispersed with stirring;

With the colostrum ultrasonic 5min that under power 400W, pops one's head in, keep 70 ℃, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product.Median size is 146nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 16:

The preparation of refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, Comprito1888 ATO 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are evenly made water 90 ℃ of following dispersed with stirring;

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 276nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 17:

Refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4.0g that this nanoparticle composition is an effective dose, Comprito1888 ATO 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

A, in preparing tank, recipe quantity emulsifying agent, assistant for emulsifying agent and water are evenly made water 85 ℃ of following dispersed with stirring;

C, the disposable quick adding lipid phase of water, high-speed stirring (6000rpm) 3min makes colostrum; D, with the colostrum ultrasonic 5min that under power 400W, pops one's head in, keep 70 ℃, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product.Median size is 321nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 18:

The preparation of refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, tripalmitin 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

C, the disposable quick adding lipid phase of water, high-speed stirring (12000rpm) 3min makes colostrum;

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 244nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 19:

Refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, tripalmitin 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

C, the disposable quick adding lipid phase of water, high-speed stirring (28000rpm) 3min makes colostrum;

D, with the colostrum ultrasonic 5min that under power 400W, pops one's head in, keep 70 ℃, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product.Median size is 351nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 20:

Refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, glyceryl monostearate 2.0g, polyoxyethylene glycol 12-hydroxy stearic acid ester 0.2g, poloxamer 1880.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

C, the disposable quick adding lipid phase of water, high-speed stirring (10000rpm) 3min makes colostrum;

D, with the colostrum ultrasonic 5min that under power 400W, pops one's head in, keep 70 ℃, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product.Median size is 321nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 21:

The preparation of refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 6g that this nanoparticle composition is an effective dose, glyceryl monostearate 4g, Yelkin TTS 0.6g, poloxamer 188 3.0g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 176nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 22:

The preparation of refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 2g that this nanoparticle composition is an effective dose, myristin 2.0g, Yelkin TTS 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 196nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

Embodiment 23:

The preparation of refined garlic oil solid lipid nanoparticle of the present invention, the Oleum Bulbus Allii 4g that this nanoparticle composition is an effective dose, glyceryl monostearate 2.0g, Sodium glycocholate 0.2g, poloxamer 188 0.6g, all the other are water for injection, altogether 100ml.

Above-mentioned solid lipid nanoparticle preparation method is as follows:

D, cross homogeneous instrument or microjet instrument, regulate homogenize pressure,, obtain uniform emulsion, cross millipore filtration, reduce to room temperature, promptly get this product solution homogenize 10 times repeatedly to 100MPa.Median size is 126nm, does not contain the breast grain greater than 5 μ m.The gained solid lipid nanoparticle can be injected also can be oral.

As follows to the Oleum Bulbus Allii intravenous injection solid lipid nanoparticle GC finger printing that the present invention makes:

GC conditions, chromatographic column: HP-5 (30m * 320 μ m * 0.25 μ m); Carrier gas: N ₂, flow velocity is 1.0mlmin-1; Injector temperature: 250 ℃; 60～220 ℃ of column temperatures, temperature programming, 8 ℃/min of temperature rise rate; Detector: FID, sensing chamber's temperature: 300 ℃, tetrahydrofuran (THF) is interior mark.With this understanding, the precision of determining fingerprint pattern, stability, repeated up to specification.Get the present invention and make 10 batches in sample, measure its finger printing, the results are shown in Figure 6.

Through traditional Chinese medicine fingerprint similarity evaluation system handles, set up GC finger printing common pattern, as shown in Figure 7, i.e. standard finger-print, wherein S represents interior mark; 11 expression garlicins.In standard finger-print, determine 17 of total fingerprint peakses, retention time (min) is: 4.50～4.68,4.70～4.80,5.40～5.58,6.00～6.20,7.90～8.10,8.20～8.34,8.35～8.45,9.00～9.20,10.00～10.29,10.30～10.45,11.70～11.90,11.91～12.10,12.12～12.30,12.80～13.10,14.90～15.20 (interior mark peaks), 15.00～16.00,20.0～21.0,23.0～25.0

The external antifungic action test of the medicine that the present invention is made is as follows:

1., bacterial classification

Candida albicans 2 strains (Liaoning Province medicine inspecting institute provides); Oidium tropicale 1 strain, Cryptococcus neoformans 1 strain, aspergillus fumigatus 1 strain (Academy of Medical Sciences, Nanjing tetter hospital).

