CN108379273A - Dihydrotanshinone I is preparing application and preparation method in treating multidrug-resistant carcinoma drug - Google Patents
Dihydrotanshinone I is preparing application and preparation method in treating multidrug-resistant carcinoma drug Download PDFInfo
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Abstract
The present invention relates to natural medicine fields, disclose dihydrotanshinone I and are preparing application in treating multidrug-resistant carcinoma drug and preparation method thereof and pharmaceutical composition.The invention discloses dihydrotanshinone Is to significantly inhibit effect to what cells of resistant tumors had, its mechanism is not influenced by P glycoprotein to the killing of multidrug resistance tumor cells, and then intracellular drug concentration is able to maintain that in normal level, to overcome multi-drug resistance of the tumor.And currently used representational antitumor drug is substantially better than to the lethal effect of multidrug-resistant carcinoma cell.The multidrug-resistant carcinoma celelular mechanism that dihydrotanshinone I kills the expression of P glycoprotein height is to lure Apoptosis into, it is consistent with its parent's susceptibility cell, and dihydrotanshinone I has low toxicity, therefore, dihydrotanshinone I has good potential applicability in clinical practice to multidrug-resistant carcinoma.
Description
Technical field
The present invention relates to dihydrotanshinone Is in the application of anti-tumor aspect, belongs to natural medicine field, more particularly to dihydro
Tanshinone I is preparing the application in treating multidrug-resistant carcinoma drug.
Background technology
The incidence and its death rate of malignant tumour are in ascendant trend year by year, multidrug resistance (Multidrug
Resistance, MDR) it is the one of the major reasons that chemotherapy of tumors fails, it is different to configurations, mechanism to show as tumour cell
A variety of drugs the characteristics of generating crossing drug resistant.American Cancer Society estimates that the death of 90% or more tumor patient is in difference
It is influenced by drug resistance in degree.Therefore it is clinical urgent problem to be solved to overcome the multidrug resistance of tumour.
The Mechanism Study of multidrug resistance relates generally to P- glycoprotein (P-glycoprotein, P-gp) on tumor cell membrane
It is overexpressed, P- glycoprotein is overexpressed the outer row that can cause cells against neoplastic drug, makes to enter the drug in cell and reduces, tumour
Cell generates multidrug resistance, therefore studies more using P- glycoprotein as the drug resistant target spot of reverse multidrug.But this albuminoid not only exists
Overexpression in mdr cell also plays normal physiological function, therefore P- sugar in the metabolism internal organs of human body, barrier system etc.
Protein inhibitor is difficult to reach the requirement of clinical application.
Meanwhile the chemotherapeutics toxicity that clinically uses at present is big, side effect is strong, patient is difficult to bear to be also current tumour
One main problem for the treatment of.
Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bge.) belongs to labiate, first recorded in《Sheng Nong's herbal classic》, it is clinical
In most common Chinese medicine promoting blood circulation and removing blood stasis.Pertinent literature report Radix Salviae Miltiorrhizae, which has, to be increased coronary blood flow, coronary artery dilator, prevents
The Cardiovasculars such as myocardial ischemia, and for treating a variety of diseases such as gastric cancer, liver cancer, cervical carcinoma.Tanshinone component has
Antiatherosclerosis reduces myocardial oxygen consumption and the multiple biological activities such as antibacterial, anti-oxidant, anti-inflammatory and application value, especially
It is especially prominent in terms of antitumor activity.
Dihydrotanshinone I by inhibit cell Proliferation, promote Apoptosis and differentiation to play antitumor action and
Significant antibacterial activity etc., is antineoplastic component in Radix Salviae Miltiorrhizae.But it not yet finds treat tumour multiple medicine about dihydrotanshinone I at present
The report of drug resistance and application.
Invention content
The technical problem to be solved by the present invention is to for the drawbacks described above in the presence of current oncotherapy, provide dihydro
Tanshinone I is preparing the application in treating multi-drug resistance of the tumor drug, and dihydrotanshinone I is to cells of resistant tumors with notable
Inhibiting effect, mechanism is not influenced by P- glycoprotein to the killing of multidrug resistance tumor cells, and to multidrug resistance
The lethal effect of property tumour cell is substantially better than currently used representational antitumor drug.
On the other hand, the present invention also provides a kind of dihydrotanshinone Is being used to prepare treatment multidrug-resistant carcinoma drug
Preparation method.
In another aspect, the present invention also provides a kind of medicines containing the dihydrotanshinone I for being useful for treatment multidrug-resistant carcinoma
Compositions.
In order to achieve the above object, the present invention uses following technical scheme：
The present invention provides dihydrotanshinone I and is preparing the application in treating multidrug-resistant carcinoma drug.
Further, the tumour includes various entity tumors and the overexpression of P- glycoprotein of P- glycoprotein overexpression
Hematological system tumor.
Further, the multidrug-resistant carcinoma includes that multidrug resistance lung cancer, multidrug resistance cancer of pancreas, multiple medicine are resistance to
Pharmacological property colon cancer, multidrug resistance liver cancer, multidrug resistance prostate cancer, multidrug resistance kidney, multidrug resistance gastric cancer or
Multidrug resistance breast cancer.
