CN1981785A - Antineoplastic Chinese medicinal preparation - Google Patents
Antineoplastic Chinese medicinal preparation Download PDFInfo
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- CN1981785A CN1981785A CNA200510111547XA CN200510111547A CN1981785A CN 1981785 A CN1981785 A CN 1981785A CN A200510111547X A CNA200510111547X A CN A200510111547XA CN 200510111547 A CN200510111547 A CN 200510111547A CN 1981785 A CN1981785 A CN 1981785A
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- chinese medicine
- fructus bruceae
- ydz
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- supercritical extract
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Abstract
An antineoplastic Chinese medicine is mainly prepared from brucea fruit through supercritical extracting. It has high curative effect and high stability.
Description
Technical field
The present invention relates to a kind of Chinese medicine preparation, relate in particular to a kind of antineoplastic Chinese medicine preparation.
Background technology
Fructus Bruceae is the dry mature fruit of Simarubaceae brucea plant (Brucea Jaranica L Merr).Gather during fruit maturation autumn, removes impurity, dries.Be distributed in Taiwan, Fujian, Guangdong, Hainan, Guangxi, Yunnan.Has heat-clearing and toxic substances removing.Preventing the attack (or recurrence) of malaria, dysentery relieving, the effect of corrosion wart is used for dysentery, malaria, external treatment wart, antineoplaston.Bibliographical information is arranged, and the Fructus Bruceae alcohol extract is to KB cell KB, Walker-256 sarcoma and P
388Lymphocytic leukemia has obvious inhibitory action; Oleum Fructus Bruceae is to ehrlich carcinoma, ascitic type liver cancer, S
37, S
180, u
14Deng significant anti-cancer activity is all arranged.The clinical treatment that can be used for gastrointestinal cancer, pulmonary carcinoma, hepatocarcinoma, breast carcinoma.
Nanotechnology is advanced by leaps and bounds, and has begun to be applied to drug delivery systems such as biomedicine field such as capsule of nano.Capsule of nano (nanocapsules) is called for short nano-capsule/nanocapsule, is meant the capsule of particle diameter between 10～1000nm.Nano-capsule can be used as drug delivery system on the one hand with the advantage of microcapsule and nanoparticle, the release of control internal package medicine.On the other hand, small size causes its tangible skin effect, is embodied in good biocompatibility, easily absorbs characteristics such as easy migration in vivo.The problem that has new drug more than half to have dissolving and absorb at present.Adopt the nano-capsule technology, can increase effective contact area of medicine and gastrointestinal fluid to a great extent, improve the absorption rate of medicine.In addition, special nanometer medicine bag can also make medicine reasonably discharge at affected area, improves drug targeting, has avoided the ill effect of medicine to other positions of human body.Therefore, the nano-capsule technology is subjected to the extensive concern of Chinese scholars in recent years, and a large amount of innovation researches just around its expansion, indicates that tentatively Chinese medicine nano-capsule preparation technique has a good application prospect in the modernization of Chinese medicine.
Because the Fructus Bruceae clinical preparation only has oral latex emulsion and two kinds of dosage forms of intravenous injection oil breast, and crude oil mostly is the crude extract of Fructus Bruceae, active constituent content is low, the impurity constituents is many, the preparation difficulty is increased, the preparation dose is big, poor stability between the storage life, and this brings a lot of uncertain factors for the judge of the clinical expansion result of use of Fructus Bruceae.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of antineoplastic Chinese medicine preparation, and this medicine antitumous effect is remarkable, and toxicity is less, and process stabilizing, can carry out large-scale industrial production.
For solving the problems of the technologies described above, a kind of antineoplastic Chinese medicine preparation of the present invention mainly comprises the Fructus Bruceae supercritical extract.
The present invention selects the Fructus Bruceae medical material that meets 2005 editions standards of pharmacopoeia of China for use, is ground into coarse powder, obtains extract through two step carbon dioxide supercritical fluid extraction, wherein first step extraction, extracting pressure is 15～20MPa, 35～50 ℃ of extraction temperature, and the extraction flow velocity is 0.6～4.8kg/m
2S, the extraction time is 1～3 hour; The extraction of second step adds 1～5 times of amount ethanol and does entrainer, and extracting pressure is 20～25MPa, 35～50 ℃ of extraction temperature, and the extraction flow velocity is 1.2～4.8kg/m
2S, the extraction time is 1～3 hour, reclaims ethanol, two parts merging promptly gets the Fructus Bruceae supercritical extract.
A kind of antineoplastic Chinese medicine preparation of the present invention can directly be made with conventional adjuvant by the Fructus Bruceae supercritical extract, perhaps by behind the described Fructus Bruceae supercritical extract process microencapsulated encapsulate, forms nanocapsule, makes with the adjuvant of routine again.Preferred version is with behind the Fructus Bruceae supercritical extract process microencapsulated encapsulate, forms nanocapsule, makes with the adjuvant of routine again.
A kind of antineoplastic Chinese medicine preparation of the present invention can be injectable powder, injection, tablet, capsule, granule, drop pill, external use plaster, gel or ointment.
With above-mentioned Fructus Bruceae supercritical extract as raw material, adopt the preparation method of improved efficient microcapsule, making with the Fructus Bruceae supercritical extract is capsule heart component, with the pharmaceutical polymers is the nanocapsule of the particle diameter of capsule material at the controlled yardstick homogeneous of the following scope of 500nm, as be prepared into about particle diameter 100nm, the nanocapsule of size homogeneous under certain emulsifying homogenizing encystation condition, obtains the nanocapsule of size homogeneous.Said emulsifying processing condition be meant by additives such as emulsifying agent or with the high pressure homogenizer synergy under, can disperse to obtain the size homogeneous, particle stability is good, the emulsion system of not reuniting behind autohemagglutination or cross-linked polymeric, obtains the nanocapsule of requirement of the present invention.
