CN105198844A - Novel limonin compound as well as preparation method and medical application thereof - Google Patents
Novel limonin compound as well as preparation method and medical application thereof Download PDFInfo
- Publication number
- CN105198844A CN105198844A CN201510690656.5A CN201510690656A CN105198844A CN 105198844 A CN105198844 A CN 105198844A CN 201510690656 A CN201510690656 A CN 201510690656A CN 105198844 A CN105198844 A CN 105198844A
- Authority
- CN
- China
- Prior art keywords
- compound
- cell
- preparation
- extract
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D307/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
Abstract
The invention discloses a novel limonin compound as well as a preparation method and medical application thereof. The compound is reported for the first time, is a limonin compound with a novel structure, and can be extracted, separated and purified from the dried whole herb of Grace. In vitro tests prove that the compound can inhibit the proliferation of gastric carcinoma cells, and promote the apoptosis of the cells, and the growth inhibiting effect on the gastric carcinoma cells is strengthened gradually along with the increase of concentration, these findings provide a basis for a clinical research of drug of the compound (I) for curing gastric carcinoma, and the compound can be developed into a drug for curing the gastric carcinoma.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry herb of rue, be separated obtain a kind of and there is limonoid for the treatment of Effect on Gastric Cancer and preparation method thereof.
Background technology
Rue RutagarveolensL., another name Root of Lindley Butterflybush, vanilla, Remote Lemongrass Herb, little Herba Cymbopogonis Citrari, wild Remote Lemongrass Herb, lime grass, stinkweed are Rutaceae per nnial herb, herb hyoscine, in the whole world, many areas are that medicinal object is extensively cultivated, and the whole year can adopt, and clean and dry in the shade or using fresh herb.Pungent, micro-hardship, cool.Clearing heat and detoxicating, blood stasis removing analgesic.For cold, fever, toothache, menoxenia, infantile eczema; Sore furuncle poison is controlled in external application, wound.
Modern study shows, this plant contains the compounds such as acridone alkaloid, tonka bean camphor, flavones, obacalactone and lignanoid.Known Natural acridone alkaloids only derives from rutaceae.
Rue as stimulating the menstrual flow and aborticide, is also used for the treatment of the cardio palmus that cardiovascular disorder causes by the traditional medicine of some countries.Modern study shows that rue has the various different pharmacologically actives such as antibacterial, spasmolysis, excited uterus.Alkaloid and furocoumarin(e) have reversible spasmolysis, anti-inflammatory and anti-histamine activity.Furocoumarin(e) class also have photosensitive, light is malicious and mutagenesis, can cause skin adverse reaction.In addition, rue tonka bean camphor has antifertility and anti-implantation effect.Violaguercitrin has spasmolysis, and it can reduce capillary permeability and fragility.Rue extract has the effect delaying cell mitogen, this antimitotic characteristic, relevant with the xanthotoxin contained by it.In addition, rue also has the effects such as anti-inflammatory, antimycotic, arteriosclerosis.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dry herb of rue, be separated obtain a kind of there is limonoid for the treatment of Effect on Gastric Cancer and preparation method thereof.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: the dry herb of rue is pulverized by (a), extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 75% ethanol elution, 10 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
Further, described alcohol heat reflux extracts the alcohol concn adopted is 80%.
A kind of pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine of preparation treatment cancer of the stomach.