Further, the multidrug-resistant carcinoma include to anthracyclines (adriamycin, daunorubicin, table Ah
Mycin), with micro-pipe be targeting chemotherapeutics (vincristine, taxol, etoposide, homoharringtonine, mitomycin
C it is) and with topoisomerase chemotherapeutics (fluorouracil, methotrexate (MTX)) the drug resistant resistant tumors targeted.
On the other hand, the present invention provides a kind of dihydrotanshinone I being used to prepare treatment multidrug-resistant carcinoma drug
Preparation method includes the following steps：
1) Radix Salviae Miltiorrhizae grinding medicinal materials are taken, the organic solvent that certain multiple is added thereto carries out refluxing extraction or ultrasound, immersion
Extracting solution is obtained, extracting solution is filtered, merges extracting solution filtrate, receives and do after recycling design, obtain crude extract；
2) crude extract is subjected to gradient elution by reversed phase chromatography filler, obtains active component；
3) active component is obtained into dihydrotanshinone I by normal-phase chromatography filler ladder degree of progress elution.
Further, in step 1), the dosage of organic solvent is 5-10 times of crude drug amount, wherein it is preferred that 8 times；It is described organic molten
Agent includes at least one of ether, petroleum ether, chloroform, acetone, ethyl acetate；Wherein preferred ether, petroleum ether；Most preferably stone
Oily ether；
Further, in step 2), reversed phase chromatography filler be C18 fillers, mobile phase be 20%-60% acetonitrile solutions with
10 times of column volume gradient elutions；
Further, in step 3), normal-phase chromatography filler is purification on normal-phase silica gel filler, and mobile phase is that volume ratio is (10:90)-
(60:40) petroleum ether：Ethyl acetate is with 10 times of column volume gradient elutions.
On the other hand, the present invention provides a kind of pharmaceutical composition for treating multidrug-resistant carcinoma comprising above-mentioned
The dihydrotanshinone I of pharmacy effective dose made from preparation method.
Further, described pharmaceutical composition is chemotherapeutics, the dihydrotanshinone I as in the chemotherapeutics only
One active principle.
Further, dosage form made of the pharmaceutical composition is liquid preparation, solid pharmaceutical preparation, including injection, jelly
Dry agent, granule, tablet, electuary, capsule and pill, capsule, pill, oral cavity disintegration preparation, sustained release preparation, controlled release preparation or various particles are given
Medicine system.
Further, dosage ranging from 0.025mg/kg-2.5mg/kg of the dihydrotanshinone I to human body；It is wherein excellent
The dosage of choosing ranging from 0.125mg/kg-1mg/kg；It can be according to factors such as state of an illness weight, form of administration and patient ages
Adjustment changes.
Compared with prior art, the invention has the advantages that：
1, the invention discloses a kind of compound pharmaceutic usages, the more particularly to pharmaceutic usage of dihydrotanshinone I：
Prepare the application in treatment multidrug-resistant carcinoma drug；
2, various acceptable preparation shapes medically can be made in the dihydrotanshinone I that the present invention obtains with pharmaceutic adjuvant
Formula, be used for above-mentioned multi-drug resistance of the tumor treatment, for example, injection, freeze-dried, tablet, capsule, oral liquid,
Granule etc.；
3, Primary Study is carried out to the pharmacodynamics part of pharmaceutical preparation of the present invention, the results showed that：In cell experiment, dihydro
Tanshinone I can significantly inhibit the growth of a variety of resistant tumors cells, and inhibition, which is substantially better than taxol etc. and shows clinic, to be made
Use drug；In testing in vivo, dihydrotanshinone I can significantly inhibit life of the transplanting resistant tumors cell in nude mouse
It is long；Mouse Acute Toxicity experimental result shows that after mouse administration, death does not occur for no obvious adverse reaction；Observation period terminates
When, it puts to death whole survival mices and is dissected, intraperitoneal each internal organs show no obvious abnormalities variation.It is above-mentioned it is demonstrated experimentally that dihydro
Tanshinone I can significantly inhibit multidrug-resistant carcinoma growth, and toxicity is smaller, have good potential applicability in clinical practice.
Description of the drawings
Fig. 1 is dihydrotanshinone I nucleus magnetic hydrogen spectrum figure in embodiment 2.
Fig. 2 is dihydrotanshinone I nuclear-magnetism carbon spectrogram in embodiment 2.
Fig. 3 A are dihydrotanshinone I HMBC nuclear-magnetism figures in embodiment 2.
Fig. 3 B are dihydrotanshinone I HMBC nuclear-magnetism figures in embodiment 2.
Fig. 4 is dihydrotanshinone I infared spectrum in embodiment 2.
Fig. 5 is dihydrotanshinone I uv-spectrogram in embodiment 2.
Fig. 6 is that dihydrotanshinone I acts on experimental group and blank after MCF-7/MDR and its parental cell MCF-7 in embodiment 5
The morphological observation figure of group.Fig. 7 A are that Annexin V-FITC/PI double-stainings detection MCF-7/MDR is thin in embodiment 6
Born of the same parents' apoptosis figure；
In figure：Q1- non-viable non-apoptotic cells；Q2- non-viable apoptotic cells；Q3- normal cells；Q4- viable apoptotic cells.
