CN103463377A - Method for preparing aloe freeze-dried powder injection by using aloe powder - Google Patents
Method for preparing aloe freeze-dried powder injection by using aloe powder Download PDFInfo
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Abstract
The invention discloses a method for preparing an aloe freeze-dried powder injection by using aloe powder. The method comprises the following steps of: firstly, blending the aloe powder and distilled water in the ratio of 100:2000 in parts by weight, after the aloe powder is completely dissolved, standing through night for separation, filtering and clarifying; regulating the pH to the range from 6.3 to 6.5 by using hydrochloric acid, heating to the range from 115 to 118 DEG C for sterilizing for 30 minutes, and then naturally cooling; regulating the pH of the cooled liquid to the range from 4.2 to 4.5, performing ultrafiltration through an ultrafiltration membrane, thus obtaining a medicine-containing solution having the molecular weight not greater than 10000 Da through interception; and finally, separately packaging the liquid medicine into ampoule bottles for freeze-drying. The method provided by the invention has the advantages that the traditional application range of the aloe as products for detoxifying, beautifying and defaecating is broken through, and the modern medical method is utilized to prepare the aloe into the freeze-dried powder injection so that the medicinal field of the aloe is expanded to the aspect of preventing or treating diseases such as tumor and liver injury; and tests proves that the aloe freeze-dried powder injection prepared by the method provided by the invention is outstanding in effect and free of toxic and side effects.
Description
Technical field
The present invention relates to aloe product, especially relate to a kind of method of using Aloe powder to prepare the Aloe injection freeze-dried powder injection.
Background technology
Aloe (Aloe) is a kind of herbaceous plant of perennial Liliaceae meat, is a kind of plant that has edible, beauty treatment, health care, medicine and the function such as view and admire.The Aloe complex chemical composition, the composition of having found surpasses kind more than 160, mainly contains the effective active compositions such as polysaccharide, anthraquinone analog compound, organic acid, aminoacid, enzyme and polypeptide, vitamin, steroid, mineral, trace element.The active component content of different cultivars Aloe has significant difference, even same kind, the different places of production, different growing stages, different collecting seasons, different processing methods, the active component content of Aloe also has notable difference.For the ease of end product processing, use, transportation and selling, usually Fresh leaf of aloe is extracted, concentrated and dry.Lyophilized powder and spray drying powder are one of principal modes of Aloe powder.
The Aloe industry only has the basic materials such as Aloe (Aloe fourth) canned food, aloe liquid, aloe leaf powder, aloe gel powder at present, can manufacture the oral aloe products such as Aloe glue, aloe capsule, Aloe tablet, Aloe extract.Oral aloe products has eliminating toxin and beautifying the skin, and the logical the intestines and stomach of profit, strengthen the effects such as immunologic function, but actual effect mainly concentrates on bowel relaxing functions, and other human body diseases are not obviously improved and therapeutical effect.
Summary of the invention
The object of the present invention is to provide a kind of method of using Aloe powder to prepare the Aloe injection freeze-dried powder injection.
For achieving the above object, the present invention can take following technical proposals:
Use Aloe powder of the present invention prepares the method for Aloe injection freeze-dried powder injection, comprises the steps:
The first step, prepare Aloe powder and distilled water in the ratio of 100:2000 weight portion, obtain 5% aloe water solution;
Second step, evenly make Aloe powder dissolve fully the aloe water solution stirring, and standing over night is separated;
The 3rd step, the supernatant liquid filtering clarification after second step is separated;
The 4th step, the filtrate that the 3rd step is obtained is adjusted to 6.3-6.5 with hydrochloric acid by pH value, is heated to 115 ℃ of-118 ℃ of sterilizings 30 minutes, then natural cooling;
The 5th step, the pH value of the liquid coolant the 4th step obtained with hydrochloric acid is adjusted to 4.2-4.5, through the ultrafilter membrane ultrafiltration, holds back and obtains the drug solns that contains that molecular weight is not more than 10000Da;
The 6th step, the medicinal liquid that the 5th step is obtained is divided in lyophilizing in ampoule bottle and gets final product.
The preparation method of described Aloe powder is:
The first step, soak 30-60 minute by the fresh aloe leaf, is ground into the Aloe juice after cleaning up;
Second step, pour the Aloe juice in the reacting by heating tank into and import and be steam heated to 8-12 ℃, adds pectase or protease by 0.125% of Aloe juice gross weight simultaneously, after 50-60 minute, temperature risen to 75-80 ℃, and inactivation treatment is carried out in insulation in 3-5 hour;
The 3rd step, by second step after inactivation treatment solution filter, concentrated final vacuum drying or lyophilization to obtain outward appearance be flaxen Aloe powder finished product.
The invention has the advantages that and broken through traditional scope of application as eliminating toxin and beautifying the skin relieving constipation product by Aloe, use the modern medicine and pharmacology means, Aloe is made to the injection lyophilized powder, make its medicinal field widen control or the treatment aspect to diseases such as tumor, hepatic injury, evidence, Aloe injection lyophilized powder therapeutic effect prepared by the present invention is given prominence to and is had no side effect.
The accompanying drawing explanation
Fig. 1 is J01 ear edge slice map apart from 1cm place, injection site in vascular stimulation test.
Fig. 2 is J02 ear edge slice map apart from 1cm place, injection site in vascular stimulation test.
Fig. 3 is that J01 and J02 are on suppressing the impact of gross tumor volume size the body mice.
