CN100512805C - Sesquiterpene injection, preparing method and use thereof - Google Patents
Sesquiterpene injection, preparing method and use thereof Download PDFInfo
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- CN100512805C CN100512805C CNB02117086XA CN02117086A CN100512805C CN 100512805 C CN100512805 C CN 100512805C CN B02117086X A CNB02117086X A CN B02117086XA CN 02117086 A CN02117086 A CN 02117086A CN 100512805 C CN100512805 C CN 100512805C
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Abstract
The invention refers to a new drug form of plant extraction materials, concretely refers to their nano lipid drug form. Front body lipid drug form, long-circulation lipid drug form, long-circulation nano lipid form, nano micro-milk drug form, vein milk drug form, lipid nano ball drug form and fat milk drug form.
Description
Technical field:
The present invention relates to contain the new injection type of the plant extract of sesquiterpene, liposome dosage form more specifically to these preparations, the nanometer liposome dosage form, the pro-liposome dosage form, the long circulating liposomes dosage form, long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form and fat milk dosage form.The invention still further relates to the preparation method and the purposes of these dosage forms.
Background technology:
In recent years, the many research worker in the anticancer natural drug research field all are devoted to study the active anticancer of paclitaxel, the various isomers of elemene, Rhizoma Curcumae Longae extract, common turmeric extract etc. and the various dosage forms of these medicines.
Elemene is a kind of sesquiterpenoids that is present in the certain plants, and Latin is called Elemenum, English Elemene by name, and chemical name is 1-methyl isophthalic acid-vinyl-2,4-diisopropenyl cyclohexane extraction, molecular formula C
15H
24Elemene has multiple isomer, beta-elemene in these isomers, and δ-elemene, γ-elemene have been proved active anticancer.Elemene is the chemical compound that can extract from the volatile oil of plant RADIX CURCUMAE (Curcuma Wenyujin Y.H.Chen et C.ling) rhizome (being commonly called as warm Rhizoma Curcumae), or the chemical compound that from the oil of plant Herba Cymbopogonis Citrari (Cymbopoqon citratus (DC.) Stapt), extracts, for example remove the remaining afterwards mixing grease of citral, citronellol from citronella oil, the chemical compound of extraction is actually the elemene isomer mixture that comprises beta-elemene.
Above-mentioned plant RADIX CURCUMAE and other two kinds of plant Rhizoma Curcumae and Rhizoma Curcumae Longaes that can be used as the natural drug use belong to the zingiberaceous plant monoid, originate in provinces such as Chinese Guangdong, Sichuan, Fujian, Guangxi, Zhejiang.The plant that comprises elemene has kinds more than 50 such as RADIX CURCUMAE, Herba Cymbopogonis Citrari, Rhizoma Acori Calami, Radix Inulae, Radix Ginseng, and wherein Herba Cymbopogonis Citrari is, resource competent plant wide in China's distribution.At present, the Elemene vinyl chemical compound mainly extracts from RADIX CURCUMAE oil and citronella oil.
There are many documents to relate to the herbal medicinal product of the anti-curing cancers of direct employing natural drug preparation, for example the open CN1216256A of Chinese patent discloses the herbal medicinal product of treatment hepatocarcinoma, it comprises contains Herba Hedyotidis Diffusae, Rhizoma Curcumae Longae, the extract of Rhizoma Polygoni Cuspidati and Radix Sophorae Tonkinensis as the Injectable composition (A) of main natural drug and the extract that contains Herba Hedyotidis Diffusae, Rhizoma Paridis, Rhizoma Polygoni Cuspidati, Radix Sophorae Tonkinensis, Radix Gentianae, Radix Et Rhizoma Rhei, Fructus Forsythiae, Radix Paeoniae Rubra, Rhizoma Curcumae Longae and Rhizoma Acori Graminei as the main Orally administered composition (B) of natural drug.
Curcumin preparation is disclosed in the open CN1302559 of Chinese patent; it is by preparing in the blendable organic solvent of water or the curcumin molecular dispersoid in the mixture of water and the blendable organic solvent of water; add protecting colloid aqueous solution, the hydrophobic millimicro decentralized photo that forms mutually of curcumin to this solution then.Final dosage form mainly is aqueous dispersion or dry powder, and particle diameter is less than 10 μ m in the preferred case.
The method of extracting beta-elemene by the secondary fractional distillation from citronella oil is disclosed in CN1200266A.
Disclose the Jiangyu injecta that is used for the treatment of malignant tumor in the open CN1247756A of Chinese patent, wherein the weight ratio of Rhizoma Curcumae Longae and Radix Curcumae is 50-60: 50-40, and this injection is a kind of simple mixture.
The open CN1076613A of Chinese patent relates to elemene emulsion injection and preparation method thereof, and this injection is a kind of emulsion, and mean diameter generally is no more than 15 microns, is no more than 2 microns especially.
The open CN1244389A of Chinese patent relates to elemene injection and preparation method thereof, and this injection is a kind of simple mixtures, is not liposome.
The open CN1110134A of Chinese patent relates to liposome preparation technology and preparation, the elemene liposome injecta of wherein having described the elemene liposome injecta preparation method especially and having obtained by this method, the envelop rate of elemene reaches more than 85%, but does not provide the data of the mean diameter of related lipase plastid.Can judge that from preparation method this liposome has bigger mean diameter.
The open CN1221607A of Chinese patent relates to and utilizes CO
2Critical or supercritical process prepare the technology of liposoluble medicinal liposome and prepared elemene liposome injecta, though the envelop rate of the liposome of elemene is very high, the mean diameter of liposome is equally too big.
More than the disclosed preparation of these documents on bin stability and active anticancer, all exist deficiency.
The various derivants and the elemene metal complex of elemene are also disclosed in some patent documentations in addition.For example, a kind of elemene metal complex and its use as cancer therapy drug are disclosed in CN11531158A.CN1153167A relates to elemene derivatives containing nitrogen and as the purposes of cancer therapy drug.CN1153168A relates to elemene hydroxyls derivs and as the purposes of cancer therapy drug.
Summary of the invention:
The purpose of this invention is to provide extract injection, it contains: the extract with total sesquiterpene alkene class content 〉=70wt% that extracts from the plant that contains sesquiterpene, surfactant, and water-bearing media, this injection is a kind of aqueous dispersion, the mean diameter of decentralized photo≤1 micron, the amount of surfactant is the 1-5wt% of total injection, the concentration of total sesquiterpene alkene class (active component) is 1-10mg/ml in injection.More preferably, this water-bearing media is a water.
Another object of the present invention provides the new injection type of extract, liposome dosage form more specifically to this extract, the nanometer liposome dosage form, the pro-liposome dosage form, the long circulating liposomes dosage form, long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form or fat milk dosage form.
Another object of the present invention provides the preparation method of these injection types.
The preparation that a further object of the present invention provides these novel forms is used to prepare the purposes of the drug injection of treatment cancer or cerebral infarction, these injection types are used for the purposes of tumor remission pain, and the preparation of these novel forms is used to suppress and/or treat the purposes of tumor.
The invention provides the new injection type of these extracts.With from SHULANHUA, Fructus Tonnae Sinensis, Caulis Piperis Kadsurae, Flos Caryophylli, Myrrha, the Hemerocallis citrina Baroni Cuculus polioephalus, caraway, Herba thymi vulgaris, parsley, Herba Pogostemonis, Atractylodes lancea (Thunb.) DC., Fructus Cnidii, Flos lupuli (Flos Humuli Lupuli), sandalwood, bergamot, opoponax, Cupressus funebris Endl., Oleum Terebinthinae, Herba Cymbopogonis Citrari, Radix Ginseng, RADIX CURCUMAE, the extract that extracts in the quintessence oil that Rhizoma Curcumae Longae etc. obtain (or being called volatile oil) is prepared into its nanometer liposome dosage form respectively as raw material, the pro-liposome dosage form, the long circulating liposomes dosage form, the long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form and fat milk dosage form.The mean diameter of these dosage forms is generally less than or equals 1 micron, even is less than or equal to 100 nanometers.
Description of drawings:
Fig. 1 is the transmission electron microscope photo of the injection 1 of embodiment 1.
Detailed description of the present invention:
Known in the prior art field that some isomers of elemene, Turmeric P.E, common turmeric extract possess the effect that suppresses tumour, some documents disclose the various dosage forms (or formulation) of these natural drugs, preparations such as liposome, emulsion, Fat Emulsion. But, because the average grain diameter of these formulations is generally larger, can cause the various reactions in the human or animal body after being expelled in the human or animal body, for example excitant is large, and haemolysis sometimes occurs.