Fig. 7 B are that Annexin V-FITC/PI double-stainings detect MCF-7/MDR Apoptosis assay figures in embodiment 6.
Fig. 8 is the P- glycoprotein tables that Western Blot detect MCF-7/MDR and its parental cell MCF-7 in embodiment 7
Up to situation.
Fig. 9 is that mouse dissects tumor mass in embodiment 9.
Figure 10 is mouse paraffin section result in embodiment 9.
In conjunction with attached drawing, the invention will be further described with specific embodiment.
Specific implementation mode
In recent years, this laboratory by Radix Salviae Miltiorrhizae antineoplastic component the study found that dihydrotanshinone I to multidrug resistance
Tumour has good inhibiting effect, and toxic side effect is small, has good potential applicability in clinical practice.
Dihydrotanshinone I is a kind of fat-soluble effective monomer component of Radix Salviae Miltiorrhizae, English name：Dihydrotanshinone
I, molecular weight 278.30, molecular formula：C18H14O3, chemical constitution is as follows：
Multidrug resistance cell derives from susceptibility cell, and difference lies in the former high express P-glycoproteins for the two, therefore the former
There is multidrug resistance to kinds of tumors chemotherapeutics, and the latter is without multidrug resistance.
Dihydrotanshinone I significantly inhibits effect to what cells of resistant tumors had, and mechanism is thin to multidrug resistance of tumor
The killing of born of the same parents is not influenced by P- glycoprotein, and then intracellular drug concentration is able to maintain that in normal level, to overcome tumour more
Medicine drug resistance.And dihydrotanshinone I is substantially better than the lethal effect of multidrug-resistant carcinoma cell currently used has generation
The antitumor drug of table.
The multidrug-resistant carcinoma celelular mechanism that dihydrotanshinone I kills the expression of P- glycoprotein height is to lure Apoptosis into,
It is consistent with its parent's susceptibility cell；And dihydrotanshinone I has low toxicity.
It is used to prepare the preparation method of the dihydrotanshinone I for the treatment of multidrug-resistant carcinoma drug, using first reverse phase separation
The method that target effective position, rear positive purifying active component obtain target product, has the advantage that：
(1) reverse phase separation target effective position is first used, is that content is extremely low in Radix Salviae Miltiorrhizae due to the target product, belongs to strong
Polar fraction is first detached using reversed phase chromatography filler, can first by a large amount of non-targeted ingredient in extract, as tanshinone IIA,
Cryptotanshinone removes, and so that target product is not dispersed in other ingredients during isolating and purifying, reduces the loss of target component；
(2) target product is obtained using positive phase filling purifying active component afterwards, be because in active component, other ingredients with
Target product polarity is much like, it is difficult to detach, normal-phase chromatography filler is different from reversed phase chromatography filler separating mechanism, positive and reverse phase
Being applied in combination for chromatography, can greatly improve the efficiency of separation.
Below will by specific embodiment, invention is further described in detail, but the present invention be not limited in it is below
Embodiment.
Embodiment 1：The preparation process of dihydrotanshinone I
(1) it after Radix Salviae Miltiorrhizae crude drug crushes, takes 20 kilograms of grinding medicinal materials that the petroleum ether soak extraction of 10 times of amounts is added, is received after filtering
Collect filtrate revolving recycling, recycling design obtains crude extract I 200g；
(2) after crude extract I and C18 reversed phase chromatography fillers being mixed sample, mobile phase is with 20%-60% acetonitrile solutions with 10
Times column volume gradient elution collects target site, obtains crude extract II 26g；
(3) crude extract II is utilized into normal-phase chromatography silicagel column, mobile phase petroleum ether：Ethyl acetate=(1:9)-(3:2)
With 10 times of column volume gradient elutions, dihydrotanshinone I 10g is obtained.
Embodiment 2：The structural characterization for the dihydrotanshinone I being prepared
2.1 dihydrotanshinone I fusing points detect.
1 gained sample of Example is appropriate, is placed in fusing point test capillary, taps tube wall, is vertically placed on surface plate
On, capillary is made freely to fall from suitable for reading be put into, is repeated several times, powder is made closely to concentrate at the sealing end of capillary.It is packed into
The height of sample is 3mm.
Separately thermometer is put into and is contained in the container for passing warm liquid, the distance from bottom of the bottom end and container in thermometer mercury bulb is made
More than 2.5cm；It is added and passes warm liquid so that the liquid level after the warm liquid of biography is heated is at the sub-dip line of thermometer.Warm liquid heating will be passed, will be waited for
When temperature rise is to than about low 10 DEG C of defined fusing point lower bound, the capillary equipped with sample is immersed and passes warm liquid, is attached to temperature
On meter, position must make the content part of capillary be in the middle part of thermometer tribute ball；Continue to heat, it is every point to adjust heating rate
Clock rises 1.0-1.5 DEG C, and when heating, which must be stirred continuously, makes biography temperature liquid temperature keep uniform, and record test sample is in incipient melting to fine melt
Temperature, replication 3 times takes its average value.The results are shown in Table 1：
1 dihydrotanshinone I fusing point testing result of table
| Measure number | For the first time | Second | For the third time | Average value |
| Temperature DEG C | 224.5 | 225 | 225 | 224.83±0.29 |
2.2 dihydrotanshinone I nucleus magnetic hydrogen spectrum figures are as shown in Figure 1, δ 1.286 (d, 3H), δ 2.679 (s, 3H), δ 3.507-
3.566 (br.m, 1H), δ 4.470 (t, 1H), δ 5.011 (t, 1H), 7.490-7.783 (br, 3H), 8.438 (d, 1H), 9.128
(d,1H)。
2.3 dihydrotanshinone I nuclear-magnetism carbon spectrograms are as shown in Figure 2.
