CN105198951A - Tetracyclic diterpenoid compound and preparation method as well as application thereof - Google Patents
Tetracyclic diterpenoid compound and preparation method as well as application thereof Download PDFInfo
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Abstract
The invention discloses a tetracyclic diterpenoid compound and a preparation method as well as application thereof. The tetracyclic diterpenoid compound is obtained through extractum extracting, organic solvent leaching, column chromatography on silica gel and high pressure liquid chromatography separation by taking Araliadumetorum Hand.-Mazz roots as a material. The compound has a molecular formula of C20H30O3, is named as dumetorumane B, and has the following structural formula as shown in the description. The preparation method means that the tetranuclear diterpenoid is obtained through extractum extracting, organic solvent leaching, column chromatography on silica gel and high pressure liquid chromatography separation by taking the Araliadumetorum Hand.-Mazz roots as the material. The application is application of the tetranuclear diterpenoid to preparation of drugs for preventing and/or treating tumour and to preparation of drugs for preventing and/or treating human lung adenocarcinoma, human acute promyelocytic leukemia or human breast cancer. The tetranuclear diterpenoid has a remarkable inhibiting effect on cell strains of human lung adenocarcinoma, human acute promyelocytic leukemia or human breast cancer, has good antitumor activity indicatively, and can serve as an antitumor activity component or a lead compound.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, be specifically related to a kind of tetracyclic diterpene compound and preparation method thereof and application.
Background technology
The situation is tense for current global pathogenesis of cancer, and incidence and mortality, in continuing ascendant trend, has become serious harm human health, caused the first foul disease of modern humans's death and chronic disease.The World Health Organization issues report in February, 2014, predicts that global cases of cancer will present rapid growth situation year by year, and by 1,400 ten thousand people of 2012,1,900 ten thousand people of cumulative year after year to 2025 year, will reach 2,400 ten thousand people by 2035.This tissue statistic data also shows, and the annual newly-increased cases of cancer about 3,500,000 of China, about has 2,500,000 people therefore dead.Therefore find effective cancer therapy drug and become the task of top priority.In cancer therapy drug research, the studies and clinical application of chemotherapeutics obtains remarkable progress.As metal complexes platinum complex, organic germanium compounds etc., although have strong cancer resistance, there is toxic side effect, the defects such as poorly water-soluble in the cancer therapy drug of chemosynthesis.In recent years, China and foreign countries researchist turns one's attention to aboundresources, the herbal medicine of pure natural, ethnic drug gradually, ites is desirable to therefrom to find new cancer therapy drug.From herbal medicine, ethnic drug, find a series of natural anti-cancer drugs such as taxol, camptothecine, kind of harringtonine as medicinal materials, vincristine(VCR), podophyllotoxin, Elemenum Emulsion at present, and have many new product listings every year.These natural drugs have all showed good antitumour activity and have been widely used clinically, also day by day prove that screening antineoplastic drugs is a very promising direction from natural product.
Root of Blackfruit Aralia (
araliadumetorumhand.-Mazz) be that (Araliaceae) Aralia fogfruit, claims in Yunnan " ox horn seven " Araliaceae, " white nine bursts of oxen ", " honey oil ginseng ".Among the peoplely to be used as medicine with root, to enter spleen, stomach two warp.Herbal medicine, 2001,32 (9): 847-850 reports, this plant has effect of invigorating the spleen and replenishing QI, eliminating the toxic material out by tonification, anti-inflammatory, for diseases such as lymphadenitis, chronic suppurative osteomyelisis and furuncle carbuncles.This platymiscium chemical composition is mainly containing diterpene, triterpenoid saponin, tonka bean camphor, flavones, lignanoid and sterol.Diterpene-kind compound in this platymiscium has antitumor, anti-inflammatory isoreactivity.The present invention attempts to do further further investigation to the antitumor action of diterpene-kind compound in root of Blackfruit Aralia, to finding cytotoxic activity natural product wherein, provides foundation for screening antitumor drug that is efficient, low toxicity.
Summary of the invention
The first object of the present invention is to provide a kind of tetracyclic diterpene compound; Second object is the preparation method providing described tetracyclic diterpene compound; 3rd object is the application providing described tetracyclic diterpene compound.