The inventor is through years of researches work, discovery with sesquiterpene compounds or the extract that contains sesquiterpene that from plant, extracts make average grain diameter≤1 micron or≤the various injection types of 100 nanometers, comprising Lipidosome, the nano liposomes formulation, the proliposome formulation, the long circulating liposome formulation, the long-circulating nanoliposome formulation, nanometer microemulsion type, vein emulsion type, lipid nanometer microballoon formulation, micro-emulsion etc., can overcome the problems in the ejection preparation of prior art, these formulations especially of the present invention have greatly reduced excitant in intravenous injection is used, and their stability is further enhanced. Novel form of the present invention has significantly improved the drug effect of pharmaceutical preparation, but the mechanism of the inhibition cancer cell of these formulations also is not fully aware of at present. The inventor makes following supposition according to a large amount of result of the tests: because preparation of the present invention has little average grain diameter and has special surface nature (hydrophile/lipophile balance for example, surface charge etc.), so that the medicine in these preparations (or active component) has stronger affinity to cancer cell, can fully be attached on the cancer cell, allow active component directly act on cancer cell, reach the effect that suppresses or kill cancer cell. In addition, find that the preparation of nano particle size is rapid-action, prevents that from there is certain effect the cancer metastasis aspect.
Simultaneously, although preparation of the present invention has little average grain diameter, but the preparation of the various formulations that obtain has high stability simultaneously, and they coagulation problems can not occur in storing for a long time, do not reduce stability so that can store the long period.
The invention provides extract injection, it contains: the extract with total sesquiterpene alkene class content 〉=70wt% that extracts from plant, surfactant, and water-bearing media, this injection is a kind of water-borne dispersions, the average grain diameter of decentralized photo≤1 micron, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, the concentration of total sesquiterpene alkene class (active component) is 1-10mg/ml in injection, wherein get rid of RADIX CURCUMAE, turmeric in these plants, and total elemene content of isomer＜70wt% of extract, preferred＜60wt%.
The invention provides another kind of extract injection, identical with above-mentioned injection, just plant is RADIX CURCUMAE or turmeric. The present invention also provides a kind of extract injection, and is identical with above-mentioned the first injection, and just extract contains 〉=total elemene isomers of 70wt%, and the concentration of the total elemene isomers in this injection is 1-10mg/ml.
The invention provides the preparation method of said extracted composition injection, it comprises that the extract that extracts with total sesquiterpene alkene class content 〉=70wt% as raw material, adds surfactant from plant, utilize Rotary Evaporators film forming or CO
2Critical or supercritical process comes mix homogeneously, add water for injection, carrying out high pressure homogenizing handles and/or ultrasonic Treatment, handle till the stabilized aqueous dispersion that obtains particle diameter of dispersing phase≤1 micron always, thereby make injection, wherein the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, and the ultimate density of total sesquiterpene bisabolene isomer body is 1～10mg/ml in the injection.
The sesquiterpene that extracts from plant (sometimes being called active component here) that can be used for making novel form in the present invention consists essentially of whole sesquiterpene compounds (C15H24), the mixture of sesquiterpene compounds or the sesquiterpene extract that from plant, extracts, extracting product for these requires the purity of sesquiterpene compounds more than 70%, preferably more than 75%, more preferably more than 85%, particularly preferably in more than 90%, even particularly preferably in more than 95%.
It is to be noted, the various extracts that prepare the preparation of various dosage forms as initiation material of the present invention can use product such as the elemene product (extract) that is purchased in the present invention, beta-elemene product (extract), Rhizoma Curcumae Longae extract or common turmeric extract, and from SHULANHUA, Fructus Tonnae Sinensis, Caulis Piperis Kadsurae, Flos Caryophylli, Myrrha, the Hemerocallis citrina Baroni Cuculus polioephalus, caraway, Herba thymi vulgaris, parsley, Herba Pogostemonis, Atractylodes lancea (Thunb.) DC., Fructus Cnidii, Flos lupuli (Flos Humuli Lupuli), sandalwood, bergamot, opoponax, Cupressus funebris Endl., Oleum Terebinthinae, Herba Cymbopogonis Citrari, the extract product that extracts in the volatile oil of Radix Ginseng etc. (or quintessence oil).If need oneself these extract of preparation in this application, the various volatile oil that are used to prepare these extracts can use and be purchased product, also can use the volatile oil that obtains from plant by common steam distillation or CO2 super critical extraction.Each extract can be used as initiation material of the present invention, and any two or more admixtures in any ratio also can be used as initiation material of the present invention in these extracts.
Sesquiterpene comprises elemene (elemene), curcumene (curcumene), zingiberene (zingiberene), bourbonene (bourbonene), humulene (humulene), Acacia farnesiana Willd. ellagic acid (farnesene), cadinene (cadinene), selinene (selinene), maaliene (maaliene), santalene (santalene), patchoulene (patchonlene), caryophyllene (be also referred to as Flos Caryophylli alkene, caryophyllene), gima ethylenic (big myrcene, germacrene), Cubeb oil alkene (cubebene), cedrene (cedrene), longifolene (longifolene), bisabolene (or bisabolene, bisabolene), bergaptene (bergamotene), gurjunene (gurjunene), lignum-vitae alkene (guaiene), ylangene (ylangene), isocaryophyllene (isocaryophyllene), long pinene (longipinene), Rhizoma Acori Calami alkene (calarene), wood sieve alkene (muurolene), acoradiene (acoradiene), sesquiphellandrene (β-sesquiphellandrene) etc.
In principle, all extracts that contain the above sesquiterpene of 60wt% that extract from plant all can be used for preparing novel form of the present invention, and sesquiterpene is the active component of all dosage forms.The extract that contains the above sesquiterpene of 70wt% is ideal, the extract that contains the above sesquiterpene of 75wt% is preferred, the extract that contains the above sesquiterpene of 85wt% is preferred, contain 90wt% above or even the extract that contains the above sesquiterpene of 95wt% be particularly preferred.
Tree orchid (claiming Flos Aglaiae Odoratae or Caulis seu folium chloranthi spicati (Chloranthus spicatus(Thunb.) Mak.) again) mainly originates in Camphor tree state and Foochow of Fujian China, and Guangdong and Yunnan also produce.The quintessence oil (or being called volatile oil) that obtains from SHULANHUA (Aglaia odorata Lour) mainly contains four kinds of sesquiterpenes, i.e. α-humulene, beta-elemene, β-Flos Caryophylli alkene, with tree orchid alkene (aglaiene) (referring to chemical journal, supplementary issue in 1981,248 pages).The composition of Fructus Tonnae Sinensis volatile oil (seeds of Toona sinensis) is referring to " GC-MS analysis " article of the Fructus Tonnae Sinensis volatile oil of Chinese Pharmaceutical Journal the 37th the 2nd phase of volume of February in 2002.Hemerocallis citrina Baroni Cuculus polioephalus (Rhododendron authopogonoides Maxim) volatilization oil composition is referring to " research of Hemerocallis citrina Baroni Cuculus polioephalus chemical composition of volatile oil " article of the 38th volume the 2nd phase chemistry journal April in 1980.The composition of ginseng essential oil (panax ginseng C.A.Mey) many documents have report (for example referring to＜conventional Chinese medicine composition and pharmacology handbook the 29th page, Chinese Medicine science and technology publishing house).
The explanation of term:
Described in the present invention particle diameter or granularity are meant the particle diameter or the granularity of decentralized photo.
In this application, extract refers to that the volatile oil (or quintessence oil) that obtains is through the smart distilled component of vacuum material filling type rectifier unit or the component made from extra care out through the molecular distillation instrument from plant, detect through GC-MS, extract contains above, the preferred 70wt% of 60wt% above, more preferably above, preferred especially 80wt% of 75wt% even the more preferably sesquiterpene of 85wt%.Wherein sesquiterpene is meant and comprises whole sesquiterpene isomers.
Modify if before dosage form, add " nanometer ", then refer to mean diameter≤150 nanometers of this dosage form, in ideal conditions mean diameter≤100 nanometers.Injection and ejection preparation are used interchangeably.
The liposome dosage form is meant the Lipid Bilayer Structure of being made up of phospholipid and cholesterol etc. that is dispersed or suspended in the water-bearing media in the present invention, and this Lipid Bilayer Structure is similar to membrane structure.
The nanometer liposome dosage form is meant that in the present invention mean diameter is less than or equal to the liposome of 150 nanometers.
The pro-liposome dosage form is meant in the present invention on the prescription basis of liposome and adds excipient again, by the prepared lyophilized powder of vacuum lyophilization (vacuum freeze-drying) method.This lyophilized powder by adding water for injection, through jolting or vibration, made it to revert to the liposome form before clinical practice.
The long circulating liposomes dosage form, briefly, being meant in the present invention increases the prepared liposome of Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE again on the prescription basis of liposome.The time of playing effectiveness in vivo than common liposome after it is expelled in the body is longer, or eliminates slowly in vivo.
Nanometer microemulsion type refers to mean diameter≤150 nanometers, in the ideal case≤100 the Emulsion of nanometer.
The vein emulsion type refers to the injection breast of mean diameter≤1 micron in the present invention.
Lipid nanometer microsphere dosage form is meant the fat milk of mean diameter≤150 nanometers in the present invention.
The fat milk dosage form requires its mean diameter≤1 micron in the present invention; In the China national pharmacopeia, also stipulate mean diameter≤1 micron in addition.
Nm is meant nanometer, and μ is meant micron.Purity is meant percent purity by weight in this application, except as otherwise noted.