The application of described pharmaceutical composition in the medicine of preparation treatment cancer of the stomach.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
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.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry herb (10kg) of rue is pulverized by (a), (25L × 3 time) are extracted with 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (3L), use sherwood oil (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated propyl carbinol (3L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 type macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 75% ethanol elution, 10 column volumes again, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract (157g); C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, successively with volume ratio be 75:1 (8 column volumes), the methylene chloride-methanol gradient elution of 45:1 (8 column volumes), 20:1 (8 column volumes), 12:1 (8 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (49g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (8 column volumes), the methylene chloride-methanol gradient elution of 12:1 (10 column volumes) and 5:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (25g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (35mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z697.3202, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
36h
50o
12, degree of unsaturation is 12.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-1 (5.39, d, J=4.0), H-2 (2.39, dd, J=14.8, 4.0), H-2 (2.87, m), H-5 (2.34, d, J=7.5), H-6 (2.13, m), H-6 (3.25, m), H-9 (3.33, d, J=7.8), H-11 (5.29, dd, J=11.0, 7.8), H-12 (5.59, d, J=11.0), H-15 (3.67, s), H-16 (1.71, t, J=12.0), H-16 (2.10, m), H-17 (2.89, m), H-18 (0.87, s), H-19 (1.56, s), H-21 (6.96, s), H-22 (6.05, s), H-23 (7.18, s), H-28 (0.92, s), H-29 (1.06, s), H-30 (4.97, s), H-30 (5.28, s), 7-OCH
3(3.49, s), 1-OAc (1.88, s), and 11-OAc (1.89, s), H-2 ' (1.77, m), H-3 ' (0.96, m), and H-3 ' (1.27, m), H-4 ' (0.71, t, J=7.5), H-5 ' (0.73, d, J=7.3), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 150Hz): 77.6 (CH, 1-C), 36.7 (CH
2, 2-C), 211.5 (C, 3-C), 49.1 (C, 4-C), 44.2 (CH, 5-C), 31.1 (CH
2, 6-C), 174.4 (C, 7-C), 134.2 (C, 8-C), 50.3 (CH, 9-C), 46.0 (C, 10-C), 71.8 (CH, 11-C), 73.9 (CH, 12-C), 45.1 (C, 13-C), 70.6 (C, 14-C), 59.2 (CH, 15-C), 33.4 (CH
2, 16-C), 37.3 (CH, 17-C), 13.0 (CH
3, 18-C), 16.8 (CH
3, 19-C), 121.8 (C, 20-C), 139.9 (CH, 21-C), 110.7 (CH, 22-C), 142.1 (CH, 23-C), 24.7 (CH
3, 28-C), 23.8 (CH
3, 29-C), 122.9 (CH
2, 30-C), 51.1 (CH
3, 7-OCH
3), 169.5 (C, 1-OAc), 20.4 (CH
3, 1-OAc), 170.1 (C, 11-OAc), 21.1 (CH
3, 11-OAc), 175.2 (C, 1 '-C), 40.2 (CH, 2 '-C), 25.3 (CH
2, 3 '-C), 11.8 (CH
3, 4 '-C), 15.1 (CH
3, 5 '-C), carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains hydroxyl (3442cm
-1) and carbonyl (1738cm
-1) group.
13cNMR spectrum and DEPT spectrum show 36 carbon signals, comprising nine methyl, five methylene radical (an alkene carbon), 11 methyne (three alkene carbon, four containing oxygen carbon), and 11 quaternary carbons (a ketone carbonyl carbon, four ester carbonyl group carbon, two alkene carbon, one containing oxygen carbon).
1h and
13cNMR nuclear magnetic data analyzes the limonoid that this compound known is the open loop of a B ring.In HMBC spectrum, Me-19 (δ H1.56, s) and H-9 (δ H3.33, d, J=7.8Hz) and C-1 (δ C77.6); The dependency of H-1 (δ H5.39, d, J=4.0Hz) and H-11 (δ H5.29, dd, J=11.0,7.8Hz) its corresponding acetyl carbonyl (δ C169.5,170.1), and
1h-
1h-1 and H in HCOSY spectrum
2-2 (δ H2.39, dd, J=14.8,4.0Hz; 2.87, intersection peak m), shows that C-1 and C-11 (δ C71.8) position is connected with an acetoxyl group respectively.In HMBC spectrum, with the dependency of C-3 (δ C211.5), H-1, Me-28 (δ H0.92, s) and Me-29 (δ H1.06, s) show that C-3 is ketone carbonyl.The monosubstituted furan nucleus of a β [δ C121.8 (C-20) is contained by nuclear magnetic data this compound known, 139.9 (C-21), 110.7 (C-22) and 142.1 (C-23)], according to H-17 (δ H2.89 in HMBC spectrum, and C-20 m), the dependency of C-21 and C-22, deducibility furan nucleus is positioned on C-17 position.In addition, this compound contains a 2-methylbutyryl oxygen based structures.In HMBC spectrum, Me-4 ' (δ H0.71, t, J=7.5Hz) and C-2 ' (δ C40.2); Me-5 ' (δ H0.73, d, J=7.3Hz) and C-3 ' (δ C25.3); Me-5 ' and C-1 ' (δ C175.2); And the dependency of H-12 (δ H5.59, d, J=11.0Hz) and C-1 ' shows that 2-methylbutyryl oxygen based structures is positioned on C-12 (δ C73.9) position.In ROESY spectrum, Me-19 and Me-29 and H-12; And H-12 and H-17 dependency shows that they are beta comfiguration.In addition, H-9 (δ H3.33, d, J=7.8Hz) and H-1; H-9 and H-11; H-9 and H-5 (δ H2.34, d, J=7.5Hz); The intersection peak of H-5 and Me-28 and H-11 and Me-18 (δ H0.87, s) shows that they are α-configuration.These results and key proton (J
9,11=7.8Hz and J
11,12=11.0Hz) between larger coupling constant match.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and ROESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental 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 method purity is greater than 98%.PDTC, trypsinase, MTT, dimethyl sulfoxide (DMSO) (DMSO), agarose, Glacial acetic acid are purchased from Sigma Co., USA.RPMI-1640 substratum, PBS phosphate buffer soln are purchased from GIBCO company of the U.S..Foetal calf serum is purchased from Shandong Yin Xiang great achievement company.Trypan blue (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), TUNEL test kit (Kai Ji biotechnology Development Co., Ltd), DAB colouring reagents box (Beijing biotech firm of Zhong Shan Golden Bridge), formaldehyde (Fisher company of the U.S.), catalase (new fine chemistry industry development centre, sky, Tianjin).