2.4 dihydrotanshinone I HMBC nuclear-magnetisms figures are as shown in Fig. 3 A, Fig. 3 B.
2.5 dihydrotanshinone I infared spectrums are as shown in Figure 4.
2.6 dihydrotanshinone I uv-spectrograms are as shown in Figure 5.
Embodiment 3：Compared with the proliferation inhibiting effect of the anti-multidrug resistance cell of the drugs such as dihydrotanshinone I and taxol
Experimental method：Using multidrug resistant cells MCF-7/MDR and its parental cell MCF-7 as object, it is red to investigate dihydro
Join influence of the drugs such as ketone I and taxol to the cell inhibitory effect.
The good two kinds of cells in growth period of growing way form are taken, with 105The cell in/hole is several in 96 orifice plates, often
100 μ L of hole.Be put into incubator be incubated 18-24 hours it is adherent after, MCF-7 groups of cells is separately added into each liquid sample, final concentration
Respectively 4.5,2.25,1.13,0.56,0.28 μm of ol/L；MCF-7/MDR groups addition liquid sample final concentration is as shown in table 2, often
Parallel 3 parallel holes of concentration are put into after being incubated 48 hours in incubator and remove culture medium, washed once with PBS, add 100 μ L
Serum free medium containing 0.5mg/mL MTT is incubated 3-4 hours；Carefully remove supernatant, 100 μ L DMSO is added, fully
It is detected with microplate reader after dissolving, Detection wavelength 570nm, reference wavelength 630nm.
Survival rate (fu)=1- (OD570-OD630)Medicine group/(OD570-OD630)Control group
Inhibiting rate (fa)=1- survival rates (fu)
Dosage is Y=a+bX with cell survival rate relational expression
Wherein Y is survival rate, and X is dosage.Different dosing dosage corresponds to when can measure drug administration by experiment
Cell survival rate.Dosage and cell survival rate data are substituted into above-mentioned equation, linear fit, meter are carried out to this group of data
Corresponding a values, b values are calculated to get to the equation Y=a+bX of dosage and survival rate relationship corresponding to the drug.
So as to find out the IC of administration cell50Value.
Drug resistance multiple：RF=IC50 mdr cells/IC50 sensitive cells
Experimental result：As shown in table 3, the RF values 0.72 ± 0.4 of dihydrotanshinone I, substantially less than adriamycin 21.86 ±
0.81, a variety of existing clinical medicines such as taxol 51.36 ± 1.84, cis-platinum 63.08 ± 0.97.
Description of test：Dihydrotanshinone I has significant proliferation inhibiting effect to multidrug resistance cell；Dihydrotanshinone I
The drug that existing Clinical practice inhibits tumour is better than to the proliferation inhibiting effect of multidrug resistance cell.
Each administration sample administration concentration of 2 MCF-7/MDR groups of table
3 dihydrotanshinone I of table is with various clinical antitumor drug to mdr cell and parental cell IC50Value comparison
Embodiment 4：The growth inhibition of six kinds of multidrug resistant cells of dihydrotanshinone I pair and its parental cell acts on
Experimental method：With multidrug resistant cells MCF-7/MDR, A549/Taxol, SW1990/GEM, HCT-8/5Fu,
SGC7901/DDP, BEL-7402/ADM and its parental cell MCF-7, A549, SW1990, HCT-8, SGC7901, BEL-7402
For object, influence of the dihydrotanshinone I to the cell Proliferation is investigated.
The good two kinds of cells in growth period of growing way form are taken, with 105The cell in/hole is several in 96 orifice plates, often
100 μ L of hole.Be put into incubator be incubated 18-24 hours it is adherent after, be separately added into final concentration of 4.5,2.25,1.13,0.56,
The dihydrotanshinone I solution of 0.28 μm of ol/L is put into after being incubated 48 hours in incubator and removes per parallel 3 parallel hole of concentration
Culture medium is washed with PBS and once adds the serum free medium that 100 μ L contain 0.5mg/mL MTT and be incubated 3-4 hours；Carefully
Ground removes supernatant, and 100 μ L DMSO are added, are fully detected with microplate reader after dissolving, Detection wavelength 570nm, reference wavelength
630nm。
Survival rate (fu)=1- (OD570-OD630)Medicine group/(OD570-OD630)Control group
Inhibiting rate (fa)=1- survival rates (fu)
Dosage is Y=a+bX with cell survival rate relational expression
Wherein Y is survival rate, and X is dosage.Different dosing dosage corresponds to when can measure drug administration by experiment
Cell survival rate.Dosage and cell survival rate data are substituted into above-mentioned equation, linear fit, meter are carried out to this group of data
Corresponding a values, b values are calculated to get to the equation Y=a+bX of dosage and survival rate relationship corresponding to the drug.
