CN102743376A - Costustoot pharmaceutical composition and medicinal application - Google Patents
Costustoot pharmaceutical composition and medicinal application Download PDFInfo
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- CN102743376A CN102743376A CN2012101850714A CN201210185071A CN102743376A CN 102743376 A CN102743376 A CN 102743376A CN 2012101850714 A CN2012101850714 A CN 2012101850714A CN 201210185071 A CN201210185071 A CN 201210185071A CN 102743376 A CN102743376 A CN 102743376A
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Abstract
The invention relates to the medicinal field, and concretely discloses a costustoot pharmaceutical composition and a medicinal application. The pharmaceutical composition of the present invention is composed of costunolide and dehydrocostus lactone; and the invention also discloses a costustoot volatile oil and a medicinal preparation, and the application of the costustoot volatile oil and the medicinal preparation by being taken as antitumor drugs. In the pharmaceutical composition, the volatile oil and the medicinal preparation, costunolide and dehydrocostus lactone have substantial synergism, the toxic and side effects are small, the pharmaceutical composition, the volatile oil and the medicinal preparation have good clinic application prospect, the preparation method is simple, the cost is low, and the pharmaceutical composition, the volatile oil and the medicinal preparation are suitable for requirements of industrial production.
Description
Technical field
The present invention relates to field of medicaments, specifically disclose a kind of volatile oil of Radix Aucklandiae and pharmaceutical composition thereof, and their method for preparing and purposes.
Background technology
Traditional medicine volatile oil (volatile oil) is also claimed quintessence oil (essential oil); It is in the Chinese medicine with the general name of the not miscible volatility oily composition of water; Contained chemical constituent more complicated, have cough-relieving clinically, relieving asthma, analgesia, analgesic, diuresis, be good for the stomach, multiple efficacies such as antibiotic, parasite killing and heart tonifying.
The Radix Aucklandiae is the dry root of Compositae herbaceos perennial Radix Aucklandiae Saussurea lappa Decne, and main product is claimed Saussurea lappa Clarke again in Yunnan.The research of traditional medicine and modern pharmacology shows that the Radix Aucklandiae has: invigorating the spleen and regulating the stomach, promoting the flow of QI for relieving the stagnancy, analgesia, improve gastric ulcer, promote gallbladder to shrink, anti-inflammatory, improve effects such as heart infarction and angina pectoris.
The main effective ingredient of the Radix Aucklandiae is present in the volatile oil of Radix Aucklandiae, is to be the mixture of master's multiple sesquiterpenoids with costunolide (costunolide) and dehydrocostuslactone (dehydrocostus lactone).Lee etc. inquire into the Anticancer Effect and Mechanism of costunolide first, and experimental result shows, thereby costunolide discharges the induced strong cancer cell-apoptosis through the penetrating transfer of mitochondrion and the cytochrome C of inducing the ROS mediation.Wang Lu etc. have separated 18 kinds of sesquiterpenoidss in the volatile oil of Radix Aucklandiae, and carry out the vitro cytotoxicity test with 6 kinds of people source tumor cells, and the result shows: △
11 (13)The exocyclic double bond structure possibly be the important structure unit that Radix Aucklandiae sesquiterpenoid has remarkable anti-tumor activity; Radix Aucklandiae sesquiterpenoid 48h under the concentration of 100 μ mol/L with this kind construction unit all has stronger inhibitory action to 6 kinds of tumor cell lines; Suppression ratio is mostly at 70-90%, and inhibitory action strengthens along with increasing of concentration basically.
The extracorporeal anti-tumor research about costunolide in the volatile oil of Radix Aucklandiae and dehydrocostuslactone monomeric compound at present has bibliographical information, but does not all appear in the newspapers about the anti-tumor medical application of volatile oil of Radix Aucklandiae and with the medical usage of costunolide and dehydrocostuslactone drug combination.
Summary of the invention
The object of the present invention is to provide a kind of pharmaceutical preparation that is used to treat pharmaceutical composition, the volatile oil of Radix Aucklandiae of tumor and is used to treat tumor; In pharmaceutical composition of the present invention, volatile oil and the pharmaceutical preparation; Costunolide and dehydrocostuslactone have significant synergism, and stronger anti-tumor activity and lower toxicity are arranged.
First aspect present invention discloses a kind of pharmaceutical composition that is used to treat tumor, and said pharmaceutical composition is made up of costunolide and dehydrocostuslactone.
More excellent, the mass ratio of said costunolide and dehydrocostuslactone is 1:0.4～2.5.
More excellent, the mass ratio of said costunolide and dehydrocostuslactone is 1:1～2.2.
Optimum, the mass ratio of said costunolide and dehydrocostuslactone is 1:1.
One of innovative point of the present invention is the synergistic antitumor effect that discloses costunolide and dehydrocostuslactone, and the coefficient compositions of costunolide and dehydrocostuslactone has better anti-tumor activity and lower toxicity than costunolide or independent use of dehydrocostuslactone.
Second aspect present invention discloses a kind of volatile oil of Radix Aucklandiae, and for taking the Radix Aucklandiae as the acquisition of raw material effective component extracting, said effective ingredient is costunolide and dehydrocostuslactone.
More excellent, said effective ingredient accounts for 30～80% of volatile oil of Radix Aucklandiae gross mass.
Further, said effective ingredient accounts for 45～75% of volatile oil of Radix Aucklandiae gross mass; Further, said effective ingredient accounts for 55～75% of volatile oil of Radix Aucklandiae gross mass; Optimum, said effective ingredient accounts for 70～75% of volatile oil of Radix Aucklandiae gross mass.
Further, said being extracted as adopts normal hexane or petroleum ether to extract as extractant.
Further, said extraction is specially: get an amount of exsiccant Radix Aucklandiae, pulverizing, after soaking as extractant with normal hexane or petroleum ether, supersound extraction is filtered, and removes the extractant in the filtrating, obtains said volatile oil of Radix Aucklandiae.
More excellent, said extractant consumption is 5～20 times of Radix Aucklandiae quality.Optimum, said extractant consumption is 10 times of Radix Aucklandiae quality.
More excellent, said extractant soak time is 0.5～2 hour.Optimum, said extractant soak time is 1 hour.
More excellent, the said supersound extraction time is 0.5～2 hour.Optimum, the said supersound extraction time is 1 hour.
In order to reach the purpose of abundant extraction, can carry out repeated ultrasonic to the Radix Aucklandiae and extract, each supersound extraction after-filtration; With filtering residue reuse normal hexane or Petroleum ether extraction 1～3 time; Merge multiple extraction filtrating, remove the extractant in the filtrating, obtain volatile oil of Radix Aucklandiae according to the invention.
Extractant can be removed through the method that Rotary Evaporators volatilizes in the filtrating.
The inventor has carried out system's contrast and analysis-by-synthesis to the anti-tumor activity of volatile oil of Radix Aucklandiae; Find that the volatile oil that the Radix Aucklandiae comes out through normal hexane extraction has the medicinal character of optimum; Show good antineoplastic activity and low toxicity, the suitable especially antitumor drug that is prepared into.
In volatile oil of Radix Aucklandiae, costunolide and dehydrocostuslactone show synergism, have effectively suppressed tumor propagation, have obtained tumor killing effect preferably; And volatile oil of Radix Aucklandiae not only toxic and side effects is less, can also strengthen the body constitution of nude mice, and tumor killing effect surpasss the expectation; Therefore, when from the Radix Aucklandiae, extracting volatile oil, also need not spend big strength the two is separated purification separately, so not only can reduce the cost of medicine greatly, also can obtain extraordinary drug effect simultaneously.
Third aspect present invention discloses a kind of pharmaceutical preparation that is used to treat tumor, comprises the pharmaceutical composition of the present invention or the volatile oil of Radix Aucklandiae of the present invention of safe and effective amount.
More excellent, also comprise pharmaceutically acceptable carrier in the said pharmaceutical preparation.
Further, the quality of said pharmaceutical composition or volatile oil of Radix Aucklandiae accounts for 1～99% of said pharmaceutical preparation gross mass, and surplus is pharmaceutically acceptable carrier.Further, the quality of said pharmaceutical composition or volatile oil of Radix Aucklandiae accounts for 3%～75% of said pharmaceutical preparation gross mass.
Pharmaceutically acceptable carrier is various adjuvant and/or excipient pharmaceutically commonly used, includes, but is not limited to saccharide (like lactose, dextrose plus saccharose), starch (like corn starch and potato starch), cellulose and derivant thereof (like sodium carboxymethyl cellulose, ethyl cellulose and methylcellulose); Tragacanth gum powder, Fructus Hordei Germinatus, gelatin, Talcum; Kollag (like stearic acid and magnesium stearate), calcium sulfate, vegetable oil; Like Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil, polyhydric alcohol (like propylene glycol, glycerol, Sorbitol, mannitol and Polyethylene Glycol), alginic acid; Emulsifying agent (like Tween, polyoxyethylene castor oil), wetting agent (like sodium lauryl sulfate), coloring agent; Flavoring agent, tablet agent, stabilizing agent, antioxidant; Antiseptic, apirogen water waits and oozes saline solution and phosphate buffer etc.; This carrier can improve stability, activity and the biological effectiveness etc. of prescription as required.
Pharmaceutical preparation of the present invention can be processed the dosage form of any routine according to the universal method on the pharmaceutics.Preferably, pharmaceutical preparation according to the invention is injection.
The method for preparing of above-mentioned injection is: in said pharmaceutical composition, add dehydrated alcohol, the excusing from death dissolving; Add polyoxyethylene castor oil then, fully mix; In mixture, add normal saline at last, mixing, sterilization obtain said injection.
More excellent, the volume ratio of said dehydrated alcohol, polyoxyethylene castor oil and normal saline is 1～3:1～3:4～8.
Optimum, the volume ratio of said dehydrated alcohol, polyoxyethylene castor oil and normal saline is 1:1:8.
Fourth aspect present invention discloses pharmaceutical composition according to the invention, volatile oil of Radix Aucklandiae or the pharmaceutical preparation purposes in the preparation anti-tumor medicine.
More excellent, said tumor is selected from the arbitrary of breast carcinoma, cervical cancer or leukemia.
The present invention adopts two-dimensional gas chromatography mass spectrometry and two kinds of methods of liquid chromatograph that the effective ingredient in the volatile oil of Radix Aucklandiae is carried out qualitative and quantitative analysis; Wherein, What the two-dimensional gas chromatography mass spectrometry adopted is that the peak area normalization method is quantitative, and the liquid chromatography employing is that external standard method is quantitative.
The present invention has significant synergism in vivo through experiment confirm costunolide and two kinds of compositions of dehydrocostuslactone, and toxicity is lower, has the good clinical Application Prospect; And the present invention also provides a kind of volatile oil of Radix Aucklandiae and preparation method thereof; The volatile oil of Radix Aucklandiae of the present invention preparation has the body constitution that good antineoplastic activity, toxic and side effects are little, can strengthen subject, and method for preparing is convenient, cost is low, be fit to need of industrial production.
Description of drawings
Fig. 1: hydrodistillation extracts the volatile oil of Radix Aucklandiae two dimension GC-MS figure that obtains
Fig. 2: the volatile oil of Radix Aucklandiae two dimension GC-MS figure that normal hexane extraction obtains
Fig. 3: the volatile oil of Radix Aucklandiae two dimension GC-MS figure that Petroleum ether extraction obtains
Fig. 4: the volatile oil of Radix Aucklandiae of Different Extraction Method preparation is to the influence of MCF-7 cell growth
Fig. 5: costunolide is to MCF-7, Hela, the influence of HL-60 and the growth of K562 cell
Fig. 6: dehydrocostuslactone is to MCF-7, Hela, the influence of HL-60 and the growth of K562 cell
Fig. 7: costunolide and dehydrocostuslactone synergy be to MCF-7, Hela, the influence of HL-60 and the growth of K562 cell
Fig. 8: volatile oil of Radix Aucklandiae is to MCF-7, Hela, the influence of HL-60 and the growth of K562 cell
Fig. 9: MCF-7 tumor bearing nude mice growth of tumor curve after the pharmaceutical intervention
Figure 10: the body weight curve of MCF-7 tumor bearing nude mice after the pharmaceutical intervention
The specific embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term that uses in the embodiment of the invention is in order to describe specific specific embodiments, rather than in order to limit protection scope of the present invention.
When embodiment provides numerical range, only if should be understood that the present invention explanation is arranged in addition, any one numerical value all can be selected for use between two end points of each numerical range and two end points.Only if definition in addition, all technology used among the present invention and the same meaning of scientific terminology and those skilled in the art of the present technique's common sense.The concrete grammar that in embodiment, uses, equipment, the material; According to the technical staff in present technique field grasp and record of the present invention, can also use with the method described in the embodiment of the invention, equipment, material is similar or any method, equipment and the material of the prior art that is equal to are realized the present invention to prior art.
Unless otherwise indicated, disclosed experimental technique, detection method, method for preparing all adopt the routine techniques of conventional molecular biology, biochemistry, analytical chemistry, cell culture technology and association area of present technique field among the present invention.
The preparation of embodiment 1 pharmaceutical composition, volatile oil of Radix Aucklandiae and pharmaceutical preparation
One, preparation of drug combination:
Method 1: get 100mg costunolide and 100mg dehydrocostuslactone, mix homogeneously prepares pharmaceutical composition.
Method 2: get 1g costunolide and 400mg dehydrocostuslactone, mix homogeneously prepares pharmaceutical composition.
Method 3: get 300mg costunolide and 700mg dehydrocostuslactone, mix homogeneously prepares pharmaceutical composition.
Method 4: get 400mg costunolide and 1g dehydrocostuslactone, mix homogeneously prepares pharmaceutical composition.
Two, the preparation of volatile oil of Radix Aucklandiae:
Method 5:
Get exsiccant Radix Aucklandiae medical material 50g, suitably pulverize, in the beaker of the 1000ml that packs into; Soaked after 1 hour supersound extraction 1 hour with 10 times of amount normal hexane, filter back residue reuse normal hexane and repeat to extract twice, when extracting at every turn; The consumption of normal hexane is 10 times of amounts, and the supersound extraction time is 1 hour, merges three times filtrating; On Rotary Evaporators, volatilize normal hexane, obtain volatile oil of Radix Aucklandiae 1.9g, liquid chromatography records that costunolide and dehydrocostuslactone content summation are 70.70% in the volatile oil of Radix Aucklandiae; Wherein, the mass ratio of costunolide and dehydrocostuslactone is 32:68.
Method 6:
Get exsiccant Radix Aucklandiae medical material 50g, suitably pulverize, in the beaker of the 1000ml that packs into; Soaked after 1 hour supersound extraction 0.5 hour with 20 times of amount petroleum ether, filter back residue reuse petroleum ether and repeat to extract twice, when extracting at every turn; The consumption of petroleum ether is 5 times of amounts, and the supersound extraction time is 2 hours, merges three times filtrating; At the dried petroleum ether of Rotary Evaporators Back stroke, obtain volatile oil of Radix Aucklandiae 1.87g, liquid chromatography records that costunolide and dehydrocostuslactone content summation are 72.5% in the volatile oil of Radix Aucklandiae; Wherein, the mass ratio of costunolide and dehydrocostuslactone is 31:69.Method 7:
Get exsiccant Radix Aucklandiae medical material 50g, suitably pulverize, in the beaker of the 1000ml that packs into; Soaked after 2 hours supersound extraction 1 hour with 10 times of amount normal hexane, filter back residue reuse normal hexane and repeat to extract twice, when extracting at every turn; The consumption of normal hexane is 20 times of amounts, and the supersound extraction time is 2 hours, merges three times filtrating; On Rotary Evaporators, volatilize normal hexane, obtain volatile oil of Radix Aucklandiae 1.91g, liquid chromatography records that costunolide and dehydrocostuslactone content summation are 83.7% in the volatile oil of Radix Aucklandiae; Wherein, the mass ratio of costunolide and dehydrocostuslactone is 29:71.
Method 8:
Get exsiccant Radix Aucklandiae medical material 50g; Suitably pulverize, in the beaker of the 1000ml that packs into, with 10 times of amount petroleum ether immersion supersound extraction 0.5 hour after 0.5 hour; Filter; To filtrate at the dried petroleum ether of Rotary Evaporators Back stroke, and obtain volatile oil of Radix Aucklandiae 1.17g, liquid chromatography records that costunolide and dehydrocostuslactone content summation are 33.2% in the volatile oil of Radix Aucklandiae; Wherein, the mass ratio of costunolide and dehydrocostuslactone is 1:2.5.
Method 9:
Get exsiccant Radix Aucklandiae medical material 50g; Suitably pulverize, in the round-bottomed flask of the 1000ml that packs into, measure distilled water immersions after 12 hours with 15 times; Extracted 6 hours with hydrodistillation; Collect volatile oil and weigh after with anhydrous sodium sulfate drying, obtain volatile oil of Radix Aucklandiae 0.23g, liquid chromatography records that costunolide and dehydrocostuslactone content summation are 12.5% in the volatile oil of Radix Aucklandiae.
Three, the preparation of pharmaceutical preparation:
Method 10:
Take by weighing the volatile oil of Radix Aucklandiae 150mg of method 5 preparations, add the 5ml dehydrated alcohol, ultrasonic dissolution; Add the 5ml polyoxyethylene castor oil, abundant mixing adds the normal saline of 40ml; Ultrasonic, pack sterilization behind the whirlpool mixing; Get aseptic parenteral solution, the concentration of volatile oil of Radix Aucklandiae is 3mg/mL in this aseptic parenteral solution, and costunolide and dehydrocostuslactone total concentration are 2.1mg/mL.
Method 11:
Take by weighing the pharmaceutical composition 150mg of method 1 preparation, add the 5ml dehydrated alcohol, ultrasonic dissolution; Add the 5ml polyoxyethylene castor oil, abundant mixing adds the normal saline of 40ml; Ultrasonic, pack sterilization behind the whirlpool mixing; Promptly get aseptic parenteral solution, the total concentration of costunolide and dehydrocostuslactone is 3mg/mL in this aseptic parenteral solution.
1, experiment material:
1.1 cell strain is used in experiment: human breast cancer cell MCF-7.
1.2 the volatile oil of Radix Aucklandiae that Different Extraction Method obtains:
Method A: with embodiment 1 method 5, the volatile oil of Radix Aucklandiae of acquisition is labeled as volatile oil 1.
Method B: with embodiment 1 method 9, the volatile oil of Radix Aucklandiae of acquisition is labeled as volatile oil 2.
Method C: with embodiment 1 method 6, the volatile oil of Radix Aucklandiae of acquisition is labeled as volatile oil 3.
2, experimental technique
2.1 the analysis of the volatile oil of Radix Aucklandiae composition that Different Extraction Method obtains
The content of costunolide and dehydrocostuslactone in the volatile oil of Radix Aucklandiae that two-dimensional gas chromatography mass spectrometry method and liquid chromatography analysis distinct methods obtain.
2.2 the cell in vitro of the volatile oil of Radix Aucklandiae cancer suppressing action that Different Extraction Method obtains experiment
Get and be in human breast cancer cell MCF-7 in good condition exponential phase of growth, after 0.25% trypsinization, process cell suspension behind the counting, cell density is adjusted to 4 * 10
4Individual/ml, be inoculated on 96 orifice plates, a zeroing hole is established in 90 μ l/ holes, places constant temperature CO
2Cultivated in the incubator 8 hours, and treated to add behind the cell attachment receiving the reagent thing, 10 μ l/ holes receive the reagent thing to establish six concentration, and each concentration is established three multiple holes.Cultivate after 48 hours in the cell culture incubator, MTT is added in 96 orifice plates, 15 μ l/ holes continue to cultivate 4 hours, inhale and remove supernatant, add DMSO, 150 μ l/ holes, dull and stereotyped shaking table concussion 5 minutes.Measure the absorbance in every hole at wavelength 570nm place with ELIASA, and calculate cell inhibitory rate, the experiment triplicate.
Cell inhibitory rate %=(negative control group OD value-tried thing group OD value)/(negative control group OD value-zeroing hole OD value)
3, experiment conclusion
3.1 the content analysis of costunolide and dehydrocostuslactone in the volatile oil of Radix Aucklandiae
In the GC-MS analysis chart (Fig. 1-3), each bright spot is represented a component, and the high more expression relative amount of brightness is high more, and the several bright spots in the black circle are represented the isomers of dehydrocostuslactone and costunolide and costunolide; Experimental data is seen table 1.
The component analysis of the volatile oil of Radix Aucklandiae that the different method for preparinies of table 1 obtain
Can know by table 1; The volatile oil 1 of normal hexane extraction and volatile oil 3 compositions of Petroleum ether extraction and relative percentage composition are much at one; And have than big difference with volatile oil 2 that hydrodistillation extracts: dehydrocostuslactone content has only 18% of dehydrocostuslactone content in the volatile oil 1 of normal hexane extraction, the content of costunolide and five isomerss thereof to have only 15% of costunolide in the volatile oil 1 and five isomerism body burdens thereof in the volatile oil 2 that hydrodistillation extracts; The total content of costunolide and dehydrocostuslactone is about 17.5% of costunolide and dehydrocostuslactone total amount in the volatile oil of Radix Aucklandiae of normal hexane or Petroleum ether extraction in the volatile oil 2 that hydrodistillation extracts.
3.2 the experiment in vitro result of the volatile oil of Radix Aucklandiae cancer suppressing action of distinct methods preparation
Can know by Fig. 4 result, the volatile oil of Radix Aucklandiae that three kinds of methods obtain to human breast cancer cell MCF-7 external all inhibited; Wherein, the effect that the volatile oil of Radix Aucklandiae of petroleum ether and normal hexane extraction (volatile oil 1 and volatile oil 3) suppresses tumor cell is close, and significantly is superior to the volatile oil of Radix Aucklandiae (volatile oil 2) that hydrodistillation extracts.
Can see that from the two-dimentional GC-MS figure of volatile oil of Radix Aucklandiae the volatile oil of Radix Aucklandiae composition of petroleum ether and normal hexane extraction is with percentage composition is very approaching relatively, so they suppress the effect of tumor, and also ten minutes is approaching.And the composition of the volatile oil of Radix Aucklandiae that water extraction obtains is with percentage composition and other two kinds of volatile oil have bigger difference relatively; Be mainly reflected in; Water is carried the volatile oil of Radix Aucklandiae of the content of costunolide in the volatile oil and five kinds of isomerss and dehydrocostuslactone far below normal hexane and Petroleum ether extraction; Therefore, though the volatile oil of water extraction preparation also possesses tumor-inhibiting action, not as normal hexane and Petroleum ether extraction method.
Extracorporeal anti-tumor effect in conjunction with three kinds of volatile oil is analyzed, and we can reach a conclusion basically: costunolide and five kinds of isomerss thereof and dehydrocostuslactone are the main anti-tumor active ingredients of volatile oil of Radix Aucklandiae.
1, experiment material
1.1 cell strain is used in experiment
Human breast cancer cell MCF-7, human cervical carcinoma cell Hela, human promyelocytic leukemia cell HL-60, people's chronic granulocytic leukemia cell K562.
1.2 receive the reagent thing
Mark C: get costunolide (available from source, Shanghai leaf bio tech ltd) 150mg, add the 5ml dehydrated alcohol, ultrasonic dissolution adds the 5ml polyoxyethylene castor oil; Abundant mixing adds the normal saline of 40ml, and is ultrasonic, packs behind the whirlpool mixing; Sterilization promptly gets aseptic parenteral solution, is designated as mark C.
Mark D: remove hydrogen constuslactone (available from source, Shanghai leaf bio tech ltd) 150mg, add the 5ml dehydrated alcohol, ultrasonic dissolution adds the 5ml polyoxyethylene castor oil; Abundant mixing adds the normal saline of 40ml, and is ultrasonic, packs behind the whirlpool mixing; Sterilization promptly gets aseptic parenteral solution, is designated as mark D.
Preparation 1: the injection of getting 10 preparations of embodiment 1 method is designated as preparation 1.
Preparation 2: the injection of getting 11 preparations of embodiment 1 method is designated as preparation 2.
2, experimental technique
2.1 the cell of adherent growth (MCF-7 cell, Hela cell):
Get and be in cell in good condition exponential phase of growth, after 0.25% trypsinization, process cell suspension behind the counting, cell density is adjusted to 4 * 10
4Individual/ml, be inoculated on 96 orifice plates, a zeroing hole is established in 90 μ l/ holes, places constant temperature CO
2Cultivated in the incubator 8 hours, and treated to add behind the cell attachment receiving the reagent thing, 10 μ l/ holes receive the reagent thing to establish six concentration, and each concentration is established three multiple holes.Cultivate after 48 hours in the cell culture incubator, MTT is added in 96 orifice plates, 15 μ l/ holes continue to cultivate 4 hours, inhale and remove supernatant, add DMSO, 150 μ l/ holes, dull and stereotyped shaking table concussion 5 minutes.Measure the absorbance in every hole at wavelength 570nm place with ELIASA, and calculate cell inhibitory rate, the experiment triplicate.
2.2 the cell of suspension growth (HL-60 cell, K562 cell):
Get and be in cell in good condition exponential phase of growth, cell density is adjusted to 4 * 10
4Individual/ml, be inoculated on 96 orifice plates, a zeroing hole is established in 90 μ l/ holes, places constant temperature CO
2Cultivated 8 hours in the incubator, adding receives the reagent thing, and 10 μ l/ holes receive the reagent thing to establish six concentration, and each concentration is established three multiple holes.Cultivate after 48 hours in the cell culture incubator, MTT is added in 96 orifice plates, 15 μ l/ holes continue to cultivate 4 hours, add three liquid, and 100 μ l/ holes placed incubator 12 hours.Measure the absorbance in every hole at wavelength 570nm place with ELIASA, and calculate cell inhibitory rate, the experiment triplicate.
Cell inhibitory rate %=(negative control group OD value-tried thing group OD value)/(negative control group OD value-zeroing hole OD value)
3, experimental result
Experimental result is seen Fig. 5-8, and table 2 is seen in interpretation, can be known by table 2:
(1) mark C, mark D, preparation 1,2 couples of MCF-7 of preparation, Hela, HL-60, four kinds of tumor cells of K562 are external all inhibited, and the most obvious to MCF-7 and the effect of K562 cell inhibiting.
(2) IC of 2 couples of MCF-7 of preparation and Hela cell inhibition
50The IC that value acts on separately between mark C and mark D
50Between the value, single costunolide and dehydrocostuslactone are when cell in vitro is tested from this data analysis, and synergism is not obvious; But 2 couples of MCF-7 of (0.25mg/L and 0.5mg/L) preparation and Hela cell inhibiting rate all are higher than mark C and mark the suppression ratio that D does the time spent separately when low concentration; Therefore costunolide and dehydrocostuslactone are when low concentration; Have synergism probably, but this synergism is not observed in HL-60 and K562 cell experiment.
(3) IC of 1 pair of MCF-7 breast cancer cell inhibition of preparation
50Value is greater than the IC of preparation 2
50Value, so the extracorporeal anti-tumor effect of preparation 2 is superior to preparation 1; Owing to preparation 1 is to be come by the volatile oil of Radix Aucklandiae preparation; Therefore it is except containing costunolide and dehydrocostuslactone; Also contain other compositions; And 2 in preparation contains costunolide and dehydrocostuslactone, so we infer in the volatile oil of Radix Aucklandiae between the costunolide and dehydrocostuslactone and other composition external do not possess collaborative antitumor action.
Experiment in vitro is the result show; The compositions of costunolide and dehydrocostuslactone is inhibited to cancerous cell; But both synergism are also not obvious in the cell in vitro experiment, in MCF-7 and Hela cell experiment, the synergism phenomenon occurs when having only low concentration.
Table 2 receives the influence of reagent thing to four kinds of tumor cell proliferations
The experiment of embodiment 4 anti-tumor in vivo
1, experiment material
1.1 cell strain is used in experiment
Human breast cancer cell MCF-7
1.2 laboratory animal
The female BALB/c nude mice in 4 ages in week, body weight 15.0 ± 1.0g
1.3 receive the reagent thing
Mark C, mark D, preparation 1, preparation 2 are all with embodiment 3.
2, experimental technique
Human breast carcinoma MCF-7 cell line is carried out routine cultivate, get active good cell of propagation phase, process 1 * 10 with normal saline
6The cell suspension of/ml, press 0.2ml/ dose inoculation in female nude mice right side flank subcutaneous, inoculate 10; After treating that tumor is grown to certain size, select the comparatively tumor piece of homogeneous of size, peel off on one's body from nude mice; Aseptic condition is transferred and is placed normal saline, discards tumor piece core after with little operating scissors it being broken, and remainder is cut into the tubercle piece of the uniform 2mm * 2mm that tries one's best * 2mm size; It is subcutaneous to be inoculated in nude mice right side flank, inoculates 80.
Surveyed the body weight and the size of tumor piece of a nude mice at a distance from two days, when treating that tumor block length and wide meansigma methods are about 5mm, select the tumor block size comparatively 60 nude mices of homogeneous carry out administration and test.To test nude mice and be divided into five groups; Every group 10; Intraperitoneal injection, each is organized dosage and is: costunolide group (mark C), dehydrocostuslactone group (mark D), 1 group in preparation, preparation are 30mg/kg for 2 groups, and positive control paclitaxel group is 15mg/kg; The normal saline of negative control group injection equivalent, the administration volume of each group is 10ml/kg.At a distance from injection in a day once, totally 10 times, the size and the nude mice body weight of measurement tumor are drawn the growth curve of tumor and the body weight curve of nude mice before the per injection.Last administration second day is plucked eyeball after measurement mice body weight and the tumor size and is got blood, and takes off vertebra and put to death mouse; Dissecting taking-up tumor piece weighs; Be calculated as follows the suppression ratio of tumor: tumor control rate=(C-T)/and C * 100, T is that the average tumor of administration group is heavy in the formula, the average tumor of the negative matched group of C is heavy; Experimental result is seen table 3, and the body weight curve of the growth curve of tumor and nude mice is seen Fig. 9 and 10.
Table 3 medicine is to the influence (n=10) of MCF-7 tumor bearing nude mice tumor growth
*: utmost point significant difference is arranged with the normal saline group; The paclitaxel group is each dead 1 mouse after administration the 9th, 10 times.
Estimate from tumor body size: the tumour inhibiting rate of paclitaxel group is up to 63.13%, and 1 group of tumour inhibiting rate with 2 groups in preparation of preparation is more or less the same, and is respectively 36.6% and 33.8%, and mark C is more or less the same with the tumour inhibiting rate of mark D group, is respectively 16.5% and 17.8%.Body weight change before and after the tumor bearing nude mice administration is estimated: have only 1 group of nude mice body weight of preparation to increase, behind the drug treatment 10 times weight increase 1.9g; And other respectively organizes body weight and is decline, and descending minimum is 2 groups in preparation (0.2g), and descending maximum is paclitaxel group (1.6g), and Ramulus et folium taxi cuspidatae group each dead 1 nude mice after administration the 9th, 10 times.
According to interpretation in the upper body; Can obtain as drawing a conclusion: all have cooperative effect when the costunolide of preparation 1 and preparation 2 and dehydrocostuslactone synergy, the tumour inhibiting rate that its tumour inhibiting rate obviously is superior to using costunolide (mark C group) separately or uses dehydrocostuslactone group (mark D group) separately; The general toxicity of 1 pair of nude mice of preparation is minimum, even also possibly strengthen the body constitution of nude mice, makes the tumor bearing nude mice of this group obviously healthy and strong than other group; Under this experiment condition, the tumor killing effect of paclitaxel is best, but its toxic and side effects is also the strongest.Almost not too big variation before nude mice body weight that preparation is 2 groups and the treatment; And the nude mouse escheat of 1 group in preparation has obvious increase; And the nude mice of paclitaxel group treatment back weight loss is very severe, and also dead two of treatment later stage, this all point out preparation 1 and preparation 2 to the toxic and side effects of nude mice far below paclitaxel; If therefore want to improve the tumour inhibiting rate of preparation 1 and preparation 2, can suitably improve the dosage of administration.
In a word, in pharmaceutical composition of the present invention and the volatile oil of Radix Aucklandiae, show the obvious synergistic effect during costunolide and dehydrocostuslactone are tested in vivo, effectively suppressed tumor propagation, obtained tumor killing effect preferably; And when costunolide and dehydrocostuslactone synergism, the toxic and side effects of using separately is littler; In addition; No matter be preparation 1 or preparation 2; They have comprised the synergism of costunolide and dehydrocostuslactone, therefore when from the Radix Aucklandiae, extracting preparation, also need not spend big strength purified the two independent separation, so not only can reduce the cost of medicine greatly; Simultaneously also can obtain extraordinary drug effect, so costunolide and the symphyogenetic pharmaceutical preparation of dehydrocostuslactone and volatile oil of Radix Aucklandiae all has good application prospects in field of cancer.
Claims (15)
1. a pharmaceutical composition that is used to treat tumor is characterized in that, said pharmaceutical composition is made up of costunolide and dehydrocostuslactone.
2. pharmaceutical composition as claimed in claim 1 is characterized in that, the mass ratio of said costunolide and dehydrocostuslactone is 1:0.4～2.5.
3. pharmaceutical composition as claimed in claim 1 is characterized in that, the mass ratio of said costunolide and dehydrocostuslactone is 1:1～2.2.
4. volatile oil of Radix Aucklandiae, for being that the raw material effective component extracting obtains with the Radix Aucklandiae, said effective ingredient is costunolide and dehydrocostuslactone.
5. volatile oil of Radix Aucklandiae as claimed in claim 4 is characterized in that said effective ingredient accounts for 30～80% of volatile oil of Radix Aucklandiae gross mass.
6. volatile oil of Radix Aucklandiae as claimed in claim 4 is characterized in that said effective ingredient accounts for 45～75% of volatile oil of Radix Aucklandiae gross mass.
7. volatile oil of Radix Aucklandiae as claimed in claim 4 is characterized in that said effective ingredient accounts for 55～75% of volatile oil of Radix Aucklandiae gross mass.
8. like the described volatile oil of Radix Aucklandiae of the arbitrary claim of claim 4-7, it is characterized in that said being extracted as adopts normal hexane or petroleum ether to extract as extractant.
9. volatile oil of Radix Aucklandiae as claimed in claim 8 is characterized in that, said extraction is specially: get an amount of exsiccant Radix Aucklandiae, pulverizing; After soaking as extractant with normal hexane or petroleum ether, supersound extraction is filtered; Remove the extractant in the filtrating, obtain said volatile oil of Radix Aucklandiae.
10. volatile oil of Radix Aucklandiae as claimed in claim 9 is characterized in that, said extractant consumption is 5～20 times of Radix Aucklandiae quality, and soak time is 0.5～2 hour, and the supersound extraction time is 0.5～2 hour, removes the extractant in the filtrating through revolving the steaming method.
11. a pharmaceutical preparation that is used to treat tumor comprises the described pharmaceutical composition of the arbitrary claim of claim 1-3 or the described volatile oil of Radix Aucklandiae of the arbitrary claim of claim 4-10 of safe and effective amount.
12. pharmaceutical preparation as claimed in claim 11 is characterized in that, also comprises pharmaceutically acceptable carrier in the said pharmaceutical preparation.
13., it is characterized in that said pharmaceutical preparation is injection like claim 11 or the described pharmaceutical preparation of 12 arbitrary claim.
14. the said pharmaceutical composition of the arbitrary claim of claim 1-3, the said volatile oil of Radix Aucklandiae of the arbitrary claim of claim 4-10 or the said pharmaceutical preparation of the arbitrary claim of the claim 11-13 purposes in preparation treatment anti-tumor medicine.
15. purposes as claimed in claim 14 is characterized in that, said tumor is selected from the arbitrary of breast carcinoma, cervical cancer or leukemia.
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| CN2012101850714A CN102743376B (en) | 2012-06-06 | 2012-06-06 | Costustoot pharmaceutical composition and medicinal application |
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| CN2012101850714A CN102743376B (en) | 2012-06-06 | 2012-06-06 | Costustoot pharmaceutical composition and medicinal application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830117A (en) * | 2014-03-26 | 2014-06-04 | 清华大学 | Costuslactone composition for preventing skin photoaging and application method thereof |
| CN113521061A (en) * | 2021-08-17 | 2021-10-22 | 张茗涵 | Application of traditional Chinese medicine costustoot extract in preparation of medicine for treating pediatric tumors |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1768774A (en) * | 2004-10-20 | 2006-05-10 | 山东绿叶制药有限公司 | Aucklandia root oil containing emulsion, its preparation process and use |
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2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1768774A (en) * | 2004-10-20 | 2006-05-10 | 山东绿叶制药有限公司 | Aucklandia root oil containing emulsion, its preparation process and use |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830117A (en) * | 2014-03-26 | 2014-06-04 | 清华大学 | Costuslactone composition for preventing skin photoaging and application method thereof |
| CN103830117B (en) * | 2014-03-26 | 2016-08-24 | 清华大学 | A kind of constuslactone compositions preventing skin photoage and using method thereof |
| CN113521061A (en) * | 2021-08-17 | 2021-10-22 | 张茗涵 | Application of traditional Chinese medicine costustoot extract in preparation of medicine for treating pediatric tumors |
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| CN102743376B (en) | 2013-11-27 |
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