CN103664986B - Antineoplastic compound extracted from gamboge and preparation method thereof and purposes - Google Patents
Antineoplastic compound extracted from gamboge and preparation method thereof and purposes Download PDFInfo
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Abstract
The present invention relates to antineoplastic compound extracted from gamboge and preparation method thereof and purposes.Described compound method for making is: (1) is got gamboge medicinal material ethyl acetate and extracted, and extract is separated through silica gel column chromatography, and petroleum ether-ethyl acetate gradient elution obtains component A ~ K; (2) discard oil component and morellic acid component, then analyze through HPLC-PDA-MS deduplication, obtain component I; (3) component I is separated through silica gel column chromatography again, and chloroform-methanol gradient elution, obtains flow point I-3; (4) get I-3 through anti-phase RP-18 chromatographic separation, acetonitrile-water gradient, obtains Arius and divides I-3C; (5) purified, the acetonitrile of I-3C, obtains the compounds of this invention.Compound of the present invention can be used for the treatment of tumour.
Description
Technical field
The present invention relates to medical art, particularly a kind of extraction and isolation from Chinese medicinal materials gamboge compound, prepare the method for described compound, comprise the medical composition of described compound, and use described compound to prepare the purposes of antitumor drug.
Background technology
Gamboge is that the trunk of Garcinia maingayii gamboge (GarciniahanburyiHookf.) is hurt the colloidal resin of rear outflow, has another name called extra large rattan, beautiful yellow, the moon is yellow, cured Huang etc.Originate in South East Asia, there is introducing culture on such as the ground such as Yunnan, Guangxi, Guangdong, China some areas.
Gamboge begins to be loaded in " Haiyao Bencao, Oversea Materia Medica ", and it is cold in nature, and taste is sour, puckery, pungent, poisonous, and have effect of broken blood dissipating bind, removing toxic substances, hemostasis, desinsection, the traditional Chinese medical science is used for controlling swollen ulcer drug, and stubborn dermatitis dislikes sore, bleeding due to trauma, noma decayed tooth, burn.
At present, reported to be divided into from gamboge from identify more than 20 compounds, be mostly xanthone (xanthone) compounds containing bridged ring, i.e. gambogic acid, Mo Lilin class, morellin class, and gamboge aldehyde, Betulinic acid etc. multiple compounds.
Modern pharmacology research shows, Resina garciniae extract has the anti-microbial activity of wide spectrum, has stronger bacteriostatic action to streptococcus aureus, Pseudomonas aeruginosa etc.And pharmacological evaluation and clinical experience prove, it has antitumor action, effectively can breed by Tumor suppression, have significant curative effect to kinds of tumors.
Contriver extracts and obtains having bioactive new compound 32,33-dihydroxyl morellic acid (formula (1) compound) from gamboge, has more superior anti-tumor activity compared with morellic acid.
Summary of the invention
The invention provides a kind of from gamboge extraction and isolation there is bioactive formula (1) compound, and provide this compound preparation method and preparation tumor purposes.
Formula (1) compound has following formula:
Present invention also offers the preparation method of this formula (1) compound, concrete steps are:
(1) get gamboge medicinal material ethyl acetate to extract, extract is separated through silica gel column chromatography, petroleum ether-ethyl acetate gradient elution, obtains component A ~ K successively;
(2) discard oil component A and B and morellic acid D and component E, analyze through HPLC-PDA-MS deduplication, obtain component I;
(3) component I is separated through silica gel column chromatography again, chloroform-methanol gradient elution, and the flow velocity of elutriant is 1-5ml/min, and every 25 ~ 35ml collects a flow point, merges the 11st part to the 16th part elutriant, obtains I-3;
(4) get I-3 through anti-phase RP-18 chromatographic separation, acetonitrile-water gradient, the flow velocity of elutriant is 1-5ml/min, and every 15 ~ 25ml collects a flow point, merges the 5th part to the 9th part elutriant, obtains Arius and divide I-3C;
(5) purified, the acetonitrile isocratic elution of I-3C, obtains formula (1) compound.
Preferably, the ratio of step (1) PetroChina Company Limited. ether-ethyl acetate 50 ~ 0:1 by volume carries out gradient elution;
Preferred, petroleum ether-ethyl acetate is divided into six gradients to carry out wash-out, is respectively 50:1,10:1,5:1,2:1,1:1,0:1.
