CN106491671B - A kind of bruceolic oil emulsion, preparation method and application - Google Patents
A kind of bruceolic oil emulsion, preparation method and application Download PDFInfo
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- CN106491671B CN106491671B CN201610950514.2A CN201610950514A CN106491671B CN 106491671 B CN106491671 B CN 106491671B CN 201610950514 A CN201610950514 A CN 201610950514A CN 106491671 B CN106491671 B CN 106491671B
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/185—Magnoliopsida (dicotyledons)
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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Abstract
The present invention relates to a kind of bruceolic oil emulsions, preparation method and application, belong to technical field of traditional Chinese medicine preparation.The type and dosage that the present invention passes through change auxiliary material, devise a kind of novel bruceolic oil emulsion, the auxiliary material price that the dosage form is selected is low, auxiliary material usage amount is small, and the antitumous effect of novel form is more preferable, consisting of: brucea fruit oil 0.5ml, Tween-80 0.12ml, glycerol 0.125ml, surplus are that sterile water complements to 5ml.Bruceolic oil emulsion in the present invention, its antitumous effect is better than common injecting Java brucea fruit oil emulsion injection agent currently on the market, and Tween-80 is the half of Fabaceous Lecithin dosage in existing product as the usage amount of auxiliary material, the dosage of Tween-80 is extremely low, meets the requirement in injection about Tween-80 dosage.
Description
Technical field
The present invention relates to a kind of bruceolic oil emulsions, preparation method and application, belong to technical field of traditional Chinese medicine preparation.
Background technique
Java brucea is ripening fruits also known as the crow of Simarubaceae brucea plant Bruceajavanica (L.) Merr
Gallbladder, khosem etc. is bitter, cold in nature, slightly poisonous, returns large intestine, Liver Channel.Java brucea has clearing heat and detoxicating, preventing malaria, stop dysentery, corrosion
The effect of superfluous defect.For treating the diseases such as hot dysentery with bloody stool or hematochezia, malaria, corn.Modern pharmacology research proves to contain in Java brucea
There is an a large amount of kusulactone class compound, this kind of compound has anti-malarial, anti-amoebic dysentery, antifeedant, insecticide, anti-inflammatory
With the bioactivity such as anticancer.Brucea fruit oil is primarily present in java brucea seed, by saccharide converted in Java brucea maturation and
At.Java brucea oil content is about 20%, is mainly made of oleic acid, linoleic acid, stearic acid and palmitinic acid etc..Brucea fruit oil is facing
It is mainly used for auxiliary for treating cancer on bed, kinds of tumors is had a better effect, such as lung cancer, gastric cancer, liver cancer, colon cancer, straight
Intestinal cancer, breast cancer, cancer of the esophagus etc..
Currently, brucea fruit oil as injection main dosage form be Emulsion of Seed Oils From Brucea Javanica, auxiliary material be mainly Fabaceous Lecithin and
Glycerol, wherein the price of the Fabaceous Lecithin of Fabaceous Lecithin is high, and usage amount is larger, and the main anti-tumor effective component of the dosage form is crow
Courage oil, the effect of auxiliary material is only to keep dosage form stability, to therapeutic effect without contribution.
Summary of the invention
The purpose of the present invention is type and dosage in view of the above-mentioned problems, by changing auxiliary material, is devised a kind of novel
Bruceolic oil emulsion, the dosage form select auxiliary material price it is low, auxiliary material usage amount is small, and the antitumous effect of novel form is more preferable.
Summary of the invention one of the invention is a kind of bruceolic oil emulsion, consisting of: brucea fruit oil 0.5ml, tween-
800.12ml, glycerol 0.125ml, surplus are that sterile water complements to 5ml.