In the present invention, if with 30 meters left and right sides length capillary gas chromatography-GC-MS (Bio-Rad FTS-65A, HP5890II, chromatographic column: BP-5) the Detection and Extraction thing contains 〉=70wt% elemene (comprising various elemene mixture of isomers) and containing＜the 85wt% beta-elemene, then this extract is called for short elemene, contain 〉=the 85wt% beta-elemene as berry extract, then this extract is called for short beta-elemene.
Employed in this application initiation material is an extract, and its total sesquiterpene alkene class content 〉=70wt% it is desirable to 75wt%, and is preferred 〉=80wt%, more preferably 〉=and 85wt%, preferred 〉=90wt%, more preferred 〉=95wt% especially.
Use about 30 meters (more than at least 24 meters) long capillary gas chromatograph-mass spectrometer (GC-MS) Detection and Extraction things described here, contain 〉=extract of 70wt% sesquiterpene just can be used among the present invention.
Employed extract raw material, reagent have no particular limits among the application, as long as this extract is to extract from natural plants and total sesquiterpene alkene class purity 〉=70wt% just can use in the present invention, better is, this purity 〉=75wt%, better 〉=80wt%, preferably 〉=85wt%, preferred 〉=90wt% especially.
Except as otherwise noted, otherwise other composition of Shi Yonging requires to meet national medicinal standard as cholesterol, soybean phospholipid, lecithin, glycerol, linoleic acid, Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, aliphatic amine, Semen sojae atricolor wet goods in the present invention, solvent also should meet national medicinal standard, and these are mainly for the consideration of healthy aspect; Similarly, modify with " making with extra care " for some raw materials, show that this raw material must meet national medicinal standard, for example refining soybean phospholipid is the available product of injection, and refined soybean oil is the available product of injection.
Except as otherwise noted, otherwise the employed gas chromatograph-mass spectrometer (GC-MS) of chromatography in this application is: Bio-Rad FTS-65A, and HP5890II, chromatographic column (about 30 meters long) is: BP-5.
Various dosage form of the present invention can be used for the treatment of purpose with intravenous injection, topical mode.
The preferred embodiments of the invention
In following embodiments, mainly use extract " elemene ", extract " beta-elemene ", Rhizoma Curcumae Longae extract, common turmeric extract as raw material, can also use the extract of SHULANHUA, Fructus Tonnae Sinensis, Caulis Piperis Kadsurae, Flos Caryophylli, Myrrha, Hemerocallis citrina Baroni Cuculus polioephalus, caraway, Herba thymi vulgaris, parsley, Herba Pogostemonis, Atractylodes lancea (Thunb.) DC., Fructus Cnidii, Flos lupuli (Flos Humuli Lupuli), sandalwood, bergamot, opoponax, Cupressus funebris Endl., Oleum Terebinthinae and Herba Cymbopogonis Citrari, Radix Ginseng etc., and any two or more admixtures in any ratio in the middle of them.Obviously, higher (product for example 〉=85wt%) can both be as raw material, and utilizes synthetic various sesquiterpenes of chemical method and derivant thereof can both be used for the present invention for the purity of wherein a certain sesquiterpene isomer.
The invention provides the preparation method of liposome or nanometer liposome, it comprises: with extract and soybean phospholipid, cholesterol with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes; Add solvent (as ether or acetone or chloroform), addition is enough to make mixture fully to dissolve, insert and remove the solvent film forming on the Rotary Evaporators, add the water for injection (calculating this surplus) (for example utilizing water for injection to be supplemented to 5000ml) of surplus according to the ultimate density that will prepare, then with material as a batch of material or insert in the ultrasonic cell disruptor of routine and carry out ultrasonic Treatment in batches; Sampling utilizes microscope, Coulter instrument, the ultramicroscope of band scale to check mean diameter, at the mean diameter that reaches regulation for example≤1 micron (liposome), or≤100 nanometers (nanometer liposome) after, regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density (weight of sesquiterpene active component (mg)/total volumes of formulation (ml)) of utilizing chromatograph (gas phase or liquid phase) to record active component is 1～10mg/ml, is preferably 2～7.5mg/ml is more typically 5～7.5mg/ml.
The envelop rate of nanometer liposome detects: detect with sieve method, promptly, utilize the SephadexG25 tomographic system, the extract nano-liposome preparation is divided into two stream parts, be extract nano-liposome stream part and free extract flow part, detect the extractive content that contains in two stream parts through gas chromatograph.The extractive content ÷ of envelop rate computing formula=extract nano-liposome stream part (extractive content+free extractive content of extract nano-liposome stream part).Whether microscope or transmission electron microscope checking are liposome.
The invention provides the preparation method of pro-liposome: with extract and refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to be supplemented to for example 5000ml, then with material as a batch of material or insert ultrasonic cell disruptor and carry out supersound process in batches, sampling utilizes the microscope or the Coulter instrument of band scale to check mean diameter, behind mean diameter≤1 micron, regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, the excipient (for example lactose or average molecular weight Mw are the low molecular dextran of about 4000-40000) that adds the 1-2wt% that accounts for total amount of formulation, 100 ℃ of vapor stream sterilizations, lyophilization under the aseptic condition, embedding.Adding injection water before using is 1～10mg/ml to reach final activity component concentration (weight of sesquiterpene active component (mg)/volumes of formulation (ml)), is preferably 2～8mg/ml, and more preferably 2～7.5mg/ml is more typically 5～7.5mg/ml.
The detection of envelop rate: use the sieve method detection method, that is: according to the final formulation concentrations (mg/ml) that will prepare, the water jolting of a certain amount of extract proliposome preparation being added the surplus volume earlier is even, become liposome this moment again, utilize Sephadex G25 tomographic system to be divided into two stream parts, be extract liposome stream part and free extract flow part, detect the extract amount that contains in two stream parts through gas chromatograph.The extractive content ÷ of envelop rate computing formula=extract liposome stream part (extractive content+free extract amount of extract liposome stream part).Whether microscope or transmission electron microscope confirm is pro-liposome.
The invention provides the preparation method of long circulation extract nano-liposome, this method comprises: with extract and refining soybean phospholipid, Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE, cholesterol is with weight ratio 0.5-1.5:1.5-3.5:0.3-1.2:0.8-2.0, preferably with weight ratio 0.8-1.2:1.8-3.0:0.4-1.0:1.2-1.8, more preferably with weight ratio 0.9-1.1:2.4-2.8:0.6-1.0:1.2-1.6, especially preferably mix with weight ratio 1:2.6-2.7:0.7-0.9:1.3-1.5, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to be supplemented to for example 5000ml, then with material as a collection of or insert conventional ultrasonic cell disruptor and carry out ultrasonic Treatment in batches, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density (weight of sesquiterpene active component (mg)/total volumes of formulation (ml)) that (gas phase or liquid phase) chromatography records active component is 1～10mg/ml, be preferably 2～7.5mg/ml, more preferably 2～8mg/ml is more typically 5～7.5mg/ml.For Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE, wherein " 2000 " refer to the mean molecule quantity (dalton) of Polyethylene Glycol segment.
The detection of the envelop rate of this injection is identical with the envelop rate detection method of the lipidosome injection of front.
The invention provides the preparation method of electronegative and positively charged extract nano microemulsion:
Preparation method 1: the preparation of electronegative nanometer microemulsion
With extract and refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to replenish for example 5000ml, insert conventional ultrasonic cell disruptor then and carry out ultrasonic Treatment, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density of active component (weight of sesquiterpene active component (mg)/total volumes of formulation (ml)) is 1～10mg/ml, be preferably 2～7.5mg/ml, be more typically 5～7.5mg/ml.Microscope, transmission electron microscope detect, and confirm whether be the nanometer microemulsion.
Preparation method 2: the preparation of positively charged nanometer microemulsion
With extract, refined lecithin, C14-C22 straight or branched aliphatic amine, cholesterol is with weight ratio 0.5-1.5:2.5-3.5:0.2-0.6:1.0-2.0, preferred 0.8-1.2:2.5-3.0:0.3-0.5:1.2-1.8, more preferably 0.9-1.1:2.7-2.9:0.3-0.5:1.4-1.6, preferred especially 1:2.8:0.4:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the densitometer that will prepare) that adds surplus then is to be supplemented to for example 5000ml, insert conventional ultrasonic cell disruptor then and carry out supersound process, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 7.5～9.0, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density of sesquiterpene active component is 1～10mg/ml, be preferably 2～7.5mg/ml, be more typically 5～7.5mg/ml.Microscope, transmission electron microscope detect, and confirm whether be the nanometer microemulsion.
