CN1883556A - A nano preparation of 'Kang'ai' injection and method for preparing same - Google Patents
A nano preparation of 'Kang'ai' injection and method for preparing same Download PDFInfo
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Abstract
Disclosed is a nano preparation for injection and its preparing process, wherein the preparation is prepared from the effective positions of Kang'ai and right amount of auxiliary materials, and can be made into the dosage forms of liquid injection, powder injections and transfusions.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicine pharmacy, be specifically related to a kind of nano preparation of ' Kang ' ai ' injection and preparation method thereof.
Background technology
Nanometer Chinese medicine mainly is meant what the utilization nanotechnology was made, and particle diameter is less than middle pharmaceutically active ingredient, effective site, former medicine and the compound preparation of 100 nanometers.Living organism is the process of a complexity to absorption, the metabolism of medicine, and it is also closely related with the physical state of said preparation that the pharmacodynamics effect that Chinese medicine preparation produces can not only be belonged to the distinctive chemical composition of medicine.Therefore, the physical state of change pharmaceutical preparation is a kind of effective ways of new drug development.Aspect the change physical state, the unit size that changes medicine is highly effective.Owing to quantum size effect and skin effect, nanoparticle presents novel physical chemistry and biological characteristics when particle size enters nanometer scale.Applying nano technology that Here it is may make pharmaceutically active and bioavailability improve and even produces new characteristic according to the place in Chinese medicine research.
Compare with traditional Chinese medicine, nanometer Chinese medicine has following characteristics: 1. improved the availability of medicine, reduced dosage.2. strengthen the targeting of medicine.3. have slow-release function, the Chinese medicine nanoparticle is carried out certain finishing after, may make Chinese medicine have slow releasing function.4. present new drug effect, widen former medicine indication, when Chinese medicine is machined to nano-scale,, thereby can make Chinese medicine present the function that makes new advances because effects such as its quantum size cause the change of its physics, chemical characteristic.5. enrich the dosage form selection of Chinese medicine, promote traditional route of administration.
Problem below nanotechnology at present exists in Chinese medicine research.First, the preparation difficulty of nanometer Chinese medicine: though China has prepared some nano level Chinese medicines, because herbal species is various, complicated component, different compositions is owing to the difference of its site of action, mechanism of action and effect emergency requires its size, carrier components and dosage all possible different.Nanometer Chinese medicine should have its a cover quality standard simultaneously, makes it produce standardization.The second, the toxic and side effects of nanometer Chinese medicine: medicine is made nanoparticle, and significant variation has all taken place for its physical property and chemical property, and whether these change has all relevant research report of toxicity to human body.But, whether can participate in directly or, there is no about report as the normal chemical reaction of catalyst upset body because the particularity of nano material except penetrable skin, also can enter in the organelle.In addition, nanometer Chinese medicine can also be by the barrier system of body, and whether it can influence the central nervous system, the growth of the generation of sperm and vigor thereof, fetus etc., and these problems all can't be avoided.The 3rd, Chinese medicine is after nanoscale is pulverized, and nano-particle surface is long-pending to become big, because the activity of nano-particle surface makes them be easy to reunite together, this brings very big difficulty for the collection of nanoparticle and the stability of drug effect thereof.The 4th, cost improves: because the restriction of prior art level and equipment causes the cost of nanometer Chinese medicine in preparation will exceed much than the preparation of Chinese medicine.
The research of nanometer Chinese medicine at present mainly concentrates on and utilizes nanotechnology that the clearer and more definite monomer effective ingredient of minority composition is carried out nanometer to handle and make nanometer formulation, or crude drug directly is ground into nanoscale, nanometer formulation to most of Chinese medicine is studied also seldom, mainly be because middle pharmaceutically active ingredient and effective site particularly in the Chinese medicine compound effective site itself be exactly one " black box ", real effective ingredient or the effective site research that plays pharmacological action itself is exactly a difficult problem in the Chinese medicine, and because the Chinese medicine ingredients more complicated, so it is prepared into difficult more that nanometer formulation need overcome, therefore, Chinese medicine nanometer formulation and technology are medical scientific research worker's important subject.
The Chinese medicine injection that the Conair injection is made up of the Radix Astragali, Radix Ginseng, kurarinone has QI invigorating and sets upright, the enhancing human body immunity function.Be used for primary hepatocarcinoma, rectal cancer, malignant lymphoma, gynecological tumor; Low leukocyte counts that a variety of causes causes and minimizing disease.The treatment of chronic hepatitis B.Pharmacological research shows that the Conair injection has following pharmacological action: (1) is kill cancer cell (dwindling lump) directly: (2) can cut off the synthetic of cancerous cell NDA strand, anticancer growth (control and the stable state of an illness); (3) erosion of health invigorating, raising cancerous cell; (4) have pain relieving, emesis, anti-diarrhea effect, and quick leukocyte increasing effect; Clinical data shows: the Conair injection has specially good effect for primary hepatocarcinoma, rectal cancer, malignant lymphoma, gynecological tumor etc.