Described pharmaceutical polymers comprises the macromolecular material of natural macromolecular material, semi-synthetic macromolecular material and synthetic.Wherein natural macromolecular material comprises gelatin, arabic gum, alginate, protide, starch based, but commonly used be gelatin and starch based because this two classes material degradation is good and production cost is lower; What wherein semi-synthetic macromolecular material was commonly used is cellulose family, polyvinylpyrrolidone, because their encystation technology is comparatively simple, easy to operate; What the macromolecular material of synthetic was commonly used is polylactic acid-based and Polyalkylcyanoacrylanano class material, wherein polylactic acid-based material comprises polylactic acid, polylactic acid copolymerization glycolic and polycaprolactone, with the polylactic acid is best, and these materials have favorable tissue intermiscibility, low toxin; Polyalkylcyanoacrylanano family macromolecule material wherein, comprise methyl ester, ethyl ester, butyl ester, isobutyl ester and own ester, wherein butyl ester is best, because paracyanogen base butyl acrylate has biological degradability, toxicity is little and many-sided advantage such as better tolerance in vivo, is subjected to extensive utilization.
Because the difference of nanocapsule coating material, its concrete method for making is distinguishing, and it can adopt a kind of in emulsion polymerization method, natural polymer method or spraying (in the liquid) seasoning.Its preparation method is as follows: earlier the Fructus Bruceae supercritical extract is dissolved in the solvent, be mixed into " oil phase " of homogeneous then with the solution of Polyalkylcyanoacrylanano class pharmaceutical polymers, continue under agitation oil phase, slowly inject the aqueous phase that contains additives with syringe, control mixing speed, solution temperature and pH value, capsule material and capsule core material ratio, mixing time, make particle size in the following scope regulation and control of 500nm, by the macromolecular material monomer polymerization, packaging medicine directly forms nanocapsule.Perhaps earlier the Fructus Bruceae supercritical extract is dissolved in the solvent, be mixed into " oil phase " of homogeneous then with the solution of water-insoluble pharmaceutical polymers, continue under agitation oil phase, slowly be added to the aqueous phase that contains additives, form a kind of emulsion, also the above-mentioned Fructus Bruceae supercritical extract that is dissolved in the solvent can be joined the aqueous phase that contains water solublity pharmaceutical polymers and additives in addition, under agitation form emulsion, carrying out homogenizing by high pressure homogenizer again handles, the pressure and temperature of regulation and control material in homogenizer in the high pressure homogenize process, and internal recycle number of times, particle size in the emulsion of handling by homogenizing is regulated and control in the following scope of 500nm, form the ultra-fine emulsion of stable homogeneous, finish embedding at last, polymerization or be cross-linked into nanocapsule.
The consumption of Chinese medicine preparation of the present invention and the course of treatment can be done suitably to adjust according to the light and heavy degree of dosage form, patient's age, disease, but are generally oral 3 times of every day, each 2 grams, with 1 month be a course of treatment, can use 3 courses of treatment or longer time continuously.
The present invention has following beneficial effect: Chinese medicine preparation of the present invention is compared with traditional Oleum Fructus Bruceae and preparation thereof, because wherein effective site is complete, content is higher, so corresponding improving a lot of antitumor efficacy of Fructus Bruceae supercritical extract and nanocapsule thereof, be convenient to the effect of clinical administration and science judgment medicine, move towards the international market thereby help the Fructus Bruceae preparation.In addition, the preparation of being made by the nanocapsule of Fructus Bruceae supercritical extract has efficiently, low toxicity, absorption is fast, utilization rate is high advantage, and determined curative effect when the treatment tumor is easy to use, has no side effect, and more helps applying.
Description of drawings
Fig. 1 is the influence sketch map of YDZ pastille serum to the HepG2 cell viability, and wherein, YDZ is the Fructus Bruceae supercritical extract, and HepG2 is a hepatoma cell strain.
The specific embodiment
The invention will be further elaborated by the following examples:
Embodiment 1: the preparation of Fructus Bruceae supercritical extract
Take by weighing Fructus Bruceae coarse powder 5kg, put in the extractor, start the CO 2 high pressure pump, uniting adjusting by counterbalance valve and high-pressure pump rotating speed makes the pressure of extractor and the flow of carbon dioxide maintain setup parameter, wherein first step extraction, extracting pressure is 20MPa, 35 ℃ of extraction temperature, and the extraction flow velocity is 4.8kg/m
2S, the extraction time is 1 hour; The extraction of second step adds 4 times of amount ethanol and does entrainer, and extracting pressure is 25MPa, 35 ℃ of extraction temperature, and the extraction flow velocity is 4.8kg/m
2S, the extraction time is 1 hour, reclaims ethanol, two parts merging promptly gets good fluidity, the Fructus Bruceae supercritical extract that color is darker.
Embodiment 2: the preparation of Fructus Bruceae supercritical extract
Take by weighing Fructus Bruceae coarse powder 5kg, put in the extractor, start the CO 2 high pressure pump, uniting adjusting by counterbalance valve and high-pressure pump rotating speed makes the pressure of extractor and the flow of carbon dioxide maintain setup parameter, wherein first step extraction, extracting pressure is 15MPa, 50 ℃ of extraction temperature, and the extraction flow velocity is 0.6kg/m
2S, the extraction time is 3 hours; The extraction of second step adds 5 times of amount ethanol and does entrainer, and extracting pressure is 20MPa, 50 ℃ of extraction temperature, and the extraction flow velocity is 1.2kg/m
2S, the extraction time is 3 hours, reclaims ethanol, two parts merging promptly gets good fluidity, the Fructus Bruceae supercritical extract that color is darker.
