CN100349582C - Oil seed bruceae lyophilized powder for injection and its preparing method - Google Patents
Oil seed bruceae lyophilized powder for injection and its preparing method Download PDFInfo
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- CN100349582C CN100349582C CNB2005101018412A CN200510101841A CN100349582C CN 100349582 C CN100349582 C CN 100349582C CN B2005101018412 A CNB2005101018412 A CN B2005101018412A CN 200510101841 A CN200510101841 A CN 200510101841A CN 100349582 C CN100349582 C CN 100349582C
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Abstract
The present invention relates to oil seed bruceae lyophilized powder for injection, which is characterized in that the oil seed bruceae is further purified by the application of novel macroporous resin so that toxic ingredients in the oil seed bruceae can be effectively removed. The oil seed bruceae lyophilized powder for injection is prepared by using cyclodextrin clathration technology and lyophilizing technology, and the problems of past oil and milk preparations are solved. The oil seed bruceae lyophilized powder for injection, which is provided by the present invention, has simple processing steps. The transition rate of active components of oleic acid and linoleic acid is enhanced by using novel macroporous resin. The toxic ingredients in the oil seed bruceae are effectively removed, and safety is enhanced. The oil seed bruceae lyophilized powder for injection, which is provided by the present invention, has stable quality and is beneficial to transportation and conservation and has the advantages of advanced technology and outstanding curative effects.
Description
Technical field:
The present invention relates to the pure Chinese medicine Fel Anas domestica of a kind of antineoplastic seed oil injection type medicine, particularly a kind of oil seed bruceae lyophilized powder for injection and preparation method thereof.
Background technology:
Fructus Bruceae is Simarubaceae brucea plant Fructus Bruceae (another name Semen Sophorae Flavescentis) fruit, evergreen big shrub or dungarunga, and up to 3 meters, Herb is all by yellow pubescence.The odd number winglike compound leaf, alternate, has long handle by long 10-30 centimetre; Lobule 5-11 piece, to life, long ovum shape lanceolar, long 5-10 centimetre, wide 2-4 centimetre, tip is point gradually, base portion wedge shape or anisopleural oblique circle, there is the triangle bastard at the edge, above green, below light green.Circular cone cyme axil is given birth to, dioecism, the long 10-30 of staminate inflorescence centimetre, the long 4-18 of female inflorescence centimetre; Spend minimum, reddish yellow; Male flower sepal 4, lanceolar, petal 4, the wire lanceolar, stamen 3, female flower sepal 4, triangle, petal 4, oval shape lanceolar, ovary is made up of 4 carpels, and big portion is from life, and the bottom is surrounded by floral disc, and style is curved down, and stigma is long pointed.Drupe is long avette, and tip omits to excurvation, black when ripe, bossed reticulate pattern.The florescence 3-8 month.The fruit phase 4-9 month (Hainan Island, Guangxi).Be grown in meadow, the bushes and the roadside area without shade.Ground such as distribution Fujian, Guangxi, Yunnan, Taiwan, Guangdong.The fruit of drying and ripening is Long Circle or avette, and two is point slightly, long 6-10 millimeter, diameter 3-7 millimeter.The surface grey black has irregular polygonal reticulate pattern, and there is the fruit stem trace of depression the bottom.Outer chitin is hard and crisp, breaks back inner face bois de rose or lark, and is smooth and glossy.In the yellow-white kernel is arranged, be avette, long 4-7 millimeter, diameter 3-5 millimeter, the thin film of outsourcing shirring is rich in oiliness.Odorless, flavor are extremely bitter., oiliness sufficient person white with matter heavily fortified point, core is good.
Fructus Bruceae in China's application among the people, has heat clearing away very early, dampness, parasite killing, antidotal effect.Control dysentery, chronic diarrhea, malaria, hemorrhoid, furunculosis, wart, clavus." removing heat from blood goes spleen man skin ulcer, the reason traumatic injury to " the SHENGCAO property of medicine fully will " record Fructus Bruceae." detailed outline is picked up any lost article from the road " record Fructus Bruceae " is controlled dysentery, hemorrhoid." " Guangxi Chinese medicinal herbal " record Fructus Bruceae: " control infantile malnutrition.Skin tumour is controlled in external (oil also can)." since nineteen fifty, many scholars are with Fel Anas domestica seed oils treatment papillomas of external auditory meatus, laryngeal papillomatosis, cervix uteri tumor etc.Having report to use Fructus Bruceae treatment cervix uteri tumor in 1972 succeeds.Decades, scientific circles carried out a large amount of research to Fructus Bruceae subsequently, and modern study is the result show: the liposoluble constituent Fel Anas domestica seed oil of Fructus Bruceae is an effective ingredient, and wherein oleic acid, linoleic acid are considered to the antineoplastic active component; Fructus Bruceae is water-soluble, the part of tool bitterness is its toxic component.
