CN105943542A - Medicine composition for treating stomach cancer - Google Patents
Medicine composition for treating stomach cancer Download PDFInfo
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- CN105943542A CN105943542A CN201610356227.9A CN201610356227A CN105943542A CN 105943542 A CN105943542 A CN 105943542A CN 201610356227 A CN201610356227 A CN 201610356227A CN 105943542 A CN105943542 A CN 105943542A
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- 0 C[C@](CC[C@@]([C@@]1(*)[C@](C2)OC)([C@@](C)(CC3=O)[C@]2(C)C(C)(C)C3=O)O2)([C@]1(CC1)C2=O)C1=C(C)CCC=C(C)C Chemical compound C[C@](CC[C@@]([C@@]1(*)[C@](C2)OC)([C@@](C)(CC3=O)[C@]2(C)C(C)(C)C3=O)O2)([C@]1(CC1)C2=O)C1=C(C)CCC=C(C)C 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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- C—CHEMISTRY; METALLURGY
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- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention discloses a medicine composition for treating stomach cancer. The medicine composition consists of a component A of a compound (I) and PDTC (pyrrolidine dithiocarbamate), wherein the mass ratio of component A and PDTC is 1:(0.5-2); the compound (I) is separated and extracted from mignonettetree. The medicine composition has the advantages that after applying, the effect of promoting apoptosis of stomach cancer cells is more obvious; compared with single usage, the growth inhibiting rate of cells and the apoptosis index are improved; compared with single usage of two medicines, the inhibiting function on stomach cancer cells by the joint usage of PDTC and compound (I) is obviously improved; the two medicines are not traditional chemical treatment medicines, the injury to organs is little, and the medicine-resistant property of tumor cells on traditional chemical treatment medicines is avoided.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of pharmaceutical composition treating gastric cancer.
Background technology
Mignonettetree Lawsonia inermis, Lythraceae Lythtaceae mignonettetree platymiscium, is again Flos Impatientis, fingernail leaf, hands
First wood, fingernail wood, Gan Jiashu, its leaf, flower, fruit, seed can serve as Chinese medicine and use.Lawsonia to diarrhoea,
Dysentery, leprosy, scabies have good curative effect；Flower can be used to treat headache, fever, allergy, anemia, insomnia etc.；Seed
Effective to fever, insomnia, dysentery, diarrhoea and intellectual deficiency；Bark can treat spleen enlargement and dermatosis chronic disease；Root can be controlled
Treat leprosy.Mignonettetree main product in the torrid zone, subtropical zone, be widely present in the Middle East, north African, in China Guangdong, Guangxi, Fujian,
Also there is cultivation the southern areas such as Taiwan.As far back as 304 years Christian eras Shanxi check and be contained in " south vegetation shape " book and be called mignonettetree,
1964 version " Uygur medicine medical materials " are called Hina or Mihid.
Modern study finds that mignonettetree contains polytype compounds such as quinone, Phenylpropanoid Glycosides, flavone, triterpene, phenolic acid and fatty acid.
Pharmacological research shows that mignonettetree has antibacterial, the pharmacologically active widely such as antitumor, antioxidation, parasiticide, has the biggest
Value of exploiting and utilizing, therefore suffer from the extensive concern of scholars.
Gastric cancer ranks first in the various malignant tumor of China, and incidence gastric cancer has obvious region difference, in the northwest of China and east
Coastal area, portion incidence gastric cancer rate is higher than southern area is evident as.Sending out well the age more than 50 years old, the ratio of men and women's sickness rate is 2:
1.The prognosis of gastric cancer is relevant with the pathological staging of gastric cancer, position, organization type, biological behaviour and remedy measures.
Summary of the invention
It is an object of the invention to provide a kind of pharmaceutical composition treating gastric cancer, stomach cancer cell can have been played by this pharmaceutical composition
The inhibitory action of effect.
The above-mentioned purpose of the present invention is achieved by techniques below scheme:
A kind of pharmaceutical composition treating gastric cancer, component A and PDTC by following compound (I) form,
Described component A is 1:0.5～2 with the mass ratio of PDTC.