The specific embodiment
The method that use Aloe powder of the present invention prepares the Aloe injection freeze-dried powder injection comprises the steps:
The first step, soak 30-60 minute by the fresh aloe leaf, cleans up rear employing food, with the rustless steel disintegrating machine, it is ground into to the Aloe juice; The Folium Aloe peeling can be pulverized during pulverizing, or Folium Aloe not removed the peel to full leaf and pulverized;
Second step, pour the Aloe juice in the reacting by heating tank into and import and be steam heated to 8-12 ℃, adds pectase or protease by 0.125% of Aloe juice gross weight simultaneously, to promote the degraded of Aloe, after 50-60 minute, after temperature is risen to 75-80 ℃, inactivation treatment is carried out in insulation in 3-5 hour;
The 3rd step, by second step after inactivation treatment solution adopt shake sieve, kieselguhr or plate-and-frame filtration, also two kinds of modes can be combined and filtered, if need decolouring, also can add the active carbon processing of decolouring; Concentrated rear (soluble solid of concentrated juice is controlled in 10%) adopts the method for spray drying or lyophilizing to be made into outward appearance is flaxen Aloe powder basic material;
The 4th step, prepared above-mentioned Aloe powder basic material 100g and distilled water 2000ml (can amplify in this ratio during commercial production), obtains 5% aloe water solution;
The 5th step, by the aloe water solution stirring evenly or ultrasonic dissolution 15 minutes, dissolve Aloe powder fully and obtain light brown solution, and standing over night is separated; (can use the magnificent high pressure homogenize machine equipment in east, Shanghai);
The 6th step, the supernatant clarification filtration after the 5th step is separated;
The 7th step, the filtrate that the 6th step is obtained is adjusted to 6.3-6.5 with 37% hydrochloric acid by pH value, is heated to 115 ℃ of-118 ℃ of high temperature sterilizes 30 minutes, then natural cooling;
The 8th step, the pH value of the liquid coolant that the hydrochloric acid with 37% obtains the 7th step is adjusted to 4.2-4.5, use ultrafiltration technology, through the ultrafilter membrane ultrafiltration, hold back obtain molecular weight be not more than 10000Da containing drug solns (the full-automatic membrance separation ultrafiltration apparatus that commercial production can be used Hefei Fengyun Membrane Technology Co., Ltd. to produce, laboratory is used No. 4 pans);
The 9th step, the medicinal liquid that the 8th step is obtained is divided in the ampoule bottle of 10 milliliters ,-30 ℃ of lyophilizing can obtain Aloe injection injectable powder (the JDG-100F fully automatic vacuum freeze dryer that can use Lanzhou Kejin Vacuum Freeze Drying Technology Co., Ltd. to produce).
During use, Aloe injection freeze-dried powder injection prepared by the present invention dissolves into injection with 0.9% normal saline dilution and gets final product.
Aloe injection freeze-dried powder injection prepared by the present invention is divided into two kinds:
1, the injection freeze-dried powder injection that adopts the peeling Aloe to make is light brown, specification: every bottle containing solubility Aloe effective ingredient 0.75 gram, hereinafter to be referred as J01.
2, adopt and do not remove the peel the injection freeze-dried powder injection that Aloe (aloe leaf) makes, be brown, specification: every bottle containing solubility Aloe effective ingredient 0.9 gram, hereinafter to be referred as J02.
The clinical safety result of the test of the Aloe injection freeze-dried powder injection that, prepared by the present invention is as follows:
1, vascular stimulation test
1) experiment purpose
Aloe injection freeze-dried powder injection prepared by the main observation of this experiment the present invention irritative response to the rabbit blood vessel.
2) tested medicine
The J01-injection freeze-dried powder injection, be light brown, specification: every bottle containing soluble component 0.75 gram.
The J02-injection freeze-dried powder injection is brown, specification: every bottle containing soluble component 0.9 gram.
3) laboratory animal
White big ear rabbit, the male and female dual-purpose, body weight 2.0-2.5kg, provided by Qinglongshan animal reproduction field, Jiangning county, the animal quality certification number: SCXK (Soviet Union): 2012-0008.
4) test method
First with the injection normal saline, lyophilized injectable powder is dissolved standby; Get 3 of health, the undamaged white big ear rabbits of ear edge, respectively at the left side auricular vein, with sterile working's method injection J01 and J02 1ml/kg single or multiple (once a day, continuous three days), in 2min, injection is complete.Right side is with 5% glucose injection of method injection equivalent.The result of variations that after completing respectively at administration, 5min, 30min, 2h, 4h, 8h, 24h observe administration part and blood vessel.Once a day, the single cage of animal is raised, continuous three days.
5) observation index
The red and swollen situation of the vein blood vessel of perusal administration part and surrounding tissue after administration.Last injection J01 and J02 are by the animal sacrificed by exsanguination after 24 hours, and respectively at injection part proximal part 1.5 to 3 centimeters clip ear edge, sample 10% formaldehyde is fixed, and the conventional organization section, observe and have or not thrombosis, endothelial injury and other pathological changes.
6) experimental result
In Table 1, table 2, the injection of rabbit single or multiple, left and right ear blood vessel is all without hyperemia, surrounding tissue is all without edema.The pathology section examination result shows: the significant stimulation reactions such as inorganization degeneration or necrosis.
The Local irritation study of table 1. J01 and J02 single-dose (n=3)
The Local irritation study of table 2. J01 and J02 multiple dosing (n=3)
7) conclusion
J01 and J02 are to rabbit blood vessel nonirritant.See Fig. 1, Fig. 2 apart from 1cm place, injection site man rabbit ear edge pathological section.