Embodiment 3: the preparation of Fructus Bruceae supercritical extract
Take by weighing Fructus Bruceae coarse powder 5kg, put in the extractor, start the CO 2 high pressure pump, uniting adjusting by counterbalance valve and high-pressure pump rotating speed makes the pressure of extractor and the flow of carbon dioxide maintain setup parameter, wherein first step extraction, extracting pressure is 18MPa, 40 ℃ of extraction temperature, and the extraction flow velocity is 1.8kg/m
2.s, the extraction time is 1.5 hours; The extraction of second step adds 1 times of amount ethanol and does entrainer, and extracting pressure is 23MPa, 45 ℃ of extraction temperature, and the extraction flow velocity is 2.4kg/m
2S, the extraction time is 2 hours, reclaims ethanol, two parts merging promptly gets good fluidity, the Fructus Bruceae supercritical extract that color is darker.
Embodiment 4: emulsion polymerization method prepares Fructus Bruceae supercritical extract nanocapsule
1. write out a prescription:
Oil phase: dehydrated alcohol 50ml paracyanogen base butyl acrylate 0.5g
Fructus Bruceae supercritical extract (embodiment 1) 3.0g
Water: water 200ml glucosan 2.0g polysorbas20 1.0g
2. preparation technology: get paracyanogen base butyl acrylate and add in the ethanol and be uniformly dispersed into white emulsion, add the Fructus Bruceae supercritical extract, be uniformly dispersed.Refrigerator and cooled is but to 20 ℃.Other gets polysorbas20 and glucosan and adds in the entry after the dissolving, and refrigerator and cooled is but to 20 ℃.Taking out the back is 3 with dilute hydrochloric acid adjusting pH value, stirs water, and making rotating speed is 600rpm, and oil phase is injected into aqueous phase.Sand core funnel filtration after 2 hours is stirred in the back continuation that finishes, and filtrate is emulsus, measures particle diameter about 100 nanometers, solidifies promptly with the mannitol spray drying.
Embodiment 5: the natural polymer legal system is equipped with Fructus Bruceae supercritical extract nanocapsule
1. write out a prescription:
Oil phase: Oleum sesami 100ml gelatin solution 100ml (1000g/L)
Fructus Bruceae supercritical extract (embodiment 2) 3.0g
Water: water 200ml sodium sulfate 2.0g Tween 80 1.0g
2. preparation technology: get gelatin solution, add the Fructus Bruceae supercritical extract, emulsifying is even in Oleum sesami.The emulsion that forms is cooled off in ice-water bath, make the gelatin complete gelation, with acetone diluted and flush away Oleum sesami.Other gets Tween 80 and sodium sulfate and adds in the entry after the dissolving, in refrigerator and cooled but to 10 ℃.Take out the biphase mixing in back, advance the high pressure homogenizer homogenizing, controlled pressure is 400bar, 25 ℃ of temperature, and internal recycle 3 times reclaims acetone, crosses the microporous filter membrane of 0.45 μ m, measures particle diameter about 200 nanometers, solidifies promptly by lyophilization or spray drying with mannitol.
Embodiment 6: semi-synthetic macromolecule spray drying method for preparation Fructus Bruceae supercritical extract nanocapsule
1. write out a prescription:
Oil phase: dehydrated alcohol 100ml phosphatidase 11 70 2g
Fructus Bruceae supercritical extract (embodiment 3) 80g
Water: water 300ml polyvinylpyrrolidone K30 10g
Tween 80 5g mannitol 80g
2. preparation technology: get phosphatidase 11 70 and Fructus Bruceae supercritical extract, use anhydrous alcohol solution, as oil phase.Other gets Tween 80, polyvinylpyrrolidone, mannitol and adds in the entry and dissolve, as water.With biphase mixing, advance the high pressure homogenizer homogenizing, controlled pressure is 300bar, 25 ℃ of temperature, internal recycle 4 times, measuring particle diameter is about 50nm, lyophilization or spray drying are solidified promptly.
Embodiment 7: the synthetic high polymer intra-liquid desiccation method prepares Fructus Bruceae supercritical extract nanocapsule
1. write out a prescription:
Oil phase: acetone 15ml polylactic acid 0.1g lecithin 0.2g
Fructus Bruceae supercritical extract (embodiment 3) 1g
Water: water 500ml poloxamer 1g
2. preparation technology: get polylactic acid and lecithin acetone solution, add the Fructus Bruceae supercritical extract, mix homogeneously is as oil phase.Other gets poloxamer and adds in the entry and dissolve, as water.With biphase slow mixing, advance the high pressure homogenizer homogenizing, controlled pressure is 800bar, 20 ℃ of temperature, internal recycle 3 times reclaims acetone, and is centrifugal, removes supernatant, and lyophilization is solidified promptly, and measuring particle diameter is about 150nm.
Embodiment 8: the preparation of injectable powder
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule (real 240g
Execute example 6)
Glucose 120g
Mannitol 240g
Vitamin C 100g
Distilled water adds to 1000ml
2. preparation technology: Fructus Bruceae supercritical extract nanocapsule and glucose, mannitol, vitamin C are added in the distilled water dissolving evenly, and the encapsulation lyophilization promptly.