The first object of the present invention is achieved in that described tetracyclic diterpene compound is with root of Blackfruit Aralia root for raw material, and obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, this compound molecule formula is C
20h
30o
3, called after dumetorumaneB, has following structural formula:
。
The second object of the present invention is achieved in that with root of Blackfruit Aralia root for raw material, is separated and obtains, be specially through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography:
A, medicinal extract extract: particle root of Blackfruit Aralia root dried and crushed being obtained 0.05 ~ 0.15cm size, concentration expressed in percentage by volume 80 ~ 95% ethanolic soln adding root of Blackfruit Aralia root weight ratio 4 ~ 8 times refluxing extraction 3 ~ 5 times at 65 ~ 74 DEG C, each 1 ~ 3h, united extraction liquid, extracting liquid filtering, concentrating under reduced pressure extracting solution to proportion is 1.1 ~ 1.3 obtain medicinal extract a;
B, organic solvent extraction: the water adding weight ratio 2 ~ 4 times amount in medicinal extract a, extract with the isopyknic sherwood oil of water, ethyl acetate and n-butanol solvent successively, often kind of solvent extraction 4 ~ 6 times, boils off solvent and obtains petroleum ether extract b, acetic acid ethyl ester extract c and n-butyl alcohol extract d respectively;
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 orders, and consumption is extract c weight 6 ~ 8 times amount; With the chloroform-methanol organic solvent gradient elution that volume ratio is 30:1 ~ 0:1, collect gradient eluent, concentrate, through TLC monitoring, merge identical part;
2) by 1) in carry out silicagel column on elutriant e that wash-out obtains with the chloroform-methanol organic solvent of 15:1 proportioning, dress post silica gel is 200 ~ 300 orders, and consumption is elutriant e volume 6 ~ 8 times amount; With the chloroform methanol organic solvent gradient elution of volume ratio 25:1 ~ 5:1, collect gradient eluent, concentrate, detect through TLC, merge identical part;
D, high performance liquid chromatography are separated: by step C 2) in carry out with the chloroform-methanol organic solvent of 20:1 proportioning the elutriant that wash-out obtains and adopt half preparative high-performance liquid chromatographic instrument separation and purification, obtain described tetracyclic diterpene compound dumetorumaneB.
Tetracyclic diterpene compound of the present invention is separated first, is defined as tetracyclic diterpene compound by nucleus magnetic resonance and other spectroscopic technique measuring methods, and characterizes its concrete structure and be:
Compound dumetorumaneB, is white amorphous powder (solvent is chloroform),
=+43.5 ° (
c0.45, CHCl
3); IR (KBr) spectrum is at 3345,2969,1711,1652,1123,1067cm
-1there is absorption, show in molecule, to there is carboxyl, hydroxyl and double bond.HRESI-MS (accompanying drawing 1) shows its quasi-molecular ion peak m/z317.2131 [M-H]
-(calcd.317.2116), determine that its molecular formula is C in conjunction with NMR spectrum
20h
30o
3, degree of unsaturation is 6.
1(accompanying drawing 2, attribution data is in Table for HNMR
1) display molecule in have 3 methyl singlets, be respectively δ
h1.25 (3H, s), 1.18 (3H, s) and 0.81 (3H, s)), there is 1 even Oxymethylene peak (δ
h3.72 (1H, dd, J=11.2,7.2Hz); 3.60 (1H, dd, J=11.2,8.0Hz));
13(accompanying drawing 3 and accompanying drawing 4, attribution data is in Table for CNMR
1) there are 20 carbon in display molecule, wherein there are 6 quaternary carbons, 3 methynes, 8 methylene radical, and 3 methyl.In conjunction with HSQC Correlated Spectroscopy (accompanying drawing 5), and documents data susceptible of proof, this compound and known compound acasianeA structural similitude, be the diterpene-kind compound containing 1 little ring of ternary, distinguished more than being in molecule 1 carbonyl (δ
c182.7) and 1 double bond (δ
c127.8 and 135.5) 1 even Oxymethylene (δ, has been lacked
c63.8) and 2 methyne (δ
c56.3 and 37.1); C-18(carbonyl is confirmed further through HMBC relevant (accompanying drawing 6)) be connected with C-4 position, C-8 and C-9 position is a double bond.Further through HMBC relevant (accompanying drawing 6), COSY relevant (accompanying drawing 7) and ROESY (accompanying drawing 8) and with document (LinA.S., LinC.R., DuY.C., L ü bkenT., ChiangM.Y., ChenI.H., WuC.C., HwangT.L., ChenS.L., YenM.H., ChangF.R., WuY.C.AcasianeAandBandfarnesiraneAandB, diterpenederivativesfromtherootsof
acaciafarnesiana.PlantaMed2009,75:256-261.) contrast confirmation, the structure of compound is finally confirmed, and compound has following structural formula:
Confirm that this compound is new compound by SCIFINDER database retrieval and literature query.