So as to find out the IC to administration cell50Value.
Experimental result：As shown in table 4, the results showed that dihydrotanshinone I acts on MCF-7/MDR and its parental cell MCF-
The half-inhibition concentration IC of 7 cell 48h50It is 0.68 ± 0.08 μm of ol/L and 0.95 ± 0.04 μm of ol/L respectively, illustrates dihydro pellet
Ginseng I pairs of two class cells of ketone all have similar proliferation inhibiting effect；In other five kinds of drug-resistant cell strains and parental cell strain
It embodies, two class cell of dihydrotanshinone I pair also has similar proliferation inhibiting effect.Prove multidrug resistant cells to dihydro
Tanshinone I does not have tolerance.
The IC of a variety of multidrug resistant cells of 4 dihydrotanshinone I of table and its parental cell50Value
| Drug-resistant cell strain | IC50(μmol/L) | Parental cell strain | IC50(μmol/L) |
| MCF-7/MDR | 0.68±0.08 | MCF-7 | 0.95±0.04 |
| A549/Taxol | 0.88±0.03 | A549 | 0.84±0.05 |
| SW1990/GEM | 0.94±0.01 | SW1990 | 1.14±0.01 |
| HCT-8/5-Fu | 0.74±0.07 | HCT-8 | 0.84±0.04 |
| SGC7901/DDP | 1.04±0.08 | SGC7901 | 1.03±0.08 |
| BEL-7402/ADM | 0.75±0.03 | BEL-7402 | 0.86±0.07 |
Embodiment 5：Morphological observation
Experimental method：This experiment uses dyestuff Hochest 33258, investigates dihydrotanshinone I to MCF-7/MDR and MCF-
The morphologic influence of 7 cells.
Testing principle：Hochest 33258 is the bonding agent of DNA, but it can penetrate the cell membrane of living cells, normal thin
Born of the same parents will present uniform light blue, and sapphirine is presented in apoptotic cell is then assembled because dyeing due to.
Concrete operations are as follows：
With 5 × 104The cell number in/hole is by cell kind in 24 orifice plates, and final concentration of 2 μ is added in experimental group after 18-24h
The complete medium of not drug containing is added in the dihydrotanshinone I of mol/L, blank group；After cultivating 48h, 5 μ L Hochest are added
33258 (making its final concentration of 10 μ g/mL) shook even rear room temperature and are protected from light dyeing 10min, and then carried out fluorescence microscopy observation.
Experimental result：As shown in fig. 6, no matter dihydrotanshinone I is to MCF-7/MDR or parental cell MCF-7, with blank
Group is compared, in experimental group, the visible bright blue fluorescence point generated by Apoptosis.Illustrate dihydrotanshinone I to MCF-7/MDR
And its parental cell MCF-7 lethal effects are to induce its apoptosis.
Embodiment 6：Annexin V-FITC/PI double-stainings detect MCF-7/MDR Apoptosis
Experimental method：This experiment uses Annexin V-FITC/PI double-stainings, further verifies dihydrotanshinone I and lures
The apoptosis of MCF-7/MDR cells is led.
Testing principle：In normal cell, phosphatidylserine is only distributed in the inside of cell membrane lipid bilayer, cell hair
Raw apoptosis earliest period, membrane phospholipid acyl serine in adipose membrane by turning on one's side outward, to be tied with cardiolipin binding protein AnnexinV phases
It closes, at this point, cell membrane keeps complete so that PI cannot enter cell；But the cell and dead cell of apoptosis middle and advanced stage are due to cell
The increase of membrane permeability, PI can be such that nucleus incarnadines through cell membrane.
Concrete operations are as follows：
(1) with 5 × 105The cell number in/hole is by MCF-7/MDR cells kind in the plate of 6cm, and administration group adds after 18-24h
Enter the dihydrotanshinone I of final concentration of 2 μm of ol/L；
(2) after drug effect 48h, after pancreatin digestion, 1000rpm centrifuges 5min and collects cell；
(3) cell is washed twice with the PBS being pre-chilled on ice；
(4) the Binding Buffer that 100 μ L contain 2 μ L Annexin-V are added, soft dispels cell, is positioned over ice
Upper incubation 10min；
After the nylon net filter of (5) 300 mesh, the physiological saline that 100 μ L contain 2 μ L PI is added and is protected from light incubation 2-3min, stream
Formula cell instrument tests and analyzes.
Experimental result：If Fig. 7 A, B are shown, when it is 2 μm of ol/L that cell is through sample administration concentration, administration group dihydro Radix Salviae Miltiorrhizae
Ketone I makes cell respectively reach 12.5 and 15.7 in the apoptosis quantity of Q2 and Q4, hence it is evident that is higher than the 2.4 and 6.9 of blank group, illustrates two
It is in apoptotic state that hydrogen Tanshinone I, which can induce MCF-7/MDR cell majorities,.