WD-9403C uv analyzer (German Biometra company), grads PCR instrument (German Biometra company), Labworks image acquisition and analysis software (Shanghai Tian Neng company), electrophoresis apparatus (Beijing 6 1), electronic balance (Shanghai precision instrumentation company limited), RKI-1002 type CO2gas incubator (Japanese Ikemoto company), Bechtop (Suzhou purification experimental installation company limited), ultracentrifuge (Beijing medical apparatus and instruments factory), low speed centrifuge (Beijing medical apparatus and instruments factory), WilovertS type inverted microscope (Japanese Olympus company), vertical pressure steam sterilizer (Shanghai Bo Xun Industrial Co., Ltd.), cell counting count board (Shanghai precision instrument company), ultrapure water water purifior (Britain PURELABulTRAGENETIC).
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 placed in rapidly 37 DEG C ~ 42 DEG C, in 75% alcohol, then move in equality of temperature water-bath; (2) rock l ~ 3min cryopreservation tube gently, frozen storing liquid is melted, move to rapidly in super clean bench; (3) frozen storing liquid containing cell is sucked sterile centrifugation tube, add appropriate RPIM-1640 substratum; (4) put into low speed centrifuge, centrifugal 3 minutes of 800rpm/min after piping and druming mixing, remove supernatant liquor; (5) adding appropriate substratum blows even, is inoculated in 10mL culture dish by cell suspension according to concentration; (6) incubator be placed in containing 5% carbonic acid gas, 37 DEG C of saturated humidities is cultivated.
1.2 passage
(1), when the cell attachment in culture dish about 80%, in super clean bench, cell in several culture dish is blown and beaten, to blow dead cell off with the substratum that pipette, extract is old; (2) suck old substratum with transfer pipet, add appropriate 0.25% trypsin digestion cell, be placed in 30 DEG C of incubators and digest; (3) observe under culture dish being placed in after about 3min inverted microscope, retraction shinny until cell periphery then illustrates that cell departs from from wall after becoming circle; (4) in super clean bench, pancreatin is sucked rapidly.Add appropriate substratum and repeatedly blow and beat cell, make cell depart from culture dish completely; (5) cell suspension is seeded in different culture dish respectively by concentration, supplies substratum; (6) be again placed in containing 5%CO
2, 37 DEG C of saturated humidities incubator in cultivate.
2, cell counting
(1) get out cell counting count board and cover glass, the two is all clean by alcohol wipe, is covered by cover glass on tally; (2) after alcohol volatilization, the appropriate cell suspension of sucking-off, drips at cover glass edge, makes suspension be full of between cover glass and tally, notes the glass guide channel not overflowing cover glass and both sides, counting after leaving standstill; (3) find 4 large lattice under the microscope, each large lattice are divided into again 16 little lattice, count the cell count in 4 large lattice respectively, average.On the left of line ball cytometer and top, on the right side of disregarding and below; (4) average cell number × 10000 of cell concn (individual/mL)=each grid; (5) cell suspension is diluted to experiment desired concn.
3, cell viability measures
(1) SGC-7901 stomach cancer cell suspension 0.9mL to be determined is got; (2) in cell suspension, add trypan blue piping and druming mixing; (3) adopt cell counting count board blind counting at least 200 cells, observe under inverted microscope; (4) Microscopic observation cell dyeing situation, dye light blue person in cell for dead cell, the person of being unstained is viable cell, with the vigor (%) of the per-cent of total cellular score shared by viable cell reflection cell.