Further, described compound (I) separation and Extraction from mignonettetree obtains, and comprises following operating procedure:
A the dried leaves of mignonettetree is pulverized by (), with 75～85% alcohol heat reflux extraction, united extraction liquid, be concentrated into without alcohol taste,
Successively with petroleum ether, ethyl acetate and water saturated n-butanol extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract
And n-butyl alcohol extract；
B acetic acid ethyl ester extract macroporous resin remove impurity in () step (a), first with 8 column volumes of 15% ethanol elution, then uses
75% ethanol elution 10 column volume, collects 75% ethanol elution, and concentrating under reduced pressure obtains 75% ethanol elution thing extractum；
In (c) step (b) 75% ethanol elution extractum with purification on normal-phase silica gel separate, successively with volume ratio be 85:1,45:1,20:1,
The methylene chloride-methanol gradient elution of 10:1 and 1:1 obtains 5 components；
D in () step (c), component 3 separates further by purification on normal-phase silica gel, be 25:1,20:1 and 10:1 by volume ratio successively
Methylene chloride-methanol gradient elution obtains 3 components；
E reverse phase silica gel that in () step (d), component 2 is bonded by octadecylsilane separates, and is 75% by concentration expressed in percentage by volume
Methanol aqueous solution isocratic elution, collects 8～10 column volume eluents, and eluent is concentrated under reduced pressure to give pure compound (I).
Further, in step (a), extract with 80% alcohol heat reflux, united extraction liquid.
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
A kind of medicine treating gastric cancer, wherein contains the aforementioned pharmaceutical compositions of therapeutically effective amount and pharmaceutically acceptable carrier.
Pharmaceutical composition of the present invention as treatment gastric cancer medicine time, can directly use, or add pharmaceutically acceptable load
Body.This medicine contains the pharmaceutical composition of the present invention of therapeutically effective amount, remaining be the most acceptable, to humans and animals without
Malicious and inert pharmaceutically suitable carrier and/or excipient.
Described pharmaceutically suitable carrier or excipient are that one or more are selected from solid, semisolid and liquid diluent, filler and medicine
Tetramune adjuvant.The pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention can be by oral
Or the form of injection is applied to need the patient for the treatment of.During for being administered orally, tablet, slow releasing tablet, controlled release tablet, glue can be made into
Capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc.；During for injecting, can be made into the water of sterilizing
Property or oily solution, aseptic powder injection, liposome or Emulsion etc..
Compared with prior art, beneficial effects of the present invention is embodied in:
Promote after the pharmaceutical composition application of the present invention that the apoptosis effect of stomach cancer cell is more notable, the growth inhibition ratio of cell and apoptosis
Index the most independent medication group raises.In the present invention, PDTC and (I) two kind of Drug combination of compound are to stomach cancer cell
Inhibitory action significantly improves when being used alone relative to two kinds of medicines, and two kinds of medicines are the chemotherapeutics of non-traditional meaning, right
The infringement of body is little, and avoids the tumor cell drug resistance to classic chemotherapy medicine.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula；
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.To the greatest extent
The present invention is explained in detail by pipe with reference to preferred embodiment, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
Embodiment 1: a kind of pharmaceutical composition treating gastric cancer, it is made up of component A and the PDTC of compound (I),
Compound (I) is 1:1 with the mass ratio of PDTC, during preparation, and will both mix homogeneously.
PDTC molecular weight is 164.29, and molecular formula is C5H9NS2·NH3, CAS Number:5108-96-3.PDTC is one
Plant and can suppress the activation of NF-kB in various kinds of cell with the NF-kB activation inhibitor of permeabilized cells film.PDTC is also
A kind of antioxidant, is also a kind of metal ion chelation agent, and can precipitation of heavy metals in an acidic solution.PDTC is permissible
Induced rat smooth muscle cell generation apoptosis, it is also possible to suppression leukaemia HL-60 occurs apoptosis.