Preferably, the ratio of chloroform-methanol 50 ~ 10:1 by volume carries out gradient elution in step (3);
Preferably, the ratio of acetonitrile-water 40 ~ 90:60 ~ 10 by volume carries out gradient elution in step (4);
Preferred, acetonitrile-water is divided into five gradients to carry out wash-out, is respectively 40:60,50:50,60:40,80:20,90:10.
Preferably, in step (5), the concentration of acetonitrile is 50% ~ 80%;
Preferred, the concentration of acetonitrile is 60% ~ 70%.
In the gradient elution of wherein said step (1), every 30ml collects a flow point, and every 7 parts of elutriants merge, and the flow velocity of the elutriant of gradient elution is 1-5ml/min, and preferable flow rate is 2ml/min.
In wherein said step (3), the flow velocity of the elutriant of gradient elution is preferably 2ml/min.
In wherein said step (4), the flow velocity of the elutriant of gradient elution is preferably 2ml/min.
Contriver by physico-chemical property and the Modern spectroscopy section of learning to do (1H-NMR(Fig. 2),
13c-NMR(Fig. 3), HR-ESI-MS,
1h-
1hCOSY with HSQC) carry out Structural Identification to being separated the compound obtained, be confirmed that it is structure compound as the formula (1).
In one aspect, formula of the present invention (1) compound may be used for treating tumor disease.Contriver finds that the compounds of this invention has the inhibit activities of wide spectrum to tumour cell.Described tumor disease comprises the group being selected from following composition: nasopharyngeal carcinoma, mammary cancer, liver cancer, carcinoma of the pancreas and cervical cancer.
Another aspect, present invention also offers the pharmaceutical composition for the treatment of tumor disease, it comprises formula (1) compound for the treatment of significant quantity, and at least one is selected from following pharmaceutically acceptable non-active ingredient: pharmaceutically acceptable thinner, pharmaceutically acceptable vehicle and pharmaceutically acceptable supporting agent.
Accompanying drawing explanation
Fig. 1 is formula (1) structural formula of compound;
Fig. 2 is the 1H-NMR spectrum of formula (1) compound;
Fig. 3 is the 13C-NMR spectrum of formula (1) compound.
Embodiment
compound
Formula (1) compound has following structure:
medical composition/composite
Be applicable to administration routes include, but is not limited to per os, intravenously, rectum, aerosol, parenteral, eye, lung, through mucous membrane, through skin, vagina, ear, intranasal, intramuscular injection, subcutaneous injection and topical administration.In addition, only for example, parenteral transmission comprises intramuscular, subcutaneous, intravenously, intramedullary injection, and in sheath, the directly interior and nasal injection of indoor, intraperitoneal, lymph.
In certain embodiments, compound as herein described with local but not systemic manner administration.In other embodiments, compound as herein described provides to discharge formulation, prolongation release formulation or middle release formulation fast.In other embodiments, compound as herein described is local administration.
In certain embodiments, compound as herein described is through being allocated as medical composition.In a particular embodiment, medical composition in the usual way, use one or more physiologically acceptable supporting agents to allocate, described physiologically acceptable supporting agent comprises vehicle and auxiliary agent, and it contributes to active compound being processed as the preparation that can be used for pharmacy.Suitable composite depends on selected dosing way.
Medical composition refers to the mixture of formula (1) compound and other chemical composition, and described chemical composition is as being supporting agent, stablizer, thinner, dispersion agent, suspension agent, thickening material and/or vehicle.In certain embodiments, medical composition contributes to Mammals administration compound.
In certain embodiments, compound as herein described is through allocating for oral administration.Compound as herein described is allocated with oral dosage form, and only for example, described oral dosage form comprises tablet, pulvis, pill, sugar-coat ingot, capsule, liquid, gel, syrup, elixir, slurries, suspension and analogue thereof.
In certain embodiments, described medical composition is tablet, capsule, powder ampoule agent for injection, injection, pill and slow releasing tablet.
In one embodiment, formula (1) compound is allocated in the aqueous solution.In a particular embodiment, only for example, the aqueous solution is selected from physiological compatibility damping fluid, as Han Shi liquid (Hank'ssolution), ringer's solution (Ringer'ssolution) or normal saline buffer solution.