Summary of the invention two of the invention is a kind of method for preparing bruceolic oil emulsion, the specific steps are as follows:
Tween-80 0.12ml, glycerol 0.125mL are added in brucea fruit oil 0.5ml, after mixing evenly, then is gradually added into nothing
Bacterium water is stirred evenly and is sub-packed in 10mL centrifuge tube after filtering, sealed to 5mL.
Sterilizing to the used time up to 10% brucea fruit oil through miillpore filter, (mL/mL:1mL medical fluid is dissolved in 1mL sterile distilled water
In) oil emu finished product.
Summary of the invention three of the invention is bruceolic oil emulsion application in preparation of anti-tumor drugs.
The anti-tumor drug refers to the anti-tumor drug for inhibiting proto-oncogene ras access excessive activation.
Beneficial effect
It is detected by anticancer experiment in vitro, the bruceolic oil emulsion in the present invention, antitumous effect is better than existing market
Upper common injecting Java brucea fruit oil emulsion injection agent, the dosage of Tween-80 is extremely low, meets the requirement in injection about Tween-80 dosage,
It ensure that the efficient of bruceolic oil emulsion, safety, low price in the present invention, there is great application and development prospect.
Specific embodiment
1 instrument, reagent and experimental material
1.1 key instrument
XYJ80-2 electric centrifuge (Hengfeng instrument plant, Jintan City), THZ-80 gas bath constant temperature oscillator (Jintan City's Hengfeng instrument
Device factory), GR60DR type high-temperature sterilization pot (Zhi Wei Instrument Ltd.), (power health biologic medical science and technology is holding for B2 Biohazard Safety Equipment
Co., Ltd), SY-2OOO type rotary evaporator (Shanghai Ya Rong biochemical instrument Co., Ltd), inverted biologic microscope (Chongqing light
Electric instrument parent company), CHZQF (60A) type gas bath constant-temperature table, DZF-6020MBE type vacuum oven, assay balance (Beijing
Sai Duolisi instrument system Co., Ltd).
1.2 experiment reagent
Petroleum ether (30%~60%) (Tianjin all generations Chemical Co., Ltd.), dehydrated alcohol (analysis is pure) (Tianjin hundred
Generation Chemical Co., Ltd.), Tween-80 (Tianjin recovery fining research institute), glycerine (Yantai City's limited public affairs of chemical industry in pairs
Department), potassium dihydrogen phosphate (Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), (chemical industry is limited in pairs for Yantai City for disodium hydrogen phosphate
Company), dipotassium hydrogen phosphate (Yantai City in pairs Chemical Co., Ltd.)
1.3 experimental materials:
(in March, 2016 is purchased from the Yellow River medicinal material market to fresh Java brucea, through the big identification of Gansu pharmaceutical college, university of TCM Wang Ming
For certified products)；Caenorhabditis elegans: MT2124, genotype: Let-60 (n1046sd, gf) IV, by Howard Hughes
MedicaL Institute give；Escherichia coli: E.coiL OP50, uracil leaky mutant, as Caenorhabditis elegans
Food, be purchased from CGC (Caenorhabditis Genetics Center).
M9 buffer: Na containing 15g in every liter of buffer2HPO4·12H2O, 3g KH2PO4, 5g NaCl, 0.25g
MgSO4·7H2O。
The preparation of 1 brucea fruit oil of embodiment
It is oily matter according to Fructus Bruceae extract main component oleic acid, is generally extracted using organic solvent extraction, often
Solvent is mainly petroleum ether.Java brucea raw material is strictly selected, since Java brucea pericarp is hard and fine and close, and its fat oil
Contained in needing for Java brucea pericarp and seed to be crushed in java brucea seed, before extraction.Precision weighs 500g, is placed in leaching medicine pot, with 1:6
3000mL petroleum is added in the solvent volume ratio of (Kg/L, wherein " 1 " is Java Brucea Fruit weight Kg, " 6 " are solvent volume L)
Ether is sealed immersion for 24 hours at room temperature, during which periodic agitation is needed sufficiently to dissolve in favor of effective component, then carry out vacuum
Suction filtration obtains filtrate and filter residue, and filtrate is spare, and filter residue adds former solvent (petroleum ether) same volume 3000mL, impregnates for 24 hours, takes out
Filter, merges 2 filtrates, and the grease that solvent obtains brown, as brucea fruit oil is separately recovered, and weighs, and be placed on -18
It is saved in DEG C refrigerator.By extracting, brucea fruit oil is obtained altogether and extracts 46.25g, brucea fruit oil content is 9.25%, extract face
Color is that brown is clear and bright.