Embodiment 7：The P- P-glycoprotein expression situations of Western blot detection two kinds of cells of MCF-7/MDR and MCF-7
Experimental method：
Take two kinds of cells of well-grown MCF-7/MDR in exponential phase of growth and MCF-7, adherent growth for 24 hours after, pancreas
Enzymic digestion, 1500rpm centrifuge 5min and collect cell, extract albumen.
Protein extraction concrete operations are as follows：
(1) PBS washs cell twice, for the last time absorbs totally PBS, suitable cell pyrolysis liquid (1mL is added
10 μ L inhibitors of phosphatases, 1 μ L protease inhibitors and 5 μ L 100mM PMSF are added in Lysis Buffer)；
(2) be vortexed concussion 30s, cooled on ice 5min, repeats 3-5 times；
(3) 14000rpm, 4 DEG C of centrifugation 15min；It takes in supernatant to the centrifuge tube of new sterilized 1.5mL to get albumen
Sample carries out subsequent experimental.
Then use BCA protein quantification kit measurement protein contents, concrete operations as follows：
(1) by after dilution BCA and protein sample be added in 96 orifice plates, per 25 μ L of hole；
(2) (BCA Reagent A and Reagent B are with 50 for 200 μ L reaction solutions of addition:1 ratio mixing)；
30min, microplate reader detection, Detection wavelength 562nm are incubated in (3) 37 DEG C of constant incubators.
(4) it after measuring OD values, is fitted to obtain the linear equation of albumen OD values and concentration according to the concentration of BCA and OD values, and
The OD values for substituting into sample afterwards, to calculate the concentration of protein sample.
Add lysate so that the concentration of all protein samples is consistent, and suitable 5 × Loading buffer are added, boil
5min is boiled, albuminous degeneration is made.
It is stored for future use with the centrifuge tube of 1.5mL, is dispensed according to the protein content of every pipe 80-100 μ g, be stored in -20 DEG C, most
Two weeks can be stored long.
The isometric loading rear electrophoresis of albumen, concrete operations are as follows：
(1) resolving gel concentration is selected, separation gel is configured, is added after preparing in the plastic plate fixed, shakes and puts down, is added appropriate
Single water that steams separation gel is flattened, be placed at room temperature for 30-40min；
(2) after gelling to be separated is good, the water on upper layer is outwelled, the concentration glue prepared is added, is inserted into the comb of 1.0mm,
Concentration glue is added again with glue-leakage-resistant, is placed at room temperature for 20-30min；
(3) glue coagulated is put into protein electrophoresis slot, fills it up with the electrophoretic buffer being pre-chilled, lightly pulls out comb
After falling, with the syringe needle of 1mL by crooked glue hole righting；
(4) loading, per hole 80-100 μ g albumen samples, electrophoresis, 70V runs 30min, and then 120V runs 1-1.5h.
After electrophoresis, according to the size of destination protein, on the basis of pre-dyed Marker, by MrObject tapeGlue at ± 10kDa
It cuts.Then glue is immersed in the transferring film buffer solution being pre-chilled, prepares transferring film.
Transferring film concrete operations are as follows：
(1) according to the size (Mr of target molecule>22kDa then selects the pvdf membrane in 0.45 μm of aperture；Mr<22kDa is then selected
With the pvdf membrane in 0.22 μm of aperture), it cuts corresponding pvdf membrane (wide 1mm more each than glue) and marks, 5min, transferring film are impregnated in methanol
5min is impregnated in buffer solution；
(2) 2 groups of filter paper items (wide 1mm more each than film) are cut, every group 3 layers, are dipped in transferring film buffer solution；
(3) three layers of filter paper, film, glue, three layers of filter paper are followed successively by by black to white；One layer is often folded, then rolles over two with glass bar
Three bouts are used in combination filter paper to blot its edge to drive bubble away；After all having taken, then with clean filter paper its edge is blotted,
And ensure that lower three layers of filter paper should not be overlapped with upper three one-tenth filter paper, to prevent short circuit；
(4) transferring film is less than half-dried turn of the use of 70kDa, and (1kDa, 1min) is arranged according to molecular weight of albumen in the transferring film time；Greatly
In wet turn of the carry out of 70kDa, the transferring film time is 2.5h.
1-2h is closed in confining liquid, 4 DEG C of refrigerators of primary antibody are incubated overnight.TBST is washed three times, each 10min.Secondary antibody room temperature is incubated
1h is educated, TBST is washed four times, each 15min.ECL chemiluminescent agents are incubated 5min, and tabletting, develop 5min, is fixed 5min, uses software
Gel-pro 32 carries out gray analysis.
Experimental result：As shown in figure 8, MCF-7/MDR is compared with MCF-7 cells, MCF-7/MDR cell P- glycoprotein sugar eggs
White expression is apparently higher than MCF-7 cells, and in vitro in cell experiment and internal nude mice model tumor experiment, dihydrotanshinone
I has apparent inhibition to MCF-7/MDR and MCF-7 cells.It can be seen that dihydrotanshinone I kills tumour cell
Wound and inhibition drug effect are not influenced by tumor multi-medicine drug-resistant, are not influenced by tumour cell P- glycoprotein especially.Even tumour
The high express P-glycoprotein of cell, makes the drug effect of a variety of antitumor drugs substantially reduce, and does not influence the antitumor of dihydrotanshinone I still
Drug effect.