Table 1 the compounds of this invention
1h-and
13c-NMR (CDCl
3) data
The third object of the present invention is achieved in that described tetracyclic diterpene compound is preparing the application prevented and/or treated in tumour medicine.
Described tetracyclic diterpene compound is preparing the application prevented and/or treated in human lung adenocarcinoma, people's acute promyelocytic leukemic or human breast carcinoma medicine.
Tetracyclic diterpene compound of the present invention has carried out cytotoxic activity test, it is active that experimental result demonstrates good suppression human lung adenocarcinoma, people's acute promyelocytic leukemic and Breast cancer lines, can be new compound or lead compound that medical industry provides using value.
Advantage of the present invention:
1, the tetracyclic diterpene compound in the present invention has significant restraining effect to human lung adenocarcinoma, people's acute promyelocytic leukemic or Breast cancer lines, points out it to have good antitumour activity, can as anticancer active constituent or lead compound.
2, the tetracyclic diterpene compound in the present invention can be naturally occurring organic compound, and raw material sources are extensive, and compound preparation manipulation flow process is simple, and the compound purity obtained is high, and suitability for industrialized production subsequently easily realizes.
Accompanying drawing explanation
Fig. 1 is the compounds of this invention high resolution mass spectrum (HRESI-MS);
Fig. 2 be the compounds of this invention proton nmr spectra (
1hNMR);
Fig. 3 be the compounds of this invention carbon-13 nmr spectra (
13cNMR);
Fig. 4 is the DEPT spectrum of the compounds of this invention;
Fig. 5 is the HSQC Correlated Spectroscopy of the compounds of this invention;
Fig. 6 is the HMBC Correlated Spectroscopy of the compounds of this invention;
Fig. 7 is the COSY Correlated Spectroscopy of the compounds of this invention;
Fig. 8 is the ROESY Correlated Spectroscopy of the compounds of this invention.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or improvement, all fall into protection scope of the present invention.
Tetracyclic diterpene compound of the present invention is with root of Blackfruit Aralia root for raw material, and obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, this compound molecule formula is C
20h
30o
3, called after dumetorumaneB, has following structural formula:
。
The preparation method of tetracyclic diterpene compound of the present invention is with root of Blackfruit Aralia root for raw material, is separated and obtains, be specially through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography:
A, medicinal extract extract: particle root of Blackfruit Aralia root dried and crushed being obtained 0.05 ~ 0.15cm size, concentration expressed in percentage by volume 80 ~ 95% ethanolic soln adding root of Blackfruit Aralia root weight ratio 4 ~ 8 times refluxing extraction 3 ~ 5 times at 65 ~ 74 DEG C, each 1 ~ 3h, united extraction liquid, extracting liquid filtering, concentrating under reduced pressure extracting solution to proportion is 1.1 ~ 1.3 obtain medicinal extract a;
B, organic solvent extraction: the water adding weight ratio 2 ~ 4 times amount in medicinal extract a, extract with the isopyknic sherwood oil of water, ethyl acetate and n-butanol solvent successively, often kind of solvent extraction 4 ~ 6 times, boils off solvent and obtains petroleum ether extract b, acetic acid ethyl ester extract c and n-butyl alcohol extract d respectively;
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 orders, and consumption is extract c weight 6 ~ 8 times amount; With the chloroform-methanol organic solvent gradient elution that volume ratio is 30:1 ~ 0:1, collect gradient eluent, concentrate, through TLC monitoring, merge identical part;
2) by 1) in carry out silicagel column on elutriant e that wash-out obtains with the chloroform-methanol organic solvent of 15:1 proportioning, dress post silica gel is 200 ~ 300 orders, and consumption is elutriant e volume 6 ~ 8 times amount; With the chloroform methanol organic solvent gradient elution of volume ratio 25:1 ~ 5:1, collect gradient eluent, concentrate, detect through TLC, merge identical part;
D, high performance liquid chromatography are separated: by step C 2) in carry out with the chloroform-methanol organic solvent of 20:1 proportioning the elutriant that wash-out obtains and adopt half preparative high-performance liquid chromatographic instrument separation and purification, obtain described tetracyclic diterpene compound dumetorumaneB.
Filtration described in step A is filtered through the filter paper of 80 ~ 120 μm.
Concentrating under reduced pressure described in step A carries out concentrating under reduced pressure with Rotary Evaporators under the temperature condition below 50 DEG C.
The solvent that boils off described in step B adopts Rotary Evaporators to boil off solvent.
Step C 1) described in the volume proportion of chloroform-methanol organic solvent be 30:1,25:1,20:1,15:1,10:1,5:1,2:1 and 0:1.
Step C 2) described in the volume proportion of chloroform-methanol organic solvent be 25:1,20:1,15:1,10:1,8:1 and 5:1.
Employing half preparative high-performance liquid chromatographic instrument separation and purification described in D step, be specially step C 2) in carry out elutriant that wash-out obtains through half preparative high-performance liquid chromatographic instrument separation and purification with the chloroform-methanol organic solvent of 20:1 proportioning, take volume proportion as the acetonitrile-water of 60:40 be moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, obtains described tetracyclic diterpene compound dumetorumaneB;
The application being applied as described tetracyclic diterpene compound and preventing and/or treating in tumour medicine in preparation of the present invention.
The application being applied as described tetracyclic diterpene compound and preventing and/or treating in human lung adenocarcinoma, people's acute promyelocytic leukemic or human breast carcinoma medicine in preparation of the present invention.
With specific embodiment, the present invention will be further described below:
The preparation of embodiment 1 compound
Material source: root of Blackfruit Aralia is adopted in Kunming, Yunnan, is accredited as through national medicine institute of Yunnan Institute for nationalities associate professor Yang Qingsong
araliadumetorumhand.-Mazz, Saving specimen is in national medicine institute of Yunnan Institute for nationalities specimen museum.
Adopt following steps:
A. pulverize: particle 10Kg root of Blackfruit Aralia root dried and crushed being become particle diameter 0.1cm size, obtains root of Blackfruit Aralia powder, for subsequent use;
B. refluxing extraction: by root of Blackfruit Aralia powder obtained in step a refluxing extraction 4 times at the temperature of 65 DEG C, each 2 hours, each extraction using alcohol by 60Kg80% concentration, merged ethanol extract, for subsequent use;
C, concentrated: the ethanol extract obtained by step b filters through 80-120 urn filter and carry out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1380g, for subsequent use;
D, extraction: 1380g medicinal extract is suspended in 4500ml water, extract with 4500ml sherwood oil, 4500ml ethyl acetate and 4500ml propyl carbinol successively, often kind of solvent extraction 5 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 390g, 112g, 405g respectively.
E. be separated: by the acetic acid ethyl ester extract that obtains in steps d through 100-200 order silica gel column chromatography, obtain Fr.1-Fr with the chloroform-methanol gradient elution of volume ratio 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1
.8 totally 8 components, Fr.1 is 8.8g, Fr.2 is 8.1g, Fr.3 is 3.2g, Fr.4 be 15.7g, Fr.5 is 34.3g, Fr.6 is 6.2g, Fr.7 be 5.1g, Fr.8 is 21.5g, Fr.4 is through 200-300 order silica gel column chromatography, and the chloroform with volume ratio 25:1,20:1,15:1,10:1,8:1,5:1: methanol elution gradient, obtains component Fr.4A-Fr.4F totally 6 components, Fr.4A is 1.0g, Fr.4B be 1.9g, Fr.4C is 0.6g, Fr.4D is 3.6g, Fr.4E be 1.5g, Fr.4F is 3.1g.Fr.4B is through half preparative high-performance liquid chromatographic instrument, using acetonitrile-water (volume ratio 60:40) as moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, and UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, prepares white amorphous powder type target compound dumetorumaneB(12mg).
The preparation of embodiment 2 compound
Adopt following steps:
A. pulverize: particle 10Kg root of Blackfruit Aralia root dried and crushed being become particle diameter 0.1cm size, obtains root of Blackfruit Aralia powder, for subsequent use;
B. refluxing extraction: by root of Blackfruit Aralia powder obtained in step a refluxing extraction 3 times at the temperature of 67 DEG C, each 2.5 hours, each extraction using alcohol by 60Kg84% concentration, merged ethanol extract, for subsequent use;
C, concentrated: the ethanol extract obtained by step b filters through 80-120 urn filter and carry out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1340g, for subsequent use;
D, extraction: 1340g medicinal extract is suspended in 4000ml water, extract with 4000ml sherwood oil, 4000ml ethyl acetate and 4000ml propyl carbinol successively, often kind of solvent extraction 4 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 372g, 103g, 396g respectively.
E. be separated: by the acetic acid ethyl ester extract that obtains in steps d through 100-200 order silica gel column chromatography, obtain Fr.1-Fr with the chloroform-methanol gradient elution of volume ratio 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1
.8 totally 8 components, Fr.1 is 7.6g, Fr.2 is 6.8g, Fr.3 is 3.0g, Fr.4 be 14.5g, Fr.5 is 32.4g, Fr.6 is 5.3g, Fr.7 be 4.8g, Fr.8 is 18.7g, Fr.4 is through 200-300 order silica gel column chromatography, and the chloroform with volume ratio 25:1,20:1,15:1,10:1,8:1,5:1: methanol elution gradient, obtains component Fr.4A-Fr.4F totally 6 components, Fr.4A is 0.9g, Fr.4B be 1.7g, Fr.4C is 0.5g, Fr.4D is 3.4g, Fr.4E be 1.4g, Fr.4F is 3.0g.Fr.4B is through half preparative high-performance liquid chromatographic instrument, using acetonitrile-water (volume ratio 60:40) as moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, and UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, prepares white amorphous powder type target compound dumetorumaneB(14mg).
The preparation of embodiment 3 compound
Adopt following steps:
A. pulverize: particle 10Kg root of Blackfruit Aralia root dried and crushed being become particle diameter 0.1cm size, obtains root of Blackfruit Aralia powder, for subsequent use;
B. refluxing extraction: by root of Blackfruit Aralia powder obtained in step a refluxing extraction 5 times at the temperature of 69 DEG C, each 2 hours, each extraction using alcohol by 60Kg88% concentration, merged ethanol extract, for subsequent use;
C, concentrated: the ethanol extract obtained by step b filters through 80-120 urn filter and carry out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1410g, for subsequent use;
D, extraction: 1410g medicinal extract is suspended in 4500ml water, extract with 4500ml sherwood oil, 4500ml ethyl acetate and 4500ml propyl carbinol successively, often kind of solvent extraction 5 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 395g, 117g, 411g respectively.
E. be separated: by the acetic acid ethyl ester extract that obtains in steps d through 100-200 order silica gel column chromatography, obtain Fr.1-Fr with the chloroform-methanol gradient elution of volume ratio 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1
.8 totally 8 components, Fr.1 is 9.1g, Fr.2 is 8.4g, Fr.3 is 3.4g, Fr.4 be 15.9g, Fr.5 is 35.6g, Fr.6 is 6.4g, Fr.7 be 5.3g, Fr.8 is 22.6g, Fr.4 is through 200-300 order silica gel column chromatography, and the chloroform with volume ratio 25:1,20:1,15:1,10:1,8:1,5:1: methanol elution gradient, obtains component Fr.4A-Fr.4F totally 6 components, Fr.4A is 1.1g, Fr.4B be 2.1g, Fr.4C is 0.7g, Fr.4D is 3.7g, Fr.4E be 1.6g, Fr.4F is 3.9g.Fr.4B is through half preparative high-performance liquid chromatographic instrument, using acetonitrile-water (volume ratio 60:40) as moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, and UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, prepares white amorphous powder type target compound dumetorumaneB(15mg).
The preparation of embodiment 4 compound
Adopt following steps:
A. pulverize: particle 10Kg root of Blackfruit Aralia root dried and crushed being become particle diameter 0.1cm size, obtains root of Blackfruit Aralia powder, for subsequent use;
B. refluxing extraction: by root of Blackfruit Aralia powder obtained in step a refluxing extraction 4 times at the temperature of 71 DEG C, each 2 hours, each extraction using alcohol by 60Kg92% concentration, merged ethanol extract, for subsequent use;
C, concentrated: the ethanol extract obtained by step b filters through 80-120 urn filter and carry out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1480g, for subsequent use;
D, extraction: 1480g medicinal extract is suspended in 5000ml water, extract with 5000ml sherwood oil, 5000ml ethyl acetate and 5000ml propyl carbinol successively, often kind of solvent extraction 6 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 387g, 111g, 408g respectively.
E. be separated: by the acetic acid ethyl ester extract that obtains in steps d through 100-200 order silica gel column chromatography, obtain Fr.1-Fr with the chloroform-methanol gradient elution of volume ratio 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1
.8 totally 8 components, Fr.1 is 8.2g, Fr.2 is 7.5g, Fr.3 is 3.0g, Fr.4 be 16.2g, Fr.5 is 34.4g, Fr.6 is 6.1g, Fr.7 be 5.0g, Fr.8 is 22.3g, Fr.4 is through 200-300 order silica gel column chromatography, and the chloroform with volume ratio 25:1,20:1,15:1,10:1,8:1,5:1: methanol elution gradient, obtains component Fr.4A-Fr.4F totally 6 components, Fr.4A is 1.1g, Fr.4B be 2.2g, Fr.4C is 0.7g, Fr.4D is 3.9g, Fr.4E be 1.8g, Fr.4F is 3.6g.Fr.4B is through half preparative high-performance liquid chromatographic instrument, using acetonitrile-water (volume ratio 60:40) as moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, and UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, prepares white amorphous powder type target compound dumetorumaneB(17mg).
The preparation of embodiment 5 compound
Adopt following steps:
A. pulverize: particle 10Kg root of Blackfruit Aralia root dried and crushed being become particle diameter 0.1cm size, obtains root of Blackfruit Aralia powder, for subsequent use;
B. refluxing extraction: by root of Blackfruit Aralia powder obtained in step a refluxing extraction 5 times at the temperature of 74 DEG C, each 2 hours, each extraction using alcohol by 60Kg95% concentration, merged ethanol extract, for subsequent use;
C, concentrated: the ethanol extract obtained by step b filters through 80-120 urn filter and carry out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1520g, for subsequent use;
D, extraction: 1520g medicinal extract is suspended in 4500ml water, extract with 4500ml sherwood oil, 4500ml ethyl acetate and 4500ml propyl carbinol successively, often kind of solvent extraction 6 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 420g, 130g, 450g respectively.
E. be separated: by the acetic acid ethyl ester extract that obtains in steps d through 100-200 order silica gel column chromatography, obtain Fr.1-Fr with the chloroform-methanol gradient elution of volume ratio 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1
.8 totally 8 components, Fr.1 is 10.5g, Fr.2 is 8.8g, Fr.3 is 3.5g, Fr.4 be 17.0g, Fr.5 is 38.5g, Fr.6 is 7.0g, Fr.7 be 5.6g, Fr.8 is 25.3g, Fr.4 is through 200-300 order silica gel column chromatography, and the chloroform with volume ratio 25:1,20:1,15:1,10:1,8:1,5:1: methanol elution gradient, obtains component Fr.4A-Fr.4F totally 6 components, Fr.4A is 1.2g, Fr.4B be 2.4g, Fr.4C is 0.8g, Fr.4D is 4.1g, Fr.4E be 1.9g, Fr.4F is 3.8g.Fr.4B is through half preparative high-performance liquid chromatographic instrument, using acetonitrile-water (volume ratio 60:40) as moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, and UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, prepares white amorphous powder type target compound dumetorumaneB(19mg).
The Structural Identification of embodiment 6 the compounds of this invention
Compound dumetorumaneB(root of Blackfruit Aralia diterpenoid acid B prepared by Example 1), be white amorphous powder (solvent is chloroform); Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique qualification structure.
=+43.5 ° (
c0.45, CHCl
3); IR (KBr) spectrum is at 3345,2969,1711,1652,1123,1067cm
-1there is absorption, show in molecule, to there is carboxyl, hydroxyl and double bond.HRESI-MS (accompanying drawing 1) shows its quasi-molecular ion peak m/z317.2131 [M-H]
-(calcd.317.2116), determine that its molecular formula is C in conjunction with NMR spectrum
20h
30o
3, degree of unsaturation is 6.
1(accompanying drawing 2, attribution data is in Table for HNMR
1) display molecule in have 3 methyl singlets, be respectively δ
h1.25 (3H, s), 1.18 (3H, s) and 0.81 (3H, s)), there is 1 even Oxymethylene peak (δ
h3.72 (1H, dd, J=11.2,7.2Hz); 3.60 (1H, dd, J=11.2,8.0Hz));
13(accompanying drawing 3 and accompanying drawing 4, attribution data is in Table for CNMR
1) there are 20 carbon in display molecule, wherein there are 6 quaternary carbons, 3 methynes, 8 methylene radical, and 3 methyl.In conjunction with HSQC Correlated Spectroscopy (accompanying drawing 5), and documents data susceptible of proof, this compound and known compound acasianeA structural similitude, be the diterpene-kind compound containing 1 little ring of ternary, distinguished more than being in molecule 1 carbonyl (δ
c182.7) and 1 double bond (δ
c127.8 and 135.5) 1 even Oxymethylene (δ, has been lacked
c63.8) and 2 methyne (δ
c56.3 and 37.1); C-18(carbonyl is confirmed further through HMBC relevant (accompanying drawing 6)) be connected with C-4 position, C-8 and C-9 position is a double bond.Further through HMBC relevant (accompanying drawing 6), COSY relevant (accompanying drawing 7) and ROESY (accompanying drawing 8) and with document (LinA.S., LinC.R., DuY.C., L ü bkenT., ChiangM.Y., ChenI.H., WuC.C., HwangT.L., ChenS.L., YenM.H., ChangF.R., WuY.C.AcasianeAandBandfarnesiraneAandB, diterpenederivativesfromtherootsof
acaciafarnesiana.PlantaMed2009,75:256-261.) contrast confirmation, the structure of compound is finally confirmed, called after dumetorumaneB.
Embodiment 7
Compound prepared by Example 2 is yellow jelly; Carry out structure determination by the method in embodiment 6, result is: its structure is with embodiment 6, and molecular formula is C
20h
30o
3.Confirm that compound prepared by embodiment 2 is described tetracyclic diterpene compound dumetorumaneB.
Embodiment 8
Compound prepared by Example 3 is yellow jelly; Carry out structure determination by the method in embodiment 6, result is: its structure is with embodiment 6, and molecular formula is C
20h
30o
3.Confirm that compound prepared by embodiment 3 is described tetracyclic diterpene compound dumetorumaneB.
Embodiment 9
Compound prepared by Example 4 is yellow jelly; Carry out structure determination by the method in embodiment 6, result is: its structure is with embodiment 6, and molecular formula is C
20h
30o
3.Confirm that compound prepared by embodiment 4 is described tetracyclic diterpene compound dumetorumaneB.
Embodiment 10
Compound prepared by Example 5 is yellow jelly; Carry out structure determination by the method in embodiment 6, result is: its structure is with embodiment 6, and molecular formula is C
20h
30o
3.Confirm that compound prepared by embodiment 5 is described tetracyclic diterpene compound dumetorumaneB.
Embodiment 11 the compounds of this invention anti-tumor activity detects
Arbitrary tetracyclic diterpene compound prepared by Example 1 ~ 5 carries out anti-tumor activity detection experiment, and test situation is as follows:
Improvement mtt assay is adopted to evaluate tetracyclic diterpene compound to the increment restraining effect of tumour cell;
Get tumor cell suspension for subsequent use and be inoculated in 96 well culture plates, 90 μ L/ holes, add the given the test agent of different concns after continuing to cultivate 24h, 10 μ L/ holes, make its final concentration be 100,10,1,0.1,0.01 μ g/mL, and each concentration all establishes 3 multiple holes.Cis-platinum (DDP) is positive control, and equal-volume RPMI1640 substratum is negative control.When setting compound 100,10 μ g/mL, corresponding DMSO concentration is as Vehicle controls simultaneously, to eliminate the impact of DMSO cell growth.Continue after dosing to be placed in 37 DEG C, 5%CO
272h cultivated by incubator, and every hole adds MTT(5mg/mL) 20 μ L, continue to cultivate 4h, every hole adds three liquid [10%SDS-5% isopropylcarbinol-0.012mol/LHCl(W/V/V)] 100 μ L Rong Xie formazans.Under 570nm wavelength, the OD value in each hole is measured by microplate reader.
By following formulae discovery cell proliferation inhibiting rate:
Inhibiting rate (%)=(OD value control wells-OD value dosing holes)/OD value control wells × 100%
Data statistic analysis: adopt MicrosoftExcel2003 to calculate inhibiting rate, adopt logit method, SPSS11.5 software package calculation of half inhibitory concentration IC
50.
Calculation of half inhibitory concentration IC
50, to the half-inhibition concentration (IC of human lung adenocarcinoma cell line (A549), human acute myeloid leukaemia strain (NB4) and Breast cancer lines (MCF7)
50) measurement result is respectively 6.7 ± 0.5 μMs, 4.2 ± 0.3 μMs, 5.3 ± 0.6 μMs, shows that compound of the present invention has good anti-tumor activity.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, the equivalent replacement of all any amendments done within the spirit and principles in the present invention and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a tetracyclic diterpene compound, is characterized in that described tetracyclic diterpene compound is with root of Blackfruit Aralia root for raw material, and obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, this compound molecule formula is C
20h
30o
3, called after dumetorumaneB, has following structural formula:
。
2. a preparation method for tetracyclic diterpene compound according to claim 1, it is characterized in that with root of Blackfruit Aralia root for raw material, is separated and obtains, be specially through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography:
A, medicinal extract extract: particle root of Blackfruit Aralia root dried and crushed being obtained 0.05 ~ 0.15cm size, concentration expressed in percentage by volume 80 ~ 95% ethanolic soln adding root of Blackfruit Aralia root weight ratio 4 ~ 8 times refluxing extraction 3 ~ 5 times at 65 ~ 74 DEG C, each 1 ~ 3h, united extraction liquid, extracting liquid filtering, concentrating under reduced pressure extracting solution to proportion is 1.1 ~ 1.3 obtain medicinal extract a;
B, organic solvent extraction: the water adding weight ratio 2 ~ 4 times amount in medicinal extract a, extract with the isopyknic sherwood oil of water, ethyl acetate and n-butanol solvent successively, often kind of solvent extraction 4 ~ 6 times, boils off solvent and obtains petroleum ether extract b, acetic acid ethyl ester extract c and n-butyl alcohol extract d respectively;
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 orders, and consumption is extract c weight 6 ~ 8 times amount; With the chloroform-methanol organic solvent gradient elution that volume ratio is 30:1 ~ 0:1, collect gradient eluent, concentrate, through TLC monitoring, merge identical part;
2) by 1) in carry out silicagel column on elutriant e that wash-out obtains with the chloroform-methanol organic solvent of 15:1 proportioning, dress post silica gel is 200 ~ 300 orders, and consumption is elutriant e volume 6 ~ 8 times amount; With the chloroform methanol organic solvent gradient elution of volume ratio 25:1 ~ 5:1, collect gradient eluent, concentrate, detect through TLC, merge identical part;
D, high performance liquid chromatography are separated: by step C 2) in carry out with the chloroform-methanol organic solvent of 20:1 proportioning the elutriant that wash-out obtains and adopt half preparative high-performance liquid chromatographic instrument separation and purification, obtain described tetracyclic diterpene compound dumetorumaneB.
3. the preparation method of tetracyclic diterpene compound according to claim 2, the filtration that it is characterized in that described in step A is filtered through the filter paper of 80 ~ 120 μm.
4. the preparation method of tetracyclic diterpene compound according to claim 2, the concentrating under reduced pressure that it is characterized in that described in step A carries out concentrating under reduced pressure with Rotary Evaporators under the temperature condition below 50 DEG C.
5. the preparation method of tetracyclic diterpene compound according to claim 2, is characterized in that the solvent that boils off described in step B adopts Rotary Evaporators to boil off solvent.
6. the preparation method of tetracyclic diterpene compound according to claim 2, is characterized in that step C 1) described in the volume proportion of chloroform-methanol organic solvent be 30:1,25:1,20:1,15:1,10:1,5:1,2:1 and 0:1.
7. the preparation method of tetracyclic diterpene compound according to claim 2, is characterized in that step C 2) described in the volume proportion of chloroform-methanol organic solvent be 25:1,20:1,15:1,10:1,8:1 and 5:1.
8. the preparation method of tetracyclic diterpene compound according to claim 2, it is characterized in that the employing half preparative high-performance liquid chromatographic instrument separation and purification described in D step, take volume proportion as the acetonitrile-water of 60:40 be moving phase, flow velocity 2.5ml/min, 250 × 10mm, the C18 of 5 μm is stationary phase, UV-detector determined wavelength is 210nm, each sample introduction 50 μ L, collect the chromatographic peak of 35 ~ 37min, repeatedly cumulative rear evaporate to dryness, obtains described tetracyclic diterpene compound dumetorumaneB.
9. a tetracyclic diterpene compound according to claim 1 is preparing the application prevented and/or treated in tumour medicine.
10. a tetracyclic diterpene compound according to claim 1 is preparing the application prevented and/or treated in human lung adenocarcinoma, people's acute promyelocytic leukemic or human breast carcinoma medicine.
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| CN105801593A (en) * | 2016-03-31 | 2016-07-27 | 上海同田生物技术股份有限公司 | Decursitin preparing method |
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| CN108530281A (en) * | 2018-06-11 | 2018-09-14 | 云南大学 | A kind of isopimarane type forskolin, medical composition and its use |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105801593A (en) * | 2016-03-31 | 2016-07-27 | 上海同田生物技术股份有限公司 | Decursitin preparing method |
| CN106431998A (en) * | 2016-09-27 | 2017-02-22 | 中国林业科学研究院林产化学工业研究所 | N-[4-(isopimaric acid acylamino)phenyl]arylsulfonamide compounds and preparation method and anticancer activity application thereof |
| CN106431998B (en) * | 2016-09-27 | 2017-12-19 | 中国林业科学研究院林产化学工业研究所 | N [4 (different Korean pine amide groups) phenyl] aromatic sulfuryl amine class compound and preparation method thereof and active anticancer application |
| CN108530281A (en) * | 2018-06-11 | 2018-09-14 | 云南大学 | A kind of isopimarane type forskolin, medical composition and its use |
| CN108530281B (en) * | 2018-06-11 | 2021-02-02 | 云南大学 | Isopimarane diterpene derivative, pharmaceutical composition and application thereof |
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