Compound (I) separates preparation and structural identification
Reagent source: ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, purchased from Shanghai Ling Feng chemistry
Reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dried leaves (10kg) of mignonettetree is pulverized by (a), extracts (25L × 3 time) with 80% alcohol heat reflux,
United extraction liquid, is concentrated into without alcohol taste (3L), satisfies with petroleum ether (3L × 3 time), ethyl acetate (3L × 3 time) and water successively
N-butyl alcohol (3L × 3 time) extraction of sum, respectively obtains petroleum ether extract, acetic acid ethyl ester extract (398g) and n-butyl alcohol extraction
Take thing；B acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in () step (a), first with 8 posts of 15% ethanol elution
Volume, then with 10 column volumes of 75% ethanol elution, collect 75% ethanol elution, concentrating under reduced pressure obtains 75% ethanol elution thing
Extractum (163g)；C in () step (b), 75% ethanol elution extractum 200-300 mesh purification on normal-phase silica gel separates, use volume successively
Ratio is 85:1 (10 column volumes), 45:1 (9 column volumes), 20:1 (8 column volumes), 10:1 (8 column volumes) and 1:1
The methylene chloride-methanol gradient elution of (5 column volumes) obtains 5 components；D in () step (c), component 3 (49g) is used
200-300 mesh purification on normal-phase silica gel separates further, successively with volume ratio be 25:1 (8 column volumes), 20:1 (10 column volumes)
3 components are obtained with the methylene chloride-methanol gradient elution of 10:1 (5 column volumes)；Component 2 (31g) in (e) step (d)
Separate with the reverse phase silica gel ODS-C18 of octadecylsilane bonding, wash with the methanol aqueous solution that concentration expressed in percentage by volume is 75% is isocratic
De-, collect 8～10 column volume eluents, eluent is concentrated under reduced pressure to give pure compound (I) (44mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z 523.3018, can obtain molecular formula in conjunction with nuclear-magnetism feature is
C30H44O6, degree of unsaturation is 9.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6, 500.13MHz): H-1 (1.95,
D, J=5.0), and H-1 (1.59, d, J=5.0), H-5 (1.36, m), H-6 (1.83, m), H-6 (1.79, m), H-7
(2.98, d, J=6.4), H-11 (1.86, m), H-11 (1.98, m), H-12 (1.49, m), H-12 (1.94, m),
H-15 (1.45, m), H-15 (1.17, ddd, J=12.5,12.5,5.3), H-16 (1.28, m), H-16 (2.06, m),
H-17 (1.57, m), H-18 (0.94, s), H-19 (1.11, s), H-20 (1.33, m), H-21 (0.80, d, J=6.6),
H-22 (0.97, m), H-22 (1.32, m), H-23 (1.76, m), H-23 (1.94, m), H-24 (4.98, tt, J=7.1,
1.4), and H-26 (1.51, s), H-27 (1.59, s), H-28 (1.01, s), H-29 (0.97, s)；Carbon-13 nmr spectra number
According to δC(ppm, DMSO-d6, 176.04MHz): 37.1 (CH2, 1-C), 201.1 (C, 2-C), 204.8 (C, 3-C),
47.6 (C, 4-C), 41.8 (CH, 5-C), 20.2 (CH2, 6-C), 53.0 (CH, 7-C), 64.1 (C, 8-C), 84.9
(C, 9-C), 36.4 (C, 10-C), 22.1 (CH2, 11-C), 33.4 (CH2, 12-C), 45.2 (C, 13-C), 58.7
(C, 14-C), 19.8 (CH2, 15-C), 26.9 (CH2, 16-C), 51.6 (CH, 17-C), 13.3 (CH3, 18-C),
15.7(CH3, 19-C), 33.8 (CH, 20-C), 17.6 (CH3, 21-C), 35.0 (CH2, 22-C), 24.1 (CH2,
23-C), 123.2 (CH, 24-C), 130.8 (C, 25-C), 17.2 (CH3, 26-C), 25.1 (CH3, 27-C), 25.2
(CH3, 28-C), 21.7 (CH3, 29-C), 176.1 (C, 30-C)；Carbon atom labelling sees Fig. 1.IR spectrum demonstrates
Ketone group (1706cm-1) and gamma lactone (1769cm-1) absorption band.1H H NMR spectroscopy shows containing six tertiary methyl signals δ H0.94
(Me-18), 1.11 (Me-19), 1.51 (Me-26), 1.59 (Me-27), 1.01 (Me-28) and 0.97 (Me-29)；
And bimodal signal δ H0.80 (Me-21, J=6.6Hz), illustrate that this compound is triterpenoid compound.13C NMR and
DEPT spectrum 30 carbon signals of display, including seven methyl, five methines (an alkene carbon, an oxygen-containing methine),