4, MTT detects inhibitory rate of cell growth
Experiment grouping: 1. negative control group; 2. compound (I) group 1, compound (I) (50 μm of ol/L); 3. compound (I) group 2, compound (I) (100 μm of ol/L); 4. PDTC group 1, PDTC (50 μm of ol/L); 5. PDTC group 2, PDTC (100 μm of ol/L); 6. drug combination group 1, compound (I) (25 μm of ol/L)+PDTC (25 μm of ol/L); 7. drug combination group 2, compound (I) (50 μm of ol/L)+PDTC (50 μm of ol/L).
(1) the SGC-7901 cell of taking the logarithm vegetative period, with cell counting count board counting, adjustment cell concn is 5 × l0
5/ mL; (2) sample injector is used to be inoculated in 96 orifice plates with every hole 100 μ L.Only be inoculated into 60 middle holes when noting inoculation, periphery 36 hole is filled with PBS, spreads 3 96 orifice plates simultaneously; (3) 96 orifice plates completed are placed in 37 DEG C, 5%CO
2in cell culture incubator, take out after 24h cell attachment; (4) 96 orifice plates are divided into compound (I) group, (0,50,100 μm of ol/L), PDTC group (0,50,100 μm of ol/L), group (0/0,25/25,50/50 μm of ol/L) combined by two medicines, sets up not celliferous blank group (only adding substratum) to give different treatment respectively simultaneously; (5) each concentration of each medicine establishes 5 multiple holes, cultivates 24,48 and 72h respectively; (6) respectively at specific end time point taking-up 96 orifice plate, every hole adds MTT (5g/L) solution 20 μ L, puts back to incubator and continues to cultivate 4h, stop cultivating.(7) carefully suck supernatant liquor in hole, every hole adds 150 μ LDMSO, and plate shaker vibration 10min makes crystallization fully dissolve, and basis of microscopic observation particle disappears.(8) measure optical density(OD) (OD) value in each hole in microplate reader 490nm wavelength place, calculate the inhibitory rate of cell growth of each time point.Inhibiting rate=[1-(dosing group OD value-blank group OD value)/(negative control group OD value-blank group OD value)] × 100%.
5, TUNEL method detects natural death of cerebral cells index
The preparation of 5.1 cell climbing sheets
(1) process of cover glass: cover glass is placed in vitriol oil soaked overnight, next day first with tap water for several times, then be placed in dehydrated alcohol and soak 4h, then clean with deionized water rinsing, be placed on for drying the sterilization of laggard horizontal high voltage in dry case, it is for subsequent use that super clean bench is directly put in taking-up.6 orifice plates are placed in uviolizing 30min in super clean bench; (2) cover glass is placed: after the position preparing to put cover glass in every hole of six orifice plates instills a small amount of substratum, place cover glass again, the tension force making cover glass and orifice plate examine substratum is bonded together, when preventing from adding cell suspension, cover glass hikes up, and causes double-layer cell adherent; (3) the SGC-7901 cell of taking the logarithm vegetative period, cell suspension is blown and beaten in digestion, is l × 10 with cell counting count board adjustment cell concn
6/ mL.(4) add 1mL cell suspension respectively with every hole that sample injector is being placed with cover glass, spread 3 plates simultaneously, be placed in 37 DEG C, 5%CO
2cultivate 24h in incubator and treat cell attachment; In super clean bench, negative control group and experimental group is divided into give different treatment respectively 6 orifice plates after (5) 24 hour cells are adherent, negative control group adds 1mL substratum, experimental group is further divided into compound (I) group, PDTC group and drug combination group 3 subgroups, and every hole adds 1mL medicine.Compound (I) group final concentration be 100 μm of ol/L, PDTC group final concentrations is 100 μm of ol/L, and it is respectively 50 μm of ol/L that two medicines combine that to organize final concentration be two kinds of medicines.Often group establishes 2 multiple holes.(6) 37 DEG C are placed in, 5%CO
2cell culture incubator in continue cultivate.