In other embodiments, formula (1) compound is used for through Mucosal administration through allotment.In a particular embodiment, the permeate agent be suitable for for permeability barrier is comprised through mucous membrane composite.
Be used in other embodiment of other parenteral injection through allotment at compound as herein described, suitable composite comprises water-based or non-aqueous solution.
In certain embodiments; by mixing one or more solid excipients and one or more compounds as herein described, optionally grinding gained mixture and adding where necessary and be applicable to auxiliary agent post-treatment granular mixture and obtain pharmaceutical preparation for per os use to obtain tablet or pill.Specifically, suitable vehicle is weighting agent, as sugar, comprises lactose, sucrose, N.F,USP MANNITOL or sorbyl alcohol; Cellulose preparation, as W-Gum, wheat starch, rice starch, yam starch, gelatin, tragacanth, methylcellulose gum, Microcrystalline Cellulose, Vltra tears, Xylo-Mucine; Or other material, as polyvinylpyrrolidone (PVP or polyvidone) or calcium phosphate.In a particular embodiment, optionally disintegrating agent is added.Only for example, disintegrating agent comprises croscarmellose sodium, polyvinylpyrrolidone, agar or Lalgine or its salt, as sodium alginate.
Oral dosage form also comprises the cooperation of being made up of gelatin and inserts capsule, and the soft seal capsule be made up of gelatin and softening agent (as glycerine or sorbyl alcohol).In a particular embodiment, the mixture inserting capsule and contain activeconstituents and one or more weighting agents is coordinated.Only for example, weighting agent comprises lactose, as tackiness agents such as starch and/or as the lubricant such as talcum or Magnesium Stearate, and the optional stablizer used.In other embodiments, soft capsule contains one or more active compounds dissolving or be suspended in suitable liquid.Only for example, appropriate liquid comprises one or more fatty oils, liquid paraffin or liquid polyethylene glycol.In addition, optionally stablizer is added.
In other embodiments, formula (1) compound is local administration.Can local administration composition comprise solution, suspension, lotion, gel, paste, swab, balm, breast frost or ointment.
In other embodiments, formula (1) compound is through allocating for inhalation dosing.The various forms being suitable for inhalation dosing includes, but is not limited to aerosol, spraying or pulvis.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.There is provided described example only for purpose of explanation, and not for the scope of restriction claims provided herein.
The preparation method of embodiment 1 formula (1) compound
(1) gamboge medicinal material (amount of ethyl acetate is 15 times of gamboge) is extracted by ethyl acetate, extract is separated through silica gel column chromatography, petroleum ether-ethyl acetate (50:1,10:1,5:1,2:1,1:1,0:1) six gradient elutions, flow velocity is 2ml/min, every 30ml collects a flow point, and every 7 parts of elutriants merge, and obtain component A ~ K successively;
(2), after discarding oil component A and B and morellic acid component D and E, analyze through HPLC-PDA-MS deduplication, obtain component I;
(3) component I is separated through silica gel column chromatography again, chloroform-methanol (50:1 ~ 10:1) gradient elution, and flow velocity is 2ml/min, and every 30ml collects a flow point, merges the 11st part to the 16th part elutriant, obtains I-3;
(4) get I-3 through anti-phase RP-18 chromatographic separation, acetonitrile-water (40:60,50:50,60:40,80:20,90:10) gradient elution, flow velocity is 2ml/min, and every 20ml collects a flow point, merges the 5th part to the 9th part elutriant, obtains Arius and divide I-3C;
(5) purified, the 65% acetonitrile isocratic elution of I-3C, obtains formula (1) compound.
The Structural Identification of embodiment 2 formula (1) compound
This compound is Yellow amorphous powder, [α] 20D-288.1 (c0.31, MeOH);
1hand
13cNMR data are in table 1; UV (MeOH) λ
max(log ε) 361,290nm; IR (KBr) ν
max3435,3232,2977,2929,1736,1687,1631,1593,1456,1437,1383,1333,1182,1138,1047,802cm
-1; HR-TOF-MS shows quasi-molecular ion peak m/z687.3132(C
38h
46o
10na, 685.2989).