Embodiment 9: the preparation of injection
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule (real 100g
Execute example 5)
Glycerol 22.5g
1,2-propylene glycol 100g
Vitamin E 2.5g
Cholic acid 30g
Refining Oleum Glycines 25g
Phosphatidase 13 3.3g
Distilled water adds to 1000ml
2. preparation technology: with Fructus Bruceae supercritical extract nanocapsule and glycerol, 1,2-propylene glycol, vitamin E, cholic acid add that dissolving evenly is dissolved in refining Oleum Glycines with phospholipid in the distilled water, and biphase high-pressure emulsification is even, and encapsulation promptly.
Embodiment 10: the preparation of tablet
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule (real 500g
Execute example 4)
Microcrystalline Cellulose 100g
Lactose 400g
Carboxymethyl starch sodium 50g
Pulvis Talci 25g
Magnesium stearate 25g
2. preparation technology: with Fructus Bruceae supercritical extract nanocapsule and microcrystalline Cellulose, lactose, carboxymethyl starch sodium mix homogeneously, the fluid bed one-step palletizing adds Pulvis Talci and magnesium stearate, mix homogeneously, and tabletting is packed promptly.
Embodiment 11: the preparation of capsule
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule (real 700g
Execute example 7)
Modified starch 200g
Dextrin 50g
Magnesium stearate 50g
2. preparation technology: with Fructus Bruceae supercritical extract nanocapsule and modified starch, dextrin mix homogeneously, the fluid bed one-step palletizing adds magnesium stearate, mix homogeneously, and the fill capsulation is promptly.
Embodiment 12: the preparation of granule
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule (real 500g
Execute example 4)
Microcrystalline Cellulose 200g
Lactose 200g
Carboxymethyl starch sodium 50g
Magnesium stearate 50g
2. preparation technology: with Fructus Bruceae supercritical extract nanocapsule and microcrystalline Cellulose, lactose, carboxymethyl starch sodium mix homogeneously, the fluid bed one-step palletizing adds magnesium stearate, and mix homogeneously, granule packaging are promptly.
Embodiment 13: the preparation of drop pill
1. write out a prescription:
Fructus Bruceae supercritical extract (embodiment 2) 400g
PEG6000 500g
Glycerol 100g
2. preparation technology: with Fructus Bruceae supercritical extract, glycerol and PEG6000 heating and melting, and stir, be incubated 60 ℃, splash in 10 ℃ the liquid paraffin, collect drop pill, wipe the paraffin oil on surface, packing promptly.
Embodiment 14: the preparation of external use plaster
1. write out a prescription:
Form the drug storehouse layer adhered layer
The Fructus Bruceae supercritical extract (is implemented 200g 50g
Example 1)
Polyisobutylene MML-100 300g 200g
Polyisobutylene LM-MS 350g 400g
Oleum Ricini 500g 400g
Gasoline 5000g 3000g
2. preparation technology: take by weighing each composition by drug storehouse layer prescription and adhesion layer recipe quantity, dissolving respectively on the aluminum plastic film of 70 micron thickness, is dried the drug storehouse layer solution coat or natural drying, forms the drug storehouse layer of about 60 micron thickness; On the silicon paper of 200 micron thickness, drying is made the adhesion layer of about 50 micron thickness with the adhesion layer solution coat.Adhesion layer is compound to drug storehouse layer, and the small pieces that are cut into suitable size promptly.
Embodiment 15: the preparation of gel
1. write out a prescription:
Fructus Bruceae supercritical extract nanocapsule 300g
(embodiment 4)
Carbopol 100g
Ethanol 100g
Glycerol 50g
Tween-80 2g
Ethyl hydroxybenzoate 1g
Sodium hydroxide 4g
Distilled water adds to 1000g
2. preparation technology: carbopol is mixed with Tween-80, Fructus Bruceae supercritical extract nanocapsule and 600ml water; The sodium hydroxide water dissolves the back in right amount and adds upward liquid, stirs evenly; Again ethyl hydroxybenzoate is dissolved in adding gradually behind the ethanol and stirs evenly, promptly get faint yellow clear gel.
Embodiment 16: the preparation of ointment
1. write out a prescription:
Fructus Bruceae supercritical extract (embodiment 200g
3)
Glyceryl monostearate 250g
Vaseline 100g
Sodium lauryl sulphate 20g
Azone 10g
Distilled water adds to 1000g
2. preparation technology: oil phase glyceryl monostearate, vaseline are heated to 80 ℃, Fructus Bruceae CO
2Supercritical extract, azone, sodium lauryl sulphate, water heat a little more than 80 ℃, and water slowly is added to oil phase, and the limit edged stirs, and becomes ointment gradually.
Below by test the example beneficial effect of the present invention is further elaborated:
Test example 1: Fructus Bruceae supercritical extract and research of nanocapsule extracorporeal anti-tumor function and serum pharmacological research
1.1 material
1.1.1 reagent and medicine
Fructus Bruceae supercritical extract (YDZ), YDZ nano-emulsion granule (particle diameter 500nm) (Nanocap-YDZ-500nm) provide by National Engineering and Research Center for TCM/Shanghai Traditional Chinese Medicine Pharmaceutical Technology Co., Ltd.YDZ crude drug yield 4.0%, lot number: 040709.Drug loading Nanocap-YDZ-500 is 25%, and YDZ nano-emulsion granule is dissolved in normal saline and gets the YDZ nano-emulsion; YDZ is dissolved in and contains 2% soil temperature, 80 normal saline and get the YDZ emulsion.YDZ emulsion and YDZ nano-emulsion test dose all calculate with YDZ.