The Fel Anas domestica seed oil milk vein injection of clinical development has stronger anticancer effect, is applicable to multiple malignant tumor such as pulmonary carcinoma, metastatic brain tumor, hepatocarcinoma, gynecological tumor, breast carcinoma.The clinical use of Fructus Bruceae oil emulsion injection in star system pharmaceutical factory, Guangzhou is arranged on the market now, the patent application of number of patent application 03156023, application number 200310108330 and application number 200310108329 also is described its preparation method, but because the restriction of technological means, present Fructus Bruceae oil emulsion injection and preparation method also exist following problem:
1) oily emulsion formulation inevitably exists the not good problem of stability, the profit layering may occur in storage process, causes hidden danger.
2) injection stores the transportation inconvenience, needs high temperature sterilize in the preparation process, causes loss of active ingredients such as a large amount of unsaturated fatty acids.
3) be unfavorable for and other water soluble drug compatibility in the clinical use of oily emulsion formulation, cause the inconvenience in the use.
4) toxic component of existing Fructus Bruceae oil refining method in still can not more effective removal Fel Anas domestica seed oil causes the clinical bad accident of present Fructus Bruceae oil emulsion injection to happen occasionally.
As seen, develop the dosage form of new Fel Anas domestica seed oil and the preparation method of safer refining Fel Anas domestica seed oil is provided, significant to the application of Fructus Bruceae in antitumor.
Summary of the invention:
One of purpose of the present invention is the deficiency that overcomes on the existing Fructus Bruceae oil making technology, provides a kind of removal toxic component more refining Fel Anas domestica seed oil.Another object of the present invention provides a kind of oil seed bruceae lyophilized powder for injection, overcomes the variety of issue of existing Emulsion.Another object of the present invention provides a kind of preparation method of oil seed bruceae lyophilized powder for injection.
In order to achieve the above object, the present invention has taked following technological means:
A kind of oil seed bruceae lyophilized powder for injection is made up of the cyclodextrin clathrate of refining Fel Anas domestica seed oil and the lyophilizing adjuvant of pharmaceutically acceptable amount.
The preferred beta-schardinger dextrin-of described cyclodextrin, more preferably HP-.
The preferred mannitol of described lyophilizing adjuvant.
A kind of cyclodextrin clathrate of refining Fel Anas domestica seed oil is provided, prepares by following steps:
(1) gets an amount of Fructus Bruceae fruit and be ground into coarse powder, to extraction pot;
(2) add petroleum ether, soak in hermetic container and extract twice, 8 times of amounts of each Fructus Bruceae fruit weight were extracted 6-9 hour, filtered merging filtrate;
(3) gained filtrate distillation is reclaimed petroleum ether to the greatest extent, gets slightly oil of Fructus Bruceae;
(4) the thick oil of Fructus Bruceae is crossed strong polar macroporous adsorption resin (the ADS-7 macroporous adsorbent resin that Synesis Company of Nankai University produces) with 5 hours flow velocity of each bed volume, collect effluent, effluent reduces pressure under 105 ℃ condition and led to nitrogen 3 hours, must make with extra care the Fel Anas domestica seed oil;
(5) in refining Fel Anas domestica seed oil, add the abundant mix homogeneously of isopyknic dehydrated alcohol, slowly join in the cyclodextrin aqueous solution of 10%-40%, under the condition that ultrasonic or high-speed shearing machine is sheared enclose 6-10 hour, the gained mixed solution is evaporated to the greatest extent, water for injection washing gained extractum, water lotion concentrates and vacuum drying after removing oil reservoir, promptly gets the cyclodextrin clathrate made from extra care the Fel Anas domestica seed oil.
Above the aqueous solution of the HP-of preferred 30% concentration of cyclodextrin aqueous solution in the described step (5), after the solution decompression of described enclose is concentrated into to the greatest extent, the preferred water for injection washed twice that adds 10 times of amounts, each 5 times of amounts, water lotion merges the back and adds melt paraffin, spends the night to freezer, removes the wax layer, aqueous solution concentrates and vacuum drying, promptly gets the cyclodextrin clathrate made from extra care the Fel Anas domestica seed oil.
