CN105198951B - 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 is and in particular to a kind of tetracyclic diterpene compound and its system
Preparation Method and application.
Background technology
The situation is tense for global pathogenesis of cancer at present, and incidence and mortality is in continue ascendant trend, has become as serious danger
Evil human health, the first big foul disease leading to modern humans' death and pertinacious disease.World Health Organization (WHO) issued report in 2014 2 months
Accuse, the global cases of cancer of prediction will assume swift and violent growing trend year by year, and by 14,000,000 people of 2012, cumulative year after year was to 2025
19,000,000 people, be up to 24,000,000 people by 2035.This tissue statistical data also shows, China increases cases of cancer newly about every year
3500000, there are about 2,500,000 people therefore dead.Therefore finding effective cancer therapy drug has become the task of top priority.In cancer therapy drug research
In, the studies and clinical application of chemotherapeutics is achieved with remarkable progress.The cancer therapy drug of chemosynthesis such as metal complex platinum network
Compound, organic germanium compounds etc. are although have strong cancer resistance, but there are toxic and side effects, the defect such as poorly water-soluble.In recent years, in
Outer research worker gradually turns one's attention to aboundresources, the Chinese herbal medicine of pure natural, ethnic drug it is desirable to therefrom find new anticarcinogen
Thing.It is found that paclitaxel, camptothecine, kind of harringtonine as medicinal materials, vincristine, podophyllotoxin, olive at present from Chinese herbal medicine, ethnic drug
A series of natural anti-cancer drugs such as fragrant alkene breast, and have many new product listings every year.These natural drugs are all demonstrated by well
Active anticancer and be clinically widely used, also increasingly prove that antitumor medicine screening is one from natural product
Individual very promising direction.
Radix Araliae melanocarpae (aralia dumetorumHand.-mazz it is) that Araliaceae (araliaceae) wood belongs to
Plant, claims " NIUJIAOQI " in Yunnan, " white JIUGUNIU ", " honey oil ginseng ".Among the people be used as medicine with root, enter spleen, stomach two warp.Chinese herbal medicine,
2001,32 (9): 847-850 reports, this plant has functions that invigorating the spleen and replenishing QI, eliminating the toxic material out by tonification, antiinflammatory, for lymphadenitises, chronic
The disease such as suppurative osteomyelitiss and furuncle carbuncle.This platymiscium chemical composition mainly contains diterpene, triterpene saponin, coumarin, flavone, lignanoid
And sterol.Diterpene-kind compound in this platymiscium has antitumor, antiinflammatory isoreactivity.The present invention attempts in Radix Araliae melanocarpae two
The antitumor action of terpenoid does to be furtherd investigate further, to finding cytotoxic activity natural product therein, for sieve
Choosing is efficient, the antitumor drug of low toxicity provides foundation.
Content of the invention
The first object of the present invention is to provide a kind of tetracyclic diterpene compound；Second purpose is to provide described Fourth Ring two
The preparation method of terpenoid；3rd purpose is to provide the application of described tetracyclic diterpene compound.
The first object of the present invention is achieved in that described tetracyclic diterpene compound is to be with Radix Araliae melanocarpae root
Raw material, obtains through extractum extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, this compound molecule formula
For c20h30o3, it is named as dumetorumane b, there is following structural formula:
.
The second object of the present invention is achieved in that with Radix Araliae melanocarpae root as raw material, through extractum extraction, You Jirong
Agent extraction, silica gel column chromatography, high pressure liquid chromatography separate and obtain, particularly as follows:
A, extractum extract: Radix Araliae melanocarpae root dried and crushed is obtained the granule of 0.05 ~ 0.15cm size, add black
Concentration expressed in percentage by volume 80 ~ 95% ethanol solution reflux, extract, 3 ~ 5 times at 65 ~ 74 DEG C than 4 ~ 8 times for the fruit Radix Angelicae Gigatiss root weight, often
Secondary 1 ~ 3h, united extraction liquid, extracting solution filters, and concentrating under reduced pressure extracting solution to proportion obtains extractum a for 1.1 ~ 1.3；
B, organic solvent extraction: in extractum a add weight than 2 ~ 4 times amount water, successively with petroleum ether isopyknic with water,
Ethyl acetate and n-butanol solvent are extracted, every kind of solvent extraction 4 ~ 6 times, boil off solvent respectively obtain petroleum ether extract b,
Acetic acid ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 mesh, and consumption is extract c weight 6 ~ 8
Times amount；The chloroform-methanol organic solvent gradient elution being 30:1 ~ 0:1 with volume ratio, collects gradient eluent, concentration, through tlc
Monitoring, merges identical part；
2) by 1) in silicagel column, dress on the eluent e that afforded with the chloroform-methanol organic solvent of 15:1 proportioning
Post silica gel is 200 ~ 300 mesh, and consumption is eluent e volume 6 ~ 8 times amount；Chloroform methanol organic solvent with volume ratio 25:1 ~ 5:1
Gradient elution, collects gradient eluent, concentration, through tlc detection, merges identical part；
D, high performance liquid chromatography separation: by step c 2) in eluting carried out with the chloroform-methanol organic solvent of 20:1 proportioning obtain
To eluent isolated and purified using half preparative high-performance liquid chromatographic instrument, obtain final product described tetracyclic diterpene compound
dumetorumane b.