Through literature search, do not find relevant report, show that this compound is new formula (1) compound, chemical name is 32,33-dihydroxyl morellic acid (32,33-dihydroxyepigambogicacid).
Table 1
The nuclear magnetic data of formula (1) compound (
1h (500MHz, CDCl
3),
13c (100MHz, CDCl
3) NMR)
Embodiment 3
32,33-dihydroxyl morellic acid (formula (1) compound) restraining effect to people's transplantable lung cancer
1. materials and methods
1.1 tested material
32,33-dihydroxyl morellic acid: 10mg/ml, purity 97.22%, lot number 20100121, is prepared by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.
Vinorelbine tartrate injection liquid (Gai Nuo): Jiangsu Haosen Pharmaceutical Co., Ltd, lot number 100301.
Physiological saline: Shandong Lukang Cisen Pharmaceutical Co., Ltd, lot number 1004234202.
Morellic acid: 10mg/ml, purity 98% (detecting through HPLC), lot number 20100819, is prepared by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov.
1.2 transplanted tumor
People lung cancer NCI-H460 Nude Mice, is inoculated in nude mouse by people lung cancer NCI-H460 cell strain subcutaneous and set up.Cell inoculum size is 3 × 10
6, inoculation uses after passing for 3 generations again in nude mice after forming transplanted tumor.
1.3 animal
Female BAl BIc/c nude mouse, age in days 35-40 days, body weight 18-22g, provided by experimentation on animals section of hospital general of Nanjing Military Command.Every treated animal number is negative control group 12, administration group 6.
1.4 test method
The tumor tissue getting growth animated period cuts into 1.5mm
3left and right, aseptically, is inoculated in armpit on the right side of nude mouse subcutaneous.Nude Mice vernier caliper measurement transplanted tumor diameter, treats tumor growth to 100 ~ 300mm
3after by animal random packet.Use the method measuring knurl footpath, dynamically observe the effect of tested drugs against tumor.The pendulous frequency of diameter of tumor is 3 times weekly, and each measurement also needs to claim mouse heavy simultaneously.Formula (1) compound intravenously administrable, dosage divides and is 12mg/kg, 6mg/kg, 3mg/kg, gives three times weekly.Lid promise intravenously administrable 10mg/kg, gives twice, each 0.2ml weekly, and negative control group gives normal saline simultaneously.
1.5 Testing index and method of calculation
(1) gross tumor volume (tumorvolume, TV), calculation formula is:
wherein a, b represent length and width respectively.
(2) relative tumour volume (relativetumorvolume, RTV), calculation formula is:
RTV=TV
t/TV
0
Wherein TV
0for (d during point cage administration
0) gross tumor volume, TV
tgross tumor volume during for measuring each time.
(3) Relative tumor proliferation rate T/C(%), calculation formula is:
T/C（%）=T
RTV/C
RTV×100
T
rTV: treatment group RTV; C
rTV: negative control group RTV.
Test-results is using Relative tumor proliferation rate T/C(%) as the evaluation index of anti-tumor activity.
1.6 statistical method
Experimental data represents with mean value and standard deviation, and statistical method adopts t-inspection.
2. result
The experimental treatment of formula (1) compound to people lung cancer NCI-H460 the results are shown in Table 2.
Formula (1) compound high dose group intravenously administrable has good growth-inhibiting effect to people lung cancer NCI-H460 mice-transplanted tumor, and best T/C (%) is 67.84;
Middle dosage group intravenously administrable 6mg/kg has certain growth-inhibiting effect to people lung cancer NCI-H460 Nude Mice, and best T/C (%) is 69.84;
Low dose group intravenously administrable 3mg/kg has certain growth-inhibiting effect to people lung cancer NCI-H460 mice-transplanted tumor, and best T/C (%) is respectively 72.30;
Lid promise intravenously administrable 10mg/kg has certain growth-inhibiting effect to people lung cancer NCI-H460 Nude Mice, and best T/C (%) is 56.69;
Morellic acid intravenously administrable 12mg/kg has certain growth-inhibiting effect to people lung cancer NCI-H460 Nude Mice, and best T/C (%) is 70.55.