2, hemolytic experiment
1) test objective
Aloe injection freeze-dried powder injection prepared by observation the present invention has or not the hemolytic reaction.
2) tested medicine
The J01-injection powder injection, be light brown, specification: every bottle containing soluble component 0.75 gram.
The J02-injection powder injection is brown, specification: every bottle containing soluble component 0.9 gram.
3) laboratory animal
White big ear rabbit, the male and female dual-purpose, body weight 2.0-2.5kg, provided by Qinglongshan animal reproduction field, Jiangning county, the animal quality certification number: SCXK (Soviet Union): 2012-0008.
4) test method
4.1) observation method of naked eye
First with the injection normal saline, lyophilized powder is dissolved standby; Get one of rabbit, from carotid artery blood-letting 20ml, put in conical flask and stir and remove Fibrinogen with bamboo let, make into defibrinated blood, then blood 10ml is moved into to graduated centrifuge tube, add normal saline 10ml, after mixing with 2000r/min centrifugal 5 minutes, remove supernatant, then add normal saline and mix centrifugally, 4 times to the supernatant water white transparency repeatedly.The gained erythrocyte, by its volume, is mixed with to 2% red blood cell suspension with normal saline.Get 7, test tube, numbering is arranged in test tube rack, and according to the form below 3 adds various solution, and the 6th pipe does not add need testing solution, as blank; The 7th pipe does not still add need testing solution, with distilled water, replaces normal saline, as the positive control of haemolysis, each pipe is shaken up, and in 37 ℃ of water-baths, insulation is 1 hour, after taking-up, by the subordinate list order, records result.
Result is judged
Full haemolysis: the clear and bright redness of solution, the pipe end, is acellular residual; Part haemolysis: the clear and bright redness of solution or brown, the pipe end, have a small amount of erythrocyte residual; Without haemolysis: erythrocyte all sinks, the supernatant liquid achromatism and clarity; Coagulation: though red cell agglutination appears in haemolysis not, can not disperse after jolting;
It is generally acknowledged, the preparation of haemolysis, part haemolysis appears in each pipe after all 1 hour before tee pipe or tee pipe, should not make used for intravenous injection.If any hemagglutination, can further judge by the following method true coagulation or pseudoagglutination.If condensation product can be uniformly dispersed again after test tube concussion, or condensation product is placed on microscope slide, drips 2 normal saline at the coverslip edge, examines under a microscope.The coagulation erythrocyte can be pseudoagglutination by the person of breaking up, and test sample can be for clinical.If condensation product can not shake loose or is not true cohesion by the person of breaking up on slide, test sample should not be for clinical.
Experimental result
Check each test tube in official hour, except positive controls, each test tube is showed no haemolysis and occurs.It the results are shown in Table 3, table 4, and J01 and J02 have no hemolytic reaction in the hemolytic experiment of rabbit, and J01 and J02 are without hemolytic reaction.
Table 3. J01 hemolytic test (unit: ml)
Annotate: haemolysis "+", non-haemolysis "-"
Table 4. J02 hemolytic test (unit: ml)
Annotate: haemolysis "+", non-haemolysis "-"
4.2) spectrophotography
Get 2% red cell suspension prepared in 4.1 experiments.Get three groups of test tubes, every group 7, every group # application of sample is with experiment 4.1, after application of sample, all test tubes are incubated after 1 hour in 37 ℃ of water-baths, centrifugal (2500rpm/5min), get supernatant and move in 96 orifice plates, separately adds three hole normal saline contrasts, measure trap at the 540nm place, get tee pipe mean value computation variable concentrations sample hemolysis rate.
Sample hemolysis rate (%)=(sample OD value-negative control OD value)/(positive control OD value-negative control OD value)
Take hemolysis rate<5% as qualified.
Experimental result
Experimental data shows, 1-5 pipe OD value is all close with saline tube, and respectively manages hemolysis rate and all be less than 5%.J01 and J02 react without hemolytic.The results are shown in Table 5, table 6, J01 and J02 have no hemolytic reaction in the hemolytic experiment of rabbit, and J01 and J02 are without hemolytic reaction.
Table 5. J01 hemolytic test (unit: ml)
Table 6. J02 hemolytic test (unit: ml)
3, systemic anaphylaxis test
1) test objective
Aloe injection freeze-dried powder injection prepared by observation the present invention has or not systemic anaphylaxis to Cavia porcellus.
2) tested medicine
The J01-injection freeze-dried powder injection, be light brown, specification: every bottle containing soluble component 0.75 gram.
The J02-injection freeze-dried powder injection is brown, specification: every bottle containing soluble component 0.9 gram.
3) laboratory animal
Healthy without hindering Cavia porcellus, body weight 250-300g, male and female half and half, provided by Qinglongshan animal reproduction field, Jiangning county, the animal quality certification number: SCXK (Soviet Union): 2012-0008.
4) test method
First with the injection normal saline, lyophilized powder is dissolved standby; Get that healthy body weight 250-300 gram, be divided into 4 groups without hindering 18 of Cavia porcelluss, 3 every group, intramuscular injection J01 next day of first group and second group of Cavia porcellus, 0.5 milliliter every; Intramuscular injection J02 next day of the 3rd group and the 4th group, 0.5 milliliter every; Intramuscular injection bovine serum albumin (1ml/kg) next day of the 5th group and the 6th group, as positive control.Totally three times.First and third, five groups after first administration the 14th day, 1 milliliter of corresponding solution of intravenous injection is attacked respectively.The second, four, six groups of being attacked with method in the 21st day after first administration.Observing Cavia porcellus has or not and grabs nose, perpendicular hair, dyspnea, spasm, shock, even dead allergic symptom such as grade.