The present invention also provides the preparation method of extract lipid nanospheres, and this method comprises: get extract and add in the soybean oil with soybean phospholipid, heat up and be controlled to oil phase (remaining oil phase), this oil phase is joined in the aqueous solution that contains 10-50wt% glycerol.Its concrete part by weight is: extract accounts for 0.08～0.8wt% of final preparaton total amount, soybean phospholipid 0.8～2.5wt%, and soybean oil 8-12wt%, glycerol 1.8-2.8wt%, water for injection adds to 5000ml; Preferred part by weight is: extract accounts for the 0.1wt%～0.5wt% of final preparaton total amount, soybean phospholipid 1.2wt%～2.0wt%, and soybean oil 10wt%, glycerol 2.25～2.5wt%, water for injection adds to 5000ml.High-speed stirred then, regulating PH is 4.5～6.5, passing through common high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) again handles, carry out foregoing ultrasonic Treatment then, sampling utilizes microscope, Coulter instrument or the ultramicroscope of band scale to check mean diameter, the mean diameter of its lipid ball reach setting for example≤100nm after, again through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃～120 ℃ sterilizations.The ultimate density that (gas phase or liquid phase) chromatography records the sesquiterpene active component is 1～10mg/ml, is preferably 2～7.5mg/ml, is more typically 2～5mg/ml, and better is 2.0～3.0mg/ml.Can in this final preparation, add an amount of cholesterol, linoleic acid etc. as required.
The invention provides the preparation method of extract intravenous injection breast, this method comprises: with extract, refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5, mix with the water for injection (calculating this surplus) of surplus according to the ultimate density that will prepare, high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) emulsifying with routine, sampling utilizes the microscope or the Coulter instrument of band scale to check mean diameter, the mean diameter that reaches regulation for example≤1 micron after, regulate PH, making pH value is 4.5～6.5, through 1.2 μ filtering with microporous membranes and bottling then, through 100 ℃～120 ℃ sterilizations, obtain extract vein breast, the ultimate density that (gas phase or liquid phase) chromatography records the sesquiterpene active component is 1～10mg/ml, be preferably 2～7.5mg/ml, be more typically 5～7.5mg/ml.
The invention provides the preparation method of extract fat emulsion, this method comprises: get extract and soybean phospholipid and add in the soybean oil, heat up and be controlled to oil phase (remaining oil phase), this oil phase is joined in the aqueous solution that contains 10-50wt% glycerol.Its concrete weight ratio is: extract accounts for 0.08～0.8wt% of final preparaton total amount, soybean phospholipid 0.8～2.5wt%, and soybean oil 8-12wt%, glycerol 1.8-2.8wt%, water for injection adds to 5000ml; Preferred part by weight is: extract accounts for the 0.1wt%～0.5wt% of final preparaton total amount, soybean phospholipid 1.2wt%～2.0wt%, and soybean oil 10wt%, glycerol 2.25～2.5wt%, water for injection adds to 5000ml.High-speed stirred then, regulating PH is 4.5～6.5, passes through common high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) again and handles, and makes its particle diameter less than 1 μ, again through 1.2 μ filtering with microporous membranes with bottle 100 ℃～120 ℃ sterilizations then.The ultimate density of sesquiterpene active component is 1～10mg/ml, is preferably 2～7.5mg/ml, the better 2.0～3.0mg/ml of being or be 5～7.5mg/ml.Can in this final preparation, add an amount of cholesterol, linoleic acid etc. as required.
As required, preparation of the present invention can with different size for example the microporous filter membrane of 1.2 μ, 1.0 μ, 0.9 μ, 0.8 μ, 0.7 μ, 0.6 μ, 0.5 μ etc. filter, can intercept mean diameter≤0.8 μ ,≤0.7 μ ,≤0.6 μ ,≤0.5 μ ,≤0.45 μ ,≤0.4 μ ,≤decentralized photo of 0.3 μ etc.
Below preparation example and embodiment be used to illustrate the present invention, but should not think and limit the scope of the invention.Protection scope of the present invention is defined by claim.
Preparation example 1: the molecular distillation of ginseng essential oil prepares Radix Ginseng extract
With 1000ml ginseng essential oil (contain 〉=48wt% sesquiterpene) is raw material, refining through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument, its process conditions: 35～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～90Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s (about 750ml), proceed molecular distillation.45～60 ℃ of vapo(u)rizing temperatures, distillation pressure 30～70Pa, cut open 300-320 rev/min of film speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys are collected distillation (about 220ml) respectively and are heated up in a steamer excess (about 530ml), the secondary that obtains is heated up in a steamer excess proceed molecular distillation.30～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation (about 180ml), bleed off and heat up in a steamer excess, last twice thing that slips out is merged (about 400ml) and adds to and continue to repeat distillation in the molecular distillation instrument up to utilizing about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS) (Bio-Rad FTS-65A, HP5890II, chromatographic column: BP-5) till the total sesquiterpene alkene content 〉=75wt% of Detection and Extraction thing, finally obtain the about 320ml of extract.Formulate for the detection finger printing with gas chromatography.This products known as Radix Ginseng extract.
Preparation example 2: prepare the SHULANHUA extract from SHULANHUA volatile oil
According to the method identical, replace 1000ml ginseng essential oil, the extract of total sesquiterpene alkene content 〉=75wt% of the 200ml of acquisition, this products known as SHULANHUA extract A with 1000ml SHULANHUA volatile oil (sesquiterpene content approximately〉25wt%) with preparation example 1.
Preparation example 3: the material filling type rectification under vacuum of SHULANHUA volatile oil prepares the SHULANHUA extract B
With the SHULANHUA volatile oil of 1000ml (sesquiterpene content approximately〉25wt%) through the rectification of material filling type vacuum rectifying apparatus; the material filling type vacuum rectifying apparatus is by the still of dress SHULANHUA volatile oil; stuffing rectification column (theoretical cam curve 〉=30); return channel; vacuum pump constitutes; design by new packing technology development center of Shanghai Chemical Research Inst; filler is Dixon filler (a Q net ring filler); vacuum is 1-3mmHg; collect the above fraction of 82 ℃/3mmHg; utilize about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS) (Bio-Rad FTS-65A; HP5890II; chromatographic column: BP-5) the total sesquiterpene alkene content 〉=75wt% of Detection and Extraction thing finally obtains the about 180ml of extract.Formulate for the detection finger printing with gas chromatography.This products known as SHULANHUA extract B.
Preparation example 4: obtain Hemerocallis citrina Baroni Cuculus polioephalus extract from Hemerocallis citrina Baroni Cuculus polioephalus volatile oil
According to the method identical with preparation example 1, replace the 1000ml ginseng essential oil with 1000ml Hemerocallis citrina Baroni Cuculus polioephalus volatile oil (sesquiterpene content approximately〉15wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 120ml, this products known as Hemerocallis citrina Baroni Cuculus polioephalus extract A.
According to the method identical with preparation example 3, replace 1000ml SHULANHUA volatile oil with 1000ml Hemerocallis citrina Baroni Cuculus polioephalus volatile oil (sesquiterpene content approximately〉15wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 120ml, this products known as Hemerocallis citrina Baroni Cuculus polioephalus extract B.
Preparation example 5: obtain Caulis Piperis Kadsurae extract from Caulis Piperis Kadsurae volatile oil
According to the method identical with preparation example 1, replace the 1000ml ginseng essential oil with 1000ml Caulis Piperis Kadsurae volatile oil (sesquiterpene content is approximately〉15wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 125ml, this products known as Caulis Piperis Kadsurae extract A.
According to the method identical with preparation example 3, replace 1000ml SHULANHUA volatile oil with 1000ml Caulis Piperis Kadsurae volatile oil (sesquiterpene content approximately〉15wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 124ml, this products known as Caulis Piperis Kadsurae extract B.
Preparation example 6: obtain the Fructus Tonnae Sinensis extract from Fructus Tonnae Sinensis volatile oil
According to the method identical with preparation example 1, replace the 1000ml ginseng essential oil with 1000ml Fructus Tonnae Sinensis volatile oil (sesquiterpene content is approximately〉30wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 250ml, this products known as Fructus Tonnae Sinensis extract A.
According to the method identical with preparation example 3, replace 1000ml SHULANHUA volatile oil with 1000ml Fructus Tonnae Sinensis volatile oil (sesquiterpene content approximately〉30wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 240ml, this products known as Fructus Tonnae Sinensis extract B.
Preparation example 7: obtain the Cupressus funebris Endl. extract from Cupressus funebris Endl. volatile oil
According to the method identical with preparation example 1, replace the 1000ml ginseng essential oil with 1000ml Cupressus funebris Endl. volatile oil (sesquiterpene content is approximately〉14wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 120ml, this products known as Cupressus funebris Endl. extract A.
According to the method identical with preparation example 3, replace 1000ml SHULANHUA volatile oil with 1000ml Cupressus funebris Endl. volatile oil (sesquiterpene content is approximately〉14wt%), obtain the extract of total sesquiterpene alkene content 〉=75wt% of about 120ml, this products known as Cupressus funebris Endl. extract B.