But the Conair injection is owing to contain kurarinone, in clinical practice, have the slight stimulation except the part use, also can cause dizziness, constipation when dosage is big, feel sick, untoward reaction such as allergy takes place, and kurarinone constituents poor stability, need lucifuge, mutual conversion can take place in matrine and oxymatrine in the long term store process, and these have all influenced the quality control and the clinical practice of Conair injection.
Summary of the invention
For these reasons, research worker of the present invention is through lot of experiments, the Conair effective site that extraction is obtained adds an amount of carrier and adopts process of the present invention to be prepared into nano injection formulation, the inventive method is easy, be easy to big production, the preparation particle diameter for preparing reaches nanoscale, adopt the nanometer formulation drug loading of the inventive method preparation big, good stability, and can significantly improve the targeting of medicine to tumor cell, pharmacological evaluation shows nano preparation of ' Kang ' ai ' injection more remarkable treatment effect of the present invention, and zest is little, and safety is good.
The present invention aims to provide a kind of stable, safety, good effect, has the nano preparation of ' Kang ' ai ' injection of targeting.
The present invention also provides the preparation method of above-mentioned nano preparation of ' Kang ' ai ' injection.
The present invention and Conair injection relatively is characterized in that adopting technology of the present invention that Conair effective site is prepared into the preparation that reaches nanometer particle size, adopt the nanometer formulation of the inventive method preparation to have significant targeting.
The present invention is achieved through the following technical solutions:
One, process recipes
(1) the preparation prescription weight portion consists of: Conair effective site 1.5-3 part, carrier 6-15 part.
(2) preparation of Conair effective site: extract purification according to the method for making under Conair injection [WS-11222 (ZD-1222)-2002] item, the last filtrate decompression of Radix Ginseng and Radix Scutellariae concentrates and vacuum drying, add the kurarinone mixing, obtain Conair of the present invention effective site.
(3) method one, Conair effective site is dissolved in the distilled water, add tween 80, stir, constantly adding in the pharmaceutical carrier under the stirring condition, continue to stir 30 minutes again, filter with 9-15 μ m glass hourglass, filtrate is diluted to 4 times, become the emulsion of scattering phenomenon, reuse 0.45 μ m membrane filtration promptly gets drug suspension.
Method two, pharmaceutical carrier is mixed with dehydrated alcohol, shakes up, clear solution; Get Conair effective site and be dissolved in the distilled water, add Pluronic F68 or tween 80, stir; In the time of 20 ℃, pharmaceutical carrier solution is injected in the aqueous solution of electromagnetic agitation by silica gel tube or fine needle head, form nanocapsule immediately, solution for vacuum is evaporated to about 1/5 of original volume, filter with glass sand hourglass (9-15 μ m), promptly get drug suspension.
(4) preparation of preparation
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, it is an amount of to add PVP, adds to the full amount of water for injection, and stirs evenly, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, it is an amount of to add PVP, adds to the full amount of water for injection, and stirs evenly, and adds sodium chloride or glucose accent etc. and oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add excipient, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and preparation cost invention Conair powder injection formulation.
Pharmaceutical carrier of the present invention is one or more in paracyanogen base alkyl acrylate, polylactic acid, polylactide-co-glycolide, methacrylic acid, chitosan, the gelatin etc.,
Nano injection formulation of the present invention can be nanometer aqueous injection, nano powder injection, nanometer infusion preparation.
Nanometer aqueous injection of the present invention, nanometer infusion solution add a small amount of PVP (polyvinylpyrrolidone), can improve stability of formulation, prevent the nanoparticle gathering.
The excipient of nanometer powder injection formulation of the present invention is one or both in mannitol, glucose, lactose, dextran, the glucosan.
Pharmaceutical carrier of the present invention can also adopt following method preparation:
Method one, get Conair effective site, emulsifying agent, the lipid of recipe quantity, mixing, heating and melting, polysorbate-20 or 60 is added wherein, mix with 70 ℃ distilled water, stir and be prepared into microemulsion, 65 ℃ microemulsion is poured in the syringe that can be incubated and be incubated 15 minutes, under the mechanical agitation condition, in certain hour, the microemulsion of heat is joined in the cold distilled water, promptly get drug suspension.
Method two, get Conair effective site, emulsifying agent, the lipid of recipe quantity, be heated to 80 ± 5 ℃, the aqueous solution that under stirring condition, adds an amount of uniform temp glycerol and poloxamer, make thick breast, under 80 ± 5 ℃ of conditions, carry 40MPa pressure stimulating milk secretion even 5 times, be cooled to room temperature rapidly and form the principal agent suspension with the high pressure dispersing emulsification machine.