The DMEM culture medium (Dulbecco ' s modified eagle medium) available from Gibco company, contain 2.2mg/mLNaHCO
3, 2.385mg/mL Hepes, 100U/mL penicillin, 100 μ g/mL streptomycins, 100U/mL kanamycin, 5mg/L be phenol red, pH7.2; MTT[bromination-(4,5-dimethyl-2-plug azoles base)-2,5-diphenyl tetrazole] available from Amresco company, face with the dissolving of PBS liquid; Trypsin trypsin) available from Serva company, face with the dissolving of D-Hanks liquid, contain 0.04%EDTA-2Na pH7.2; Hepes is available from Sigma company; (newborn calfserum is NCS) available from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation for new-born calf serum; Dimethyl sulfoxine (dimethylsulphoxide, DMSO), available from Ward kind chemical reagent factory forever; PBS liquid: 8.00mg/mL NaCl, 0.20mg/mL KCl, 1.56mg/mL Na
2HPO
4H
2O, 0.20mg/mL KH
2PO
4D-Hanks liquid: 8.00mg/mL NaCl, 0.40mg/mL KCl, 0.06mg/mL Na
2HPO
4H
2O, 0.06mg/mLKH
2PO
4, 0.35mg/mLNaHCO
3, Glucose 1.00mg/mL, 20mg/L be phenol red.
1.1.2 animal and cell: male mouse of kunming, body weight 20 ± 2g is provided by laboratory animal field, Green Dragon mountain, Jiangsu Province, the quality certification number: SCXK Soviet Union-2002-0018.Hepatoma cell strain HepG2 is provided by China Medicine University new medicament screen center.
1.1.3 instrument: HERA cell culture incubator, U.S. Therma Elemental company; The XSZ-D inverted biological microscope, the optical instrument factory, Chongqing; Clean bench, safe and sound company of Su Jing group; The Z323K high-speed refrigerated centrifuge, German Hermle labor tecknik company; Sunrise remote control microplate reader, Austrian Tecan GmbH company.
1.1.4 statistical analysis: use EXCEL and NDST4.4 statistical software and add up, data are used
Expression, capable t of comparable group differences or t ' check.
1.2 method and result
1.2.1YDZ with Nanocap-YDZ-500nm pastille serum preparation
Male ICR mouse, body weight 22 ± 2g is divided into 3 groups at random: YDZ30,60mg/kg group and blank group.Mice ig gives YDZ emulsion, and dosage is the 20mL/kg body weight, bid * 3.5d, 1h after the last administration of d4, pluck eyeball and get blood, leave standstill 1-2h, the centrifugal secondary of 4000rpm * 10min under 4 ℃, collect serum, 0.22 μ m microporous filter membrane malleation filtration sterilization ,-20 ℃ are freezing standby down.Be equipped with YDZ 30mg/kg pastille serum, Nanocap-YDZ-500nm 30mg/kg pastille serum and blank serum with legal system in addition.
1.2.2YDZ influence to the HepG2 cell viability
Take the logarithm the HepG2 cell inoculation of trophophase in 96 orifice plates, put 37 ℃, 5%CO with 10%NCS DMEM culture fluid
2Cultivate 24h in the incubator.Cell changes serum-free DMEM culture fluid after washing 1 time with D-Hanks liquid, establishes blank group 6 holes, and experimental group establishes 0.1,1.0,10,100,1000 a μ g/mL5 YDZ concentration, every concentration 6 holes.
After cultivating 48h, in the 96 porocyte culture plates that contain 100 μ l/ hole culture fluid, add final concentration 0.5mg/mLMTT solution 10 μ l/ holes, after continuing to cultivate 4h, the careful supernatant 90 μ l that draw add DMSO 50 μ l/ holes again, and 5min vibrates on the microoscillator, Sunrise remote control microplate reader is measured absorbance value, wavelength 570nm.Suppression ratio (%)=(1-A reality/A to) * 100%.
Table 1 YDZ is to the influence of HepG2 cell viability
| Group | Concentration (μ g/mL) | A570 | Suppression ratio (%) | IC50(μg/mL) |
| Matched group YDZ | 1000 100 10 1 | 0.262±0.039 0.050±0.005c 0.124±0.029c 0.126±0.051b 0.237±0.035 | 80.8 52.5 51.8 9.3 | 31.8 |
| 0.1 | 0.254±0.018 | 3.1 |
Ap＜0.05 b p＜0.01 cp＜0.001 is with respect to matched group
Table 1 shows, 0.1-1000 μ g/mL YDZ has in various degree inhibitory action to the HepG2 cell viability, and suppression ratio is 3.1-80.8%, and wherein 10-1000 μ g/mL YDZ effect has utmost point significance (p＜0.001-0.01), IC50 is 31.8 μ g/mL.
1.2.3YDZ pastille serum is to the influence of HepG2 cell viability
Take the logarithm the HepG2 cell inoculation of trophophase in 96 orifice plates, put in 37 ℃, 5%CO2 incubator with 10%NCS DMEM culture fluid and cultivate 24h.Cell changes serum-free DMEM culture fluid after washing 1 time with D-Hanks liquid, establishes 0.1%, 0.5%, 1.0%, 5%, 10%, 20% blank serum matched group, and experimental group establishes 0.1%, 0.5%, 1.0%, 5%, 10%, 20% 30,60mg/kg YDZ pastille serum group.