Preparation example 8: the preparation of elemene
(citronella oil is removed citral with 1000ml citronella oil Cymbopoqon citratus (DC.) Stapt submember, the mixing grease of remainder after the citronellol, the sesquiterpene that contains the 30wt% that has an appointment, available from Baihua Perfumery, Guangzhou) be raw material, through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument fractional distillation elemene, its process conditions: 30～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～90Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows are only collected and are heated up in a steamer the about 700ml of excess, will heat up in a steamer excess and carry out molecular distillation.Vapo(u)rizing temperature is 40～60 ℃, distillation pressure 20～70Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of condensation temperatures, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows are collected the about 160ml of distillation respectively and are heated up in a steamer excess with about 540ml, this is heated up in a steamer excess proceed molecular distillation.Vapo(u)rizing temperature is 30～70 ℃, distillation pressure 10～50Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of condensation temperatures, 20～25 ℃ of system temperatures, 15～30 milliliters/hour of material flows, collect the about 140ml of distillation, bleed off and heat up in a steamer excess, the secondary distillation that obtains is mixed (about 300ml), continue distillation, until with about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s (instrument: Bio-Rad FTS-65A, HP5890II, chromatographic column: BP-5) detection product total elemene isomer purity reach till the pact 〉=75wt%, the final about 220ml of elemene extract (about 200g) that obtains is called for short the elemene product.
Preparation example 9: the preparation of elemene
RADIX CURCUMAE (the Curcumawenyujin Y.H.Chen et C.Ling) volatile oil of being produced by Ruian, Zhejiang Ouhai vegetable oil refining factory with 1000ml (contain approximately〉total elemene isomer of the 7wt%) is raw material, according to the method refine same with above-mentioned preparation example 1, the final extract that obtains total elemene isomer purity pact 〉=75wt% of 80ml is called for short the elemene product.
Preparation example 10: the preparation of beta-elemene
With the above-mentioned citronella oil Cymbopoqon of 1000ml citratus (DC.) Stapt submember is raw material, through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument fractional distillation beta-elemene, its process conditions: 30～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～100Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 7～10 milliliters/hour of material flows are only collected and are heated up in a steamer the about 700ml of excess, will heat up in a steamer excess and carry out molecular distillation.Vapo(u)rizing temperature is 40～60 ℃, distillation pressure 20～70Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of condensation temperatures, 20～25 ℃ of system's insulations, 7～10 milliliters/hour of material flows are collected the about 160ml of distillation respectively and are heated up in a steamer excess with about 540ml, this is heated up in a steamer excess proceed molecular distillation.Vapo(u)rizing temperature is 30～70 ℃, distillation pressure 10～50Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of condensation temperatures, 20～25 ℃ of system temperatures, 7～10 milliliters/hour of material flows, collect the about 140ml of distillation, bleed off and heat up in a steamer excess, the secondary distillation that obtains is mixed (about 300ml), continue distillation, until with about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s (instrument: Bio-Rad FTS-65A.HP5890II, chromatographic column: BP-5) the beta-elemene purity that detects product reach 〉=85% till, finally obtain the about 120ml of extract (about 105g).Obviously, if with the further rectification of this product, then can obtain high-purity more (for example beta-elemene purity reach 〉=90wt% or even 〉=90wt%) extract, be called for short the beta-elemene product.
Preparation example 11: the preparation of beta-elemene
RADIX CURCUMAE (the Curcumawenyujin Y.H.Chen et C.Ling) volatile oil of being produced by Ruian, Zhejiang Ouhai vegetable oil refining factory with 1000ml (contain approximately〉total elemene isomer of the 7wt%) is raw material, according to the method refine same with above-mentioned preparation example 3, the final extract that obtains beta-elemene purity 〉=about 85wt% of 45ml is called for short the beta-elemene product.
Preparation example 12: the preparation of Rhizoma Curcumae Longae extract
Preparation method 1: the molecular distillation of Rhizoma Curcumae Longae volatile oil
(sesquiterpene content 〉=16wt%) is raw material with the 1000ml Rhizoma Curcumae Longae volatile oil, refining through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument, its process conditions: 35～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～90Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s (about 450ml), proceed molecular distillation.45～60 ℃ of vapo(u)rizing temperatures, distillation pressure 30～70Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys are collected the 150ml distillation respectively and are heated up in a steamer excess with about 300ml, the secondary that obtains is heated up in a steamer excess run up to q.s (750ml), proceed molecular distillation.30～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation (320ml), bleed off and heat up in a steamer excess, add to again and continue to repeat distillation in the molecular distillation instrument slipping out thing until (be about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s: Bio-Rad FTS-65A with gas chromatograph, HP5890II, chromatographic column: till total sesquiterpene alkene content 〉=75wt%, obtain the 150ml extract when BP-5) detecting.This extract detects through above-mentioned gas chromatograph, also contains about 1/20 the curcumin that is equivalent to whole sesquiterpene gross weights.Formulate for the detection finger printing with gas chromatography.This products known as Rhizoma Curcumae Longae extract A.
Preparation method 2: material filling type rectification under vacuum
With the Rhizoma Curcumae Longae volatile oil of 3000ml (sesquiterpene content 〉=16wt%) through the rectification of material filling type vacuum rectifying apparatus; the material filling type vacuum rectifying apparatus is the still of 5000ml by the volume of dress Rhizoma Curcumae Longae volatile oil; stuffing rectification column (theoretical cam curve 〉=30); return channel; vacuum pump constitutes; design by new packing technology development center of Shanghai Chemical Research Inst; filler is Dixon filler (a Q net ring filler); vacuum is 1-3mmHg; collect the above fraction of 82 ℃/3mmHg; utilize about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS) (instruments: Bio-RadFTS-65A; HP5890II; chromatographic column: BP-5) the total sesquiterpene alkene content 〉=75wt% of Detection and Extraction thing; it also contains about 1/20 the curcumin that is equivalent to whole sesquiterpene gross weights, obtains the extract of about 450ml altogether.Formulate for the detection finger printing with gas chromatography.This products known as Rhizoma Curcumae Longae extract B.
In addition, with Rhizoma Curcumae Longae through CO
2The slag that the volatile oil that supercritical extraction obtains stays (1000g), add methanol, the heating extracting obtained yellow viscous solution in 3 hours, methanol is distilled, obtain pale brown color paste, with these pastes preparation of silica gel chromatogram purification, obtain the curcumin product (13g) of purity 〉=90wt% with the chloroform eluting.Above-mentioned Rhizoma Curcumae Longae extract A and Rhizoma Curcumae Longae extract B are mixed with the 50:50 weight ratio, formed mixture is mixed in the ratio of 20:1 (weight) with this curcumin product and obtain another kind of mixture, be called Rhizoma Curcumae Longae extract C.
Preparation example 13: the preparation of common turmeric extract
Preparation method A: with the RADIX CURCUMAE volatile oil of 1000ml (contain 〉=7wt% sesquiterpene) is raw material, refining through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument, its process conditions: 35～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～90Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system temperatures, 15～30 milliliters/hour of material flows are only collected about 720ml and are heated up in a steamer excess, will heat up in a steamer excess and proceed molecular distillation.Vapo(u)rizing temperature is 45～60 ℃, distillation pressure 30～70Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, collect about 170ml distillation respectively and heat up in a steamer excess, will heat up in a steamer excess and carry out molecular distillation with about 540ml.Vapo(u)rizing temperature is 30～70 ℃, distillation pressure 10～50Pa, 300～320 rev/mins of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, collect the 160ml distillation, bleed off and heat up in a steamer excess, the secondary distillation that obtains is mixed (330ml), continue distillation, until utilize about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s (instrument: Bio-Rad FTS-65A, HP5890II, chromatographic column: BP-5) till the total sesquiterpene alkene content 〉=75wt% of Detection and Extraction thing, it also contains about 1/25 the curcumin that is equivalent to whole sesquiterpene gross weights, obtains the extract of about 70ml.Formulate for the detection finger printing with gas chromatography.This products known as common turmeric extract A.
Preparation method B. is with RADIX CURCUMAE volatile oil, through the rectification of material filling type vacuum rectifying apparatus
The material filling type vacuum rectifying apparatus is made of the still of dress 1000ml RADIX CURCUMAE volatile oil (contain 〉=7wt% sesquiterpene), stuffing rectification column (theoretical cam curve〉30), return channel, vacuum pump; manufacture and design by new packing technology development center of Shanghai Chemical Research Inst; filler is Dixon filler (a Q net ring filler); vacuum is 1～3mmHg, collects the above fraction of 82 ℃/3mmHg.Utilize the total sesquiterpene alkene content 〉=75wt% of about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS) Detection and Extraction things, obtain the extract of about 80ml.Extract detects through above-mentioned gas chromatograph-mass spectrometer (GC-MS), also contains about 1/25 the curcumin that is equivalent to whole sesquiterpene gross weights.Formulate for the detection finger printing with gas chromatography.This products known as common turmeric extract B.
In addition, the CO that learns from else's experience
2Supercritical extraction is crossed the RADIX CURCUMAE medicinal residues (1000g) of volatile oil, added the methanol reflux 3 hours, obtain brown solution, remove methanol with Rotary Evaporators, obtain brown dope, should be prepared chromatograph with silica gel by brown thickness paste,, obtain the 8g curcumin product of purity 〉=90wt% with the chloroform eluting.Above-mentioned common turmeric extract A and common turmeric extract B are mixed with the 50:50 weight ratio, formed mixture is mixed in the ratio of 20:1 (weight) with this curcumin product and obtain another kind of mixture, be called common turmeric extract C.