The present invention also provides a kind of preparation method of oil seed bruceae lyophilized powder for injection, is prepared by following steps:
(1) after the cyclodextrin clathrate of getting the refining Fel Anas domestica seed oil of recipe quantity fully is dissolved in the water for injection, adds an amount of lyophilizing adjuvant, fully dissolving;
(2) add the injection water and be adjusted to volume requiredly, adjust acid-base value to 6.0-7.0;
(3) add 0.05% active carbon after being heated to 80 ℃, kept 30 minutes;
(4) lyophilizing conventional method decarburization, fine straining, packing is jumped a queue, lyophilizing, gland, promptly.
The mannitol of preferred 3% concentration of lyophilizing adjuvant that adds in the step (1).
The preparation method of oil seed bruceae lyophilized powder for injection of the present invention has solved the water solublity problem of Fructus Bruceae oil formulation by the technology of cyclodextrin inclusion compound, for clinical use provides convenience.The dosage form of lyophilized powder is more conducive to storage, transportation and the use of preparation.The use of strong polar macroporous adsorption resin is more favourable to removing pigment and toxicity water-soluble components, and has been further purified the Fel Anas domestica seed oil, makes index components reach more than 80% of total oil mass, meets the specification requirement of Chinese medicine injection.Experimental result shows that oil seed bruceae lyophilized powder for injection provided by the invention has better kept active ingredients such as oleic acid than former Fructus Bruceae oil emulsion injection, and preparation is safer easy to use.
Specific embodiment:
Embodiment 1: the preparation of refining Fructus Bruceae oil ring cyclodextrin inclusion compound
Get 5kg Fructus Bruceae fruit and be ground into coarse powder, to extraction pot; Add the 40L petroleum ether, in hermetic container, soak under the stirring condition and extracted 7 hours, filter and emit extracting solution.Medicinal residues add the 40L petroleum ether and stirring again and soak extraction 7 hours, filter merging filtrate.Gained filtrate is reclaimed petroleum ether to the greatest extent to concentration tank, get the thick oily 620ml of Fructus Bruceae.The thick oil of Fructus Bruceae is crossed the good strong polar macroporous adsorption resin of pretreatment (the ADS-7 macroporous adsorbent resin that Synesis Company of Nankai University produces), flow velocity is each bed volume 5 hours, collect effluent, effluent reduces pressure under 105 ℃ condition and led to nitrogen 3 hours, must make with extra care Fel Anas domestica seed oil 480ml; In refining Fel Anas domestica seed oil, add the abundant mix homogeneously of 480ml dehydrated alcohol, mixed solution slowly joins under the high-speed shearing machine shearing condition in the beta-schardinger dextrin-aqueous solution of 16L 30%, and continued enclose 8 hours, the gained mixed solution is evaporated to the greatest extent, must mix extractum, add 2000ml water for injection washing gained extractum, add fused paraffin 200ml, fully stir, put cold back and places to the freezer and spend the night, remove the wax layer and filter, filtrate decompression concentrates also vacuum drying, must make with extra care the cyclodextrin clathrate 1080g of Fel Anas domestica seed oil, lot number: 040723.
Embodiment 2: the preparation of refining Fructus Bruceae oil ring cyclodextrin inclusion compound
Get 5kg Fructus Bruceae fruit and be ground into coarse powder, to extraction pot; Add the 40L petroleum ether, in hermetic container, soak under the stirring condition and extracted 8 hours, filter and emit extracting solution.Medicinal residues add the 40L petroleum ether and stirring again and soak extraction 8 hours, filter merging filtrate.Gained filtrate is reclaimed petroleum ether to the greatest extent to concentration tank, get the thick oily 630ml of Fructus Bruceae.The thick oil of Fructus Bruceae is crossed the good strong polar macroporous adsorption resin of pretreatment (the ADS-7 macroporous adsorbent resin that Synesis Company of Nankai University produces), flow velocity is each bed volume 5 hours, collect effluent, effluent reduces pressure under 105 ℃ condition and led to nitrogen 3 hours, must make with extra care Fel Anas domestica seed oil 475ml; In refining Fel Anas domestica seed oil, add the abundant mix homogeneously of 475ml dehydrated alcohol, mixed solution slowly joins under the high-speed shearing machine shearing condition in the HP-aqueous solution of 22.8L 25%, and continued enclose 8 hours, the gained mixed solution is evaporated to the greatest extent, must mix extractum, add 2000ml water for injection washing gained extractum, add fused paraffin 200ml, fully stir, put cold back and places to the freezer and spend the night, remove the wax layer and filter, filtrate decompression concentrates also vacuum drying, must make with extra care the cyclodextrin clathrate 1120g of Fel Anas domestica seed oil, lot number 040802.