After cultivating 48h, in the 96 porocyte culture plates that contain 100 μ l/ hole culture fluid, add final concentration 0.5mg/mLMTT solution 10 μ l/ holes, after continuing to cultivate 4h, the careful supernatant 90 μ l that draw add DMSO50 μ l/ hole again, and 5min vibrates on the microoscillator, Sunrise remote control microplate reader is measured absorbance value, wavelength 570nm.
Fig. 1 is the influence sketch map of YDZ pastille serum to the HepG2 cell viability, wherein (
N=4), cp＜0.001 is with respect to matched group.As shown in Figure 1, compare with blank serum matched group, under the 0.1%-1.0% experimental concentration, YDZ pastille serum does not make significant difference to the HepG2 cell viability; Under the 5.0%-10.0% experimental concentration, YDZ pastille serum has certain promotion to the HepG2 cell viability; Under 20% experimental concentration, YDZ pastille serum has extremely significantly inhibitory action (p＜0.001) to the HepG2 cell viability.
1.2.4YDZ with of the influence of YDZ nano-emulsion pastille serum to the HepG2 cell viability
Take the logarithm the HepG2 cell inoculation of trophophase in 96 orifice plates, put in 37 ℃, 5%CO2 incubator with 10%NCS DMEM culture fluid and cultivate 24h.Cell changes serum-free DMEM culture fluid after washing 1 time with D-Hanks liquid, establishes 5% and 10% blank serum matched group, experimental group establish 5% with 10%YDZ pastille serum group and Nanocap-YDZ-500nm pastille serum group.
After cultivating 48h, in the 96 porocyte culture plates that contain 100 μ l/ hole culture fluid, add final concentration 0.5mg/mLMTT solution 10 μ l/ holes, after continuing to cultivate 4h, the careful supernatant 90 μ l that draw add DMSO50 μ l/ hole again, and 5min vibrates on the microoscillator, Sunrise remote control microplate reader is measured absorbance value, wavelength 570nm.
Table 2 YDZ and YDZ nano-emulsion pastille serum to the influence of HepG2 cell viability (
N=6)
| Group | Dosage (mg/mL) | A570 | |
| 5％ | 10％ | ||
| Matched group Nanocap-YDZ-500nm YDZ | 30 30 | 0.585±0.076 0.484±0.022b# 0.583±0.095 | 0.730±0.094 0.701±0.055 0.759±0.071 |
Bp＜0.01 is with respect to matched group; #p＜0.05 is with respect to YDZ
Table 2 shows, compares with blank serum matched group, and under 5% and 10% real concentration, YDZ pastille serum is to HepG2 cell viability unrestraint effect; And wait under the medicine carrying dosage, 5% can suppress the HepG2 cell viability in various degree with 10%Nanocap-YDZ-500nm, wherein with 5%Nanocap-YDZ-500nm effect remarkable (p＜0.01); Compare with YDZ pastille serum group, 5%Nanocap-YDZ-500nm can significantly improve YDZ cytotoxicity (p＜0.05).
1.3 conclusion
Experiment in vitro shows that YDZ has Graft Versus Tumor to hepatocarcinoma HepG2 cell.Serum pharmacological discovers that the YDZ pastille serum of different experiments concentration is to HepG2 impact cell difference.Do not show under the antitumor action experimental concentration at YDZ pastille serum, wait the YDZ nano-emulsion under the YDZ dosage but to demonstrate significant Graft Versus Tumor.
Test the research of the anti-hepatocarcinoma dimensional effect of routine 2:YDZ nanocapsule serum pharmacological
2.1 material
2.1.1 reagent and medicine
Fructus Bruceae supercritical extract (YDZ), YDZ nanocapsule (Nanocap-YDZ-1-4) provide by National Engineering and Research Center for TCM/Shanghai Traditional Chinese Medicine Pharmaceutical Technology Co., Ltd.YDZ crude drug yield 4.0%, lot number: 040709.Drug loading Nanocap-YDZ is 0.8g/100mL; YDZ is dissolved in and contains 2% soil temperature, 80 normal saline and get the YDZ emulsion.YDZ emulsion and YDZ nano-capsule emulsion dosage all calculate with YDZ.
The DMEM culture medium (Dulbecco ' s modified eagle medium) available from Gibco company, contain 2.2mg/mL NaHCO
3, 2.385mg/mL Hepes, 100U/mL penicillin, 100 μ g/mL streptomycins, 100U/mL kanamycin, 5mg/L be phenol red, pH7.2; MTT[bromination-(4,5-dimethyl-2-plug azoles base)-2,5-diphenyl tetrazole] available from Amresco company, face with the dissolving of PBS liquid; Trypsin trypsin) available from Serva company, face with the dissolving of D-Hanks liquid, contain 0.04%EDTA-2Na pH7.2; Hepes is available from Sigma company; (newborn calfserum is NCS) available from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation for new-born calf serum; Dimethyl sulfoxine (dimethylsulphoxide, DMSO), available from Ward kind chemical reagent factory forever; PBS liquid: 8.00mg/mL NaCl, 0.20mg/mL KCl, 1.56mg/mL Na
2HPO
4H
2O, 0.20mg/mL KH
2PO
4D-Hanks liquid: 8.00mg/mL NaCl, 0.40mg/mL KCl, 0.06mg/mL Na
2HPO
4H
2O, 0.06mg/mLKH
2PO
4, 0.35mg/mL NaHCO
3, Glucose 1.00mg/mL, 20mg/L be phenol red.
2.1.2 animal and cell: male mouse of kunming, body weight 20 ± 2g is provided by laboratory animal field, Green Dragon mountain, Jiangsu Province, the quality certification number: SCXK Soviet Union-2002-0018.Hepatoma cell strain HepG2 is provided by China Medicine University new medicament screen center.