The preparation of 2 bruceolic oil emulsion of embodiment
Brucea fruit oil is brown color, clear and bright grease liquid, is soluble in chloroform, ether, is slightly soluble in ethyl alcohol, does not dissolve in
Water, main ingredient are oleic acid, and clinic is made Emulsion of Seed Oils From Brucea Javanica (for oil-in-water type O/W emulsion), and emulsion grain is 1 μm or so,
The uniform emulsion liquid that finished appearance is creamy white.Bruceolic oil emulsion prescription brucea fruit oil is obtained according to document and by screening
0.5mL, Tween-80 0.12mL, glycerol 0.125mL, sterile distilled water add to 5mL.Specific preparation method: brucea fruit oil 0.5mL is added
Tween-80 0.12mL, glycerol 0.125mL after mixing evenly, then are gradually added into injection water to 5mL, stir evenly and divide after filtering
Loaded in 10mL centrifuge tube, sealing, sterilizing to the used time through miillpore filter, (mL/mL:1mL medical fluid is dissolved in up to 10% brucea fruit oil
In 1mL sterile distilled water) oil emu finished product.Specific screening process: being added 0.5mL brucea fruit oil in each centrifuge tube,
Remaining reagent dosage is shown in Table 1.
The reagent preparation additional amount of 1 bruceolic oil emulsion of table
The appearance character of obtained oil cream made above is as follows:
Emulsion of Seed Oils From Brucea Javanica (for oil-in-water type O/W emulsion), through analyzing, C Emulsion of Seed Oils From Brucea Javanica solubility is preferable, emulsion
Grain is 1 μm or so, the uniform emulsion liquid that finished appearance is creamy white, therefore Emulsion of Seed Oils From Brucea Javanica used is using No. C in this experiment
Preparation method is made, i.e. brucea fruit oil 0.5ml, Tween-80 0.12ml, glycerol 0.125ml, and sterile water adds to 5ml.
Embodiment 3
Tween-80 0.12mL, cysteine hydrochloride 0.05g, glycerol 0.125mL is added in brucea fruit oil 0.5mL, stirs evenly
Afterwards, then injection water is gradually added into 5mL, stir evenly and be sub-packed in 10mL centrifuge tube after filtering, sealed, to the used time through micropore
Filter membrane sterilizes up to 10% brucea fruit oil (mL/mL:1mL medical fluid is dissolved in 1mL sterile distilled water) oil emu finished product.
Reference examples 1
In order to compare with the prior art, using Fabaceous Lecithin as the raw material for replacing Tween-80, check experiment is as follows
Table 2 prepares the additional amount of bruceolic oil emulsion using customary adjuvant
The appearance character of obtained oil cream made above is as follows:
4 Caenorhabditis elegans of embodiment
1. Escherichia coli OP50 is cultivated
Escherichia coli (OP50) are coated on LB solid medium, is placed in incubator and cultivates for 24 hours, after from incubator
OP50 culture medium is taken out, chooses 1~2 monoclonal colonies in LB liquid medium with liquid-transfering gun, by the LB liquid medium
It is placed in shaking table, 37 DEG C of revolving speed 180r/min cultures are for 24 hours.