Eight methylene and ten quaternary carbons (three carbonyl carbon, an alkene carbon, two oxygen-containing quaternary carbons).Side chain has 24 (25)-and bis-
Key (δ C130.8,123.2 and δ H4.98, tt, J=7.1,1.4Hz), illustrates that this compound is lanosterol type triterpenoid,
Me-26 and Me-27 and C-24 and C-25, Me-21 and C-17 in HMBC spectrum, C-20 and C-22, and H-24 with
The above-mentioned inference of the relevance verification of C-22, C-23, Me-26, Me-27.In HMBC spectrum, H2-1 and C-2, and H-5,
To demonstrate C-2 (δ C201.1) and C-3 (δ C204.8) be ketone carbonyl at the peak that intersects of Me-28 and Me-29 and C-3.Additionally,
One ester carbonyl group signal [δ C176.1 (C-30)] and two carbon signals [δ C84.9 (C-9) and 58.7 (C-14)] illustrate this chemical combination
There is a gamma lactone structure in thing.Me-18 and C-14 in HMBC spectrum；H2-15 and C-30；H2-11 and C-9；H-11α
The connection of this functional group of 30,9-with the relevance verification of C-8 and Me-19 and C-9.Comprehensive hydrogen spectrum, carbon spectrum, HMBC
Spectrum and NOESY compose, and document is about correlation type nuclear magnetic data, can substantially determine this compound as it is shown in figure 1, solid
Configuration is determined by ECD test further, theoretical value and experiment value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Human stomach cancer cell line SGC-7901 is purchased from Sai Er Reagent Company.Compound (I) is made by oneself, and HPLC normalization purity is more than
98%.It is public that PDTC, trypsin, MTT, dimethyl sulfoxide (DMSO), agarose, glacial acetic acid are purchased from U.S. Sigma
Department.RPMI-1640 culture medium, PBS phosphate buffered solution are purchased from GIBCO company of the U.S..Hyclone is purchased from Shandong silver
Fragrant great achievement company.Trypan blue (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), TUNEL test kit (send out by triumphant base biotechnology
Exhibition company limited), DAB colour reagent box (Beijing biotech firm of Zhong Shan Golden Bridge), formaldehyde (Fisher company of the U.S.), peroxide
Change hydrogen enzyme (new fine chemistry industry development centre, sky, Tianjin).
WD-9403C uv analyzer (Biometra company of Germany), grads PCR instrument (Biometra company of Germany), solidifying
Glue Image analysis system (Shanghai Tian Neng company), electrophresis apparatus (Beijing 6 1), (Shanghai precision instrumentation is limited for electronic balance
Company), RKI-1002 type CO2 gas incubator (Ikemoto company of Japan), (Suzhou purifies experimental facilities to superclean bench
Company limited), supercentrifuge (Beijing medical apparatus and instruments factory), low speed centrifuge (Beijing medical apparatus and instruments factory), Wilovert S type
Inverted microscope (Olympus company of Japan), vertical pressure steam sterilizer (Shanghai Bo Xun Industrial Co., Ltd.), cytometer
Number plate (Shanghai precision instrument company), ultra-pure water demineralizer (Britain PURELAB ulTRA GENETIC).
Two, test method
1, the cultivation of stomach cancer cell SGC-7901
1.1 cell recovery
(1) from liquid nitrogen container, take out cell cryopreservation tube, be immediately placed in 37 DEG C～42 DEG C, in 75% ethanol, then move to same warm water
In bath；(2) rock l～3min cryopreservation tube gently, make frozen stock solution melt, move to rapidly in super-clean bench；(3) will be containing cell
Frozen stock solution suck sterile centrifugation tube, add appropriate RPIM-1640 culture medium；(4) low speed centrifuge is put into after piping and druming mixing,
800rpm/min is centrifuged 3 minutes, removes supernatant；(5) add appropriate culture medium and blow even, cell suspension is inoculated according to concentration
In 10mL culture dish；(6) be placed in containing 5% carbon dioxide, 37 DEG C of saturated humidities incubator in cultivate.