2., medicine

Fluconazole injection (3059102, Pfizer Inc.); Oleum Bulbus Allii intravenous injection emulsion (031118, self-control); 5% glucose injection, sodium chloride injection (Pharmacy of General Hospital, Shenyang Military Command).

3., husky formula nutrient agar method is measured MIC

With coubling dilution Oleum Bulbus Allii intravenous injection emulsion, fluconazole injection are made into the medicine gradient of 6 concentration of 100 μ g/mL to 0.8 μ g/mL respectively with husky formula nutrient agar, move into plate, inoculate the bacteria suspension of 105cfu/mL respectively.Other does the culture medium inoculated bacteria suspension that does not contain medicine, as blank; The culture medium inoculated bacteria suspension that contains blank emulsion is blank auxiliary material contrast.Sample and 28 ℃ of cultivations are placed different time and are observed the fungal growth situation, with continuously doubly the highly diluted multiple of amount dilution white precipitate or muddy as MIC does not appear, the results are shown in Table 6.

Table 6 Oleum Bulbus Allii intravenous injection emulsion and fluconazole injection MICs (μ g/mL) test-results

The result shows, Oleum Bulbus Allii intravenous injection emulsion of the present invention has different restraining effect to Candida albicans, Oidium tropicale, Cryptococcus neoformans and aspergillus fumigatus, inhibitory or killing effect to aspergillus fumigatus is the most remarkable, experimental observation one week, have only anti-aspergillus fumigatus first, second concentration, asepsis growth still illustrate that garlic oils preparation can kill aspergillus fumigatus, and fluconazole is invalid to it.The negative control experiment shows blank substratum and adds blank newborn substratum does not all have antibiotic effect.

The safety testing of the medicine that the present invention is made:

Investigate security with the submicronized emulsion of Oleum Bulbus Allii preparation of the present invention, as: acute toxicity, hemolytic, blood vessel irritation, pyrogen and sensitization test etc.

1., acute toxicity test

Choose 40 of healthy mices, body weight 18～22g is divided into 4 groups at random, male and female half and half, 10 every group, the toxicity of investigation Oleum Bulbus Allii intravenous injection emulsion.The LD of Oleum Bulbus Allii intravenous injection emulsion as a result ₅₀Be 184mg/kg, LD ₅₀95% fiducial interval be: 163.8～207.0mg/kg

2., hemolytic experiment

The Oleum Bulbus Allii pungency is stronger, for a long time at a certain position quiet easily cause phlebitis, high density can cause erythrolysis.The unsaturated link(age) that is had in the phospholipid molecule easily combines the structure of generation two ethylene linkages with oxygen and oxidation causes hemolytic toxicity.Therefore this experimental observation the Oleum Bulbus Allii intravenous injection emulsion man rabbit erythrocyte is had or not haemolysis and cohesion.

Get for the examination rabbit, from arteria auricularis blood sampling 9mL, glass stick stirs gently to remove and defibrinates, make into defibrinated blood, add the sodium chloride injection of 10 times of amounts, shake up, centrifugal, remove supernatant liquor, sedimentary red corpuscle does not show red with sodium chloride injection such as method washing 3 times to supernatant liquor again, and the gained red corpuscle is made into 2% suspension with sodium chloride injection, uses for experiment.