Table 2
Formula (1) compound is to the experimental therapy of people lung cancer NCI-H460 Nude Mice
D1 divides the RTV of cage administration time P administration group to contrast *: P<0.05**:P<0.01 with the blank RTV organized
The extracorporeal anti-tumor function of embodiment 4 the compounds of this invention
1. experiment material
1.1 cell strain
CNE: human nasopharyngeal carcinoma cell line, purchased from Chinese Academy of Sciences's cell bank;
Bcap-37: Breast cancer lines, purchased from Chinese Academy of Sciences's cell bank;
HepG2: human liver cell JEG-3, purchased from Chinese Academy of Sciences's cell bank;
Bxpc-3: human pancreas cancer cell strain, purchased from Chinese Academy of Sciences's cell bank;
Hela-229: human cervical carcinoma cell lines, purchased from Chinese Academy of Sciences's cell bank.
1.2 tested materials and reagent
Formula (1) compound: purity 97.22%, lot number 20100121, is prepared by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences;
0.25% trypsinase-EDTA: GIBCO company of the U.S.;
MTT solution: purchased from this hundred remittances bio tech ltd, Beijing;
PBS damping fluid: Ji Nuo biological medicine technology company limited;
Dimethyl sulfoxide (DMSO) (DMSO): analytical pure.
1.3 experimental technique
1.3.1 Cell culture invitro
Cell Soviet Union of often restoring to norm is placed at 37 DEG C in incubator, cultivates, when Growth of Cells is to exponential phase of growth, go down to posterity with 0.25% trypsinase one DTA digestion method under 5%CO2 and saturated humidity condition.Absorb old nutrient solution in bottle, residual nutrient solution is washed away with PBS, appropriate Digestive system (0.25% trypsinase-EDTA) is added again in bottle, make Digestive system submergence all cells surface, put in 37 DEG C of incubators and hatch (time is depending on different cell), observe under putting microscope, after finding kytoplasm retraction, intercellular substance increase, the complete culture solution added immediately containing serum stops digestion, centrifugal (1000rpm, remove supernatant liquor 5min), count with after nutrient solution re-suspended cell, with cell count 3 × 10
5~ 5 × 10
5cells/mL is seeded in new culturing bottle, and be placed in incubator and cultivate with above-mentioned culture condition, 2 ~ 3d goes down to posterity once.
1.3.2MTT method detects
When Growth of Cells is to exponential phase of growth, digest, centrifugal (1000rpm, 5min) with 0.25% trypsinase one DTA, cell precipitation perfect medium adjusts cell count to be 2 × 10
4~ 3 × 10
4cells/mL, 96 holes are cultivated the every hole of version and are inoculated 190 μ L, at 37 DEG C, 5%CO
2and cultivate under saturated humidity condition, add formula (1) compound after 24h, make cumulative volume be 200 μ L, if 6 multiple holes, control wells adds PBS solution, continues to cultivate 48h, then to add 20 μ L concentration be the MTT of 5mg/mL, is placed in CO
2incubator 37 DEG C is hatched, and discards nutrient solution after 4h, and every hole adds 150 μ LDMSO, and vibration 10min, puts in enzyme micro-plate reader and detect, measure the OD value in each hole, calculates inhibiting rate.Formula (1) compound is to the IC of subject cell strain
50calculate by probit's weighted regression method with SPSS15.0 software.
Inhibiting rate calculation formula:
2. experimental result
Experimental result display (as table 3), formula (1) compound all has restraining effect to each tested tumour cell, and wherein the susceptibility of human hepatocellular carcinoma HepG2 cell strain to medicine is the highest.
Table 3
Formula (1) compound is to the IC of each subject cell strain
50
The preparation of embodiment 5 tablet
Conveniently the working method of tablet, more than mixes, wet granulation, finally adds Magnesium Stearate and mixes and be pressed into tablet, totally 50, every sheet 500mg.
The preparation of embodiment 6 capsule
Conveniently the working method of capsule, more than mixes, wet granulation, filling one-tenth capsule, totally 90, every 300mg.
The preparation of embodiment 7 pill
32,33-dihydroxyl morellic acid 10.0g
Polyethylene glycol 6000 25.0g
32,33-dihydroxyl morellic acid was pulverized 100 mesh sieves, evenly added in the polyethylene glycol 6000 matrix of melting, stir 30 minutes to evenly, with dimethyl silicone oil 100 for refrigerant, 15-4 DEG C of gradient cooling, dripping becomes ball, the agent of centrifugal segregation surface cool, obtains dripping pill 1000 ball.