Embodiment 3: the preparation of oil seed bruceae lyophilized powder for injection
Get the cyclodextrin clathrate 280g of the lot number 050802 refining Fel Anas domestica seed oil of embodiment 2 preparations, put adding 2000ml water for injection in the Agitation Tank, stir fully dissolving, put into freezer cold preservation after the cooling 24 hours.The taking-up after-filter filters, and filtrate adds 30g mannitol, and heated and stirred is fully dissolved.Add the injection water and be adjusted to 2500ml, adjust acid-base value to 6.0-7.0 (actual measurement 6.4) with 20% sodium hydroxide.Add the 1.25g active carbon after being heated to 80 ℃, kept temperature 30 minutes, the decarburization of titanium rod, 0.22 micron microporous filter membrane aseptic filtration is sub-packed in the 10ml cillin bottle, and every 4ml jumps a queue, lyophilizing, gland, that is, and lot number: 040905.
Embodiment 4: the preparation of oil seed bruceae lyophilized powder for injection
Get the cyclodextrin clathrate 280g of the lot number 050802 refining Fel Anas domestica seed oil of embodiment 2 preparations, put adding 2000ml water for injection in the Agitation Tank, stir fully dissolving, put into freezer cold preservation after the cooling 24 hours.The taking-up after-filter filters, and filtrate adds 30g mannitol, and heated and stirred is fully dissolved.Add the injection water and be adjusted to 2500ml, adjust acid-base value to 6.0-7.0 (actual measurement 6.6) with 20% sodium hydroxide.Add the 1.25g active carbon after being heated to 80 ℃, kept temperature 30 minutes, the decarburization of titanium rod, 0.22 micron microporous filter membrane aseptic filtration is sub-packed in the 10ml cillin bottle, and every 4ml jumps a queue, lyophilizing, gland, that is, and lot number: 040917.
Embodiment 5: the preparation of oil seed bruceae lyophilized powder for injection
Get the cyclodextrin clathrate 280g of the lot number 050802 refining Fel Anas domestica seed oil of embodiment 2 preparations, put adding 2000ml water for injection in the Agitation Tank, stir fully dissolving, put into freezer cold preservation after the cooling 24 hours.The taking-up after-filter filters, and filtrate adds 30g mannitol, and heated and stirred is fully dissolved.Add the injection water and be adjusted to 2500ml, adjust acid-base value to 6.0-7.0 (actual measurement 6.3) with 20% sodium hydroxide.Add the 1.25g active carbon after being heated to 80 ℃, kept temperature 30 minutes, the decarburization of titanium rod, 0.22 micron microporous filter membrane aseptic filtration is sub-packed in the 10ml cillin bottle, and every 4ml jumps a queue, lyophilizing, gland, that is, and lot number: 040929.
Embodiment 6: contrast this product oil seed bruceae lyophilized powder for injection and commercially available Fructus Bruceae oil emulsion injection
One: assay:
1) glycerol trioleate detection method: precision is measured this product 1g, puts in the 25ml measuring bottle, adds petroleum ether (60-70 ℃) and is diluted to scale, shakes up, as need testing solution.Other gets the glycerol trioleate reference substance solution, according to the thin layer chromatography test, draw need testing solution 1 μ l, reference substance solution 1 μ l, 2 μ l, the cross point be in same silica gel G ripple once quite a while respectively, with petroleum ether (60-90 ℃)-ethyl acetate (16: 1) is developing solvent, launch, take out two bars, it is smoked clear to the least bit to put in the iodine steam, take out,, take out in 105 ℃ of dry 2-3 minutes, on lamellae, cover the glass plate of identical size, use the adhesive plaster Apis all around, scan wavelength according to thin layer chromatography: λ S=400nm, λ r=650nm, measure test sample and reference substance play capital marking system, calculate, promptly.