2.1.3 instrument: HERA cell culture incubator, U.S. Therma Elemental company; The XSZ-D inverted biological microscope, the optical instrument factory, Chongqing; Clean bench, safe and sound company of Su Jing group; The Z323K high-speed refrigerated centrifuge, German Hermle labor tecknik company; Sunrise remote control microplate reader, Austrian Tecan GmbH company.
2.1.4 statistical analysis: use EXCEL and NDST4.4 statistical software and add up, data are used
Expression, capable t of comparable group differences or t ' check.
2.2 method and result
2.2.1YDZ with Nanocap-YDZ pastille serum preparation
Male ICR mouse, body weight 22 ± 2g is divided into 6 groups at random: YDZ 30mg/kg group, Nanocap-YDZ-1-430mg/kg group and blank group.Mice ig gives YDZ emulsion, and dosage is the 20mL/kg body weight, bid * 3.5d, 1h after the last administration of d4, pluck eyeball and get blood, leave standstill 1-2h, the centrifugal secondary of 4000rpm * 10min under 4 ℃, collect serum, 0.22 μ m microporous filter membrane malleation filtration sterilization ,-20 ℃ are freezing standby down.
2.2.2Nanocap-YDZ influence to the HepG2 cell viability
Take the logarithm the HepG2 cell inoculation of trophophase in 96 orifice plates, put 37 ℃, 5%CO with 10%NCS DMEM culture fluid
2Cultivate 24h in the incubator.Cell changes serum-free DMEM culture fluid after washing 1 time with D-Hanks liquid, establishes blank group 6 holes, and experimental group establishes 8,80,3 Nanocap-YDZ-1-4 concentration of 800 μ g/mL, every concentration 6 holes.
After cultivating 48h, in the 96 porocyte culture plates that contain 100 μ l/ hole culture fluid, add final concentration 0.5mg/mLMTT solution 10 μ l/ holes, after continuing to cultivate 4h, the careful supernatant 90 μ l that draw add DMSO 50 μ l/ holes again, and 5min vibrates on the microoscillator, Sunrise remote control microplate reader is measured absorbance value, wavelength 570nm.Suppression ratio (%)=(1-A reality/A to) * 100%.
Table 3 Nanocap-YDZ is to the influence of HepG2 cell viability
N=4)
| Group | Concentration (μ g/mL) | A570 | Suppression ratio (%) | IC50(μg/mL) |
| Matched group Nano-YDZ-1 Nano-YDZ-2 Nano-YDZ-3 Nano-YDZ-4 | 800 80 8 800 80 8 800 80 8 800 80 8 | 0.336±0.041 0.023±0.006c 0.046±0.028c 0.341±0.044 0.034±0.013c 0.205±0.047c 0.255±0.098 0.033±0.009c 0.298±0.060 0.301±0.026 0.038±0.011c 0.184±0.065c 0.279±0.047 | 93.1 86.3 -1.49 89.9 39.0 24.1 90.2 11.3 10.4 88.7 45.2 16.9 | 33.9 92.7 216.4 86.5 |
Ap＜0.05 b p＜0.01 cp＜0.001 is with respect to matched group
Table 3 shows, 8-800 μ g/mL Nanocap-YDZ-1-4 has inhibitory action in various degree to the HepG2 cell viability, and IC50 is respectively Nanocap-YDZ-1 33.9 μ g/mL, Nanocap-YDZ-292.7 μ g/mL, Nanocap-YDZ-3 216.4 μ g/mL and Nanocap-YDZ-4 86.5 μ g/mL.
2.2.3YDZ with of the influence of Nanocap-YDZ pastille serum to the HepG2 cell viability
Take the logarithm the HepG2 cell inoculation of trophophase in 96 orifice plates, put in 37 ℃, 5%CO2 incubator with 10%NCS DMEM culture fluid and cultivate 24h.Cell changes serum-free DMEM culture fluid after washing 1 time with D-Hanks liquid, establishes 5% and 10% blank serum matched group, experimental group establish 5% with 10%YDZ pastille serum group and Nanocap-YDZ-1-4 pastille serum group.
After cultivating 48h, in the 96 porocyte culture plates that contain 100 μ l/ hole culture fluid, add final concentration 0.5mg/mLMTT solution 10 μ l/ holes, after continuing to cultivate 4h, the careful supernatant 90 μ l that draw add DMSO50 μ l/ hole again, and 5min vibrates on the microoscillator, Sunrise remote control microplate reader is measured absorbance value, wavelength 570nm.
Table 4 YDZ and Nanocap-YDZ pastille serum are to the influence of HepG2 cell viability
N=6)
| Group | Dosage (mg/mL) | A570 | |
| 5％ | 10％ | ||
| Control group Nanocap-YDZ-1 Nanocap-YDZ-2 Nanocap-YDZ-3 Nanocap-YDZ-4 YDZ | 30 30 30 30 30 | 0.320±0.013 0.299±0.016a## 0.293±0.012b### 0.296±0.017a## 0.331±0.027 0.326±0.012 | 0.368±0.021 0.387±0.023 0.346±0.009a# 0.355±0.028 0.352±0.026 0.378±0.028 |
Ap＜0.05 b p＜0.01 is with respect to matched group; #p＜0.05 ## p＜0.01 ###p＜0.001 is with respect to YDZ
Table 4 shows, compares with blank serum matched group, and under 5% and 10% real concentration, YDZ pastille serum is to HepG2 cell viability unrestraint effect; And wait under the medicine carrying dosage, 5% can suppress the HepG2 cell viability in various degree with 10%Nanocap-YDZ-1-3, wherein with the remarkable (p＜0.05-0.01) of Nanocap-YDZ-2 effect; With YDZ pastille serum group relatively, 5% can significantly improve the YDZ cytotoxicity with 10%Nanocap-YDZ-1-3, wherein with Nanocap-YDZ-2 significant difference (p＜0.05-0.001).