Embodiment 1: the preparation of the nano-lipid body injection of Radix Ginseng extract
Radix Ginseng extract (6.5g with preparation example 1, sesquiterpene content about 76 ± 2wt%) and refining soybean phospholipid, cholesterol mixes as initiation material with weight ratio at 1: 3.2: 1.5, add ether, addition is enough to fully dissolve this mixture, insert and remove the ether film forming on the Rotary Evaporators, the water for injection (is that 5mg/ml calculates this surplus according to the ultimate density that will prepare) that adds surplus is supplemented to 1 liter (L), be divided into 3 batches then and insert ultrasonic cell disruptor (model JCS-204) supersound process that Jining, Shandong ultrasonic electronic instrument plant produces, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after mean diameter≤100 nanometers, regulate PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, (new Asia, Shanghai purifies device factory and produces through 0.8 μ microporous filter membrane, specification 0.8 μ) filters and bottling then, it is 5 ± 0.10mg/ml with the ultimate density that about 30 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s described here record active component (weight (mg) of total sesquiterpene alkene active component/volumes of formulation (ml)) that 100 ℃ of vapor stream sterilizations, the injection product that is obtained are called for short injection 1. samplings.
Detect envelop rate with sieve method, promptly, utilize Sephadex G25 tomographic system that the extract nano-liposome preparation is divided into two stream parts, promptly extract nano-liposome stream part and free extract flow part are detected the extractive content that contains in two stream parts through gas chromatograph.Envelop rate surpasses 85%.The extractive content ÷ of envelop rate computing formula=extract nano-liposome stream part [extractive content+free extract amount of extract nano-liposome stream part].Microscope, transmission electron microscope detect, and confirmation is a nanometer liposome, provides the transmission electron microscope photo (scale 200nm) of this injection in accompanying drawing 1.
Embodiment 2: the preparation of the pro-liposome of Radix Ginseng extract
According to embodiment 1 in identical method and proportioning raw materials, repeat the program of embodiment 1, just require particle diameter≤1 micron of decentralized photo after the ultrasonic Treatment and after filtering and before bottling, the lactose that adds the 1-2% that accounts for the total formulation weight amount that is obtained, utilize 100 ℃ of vapor stream sterilizations then, lyophilization under the aseptic condition, embedding (packing).Detecting the confirmation preparation with the Coulter instrument is pro-liposome.This injection product is called for short injection 2.Envelop rate is higher than 85%.Said preparation added injection water before injection is used be 5 ± 0.10mg/ml to reach final active component (total sesquiterpene alkene class) concentration.
Embodiment 3: the preparation of the pro-liposome of SHULANHUA extract
Repeat the foregoing description 2, just with preparation example 2 and 3 separately the SHULANHUA extract A and the B mixture of press the 1:1 weight ratio (sesquiterpene content about 76 ± 2wt%) replaces the Radix Ginseng extract of preparation examples 1, is about 40000 low molecular dextran replacement lactose with average molecular weight Mw.This injection is called for short injection 3.Detecting the confirmation preparation with the Coulter instrument is pro-liposome.Envelop rate is higher than 85%.Said preparation added injection water before injection is used be 5 ± 0.10mg/ml to reach final activity component concentration (weight (mg) of total sesquiterpene alkene class/volumes of formulation (ml)).
Embodiment 4: the preparation of the long-circulating nanoliposome of Hemerocallis citrina Baroni Cuculus polioephalus extract
Repeat the operational degree of embodiment 1, just initiation material is composed of the following components: Hemerocallis citrina Baroni Cuculus polioephalus extract A (6.5g, sesquiterpene content about 76 ± 2wt%): soybean phospholipid: Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE: cholesterol=1:2.6:0.8:1.4 (weight ratio).This injection is called for short injection 4.Pick test mean diameter≤100nm.The ultimate density of total sesquiterpene alkene class is 5 ± 0.10mg/ml in the preparation.Envelop rate is higher than 85%.
Embodiment 5: the preparation of the nanometer microemulsion of electronegative and positively charged extract
5A-1: the preparation of electronegative Hemerocallis citrina Baroni Cuculus polioephalus extract nano microemulsion
Repeating the program of embodiment 1, is that (6.5g, sesquiterpene content about 76 ± 2wt%) replaces Radix Ginseng extracts to the Hemerocallis citrina Baroni Cuculus polioephalus extract B of using preparation example 4, and does not measure envelop rate.The ultimate density that sampling records total sesquiterpene alkene class is 5 ± 0.10mg/ml.Confirm it is the nanometer microemulsion with the Coulter instrument.This injection product is called for short 5A-1.
5A-2: the preparation of positively charged Hemerocallis citrina Baroni Cuculus polioephalus extract nano microemulsion
Repeat the program of embodiment 1, just initiation material is made up of following: Hemerocallis citrina Baroni Cuculus polioephalus extract B (6.5g, sesquiterpene content about 76 ± 2wt%): lecithin: n-octadecane base amine: cholesterol=1:2.8:0.4:1.5 (weight ratio) does not measure envelop rate.The ultimate density that sampling records total sesquiterpene alkene class is 5 ± 0.10mg/ml.Confirm it is the nanometer microemulsion with the Coulter instrument.This injection is called for short 5A-2.
5B-1: the preparation of electronegative Caulis Piperis Kadsurae extract nanometer microemulsion
Repeat above-mentioned 5A-1, just use Caulis Piperis Kadsurae extract A (6.5g, about 76 ± 2wt%) the replacement Hemerocallis citrina Baroni Cuculus polioephalus extract B of sesquiterpene content of preparation example 5.The ultimate density that sampling records total sesquiterpene alkene class is 5 ± 0.10mg/ml.Confirm it is the nanometer microemulsion with the Coulter instrument.The injection that is obtained is called for short 5B-1.
5B-2: the preparation of positively charged Caulis Piperis Kadsurae extract nanometer microemulsion
Repeat above-mentioned 5A-2, just replace the Hemerocallis citrina Baroni Cuculus polioephalus extract B of preparation example 4 with the Caulis Piperis Kadsurae extract A of preparation example 5.The ultimate density that sampling records total sesquiterpene alkene class is 5 ± 0.10mg/ml.Confirm it is the nanometer microemulsion with the Coulter instrument.The injection that is obtained is called for short 5B-2.
Embodiment 6: the preparation of the lipid nanospheres preparation of Fructus Tonnae Sinensis extract
Get the Fructus Tonnae Sinensis extract A (2.7g of preparation example 6, sesquiterpene content about 76 ± 2wt%) adds in the soybean oil with soybean phospholipid, intensification is controlled to oil phase (promptly remaining oil phase), and this oil phase is joined in the glycerinated aqueous solution (containing 20wt% glycerol).Its concrete weight ratio is: extract accounts for 0.27% of final total formulation weight amount, soybean phospholipid 1.6%, soybean oil 10%, glycerol 2.5%, surplus is water for injection (being supplemented to 1L), high-speed stirred then, regulating PH with 1M biphosphate sodium water solution is 4.5～6.5, handle through M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer again, carry out ultrasonic Treatment according to same procedure among the embodiment 1 then, make the mean diameter≤100nm of its lipid ball, again through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃～120 ℃ sterilizations.The ultimate density that sampling records total sesquiterpene alkene class is 2 ± 0.04mg/ml.The injection that is obtained is called for short injection 6.Can in raw material, comprise an amount of cholesterol, linoleic acid etc. as required.
Embodiment 7: the preparation of the intravenous injection breast of Fructus Tonnae Sinensis extract
Fructus Tonnae Sinensis extract B (6.5g with preparation example 6, about 76 ± 2wt%), the refining soybean phospholipid of sesquiterpene content, cholesterol mix with weight ratio 1:3.2:1.5, add to 1L with water for injection, with M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer emulsifying, till usefulness Coulter instrument checks particle diameter less than 1 μ always.Then, regulate PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, through 1.2 μ filtering with microporous membranes and bottling then, through 100 ℃～120 ℃ sterilizations, obtain Fructus Tonnae Sinensis extract vein breast, the ultimate density that sampling records total sesquiterpene alkene class is 5 ± 0.10mg/ml.This injection is called for short 7.
Embodiment 8: the preparation of Cupressus funebris Endl. extract fat emulsion
Get the Cupressus funebris Endl. extract A of preparation example 7 and B mixture (2.7g by the 1:1 weight ratio, sesquiterpene content about 76 ± 2wt%) adds in the soybean oil with soybean phospholipid, intensification is controlled to oil phase, and this oil phase is joined in the glycerinated aqueous solution (containing 20wt% glycerol).Its concrete weight ratio is: the Cupressus funebris Endl. extract accounts for 0.27% of total weight of formulation, soybean phospholipid 1.2%, and soybean oil 10%, glycerol 2.5% adds to 1L with water for injection.High-speed stirred then, regulating PH with 1M biphosphate sodium water solution is 4.5～6.5, handles through M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer, until with till Coulter instrument check particle diameter≤1 μ again, again through 1.2 μ filtering with microporous membranes and bottling then, 100 ℃～120 ℃ sterilizations.The ultimate density that records total sesquiterpene alkene class is 2 ± 0.04mg/ml.The injection that is obtained is called for short 8.Can in raw material, comprise an amount of cholesterol, linoleic acid etc. as required.