5) observation index
If two groups of Cavia porcelluss, all without significant reaction, can think that the test sample hypersensitive test is qualified.Can, by table 7 classification, judge that whether it is qualified as responded.
Table 7. Cavia porcellus anaphylaxis progression
The order of | Reaction symptom | |
0 | Without |
|
1 | Only have and slightly grab nose, tremble or |
|
2 | Have several times and cough, grab nose, tremble or erect hair | |
3 | Repeatedly or continuously cough, with dyspnea or spasm tic etc. | |
4 | Spasm, tic, gatism, shock, death |
When the order of reaction reaches 2 grades (comprising 2 grades), this test sample can think that hypersensitive test is defective.
6) experimental result
Cavia porcellus is being injected latter 14 days and intravenous injection J01 on the 21st and J02 1ml for the first time, in 15 minutes, without perpendicular hair, dyspnea, sneeze, retch or s cough, without twitching, collapse or death, belongs to 0 order reaction, shows that J01 and J2 are without sensitization.The results are shown in Table 8.
The order of reaction of table 8. J01 and J02 allergic experiment (n=3)
4, pyrogen test
1) experiment purpose
The Aloe injection freeze-dried powder injection test sample (J01 and J02) that this genealogy of law prepares the present invention of doses, vein injects in the rabbit body, at the appointed time, observe the situation of rabbit fervescence, whether up to specification with the limit of judging contained pyrogen in test sample.
2) tested medicine
The J01-injection freeze-dried powder injection, be light brown, and every bottle containing soluble component 0.75 gram.
The J02-injection freeze-dried powder injection, be brown, and every bottle containing soluble component 0.9 gram.
Test syringe, the syringe needle of use and the vessel that all contacts with need testing solution, should put in baking oven and heat 30 minutes with 250 ℃ or heat 2 hours with 180 ℃, and also available other suitable methods are removed pyrogen.
3) laboratory animal
White big ear rabbit, the male and female dual-purpose, body weight 2.0-2.5kg, provided by Qinglongshan animal reproduction field, Jiangning county, the animal quality certification number: SCXK (Soviet Union): 2012-0008.
4) test method
Inspection technique: first lyophilized powder is dissolved standby with the injection normal saline; Get 6 of health, the undamaged white big ear rabbits of ear edge, measure after its normal body temperature in 15 minutes, slowly inject prescribed dose (J01 and J02 from ear vein, 1ml/kg) and be warmed to the approximately need testing solution of 38 ℃, then measured its body temperature 1 time every 1 hour by front method, survey altogether 3 times, with the highest normal body temperature that once deducts in 3 body temperature, be the rising number of degrees of this rabbit body temperature.As 1 fervescence arranged more than 0.6 ℃ or 0.6 ℃ in 3 rabbit, or 3 rabbit body temperatures raise all lower than 0.6 ℃, but the sum raise reaches more than 1.4 ℃ or 1.4 ℃, should separately get 5 rabbit retrials, and inspection method is the same.
The result judgement: in 3 rabbit of preliminary examination, fervescence is all lower than 0.6 ℃, and 3 rabbit body temperatures raise sum lower than 1.4 ℃; In 5 rabbit of retrial, the rabbit number of fervescence more than 0.6 ℃ or 0.6 ℃ only has 1, and the fervescence that preliminary examination, retrial merge 8 rabbit adds up to below 3.5 ℃ or 3.5 ℃, all thinks that the pyrogen test of test sample is up to specification.In 3 rabbit of preliminary examination, the rabbit number of fervescence more than 0.6 ℃ or 0.6 ℃ is over 1; In 5 rabbit of retrial, the rabbit number of fervescence more than 0.6 ℃ or 0.6 ℃ is over 1; The fervescence sum that merges 8 rabbit in preliminary examination, retrial surpasses 3.5 ℃, all thinks that the pyrogen test of test sample is against regulation.
5) experimental result
From table 9, result can be found out, after J01 and J02 administration in every rabbit three hours the intensification value of each time point all be less than 0.6 ℃, and the intensification sum of three rabbit all is less than 1.4 ℃ on the same group, illustrates that the pyrogen test of J01 and J02 meets the requirements.
The pyrogen test result of table 9. J01 and J02 (unit: ℃)
5, the foundation of endotoxin inspection method and endotoxin inspection
1) experiment purpose
Set up the method that tachypleus amebocyte lysate detects J01 and J02 bacterial endotoxin, whether the endotoxin index of Aloe injection freeze-dried powder injection sample prepared by detection the present invention is qualified.
2) experiment material
The J01-injection freeze-dried powder injection, be light brown, and every bottle containing soluble component 0.75 gram.
The J02-injection freeze-dried powder injection, be light brown, and every bottle containing soluble component 0.9 gram.
Be dissolved in during use in 250ml injection normal saline and instiled.
Tachypleus amebocyte lysate (sensitivity 0.125EU/ml, Zhanjiang Andusi Biology Co., Ltd., lot number: 130316)
Bacterial endotoxin checks water (10ml/Amp, endotoxin content≤0.005 EU/ml, Zhanjiang Andusi Biology Co., Ltd., lot number: W2013-2)
Bacterial endotoxin working standard (every the 180EU that tires, Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 2013-4)
Test tools made is all through moist heat sterilization, and test operation carries out in aseptic operating platform.