Embodiment 8：Preparation process for the dihydrotanshinone I pharmaceutical composition for treating multidrug-resistant carcinoma
Dihydrotanshinone I sample 9mg is made in Example 1, after the dissolving of 200 μ L absolute ethyl alcohols is added, adds 200 μ L castor oil
After ultrasonic mixing, adds 1600 μ L physiological saline solutions, A samples, a concentration of 4.5mg/mL is made.
In use, after taking 100 μ L of A samples to add 900 μ L normal saline dilutions, intravenous injection uses.
The concentration is 18g with mouse weight, and when dosage is 0.1mL, dosage is 2.5mg/kg.
Embodiment 9：Nude mice of the dihydrotanshinone I pharmaceutical composition to transplanting two kinds of tumour cells of MCF-7/MDR and MCF-7
Tumor inhibition effect
Experimental method：Two kinds of cells of well-grown MCF-7/MDR in exponential phase of growth and MCF-7 are taken, are washed with PBS
It washs 2 times, collects cell, count, (6-7) * 10 is configured to PBS6The cell suspension of cells/mL is injected with 0.1mL/ nude mice
In right oxter tumor formation, vernier caliper measurement to tumor mass is molded about 0.3*0.3cm2Start to be administered when size.
Drug sample is prepared by embodiment 8.
If blank group, positive controls taxol (2.5mg/kg), sample sets (2.5mg/kg), are administered once for two days.Even
After continuous administration 14 days, tumor is taken to weigh, measures size, paraffin section observation.
Experimental result：
By Fig. 9 nude mices dissection tumor mass as it can be seen that the nude mice with blank group, positive controls taxol compares, dihydrotanshinone I
Administration group has good inhibiting effect to two kinds of tumours of MCF-7/MDR and MCF-7.
Shown in Figure 10 paraffin section results, blood vessel is observed in dihydrotanshinone I administration group nude mouse tumor paraffin section
Surrounding tumor cells have apparent apoptosis phenomenon.
It can be learnt by table 5, table 6：It is compared with the nude mice of blank group, dihydrotanshinone I administration group is to transplanting MCF-7/MDR
And the nude mouse tumor of two kinds of cells of MCF-7 has a good inhibiting effect, in experimentation, mouse has no adverse reaction；Taxol
It is thin in transplanting multidrug resistance although positive controls have inhibiting effect in transplanting parental cell MCF-7 group nude mouse tumors
Almost without inhibiting effect in born of the same parents' MCF-7/MDR group nude mouse tumors, and during Germicidal efficacy, taxol positive controls it is naked
There is weight stagnation and slightly declines in mouse, and dietary amount declines.
It is converted according to human body and the administration equivalent dose of mouse, this effective dose for testing corresponding human administration is
0.28mg/kg.Actually active dosage can adjust as the case may be.
Table 5MCF-7 groups of cells mouse anatomical datas
| Group | Weight/g before experiment | Weight/g after experiment | Weight difference | Knurl weight/g | Tumour inhibiting rate % |
| Dihydrotanshinone I | 15.98±0.78 | 18.74±0.15 | 2.87±0.64 | 0.28±0.24 | 73.07692 |
| Taxol | 16.07±1.01 | 17.43±0.25 | 1.34±0.74 | 0.34±0.53 | 67.30769 |
| Blank control | 15.93±0.47 | 18.88±0.97 | 2.95±0.51 | 1.04±0.39 |
Table 6MCF-7/MDR groups of cells mouse anatomical datas
| Group | Weight/g before experiment | Weight/g after experiment | Weight difference | Knurl weight/g | Tumour inhibiting rate % |
| Dihydrotanshinone I | 15.41±0.54 | 18.47±0.61 | 3.00±0.35 | 0.20±0.41 | 81.81818 |
| Taxol | 16.23±0.19 | 17.77±0.37 | 1.32±0.87 | 0.87±0.64 | 20.90909 |
| Blank control | 16.08±0.39 | 18.59±0.89 | 2.67±0.64 | 1.10±0.38 |
Embodiment 10：Acute Toxicity Test in dihydrotanshinone I Mice Body
Experimental method：According to《Chemicals acute toxicity test technological guidance's principle》, Normal healthy mice is at empty stomach
After reason, such as death does not occur for disposable celiac drug administration by injection 5g/kg, preceding 2 hours close observations, record of weighing in daily, training
After supporting 14 days, takes off neck and put to death mouse, its internal organ analysis of dissection gross examination of skeletal muscle.
Experimental result：Mouse upon administration, does not occur death, and weight is compareed with blank group, normal to increase, after 14 days, solution
It cuts open and does not observe internal organ exception.Illustrate that dihydrotanshinone I toxicity is smaller, clinically there is good application prospect
Above example 3-7,9-10 explanation：
(1) dihydrotanshinone I is significantly better than taxol, adriamycin, vincristine etc. to the drug effect of multidrug resistance cell and faces
Bed antitumor drug；
(2) dihydrotanshinone I kills the drug effect of multidrug resistance tumor cells and its parental cell without significant difference, it was demonstrated that two
The drug effect of hydrogen Tanshinone I is not influenced by tumour cell P- P-glycoprotein expressions；
(3) in mouse experiment in vivo, dihydrotanshinone I is shown to the significant inhibiting effect of multidrug resistance cell, and
Toxicity is significantly lower than existing clinical antitumor agents.