5.2TUNEL method operation steps
(1) take out 6 orifice plates when 24h is cultivated in cell climbing sheet dosing, carry out following steps successively according to TUNEL specification sheets; (2) supernatant liquor abandoned in every hole is carefully inhaled; (3) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time; (4) every hole adds 1mL precooling cell stationary liquid, and 30min fixed by 4 DEG C of refrigerators.(5) stationary liquid is abandoned in suction, and each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.(6) immerse 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 thieving paper around sample.(8) each sample drips 50 μ LTdT enzyme reaction solutions, 37 DEG C, lucifuge moistening reaction 60min.(9) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.Blot with thieving paper around sample.(10) 50 μ LStreptavidin-HRP working fluids 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 LDAB working fluids are dripped, color development at room temperature reaction 10min.(13) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.(14) optical microphotograph Microscopic observation is taken pictures: random selecting 10 high power (× 100) visuals field, and each visual field counts 100 cells, calculates the mean value adjusting index of dying.Apoptotic index (AI)=(positive cell number/total cell count × 100%).The nucleus of positive cell is brown.
6, statistical analysis
Adopt SPSS11.5 to analyze, the measurement data meeting the distribution of JH state, to represent, compares between group and analyzes by Oneway-ANOVA method, and compare between two and adopt LSD-t method, P<0.05 is that difference has statistical significance.
Three, result and conclusion
1, MTT detection of drugs is to the growth inhibition ratio of SGC-7901 stomach cancer cell
MTT is repeated through 3 times, detected result shows, single drug effect is after stomach cancer cell 72h, and the medicine group of 100 μm of ol/L is all higher to the growth inhibition ratio of stomach cancer cell compared with the medicine group of 50 μm of ol/L, and difference has statistical significance (P<0.05).The compound (I) of 50 μm of ol/L is to the growth-inhibiting effect of stomach cancer cell and drug combination 25/25 μm of ol/L group no significant difference (P>0.05).The PDTC of 50 μm of ol/L is to compared with the growth-inhibiting effect of stomach cancer cell and drug combination 25/25 μm of ol/L group, and difference has statistical significance (P<0.05).Drug combination 50/50 μm of ol/L group to the growth inhibition ratio of stomach cancer cell higher than each high density list medicine group (100 μm of ol/L) growth inhibition ratio to stomach cancer cell, difference has statistical significance (P<0.001), in table 1 (* P<0.05, * * P<0.01).
2, TUNEL method detects each group of apoptotic index
Compound (I), PDTC and drug combination group all have the effect of Developing restraint to stomach cancer cell SGC-7901, each group all has statistical significance (P<0.001) with negative control group comparing difference, negative control group has the karyon of a small amount of cell to be dyed to brown, each single medicine group and drug combination group apoptotic cell comparatively all have increase compared with negative control group, the apoptotic index of drug combination group cell is the highest, more remarkable to the inhibition of stomach cancer cell.In table 2 (* * P<0.01).
Conclusion, PDTC and compound (I) two medicine act solely on stomach cancer cell all can the propagation of anticancer, promote the apoptosis of cell, along with the increase of concentration, be enhancing trend to the Developing restraint effect of stomach cancer cell, promote the apoptosis more remarkable effect of stomach cancer cell after two kinds of Drug combinations, all more independent medication group of the growth inhibition ratio of cell and apoptotic index raises.Research find PDTC and the restraining effect of compound (I) two kind of Drug combination to stomach cancer cell more remarkable, and two kinds of medicines are the chemotherapeutics of non-traditional meaning, little to the infringement of body, and avoid the resistance of tumour cell to classic chemotherapy medicine, reference can be provided for the clinical treatment of cancer of the stomach from now on.
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 of ol/L (1) | 5 | 14.87±5.19 | 16.29±4.53 | 55.70±6.67 |
| Compound (I) 50 μm of ol/L (2) | 5 | 31.00±6.36 | 59.42±7.74 | 74.32±5.84 |
| PDTC50μmol/L(3) | 5 | 24.87±9.04 | 36.83±5.21 | 40.58±7.15 |
| PDTC100μmol/L(4) | 5 | 42.86±6.28 | 44.21±8.44 | 50.31±4.63 |
| Drug combination 25/25 μm of ol/L (5) | 5 | 36.43±8.13 | 38.76±10.87 | 58.55±9.53 |
| Drug combination 50/50 μm of ol/L (6) | 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 drug effect 24h each group cell apoptotic index (n=3,
)
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:9 in itself and excipient weight.
Embodiment 4
Prepared by oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula,
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry herb of rue is pulverized by (a), extract with 75 ~ 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 75% ethanol elution, 10 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,20:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
4. the preparation method of compound according to claim 2 (I), is characterized in that: it is 80% that described alcohol heat reflux extracts the alcohol concn adopted.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the medicine of preparation treatment cancer of the stomach.