1.2 passage
(1), during the cell attachment in culture dish about 80%, train for several times with the culture medium piping and druming that pipette, extract is old in super-clean bench
Support cell in ware, to blow dead cell off；(2) suck old culture medium with pipet, add appropriate 0.25% trypsin digestion cell,
It is placed in 30 DEG C of incubators digestion；(3) after about 3min, culture dish is placed under inverted microscope observation, treat cell periphery shinny,
Retraction then illustrates after becoming round that cell departs from from wall；(4) in super-clean bench, pancreatin is sucked rapidly.Add appropriate culture medium anti-
Blow and beat cell again, make cell completely disengage from culture dish；(5) cell suspension is seeded in different culture dishs respectively by concentration,
Supply culture medium；(6) it is replaced in containing 5%CO2, 37 DEG C of saturated humidities incubator in cultivate.
2, cell counting
(1) getting out cell counting count board and coverslip, both is clean by alcohol wipe, is covered on counting chamber by coverslip；(2)
After ethanol volatilizees, the appropriate cell suspension of sucking-off, dropping, at coverslip edge, makes suspension be full of between coverslip and counting chamber,
It is careful not to overflow coverslip and the glass guide channel of both sides, counts after standing；(3) 4 big lattice, Mei Ge great are found under the microscope
Lattice are divided into again 16 little lattice, and the cell number in 4 big lattice of counting, averages respectively.On the left of line ball cytometer and top,
Disregard right side and lower section；(4) average cell number × 10000 of cell concentration (individual/mL)=each grid；(5) cell is hanged
Liquid is diluted to test desired concn.
3, cell viability measures
(1) SGC-7901 stomach cancer cell suspension 0.9mL to be determined is taken；(2) in cell suspension, trypan blue piping and druming is added mixed
Even；(3) use cell counting count board blind at least 200 cells of counting, observe under inverted microscope；(4) Microscopic observation cell
Staining conditions, intracellular dyes light blue person for dead cell, and the person of being unstained is living cells, with hundred of total cellular score shared by living cells
The vigor (%) of proportion by subtraction reflection cell.
4, MTT detects inhibitory rate of cell growth
Experiment packet: 1. negative control group；2. compound (I) group 1, compound (I) (50 μm ol/L)；3. compound
(I) group 2, compound (I) (100 μm ol/L)；4. PDTC group 1, PDTC (50 μm ol/L)；5. PDTC group 2,
PDTC(100μmol/L)；6. drug combination group 1, compound (I) (25 μm ol/L)+PDTC (25 μm ol/L)；7. join
Share medicine group 2, compound (I) (50 μm ol/L)+PDTC (50 μm ol/L).
(1) taking the logarithm the SGC-7901 cell of trophophase, count with cell counting count board, regulation cell concentration is 5 × l05/mL；
(2) sample injector is used to be inoculated in 96 orifice plates with every hole 100 μ L.Note only being inoculated into during inoculation 60 holes of centre, periphery
36 holes are filled with PBS, simultaneously 3 96 orifice plates of paving；(3) 96 orifice plates completed are placed in 37 DEG C, 5%CO2Cell is cultivated
In case, take out after 24h cell attachment；(4) 96 orifice plates are divided into compound (I) group, (0,50,100 μm ol/L),
PDTC group (0,50,100 μm ol/L), two medicines are combined group (0/0,25/25,50/50 μm ol/L), are set up without thin simultaneously
The blank group (only adding culture medium) of born of the same parents gives different disposal respectively；(5) each concentration of each medicine sets 5 multiple holes, respectively
Cultivate 24,48 and 72h；(6) taking out 96 orifice plates at point of specific end time respectively, it is molten that every hole adds MTT (5g/L)
Liquid 20 μ L, puts back to incubator and continues to cultivate 4h, terminate cultivating.(7) carefully sucking supernatant in hole, every hole adds 150 μ L
DMSO, plate shaker vibration 10min make crystallization fully dissolve, and basis of microscopic observation granule disappears.(8) in microplate reader 490nm
Measure optical density (OD) value in each hole at wavelength, calculate the inhibitory rate of cell growth of each time point.Suppression ratio=[1-(dosing group
OD value-blank group OD value)/(negative control group OD value-blank group OD value)] × 100%.
