CN104817432A - Anti-tumor drug of diterpenoid compound, and preparation method and application thereof - Google Patents

Anti-tumor drug of diterpenoid compound, and preparation method and application thereof Download PDF

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CN104817432A
CN104817432A CN201510133850.3A CN201510133850A CN104817432A CN 104817432 A CN104817432 A CN 104817432A CN 201510133850 A CN201510133850 A CN 201510133850A CN 104817432 A CN104817432 A CN 104817432A
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diterpene
ethyl acetate
cancer cell
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不公告发明人
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Xie Rui
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Abstract

The invention discloses a diterpenoid compound. A preparation method comprises the following steps: A, degreasing Chinese azalea flower with dichloromethane once or more than once, then carrying out filtration and collecting filter residue; B, extracting the Chinese azalea flower with alcohol or an alcohol solution once or more than once, then carrying out filtration, collecting a filtrate and then carrying out pressure-reduced concentration and drying so as to obtain alcohol extract; C, adding water into the alcohol extract, degreasing the alcohol extract with petroleum ether again, carrying out extraction with ethyl acetate after degreasing, extracting the obtained ethyl acetate extraction liquid with a saturated aqueous sodium bicarbonate solution and evaporating an ethyl acetate phase to dryness to obtain crude ethyl acetate extract; and D, separating and purifying the crude ethyl acetate extract by using column chromatography to obtain the purified diterpenoid compound. The diterpenoid compound provided by the invention has a strong inhibitory effect on colon cancer cells (HT-29 and HCT-8), liver cancer cells (Bcl-7402), small cell lung cancer (A549) and/or breast cancer (MCF-7).