3) experimental technique and result
3.1) sensitivity of the limulus reagent checks: check a lower experiment by 2010 editions pharmacopeia appendix XI E bacterial endotoxins test sensitivity of the limulus reagent, check that with bacterial endotoxin water is diluted to 0.25 by the bacterial endotoxin working standard, 0.125, 0.062, 0.031 the standard solution of λ, every dilution one step all mixes 30s on swirl mixing device, by testing under the bacterial endotoxins test item, each concentration is parallel does four, do 2 negative control pipes with bacterial endotoxin inspection water simultaneously, mix gently, the sealing mouth of pipe, vertically put into 37 ℃ ± 1 ℃ water bath with thermostatic control, insulation 60 ± 2min.Insulation and the process of taking should be avoided being subject to vibration and cause false negative result.Press a bacterial endotoxins test result judgement lower result of determination, calculate the geometrical mean of reaction end concentration.
The results are shown in following table 10, show that this lot number tachypleus amebocyte lysate reaction end concentration geometrical mean is 0.125 EU/ml, checking sensitivity is 0.125 EU/ml, reagent Pass Test requirement.
Table 10 sensitivity of the limulus reagent inspection test result
3.2) interference experiment of test sample J01 and J02
Determining of J01 and J02 Bacterial endotoxin limit (L): L=K/M, during the intravenously administrable approach, K is 5EU/kg; M is the people with the per kilogram of body weight maximal dose M=10ml/kg of administration per hour, the theoretical limit value of this product bacterial endotoxin, L=K/M=5/10=0.5EU/ml.
The maximum valid dilution of test sample (MVD) multiple: while selecting sensitivity to be 0.125 EU/ml tachypleus amebocyte lysate, MVD=L/ λ=0.5/0.125=4 doubly.
This test is carried out interference test, J01:C=0.75mg/ml according to minimum effectively diluted concentration; J02:C=0.9mg/ml.
Press under sensitivity of the limulus reagent inspection test item, check that with bacterial endotoxin water and J01 or J02 solution (by minimum effectively diluted concentration, J01 and J02 being diluted to respectively to 0.75mg/ml and 0.9mg/ml with the inspection water) make the bacterial endotoxin working standard respectively the endotoxin solution containing tetra-kinds of concentration of bacterial endotoxin working standard 0.250,0.125,0.062,0.031 EU/ml.Each concentration made from J02 solution with J01 with bacterial endotoxin inspection water is parallel does four, separately gets bacterial endotoxin inspection water and J01 and J02 solution and does two negative control pipes.Calculating checks the geometrical mean (E of the reaction end concentration of the endotoxin standard solution that water is made with bacterial endotoxin
s) and the geometrical mean (E of the reaction end concentration of the endotoxin solution made with J01 and J02 solution
t).Observe J01 and J02, under this concentration, this test is had or not to interference effect, for setting up the bacterial endotoxin tachypleus amebocyte lysate inspection technique of J01 and J02.
Interference experiment the results are shown in following table 11, the Es=0.125 of endotoxin standard solution, value is between 0.5 λ~2.0 λ, J01 and J02 the Et value under 0.75mg/ml and 0.9mg/ml diluted concentration respectively all between 0.5Es~2.0Es, illustrate that this crowd of J01 and J02 test noiseless effect with the method dilution to this.
Table 11 J01 and J02 interference test result
3.3) inspection of sample endotoxin
Get J01 and J02 according to said method by checking under the inspection technique item, the results are shown in Table 12, all be negative, the endotoxin passed examination.
Table 12 J01 and J02 endotoxin check result
Sample | 130316 |
Positive control | ++ |
Negative control | -- |
The J01 positive control | ++ |
The J02 positive control | ++ |
J01 ( 0.75 mg/ml ) | -- |
J02 ( 0.90 mg/ml ) | -- |
4) conclusion (of pressure testing):
Under this experiment condition, J01 and J02, through pressing the experiment of bacterial endotoxins test application directs principle, can get rid of and disturb with dilution method, and test sample is detected with bacterial endotoxins test.
Injection J01 and J02 injectable powder Bacterial endotoxin limit are 0.5 EU/ml.Injection J01 and J02 injection Bacterial Endotoxin Test adopt this medicine clinical in 4 times of liquid dilutions, noiseless to testing.This batch sample all meets the requirements by the method check.
The clinical result of use test of the Aloe injection freeze-dried powder injection that two, prepared by the present invention is as follows:
1, antitumor activity in vitro
1) experiment material
1.1 sample:
The J01-injection freeze-dried powder injection, be light brown, and 10ml is bottled, and every bottle containing Aloe composition 0.75g; J01 adds cultivating system by concentration dilution after adding sodium chloride for injection solution to dissolve;
The J02-injection freeze-dried powder injection, be brown, and 10ml is bottled, and every bottle containing soluble component 0.9 gram.J02 adds cultivating system by concentration dilution after adding sodium chloride for injection solution to dissolve.
1.2 reagent: RPMI-1640, DMEM(Gibco), calf serum (Vicente, 20130118),
Trypsin (Amresco), MTT(Sigma) ,DMSO(Sigma)。
1.3 cell strain: the MDA-MB-231(human breast cancer cell), the HPEG2(human liver cancer cell), B16F10
(melanoma cell), the A549(human lung carcinoma cell), the MCF-7(human breast cancer cell) and, this laboratory is preserved.