2) oleic acid detection method: according to gas chromatography determination
Chromatographic condition: with 5% phenyl, 95% methyl polysiloxane is immobile phase, and (25m*0.25 μ m (crosslinked): column temperature is 250 ℃ to capillary column.Theoretical cam curve is pressed the oleic acid peak and is calculated, and should be not less than 2500, and the separating degree of oleic acid and internal standard substance should be greater than 2.
Algoscopy: precision takes by weighing this product 1g, and to the 10ml volumetric flask, the accurate inner mark solution 1ml that adds adds normal hexane and is diluted to scale, shakes up.Measure 0.5 μ l such as in the gas chromatograph, measure, calculate, promptly.
3) linoleic acid detection method: according to gas chromatography determination
Chromatographic condition: with 10% phenyl, 90% methyl polysiloxane is immobile phase, and (25m*0.25 μ m (crosslinked): column temperature is 250 ℃ to capillary column.Theoretical cam curve is pressed the oleic acid peak and is calculated, and should be not less than 2500, and the separating degree of linoleic acid and internal standard substance should be greater than 2.
Algoscopy: precision takes by weighing this product 0.5g, and to the 10ml volumetric flask, the accurate inner mark solution 1ml that adds adds normal hexane and is diluted to scale, shakes up.Measure 0.5 μ l such as in the gas chromatograph, measure, calculate, promptly.
Result: get this product and commercially available Fructus Bruceae oil emulsion injection (Mingxing Pharmaceuticals Co., Ltd., Guangzhou City) detects glycerol trioleate, oleic acid and linoleic content respectively, see Table 1:
Table 1: assay comparative result
| Color and luster | The amount of glycerol trioleate (mg/ props up) | Oleic amount (mg/ props up) | Linoleic amount (mg/ props up) | |
| 040905 | Of light color | 42.3 | 12.2 | 6.0 |
| 040917 | Of light color | 41.5 | 12.6 | 6.2 |
| 040929 | Of light color | 42.8 | 12.7 | 6.8 |
| Commercially available Fructus Bruceae | Color | 13.4 | 5.5 | 2.1 |
| Oil emulsion injection | Deeply |
Show by result of the test: by the oil seed bruceae lyophilized powder for injection (three batches) of the present invention's preparation, active component oleic acid and linoleic amount are all apparently higher than the commercially available prod, and the amount of index components glycerol trioleate is higher, meets the restriction that accounts for solid content in the Chinese medicine injection specification requirement about index components.And three batch sample results show process stabilizing, good reproducibility.
Two: injection routine examination item:
[inspection]
Clarity is according to the regulation inspection of " clarity test detailed rules and regulations and criterion ", and every bottle of content adds injection water 4ml makes dissolving, should be up to specification.
Moisture is got this product, measures (an appendix IXH of Chinese Pharmacopoeia version in 2000 aquametry first method) in accordance with the law, and water content must not cross 5.0%.
Protein is got one bottle of this product, adds injection water 4ml and makes dissolving, measures 1ml, adds 1～3 of tannic acid test solution, must not become turbid.
Tannin is got one bottle of this product, adds injection water 3ml and makes dissolving, measures 1ml, adds the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, shakes up, and places 10 minutes, muddiness or precipitation must not occur.
Residue on ignition is got 2 bottles of this product, and precision adds water 6ml makes dissolving, and precision is measured 4ml, put in the crucible of ignition to constant weight, and evaporate to dryness, check (" appendix IXJ of Chinese pharmacopoeia version in 2000). in accordance with the lawLeave over residue and must not cross 1.5% (g/ml).
Particulate matter is got 4 bottles of contents of this product, adds injection water 12ml and makes dissolving, according to microscopic counting (Chinese Pharmacopoeia appendix IXR in 2000), contains the above microgranule of 10 μ m among every 1ml and must not cross 20, contains the above microgranule of 25 μ m and must not cross 2.
Heavy metal is got this product 1g, the residue of leaving over according to residue on ignition algoscopy (" appendix IXJ of Chinese pharmacopoeia version in 2000) operation gained, check (" appendix IXE second method of Chinese pharmacopoeia version in 2000), contain heavy metal and must not cross 10/1000000ths. in accordance with the law
Arsenic salt is got this product 1g, according to the residue of leaving over of residue on ignition algoscopy (" appendix IXJ of Chinese pharmacopoeia version in 2000) operation gained, check (" appendix IXF first method of Chinese pharmacopoeia version in 2000), arsenic content must not cross 2/1000000ths. in accordance with the law
Resin is got 2 bottles of contents of this product, adds injection water 6ml and makes dissolving, measures 5ml, adds chloroform 10ml jolting and extracts, divide and get chloroform solution, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, add water 3ml, mixing was placed 30 minutes, should not have the resin-like thing and separated out.