5, TUNEL method detection natural death of cerebral cells index
The preparation of 5.1 cell climbing sheets
(1) process of coverslip: coverslip is placed in soaked overnight in concentrated sulphuric acid, first rinses for several times with tap water, then puts next day
In dehydrated alcohol, soak 4h, then rinse well with deionized water, be placed on for dry case is dried the sterilization of laggard horizontal high voltage, take out
It is directly placed in super-clean bench standby.In 6 orifice plates are placed in super-clean bench, ultraviolet irradiates 30min；(2) coverslip is placed: at six orifice plates
Every hole in prepare to put the position of coverslip and instill after a small amount of culture medium and place coverslip again so that coverslip and orifice plate examine culture medium
Tension force be bonded together, when preventing from adding cell suspension, coverslip hikes up, and causes double-layer cell adherent；(3) take the logarithm trophophase
SGC-7901 cell, digestion blow and beat into cell suspension, with cell counting count board adjust cell concentration be l × 106/mL.(4) use
Sample injector is separately added into 1mL cell suspension in the every hole being placed with coverslip, spreads 3 plates simultaneously, is placed in 37 DEG C, 5%CO2Cultivate
Case is cultivated 24h and treats cell attachment；In super-clean bench, 6 orifice plates are divided into negative control group and reality after (5) 24 hour cells are adherent
Testing group and give different disposal respectively, negative control group adds 1mL culture medium, and experimental group is further divided into compound (I) group, PDTC
Group and 3 subgroups of drug combination group, every hole adds 1mL medicine.Compound (I) organizes final concentration of 100 μm ol/L, PDTC
Organizing final concentration of 100 μm ol/L, the final concentration of two kinds of medicines of group combined by two medicines is respectively 50 μm ol/L.Often group sets 2 multiple holes.(6)
It is placed in 37 DEG C, 5%CO2Cell culture incubator in continue cultivate.
5.2TUNEL method operating procedure
(1) cell climbing sheet dosing takes out 6 orifice plates when cultivating 24h, carries out following steps successively according to TUNEL description；(2)
Careful suction abandons the supernatant in every hole；(3) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time；(4)
Every hole adds 1mL pre-cooling cell fixative, and 30min fixed by 4 DEG C of refrigerators.(5) fixative is abandoned in suction, and each hole adds PBS (4 DEG C)
2mL, plate shaker rinses 5min × 3 time.(6) immersing in confining liquid, room temperature (15 DEG C～25 DEG C) closes 10min.(7)
Each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.Blot with absorbent paper around sample.(8) each
Sample drips 50 μ L TdT enzyme reaction solutions, and 37 DEG C, lucifuge is moistening reacts 60min.(9) each hole adds PBS (4 DEG C) 2mL,
Plate shaker rinses 5min × 3 time.Blot with absorbent paper around sample.(10) 50 μ L Streptavidin-HRP working solutions are dripped,
37 DEG C, lucifuge moistening reaction 30min.(11) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.
(12) 100 μ L DAB working solutions, color development at room temperature reaction 10min are dripped.(13) each hole adds PBS (4 DEG C) 2mL,
Plate shaker rinses 5min × 3 time.(14) optical microphotograph Microscopic observation is taken pictures: randomly select 10 high power (× 100) visuals field,
Each visual field 100 cells of counting, calculate the meansigma methods adjusting index of dying.Apoptotic index (AI)=(positive cell number/total cell
Number × 100%).The nucleus of positive cell is sepia.
6, statistical analysis
Use SPSS 11.5 be analyzed, meet JH state distribution measurement data to represent, compare between group and use Oneway-ANOVA
Method is analyzed, and compares employing LSD-t method two-by-two, and P < 0.05 is that difference is statistically significant.
Three, result and conclusion
1, MTT detects the medicine growth inhibition ratio to SGC-7901 stomach cancer cell
Repeating MTT through 3 times, testing result shows, after single medicine acts on stomach cancer cell 72h, and the medicine of 100 μm ol/L
Group is the highest to the growth inhibition ratio of stomach cancer cell compared with the medicine group of 50 μm ol/L, difference statistically significant (P < 0.05).50
The compound (I) of μm ol/L is to the growth inhibited effect of stomach cancer cell and drug combination 25/25 μm ol/L group no statistical difference
Meaning (P > 0.05).The PDTC of 50 μm ol/L is to the growth inhibited effect of stomach cancer cell and drug combination 25/25 μm ol/L group
Compare, difference statistically significant (P < 0.05).The growth inhibition ratio of stomach cancer cell is higher than by drug combination 50/50 μm ol/L group
Each high concentration list medicine group (100 μm ol/L) growth inhibition ratio to stomach cancer cell, difference statistically significant (P < 0.001),
It is shown in Table 1 (* P < 0.05, * * P < 0.01).