1.4 instrument: GS-15R tabletop refrigerated centrifuge (Beckman); The BP310s electronic balance
(sartorius); Microfuge microcentrifuge (Beckman); DK-8D electric heating constant temperature water bath (the upper grand experimental facilities company limited of Nereid); MS1 Minishaker (IKA); Laminar-flow rack (safe and sound company of Su Jing group); CO
2incubator (Forma scientific); Pureplus demineralizer (U.S.A); SW-CJ-1FD clean work station (safe and sound company of Su Jing group); Biotek Synerge 2 microplate reader (Biotek); Zeiss AVIVO type inverted microscope (Zeiss);
2) experimental technique
2.1) inoculating cell: the tumor cell with 0.25% trypsinization in exponential phase, with containing 10%
The RPMI1640 culture fluid of hyclone is made into the individual cells suspension, with every hole 2 * 10
4individual cell is inoculated in 96 orifice plates, and culture plate is moved into to CO
2in incubator, at 37 ℃, 5%CO
2and cultivation dosing after 24 hours under the saturated humidity condition.
2.2) dosing: parallel 6 holes of each concentration of final concentration, with after the culture medium dilution, adding (blank hole: do not add cell, only add culture fluid; Negative control hole: except not dosing beyond the region of objective existence, all the other operations are identical), cell continues to cultivate 24 hours.
2.3) colour generation: abandon supernatant, it is freshly prepared containing 0.4mg ml that every hole adds 200 μ l
-1the serum-free medium of MTT, continue to cultivate 4h, then abandons and reset and add 200 μ l DMSO dissolving MTT first hairpin precipitations, with the microoscillator vibration, mixes.
2.4) colorimetric: microplate reader is measured 490nm place optical density value (with the zeroing of blank hole), is calculated as follows
Inhibition rate of tumor cell:
Cell inhibitory rate (%)=(matched group OD value-test group OD value)/matched group OD value * 100%, and calculate
Prediction IC50 value (half-inhibition concentration).
3) experimental result
Result from following table 13-to table 17 can find out, MDA-MB-231, HPEG2, MCF-7 cell are comparatively responsive to given the test agent J01, and its IC50 value is respectively 70.6 μ g/ml, 275.3 μ g/ml and 224.6 μ g/ml; The sensitivity of B16F10 and A549 cell is lower, and its IC50 value is respectively 1857.2 μ g/ml and 965.4 μ g/ml.
Following table 18 to table 21 has shown the effect situation of J02 to tumor cell.From data, be that HPEG2, MCF-7 cell are comparatively responsive to J02 equally, its IC50 value is respectively 450.7 μ g/ml and 298.6 μ g/ml; And J02 is 1302.1 μ g/ml and 1635.4 μ g/ml to the half-inhibition concentration of B16F10 and A549 cell.
Must, J01 and J02 show stronger inhibitory action to Partial tumors cell (as HPEG2, MCF-7) in cultivating system in vitro, the inhibitory action that B16F10 and A549 are showed is lower.
2, anti-tumor in vivo test
1) experiment material
1.1 sample:
J01-injection freeze-dried powder injection (pack 10 milliliters of amperes bottled, every bottle containing Aloe composition 0.75 gram) concentration intraperitoneal injection on request after adding sodium chloride for injection solution to dissolve;
J02-injection freeze-dried powder injection (pack 10 milliliters of amperes bottled, every bottle containing Aloe composition 0.9 gram), add sodium chloride for injection solution to dissolve after on request concentration carry out intraperitoneal injection.
Gemcitabine, the 0.2g/ bottle, gift comes company.
1.2 reagent: DMEM(Gibco), calf serum (Vicente, 20130118), Trypsin (Amresco),
DMSO(Sigma),
1.3 animal: the C57BL/6 mice, 18-22g, male and female half and half, provided by Yangzhou University's comparative medicine center.
The laboratory animal quality certification: SCXK (Soviet Union) 2012-0004
1.4 cell strain: the H22 hepatoma cell strain is provided by this laboratory.
1.5 instrument: GS-15R tabletop refrigerated centrifuge (Beckman); BP310s electronic balance (sartorius);
Microfuge microcentrifuge (Beckman); DK-8D electric heating constant temperature water bath (the upper grand experimental facilities company limited of Nereid); MS1 Minishaker (IKA); Laminar-flow rack (safe and sound company of Su Jing group); CO
2incubator (Thermal); Millipore Q8 pure water instrument (Millipore, U.S.A); SW-CJ-1FD clean work station (safe and sound company of Su Jing group); Zeiss AVIVO type inverted microscope (Zeiss);
2) mice is at body tumor growth model
The good H22 hepatoma carcinoma cell with 0.25% trypsinization adherent growth, add culture fluid suspendible cell, and then 800 rpm/5min are centrifugal, abandon supernatant, then, with serum-free medium suspendible tumor cell, adjust cell number to 5 * 10
6individual/ml.Then in every mice left side inoculated tumour cell suspension 0.2 ml of the subcutaneous place of groin (approximately 10
6individual).Mice Inoculated is 60 altogether, is divided at random 6 groups, 10 every group.Inoculation starts lumbar injection and gives ZD-01 and ZD-02 next day, and continuous 21 days, the active situation of observing mice every day, measured the subcutaneous place of mice groin tumor growth situation in every three days.Put to death mice after administration finishes, strip the separation tumor tissue, with scales/electronic balance weighing, record the tumor weight in wet base.Mice is raised in the clean environment laboratory.