Oxalates is got one bottle of this product, adds injection water 3ml and makes dissolving, measures 2ml, check " appendix IXS of Chinese pharmacopoeia version in 2000), muddiness or precipitation must not appear. in accordance with the law
Aseptic one bottle of this product of getting adds injection water 3ml and makes dissolving, gets 1ml, checks (an appendix XIII of Chinese Pharmacopoeia version in 2000 B) in accordance with the law, should be up to specification.
Pyrogen is got one bottle of content of this product, adds normal saline solution 10ml and makes dissolving, by the every 1kg injection of rabbit body weight 2ml, checks (an appendix XIII of Chinese Pharmacopoeia version in 2000 A) in accordance with the law, should be up to specification.
The undue toxicity gets one bottle of content of this product, adds normal saline solution 10ml and makes dissolving, check (" two appendix XIC of Chinese pharmacopoeia version in 2000). in accordance with the lawPress the intravenous injection administration, should be up to specification.
Potassium ion is got one bottle of this product, adds injection water 3ml and makes dissolving, and precision is measured 2ml, check " appendix IXS of Chinese pharmacopoeia version in 2000), should be up to specification. in accordance with the law
Other should meet every regulation relevant under the injection item (" Chinese pharmacopoeia) appendix IU of version in 2000).
Experimental result sees Table 2:
Table 2: oil seed bruceae lyophilized powder for injection check result
| Project | Check result | ||
| Lot number: 040905 | Lot number: 040917 | Lot number: 040929 | |
| Content uniformity | Up to specification | Up to specification | Up to specification |
| Moisture | 2.26％ | 2.45％ | 2.64％ |
| Clarity | Up to specification | Up to specification | Up to specification |
| Aseptic | Up to specification | Up to specification | Up to specification |
| Protein | Do not detect | Do not detect | Do not detect |
| Tannin | Do not detect | Do not detect | Do not detect |
| Heavy metal | Do not cross 10/1000000ths | Do not cross 10/1000000ths | Do not cross 10/1000000ths |
| Arsenic salt | Do not cross 2/1000000ths | Do not cross 2/1000000ths | Do not cross 2/1000000ths |
| Oxalates | Do not detect | Do not detect | Do not detect |
| Resin | Do not detect | Do not detect | Do not detect |
| Particulate matter | Up to specification | Up to specification | Up to specification |
| Residue on ignition | 0.28％ | 0.36％ | 0.33％ |
| Potassium ion | Up to specification | Up to specification | Up to specification |
| Pyrogen | Up to specification | Up to specification | Up to specification |
| The undue toxicity | Up to specification | Up to specification | Up to specification |
| Zest | Nonirritant | Nonirritant | Nonirritant |
| Haemolysis and cohesion | No haemolysis and cohesion | No haemolysis and cohesion | No haemolysis and cohesion |
Experimental result shows, meets the requirement of Chinese medicine injection conventional sense item, steady quality by the oil seed bruceae lyophilized powder for injection of the present invention's preparation.
Embodiment 7: Oleum Fructus Bruceae is to the effect of gastric carcinoma cells BGC-A23 in vitro toxicity
The experiment medicine: Fructus Bruceae powder pin (0.5ml Oleum Fructus Bruceae /)
Normal saline
Gastric carcinoma cells (BGC-A23)
Experimental technique: with in good condition, the BGC-A23 cell that grows to exponential phase carries out cell counting with the trypan blue repelling attack, according to the cell number of counting, with cell complete medium furnishing 5 * 10
4Culture fluid is abandoned after being inoculated in and cultivating 24h in 96 well culture plates in individual/hole.Experimental group adds the Oleum Fructus Bruceae of variable concentrations, and 6 concentration of setting are respectively 0.01ml/L, 0.05ml/L, 0.10ml/L, 0.25ml/L, 0.50ml/L, 1.00ml/L; The blank group only adds the culture fluid with medicine equivalent; The zeroing group is the equivalent culture fluid, does not contain cell and medicine.Establish 6 multiple holes for every group and cultivate 8h respectively, 20h, 44h, 68h, abandon culture fluid behind the 92h, every hole adds MTT (1g/L) 50ul, continues to cultivate 4h, abandons supernatant, every hole adds 10%SDS100ul, hatch 30min for 37 ℃, microplate reader is measured each hole in the absorbance A at 570nm wavelength place (OD value), calculates suppression ratio (IR): IR (%)=[(OD with following formula
Contrast-OD
Medication)/OD
Contrast] * 100%.The results are shown in Table 3.