Method three, take by weighing emulsifying agent by prescription, add a small amount of Tween 80, in the distilled water of 70 ± 5 ℃ of high-speed stirred condition addings, treat complete fusion as water, take by weighing lipid again and add recipe quantity Conair effective site, heating and melting is as oil phase, oil phase is slowly added aqueous phase, and continue to stir ultra-sonic dispersion after 1 hour: the chamber is steady, frequency 45-50Hz, ultrasonic 5-8 minute, be cooled to room temperature rapidly and form the principal agent suspension.
The used lipid of above method can be: fatty acid glycerine esters (comprise glyceryl tristearate, tripalmitin, myristin, trilaurin, three climb over a wall acid glyceride, Witepsol W35, Witepsol H35, Witepsol H42, glyceryl monostearate) and fatty acid (as stearic acid, Palmic acid) etc.
Used emulsifying agent can be phospholipid (comprising soybean lecithin, Ovum Gallus domesticus Flavus lecithin and phosphatidylcholine etc.), Pluronic F68, poloxamer, polysorbate, cholate, alevaire etc.
The used above preparing carriers technology of the present invention is that we are through lot of experiments and preferred result, we have adopted a lot of nanotechnology technologies and have compared in research process, the technology that has is difficult to be prepared into Nano medication, even and if the technology that has is prepared into Nano medication, do not reach the targeting effect of preparation of the present invention yet.
Two, quality testing
Instrument: H-7000 type transmission electron microscope instrument (Japanese Hitachi company); Zetamaster photon correlation spectrometer (Britain Malvern company); LC-10A high performance liquid chromatograph (day island proper Tianjin); TGL-18G type table model high speed centrifuge.
Detect foundation: with reference to the method under 2005 editions two appendix XIX E of the Pharmacopoeia of the People's Republic of China guideline item.
1, morphologic observation and particle diameter
Nano medication of the present invention is carried out morphologic observation under transmission electron microscope, as seen be spherosome, size is more even, smooth surface, no adhesion.Measured 500 according to the microphotograph of Nano medication, mean diameter is 40.5nm, and maximum particle diameter is 100nm, and minimum grain size is 20.0nm, and particle size distribution meets normal distribution law.
2, envelop rate and drug loading are measured
Content assaying method with reference to kurarinone under the injection item of Conair carries out assay, and with following formula computational envelope rate and drug loading: envelop rate (%)=(dosage-free drug amount)/dosage * 100%; Weight * 100% of drug loading (%)=(dosage-free drug amount)/Nano medication.The result: in the average envelop rate of kurarinone is 92.6%, and drug loading can reach 10%-25%.
3, study on the stability
Nano medication of the present invention is placed in the bottle sealing respectively.In the environment of refrigerator (3-5 ℃), room temperature (20-25 ℃) and 37 ℃ (RH75%), place, in 0,1,2 with observe March such as outward appearance, size, redispersibility of Nano medication etc.The result there is no significant change, and it is good that the Nano medication redispersibility under 3 kinds of conditions keeps, the generation of no polymerism.The results are shown in Table 1.
Table 1 stability experiment result
| The placement condition | Observation index | Time (moon) | |||
| 0 | 1 | 2 | 3 | ||
| 3-5℃ | Mean diameter (nm) | 40.8 | 40.2 | 40.5 | 40.3 |
| 20-25℃ | Mean diameter (nm) | 40.0 | 40.5 | 41.2 | 41.0 |
| 37℃(RH75％) | Mean diameter (nm) | 41.5 | 40.8 | 40.7 | 40.4 |
4, accelerated tests assay
Conair injection (Changbai mountain, Jilin Pharmacy stock Co., Ltd); Nano preparation of ' Kang ' ai ' injection of the present invention (by preparation technology's preparation of the present invention, by Tianzhijiao Medication Development Co., Ltd., Guangdong's prepared in laboratory); Above preparation is put 40 ℃ respectively, placed 6 months under the RH75% condition, measure the content of unsettled relatively composition kurarinone in the preparation respectively, investigate variation (was 100% calculating with 0 month content) the specified content assaying method of content of Conair nanometer formulation content under the acceleration environment with reference to kurarinone under the injection item of Conair.The results are shown in Table 2.
Table 2 preparation stability test kurarinone assay result
| Sample | 0 month | January | February | March | June |
| Nano powder injection Conair, nanometer aqueous injection Conair, injection Conair, Conair nanometer infusion solution | 100％ 100％ 100％ 100％ | 98.0％ 99.7％ 99.8％ 99.7％ | 95.3％ 98.5％ 98.7％ 98.6％ | 92.5％ 97.6％ 97.5％ 97.5％ | 88.7％ 95.8％ 96.2％ 96.1％ |
Result of the test shows that the kurarinone content of Conair injection in the accelerated tests process descends obviously, and adopt the nano preparation of ' Kang ' ai ' injection of the inventive method preparation to change less, illustrate that the preparation method of Conair of the present invention nanometer formulation has significantly improved the quality and the stability of preparation, has important and practical meanings.