Tetracyclic diterpene compound of the present invention is separated first, is surveyed by nuclear magnetic resonance, NMR and other spectroscopic techniques
The method of determining is defined as tetracyclic diterpene compound, and characterizes its concrete structure and be:
Compound dumetorumane b, is white amorphous powder (solvent is chloroform),= +43.5° (c
0.45, chcl3);Ir (kbr) composes in 3345,2969,1711,1652,1123,1067 cm-1There is absorption, show
There is carboxyl, hydroxyl and double bond in molecule.Hresi-ms (accompanying drawing 1) shows its quasi-molecular ion peak m/z 317.2131 [m-
h]-(calcd. 317.2116), composes in conjunction with nmr and determines that its molecular formula is c20h30o3, degree of unsaturation is 6.1H nmr (accompanying drawing
2, attribution data is shown in Table 1) show in molecule there is 3 methyl singlets, respectively δh1.25 (3h, s), 1.18 (3h, s)
And 0.81 (3h, s)), there is 1 company's Oxymethylene peak (δh3.72 (1h,dd, j = 11.2, 7.2 hz)；3.60
(1h, dd, j=11.2,8.0 hz));1320 are had in c nmr (accompanying drawing 3 and accompanying drawing 4, attribution data is shown in Table 1) display molecule
Individual carbon, wherein has 6 quaternary carbons, 3 methines, 8 methylene, and 3 methyl.In conjunction with hsqc Correlated Spectroscopy (accompanying drawing 5), and contrast
Data in literature susceptible of proof, this compound is similar to known compound acasiane a structure, is the diterpene containing the little ring of 1 ternary
Class compound, how difference has been in molecule 1 carbonyl (δc182.7) and 1 double bond (δc127.8 and 135.5), lacked 1
Even Oxymethylene (δc63.8) and 2 methine (δc56.3 and 37.1)；Confirm c-18 through hmbc correlation (accompanying drawing 6) further
(carbonyl) is connected with c-4 position, c-8 and c-9 position is a double bond.Further through hmbc correlation (accompanying drawing 6), cosy correlation (accompanying drawing 7)
With roesy (accompanying drawing 8) and with document (lin a. s., lin c. r., du y. c., l ü bken t., chiang m.
y., chen i. h., wu c. c., hwang t. l., chen s. l., yen m. h., chang f. r., wu
y. c. acasiane a and b and farnesirane a and b, diterpene derivatives from
the roots ofacacia farnesiana. planta med 2009,75:256-261.) contrast confirmation, making
The structure of compound finally gives confirmation, and compound has a following structural formula:
Confirm that this compound is noval chemical compound by scifinder database retrieval and literature query.
Table 1 the compounds of this invention1H- and13c-nmr (cdcl3) data
The third object of the present invention is achieved in that described tetracyclic diterpene compound in preparation prevention and/or is controlled
Treat the application in tumour medicine.
Described tetracyclic diterpene compound is in preparation prevention and/or treatment human lung adenocarcinoma, people's acute promyelocytic leukemic
Or the application in human breast carcinoma medicine.
Tetracyclic diterpene compound of the present invention has carried out cytotoxic activity test, and experimental result shows good
Suppression human lung adenocarcinoma, people's acute promyelocytic leukemic and Breast cancer lines activity, can be provided with application valency for medical industry
The noval chemical compound of value or lead compound.
Advantages of the present invention:
1st, the tetracyclic diterpene compound in the present invention is thin to human lung adenocarcinoma, people's acute promyelocytic leukemic or human breast carcinoma
Born of the same parents' strain has significant inhibitory action, points out it to have good active anticancer, can be used as anticancer active constituent or guide's chemical combination
Thing.