7. the application of pharmaceutical composition according to claim 5 in the medicine of preparation treatment cancer of the stomach.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510690656.5A CN105198844A (en) | 2015-10-22 | 2015-10-22 | Novel limonin compound as well as preparation method and medical application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510690656.5A CN105198844A (en) | 2015-10-22 | 2015-10-22 | Novel limonin compound as well as preparation method and medical application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105198844A true CN105198844A (en) | 2015-12-30 |
Family
ID=54946842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510690656.5A Withdrawn CN105198844A (en) | 2015-10-22 | 2015-10-22 | Novel limonin compound as well as preparation method and medical application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105198844A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105481937A (en) * | 2016-01-14 | 2016-04-13 | 郑平珍 | Novel limonin compound as well as preparation method and medical application thereof |
| CN106008427A (en) * | 2016-07-01 | 2016-10-12 | 郑飞珍 | Novel sesquiterpene lactones compound, and preparation method and medical application thereof |
| CN107793403A (en) * | 2016-08-28 | 2018-03-13 | 成都宝科生物科技有限公司 | A kind of noval chemical compound with epoxy construction and preparation method thereof and medical usage |
-
2015
- 2015-10-22 CN CN201510690656.5A patent/CN105198844A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105481937A (en) * | 2016-01-14 | 2016-04-13 | 郑平珍 | Novel limonin compound as well as preparation method and medical application thereof |
| CN106008427A (en) * | 2016-07-01 | 2016-10-12 | 郑飞珍 | Novel sesquiterpene lactones compound, and preparation method and medical application thereof |
| CN107793403A (en) * | 2016-08-28 | 2018-03-13 | 成都宝科生物科技有限公司 | A kind of noval chemical compound with epoxy construction and preparation method thereof and medical usage |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105085539A (en) | Novel diterpenoid compound, preparation method thereof and medical application | |
| CN105198893A (en) | Diterpenoid compounds for treating stomach cancer | |
| CN104974145A (en) | Diterpene lactone compounds, pharmaceutical composition containing compounds, and preparation method and application thereof | |
| CN105330717A (en) | Novel triterpenoid and preparation method and medical application thereof | |
| CN105943532A (en) | Application of diterpenoid compound to preparation of medicament for treating liver cancer | |
| CN105085452A (en) | Sesquiterpene naphthoquinone compound as well as preparation method and medical application thereof | |
| CN105198844A (en) | Novel limonin compound as well as preparation method and medical application thereof | |
| CN105566427A (en) | Lanostane triterpene compound, and preparation method and medicinal use thereof | |
| CN106008543A (en) | Novel diterpenoid compound and preparation method thereof | |
| CN105418545A (en) | Novel isocoumarin compound and preparation method and medical application thereof | |
| CN105524075A (en) | A novel diterpene compound, a preparing method thereof and medical uses of the diterpene compound | |
| CN105418539A (en) | New meroterpenoid compound as well as preparation method and pharmaceutical application thereof | |
| CN105367536A (en) | Novel iridoid and preparation method and medical application thereof | |
| CN105237380A (en) | Triterpene compound used for treating ovarian cancer and preparation method of triterpene compound | |
| CN105153263A (en) | New limonin compound as well as preparation method and medical application thereof | |
| CN105503996A (en) | Limonin compound as well as preparation method and neuroprotective effect thereof | |
| CN106397369A (en) | Novel labdane-type diterpenoid compound, preparation method and application thereof, pharmaceutical composition and application of pharmaceutical composition | |
| CN105622629A (en) | Kaurane type diterpenoid compound for treating prostatic cancer | |
| CN106074499A (en) | The application in medicine of a kind of Crow alkane type diterpene-kind compound | |
| CN105198897A (en) | New kaurane diterpenoid compound, preparation method and medical application of compound in treating liver cancer | |
| CN105524134A (en) | Novel lanostane type triterpene compound, preparation method therefor and medicinal use of novel lanostane type triterpene compound | |
| CN105481874A (en) | Novel diterpene compound for treating ovarian cancer | |
| CN105503895A (en) | Novel diterpene compound for treating acute T lymphocytic leukemia | |
| CN105348227A (en) | Novel isocoumarins compound as well as preparation method and medical application thereof | |
| CN105198895A (en) | New limonin compound and preparing method and medical application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20151230 |