Three, pharmacology and targeting experiment
Reagent and animal: Conair injection (Changbai mountain, Jilin Pharmacy stock Co., Ltd); Nano preparation of ' Kang ' ai ' injection of the present invention (by preparation technology's preparation of the present invention, by Tianzhijiao Medication Development Co., Ltd., Guangdong's prepared in laboratory): Wistar rat, body weight 180-220g; Healthy mice, body weight 18-22g; Tumor strain: mice S
180, rat liver cancer Heps (Guangzhou Experimental Animal Center).
1. antitumor test
1.1 influence and targeting to mouse bearing liver cancer Heps
The hepatocarcinoma Heps tumor-bearing mice ascites of aseptic extraction inoculation back 7d, add 4 times of amount normal saline mixings, it is subcutaneous to inoculate sub-mice right fore axillary fossa place immediately, every inoculation 0.2ml, inoculation back 24h random packet, every group 10, the mouse tail vein injection administration, dosage is equivalent to 3g crude drug/kg, the administration volume only is all 0.2ml/, and matched group gives the equivalent normal saline, administration every day 1 time, successive administration 10d, 24h takes off neck execution mice after the last administration, strips tumor and weighs, and get tumor cell then in tissue: normal saline is the ratio homogenate of 1: 1.5 (W/V), centrifugal 10min (2000rpm) gets supernatant is measured kurarinone (in oxymatrine) with the HPLC method content.1h gets kurarinone (in the oxymatrine) concentration in the hematometry serum after the while last administration.The results are shown in Table 3, table 4.
The influence of table 3 pair transplanted hepatoma Heps mice
| Group | Tumor body weight (g) | Tumour inhibiting rate (%) |
| Normal saline group Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 2.35±0.33 1.29±0.25 1.01±0.12 | 45.1 ** 57.0 **Δ |
Annotate: compare with the normal saline group
*Compare with Conair injection group P＜0.01
ΔP＜0.01
The targeting of table 4 couple transplanted hepatoma Heps
| Group | Oxymatrine concentration in the serum (μ g/ml) | Oxymatrine concentration in the tumor cell (μ g) |
| Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 0.067 0.065 | 0.45 4.87 |
The result shows that nano preparation of ' Kang ' ai ' injection of the present invention can significantly suppress the growth of mouse bearing liver cancer Heps, have than the better pharmacological action of Conair injection, relatively serum Chinese medicine concentration is suitable for nano injection formulation of the present invention and Conair injection, and active constituent content significantly increases in the tumor cell, shows that nano injection formulation of the present invention has targeting preferably to tumor cell.
1.2 to mice S
180The inhibitory action of tumor and targeting
Get and inoculate the mice S that goes down to posterity
180, in homogenizer, add normal saline, make mice S
180The tumor homogenate, again with normal saline dilution in 1: 3, it is subcutaneous to get oxter, a 0.2ml injection mice left side then, weighs grouping in 24 hours, every group 10, administration group mice tail every day intravenously administrable once, dosage is 3g crude drug/kg, the administration volume is all 0.2ml/ only, matched group gives the equivalent normal saline, totally 7 days.Mice is put to death in drug withdrawal next day, the subcutaneous tumors piece is peeled off in the also carefulness of weighing, taking by weighing tumor in the EM50 electronic balance weighs, and calculating tumour inhibiting rate, with the tumor piece after weighing in tissue: normal saline is the ratio homogenate of 1: 1.5 (W/V), centrifugal 10min (2000rpm) gets supernatant, measures the content of the kurarinone (in oxymatrine) in tumor tissues and the serum with the HPLC method.1h gets kurarinone (in the oxymatrine) concentration in the hematometry serum after the while last administration.The results are shown in Table 5, table 6.
Table 5 couple mice S
180The tumor growth inhibitory action
| Group | Tumor body weight (g) | Tumour inhibiting rate (%) |
| Normal saline group Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 1.59±0.21 1.03±0.16 0.69±0.06 | 35.2 ** 56.6 **Δ |
Annotate: compare with the normal saline group
*Compare with the Conair injection P＜0.01
ΔP＜0.01
Table 6 couple mice S
180The targeting of tumor
| Group | Oxymatrine concentration in the serum (μ g/ml) | Oxymatrine concentration in the tumor cell (μ g) |
| Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 0.068 0.067 | 0.51 5.03 |
The result shows that nano preparation of ' Kang ' ai ' injection of the present invention can significantly suppress mouse tumor S
180Growth has than the better pharmacological action of Conair injection, and relatively serum Chinese medicine concentration is suitable for nano injection formulation of the present invention and Conair injection, and effective ingredient significantly increases in the tumor cell, shows that nano injection formulation of the present invention has targeting preferably.