2. nematode mass propgation
A cryopreservation tube MT2124 strain nematode is taken out from -80 DEG C of ultra low temperature freezers to thaw rapidly, it is big in being coated with
Enterobacteria OP50 has simultaneously grown up on the culture dish (NGM) of lawn, cultivates in 20 DEG C of biochemical cultivation cases to adult.
3. the synchronization process of Caenorhabditis elegans
The Caenorhabditis elegans in the oviposition stage is rinsed from culture dish with M9 buffer, goes to 10mL centrifugation
Guan Zhong stands about 5min or so, after nematode sedimentation, supernatant is sucked out, discards.8mL Caenorhabditis elegans lysate is added
(1moL/mLNaOH: 3.2%NaClO=1:1, V/V can be added and subtracted according to the actual situation), whirlpool shakes 4~5min, complete to nematode
It being transferred in the small centrifuge tube of 1.5mL after dead cracking entirely, 3000r/min is centrifuged 4min, it discards supernatant, precipitating addition 1~
1.5mLM9 buffer by centrifugation cleans 1 time, repeats aforesaid operations, after cleaning 2~3 times repeatedly, 1mLM9 liquid is added, centrifuge tube is set
20 DEG C of cultures in gas bath constant-temperature table take out after 5~6 days and collect polypide progress Chinese medicine acute toxicity testing and pharmacodynamics reality
It tests.
The experiment of 5 Acute toxicity of embodiment
This experiment carries out on 96 orifice plates, and when experiment, the bruceolic oil emulsion that in 100 μ L embodiments 2 prepared by C group was added in every hole
Agent, 80 μ LM9 liquid, the nematode liquid of 20 μ L.Contain nematode 70~80 in the nematode liquid of usual 20 μ L, is at the nematode of L1 phase.
Medical fluid designs 4 dosage, and every group sets 3 Duplicate Samples, and dose design is sesquialter dilution, and blank control is sterile water, negative control
It for brucea fruit oil solvent, is placed in 18 DEG C of biochemical cultivation cases and cultivates, nematode cultivates for 24 hours i.e. readable afterwards in the liquid, by 96 holes
Plate is placed under inverted microscope the Acute Toxicity ratio for observing the brucea fruit oil of nematode, and records, and experimental result is as follows:
The Acute Toxicity of brucea fruit oil
The result shows that the death rate of Caenorhabditis elegans mutant strain MT2124 is not yet as drug concentration constantly increases
Disconnected to increase, when concentration reaches 25 μ l/ml, the death rate is only 26%, and the Emulsion of Seed Oils From Brucea Javanica toxicity that the present invention develops is smaller.
6 Emulsion of Seed Oils From Brucea Javanica of embodiment is in the effect experiment of nematode
Research finds about 30% human tumor, and there are ras gene mutations, and kinds of tumors detects p21ras mistake
It is really related with the generation of malignant tumour to show that ras gene mutation and p21ras are overexpressed for expression.Due to ras signal path from
Nematode is highly conserved to people, the conservative ras albumen of let-60 gene coding, the ras egg of it and the mankind in Caenorhabditis elegans
It is white compared to the homology having.If ras is mutated, nematode can be caused to generate more vaginal orifices in Caenorhabditis elegans, in the mankind
It is then the malignant proliferation for causing cell, leads oncogenic generation.Therefore, model organism Caenorhabditis elegans ras signal path mistake
Degree activated form mutant is widely used in screening as test population and inhibits the antitumor of proto-oncogene ras access excessive activation
Drug.Therefore, present invention employs Caenorhabditis elegans mutant strains as assessment inhibition ras proto-oncogene access excessive activation medicine
The model of object.The ratio of wild type nematode is higher in group, then drug effectiveness is more significant.