3) statistical method
Each is organized the mouse tumor weight in wet base and means with M ± SD.Compare the difference between administration group and model group by the t-test method of inspection, with p<0.05, mean to have significant difference.
4) result
As can be seen from Figure 3, the model group mice is after transplanting inoculation H22 tumor cell, and the volume size of tumor is along with the prolongation of time presents the trend increased gradually, and, in the growth later stage of tumor, obviously increasing appears in the volume of tumor.J01 administration group is basic and model group indifference at first week, continues administration and finds that the growth trend of tumor is milder than model group, illustrates that the intervention of J01 produces certain effect.And, in the time of the 3rd week, J01 high and low dose administration group mouse tumor volume and matched group more all have obvious difference (p<0.01).The mouse tumor weight in wet base that this result is also in the end processed is verified (seeing the following form 22) in relatively, and J01 administration group mouse tumor weight in wet base significantly is less than matched group (p<0.01).
The intervention effect performance of J02 is basic similar to J01.After intervention, also can obviously suppress tumor at bulk-growth, but it intervenes performance than a little less than J01.J02 administration high and low dose group mouse tumor weight in wet base significantly is less than matched group (p<0.05 and p<0.01) (seeing the following form 22).
Table 22. J02 is on the impact in body mouse transplanting tumor H22 growth
Group | Dosage mg/kg | n | Tumor weight in wet base (g) |
Model group | -- | 10 | 1.899± 0.492 * |
Gemcitabine | 200 | 10 | 0.957± 0.259 ** |
The J01 low dosage | 320 | 10 | 1.194± 0.510 ** |
The J01 high dose | 640 | 8 | 1.137± 0.299 ** |
The J02 low dosage | 900 | 10 | 1.319± 0.361 ** |
The J02 high dose | 1800 | 7 | 1.353± 0.353 * |
Annotate: compare * p<0.05, * * p<0.01 with model group.
5) conclusion and discussion
Experiment shows that J01 has obvious inhibitory action to kinds of tumor cells in body, and in-vivo tumour transplantation experiments result also show J01 to tumor at bulk-growth, inhibitory action is arranged, illustrate that J01 manifests obvious antitumor action.Although J01 to the inhibitory action of the cancerous cell such as B16F10 and A549 a little less than, show that J01 may have stronger effect to some tumor, with the type of tumor, certain dependency is arranged.
In the body of J02, experimental result also shows that it has certain antitumor action.The J02 depression effect of the high dose in anti-tumor experiment in vivo is low than the effect of low dosage, and this may be relevant to some effect characteristics of Chinese medicine.Concrete needs are paid close attention to the component analysis of this tested material.
3, Aloe injection freeze-dried powder injection anti-liver injury effect research of the present invention
1) purpose
The application carbon tetrachloride causes the acute liver injury of rats model, freeze dried powder given the test agent J01 prepared by investigation the present invention and the anti-liver injury effect of J02.
2) material
Animal: male SD rat, the 250-300g left and right, provided the animal quality certification number: SCXK(Zhejiang) 2008-0033 by Zhejiang Province's Experimental Animal Center.
Reagent: carbon tetrachloride (CCl
4directly miscible in the middle of vegetable oil, with volume ratio, V/V is configured to 20%).
The J01-injection freeze-dried powder injection, be light brown, specification: every bottle containing soluble component 0.75 gram.
The J02-injection freeze-dried powder injection is brown, specification: every bottle containing soluble component 0.9 gram.Directly use the Injectable sterile physiological saline solution during administration, and be diluted to concentration used.
Diammonium glycyrrhizinate injection, specification: 10ml: 50mg, lot number: 20130407 Jiangsu Zhengda Tianqing Drug Industry Co., Ltd provide.
3) method
3.1 carbon tetrachloride causes liver injury model
Get 60 of healthy male SD rats, body weight 250-300g, sub-cage rearing.Environment is constant temperature (23 ± 2 ℃), constant humidity (55 ± 10%).Every rats by intraperitoneal injection 20% carbon tetrachloride oil solution 10ml/kg(high dose, disposable liver injury) once, cause Liver Damage in Rats.Then the carbon tetrachloride hepatic injury rat model is divided into to 6 groups at random, be respectively: model group (equal-volume 0.9% normal saline solution), diammonium glycyrrhizinate group (30mg/kg), J01 high dose group (200mg/kg), J01 low dose group (100mg/kg), J02 high dose group (240mg/kg), J02 low dose group (120mg/kg), separately set up one group of normal control rat (not inject CCl
4, give the equal-volume normal saline).Then intervened with corresponding tested medicine, continued 7 days.After administration finishes, the blood sampling of rat eye socket rear vein beard, (4 ℃, 3600rpm/10min) separation of serum, measure Biochemical Indices In Serum or-20 ℃ of preservations to low-temperature centrifugation immediately, while selecting, measures.Simultaneously, take out rapidly rat liver, perusal liver situation, and weigh and calculate the liver index.Then be fixed with 10% formaldehyde, carry out follow-up pathology HE dyeing.Within in experiment every 3 days, measure the body weight of a rat, record behavior, the active state of rat every day, the one-tenth mould situation of auxiliary judgment rat and the intervention situation of medicine.Heavy (the g)/rat body weight (g) * 100 of liver index=liver.
3.2 administering mode and time
Adopt intraperitoneal injection, 5ml/kg, once a day, administration is 7 days altogether.