1.3 to lotus tumor S
180The influence of mouse death rate and time-to-live
Get and inoculate the mice S that goes down to posterity
180, in homogenizer, add normal saline, make mice S
180The tumor homogenate, again with normal saline 1: 3 dilution, getting 0.2ml then, to inject oxter, a mice left side subcutaneous, a week behind inoculated tumour, promptly the 8th day begins by 1.1 method administrations, allow its natural death, treat the whole death of control animals after, mortality rate relatively, and the time-to-live difference of 90 days each treated animals of comparison, if administration group and matched group, 20 of every group of mices the results are shown in Table 7.
Table 7 pair mouse death rate and the influence of time-to-live
| Group | Death toll | Mortality rate | Mean survival time | Increase in life span |
| (％) | (％) | (my god) | (％) | |
| Normal saline group Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 20 12 6 | 100 60* 30* Δ | 33.0±1.7 54.5±3.5 70.8±6.2 | 65.2** 113.0** Δ |
Annotate: compare * * P＜0.01 with the normal saline group, compare with Conair injection group
ΔP＜0.05
The result shows that nano preparation of ' Kang ' ai ' injection of the present invention can significantly reduce the tumor-bearing mice mortality rate, prolongs the tumor-bearing mice time-to-live, relatively acts on more remarkable with the Conair injection.
2. murine interleukin is reduced the influence of disease
Get 50 of the Kunming mouses of body weight 18-22g, divide 5 groups at random, 10 every group, male and female half and half, i.e. normal control group, cyclophosphamide group, Conair injection group, nano preparation of ' Kang ' ai ' injection group of the present invention.Except that the normal control group, all give cyclophosphamide 100g/kg lumbar injection, every day 1 time, continuous 3 days, each administration group of while is intravenously administrable respectively, and dosage is equivalent to crude drug 3g/kg, administration every day 2 times, continuous 8 days, get blood by the eye socket vein respectively at the 4th day and the 8th day, the microscopy total white blood cells the results are shown in Table 8.
Table 8 pair murine interleukin reduces the influence of disease
| Group | Leukocyte number (10 9/L) | |
| The 4th day | The 8th day | |
| Normal control group cyclophosphamide group Conair injection group nano preparation of ' Kang ' ai ' injection group of the present invention | 8.42±1.56 3.05±0.43 ** 4.28±0.63 5.17±0.75 | 8.95±1.78 4.02±0.50 ** 6.13±0.95 Δ 8.39±1.22 ΔΔ |
Annotate: compare with the normal control group
*Compare with Conair injection group P＜0.01
ΔP＜0.05,
The Δ ΔP＜0.01
Experimental result shows that for the leukopenia of caused by cyclophosphamide, nano preparation of ' Kang ' ai ' injection group of the present invention and Conair injection relatively have better function of increasing leukocyte.
3. to the influence of rat acute hepatic injury due to the carbon tetrachloride
Get 60 of the Wistar rats of body weight 180-220g, male and female half and half are divided into normal saline group, model group, Conair injection group, of the present invention group at random.Reference literature method (Li Yikui, Deng. the herbal pharmacology experimental methodology. Shanghai: Shanghai science tech publishing house, 1991:834), except that the normal saline group, each group of model and administration is all with 15% carbon tetrachloride olive oil solution, press the 2ml/kg body weight, every other day subcutaneous injection is once injected 4 times continuously.Simultaneously, normal saline group intravenous injection normal saline, administration group intravenous injection Conair injection and nano preparation of ' Kang ' ai ' injection of the present invention, dosage is equivalent to 2.5g crude drug/kg, continuous 7 days, behind the last administration 24h, gather blood, separation of serum, measure glutamate pyruvate transaminase (SGPT), glutamic oxaloacetic transaminase, GOT (SGOT) and lactic acid deaminase (LDH), the results are shown in Table 9, and get the liver sample, 10% formalin is fixed, and does the pathology cut sections for microscopic examination.
Table 9 couple CCl
4The influence of hepatic injury
| Group | SGPT | SGOT | LDH |
| Normal saline group model group Chelerythrine hydrochloride group chelerythrine arginine complex group | 43.12±6.50 93.45±9.23 ** 67.57±6.46 Δ 45.08±6.71 ΔΔ | 35.71±4.53 103.29±13.4 ** 84.51±12.5 67.83±10.5 Δ | 185.76±30.6 233.92±35.8 ** 180.13±25.0 Δ 154.84±20.7 ΔΔ |
Annotate: compare with the normal saline group,
*Compare with the model group group p＜0.01,
ΔP＜0.05,
The Δ ΔP＜0.01
The result shows: nano preparation of ' Kang ' ai ' injection of the present invention and Conair injection relatively have the effect of better reduction Rats with Acute Liver Injury serum SGOT and LDH content, and nano preparation of ' Kang ' ai ' injection group SGPT content of the present invention also has remarkable decline.Each administration group of also finding pathological examination all can significantly suppress rat liver inflammatory reaction due to the carbon tetrachloride, and nano preparation of ' Kang ' ai ' injection group wherein of the present invention has in vain than the more significant effect of Conair injection.