The preparation of embodiment 8 powder ampoule agent for injection
32,33-dihydroxyl morellic acid 1.0g
Hydrochloric acid is appropriate
N.F,USP MANNITOL 50.0g
Conveniently the operation of freeze-dried powder is carried out, and 32,33-dihydroxyl morellic acid adds 800ml water for injection and dissolves, and adds N.F,USP MANNITOL, is settled to 1000ml, between adjust ph to 5.0-6.5, and Sterile Filtration, lyophilize and get final product.
The preparation of embodiment 9 injection
32,33-dihydroxyl morellic acid is dissolved in dehydrated alcohol, adds 20% polyoxyl castor oil mixing, reduction vaporization removing ethanol, adds appropriate water for injection and is mixed into clear transparent solutions, through filtering with microporous membrane, and coating-dividing sealing, flowing steam sterilization and get final product.
The preparation of embodiment 10 slow releasing tablet
32,33-dihydroxyl morellic acid and polyvidone are dissolved in a small amount of ethanol, reduction vaporization ethanol, 100 mesh sieves crossed by gained solid; Above-mentioned solid and lactose, hypromellose are crossed 60 mesh sieves mix, add 3% HPMC (E5) aqueous solution softwood processed in right amount, cross 20 mesh sieves and granulate, forced air drying.Dry particle crosses the whole grain of 20 mesh sieve, adds the talcum powder of recipe quantity, mixing, compressing tablet and get final product.
In sum, the present invention can draw following specific embodiments:
1. formula (1) compound:
2. a medical composition, it comprises the compound according to claim 1 for the treatment of significant quantity, and at least one is selected from following pharmaceutically acceptable non-active ingredient: pharmaceutically acceptable thinner, pharmaceutically acceptable vehicle and pharmaceutically acceptable supporting agent.
3. the medical composition according to scheme 2, wherein said medical composition is tablet, capsule, powder ampoule agent for injection, injection, pill and slow releasing tablet.
4., according to the purposes of the compound described in scheme 1 to 3 or medical composition, it is for the preparation of the medicine being used for the treatment of tumor disease.
5. the purposes according to scheme 4, described tumor disease is selected from the group of following composition: nasopharyngeal carcinoma, mammary cancer, liver cancer, carcinoma of the pancreas and cervical cancer.
6. the purposes according to scheme 5, described tumor disease is liver cancer.
7. prepare a method for the compound according to scheme 1, it comprises the following steps:
(1) get gamboge medicinal material ethyl acetate to extract, extract is separated through silica gel column chromatography, petroleum ether-ethyl acetate gradient elution, obtains component A ~ K successively;
(2) discard oil component A and B and morellic acid D and component E, analyze through HPLC-PDA-MS deduplication, obtain component I;
(3) component I is separated through silica gel column chromatography again, chloroform-methanol gradient elution, and the flow velocity of elutriant is 1-5ml/min, and every 25 ~ 35ml collects a flow point, merges the 11st part to the 16th part elutriant, obtains I-3;
(4) get I-3 through anti-phase RP-18 chromatographic separation, acetonitrile-water gradient, the flow velocity of elutriant is 1-5ml/min, and every 15 ~ 25ml collects a flow point, merges the 5th part to the 9th part elutriant, obtains Arius and divide I-3C;
(5) purified, the acetonitrile isocratic elution of I-3C, obtains formula (1) compound.
8. the method according to scheme 7, the ratio of wherein said step (1) PetroChina Company Limited. ether-ethyl acetate 50 ~ 0:1 by volume carries out gradient elution.
9. the method according to scheme 8, wherein said petroleum ether-ethyl acetate is divided into six gradients to carry out wash-out, is respectively 50:1,10:1,5:1,2:1,1:1,0:1.
10. the method according to scheme 7, the ratio of chloroform-methanol 50 ~ 10:1 by volume carries out gradient elution in wherein said step (3).
11. methods according to scheme 7, in wherein said step (4), the ratio of acetonitrile-water 40 ~ 90:60 ~ 10 by volume carries out gradient elution.