2, each group of apoptotic index of TUNEL method detection
Compound (I), PDTC and drug combination group all have the effect of Developing restraint to stomach cancer cell SGC-7901, each group with
Negative control group comparing difference the most statistically significant (P < 0.001), it is brown that negative control group has the karyon of a small amount of cell to be dyed to
Color, each single medicine group all has increase, the apoptosis of drug combination group cell compared with drug combination group apoptotic cell relatively negative control group
Index is the highest, more notable to the inhibition of stomach cancer cell.It is shown in Table 2 (* * P < 0.01).
Conclusion, PDTC and compound (I) two medicine act solely on stomach cancer cell and all can promote with the propagation of anticancer
The apoptosis of cell, along with the increase of concentration, to the Developing restraint effect of stomach cancer cell in strengthening trend, two kinds of Drug combinations
The apoptosis effect of rear promotion stomach cancer cell is more notable, and the growth inhibition ratio of cell and apoptotic index the most independent medication group raise.Grind
Study carefully and find that PDTC and (I) two kind of Drug combination of compound are more notable to the inhibitory action of stomach cancer cell, and two kinds of medicines
Being the chemotherapeutics of non-traditional meaning, the infringement to body is little, and avoids the tumor cell drug resistance to classic chemotherapy medicine
Property, can be that the clinical treatment of gastric cancer from now on provides reference.
Table 1 each medicine group different time points to the growth inhibition ratio of stomach cancer cell (n=5,)
| Group | n | 24h | 48h | 72h |
| Compound (I) 50 μm ol/L is 2. | 5 | 14.87±5.19 | 16.29±4.53 | 55.70±6.67 |
| Compound (I) 100 μm ol/L is 3. | 5 | 31.00±6.36 | 59.42±7.74 | 74.32±5.84 |
| PDTC 50μmol/L④ | 5 | 24.87±9.04 | 36.83±5.21 | 40.58±7.15 |
| PDTC 100μmol/L⑤ | 5 | 42.86±6.28 | 44.21±8.44 | 50.31±4.63 |
| Drug combination 25/25 μm ol/L is 6. | 5 | 36.43±8.13 | 38.76±10.87 | 58.55±9.53 |
| Drug combination 50/50 μm ol/L is 7. | 5 | 49.38±2.24 | 74.56±10.59 | 96.12±2.25 |
| F | 17.918** | 29.646** | 47.625** | |
| P(1):(2) | 0.001 | <0.001 | <0.001 | |
| (3):(4) | <0.001 | 0.169 | 0.025 | |
| (5):(6) | 0.005 | <0.001 | <0.001 | |
| (1):(5) | <0.001 | <0.001 | 0.49 | |
| (3):(5) | 0.01 | 0.713 | <0.001 | |
| (2):(6) | <0.001 | 0.008 | <0.001 | |
| (4):(6) | 0.13 | <0.001 | <0.001 |
After table 2 medicine effect 24h each group cell apoptotic index (n=3,)
Embodiment 2: a kind of pharmaceutical composition treating gastric cancer, it is made up of component A and the PDTC of compound (I),
Compound (I) is 1:0.5 with the mass ratio of PDTC.
Embodiment 3: a kind of pharmaceutical composition treating gastric cancer, it is made up of component A and the PDTC of compound (I),
Compound (I) is 1:2 with the mass ratio of PDTC.
Embodiment 4: a kind of pharmaceutical composition treating gastric cancer, it is made up of component A and the PDTC of compound (I),
Compound (I) is 1:1.5 with the mass ratio of PDTC.
Embodiment 5
The preparation of tablet: first prepare pharmaceutical composition by embodiment 1～4 any embodiment method, recycling organic acid such as tartaric acid,
Or the salt that citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by it with excipient weight ratio it is
The ratio of 1:9 adds excipient, pelletizing press sheet.