Table 3 Oleum Fructus Bruceae is to the effect of gastric carcinoma cells BGC-A23 in vitro toxicity
| The variable concentrations group | Hole count | Different time suppression ratio (%) | ||||
| 8h | 20h | 44h | 68h | 92h | ||
| 0.01ml/L 0.05ml/L 0.10ml/L 0.25ml/L 0.50ml/L 1.00ml/L | 8 8 8 8 8 8 | 10.3±2.3 15.6±2.6 20.4±2.5 29.1±2.6 38.8±3.0 40.2±3.2 | 18.6±3.3 25.4±3.5 35.4±3.4 41.3±3.6 49.5±3.9 58.7±4.4 | 23.5±3.2 34.1±4.0 42.4±3.7 50.6±4.5 62.4±4.2 76.8±5.7 | 30.5±3.7 37.2±3.8 49.4±4.2 61.4±4.5 74.5±4.9 86.7±6.3 | 35.4±4.0 40.5±4.1 60.2±4.5 72.4±4.8 85.6±5.8 92.3±6.7 |
Embodiment 8: Oleum Fructus Bruceae is to the intravital inhibitory action of gastric carcinoma cells BGC-A23
Fructus Bruceae powder pin (0.5ml Oleum Fructus Bruceae /)
Normal saline
Fluorouracil group (5-FU)
Gastric carcinoma cells (BGC-A23)
Laboratory animal: 60 of Kunming mouses, male and female half and half.
Experimental technique: get frozen BGC-A23 cell, cell suspension is made with normal saline in the recovery back, and platform is expected blue repelling attack living cell counting number, by 2 * 10
6Cell inoculation is in 10 mouse peritoneals, and after about 7～10 days, obvious ascites appears in mice.Get the mice of above-mentioned abdominal cavity inoculated tumour cell, the sterilization abdominal part, aseptic extraction peritoneal fluid, with the 1000r/min rotating speed centrifugal 5 minutes, supernatant discarded, normal saline washes twice, and platform is expected blue repelling attack living cell counting number.Be made into 1 * 10 with normal saline
7Cell/ml cell suspension, right side of mice oxter subcutaneous injection 0.2ml/ are only.Mouse hypodermic inoculation second day is divided into 5 groups at random with it, and 10 every group, the high, medium and low dosage group of Oleum Fructus Bruceae is pressed 0.6ml/kg respectively, 0.4ml/kg, and 0.2ml/kg irritates the Oleum Fructus Bruceae suspension that stomach gives respective concentration.Every day 1 time, continuous 10 days; Fluorouracil group (5-FU) is pressed 20mg/kg dosage lumbar injection, every day 1 time, continuous 7 days.The blank group was irritated the normal saline that stomach gives equal volume in continuous 10 days.After the last administration second day, separate tumor mass, weigh, calculate tumour inhibiting rate.Tumour inhibiting rate (%)=[(tumor is heavy
Contrast-tumor is heavy
Medication)/tumor is heavy
Contrast] * 100% the results are shown in Table 4.
Table 4 Oleum Fructus Bruceae is to the intravital inhibitory action of gastric carcinoma cells BGC-A23
| Group | Dosage | Example number (only) | Tumor heavy (g) | Tumour inhibiting rate (%) |
| Blank group Fructus Bruceae (height) Fructus Bruceae (in) Fructus Bruceae (low) 5-FU | - 0.6ml/kg 0.4ml/kg 0.2ml/kg 0.02g/kg | 10 10 10 10 10 | 1.623±0.454 0.929±0.441 ** 0.956±0.385 ** 1.037±0.400 ** 0.838±0.455 ** | - 42.7 41.1 36.1 48.3 |
Annotate: contrast with the blank group
*P＜0.01
Claims (10)
1. the cyclodextrin clathrate of a refining Fel Anas domestica seed oil is prepared by following steps:
(1) gets an amount of Fructus Bruceae fruit and be ground into coarse powder, to extraction pot;
(2) add petroleum ether, in hermetic container, soak and extract twice, add 8 times of amounts of Fructus Bruceae fruit weight at every turn, extracted 6-9 hour, filter merging filtrate;
(3) gained filtrate distillation is reclaimed petroleum ether to the greatest extent, gets slightly oil of Fructus Bruceae;
(4) the thick oil of Fructus Bruceae is crossed strong polar macroporous adsorption resin with 5 hours flow velocity of each bed volume, collect effluent, effluent reduces pressure under 105 ℃ condition and led to nitrogen 3 hours, must make with extra care the Fel Anas domestica seed oil;
(5) in refining Fel Anas domestica seed oil, add the abundant mix homogeneously of isopyknic dehydrated alcohol, slowly join in the cyclodextrin aqueous solution of 10%-40%, under the condition that ultrasonic or high-speed shearing machine is sheared enclose 6-10 hour, the gained mixed solution is evaporated to the greatest extent, water for injection washing gained extractum, water lotion concentrates and vacuum drying after removing oil reservoir, promptly gets the cyclodextrin clathrate made from extra care the Fel Anas domestica seed oil.