12. methods as described in scheme 11, wherein acetonitrile-water is divided into five gradients to carry out wash-out, is respectively 40:60,50:50,60:40,80:20,90:10.
13. methods as described in scheme 7, in wherein said step (5), the concentration of acetonitrile is 50% ~ 80%.
14. methods according to scheme 13, the concentration of wherein said acetonitrile is 60% ~ 70%.
15. methods according to scheme 14, the concentration of wherein said acetonitrile is 65%.
16. methods according to scheme 7, in the gradient elution of wherein said step (1), every 30ml collects a flow point, and every 7 parts of elutriants merge.
17. methods according to scheme 7, in wherein said step (1), the flow velocity of the elutriant of gradient elution is 1-5ml/min.
18. methods according to scheme 17, the flow velocity of wherein said elutriant is 2ml/min.
19. methods according to scheme 7, in wherein said step (3), the flow velocity of the elutriant of gradient elution is 2ml/min.
20. methods according to scheme 7, in wherein said step (4), the flow velocity of the elutriant of gradient elution is 2ml/min.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. a method for preparation formula (1) compound, it comprises the following steps:
(1) get gamboge medicinal material ethyl acetate to extract, extract is separated through silica gel column chromatography, petroleum ether-ethyl acetate gradient elution, obtains component A ~ K successively;
(2) discard oil component A and B and morellic acid D and component E, analyze through HPLC-PDA-MS deduplication, obtain component I;
(3) component I is separated through silica gel column chromatography again, chloroform-methanol gradient elution, and the flow velocity of elutriant is 1-5ml/min, and every 25 ~ 35ml collects a flow point, merges the 11st part to the 16th part elutriant, obtains I-3;
(4) get I-3 through anti-phase RP-18 chromatographic separation, acetonitrile-water gradient, the flow velocity of elutriant is 1-5ml/min, and every 15 ~ 25ml collects a flow point, merges the 5th part to the 9th part elutriant, obtains Arius and divide I-3C;
(5) purified, the acetonitrile isocratic elution of I-3C, obtain formula (1) compound, the structural formula of described formula (1) compound is
2. method according to claim 1, the ratio of wherein said step (1) PetroChina Company Limited. ether-ethyl acetate 50 ~ 0:1 by volume carries out gradient elution.
3. method according to claim 2, wherein said petroleum ether-ethyl acetate is divided into six gradients to carry out wash-out, is respectively 50:1,10:1,5:1,2:1,1:1,0:1.
4. method according to claim 1, the ratio of chloroform-methanol 50 ~ 10:1 by volume carries out gradient elution in wherein said step (3).
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| CN103664984A (en) * | 2013-11-28 | 2014-03-26 | 江苏康缘药业股份有限公司 | Antineoplastic compound extracted from gamboge, and preparation method and application thereof |
| CN108959855B (en) * | 2018-04-25 | 2021-05-18 | 上海药明康德新药开发有限公司 | Computer coding method for screening reagent of DNA coding compound library |
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| EP2395007A1 (en) * | 2010-06-11 | 2011-12-14 | Taiwan Sunpan Biotechnology Development Co., Ltd. | Fractionated products obtained from gamboge resin, and medical uses of the same |
| CN102276621A (en) * | 2010-06-11 | 2011-12-14 | 台湾森本生物科技开发股份有限公司 | Compound separated from gamboge resin and derivative thereof and pharmaceutical composition comprising compound and derivative |
| CN103664984A (en) * | 2013-11-28 | 2014-03-26 | 江苏康缘药业股份有限公司 | Antineoplastic compound extracted from gamboge, and preparation method and application thereof |
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| EP2395007A1 (en) * | 2010-06-11 | 2011-12-14 | Taiwan Sunpan Biotechnology Development Co., Ltd. | Fractionated products obtained from gamboge resin, and medical uses of the same |
| CN102276621A (en) * | 2010-06-11 | 2011-12-14 | 台湾森本生物科技开发股份有限公司 | Compound separated from gamboge resin and derivative thereof and pharmaceutical composition comprising compound and derivative |
| CN103664984A (en) * | 2013-11-28 | 2014-03-26 | 江苏康缘药业股份有限公司 | Antineoplastic compound extracted from gamboge, and preparation method and application thereof |
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