Material: Caenorhabditis elegans
It is carried out on 96 orifice plates, when experiment, every hole was added 140 μ LS basal liquids, 20 μ L culture solutions, 20 μ L medical fluids and 20 μ L's
Nematode liquid.Contain nematode 70~80 in the nematode liquid of usual 20 μ L, is at the nematode of L1 phase.Medical fluid is brucea fruit oil, reality
Apply the Emulsion of Seed Oils From Brucea Javanica of the preparation of example two, Tween-80, purchase oleum fructus bruceae injection agent be positive control, sterile water is negative
Control, every group sets 3 repetitions, is placed in 18 DEG C of biochemical cultivation cases and cultivates, and nematode is cultivated 5 days or so in the liquid and grows into
Nematode in every hole is all sucked out with liquid-transfering gun, is placed on planktonic organism tally, 3~5 drop (about 10 μ L) NaN are added by worm3
Solution is anaesthetized to nematode, is placed under inverted microscope when body swing is slower and is observed the WT ratio of nematode, and recorded.
The experimental results are shown inthe following table
The experimental results showed that Tween-80 and sterile water act on ras gene unrestraint, WT ratio is lower, contains crow
In the medical fluid of courage ingredient, WT ratio is increased, and shows that brucea fruit oil significantly inhibits ras gene,
In, the WT ratio highest in Emulsion of Seed Oils From Brucea Javanica experimental group in the present invention is 80% or more, and oleum fructus bruceae injection agent is real
Test group secondly, brucea fruit oil is minimum, show that the stability of this one dosage type low temperature of Emulsion of Seed Oils From Brucea Javanica is better than single brucea fruit oil, and
And using this novel form of Tween-80 substitution Fabaceous Lecithin in the present invention, drug effect is more preferable, is better than traditional oils emulsion-type.
Claims (4)
1. a kind of bruceolic oil emulsion, which is characterized in that composition are as follows: brucea fruit oil 0.5mL, Tween-80 0.12mL, glycerol
0.125mL, surplus are that sterile water complements to 5mL.
2. the preparation method of bruceolic oil emulsion described in claim 1 includes the following steps: to be added in brucea fruit oil 0.5mL and spit
Temperature -80 0.12 mL, glycerol 0.125mL, after mixing evenly, then are gradually added into sterile water to 5mL, stir evenly after filtering
It is sub-packed in 10mL centrifuge tube, seals.
3. bruceolic oil emulsion application in preparation of anti-tumor drugs described in claim 1.
4. application according to claim 3, which is characterized in that the anti-tumor drug, which refers to, inhibits proto-oncogene ras
The anti-tumor drug of access excessive activation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101683367A (en) * | 2006-08-22 | 2010-03-31 | 浙江九旭药业有限公司 | Method for preparing emulsified medicinal composition containing oleum fructus bruceae |
| CN102429935A (en) * | 2010-09-29 | 2012-05-02 | 成都中医药大学 | Brucea fruit oil self-emulsifying preparation and preparation method thereof |
| CN103271955A (en) * | 2013-05-24 | 2013-09-04 | 李宏 | Brucea javanica oil emulsion composition |
| CN104706689A (en) * | 2013-12-16 | 2015-06-17 | 天津迈迪瑞康生物医药科技有限公司 | Oleum fructus bruceae fat emulsion concentrated solution, preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101683367A (en) * | 2006-08-22 | 2010-03-31 | 浙江九旭药业有限公司 | Method for preparing emulsified medicinal composition containing oleum fructus bruceae |
| CN102429935A (en) * | 2010-09-29 | 2012-05-02 | 成都中医药大学 | Brucea fruit oil self-emulsifying preparation and preparation method thereof |
| CN103271955A (en) * | 2013-05-24 | 2013-09-04 | 李宏 | Brucea javanica oil emulsion composition |
| CN104706689A (en) * | 2013-12-16 | 2015-06-17 | 天津迈迪瑞康生物医药科技有限公司 | Oleum fructus bruceae fat emulsion concentrated solution, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| 鸦胆子油自乳化制剂处方研究;刘彦 等;《第二军医大学学报》;20090930;第30卷(第9期);第1069-1073页 |
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