Embodiment 9:CO
2Supercritical methanol technology prepares nanometer liposome
Injection 9A: the chloroform that will account for the 10wt% of cholesterol weight adds to be made it in the cholesterol (0.8125g) to soak into, SHULANHUA extract A (0.65g with soybean phospholipid and preparation example 2, sesquiterpene content about 76 ± 2wt%) adds and wherein mixes (extract: soybean phospholipid: cholesterol=weight ratio 1.2:3.2:1.5), the mixture that is obtained is put into CO
2In the extractor of supercritical extraction instrument (Han Wei mechanical ﹠ electrical corporation in Guangzhou produces, model SFE100ml), feed CO
2(pressure 50-80kg), 50 ℃ of system temperatures carry out critical or supercritical dispersion, make the mixture uniform mixing, and slowly decompression discharges CO then
2, allow CO
2Chloroform is taken out of, take out extractor, the water for injection that adds surplus in extractor reaches 100mL, stirs with agitator, formed dispersion is handled with above-mentioned ultrasonic equipment, until till particle diameter of dispersing phase≤100 nanometers of liposome (obtaining nanometer liposome).Then liposome is regulated PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, filters and bottling then through 0.8 μ microporous filter membrane (new Asia, Shanghai purifies device factory and produces, specification 0.8 μ), 100 ℃ of vapor stream sterilizations, the injection that is obtained is called for short injection 9A.Sampling is 5 ± 0.10mg/ml with the ultimate density that gas chromatography records total sesquiterpene alkene class.Envelop rate is higher than 88%.
9B: repeating above-mentioned 9A, is that (0.65g, sesquiterpene content about 76 ± 2wt%) replaces the SHULANHUA extract A of preparation examples 2 to the Hemerocallis citrina Baroni Cuculus polioephalus extract A of using preparation example 4, makes nanometer liposome.The ultimate density that records total sesquiterpene alkene class in preparation is 5 ± 0.10mg/ml.The injection that is obtained is called for short 9B.Envelop rate is higher than 88%.
9C: repeat above-mentioned 9A, just use mixture (the 1:1:1 weight ratio of Radix Ginseng extract (preparation example 1), Fructus Tonnae Sinensis extract A (preparation example 6) and Cupressus funebris Endl. extract A (preparation example 7), 0.65g, sesquiterpene content about 76 ± 2wt%)) the SHULANHUA extract A of replacement preparation example 2 makes nanometer liposome.The ultimate density that records total sesquiterpene alkene class in preparation is 5 ± 0.10mg/ml.The injection that is obtained is called for short 9C.Envelop rate is higher than 88%.
9D: repeat above-mentioned 9A, (0.65g, sesquiterpene content about 76 ± 2wt%) replaces the SHULANHUA extract A of preparation examples 2, makes nanometer liposome just to use preparation example 8 and 9 elemene product separately to press the mixture of 1:1 weight ratio.The ultimate density that records total sesquiterpene alkene class in preparation is 5 ± 0.10mg/ml.The injection that is obtained is called for short 9D.Envelop rate is higher than 88%.
9E: repeat above-mentioned 9A, (0.58g, beta-elemene content about 86 ± 2wt%) replaces the SHULANHUA extract A of preparation examples 2, makes nanometer liposome just to use preparation example 10 and 11 beta-elemene product separately to press the mixture of 1: 1 weight ratio.The ultimate density that records beta-elemene in preparation is 5 ± 0.10mg/ml.The injection that is obtained is called for short 9E.Envelop rate is higher than 88%.
In addition, press the mixture of 1:1 weight ratio with Rhizoma Curcumae Longae extract A and Rhizoma Curcumae Longae extract B, common turmeric extract A and common turmeric extract B press the mixture of 1:1 weight ratio, or common turmeric extract C (they are 0.65g separately, sesquiterpene content about 76 ± 2wt%) replaces the SHULANHUA extract A of preparation example 2, repeat above-mentioned 9A, obtain injection 9F, 9G and 9H respectively, the ultimate density of total sesquiterpene alkene class is 5 ± 0.10mg/ml.Envelop rate all is higher than 88%.
Pharmacodynamics test
The anti-tumor in vivo Evaluation on effect method of pharmaceutical preparation has had description (for example referring to CN1153168A) in above some patent documentation of enumerating.
In the present invention, the preparation of various dosage forms is to human tumor heteroplastic transplantation model QGY hepatocarcinoma, the MKN-45 efficacy in treating gastric carcinoma of subcutaneous vaccination, can be by dosage with 80mg (sesquiterpene)/kg (body weight), 40mg/kg and 20mg/kg, every day, the tail intravenously administrable was 1 time, 10 days therapeutic scheme of successive administration confirms wherein there is not the group in contrast of administration.The relevant regulations " the evaluating drug effect way of antitumor drug " of the National Drug Administration that the calculating of concrete operating process and tumor control rate (tumour inhibiting rate) (referred to before the application's the applying date) according to is at present implemented.
Tumour inhibiting rate %=[(does not have the tumor weight of model of tumor weight-administration of the model of administration)/do not have a tumor weight of the model of administration] * 100%
The tumour inhibiting rate of table 1. human tumor heteroplastic transplantation model QGY hepatocarcinoma
| Injection | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate |
| 1 | 20 | 30.6％ | 40 | 39.3％ | 80 | 45.6％ |
| 2 | 20 | 30.1％ | 40 | 37.5％ | 80 | 44.0％ |
| 3 | 20 | 29.2％ | 40 | 39.2％ | 80 | 44.5％ |
| 4 | 20 | 29.8％ | 40 | 38.2％ | 80 | 44.4％ |
| 5A-1 | 20 | 30.4％ | 40 | 38.5％ | 80 | 46.0％ |
| 5A-2 | 20 | 31.2％ | 40 | 37.9％ | 80 | 46.6％ |
| 5B-1 | 20 | 30.6％ | 40 | 37.4％ | 80 | 45.3％ |
| 5B-2 | 20 | 29.4％ | 40 | 38.2％ | 80 | 44.9％ |
| 6 | 20 | 29.3％ | 40 | 38.7％ | 80 | 44.3％ |
| 7 | 20 | 29.5％ | 40 | 38.2％ | 80 | 44.5％ |
| 8 | 20 | 29.8％ | 40 | 37.8％ | 80 | 43.6％ |
| 9A | 20 | 29.4％ | 40 | 37.4％ | 80 | 44.0％ |
| 9B | 20 | 29.6％ | 40 | 37.6％ | 80 | 43.6％ |
| 9C | 20 | 29.4％ | 40 | 37.2％ | 80 | 44.6％ |
| 9D | 20 | 30.4％ | 40 | 38.5％ | 80 | 46.0％ |
| 9E | 20 | 31.2％ | 40 | 37.8％ | 80 | 46.6％ |
| 9F | 20 | 30.5％ | 40 | 37.4％ | 80 | 45.5％ |
| 9G | 20 | 29.6％ | 40 | 37.6％ | 80 | 43.6％ |
| 9H | 20 | 29.4％ | 40 | 38.2％ | 80 | 44.9％ |
The tumour inhibiting rate of table 2. human tumor heteroplastic transplantation model MKN-45 gastric cancer
| Injection | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate |
| 1 | 20 | 28.9％ | 40 | 39.3％ | 80 | 45.6％ |
| 2 | 20 | 29.1％ | 40 | 39.1％ | 80 | 45.0％ |
| 3 | 20 | 29.3％ | 40 | 37.5％ | 80 | 44.0％ |
| 4 | 20 | 28.4％ | 40 | 38.5％ | 80 | 43.2％ |
| 5A-1 | 20 | 29.1％ | 40 | 39.2％ | 80 | 44.5％ |
| 5A-2 | 20 | 29.0％ | 40 | 38.2％ | 80 | 44.4％ |
| 5B-1 | 20 | 29.4％ | 40 | 38.5％ | 80 | 46.0％ |
| 5B-2 | 20 | 29.5％ | 40 | 37.9％ | 80 | 46.6％ |
| 6 | 20 | 28.8％ | 40 | 37.4％ | 80 | 45.3％ |
| 7 | 20 | 28.3％ | 40 | 38.2％ | 80 | 44.9％ |
| 8 | 20 | 29.4％ | 40 | 38.5％ | 80 | 46.0％ |
| 9A | 20 | 29.5％ | 40 | 37.9％ | 80 | 46.6％ |
| 9B | 20 | 28.8％ | 40 | 37.4％ | 80 | 45.3％ |
| 9C | 20 | 28.3％ | 40 | 38.2％ | 80 | 49.9％ |
| 9D | 20 | 29.3％ | 40 | 38.5％ | 80 | 44.0％ |
| 9E | 20 | 28.4％ | 40 | 38.5％ | 80 | 43.2％ |
| 9F | 20 | 29.6％ | 40 | 39.2％ | 80 | 47.5％ |
| 9G | 20 | 29.0％ | 40 | 39.2％ | 80 | 48.4％ |
| 9H | 20 | 29.4％ | 40 | 38.5％ | 80 | 46.0％ |
Table 3: to the curative effect (tumour inhibiting rate) of intracranial in-situ inoculating mice cerebroma G422 model.