4. blood vessel irritation is investigated
Get 8 of rabbit, divide equally 2 groups at random, every group 4, respectively at left ear rabbit is placed holder, and head is fixed with a rabbit fixer, with ethanol with skin degerming after, in right side auricular vein injection Conair injection and nano preparation of ' Kang ' ai ' injection of the present invention, the normal saline of injecting same volume in the opposite side corresponding position is injected continuous 1 week every day 1 time in contrast, observe the reaction of rabbit ear edge vein, observe the injection site blood vessel every day and have or not rubescent, edema, have or not oozing of blood on every side, touch blood vessel and have or not the hardening phenomenon, left and right sides ear comparative observation.24h after the last administration with sacrifice of animal, takes off two ears, carries out the tissue slice inspection, and the section position is the entad end at inserting needle position, apart from inserting needle position 1-4cm, divides 1-2.5cm and two sections samplings of 2.5-4cm.
Observation index and standards of grading: observational technique comprises perusal and microscopically pathological observation.Observe the variation of administration blood vessel and surrounding tissue thereof, microscopically is observed the pathological change situation that auricular vein has or not thrombosis, endothelial injury and blood vessel surrounding tissue.To every index scoring.1. blood vessel changes.Perusal does not have obvious congested note 0 minute, and mild hyperaemia is rubescent, the clear note of lines 1 minute, and congested rubescent, the unclear note of lines 2 minutes, is aubergine note 3 minutes at severe hyperemia; The complete note of microscopic examination blood vessel endothelium and blood vessel wall 0 minute has endothelial injury note 1 minute, and thromboembolism note 2 minutes are arranged, angiorrhexis note 3 minutes.2. the blood vessel surrounding tissue changes.Perusal does not have obvious edema note 0 minute, slight edema note 1 minute, and obviously the edema note is 2 minutes, serious edema note 3 minutes; The normal note of microscopic examination surrounding tissue 0 minute, edema note 1 minute, hemorrhage note 2 minutes has inflammatory cell infiltration note 3 minutes.Every group of every observation index score of 4 rabbit added up, calculate and respectively organize the average value, see Table 10.
Table 10 irritation test result
| Group | The zest scoring |
| Conair injection group nano preparation of ' Kang ' ai ' injection of the present invention | 1.5 0.5 |
By above experimental result as can be known: nano preparation of ' Kang ' ai ' injection of the present invention and Conair injection comparison stimulus have obvious improvement.
5. hemolytic toxicity is investigated
The preparation of 2% red blood cell suspension: get rabbit ear edge vein and get blood 10-20ml, put into the conical flask that fills bead, Fibrinogen is removed in jolting 10 minutes, makes to become to take off fine blood.Add the normal saline solution of 10 times of amounts, shake up, centrifugal, remove supernatant, sedimentary erythrocyte reuse normal saline solution washing 2-3 time, when supernatant does not take on a red color till.It is 2% suspension that the erythrocyte of gained is made into concentration with normal saline, promptly.
Test method: get 6 in test tube, add 2% red blood cell suspension and normal saline solution successively by the proportional quantity in the table, mixing was placed 30 minutes in 37 ℃ of calorstats, added not commensurability medicinal liquid (is blank with the 6th pipe) respectively, after shaking up, put in 37 ℃ of calorstats, beginning was observed 1 time every 15 minutes, after 1 hour, observed 1 time every 1 hour, observed altogether 2 hours.The results are shown in Table 11.
Table 11 hemolytic experiment result
| The test tube numbering | 2% red blood cell suspension (ml) | Normal saline solution (ml) | Medicinal liquid (ml) |
| 1 2 3 4 5 6 | 2.5 2.5 2.5 2.5 2.5 2.5 | 2.0 2.1 2.2 2.3 2.4 2.5 | 0.5 0.4 0.3 0.2 0.1 0 |
The result: be as the criterion with the 3rd test tube, each Guan Junwei dyes redness, and microscopically is observed and do not seen that erythrocyte fragmentation is arranged, and not haemolysis of this product is described, safety is good.
6. acute toxicity testing
Get 80 of Wistar rats, male and female half and half.Fasting 24h divides 4 groups at random, 20 every group.Be condensed into identical crude drug concentration, each group is the tail vein injection medicine respectively, and dosage is equivalent to 150g crude drug/kg by the conversion of crude drug amount, administration every day 3 times, and continuous 7 days, observe the dead mouse situation, the results are shown in Table 12.
Table 12 acute toxicity testing result
| Group | Number of animals | Death toll | Mortality rate (%) |
| Nano powder injection Conair of the present invention, nanometer aqueous injection Conair of the present invention, injection group Conair of the present invention, Conair nanometer infusion solution | 20 20 20 20 | 7 4 3 4 | 35 20 15 20 |
The acute toxicity testing result shows nano preparation of ' Kang ' ai ' injection of the present invention and Conair injection, and relatively safety is better.