Get 7 in test tube, add 2% red blood cell suspension and sodium chloride injection successively, behind the mixing, in 37 ℃ of water-baths, place 10min, the Oleum Bulbus Allii intravenous injection emulsion is added this dilute liquid medicine 0.5mL, 0.4mL, 0.3mL, 0.2mL, 0.1mL respectively in 1～No. 5 pipe, No. 6 pipe adds sodium chloride injection 2.5mL, No. 7 pipe adds sterilized water 2.5mL, makes respectively to manage final volume and be 5mL, gently behind the mixing, put in 37 ℃ of water-baths, observe the haemolysis situation respectively at different time.Be transparent redness as solution, promptly represent haemolysis, as the red-brown flocks is arranged in the solution, expression has erythroagglutination.If any the red blood cell condensation phenomenon, should shake test tube or examine under a microscope, judgement is true cohesion or pseudo agglutination.

Observe at the appointed time, find 1～No. 6 pipe supernatant liquid water white transparency of Oleum Bulbus Allii intravenous injection emulsion, red corpuscle is sunken to the pipe end gradually, No. 7 the pipe supernatant liquid takes on a red color transparent, above-mentioned each Guan Junwu reddish-brown flocks, also not show cell condensation effect shows that the Oleum Bulbus Allii intravenous injection emulsion does not have hemolytic action.

3., blood vessel irritation test

With 5% glucose injection dilution Oleum Bulbus Allii intravenous injection emulsion, administration concentration is equivalent to the maximum dosage-feeding of clinical fungi infestation.Get 4 of body weight 2.0～2.5kg healthy rabbits, the male and female dual-purpose is divided into two groups immediately.Rabbit is placed in the fixer.Rabbit left side auricular vein instillation Oleum Bulbus Allii intravenous injection emulsion 20mL/kg, auris dextra edge intravenous drip 5% glucose injection 20mL/kg, drip velocity is 20 (1mL)/min, every morning instils once, for three days on end, observe the auricular vein reaction of a rabbit left side, and 72h, 14 days put to death rabbit after the last administration, get the injection site or, carry out the tissue slice inspection away from injection site 5cm place auricular vein.

Observe after reaching administration during each the instillation, visual inspection sees that left auricular vein inserting needle place does not have local excitation reactions such as blood vessel is rubescent, swollen, heat, histopathological examination control group and medication group skin, subcutis form are normal, the auricular vein vascular endothelial cell is arranged normal, does not see that level increases, the change of arrangement disorder, no mural thrombus in the official jargon, no cell infiltration, tube wall do not have and thicken, and do not see morphological changes such as obvious sex change, necrosis and inflammation around the tube wall.The result shows that the Oleum Bulbus Allii intravenous injection emulsion does not have obvious blood vessel irritation.The pathological section photo is seen Fig. 8, Fig. 9, Figure 10.

4., pyrogen test

According to " Chinese pharmacopoeia version pyrogen test in 2005 is observed rabbit fervescence situation, judges whether Oleum Bulbus Allii intravenous injection emulsion pyrogen limit of the present invention is qualified.

Get 3 of qualified rabbit, 15min after measuring its normal body temperature slowly injects the Oleum Bulbus Allii intravenous injection emulsion diluent 10mLkg that is preheated to 37 ℃ from auricular vein ^-1(suitable Oleum Bulbus Allii 1.5mgkg ^-1), survey rabbit anus temperature 1 time every 30min, survey altogether 6 times.The result shows that every rabbit body temperature raises and all is lower than 0.6 ℃, and 3 rabbit body temperature rising summations are lower than 1.4 ℃, illustrates that Oleum Bulbus Allii intravenous injection emulsion pyrogen limit of the present invention meets the requirements, and can use safely.