These results illustrate the medicine for the multi-drug resistance of the tumor that there is dihydrotanshinone I wide anti-P- glycoprotein height to express
Object prepares and the foreground of clinical application.
The invention is not limited in the above embodiments, if not departing from the present invention to the various changes or modifications of the present invention
Spirit and scope, if these modification and variations belong within the scope of the claim and equivalent technologies of the present invention, then this hair
It is bright to be also intended to comprising these changes and change.
Claims (10)
1. dihydrotanshinone I is preparing the application in treating multidrug-resistant carcinoma drug.
2. application according to claim 1, it is characterised in that：The tumour includes the various realities of P- glycoprotein overexpression
Body tumour and the hematological system tumor of P- glycoprotein overexpression.
3. application according to claim 1, it is characterised in that：The multidrug-resistant carcinoma includes multidrug resistance lung
Cancer, multidrug resistance cancer of pancreas, multidrug resistance colon cancer, multidrug resistance liver cancer, multidrug resistance prostate cancer, multiple medicine are resistance to
Pharmacological property kidney, multidrug resistance gastric cancer and multidrug resistance breast cancer.
4. application according to claim 1, it is characterised in that：The multidrug-resistant carcinoma includes to anthracycline chemotherapy medicine
Object, with micro-pipe be targeting chemotherapeutics and with topoisomerase be targeting the drug resistant resistant tumors of chemotherapeutics.
5. a kind of preparation method for the dihydrotanshinone I being used to prepare treatment multidrug-resistant carcinoma drug, which is characterized in that packet
Include the following steps：
1) Radix Salviae Miltiorrhizae grinding medicinal materials are taken, the organic solvent that certain times of amount is added thereto carries out refluxing extraction or ultrasound, impregnates to carry
Liquid is taken, extracting solution is filtered, merges extracting solution filtrate, receives and do after recycling design, obtain crude extract；
2) crude extract is subjected to gradient elution by reversed phase chromatography filler, obtains active component；
3) active component is subjected to gradient elution by normal-phase chromatography filler, obtains dihydrotanshinone I.
6. preparation method according to claim 5, it is characterised in that：
In step 1), the dosage of organic solvent is 5-10 times of crude drug amount, wherein it is preferred that 8 times；The organic solvent includes ether, stone
At least one of oily ether, chloroform, acetone, ethyl acetate；
In step 2), reversed phase chromatography filler is C18 fillers, and mobile phase is 20%-60% acetonitrile solutions with 10 times of column volume ladders
Degree elution；
In step 3), normal-phase chromatography filler is purification on normal-phase silica gel filler, and mobile phase is that volume ratio is (10:90)-(60:40) oil
Ether：Ethyl acetate is with 10 times of column volume gradient elutions.
7. a kind of pharmaceutical composition for treating multidrug-resistant carcinoma, it is characterised in that：It includes claim 5 or 6 institutes
The dihydrotanshinone I of pharmacy effective dose made from the preparation method stated.
8. pharmaceutical composition according to claim 7, it is characterised in that：Described pharmaceutical composition is chemotherapeutics, described
Dihydrotanshinone I is as unique active principle in the chemotherapeutics.
9. pharmaceutical composition according to claim 7, it is characterised in that：Dosage form made of described pharmaceutical composition is liquid
Preparation, solid pharmaceutical preparation, including it is injection, freeze-dried, granule, tablet, electuary, capsule and pill, capsule, pill, oral cavity disintegration preparation, slow
Release formulation, controlled release preparation or various particulate delivery systems.
10. pharmaceutical composition according to claim 7, it is characterised in that：Dosage model of the dihydrotanshinone I to human body
It encloses for 0.025mg/kg-2.5mg/kg；Wherein preferred dosage ranging from 0.125mg/kg-1mg/kg；It can be according to the state of an illness
The adjustment of the factors such as weight, form of administration and patient age changes.
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Cited By (3)
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| CN110759881A (en) * | 2019-08-08 | 2020-02-07 | 广州医科大学附属第二医院 | Src inhibitor and application thereof |
| CN115212218A (en) * | 2022-06-14 | 2022-10-21 | 天津中医药大学 | Application of dihydrotanshinone I in inhibiting interaction between blood platelet and tumor cell |
| CN116514896A (en) * | 2023-03-27 | 2023-08-01 | 浙江大学 | Synthesis method of dihydrotanshinone I |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110759881A (en) * | 2019-08-08 | 2020-02-07 | 广州医科大学附属第二医院 | Src inhibitor and application thereof |
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| CN115212218A (en) * | 2022-06-14 | 2022-10-21 | 天津中医药大学 | Application of dihydrotanshinone I in inhibiting interaction between blood platelet and tumor cell |
| CN116514896A (en) * | 2023-03-27 | 2023-08-01 | 浙江大学 | Synthesis method of dihydrotanshinone I |
| CN116514896B (en) * | 2023-03-27 | 2024-10-01 | 浙江大学 | Synthesis method of dihydrotanshinone I |
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Application publication date: 20180810 |