2nd, the tetracyclic diterpene compound in the present invention can be naturally occurring organic compound, and raw material sources are extensive, and
And compound preparation manipulation flow process is simple, the compound purity being obtained is high, and subsequent industrialized production is easily realized.
Brief description
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 (1h nmr) ；
Fig. 3 be the compounds of this invention carbon-13 nmr spectra (13c nmr) ；
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.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but never in any form to the present invention in addition
Limit, based on present invention teach that any conversion of being made or improvement, each fall within protection scope of the present invention.
Tetracyclic diterpene compound of the present invention, is with Radix Araliae melanocarpae root as raw material, through extractum extraction, You Jirong
Agent extraction, silica gel column chromatography, high pressure liquid chromatography separation obtain, and this compound molecule formula is c20h30o3, it is named as
Dumetorumane b, has a following structural formula:
.
The preparation method of tetracyclic diterpene compound of the present invention, is with Radix Araliae melanocarpae root as raw material, through extractum
Extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separate and obtain, particularly as follows:
A, extractum extract: Radix Araliae melanocarpae root dried and crushed is obtained the granule of 0.05 ~ 0.15cm size, add black
Concentration expressed in percentage by volume 80 ~ 95% ethanol solution reflux, extract, 3 ~ 5 times at 65 ~ 74 DEG C than 4 ~ 8 times for the fruit Radix Angelicae Gigatiss root weight, often
Secondary 1 ~ 3h, united extraction liquid, extracting solution filters, and concentrating under reduced pressure extracting solution to proportion obtains extractum a for 1.1 ~ 1.3；
B, organic solvent extraction: in extractum a add weight than 2 ~ 4 times amount water, successively with petroleum ether isopyknic with water,
Ethyl acetate and n-butanol solvent are extracted, every kind of solvent extraction 4 ~ 6 times, boil off solvent respectively obtain petroleum ether extract b,
Acetic acid ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 mesh, and consumption is extract c weight 6 ~ 8
Times amount；The chloroform-methanol organic solvent gradient elution being 30:1 ~ 0:1 with volume ratio, collects gradient eluent, concentration, through tlc
Monitoring, merges identical part；
2) by 1) in silicagel column, dress on the eluent e that afforded with the chloroform-methanol organic solvent of 15:1 proportioning
Post silica gel is 200 ~ 300 mesh, and consumption is eluent e volume 6 ~ 8 times amount；Chloroform methanol organic solvent with volume ratio 25:1 ~ 5:1
Gradient elution, collects gradient eluent, concentration, through tlc detection, merges identical part；
D, high performance liquid chromatography separation: by step c 2) in eluting carried out with the chloroform-methanol organic solvent of 20:1 proportioning obtain
To eluent isolated and purified using half preparative high-performance liquid chromatographic instrument, obtain final product described tetracyclic diterpene compound
dumetorumane b.
Filtration described in a step is to be filtered through 80 ~ 120 μm of filter paper.
Concentrating under reduced pressure described in a step is to carry out concentrating under reduced pressure with Rotary Evaporators under temperature conditionss below 50 DEG C.
The solvent that boils off described in b step is to boil off solvent using Rotary Evaporators.
Step c 1) described in chloroform-methanol organic solvent volume proportion be 30:1,25:1,20:1,15:1,10:1,
5:1,2:1 and 0:1.
Step c 2) described in chloroform-methanol organic solvent volume proportion be 25:1,20:1,15:1,10:1,8:1 and
5:1.
Being isolated and purified using half preparative high-performance liquid chromatographic instrument described in Step d, specially by step c 2) in joined with 20:1
The eluent that the chloroform-methanol organic solvent of ratio is afforded isolates and purifies through half preparative high-performance liquid chromatographic instrument, with body
Long-pending proportioning is the acetonitrile-water of 60:40 is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, and the c18 of 5 m is fixing phase,
UV-detector Detection wavelength is 210nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, is evaporated after repeatedly adding up,
Obtain described tetracyclic diterpene compound dumetorumane b；
The application of the present invention is described tetracyclic diterpene compound answering in preparation prevention and/or tumor
With.
The application of the present invention is that described tetracyclic diterpene compound in preparation prevention and/or treats human lung adenocarcinoma, people suddenly
Property early children's grain leukemia or human breast carcinoma medicine in application.
With specific embodiment, the present invention will be further described below:
The preparation of embodiment 1 compound
Material source: Radix Araliae melanocarpae is adopted in Kunming, Yunnan, through the secondary religion of national medicine institute of Yunnan Institute for nationalities poplar Lignum Pini Nodi
Award and be accredited asaralia dumetorumHand.-mazz, Saving specimen is in national medicine institute of Yunnan Institute for nationalities specimen museum.
Using following steps:
A. pulverize: 10kg Radix Araliae melanocarpae root dried and crushed is become the granule of particle diameter 0.1cm size, obtain Radix Araliae melanocarpae
Powder, standby；
B. reflux, extract: Radix Araliae melanocarpae powder reflux, extract, 4 times at a temperature of 65 DEG C being obtained in step a, every time 2
Hour, use the ethanol extraction of 60kg 80% concentration every time, merge ethanol extract, standby；
C, concentration: the ethanol extract that step b is obtained filters through 80-120 urn filter and uses at a temperature of 50 DEG C
Rotary Evaporators carry out concentrating under reduced pressure, to proportion be 1.2 when, obtain extractum 1380g, standby；
D, extraction: 1380g extractum is suspended in 4500ml water, uses 4500ml petroleum ether, 4500ml ethyl acetate successively
And 4500ml n-butyl alcohol is extracted, every kind of solvent extraction 5 times, then boil off solvent with Rotary Evaporators, respectively petroleum ether,
Ethyl acetate and n-butyl alcohol extract 390 g, 112g, 405g.
E. separate: by the acetic acid ethyl ester extract being obtained in step d through 100-200 mesh silica gel column chromatography, use volume ratio
The chloroform-methanol gradient elution of 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1 obtains fr.1-fr.8 totally 8 groups
Point, fr.1 is 8.8g, and fr.2 is 8.1g, and fr.3 is 3.2g, and fr.4 is 15.7g, and fr.5 is 34.3g, and fr.6 is 6.2g, and fr.7 is
5.1g, fr.8 is 21.5g, fr.4 through 200-300 mesh silica gel column chromatography, with volume ratio 25:1,20:1,15:1,10:1,8:1,
The chloroform of 5:1: methanol elution gradient, obtain component fr.4a-fr.4f totally 6 components, fr.4a is 1.0g, fr.4b is 1.9g,
Fr.4c is 0.6g, and fr.4d is 3.6g, and fr.4e is 1.5g, and fr.4f is 3.1g.Fr.4b through half preparative high-performance liquid chromatographic instrument,
Using acetonitrile-water (volume ratio 60:40) as mobile phase, flow velocity 2.5ml/min, 250 × 10mm, the c18 of 5 m is to fix
Phase, UV-detector Detection wavelength is 210 nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, steams after repeatedly adding up
Dry, prepare white amorphous powder formula target compound dumetorumane b(12 mg).
The preparation of embodiment 2 compound
Using following steps:
A. pulverize: 10kg Radix Araliae melanocarpae root dried and crushed is become the granule of particle diameter 0.1cm size, obtain Radix Araliae melanocarpae
Powder, standby；
B. reflux, extract: Radix Araliae melanocarpae powder reflux, extract, 3 times at a temperature of 67 DEG C being obtained in step a, every time
2.5 hours, use the ethanol extraction of 60kg 84% concentration every time, merge ethanol extract, standby；
C, concentration: the ethanol extract that step b is obtained filters through 80-120 urn filter and uses at a temperature of 50 DEG C
Rotary Evaporators carry out concentrating under reduced pressure, to proportion be 1.2 when, obtain extractum 1340g, standby；
D, extraction: 1340g extractum is suspended in 4000ml water, uses 4000ml petroleum ether, 4000ml ethyl acetate successively
And 4000ml n-butyl alcohol is extracted, every kind of solvent extraction 4 times, then boil off solvent with Rotary Evaporators, respectively petroleum ether,
Ethyl acetate and n-butyl alcohol extract 372 g, 103g, 396g.
E. separate: by the acetic acid ethyl ester extract being obtained in step d through 100-200 mesh silica gel column chromatography, use volume ratio
The chloroform-methanol gradient elution of 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1 obtains fr.1-fr.8 totally 8 groups
Point, fr.1 is 7.6g, and fr.2 is 6.8g, and fr.3 is 3.0g, and fr.4 is 14.5g, and fr.5 is 32.4g, and fr.6 is 5.3g, and fr.7 is
4.8g, fr.8 are 18.7g, fr.4 through 200-300 mesh silica gel column chromatography, with volume ratio 25:1,20:1,15:1,10:1,8:1,
The chloroform of 5:1: methanol elution gradient, obtain component fr.4a-fr.4f totally 6 components, fr.4a is 0.9g, fr.4b is 1.7g,
Fr.4c is 0.5g, and fr.4d is 3.4g, and fr.4e is 1.4g, and fr.4f is 3.0g.Fr.4b through half preparative high-performance liquid chromatographic instrument,
Using acetonitrile-water (volume ratio 60:40) as mobile phase, flow velocity 2.5ml/min, 250 × 10mm, the c18 of 5 m is to fix
Phase, UV-detector Detection wavelength is 210 nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, steams after repeatedly adding up
Dry, prepare white amorphous powder formula target compound dumetorumane b(14 mg).
The preparation of embodiment 3 compound
Using following steps:
A. pulverize: 10kg Radix Araliae melanocarpae root dried and crushed is become the granule of particle diameter 0.1cm size, obtain Radix Araliae melanocarpae
Powder, standby；
B. reflux, extract: Radix Araliae melanocarpae powder reflux, extract, 5 times at a temperature of 69 DEG C being obtained in step a, every time 2
Hour, use the ethanol extraction of 60kg 88% concentration every time, merge ethanol extract, standby；
C, concentration: the ethanol extract that step b is obtained filters through 80-120 urn filter and uses at a temperature of 50 DEG C
Rotary Evaporators carry out concentrating under reduced pressure, to proportion be 1.2 when, obtain extractum 1410g, standby；
D, extraction: 1410g extractum is suspended in 4500ml water, uses 4500ml petroleum ether, 4500ml ethyl acetate successively
And 4500ml n-butyl alcohol is extracted, every kind of solvent extraction 5 times, then boil off solvent with Rotary Evaporators, respectively petroleum ether,
Ethyl acetate and n-butyl alcohol extract 395 g, 117g, 411g.
E. separate: by the acetic acid ethyl ester extract being obtained in step d through 100-200 mesh silica gel column chromatography, use volume ratio
The chloroform-methanol gradient elution of 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1 obtains fr.1-fr.8 totally 8 groups
Point, fr.1 is 9.1g, and fr.2 is 8.4g, and fr.3 is 3.4g, and fr.4 is 15.9g, and fr.5 is 35.6g, and fr.6 is 6.4g, and fr.7 is
5.3g, fr.8 are 22.6g, fr.4 through 200-300 mesh silica gel column chromatography, with volume ratio 25:1,20:1,15:1,10:1,8:1,
The chloroform of 5:1: methanol elution gradient, obtain component fr.4a-fr.4f totally 6 components, fr.4a is 1.1g, fr.4b is 2.1g,
Fr.4c is 0.7g, and fr.4d is 3.7g, and fr.4e is 1.6g, and fr.4f is 3.9g.Fr.4b through half preparative high-performance liquid chromatographic instrument,
Using acetonitrile-water (volume ratio 60:40) as mobile phase, the c18 of flow velocity 2.5 ml/min, 250 × 10mm, 5 m is to fix
Phase, UV-detector Detection wavelength is 210 nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, steams after repeatedly adding up
Dry, prepare white amorphous powder formula target compound dumetorumane b(15 mg).
The preparation of embodiment 4 compound
Using following steps:
A. pulverize: 10kg Radix Araliae melanocarpae root dried and crushed is become the granule of particle diameter 0.1cm size, obtain Radix Araliae melanocarpae
Powder, standby；
B. reflux, extract: Radix Araliae melanocarpae powder reflux, extract, 4 times at a temperature of 71 DEG C being obtained in step a, every time 2
Hour, use the ethanol extraction of 60kg 92% concentration every time, merge ethanol extract, standby；
C, concentration: the ethanol extract that step b is obtained filters through 80-120 urn filter and uses at a temperature of 50 DEG C
Rotary Evaporators carry out concentrating under reduced pressure, to proportion be 1.2 when, obtain extractum 1480g, standby；
D, extraction: 1480g extractum is suspended in 5000ml water, uses 5000ml petroleum ether, 5000ml ethyl acetate successively
And 5000ml n-butyl alcohol is extracted, every kind of solvent extraction 6 times, then boil off solvent with Rotary Evaporators, respectively petroleum ether,
Ethyl acetate and n-butyl alcohol extract 387 g, 111g, 408g.
E. separate: by the acetic acid ethyl ester extract being obtained in step d through 100-200 mesh silica gel column chromatography, use volume ratio
The chloroform-methanol gradient elution of 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1 obtains fr.1-fr.8 totally 8 groups
Point, fr.1 is 8.2g, and fr.2 is 7.5g, and fr.3 is 3.0g, and fr.4 is 16.2g, and fr.5 is 34.4g, and fr.6 is 6.1g, and fr.7 is
5.0g, fr.8 are 22.3g, fr.4 through 200-300 mesh silica gel column chromatography, with volume ratio 25:1,20:1,15:1,10:1,8:1,
The chloroform of 5:1: methanol elution gradient, obtain component fr.4a-fr.4f totally 6 components, fr.4a is 1.1g, fr.4b is 2.2g,
Fr.4c is 0.7g, and fr.4d is 3.9g, and fr.4e is 1.8g, and fr.4f is 3.6g.Fr.4b through half preparative high-performance liquid chromatographic instrument,
Using acetonitrile-water (volume ratio 60:40) as mobile phase, flow velocity 2.5ml/min, 250 × 10mm, the c18 of 5 m is to fix
Phase, UV-detector Detection wavelength is 210 nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, steams after repeatedly adding up
Dry, prepare white amorphous powder formula target compound dumetorumane b(17 mg).
The preparation of embodiment 5 compound
Using following steps:
A. pulverize: 10kg Radix Araliae melanocarpae root dried and crushed is become the granule of particle diameter 0.1cm size, obtain Radix Araliae melanocarpae
Powder, standby；
B. reflux, extract: Radix Araliae melanocarpae powder reflux, extract, 5 times at a temperature of 74 DEG C being obtained in step a, every time 2
Hour, use the ethanol extraction of 60kg 95% concentration every time, merge ethanol extract, standby；
C, concentration: the ethanol extract that step b is obtained filters through 80-120 urn filter and uses at a temperature of 50 DEG C
Rotary Evaporators carry out concentrating under reduced pressure, to proportion be 1.2 when, obtain extractum 1520g, standby；
D, extraction: 1520g extractum is suspended in 4500ml water, uses 4500ml petroleum ether, 4500ml ethyl acetate successively
And 4500ml n-butyl alcohol is extracted, every kind of solvent extraction 6 times, then boil off solvent with Rotary Evaporators, respectively petroleum ether,
Ethyl acetate and n-butyl alcohol extract 420 g, 130g, 450g.
E. separate: by the acetic acid ethyl ester extract being obtained in step d through 100-200 mesh silica gel column chromatography, use volume ratio
The chloroform-methanol gradient elution of 30:1,25:1,20:1,15:1,10:1,5:1,2:1,0:1 obtains fr.1-fr.8 totally 8 groups
Point, fr.1 is 10.5g, and fr.2 is 8.8g, and fr.3 is 3.5g, and fr.4 is 17.0g, and fr.5 is 38.5g, and fr.6 is 7.0g, fr.7
For 5.6g, fr.8 is 25.3g, fr.4 through 200-300 mesh silica gel column chromatography, with volume ratio 25:1,20:1,15:1,10:1,8:
1st, the chloroform of 5:1: methanol elution gradient, obtain component fr.4a-fr.4f totally 6 components, fr.4a is 1.2g, fr.4b is
2.4g, fr.4c are 0.8g, and fr.4d is 4.1g, and fr.4e is 1.9g, and fr.4f is 3.8g.Fr.4b is through partly preparing high-efficient liquid phase color
Spectrometer, using acetonitrile-water (volume ratio 60:40) as mobile phase, flow velocity 2.5ml/min, 250 × 10mm, the c18 of 5 m is
Fixing phase, UV-detector Detection wavelength is 210 nm, each sample introduction 50 l, collects the chromatographic peak of 35 ~ 37min, repeatedly adds up
After be evaporated, prepare white amorphous powder formula target compound dumetorumane b(19 mg).
The Structural Identification of embodiment 6 the compounds of this invention
The compound dumetorumane b(Radix Araliae melanocarpae diterpenoid acid b) of Example 1 preparation, is white amorphous powder
End (solvent is chloroform)；Assay method is: uses nuclear magnetic resonance, NMR, identifies structure in conjunction with other spectroscopic techniques.
= +43.5° (c0.45, chcl3);Ir (kbr) spectrum 3345,2969,1711,1652,
1123, 1067 cm-1There is absorption, show in molecule, there is carboxyl, hydroxyl and double bond.Hresi-ms (accompanying drawing 1) shows its standard point
Daughter ion peak m/z 317.2131 [m-h]-(calcd. 317.2116), composes in conjunction with nmr and determines that its molecular formula is c20h30o3,
Degree of unsaturation is 6.1There are 3 methyl singlets, respectively δ in h nmr (accompanying drawing 2, attribution data is shown in Table 1) display moleculeh1.25
(3h, s), 1.18 (3h s) and 0.81 (3h, s)), has 1 company's Oxymethylene peak (δh3.72 (1h,dd, j =
11.2, 7.2 hz)；3.60 (1h, dd, j=11.2,8.0 hz));13C nmr (accompanying drawing 3 and accompanying drawing 4, attribution data
It is shown in Table 1) show in molecule there is 20 carbon, wherein there are 6 quaternary carbons, 3 methines, 8 methylene, and 3 methyl.In conjunction with hsqc
Correlated Spectroscopy (accompanying drawing 5), and documents data, it can be confirmed that this compound is similar to known compound acasiane a structure, is
Diterpene-kind compound containing the little ring of 1 ternary, difference has been in molecule 1 carbonyl (δ manyc182.7) and 1 double bond (δc
127.8 and 135.5), lacked 1 company Oxymethylene (δc63.8) and 2 methine (δc56.3 and 37.1)；Warp further
Hmbc correlation (accompanying drawing 6) confirms c-18(carbonyl) it is connected with c-4 position, c-8 and c-9 position is a double bond.Related through hmbc further
(accompanying drawing 6), cosy correlation (accompanying drawing 7) and roesy (accompanying drawing 8) and with document (lin a. s., lin c. r., du y. c.,
lübken t., chiang m. y., chen i. h., wu c. c., hwang t. l., chen s. l., yen
m. h., chang f. r., wu y. c. acasiane a and b and farnesirane a and b,
diterpene derivatives from the roots ofacacia farnesiana. planta med 2009,
75:256-261.) contrast confirms, makes the structure of compound finally give confirmation, is named as dumetorumane b.
Embodiment 7
The compound of Example 2 preparation, is yellow jelly；Carry out structure determination, result by the method in embodiment 6
For: with embodiment 6, molecular formula is c to its structure20h30o3.Confirm that compound prepared by embodiment 2 is described tetracyclic diterpene class
Compound dumetorumane b.
Embodiment 8
The compound of Example 3 preparation, is yellow jelly；Carry out structure determination, result by the method in embodiment 6
For: with embodiment 6, molecular formula is c to its structure20h30o3.Confirm that compound prepared by embodiment 3 is described tetracyclic diterpene class
Compound dumetorumane b.
Embodiment 9
The compound of Example 4 preparation, is yellow jelly；Carry out structure determination, result by the method in embodiment 6
For: with embodiment 6, molecular formula is c to its structure20h30o3.Confirm that compound prepared by embodiment 4 is described tetracyclic diterpene class
Compound dumetorumane b.
Embodiment 10
The compound of Example 5 preparation, is yellow jelly；Carry out structure determination, result by the method in embodiment 6
For: with embodiment 6, molecular formula is c to its structure20h30o3.Confirm that compound prepared by embodiment 5 is described tetracyclic diterpene class
Compound dumetorumane b.
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 test, tests feelings
Condition is as follows:
The increment inhibitory action to tumor cell for the tetracyclic diterpene compound is evaluated using improvement mtt method；
Take standby tumor cell suspension to be inoculated in 96 well culture plates, 90 l/ holes, add not after continuing culture 24 h
With the given the test agent of concentration, 10 l/ holes so as to final concentration of 100,10,1,0.1,0.01 g/ml, each concentration is all provided with 3
Individual multiple holes.Cisplatin (ddp) is positive control, and equal-volume rpmi1640 culture medium is negative control.Set compound 100,10 simultaneously
During g/ml, corresponding dmso concentration is Vehicle controls, to eliminate the impact of dmso cell growth.Continue after dosing to be placed in 37
DEG C, 5% co2Incubator cultivates 72 h, and every hole adds mtt(5 mg/ml) 20 l, continue culture 4 h, every hole adds three liquid
[10%sds-5% isobutanol 0.012 mol/l hcl(w/v/v)] 100 l dissolving first.With microplate reader in 570 nm wavelength
The lower od value measuring each hole.
By following equation calculating cell proliferation suppression ratio:
Suppression ratio (%)=(od value control wells od value dosing holes)/od value control wells × 100%
Data statistic analysis: suppression ratio is calculated using microsoft excel 2003, using logit method,
Spss11.5 software kit calculation of half inhibitory concentration ic50.
Calculation of half inhibitory concentration ic50, to human lung adenocarcinoma cell line (a549), human acute myeloid leukaemia strain
(nb4) and Breast cancer lines (mcf7) half-inhibition concentration (ic50) measurement result respectively 6.7 ± 0.5 m, 4.2
± 0.3 m, 5.3 ± 0.6 m, show that the compound of the present invention has preferable anti-tumor activity.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification equivalent made within god and principle and improvement etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of tetracyclic diterpene compound preparation method is it is characterised in that described tetracyclic diterpene compound is with Radix osteomelis schwerinais soil
Angelica root is raw material, obtains through extractum extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, this chemical combination
Thing molecular formula is c20h30o3, it is named as dumetorumane b, there is following structural formula:
；
Preparation method particularly as follows:
A, extractum extract: Radix Araliae melanocarpae root dried and crushed is obtained the granule of 0.05 ~ 0.15cm size, add Radix osteomelis schwerinais soil
Concentration expressed in percentage by volume 80 ~ 95% ethanol solution reflux, extract, 3 ~ 5 times at 65 ~ 74 DEG C than 4 ~ 8 times for the angelica root weight, every time 1 ~
3h, united extraction liquid, extracting solution filters, and concentrating under reduced pressure extracting solution to proportion obtains extractum a for 1.1 ~ 1.3；
B, organic solvent extraction: add the water that weight is than 2 ~ 4 times amount in extractum a, successively with petroleum ether isopyknic with water, acetic acid
Ethyl ester and n-butanol solvent are extracted, every kind of solvent extraction 4 ~ 6 times, boil off solvent and respectively obtain petroleum ether extract b, acetic acid
Ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography:
1) by silicagel column on acetic acid ethyl ester extract c, dress post silica gel is 100 ~ 200 mesh, and consumption is extract c weight 6 ~ 8 times amount；
The chloroform-methanol organic solvent gradient elution being 30:1 ~ 0:1 with volume ratio, collects gradient eluent, concentration, through tlc monitoring,
Merge identical part；
2) by 1) in silicagel column on the eluent e that afforded with the chloroform-methanol organic solvent of 15:1 proportioning, fill post silicon
Glue is 200 ~ 300 mesh, and consumption is eluent e volume 6 ~ 8 times amount；Chloroform methanol organic solvent gradient with volume ratio 25:1 ~ 5:1
Eluting, collects gradient eluent, concentration, through tlc detection, merges identical part；
D, high performance liquid chromatography separation: by step c 2) in afforded with the chloroform-methanol organic solvent of 20:1 proportioning
Eluent is isolated and purified using half preparative high-performance liquid chromatographic instrument, obtains final product described tetracyclic diterpene compound dumetorumane
b.
2. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that the filtration described in a step be through
80 ~ 120 μm of filter paper is filtered.
3. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that concentrating under reduced pressure described in a step
It is to carry out concentrating under reduced pressure with Rotary Evaporators under the temperature conditionss below 50 DEG C.
4. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that boiling off described in b step is molten
Agent is to boil off solvent using Rotary Evaporators.
5. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that step c 1) described in chlorine
The volume proportion of imitation-carbinol organic solvent is 30:1,25:1,20:1,15:1,10:1,5:1,2:1 and 0:1.
6. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that step c 2) described in chlorine
The volume proportion of imitation-carbinol organic solvent is 25:1,20:1,15:1,10:1,8:1 and 5:1.
7. according to claim 1 tetracyclic diterpene compound preparation method it is characterised in that described in Step d using half
Preparative high-performance liquid chromatographic instrument isolates and purifies, be acetonitrile-water with volume proportion as 60:40 as mobile phase, flow velocity 2.5ml/
Min, 250 × 10mm, 5 μm of c18 is fixing phase, and UV-detector Detection wavelength is 210nm, each sample introduction 50 μ l, collects
The chromatographic peak of 35 ~ 37min, is evaporated after repeatedly adding up, and obtains described tetracyclic diterpene compound dumetorumane b.
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