3.3 evaluation index
Serum Indexes: the ALT(glutamate pyruvate transaminase), the AST(glutamic oxaloacetic transaminase, GOT), the ALP(alkali phosphatase), the TBIL(total bilirubin), the TP(total protein), the ALB(albumin), A/G Archon ratio.
3.4 statistical method
Continuous data adopts SPSS10.0 to carry out ANOVA T-TEST check group difference.
4) experimental result
4.1 J01 and J02 intervene the perusal result after the hepatic injury rat
Perusal normal rat liver color clear, smooth surface, quality softness.The model group rat liver is greyish white, the rough surface of color, the hardening of quality compared with normal liver.J01 intervention group rat liver is faint in color, mattness, and quality is slightly hard, between two dosage groups, without obvious, distinguishes; J02 intervention group rat liver performance is distinguished to some extent with the J01 intervention group, and liver is faint in color but gloss slightly, and surface is slightly coarse, and quality is softer; Diammonium glycyrrhizinate group rat liver color is slightly dark, quality is soft.
4.2 J01 and the J02 impact on hepatic injury rat biochemical index
From detected biochemical parameter, the model group rat raises because CCl4 causes liver function index to hepatocellular destruction comprehensively, and ALT, AST, ALP, TBIL etc., all apparently higher than Normal group, relatively have significant difference (p<0.05).TP, ALB etc. obviously reduce, and the liver index significantly raises simultaneously, and liver swelling is obvious.Although J01 intervenes result demonstration 200mg/kg, 100 mg/kg two dosage see that from numerical value ALT, AST, ALP are had to certain reducing effect, and comparison test unknown significance difference (p > 0.05) (seeing the following form 23); On the TBIL(total bilirubin) impact is not quite.From data, the J02 high and low dose all has obvious reducing effect to ALT, AST, the ALP raise, and with model group, significant difference is relatively arranged; Simultaneously J02 can significantly reduce rat liver TBIL level (p<0.05 and p<0.01) (seeing the following form 24), and the liver exponent data also shows, J02 can obviously be alleviated the liver swelling of rat, and liver is had to significant protective effect (seeing the following form 25).The indicator and model groups such as the total protein of each administration intervention group, albumin, globulin and Archon compare relatively are showed no significant change (seeing the following form 23,24).
The impact of table 23. J01 on hepatic injury rat biochemical indicator
Annotate: compare $ $ p<0.01 with normal group; Compare * p<0.05, * * p<0.01 with model group.
The impact of table 24. J02 on hepatic injury rat biochemical indicator
Annotate: compare $ $ p<0.01 with normal group; Compare * p<0.05, * * p<0.01 with model group.
Table 25. J01 and the J02 impact on hepatic injury rats'liver index
Annotate: compare $ $ p<0.01 with normal group; Compare * p<0.05, * * p<0.01 with model group.
5) experiment conclusion
The conceptual data demonstration, the effect of J02 reduction transaminase, the swelling of alleviation liver is comparatively obvious, has obvious anti-liver injury effect.Although J01 has certain reducing effect to the transaminase, synthetic data sees, the effect of anti-liver injury fails obviously to embody.
Claims (2)
1. a method of using Aloe powder to prepare the Aloe injection freeze-dried powder injection, is characterized in that: comprise the steps:
The first step, prepare Aloe powder and distilled water in the ratio of 100:2000 weight portion, obtain 5% aloe water solution;
Second step, evenly make Aloe powder dissolve fully the aloe water solution stirring, and standing over night is separated;
The 3rd step, the supernatant liquid filtering clarification after second step is separated;
The 4th step, the filtrate that the 3rd step is obtained is adjusted to 6.3-6.5 with hydrochloric acid by pH value, is heated to 115 ℃ of-118 ℃ of sterilizings 30 minutes, then natural cooling;
The 5th step, the pH value of the liquid coolant the 4th step obtained with hydrochloric acid is adjusted to 4.2-4.5, through the ultrafilter membrane ultrafiltration, holds back and obtains the drug solns that contains that molecular weight is not more than 10000Da;
The 6th step, the medicinal liquid that the 5th step is obtained is divided in lyophilizing in ampoule bottle and gets final product.
2. use Aloe powder according to claim 1 prepares the method for Aloe injection freeze-dried powder injection, it is characterized in that:
The preparation method of described Aloe powder is:
The first step, soak 30-60 minute by the fresh aloe leaf, is ground into the Aloe juice after cleaning up;
Second step, pour the Aloe juice in the reacting by heating tank into and import and be steam heated to 8-12 ℃, adds pectase or protease by 0.125% of Aloe juice gross weight simultaneously, after 50-60 minute, temperature risen to 75-80 ℃, and inactivation treatment is carried out in insulation in 3-5 hour;
The 3rd step, by second step after inactivation treatment solution filter, concentrated final vacuum drying or lyophilization to obtain outward appearance be flaxen Aloe powder finished product.
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CN1411824A (en) * | 2001-10-12 | 2003-04-23 | 湖南金正方生物科技有限公司 | Biological pourpoint depression and nano concentration process for extracting aloe juice |
CN1634251A (en) * | 2004-10-11 | 2005-07-06 | 贵阳云岩西创药物科技开发有限公司 | Compound medicinal gel of aloe and its preparation method |
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CN1634251A (en) * | 2004-10-11 | 2005-07-06 | 贵阳云岩西创药物科技开发有限公司 | Compound medicinal gel of aloe and its preparation method |
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