Embodiment 6
Prepared by oral liquid: first prepare pharmaceutical composition by embodiment 1～4 any embodiment method, recycling organic acid such as tartaric acid,
Or the salt that citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, oral liquid preparation method is made routinely
Oral liquid.
Embodiment 7
Capsule or the preparation of granule: first prepare pharmaceutical composition by embodiment 1～4 any embodiment method, recycle organic
The salt that acid such as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by itself and tax
Shape agent weight ratio is that the ratio of 1:9 adds excipient, makes capsule or granule.
Embodiment 8
The preparation of injection: first prepare pharmaceutical composition by embodiment 1～4 any embodiment method, recycles organic acid such as winestone
The salt that acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, injects with water routinely,
Fine straining, injection is made in embedding sterilizing.
Embodiment 9
The preparation of aseptic powder injection: first prepare pharmaceutical composition by embodiment 1～4 any embodiment method, recycles organic acid such as wine
The salt that stone acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, is dissolved in aseptic note
Penetrating with in water, stirring makes molten, filters with aseptic suction funnel, more aseptic fine straining, is sub-packed in ampoule, after frozen drying
Aseptic sealing by fusing obtains injectable powder.
The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit protection scope of the present invention with this.
It will be understood by those within the art that, technical scheme can be modified or equivalent, and not take off
Essence and protection domain from technical solution of the present invention.
Claims (5)
1. the pharmaceutical composition treating gastric cancer, it is characterised in that by component A and the PDTC of following compound (I)
Composition,
Described component A is 1:0.5～2 with the mass ratio of PDTC.
2. the pharmaceutical composition treating gastric cancer as claimed in claim 1, it is characterised in that described compound (I) is from scattered foam
Spend middle separation and Extraction to obtain, comprise following operating procedure:
A the dried leaves of mignonettetree is pulverized by (), with 75～85% alcohol heat reflux extraction, united extraction liquid, be concentrated into without alcohol taste,
Successively with petroleum ether, ethyl acetate and water saturated n-butanol extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract
And n-butyl alcohol extract；
B acetic acid ethyl ester extract macroporous resin remove impurity in () step (a), first with 8 column volumes of 15% ethanol elution, then uses
75% ethanol elution 10 column volume, collects 75% ethanol elution, and concentrating under reduced pressure obtains 75% ethanol elution thing extractum；
In (c) step (b) 75% ethanol elution extractum with purification on normal-phase silica gel separate, successively with volume ratio be 85:1,45:1,20:1,
The methylene chloride-methanol gradient elution of 10:1 and 1:1 obtains 5 components；
D in () step (c), component 3 separates further by purification on normal-phase silica gel, be 25:1,20:1 and 10:1 by volume ratio successively
Methylene chloride-methanol gradient elution obtains 3 components；
E reverse phase silica gel that in () step (d), component 2 is bonded by octadecylsilane separates, and is 75% by concentration expressed in percentage by volume
Methanol aqueous solution isocratic elution, collects 8～10 column volume eluents, and eluent is concentrated under reduced pressure to give pure compound (I).
The pharmaceutical composition for the treatment of gastric cancer the most according to claim 2, it is characterised in that in step (a), with 80%
Alcohol heat reflux extracts, united extraction liquid.
The pharmaceutical composition for the treatment of gastric cancer the most according to claim 2, it is characterised in that described macroporous resin is AB-8
Type macroporous adsorbent resin.
5. the medicine treating gastric cancer, it is characterised in that wherein contain the drug regimen described in the claim 1 of therapeutically effective amount
Thing and pharmaceutically acceptable carrier.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869524A (en) * | 1996-11-12 | 1999-02-09 | American Home Products Corporation | Indene inhibitors of COX-2 |
| CN104478832A (en) * | 2015-01-05 | 2015-04-01 | 富阳鸿祥技术服务有限公司 | Diterpene compound, pharmaceutical composition containing same and preparation method and usage thereof |
-
2016
- 2016-05-25 CN CN201610356227.9A patent/CN105943542A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869524A (en) * | 1996-11-12 | 1999-02-09 | American Home Products Corporation | Indene inhibitors of COX-2 |
| CN104478832A (en) * | 2015-01-05 | 2015-04-01 | 富阳鸿祥技术服务有限公司 | Diterpene compound, pharmaceutical composition containing same and preparation method and usage thereof |
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