Brief summary: above The pharmacological results shows that nano preparation of ' Kang ' ai ' injection pharmacological action of the present invention significantly is better than the Conair injection, and nano preparation of ' Kang ' ai ' injection of the present invention has better targeting and safety.
Four, preparation embodiment
Embodiment 1
Get Radix Ginseng 100g, with 90% alcohol reflux three times, each 2 hours, merge extractive liquid, is evaporated to relative density and is the clear paste of 1.10-1.20 (65 ℃), and was standby.Radix Astragali 300g decocts with water secondary, each 2 hours, filters, merging filtrate, being evaporated to relative density is the clear paste of 1.10-1.20 (65 ℃), merges with the Radix Ginseng clear paste, adding ethanol makes and contains alcohol amount and reach 75%, regulate pH value to 6-7 with sodium hydroxide, left standstill 12 hours, get supernatant, reclaim ethanol, being evaporated to relative density is the clear paste of 1.10-1.15 (65 ℃), adds ethanol again and makes and contain alcohol amount and reach 85%, regulates pH value to 6-7 with sodium hydroxide, leave standstill, filter, filtrate recycling ethanol adds the injection water to 400ml to there not being the alcohol flavor, regulate pH value to 6-7 with sodium hydroxide, cold preservation, sucking filtration sterilized 30 minutes for 100 ℃.Regulate pH value to 6-7 with sodium hydroxide, it is an amount of to add active carbon, stirs evenly, and boils 15 minutes, filters, and filtrate decompression concentrates and vacuum drying, merges with kurarinone 10g, and mixing obtains Conair of the present invention effective site.
Embodiment 2
(1) Conair effective site 15g is dissolved in the 200ml distilled water, add tween 80 1g, stir, constantly adding among the paracyanogen base alkyl acrylate 60g under the stirring condition, continue to stir 30 minutes again, filter with 9-15 μ m glass sand hourglass, filtrate is diluted to 600ml, become the emulsion of scattering phenomenon, reuse 0.45 μ m membrane filtration promptly gets drug suspension.
(2) preparation of preparation
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, it is an amount of to add PVP 4g, adds the injection water to 20000ml, oozes with sodium chloride accent etc., and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost is invented Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add mannitol 50g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 3
(1) Conair effective site 20g is dissolved in the 100ml distilled water, add tween 80 1g, stir, constantly adding among the methacrylic acid 80g under the stirring condition, continue to stir 30 minutes again, filter with 9-15 μ m glass hourglass, filtrate is diluted to 600ml, become the emulsion of scattering phenomenon, reuse 0.45 μ m membrane filtration promptly gets drug suspension.
(2) preparation of preparation
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add to the full amount of water for injection, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 4g, add to the full amount of water for injection, stir evenly, adding glucose accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add glucose 60g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 4
(1) Conair effective site 30g is dissolved in the 150ml distilled water, add tween 80 2g, stir, constantly adding among the polylactic acid 150g under the stirring condition, continue to stir 30 minutes again, filter with 9-15 μ m glass hourglass, filtrate is diluted to 700ml, become the emulsion of scattering phenomenon, reuse 0.45 μ m membrane filtration promptly gets drug suspension.
(2) preparation of preparation
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, it is an amount of to add PVP, adds the injection water to 20000ml, stirs evenly, and adding sodium chloride accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add lactose 40g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 5
(1) polylactide-co-glycolide 70g is mixed with dehydrated alcohol 500ml, shake up, get clear solution; Get Conair effective site 20g and be dissolved in the 200ml distilled water, add Pluronic F68 1g, stir; In the time of 20 ℃, polylactide-co-glycolide solution is injected by silica gel tube in the aqueous solution of electromagnetic agitation, form nanocapsule immediately, solution for vacuum is evaporated to about 1/5 of original volume, filter with glass sand hourglass (9-15 μ m), promptly get drug suspension.
(2) preparation of preparation:
The preparation of hydro-acupuncture preparation:. get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 3g, add the injection water to 20000ml, stir evenly, adding glucose accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add mannitol 60g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 6
(1) polylactide-co-glycolide 60g, gelatin 40g are mixed with the 500ml dehydrated alcohol, shake up, get clear solution; Get Conair effective site 20g and be dissolved in the 200ml distilled water, add tween 80 1g, stir; In the time of 20 ℃, the solution of polylactide-co-glycolide and gelatin is injected by the fine needle head in the aqueous solution of electromagnetic agitation, form nanocapsule immediately, solution for vacuum is evaporated to about 1/5 of original volume, filter with glass sand hourglass (9-15 μ m), promptly get drug suspension.