5., sensitization test

Animal: male guinea pig, 250～350g; Medicine: Oleum Bulbus Allii intravenous injection emulsion, bovine serum albumin; Method: 24 of the healthy male guinea pigs of extracting waste, body weight 250～350g, be divided into 4 groups at random, 6 every group, be respectively Oleum Bulbus Allii intravenous injection emulsion low dose group of the present invention, Oleum Bulbus Allii intravenous injection emulsion high dose group of the present invention, positive group (bovine serum albumin), negative group (blank breast).By aseptic technique, only distinguish abdominal injection trial-product 0.5ml/, totally 3 times for every group next day of continuously.During the sensitization, observe the symptom of every animal every day.For the first time, last sensitization and excite the body weight of measuring every group every animal the same day.Injected the back the 10th day in last then, each group is got 3 cavys, and intravenous injection trial-product 1ml/ only excites respectively, observe every the animal in injection back in detail by systemic anaphylaxis reaction grading standard immediately after the administration, the appearance of symptom and extinction time are observed 3h, judge cavy anaphylaxis progression.In injecting the back the 21st day first, each group is got other 3 cavys and is excited with method, observes again.The anaphylaxis of medicine group cavy as a result is negative, and shows that Oleum Bulbus Allii intravenous injection submicron emulsion hypersensitive test is qualified,

Particle size measurement: use PSS.NICOMPTM380 among the present invention as the instrument of measuring particle diameter.

In sum, the present invention has set up a kind of process for purification of Oleum Bulbus Allii, and the refined garlic oil that is obtained becomes faint yellow by original pale brown look; Measure garlicin content greater than 37.0% with the GC method of being set up, 15 of total fingerprint peakses; With the intravenous injection emulsion of Oleum Bulbus Allii of the present invention preparation and fixedly lipid nanoparticle have external anti-deep fungal effect, and security improves, for clinical application provides reliable foundation.

Claims

1, a kind of process for purification of Oleum Bulbus Allii is characterized in that: this method is undertaken by following step:

A, get each raw material: Oleum Bulbus Allii 1: sorbent material I 0.25～1.25: sorbent material II 1～5 by following weight ratio;

B, with above-mentioned Oleum Bulbus Allii and sorbent material II mixing;

D, get solvent decompression wash-out, collect effluent liquid;

E, reclaim under reduced pressure solvent promptly get refined garlic oil;

2, a kind of with the prepared solid lipid nanoparticle agent of claim 1 described method purified Oleum Bulbus Allii, it is characterized in that: mainly contain refined garlic oil and pharmaceutical excipient, pharmaceutical excipient is mainly solid lipid, emulsifying agent, assistant for emulsifying agent composition, and the mass ratio of Oleum Bulbus Allii and pharmaceutical excipient is: 5:1～1:50; Each composition consumption of its pharmaceutical excipient is: solid lipid 1%～10% (w/v), emulsifying agent 1%～10% (w/v), assistant for emulsifying agent 0.05～5% (w/v); Other composition is a water.

3, refined garlic oil solid lipid nanoparticle according to claim 2 agent is characterized in that: described solid lipid is a kind of in glyceride, lipid acid, steroid or the wax class; Glyceride is selected from a kind of in myristin, tripalmitin, the Tristearoylglycerol; Lipid acid is selected from a kind of in stearic acid, palmitinic acid, capric acid, the behenic acid; Steroid is selected from cholesterol; The wax class is selected from a kind of in cetyl palmitate, microcrystalline wax, the whale vinegar wax; Described emulsifying agent is selected from a kind of in phospholipid, HS15, vitamin E polyethylene glycol succinic acid ester, poloxamer 188, cholic acid salt, Tweens, the short-chain alcohols; The phospholipid consumption is 0.5～5% (w/v), HS15, consumption is 0.1～5% (w/v), vitamin E polyethylene glycol succinic acid ester, consumption is 0.1～5% (w/v), poloxamer 188, consumption is 0.05～5% (w/v), cholic acid salt, consumption is 0.1%～5% (w/v), Tweens, consumption is 0.1%～5% (w/v), short-chain alcohols, and consumption is 0.05～5% (w/v); Described assistant for emulsifying agent be selected from oleic acid, sad, certain herbaceous plants with big flowers is sour, a kind of in the lauric acid, palmitinic acid, linolic acid, linolenic acid, stearic acid, docosahexenoic acid, cholic acid.

4, the preparation technology of a kind of refined garlic oil solid lipid nanoparticle as claimed in claim 2 agent, it is characterized in that: this technology is undertaken by following step: