CN104069194B - A kind of Chinese medicine composition with antitumaous effect and its production and use - Google Patents
A kind of Chinese medicine composition with antitumaous effect and its production and use Download PDFInfo
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Abstract
The invention provides a kind of Chinese medicine composition with antitumaous effect and its production and use.The bulk drug of the Chinese medicine composition of the antitumaous effect is mainly made up of the root of herbaceous peony, Radix Angelicae Sinensis；The preparation method of the Chinese medicine composition includes：Bulk drug is taken in proportion, the Solvent Extract methods mutually dissolved each other with water or with water；Or, taking bulk drug in proportion, prepared using water extraction and alcohol precipitation method；Wherein described alcohol precipitation concentration is 50 90.The purposes is the application in the Chinese medicine composition in treating cancer medicine, particularly uterine neck cancer drug is prepared.The Chinese medicine composition is made up of the bulk drug of following weight ratio：The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.2‑4）.The root of herbaceous peony of the present invention and Radix Angelicae Sinensis compatibility have coordinate repression to Hela cells, with preferable antitumaous effect.
Description
Technical field
The present invention relates to a kind of Chinese medicine composition and its production and use, and in particular to a kind of with antitumaous effect
Chinese medicine composition and its production and use, belongs to technical field of Chinese medicine.
Background technology
Cancer is the major disease for jeopardizing human health, and its fatal rate is in the trend for rising year by year, its occurrence and development
It is the complex lesions process of multifactor participation, polygenes change and Multi stage development.At present for the treatment of cancer is adopted mostly
Operative treatment combines Radiotherapy chemotherapy method, but the method is big to the wound of patient, dangerous high, and somewhat expensive, but curative effect
It is unsatisfactory.The weapon that Chinese medicine is prevented and cured diseases as China's tradition, along with progressively in-depth study and exploration in recent years, in cancer
Increasingly it is taken seriously in disease treatment.Research shows, during treatment and prevention of tumour, Chinese medicine is mainly from raising immunity of organism work(
Energy, inducing apoptosis of tumour cell, the suppression aspect such as Nasopharyngeal neoplasms and the attenuation synergistic to chemotherapy play a role.
Chinese medicine composition compatibility of the present invention rationally, can adjust negative and positive of qi and blood and function, protect marrow hemopoiesis
Function, can start self-defensive system again, improve immunity of organisms, very helpful for the auxiliary treatment of tumour.
The content of the invention
First purpose of the present invention is to provide a kind of Chinese medicine composition with antitumaous effect.
Second object of the present invention is the preparation method for providing said composition.
Third object of the present invention is the application for providing said composition in treating cancer medicine is prepared.
Fourth object of the present invention is the application for providing said composition in treatment uterine neck cancer drug is prepared.
The purpose of the present invention is achieved by the following technical solution：
A kind of Chinese medicine composition with antitumaous effect, the Chinese medicine composition are made up of the bulk drug of following weight ratio：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.2-4）.
The bulk drug of the Chinese medicine composition is preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.3-3.5）.
The bulk drug of the Chinese medicine composition is preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.4-3）.
The bulk drug of the Chinese medicine composition is preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.5-2.5）.
The bulk drug of the Chinese medicine composition is preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.5-2.2）.
The bulk drug of the Chinese medicine composition is preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：（0.5-2）.
The bulk drug of the Chinese medicine composition is more preferably：
The root of herbaceous peony：Radix Angelicae Sinensis=1：0.5；
Or, the root of herbaceous peony：Radix Angelicae Sinensis=1：1；
Or, the root of herbaceous peony：Radix Angelicae Sinensis=1：2.
Chinese medicine composition of the present invention can be produced in conventional fashion into clinically-acceptable particle, tablet, capsule, dripping pill, mouth
Take liquid, suspension, emulsion, injection.
Chinese medicine composition of the present invention can be prepared as follows：
Bulk drug is taken in proportion, the Solvent Extract methods mutually dissolved each other with water or with water；
Further, one or more being selected from the organic solvent that water mutually dissolves each other in methyl alcohol, ethanol, acetone；More
The more preferably ethanol of 50-90%；It is still more preferably the ethanol of 60-80%；Ethanol still more preferably for 70%；
Extracting method used includes decocting appointing in extraction, refluxing extraction, soak extraction, ultrasonic extraction or seepage pressure effects
Anticipate a kind of mode, or the combination of Different Extraction Method.
Chinese medicine composition of the present invention can also be adopted：Bulk drug is taken in proportion, is prepared using water extraction and alcohol precipitation method；Wherein described alcohol
Heavy concentration is 50-90%；
Further, the alcohol precipitation concentration is 60-80%；Further described alcohol precipitation concentration is 70%.
Can also be purified, refined after in above-mentioned preparation method, bulk drug is extracted, such as cross macroporous resin column；And further press
Conventional preparation process makes the acceptable any conventional formulation of pharmacy, including particle, tablet, capsule, dripping pill, oral liquid,
Suspension, emulsion, injection.
To enable above-mentioned formulation to realize, pharmaceutically acceptable auxiliary material need to be added when these formulations are prepared, for example：Filling
Agent, disintegrant, lubricant, suspending agent, adhesive, sweetener, flavouring, preservative, matrix etc..Filler includes：It is starch, pre-
Gelling starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose etc.；Disintegrant includes：Starch, pregelatinized starch, crystallite
Cellulose, sodium carboxymethyl starch, PVPP, low-substituted hydroxypropyl cellulose, Ac-Di-Sol etc.；
Lubricant includes：Magnesium stearate, lauryl sodium sulfate, talcum powder, silica etc.；Suspending agent includes：Polyvinylpyrrolidine
Ketone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose etc.；Adhesive includes：Starch slurry, polyvinylpyrrolidone,
Hydroxypropyl methyl cellulose etc.；Sweetener includes：Saccharin sodium, aspartame, sucrose, honey element, enoxolone etc.；Flavouring bag
Include：Sweetener and various essence；Preservative includes：Parabens, benzoic acid, Sodium Benzoate, sorbic acid and its esters, benzene prick bromine
Ammonium, acetic acid chloroethene are fixed, eucalyptus oil etc.；Matrix includes：PEG6000, PEG4000, insect wax etc..In above-mentioned formulation is realized
Medicine pharmacy, need to add pharmaceutically acceptable other auxiliary materials when these formulations are prepared（Fan Biting《Pharmacy of Chinese materia medica》, Shanghai section
Learn the auxiliary material that each formulation in publishing house's December in 1997 the 1st edition is recorded）.
In addition to Chinese medicine composition of the present invention is in the form of fed intake with the root of herbaceous peony, Radix Angelicae Sinensis crude drug, can also adopt with white
Chinese herbaceous peony, angelica extract（Active component）The form for feeding intake, thus it is the present invention further discloses a kind of with antitumaous effect
Drug composition：
A kind of Chinese medicine composition with antitumaous effect, the Chinese medicine composition are made up of the raw material of following weight ratio：
Radix paeoniae alba extraction：Angelica extract=1：（0.2-4）.
The bulk drug of the Chinese medicine composition is preferably：
Radix paeoniae alba extraction：Angelica extract=1：（0.3-3.5）.
The bulk drug of the Chinese medicine composition is preferably：
Radix paeoniae alba extraction：Angelica extract=1：（0.4-3）.
The bulk drug of the Chinese medicine composition is preferably：
Radix paeoniae alba extraction：Angelica extract=1：（0.5-2.5）.
The bulk drug of the Chinese medicine composition is preferably：
Radix paeoniae alba extraction：Angelica extract=1：（0.5-2.2）.
The bulk drug of the Chinese medicine composition is preferably：
Radix paeoniae alba extraction：Angelica extract=1：0.5-2.
The bulk drug of the Chinese medicine composition is more preferably：
Radix paeoniae alba extraction：Angelica extract=1：0.5；
Or, radix paeoniae alba extraction：Angelica extract=1：1；
Or, radix paeoniae alba extraction：Angelica extract=1：2.
Radix paeoniae alba extraction of the present invention and angelica extract are respectively the root of herbaceous peony, the water extract of Radix Angelicae Sinensis or are mutually dissolved each other with water
Extractive with organic solvent；Or, the respectively root of herbaceous peony, the extract obtained after return through water extract-alcohol precipitation is processed.
Further, one or more being selected from the organic solvent that water mutually dissolves each other in methyl alcohol, ethanol, acetone；More
The more preferably ethanol of 50-90%；It is still more preferably the ethanol of 60-80%；Ethanol still more preferably for 70%；
Extracting method used includes decocting appointing in extraction, refluxing extraction, soak extraction, ultrasonic extraction or seepage pressure effects
Anticipate a kind of mode, or the combination of Different Extraction Method.
Further, the alcohol precipitation concentration is 50-90%；Further preferably alcohol precipitation concentration is 60-80%；Further institute
Alcohol precipitation concentration is stated for 70%.
Water extraction and alcohol precipitation method of the present invention means that in Chinese medicine water extracting liquid addition ethanol makes to reach the alcohol content of regulation
（That is alcohol precipitation concentration）, some compositions solubility reduction precipitation precipitation, filtration in alcoholic solution take ethanol solution, reclaim ethanol and obtain
Extract, makes Aqueous extracts be able to refined method.
Test result indicate that, when the root of herbaceous peony, Radix Angelicae Sinensis, Ligusticum wallichii and cultivated land act solely on human cervical carcinoma Hela cell, the only root of herbaceous peony
There is inhibitory action to the propagation of Hela cells, inhibiting rate can reach 28.6%, and Radix Angelicae Sinensis, Ligusticum wallichii and cultivated land are without obvious inhibiting effect.
And the root of herbaceous peony plays coordinate repression (CI to Hela cells with during Radix Angelicae Sinensis compatibility<1,0.1<Fa<0.9), the root of herbaceous peony and Ligusticum wallichii compatibility or
The root of herbaceous peony plays antagonism (CI to the Proliferation Ability of Hela cells with cultivated land compatibility>1).
Effect experimental examples
The research of 1 RADIX PAEONIAE ALBA pair of experimental example
1 material
Human cervical carcinoma Hela cell is given by central laboratory of Tianjin Cancer Institute；RPMI-1640 culture mediums, the U.S.
GIBCO Products；
The each single medicine of the root of herbaceous peony, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony press the group of different proportion compatibility respectively at Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land
" medicine to " of compound is adopted and is prepared with the following method：Bulk drug is taken, 30min is soaked, adds 8 times of amount decoctings to boil three times, often
Secondary 1.5 hours, filtration, merging filtrate were centrifuged, dry, pulverize into powder.Empirically need xeraphium DMEM high glucose mediums
It is made into the reagent filtration sterilization of respective concentration.
2 experimental techniques
The impact that 2.1 root of herbaceous peonys, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land are bred to Hela cells
Hela cells are cultivated in 5%CO2 incubators, and culture medium is the RPMI-l640 containing 10% inactivation calf serum, is contained
1% dual anti-(penicillin and streptomysin).Passed on once per 2～3 days, growth period cell of taking the logarithm is used to test.24 orifice plates are per hole
5000/m1 Hela cell of inoculation, dosing culture in 24 hours after inoculation, if the root of herbaceous peony, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and negative control group.
Drug concentration is added with following formula conversion each group in vitro culture：C(μg/mL)=5D(mg/Kg/d)；C is experiment in vitro desired concn, and D is
Clinical application concentration, quantity are pressed each 9g/ of four traditional Chinese medicine and are calculated.Collect cell within 96 hours after dosing, row trypan blue is counted.
The impact that 2.2 root of herbaceous peony compatibility different pharmaceuticals are bred to Hela cells
Take the logarithm the Hela cells in growth period, pancreatin digests and blows and beats as cell suspension.Cell is collected by centrifugation, is adjusted after counting
The cell concentration of whole suspension is 1 × 104/ mL, is inoculated in 24 porocyte culture plates, per hole 1mL.Chosen according to 2.1 experimental results
There is the root of herbaceous peony of obvious inhibiting effect to Hela cells propagation, itself and other drugs are pressed into different proportion variable concentrations compatibility using work
For Hela cells, alone group of the root of herbaceous peony, Radix Angelicae Sinensis, cultivated land, Ligusticum wallichii is equipped with, the root of herbaceous peony presses 1 with Radix Angelicae Sinensis:1、2:1、1:2 ratio compatibilities are closed
1 is pressed with group, the root of herbaceous peony and cultivated land:1、2:1、1:2 ratio compatibility combination groups, the root of herbaceous peony and Ligusticum wallichii press 1:1、2:1、1:2 ratio compatibilities are closed
With group, and negative control group.Drug concentration is added with different pharmaceutical compatibility middle dose group in vitro culture with the following formula conversion root of herbaceous peony：C
(μg/mL)=5D(mg/Kg/d)；C is experiment in vitro desired concn, and D is clinical application concentration.With this concentration as intermediate concentration, point
Not upwards, downward 2 times are successively decreased, and continuously arrange 6 concentration gradients.96h, light Jing Microscopic observation each group cells are cultivated in incubator
Metamorphosis, collects cell afterwards, and row trypan blue is counted.
Joint drug effect is judged with median-effect principle：Middle effect concentration（Dm）, i.e. the calculating of half-inhibition concentration (IC50), according to
Trypan blue exclusion viable count calculates medicine group cell fu（Survival rate fraction of unaffeeted cells）And fa（Suppression
Rate fraetion of affected cells processed）
The average viable count of the average viable count/control group of fu=test groups
fa=1-fu
Middle efficacious prescriptions journey fa/fu=(D/Dm) m both sides are taken the logarithm（D is drug concentration；Dm is middle effect concentration）Obtain equation:
log(fa/fu)=mLogD-mLogDm
So as to Dx=Dm × [fa/ (l-fa)] l/m
If Y=log (fa/fu), X=logD, then linear equation is obtained:
Y=mX+a（The slope of m equations, a are equation intercept）
During Fa=0.5, X=logDm=-a/m calculates Dm values when alone medicine and use in conjunction
Calculate and reach various effect (fa=0.1,0.2,0.3 ... using this formula Dx=Dm × [fa/ (1-fa)] 1//m
0.9) when, two kinds of medicines are alone and use in conjunction required for drug concentration (D)
CI (The Combination when two medicines are shared are calculated according to the amount in week-Te Shi association index method-effect formula
Index) value
Clx=D1/Dxl+D2/Dx2
Note：Dl in formula, D2 are for, when sharing, concentration shared by two medicines when effect reaches X, Dxl, Dx2 were two prescription used times, were imitated
Should be up to respective concentration during X.Factor alpha is imitated mapping according in and is determined, when the mapping of two medicines is parallel to each other, α values are 0, otherwise is 1.
Result judgement：CI<Collaboration (synergism) effect is produced when l represents that two medicines are shared；When CI=1 represents that two medicines are shared
Produce and be added (additivity) effect；CI>Antagonism (antagonism) effect is produced when l represents that two medicines are shared.
Middle effect concentration and joint drug effect are calculated by CalcuSyn softwares and are fitted chart.
2.3 compatibility of drugs synergy Mechanism Study
Take the logarithm the Hela cells in growth period, after the digestion of 0.25% pancreatin, collect cell.Trypan Blue makees living cells meter
Number（Viable count must be more than 90%）And cell suspension is prepared, adjustment cell concentration is every hole 1 × 105, it is inoculated in the culture of 6 holes
In plate, continue culture 24h, after cell attachment, change liquid dosing, if the root of herbaceous peony is alone（250μg/mL）Group, Radix Angelicae Sinensis are alone（125μg/
mL）Group, root of herbaceous peony compatibility Radix Angelicae Sinensis（250μg/mL+125μg/mL）Group and blank control group, each experimental group correspondence addition account for training matrix
The Contained Serum of each concentration of product 10%, blank control group add equal-volume nutrient solution, collect cell, lead to after continuing culture 96h
Overflow-type cell art detects medicine on Hela cell cycles and apoptotic impact.
Take the logarithm the Hela cells in growth period, adjustment cell concentration is 5 × 104/ hole is inoculated in 12 well culture plates, is continued
Culture 24h, after cell attachment, changes liquid dosing, if the root of herbaceous peony is alone（250μg/mL）Group, Radix Angelicae Sinensis are alone（125μg/mL）Group, in vain
Chinese herbaceous peony compatibility Radix Angelicae Sinensis（250μg/mL+125μg/mL）Group and each experimental group correspondence of blank control group are added and account for each of training matrix product 10%
The Contained Serum of concentration, blank control group add equal-volume nutrient solution, and cell, the Elisa detection root of herbaceous peonys are collected after continuing culture 96h
Impact Hela cells CyclinD1 expressed with Radix Angelicae Sinensis compatibility.
3 statistical methods
Using SPSS16.0 statistics analysis software packages, experimental data is with mean ± standard deviationRepresent, in check number
Under conditions of according to normal distribution and homogeneity of variance, multigroup data compare and adopt variance analysis, and the comparison of each group group difference is used
LSD-t is checked.With P ＜ 0.05 as statistically significant.
4 experimental results
The impact that 4.1 root of herbaceous peonys, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land are bred to Hela cells
Trypan blue counting method detects different experiments group.The root of herbaceous peony has inhibitory action to the propagation of Hela cells, and inhibiting rate is reachable
To 28.6%, and the Radix Angelicae Sinensis of same dose, Ligusticum wallichii and cultivated land to Hela cells without obvious inhibiting effect（Refer to table 1）.
1 each experimental group of table is to Hela cell inhibitory rates
| Blank group | The root of herbaceous peony | Ligusticum wallichii | Radix Angelicae Sinensis | Cultivated land | |
| Crude drug g/d | 0 | 9 | 9 | 9 | 9 |
| Extract g/d | 0 | 2.15 | 2.71 | 3.77 | 4.21 |
| Drug concentration（μg/mL） | 0 | 178.92 | 225.83 | 313.85 | 350.50 |
| Inhibiting rate | 0 | *28.60 | -0.07 | 0.78 | -2.55 |
* represent and compare P with blank control group<0.01
The impact that 4.2 root of herbaceous peony compatibility different pharmaceuticals are bred to Hela cells
4.2.1 the impact that the root of herbaceous peony is bred to Hela cells with Radix Angelicae Sinensis compatibility
Trypan blue counting method detects different experiments group：The root of herbaceous peony and Radix Angelicae Sinensis are alone and share（The root of herbaceous peony：Radix Angelicae Sinensis=1:1,1:2 and 2:
1）Viable count after human cervical carcinoma Hela cell strain 96h, calculates the Fa under each concentration of each experimental group.Using Excel and
CalcuSyn softwares calculate the root of herbaceous peony, the IC50 of Radix Angelicae Sinensis is respectively 109.28 μ g/mL, 721.74 μ g/mL.Calculate the root of herbaceous peony and Radix Angelicae Sinensis is closed
Used in different effect when CI values to Hela cells, statistical analysis shows that the root of herbaceous peony presses 1 with Radix Angelicae Sinensis:1,2:1,1:During 2 ratio compatibility
Coordinate repression is played to Hela cells：CI<1(0.1<Fa<0.9)（Refer to table 2- tables 4）.
The 2 variable concentrations root of herbaceous peony of table and Radix Angelicae Sinensis are alone to cytostatic effects Fa of Hela
3 root of herbaceous peony of table and Radix Angelicae Sinensis share effect Fa cytostatic to Hela
4 root of herbaceous peony of table and CI index of the Radix Angelicae Sinensis different proportion compatibility to Hela cell growths
4.2.2 the impact that the root of herbaceous peony is bred to Hela cells with cultivated land compatibility
Trypan blue counting method detects different experiments group：The root of herbaceous peony and cultivated land are alone and share（The root of herbaceous peony：Cultivated land=1:1,1:2 and 2:
1）Viable count after human cervical carcinoma Hela cell strain 96h, calculates the cell inhibitory rate under each concentration of each experimental group.Using
Excel and CalcuSyn softwares calculate the root of herbaceous peony and cultivated land shares the association index value in different effect to Hela cells（CI）,
Statistical analysis shows that the root of herbaceous peony presses different proportion compatibility 0.1 with cultivated land<Fa<Antagonism is played to the Proliferation Ability of Hela cells when 0.9
Effect (CI>1), wherein synergy CI=0.983 (0.1 slight from only as 125 μ g/mL+ 62.5 μ g/mL of cultivated land of the root of herbaceous peony<
Fa<0.9)（Refer to table 5-7）.
The 5 variable concentrations root of herbaceous peony of table and cultivated land are alone to cytostatic effects Fa of Hela
6 root of herbaceous peony of table and cultivated land share effect Fa cytostatic to Hela
7 root of herbaceous peony of table and CI index of the cultivated land different proportion compatibility to Hela cell growths
4.2.3 the impact that the root of herbaceous peony is bred to Hela cells with Ligusticum wallichii compatibility
Trypan exclusion stain detection different experiments group acts on the viable count after Hela cell 96h, calculates Carbazole alkaloid
Rate.Each combination group CI is calculated using CalcuSyn softwares to show：The root of herbaceous peony presses 1 with Ligusticum wallichii:1, 2:It is thin to Hela during 1 ratio compatibility
Born of the same parents play antagonism inhibitory action (CI>1).The root of herbaceous peony and Ligusticum wallichii 1:It is during 2 compatibility, only light as 125 μ g/mL+ 250 μ g/mL of Ligusticum wallichii of the root of herbaceous peony
Micro- synergy, CI=0.96 (0.1<Fa<0.9) it is, remaining to be antagonistic effect (CI>1)（Refer to table 8-10）.
The 8 variable concentrations root of herbaceous peony of table and Ligusticum wallichii are alone to cytostatic effects Fa of Hela
9 root of herbaceous peony of table and Ligusticum wallichii share effect Fa cytostatic to Hela
10 root of herbaceous peony of table and CI index of the Ligusticum wallichii different proportion compatibility to Hela cell growths
4.3 compatibility of drugs synergy Mechanism Study
（1）Cell cycle result shows：Alone group of blank control group, the root of herbaceous peony, alone group of Radix Angelicae Sinensis and 250 μ g/mL of the root of herbaceous peony match somebody with somebody
The G0/G1 phase cells of 5 Radix Angelicae Sinensis, 125 μ g/mL groups are followed successively by 57.07%, 53.27%, 54.125%, 47.25%, S phase cells according to
It is secondary be 25.4%, 29.53%, 28.94%, 37.61%, G2/M phase cells be followed successively by 17.56%, 17.205%, 16.93%,
14.97%, G0/G1 and S phase cell numbers, root of herbaceous peony compatibility compare statistically significant (P when grouping with blank group<0.01)（Refer to
Table 11）.
11 different experiments group Hela cell cycle of table testing result
| Group | G0/G1（%） | S（%） | G2/M（%） |
| Blank control group | 57.04±0.81 | 25.4±1.38 | 17.56±0.85 |
| 250 μ g/mL of the root of herbaceous peony | 53.27±1.84 | 29.53±2.73 | 17.20±0.89 |
| 125 μ g/mL of Radix Angelicae Sinensis | 54.12±0.38 | 28.94±0.44 | 16.93±0.14 |
| 250 μ g/mL+ Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ g/mL | 47.25±1.80* | 37.61±2.32* | 14.97±2.24 |
* represent and compare P with blank experimental group<0.01
（2）Apoptosis result shows：Jing after processing 96h, flow cytometer measures apoptosis rate to Hela cells, as a result
Show that 250 μ g/mL groups of the root of herbaceous peony and 250 μ g/mL compatibilities Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ g/mL combination groups are compared with blank control group, difference has
Statistical significance (P<0.01).But alone with the 250 μ g/mL root of herbaceous peonys to compare, 250 μ g/mL compatibilities Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ g/mL are shared
Group fails the apoptosis (P of co-induction Hela cells<0.05)（Refer to table 12）.12 different experiments group Hela natural death of cerebral cells rate of table becomes
Change
| Group | Apoptosis rate |
| Blank control group | 6.13±0.68 |
| 125 μ g/mL of Radix Angelicae Sinensis | 6.77±0.69 |
| 250 μ g/mL of the root of herbaceous peony | *15.77±1.36 |
| 250 μ g/mL+ Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ g/mL | **10.17±1.04 |
* represent and compare * * P with blank experimental group<0.01
（3）Elisa detects the impact that the root of herbaceous peony is expressed to Hela cells CyclinD1 with Radix Angelicae Sinensis compatibility
Experimental result shows：Hela cells Jing different experiments group process 96h after, only 250 μ g/mL compatibilities Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ
In g/mL combination group Hela cells, CyclinD1 expressions compare reduction, the statistically significant (P of difference with blank control group<
0.05).And CyclinD1 expressions and blank are right in 250 alone group of the μ g/mL root of herbaceous peonys, Radix Angelicae Sinensis alone group of Hela cell of 125 μ g/mL
Compare according to group, no significant difference（P>0.05）（Refer to table 13）.
Table 13Hela cells CyclinD1 expressions Jing after different experiments group processes 96h
| Group | CyclinD1(pg/mL) |
| Blank control group | 101.00±47.27 |
| 125 μ g/mL of Radix Angelicae Sinensis | 80.19±12.74 |
| 250 μ g/mL of the root of herbaceous peony | 101.94±58.77 |
| 250 μ g/mL+ Radix Angelicae Sinensis of the root of herbaceous peony, 125 μ g/mL | *23.61±13.93 |
Compare * P with blank group<0.05
The research of 2 Elsholtzia pair of experimental example
1 material
Human cervical carcinoma Hela cell is given by central laboratory of Tianjin Cancer Institute；RPMI-1640 culture mediums, the U.S.
GIBCO Products；
The each single medicine of rheum officinale, peach kernel, leech and rheum officinale are adopted by the composition of different proportion compatibility with peach kernel, leech respectively
It is prepared by following method：Bulk drug is taken, 30min is soaked, adds 8 times of amount decoctings to boil three times, 1.5 hours every time, filtration merged
Filtrate, is centrifuged, dry, pulverize into powder.Empirically need xeraphium DMEM high glucose mediums are made into the reagent of respective concentration
And filtration sterilization.
2 experimental techniques
Hela cells are cultivated in 5%CO2 incubators, and culture medium is the RPMI-l640 that calf serum is inactivated containing l0%, is contained
1% dual anti-(penicillin and streptomysin).Passed on once per 2～3 days, growth period cell of taking the logarithm is used to test.24 orifice plates are per hole
5000/m1 Hela cell of inoculation, dosing culture in 24 hours after inoculation, choosing has obvious inhibiting effect to Hela cells propagation
Rheum officinale, by its with other drugs by different proportion variable concentrations compatibility using Hela cells are acted on, be equipped with rheum officinale, peach kernel, water
Alone group of leech, rheum officinale press 1 with peach kernel:1、2:1、1:2 ratio compatibility combination groups, rheum officinale and leech press 1:1、2:1、1:2 ratios are matched somebody with somebody
5 combination groups, and negative control group.It is dense with different pharmaceutical compatibility middle dose group in vitro culture dosing thing with following formula conversion rheum officinale
Degree：C(μg/mL)=5D(mg/Kg/d)；C is experiment in vitro desired concn, and D is clinical application concentration.It is dense as centre with this concentration
Degree, respectively upwards, downward 2 times successively decrease, 6 concentration gradients of continuous setting.96h is cultivated in incubator, light Jing Microscopic observations are each
Group cellular morphology change, collects cell afterwards, and row trypan blue is counted.
Joint drug effect is judged with median-effect principle：Middle effect concentration（Dm）, i.e. the calculating of half-inhibition concentration (IC50), according to
Trypan blue exclusion viable count calculates medicine group cell fu（Survival rate fraction of unaffeeted cells）And fa（Suppression
Rate fraetion of affected cells processed）
The average viable count of the average viable count/control group of fu=test groups
fa=1-fu
Middle efficacious prescriptions journey fa/fu=(D/Dm) m both sides are taken the logarithm（D is drug concentration；Dm is middle effect concentration）Obtain equation:
log(fa/fu)=mLogD-mLogDm
So as to Dx=Dm × [fa/ (l-fa)] l/m
If Y=log (fa/fu), X=logD, then linear equation is obtained:
Y=mX+a（The slope of m equations, a are equation intercept）
During Fa=0.5, X=logDm=-a/m calculates Dm values when alone medicine and use in conjunction
Calculate and reach various effect (fa=0.1,0.2,0.3 ... using this formula Dx=Dm × [fa/ (1-fa)] 1//m
0.9) when, two kinds of medicines are alone and use in conjunction required for drug concentration (D)
CI (The Combination when two medicines are shared are calculated according to the amount in week-Te Shi association index method-effect formula
Index) value
Clx=D1/Dxl+D2/Dx2
Note：Dl in formula, D2 are for, when sharing, concentration shared by two medicines when effect reaches X, Dxl, Dx2 were two prescription used times, were imitated
Should be up to respective concentration during X.Factor alpha is imitated mapping according in and is determined, when the mapping of two medicines is parallel to each other, α values are 0, otherwise is 1.
Result judgement：CI<Collaboration (synergism) effect is produced when l represents that two medicines are shared；When CI=1 represents that two medicines are shared
Produce and be added (additivity) effect；CI>Antagonism (antagonism) effect is produced when l represents that two medicines are shared.
Middle effect concentration and joint drug effect are calculated by CalcuSyn softwares and are fitted chart.
3 statistical methods
Using SPSS16.0 statistics analysis software packages, experimental data is with mean ± standard deviationRepresent, in check number
Under conditions of according to normal distribution and homogeneity of variance, multigroup data compare and adopt variance analysis, and the comparison of each group group difference is used
LSD-t is checked.With P ＜ 0.05 as statistically significant.
4 experimental results
The impact that 4.1 rheum officinales are bred to Hela cells with peach kernel compatibility
Rheum officinale and peach kernel are alone and share（Rheum officinale：Peach kernel=1:1,1:2 and 2:1）After human cervical carcinoma Hela cell strain 96h
Viable count, calculate the cell inhibitory rate under each concentration of each experimental group.Using Excel and CalcuSyn softwares calculate rheum officinale and
Peach kernel shares the association index value in different effect to Hela cells（CI）Statistical analysis shows that rheum officinale is with peach kernel by not on year-on-year basis
Example compatibility is 0.1<Fa<Antagonism (CI is played to the Proliferation Ability of Hela cells when 0.9>1)（Refer to table 14- tables 16）.
14 variable concentrations rheum officinale of table and peach kernel are alone to cytostatic effects Fa of Hela
15 rheum officinale of table and peach kernel share effect Fa cytostatic to Hela
16 rheum officinale of table and CI index of the peach kernel different proportion compatibility to Hela cell growths
The impact that 4.2 rheum officinales are bred to Hela cells with leech compatibility
Each combination group CI is calculated using CalcuSyn softwares to show：Rheum officinale presses 1 with leech:1,2:1,1:During 2 ratio compatibility
Antagonism inhibitory action (CI is risen to Hela cells>1)（Refer to table 17- tables 19）.
17 variable concentrations rheum officinale of table and leech are alone to cytostatic effects Fa of Hela
18 rheum officinale of table and leech share effect Fa cytostatic to Hela
19 rheum officinale of table and CI index of the leech different proportion compatibility to Hela cell growths
Specific embodiment
1 oral liquid of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 30g；
The bulk drug of recipe quantity is taken, is added water to cook 2 times, 2 hours for the first time, second 1.5 hours, collecting decoction, filtration,
Filtrate concentrates, and concentrate adds ethanol to alcohol content to 70%, stands, filtration, and filtrate recycling ethanol is extremely without alcohol taste, and is concentrated into phase
It is 1.15 to density（60℃）, add customary adjuvant to make oral liquid.
2 tablet of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 15g；
The bulk drug of recipe quantity is taken, plus 5 times of 70% alcohol refluxs of amount are extracted 2 times, every time 1.5 hours；Merge extract, filter
Cross, reclaim ethanol and concentrate；Concentrate drying under reduced pressure, is ground into fine powder, adds customary adjuvant, mixes；Dry-pressing is pelletized, compressing tablet,
Obtain final product.
3 capsule of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 60g；
Take the bulk drug of recipe quantity, plus 10 times of amount methyl alcohol ultrasonic extractions 3 times, first time 40min, the 2nd, 3 each 20min,
Merge extract, filtration, reduced pressure concentration；Concentrate drying under reduced pressure, is ground into fine powder, adds customary adjuvant, mixes；Dry-pressing system
Grain, loads capsule, obtains final product.
4 granule of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 120g；
The bulk drug of recipe quantity is taken, 30min is soaked, is added 8 times of amount decoctings to boil three times, 1.5 hours every time, is filtered,
Merging filtrate, concentration；Concentrate by macroreticular resin, first with 5 times of amount water elutions, abandon or adopt by eluent, then with adding 70% ethanol to wash
It is de-, eluent is collected, decompression recycling ethanol is to without alcohol taste；Less than 80 DEG C drying under reduced pressure, are ground into fine powder, add auxiliary material, mix；
Dry-pressing is pelletized, and obtains final product.
5 oral liquid of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 90g；
Recipe quantity bulk drug is taken, is added water to cook 2 times, 1.5 hours every time, collecting decoction, filtration, filtrate were concentrated, concentrate
Plus ethanol is stood to alcohol content to 80%, filtration, filtrate recycling ethanol is extremely without alcohol taste, and is concentrated into relative density for 1.15（60
℃）, auxiliary material is added, oral liquid is made.
6 dripping pill of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 9g；
The bulk drug of recipe quantity is taken, 8 times of amounts water retting 6h, diacolation 24h, flow velocity 2L/h are added.Percolate is collected, is centrifuged,
Upper large pore resin absorption column, first washes with water, then uses 60% ethanol elution, collects ethanol eluate, and filtrate concentration adds system
On medicine, acceptable auxiliary material makes pill according to the routine techniques of this area preparation.
7 tablet of embodiment
Prescription bulk drug is constituted：Root of herbaceous peony 30g, Radix Angelicae Sinensis 6g；
The bulk drug of recipe quantity is taken, plus 5 times of 60% alcohol refluxs of amount are extracted 2 times, every time 1.5 hours；Merge extract, filter
Cross, reclaim ethanol and concentrate；Concentrate drying under reduced pressure, is ground into fine powder, adds customary adjuvant, mixes；Dry-pressing is pelletized, compressing tablet,
Obtain final product.
Embodiment 8
Prescription bulk drug is constituted：Radix paeoniae alba extraction 30g, angelica extract 30g；
The radix paeoniae alba extraction, angelica extract be respectively the root of herbaceous peony, when 60% alcohol reflux of return through extract prepare carry
Take thing.Said extracted thing drying under reduced pressure is crushed for fine powder, mixed, add customary adjuvant to make granule.
Embodiment 9
Prescription raw material is constituted：Radix paeoniae alba extraction 30g, angelica extract 15g；
The radix paeoniae alba extraction, angelica extract are respectively the root of herbaceous peony, boil the extract for preparing when return through decocting.Will be upper
Extract drying under reduced pressure is stated, is crushed as fine powder, mixed, add customary adjuvant to make tablet.
Embodiment 10
Prescription raw material is constituted：Radix paeoniae alba extraction 30g, angelica extract 60g；
The radix paeoniae alba extraction, angelica extract be respectively the root of herbaceous peony, when return through acetone reflux extracts the extraction for preparing
Thing.Said extracted thing drying under reduced pressure is crushed for fine powder, mixed, add customary adjuvant to make capsule.
Embodiment 11
Prescription raw material is constituted：Radix paeoniae alba extraction 30g, angelica extract 90g；
The extraction that the radix paeoniae alba extraction, angelica extract are respectively the root of herbaceous peony, prepare when return through aqueous extraction-alcohol precipitation technology
Thing, its alcohol precipitation concentration are the extract that 70% ethanol solution seepage pressure effects are obtained；By said extracted thing drying under reduced pressure, it is ground into thin
Powder, adds customary adjuvant, mixes；Dry-pressing is pelletized, and compressing tablet is obtained final product.
Claims (7)
1. a kind of Chinese medicine composition for treating cervical carcinoma, it is characterised in that bulk drug of the Chinese medicine composition by following weight ratio
Composition：
The root of herbaceous peony：Radix Angelicae Sinensis=1：(0.5-2).
2. Chinese medicine composition as claimed in claim 1, it is characterised in that bulk drug of the Chinese medicine composition by following weight ratio
Composition：
The root of herbaceous peony：Radix Angelicae Sinensis=1：0.5；
Or, the root of herbaceous peony：Radix Angelicae Sinensis=1：1；
Or, the root of herbaceous peony：Radix Angelicae Sinensis=1：2.
3. as described in claim 1-2 is arbitrary Chinese medicine composition preparation method, it is characterised in that the method includes：
Bulk drug is taken in proportion, the Solvent Extract methods mutually dissolved each other with water or with water；
Or, taking bulk drug in proportion, prepared using water extraction and alcohol precipitation method；Wherein described alcohol precipitation concentration is 50-90%.
4. the preparation method of Chinese medicine composition as claimed in claim 3, it is characterised in that the organic solvent mutually dissolved each other with water
One or more in methyl alcohol, ethanol, acetone；The extracting method include decoct extraction, refluxing extraction, soak extraction,
Any one mode in ultrasonic extraction or seepage pressure effects, or the combination of Different Extraction Method.
5. a kind of Chinese medicine composition for treating cervical carcinoma, it is characterised in that raw material group of the Chinese medicine composition by following weight ratio
Into：
Radix paeoniae alba extraction：Angelica extract=1：(0.5-2)；
Wherein, the radix paeoniae alba extraction and angelica extract be respectively the root of herbaceous peony, the water extract of Radix Angelicae Sinensis or with having that water mutually dissolves each other
Machine solvent extractable matter；Or, the respectively root of herbaceous peony, the extract obtained after return through water extract-alcohol precipitation is processed.
6. Chinese medicine composition as claimed in claim 5, it is characterised in that raw material group of the Chinese medicine composition by following weight ratio
Into：
Radix paeoniae alba extraction：Angelica extract=1：0.5；
Or, radix paeoniae alba extraction：Angelica extract=1：1；
Or, radix paeoniae alba extraction：Angelica extract=1：2.
7. application of the Chinese medicine composition as described in claim 1-2,5-6 is arbitrary in treatment uterine neck cancer drug is prepared.
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| CN104544106A (en) * | 2015-02-24 | 2015-04-29 | 刘永 | Preparation method of panacis quinquefolii radix chewable tablet |
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| JP2004307458A (en) * | 2003-04-07 | 2004-11-04 | Korea Atom Energ Res Inst | Galenical composition having anticancer/immune/hematopoietic function enhancing effect and oxidative living body damage suppressing effect and manufacturing method thereof |
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