2. the cyclodextrin clathrate of refining Fel Anas domestica seed oil as claimed in claim 1 is characterized in that the cyclodextrin aqueous solution described in the step (5) is the aqueous solution of the beta-schardinger dextrin-of 30% concentration.
3. the cyclodextrin clathrate of refining Fel Anas domestica seed oil as claimed in claim 1 is characterized in that the cyclodextrin aqueous solution described in the step (5) is the aqueous solution of the HP-of 30% concentration.
4. as the cyclodextrin clathrate of claim 1 or 3 described refining Fel Anas domestica seed oils, after the solution decompression that it is characterized in that the described enclose of step (5) is concentrated into to the greatest extent, the water for injection washed twice that adds 10 times of amounts, each 5 times of amounts, water lotion merges the back and adds melt paraffin, spends the night to freezer, removes the wax layer, aqueous solution concentrates and vacuum drying, promptly gets the cyclodextrin clathrate made from extra care the Fel Anas domestica seed oil.
5. an oil seed bruceae lyophilized powder for injection is made up of the cyclodextrin clathrate of the described refining Fel Anas domestica seed oil of claim 1 and the lyophilizing adjuvant of pharmaceutically acceptable amount.
6. oil seed bruceae lyophilized powder for injection as claimed in claim 5 is characterized in that described cyclodextrin is beta-schardinger dextrin-or HP-.
7. as claim 5 or 6 described oil seed bruceae lyophilized powder for injection, it is characterized in that the lyophilizing adjuvant is a mannitol.
8. oil seed bruceae lyophilized powder for injection as claimed in claim 7 is characterized in that the concentration of mannitol before lyophilizing is 2%-5%.
9. the preparation method of the described oil seed bruceae lyophilized powder for injection of claim 5 is prepared by following steps:
(1) after the cyclodextrin clathrate of getting the described refining Fel Anas domestica seed oil of recipe quantity claim 1 fully is dissolved in the water for injection, adds an amount of lyophilizing adjuvant, fully dissolving;
(2) add the injection water and be adjusted to volume requiredly, adjust acid-base value to 6.0-7.0;
(3) add 0.05% active carbon after being heated to 80 ℃, kept 30 minutes;
(4) lyophilizing conventional method decarburization, fine straining, packing is jumped a queue, lyophilizing, gland, promptly.
10. the preparation method of oil seed bruceae lyophilized powder for injection as claimed in claim 9 is characterized in that the hydroxypropyl-beta-cyclodextrin inclusion of described raw material for refining Fel Anas domestica seed oil.
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| CNB2005101018412A CN100349582C (en) | 2005-12-06 | 2005-12-06 | Oil seed bruceae lyophilized powder for injection and its preparing method |
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| CN100349582C true CN100349582C (en) | 2007-11-21 |
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| CN111228319A (en) * | 2020-03-30 | 2020-06-05 | 广东省微生物研究所（广东省微生物分析检测中心） | Brucea javanica extract, preparation method thereof and application thereof in preventing and treating breast cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1539938A (en) * | 2003-10-28 | 2004-10-27 | 卞 力 | Method for preparing pure oleum fructus brucease |
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| CN1539938A (en) * | 2003-10-28 | 2004-10-27 | 卞 力 | Method for preparing pure oleum fructus brucease |
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| Title |
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| 马蔺子素冻干注射剂的制备及其稳定性考察 张学农.抗感染药学，第1卷第3期 2004 * |
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