2.3 conclusion
Experiment in vitro shows that the YDZ nanocapsule has Graft Versus Tumor to hepatocarcinoma HepG2 cell.Serum pharmacological is discovered, does not show under the antitumor action experimental concentration at YDZ pastille serum, waits the YDZ nanocapsule under the YDZ dosage but to demonstrate significant Graft Versus Tumor, and its action intensity may be relevant with the nanocapsule size.
Claims (9)
1. an antineoplastic Chinese medicine preparation is characterized in that, mainly comprises the Fructus Bruceae supercritical extract.
2. antineoplastic Chinese medicine preparation as claimed in claim 1, it is characterized in that, described Fructus Bruceae supercritical extract is made by following method: the Fructus Bruceae pulverizing medicinal materials is become coarse powder, earlier through carbon dioxide supercritical fluid extraction, wherein extracting pressure is 15～20MPa, 35～50 ℃ of extraction temperature, the extraction flow velocity is 0.6～4.8kg/m
2S, the extraction time is 1～3 hour; Add 1～5 times of amount ethanol again and do entrainer, through carbon dioxide supercritical fluid extraction, wherein extracting pressure is 20～25MPa, 35～50 ℃ of extraction temperature, and the extraction flow velocity is 1.2～4.8kg/m
2S, the extraction time is 1～3 hour, reclaims ethanol, two parts merging promptly gets the Fructus Bruceae supercritical extract.
3. antineoplastic Chinese medicine preparation as claimed in claim 1, it is characterized in that, described antineoplastic Chinese medicine preparation is directly made with conventional adjuvant by described Fructus Bruceae supercritical extract, perhaps by behind the described Fructus Bruceae supercritical extract process microencapsulated encapsulate, form nanocapsule, make with the adjuvant of routine again.
4. antineoplastic Chinese medicine preparation as claimed in claim 3 is characterized in that, described antineoplastic Chinese medicine preparation through behind the microencapsulated encapsulate, forms nanocapsule by described Fructus Bruceae supercritical extract, makes with the adjuvant of routine again.
5. antineoplastic Chinese medicine preparation as claimed in claim 1 is characterized in that, described antineoplastic Chinese medicine preparation is injectable powder, injection, tablet, capsule, granule, drop pill, external use plaster, gel or ointment.
6. as claim 3 or 4 described antineoplastic Chinese medicine preparation, it is characterized in that described nanocapsule is to be capsule core material with the Fructus Bruceae supercritical extract, pharmaceutical polymers is the capsule material, be furnished with additives and constitute, described additives are stabilizing agent, diluent or emulsifying agent.
7. antineoplastic Chinese medicine preparation as claimed in claim 6 is characterized in that, described stabilizing agent is a kind of or its combination in glucosan, glucose, mannitol, the dextran; Described emulsifying agent is a kind of or its combination in phospholipid, tween, span, the poloxamer.
8. antineoplastic Chinese medicine preparation as claimed in claim 6 is characterized in that, described pharmaceutical polymers is the macromolecular material of natural macromolecular material, semi-synthetic macromolecular material or synthetic; Described natural macromolecular material is gelatin, arabic gum, alginate, protide or starch based; Described semi-synthetic macromolecular material is cellulose family or polyvinylpyrrolidone; The macromolecular material of described synthetic is polylactic acid-based or Polyalkylcyanoacrylanano class material, wherein, described polylactic acid-based material is polylactic acid, polylactic acid copolymerization glycolic or polycaprolactone, and described Polyalkylcyanoacrylanano class material is methyl ester, ethyl ester, butyl ester, isobutyl ester or own ester.
9. antineoplastic Chinese medicine preparation as claimed in claim 8 is characterized in that, described polylactic acid-based material is a polylactic acid; Described Polyalkylcyanoacrylanano class material is a paracyanogen base butyl acrylate.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101874817A (en) * | 2010-05-31 | 2010-11-03 | 沈阳药科大学 | Brucea javanica oil enteric-coated microcapsule, preparation method and application thereof |
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN106491670A (en) * | 2016-10-27 | 2017-03-15 | 甘肃中医药大学 | A kind of Fructus Bruceae extract, extracting method and application |
| CN113577040A (en) * | 2021-07-19 | 2021-11-02 | 河北工程大学附属医院 | Nano-carrier loaded with traditional Chinese medicine monomers and application of nano-carrier in preparation of antitumor drugs |
-
2005
- 2005-12-15 CN CNB200510111547XA patent/CN100544738C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN101874817A (en) * | 2010-05-31 | 2010-11-03 | 沈阳药科大学 | Brucea javanica oil enteric-coated microcapsule, preparation method and application thereof |
| CN101874817B (en) * | 2010-05-31 | 2014-01-22 | 沈阳药科大学 | Brucea javanica oil enteric-coated microcapsule and preparation method |
| CN106491670A (en) * | 2016-10-27 | 2017-03-15 | 甘肃中医药大学 | A kind of Fructus Bruceae extract, extracting method and application |
| CN113577040A (en) * | 2021-07-19 | 2021-11-02 | 河北工程大学附属医院 | Nano-carrier loaded with traditional Chinese medicine monomers and application of nano-carrier in preparation of antitumor drugs |
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| CN100544738C (en) | 2009-09-30 |
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