| Injection | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate |
| 1 | 20 | 47.2％ | 40 | 59.5％ | 80 | 68.6％ |
| 2 | 20 | 46.8％ | 40 | 59.6％ | 80 | 68.5％ |
| 3 | 20 | 47.6％ | 40 | 57.2％ | 80 | 64.0％ |
| 4 | 20 | 48.6％ | 40 | 56.3％ | 80 | 63.2％ |
| 5A-1 | 20 | 49.5％ | 40 | 59.2％ | 80 | 67.5％ |
| 5A-2 | 20 | 48.4％ | 40 | 58.3％ | 80 | 66.4％ |
| 5B-1 | 20 | 49.3％ | 40 | 58.4％ | 80 | 67.0％ |
| 5B-2 | 20 | 49.4％ | 40 | 57.7％ | 80 | 67.6％ |
| 6 | 20 | 48.2％ | 40 | 57.6％ | 80 | 66.3％ |
| 7 | 20 | 49.3％ | 40 | 58.4％ | 80 | 67.9％ |
| 8 | 20 | 49.8％ | 40 | 58.7％ | 80 | 68.3％ |
| 9A | 20 | 47.8％ | 40 | 58.3％ | 80 | 65.5％ |
| 9B | 20 | 47.2％ | 40 | 56.8％ | 80 | 63.6％ |
| 9C | 20 | 46.9％ | 40 | 56.9％ | 80 | 63.5％ |
| 9D | 20 | 48.4％ | 40 | 58.3％ | 80 | 66.4％ |
| 9E | 20 | 51.3％ | 40 | 58.4％ | 80 | 69.0％ |
| 9F | 20 | 49.4％ | 40 | 58.7％ | 80 | 67.6％ |
| 9G | 20 | 49.2％ | 40 | 57.6％ | 80 | 68.3％ |
| 9H | 20 | 49.3％ | 40 | 59.4％ | 80 | 69.9％ |
Table 4: to the curative effect (tumour inhibiting rate) of subcutaneous vaccination mice cerebroma G422 model.
| Injection | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate | Dosage (mg/kg) | Tumour inhibiting rate |
| 1 | 20 | 23.1％ | 40 | 29.8％ | 80 | 35.4％ |
| 2 | 20 | 23.0％ | 40 | 29.2％ | 80 | 35.6％ |
| 3 | 20 | 22.8％ | 40 | 27.8％ | 80 | 34.0％ |
| 4 | 20 | 22.8％ | 40 | 28.5％ | 80 | 33.3％ |
| 5A-1 | 20 | 22.9％ | 40 | 29.8％ | 80 | 34.6％ |
| 5A-2 | 20 | 23.0％ | 40 | 28.7％ | 80 | 34.8％ |
| 5B-1 | 20 | 24.6％ | 40 | 28.1％ | 80 | 36.6％ |
| 5B-2 | 20 | 24.4％ | 40 | 27.9％ | 80 | 36.6％ |
| 6 | 20 | 23.2％ | 40 | 27.8％ | 80 | 35.6％ |
| 7 | 20 | 23.6％ | 40 | 28.7％ | 80 | 34.4％ |
| 8 | 20 | 23.4％ | 40 | 28.7％ | 80 | 34.8％ |
| 9A | 20 | 24.1％ | 40 | 28.2％ | 80 | 34.5％ |
| 9B | 20 | 22.4％ | 40 | 27.9％ | 80 | 33.6％ |
| 9C | 20 | 22.2％ | 40 | 27.8％ | 80 | 33.4％ |
| 9D | 20 | 22.9％ | 40 | 29.8％ | 80 | 38.6％ |
| 9E | 20 | 22.0％ | 40 | 28.7％ | 80 | 34.8％ |
| 9F | 20 | 24.6％ | 40 | 28.1％ | 80 | 38.6％ |
| 9G | 20 | 23.4％ | 40 | 28.9％ | 80 | 36.6％ |
| 9H | 20 | 23.2％ | 40 | 27.8％ | 80 | 35.6％ |
In addition, the injection of various dosage forms improves 21%～23% to the NK cells in mice and the matched group specific activity of lotus Lewis lung cancer.The injection of various dosage forms has improved 1.5～1.45 to the influence of mouse lymphocyte proliferation activity, the stimulation index of lotus Lewis lung cancer.
In a word, from above-mentioned table data as can be seen, various preparations are effective through QGY hepatocarcinoma, MKN-45 gastric cancer, mice cerebroma G422 model pharmacology test.To the Immune Function test, the NK cells in mice of lotus Lewis lung cancer improves 21%～23% with the matched group ratio.Lymphocyte increment activity influence, stimulation index have improved 1.5～1.45.
So injection can be used in and suppresses and/or the treatment tumor.In addition, through overtesting, said preparation can also be used for anticancer to be shifted, and is used for tumor remission pain, is used for prevention or treatment cerebral infarction.
Claims (9)
1, the extract injection that contains the plant of sesquiterpene, it contains: the extract with total sesquiterpene alkene class content 〉=70wt% that extracts from the plant that contains sesquiterpene, surfactant, and water-bearing media, it is characterized in that: described injection is a kind of aqueous dispersion, the mean diameter of decentralized photo≤100 nanometers, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, the ultimate density of total sesquiterpene alkene class is 1～10mg/ml in the injection.
2, according to the extract injection of claim 1, wherein the dosage form of injection is the nanometer liposome dosage form, and wherein the concentration of the total sesquiterpene alkene class of injection is 2～7.5mg/ml.
3, extract injection according to claim 2, wherein the dosage form of injection is the long-circulating nanoliposome dosage form.
4, according to the extract injection of claim 1, wherein the dosage form of injection is nanometer microemulsion type or lipid nanospheres dosage form, and wherein the concentration of the total sesquiterpene alkene class of injection is 2-7.5mg/ml.
5, according to the extract injection of claim 4, wherein the concentration of the total sesquiterpene alkene class of lipid nanospheres dosage form injection is 2.0-3.0mg/ml.
6, the preparation method of the described extract injection of claim 2, it comprises: extract is mixed with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0 with soybean phospholipid, cholesterol; A kind of solvent that adds in ether, acetone or the chloroform fully dissolves mixture, insert and remove the solvent film forming on the Rotary Evaporators, add the water for injection of surplus, then material is carried out ultrasonic Treatment till obtaining particle diameter of dispersing phase≤100 nanometers, obtain the injection of nanometer liposome dosage form.
7, the preparation method of the described extract injection of claim 3, it comprises: with extract and refining soybean phospholipid, the Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, cholesterol mixes with weight ratio 0.5-1.5:1.5-3.5:0.3-1.2:0.8-2.0, wherein the mean molecule quantity of Polyethylene Glycol segment is 2000 dalton in the Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, add ether, a kind of solvent in acetone or the chloroform fully dissolves mixture, insert and remove the solvent film forming on the Rotary Evaporators, add the water for injection of surplus, then material is carried out ultrasonic Treatment till obtaining particle diameter of dispersing phase≤100 nanometers, obtain the injection of long-circulating nanoliposome dosage form.
8, the preparation method of the described nanometer microemulsion type extract injection of claim 4, it comprises: extract is mixed with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0 with refining soybean phospholipid, cholesterol, a kind of solvent that adds in ether, acetone or the chloroform fully dissolves mixture, insert and remove the solvent film forming on the Rotary Evaporators, add the water for injection of surplus, then material is carried out ultrasonic Treatment till obtaining particle diameter of dispersing phase≤100 nanometers, obtain the injection of nanometer microemulsion type.
9, the extract injection of any one is used to prepare the purposes that suppresses or treat the medicine of cancer among the claim 1-5.
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Application publication date: 20031112 Assignee: Dalian Holley Kingkong Pharmaceutical Co., Ltd. Assignor: Xie Tian Contract record no.: 2013210000024 Denomination of invention: Sesquiterpene injection, and preparing method and use thereof Granted publication date: 20090715 License type: Exclusive License Record date: 20130329 |
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Owner name: DALIAN HOLLEY KINGKONG PHARMACEUTICAL CO., LTD. Free format text: FORMER OWNER: XIE TIAN Effective date: 20150330 |
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Free format text: CORRECT: ADDRESS; FROM: 310013 HANGZHOU, ZHEJIANG PROVINCE TO: 116100 DALIAN, LIAONING PROVINCE |
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Effective date of registration: 20150330 Address after: Jinzhou District of Dalian City, Liaoning province 116100 Chengen Street No. 8 Patentee after: Dalian Holley Kingkong Pharmaceutical Co., Ltd. Address before: Is 310013 Hangzhou Road, Zhejiang province No. 36 Luyuan Ginkgo Garden 3-1902 Patentee before: Xie Tian |
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Granted publication date: 20090715 |