(2) preparation of preparation:
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 4g, add the injection water to 20000ml, stir evenly, adding sodium chloride accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add dextran 30g and lactose 30g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 7
(1) gets Conair effective site 20g, lecithin 50g, glyceryl monostearate 40g, mixing, heating and melting, polysorbate-20 2g is added wherein, mix with 70 ℃ distilled water, stir and be prepared into microemulsion, 65 ℃ microemulsion is poured in the syringe that can be incubated and be incubated 15 minutes, under the mechanical agitation condition, in certain hour, the microemulsion of heat is joined in the cold distilled water, promptly get drug suspension.
(2) preparation of preparation:
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 4g, add the injection water to 20000ml, stir evenly, adding sodium chloride accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add dextran 30g and mannitol 30g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 8
(1) gets Conair effective site 20g, lecithin 60g, stearic acid 40g, be heated to 80 ± 5 ℃, the aqueous solution that under stirring condition, adds uniform temp glycerol 2g and poloxamer 2g, make thick breast, under 80 ± 5 ℃ of conditions, carry 40MPa pressure stimulating milk secretion even 5 times, be cooled to room temperature rapidly and form the principal agent suspension with the high pressure dispersing emulsification machine.
(2) preparation of preparation:
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 4g, add the injection water to 20000ml, stir evenly, adding sodium chloride accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add glucose 30g and mannitol 30g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Embodiment 9
(1) gets lecithin 50g, add Tween 80 1g, in the distilled water of 70 ± 5 ℃ of high-speed stirred condition addings, treat that complete fusion is as water, take by weighing the three acid glyceride 60g that climb over a wall again, add Conair effective site 20g, heating and melting slowly adds aqueous phase as oil phase with oil phase, continue to stir ultra-sonic dispersion after 1 hour: the chamber is steady, frequency 45-50Hz ultrasonic 5-8 minute, is cooled to room temperature rapidly and forms the principal agent suspension.
(2) preparation of preparation:
The preparation of hydro-acupuncture preparation: get above-mentioned suspension, add PVP 2g, add the injection water to 1000ml, stir evenly, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, add PVP 4g, add the injection water to 20000ml, stir evenly, adding sodium chloride accent etc. oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add dextran 50g, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and 500 bottles of preparation cost invention Conair powder injection formulations.
Claims (3)
1, a kind of nano preparation of ' Kang ' ai ' injection is characterized in that it is that the Conair effective site that Radix Ginseng, Radix Scutellariae extract and kurarinone are formed is equipped with the nano level ejection preparation that carrier is made, wherein Conair effective site 1.5-3 weight portion, carrier 6-15 weight portion.Its feature is that also it has targeting.
2,, it is characterized in that described nanoscale is 20-100nm according to a kind of nano preparation of ' Kang ' ai ' injection of claim 1.
3, nano preparation of ' Kang ' ai ' injection according to claim 1 and 2, its preparation method is:
(1) preparation of principal agent suspension
Method one, Conair effective site is dissolved in the distilled water, add tween 80, stir, constantly adding in the pharmaceutical carrier under the stirring condition, continue to stir 30 minutes again, filter with 9-15 μ m glass hourglass, filtrate is diluted to 4 times, become the emulsion of scattering phenomenon, reuse 0.45 μ m membrane filtration promptly gets drug suspension.
Method two, pharmaceutical carrier is mixed with dehydrated alcohol, shakes up, clear solution; Get Conair effective site and be dissolved in the distilled water, add Pluronic F68 or tween 80, stir; In the time of 20 ℃, pharmaceutical carrier solution is injected in the aqueous solution of electromagnetic agitation by silica gel tube or fine needle head, form nanocapsule immediately, solution for vacuum is evaporated to about 1/5 of original volume, filter with glass sand hourglass (9-15 μ m), promptly get drug suspension.
(2) preparation of preparation
The preparation of hydro-acupuncture preparation:. get above-mentioned suspension, it is an amount of to add PVP, adds to the full amount of water for injection, and stirs evenly, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, sterilization, preparation cost invention Conair nanometer hydro-acupuncture preparation;
The preparation of infusion preparation: get above-mentioned suspension, it is an amount of to add PVP, adds to the full amount of water for injection, and stirs evenly, and adds sodium chloride or glucose accent etc. and oozes, and adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, and sterilization, preparation cost invention Conair nanometer infusion preparation;
The preparation of powder injection formulation: get above-mentioned suspension, add excipient, add the injection water and adjust concentration, adjust pH is 6.0-7.0, with 0.22 μ m filtering with microporous membrane, lyophilization, and preparation cost invention Conair nanometer powder injection formulation.
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| CN 200510077495 CN1883556A (en) | 2005-06-24 | 2005-06-24 | A nano preparation of 'Kang'ai' injection and method for preparing same |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10925912B2 (en) * | 2017-02-22 | 2021-02-23 | Jiangsu Province Institute of Traditional Chinese Medicine | Preparation and application of ginseng derived membranous microparticles |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10925912B2 (en) * | 2017-02-22 | 2021-02-23 | Jiangsu Province Institute of Traditional Chinese Medicine | Preparation and application of ginseng derived membranous microparticles |
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