Description

A kind of antitumor drug of diterpene-kind compound and method for application thereof
Technical field
The present invention relates to natural extract field of pharmaceutical chemistry technology, particularly a kind of antitumor drug of diterpene-kind compound and method for application thereof.
Background technology
Chinese azalea (Rhododendron molle G.Don) is ericad, is distributed in China Yangtze valley to southern various places, is grown in hillside, shrubbery or thick grass.The flower of Chinese azalea, fruit and root etc. all can hyoscines.Spend and generally claim Rhododendron molle, taste is pungent, temperature, very toxic, enters Liver Channel; There is the effects such as analgesic therapy, wind dispelling and dehumidifying, for rheumatoid arthritis stubborn, traumatic fracture pain, skin stubborn dermatitis.Fruit generally claims Fructus Rhododendri Mollis or Flos Echinopsis latifolii, bitter, temperature, very toxic, enters lung, the spleen channel; Have expelling wind to alleviate pain, the effect such as relieving cough and asthma, for arthralgia due to wind-cold-dampness pathogen BI syndrome, wound, coughs and breathes heavily.Root taste is pungent, temperature, poisonous, enters the spleen channel; Have dispel the wind, cough-relieving, loose effect such as the stasis of blood and pain relieving, for arthralgia due to wind-cold-dampness pathogen BI syndrome, wound, anal fistula, tinea sore.Chemical research shows, diterpene and flavonoid compound are the main components of Chinese azalea, fruit and root etc., and wherein Diterpenoids from bulbus is principle active component, is also its toxic component.
Be separated from the flower of Chinese azalea, fruit and root at present and obtain and Diterpenoids from bulbus through structural identification is mainly divided into two large classes according to parent nucleus: grayanane type and kalmanol type.Grayanane type representative compound comprises: rhodomollein XVI, rhodomollein XVII, rhodomollein XVIII, rhodojaponin III, rhodojaponin VI (Diterpenoids from the Fruits ofRhododendron molle, J.Nat.Prod.2000,63,1214-1217); Rhodomoside A and rhodomoside B (Diterpenoid and Phenolic Glycosides from the roots ofRhododendron molle); Rhodomollein IX, rhodomollein X, rhodomollein XI, rhodomollein XII and rhodomollein XIII (Diterpenoids from the Flowers ofRhododendron molle, J.Nat.Prod.2004,67,1903-1906); Rhodomolin A and rhodomolin B (Grayanane Diterpenoids from the Flowers of Rhododendron mollewith Cytotoxic Activity against a Spodoptera frugiperda Cell Line, J.Nat.Prod.2005,68,924-926); Seco-rhodomollone (Diterpenoids from the Flowers ofRhododendron molle, J.Nat.Prod.2014,77,1185-1192).Kalmanol type representative compound comprises: rhodomollein XV, kalmanol (Diterpenoids from the Fruits ofRhododendron molle, J.Nat.Prod.2000,63,1214-1217) with rhodomollein XIV (Diterpenoids from the Flowers of Rhododendron molle, J.Nat.Prod.2004,67,1903-1906).Representative Grayanane type and kalmanol type diterpene compound structure are as shown in Figure 1.
Rhododendron molle is the dried floral of Chinese azalea, has the effect of dispelling rheumatism, faling apart stasis of blood analgesic therapy, can be used for rheumatic arthralgia, migraine and general headache, tumbling and swelling, stubborn dermatitis [Pharmacopoeia of People's Republic of China () [S], 2010 editions].Its chemical constitution study starts from the 1980s, and research contents mainly concentrates on its diterpenes chemical composition.Grayanane type and kalmanol type diterpene-kind compound obtain mainly through the column chromatography separation and purification of liquid-liquid extraction and dissimilar filler.Typical method is: the first step, and the alcoholic solution of different concns extracts; Second step, extract is scattered in water, uses sherwood oil, ethyl acetate and n-butanol extraction respectively, is divided into opposed polarity section; 3rd step, gets acetic acid ethyl ester extract normal phase silica gel column chromatography, reversed-phase silica gel column chromatography, MCI micro-porous resin column chromatography or the gel column chromatography segmentation purifying that diterpene-kind compound is concentrated.
In Rhododendron molle, the report of Diterpenoids from bulbus Pharmacological Activity Screening is less, cytotoxic activity researches.2005, Zhong etc. to report in Rhododendron molle two Grayanane type compound rhodomolins A and rhodomolins B to the cytotoxicity of SL cell line Sf-9, IC50 value is at 12-80 μ g/mL (Grayanane Diterpenoids from the Flowers of Rhododendron molle withCytotoxic Activity against a Spodoptera frugiperda Cell Line, J.Nat.Prod.2005,68,924-926).Have not yet to see the active reporter of kalmanol type Diterpene in Rhododendron molle.
Summary of the invention
Technical problem to be solved by this invention is, for in the research of Rhododendron molle effective constituent, the kalmanol type diterpene compound that isolation identification one is new from acetic acid ethyl ester extract, chemical structure is rhodomollein XXIV 5 hydroxyls and 1 hydrogen dewatered product, for reported first, called after kalmanene.Cytotoxicity test shows, this compound on tumor cell has significant cytotoxicity, relatively weak to normal cell toxic action, can as the activeconstituents preparing antitumor drug.
For solving the problems of the technologies described above, the invention provides a kind of diterpene-kind compound, its preparation method comprises:
A. by Rhododendron molle methylene dichloride one or many degreasing, filter, collect filter residue;
B. Rhododendron molle alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
C. added water by alcohol extract, after petroleum ether extraction again degreasing, with extraction into ethyl acetate, the acetic acid ethyl acetate extract of acquisition, again with saturated sodium bicarbonate aqueous solution extraction, is got ethyl acetate phase evaporate to dryness, is obtained acetic acid ethyl ester extract crude product;
D. the crude product of acquisition is carried out column chromatography separating purification, obtain the diterpene-kind compound after purifying.
The detailed step of described step D can comprise:
1. crude product is carried out column chromatography on silica gel, use ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol eluate;
2. get ethyl acetate-ethanol eluate and carry out silica gel column chromatography again, use methylene chloride-methanol isocratic elution, collect 6-8 column volume wash-out position, concentrated;
3. get step 2. enriched material carry out reversed-phase silica gel column chromatography, use water-methanol isocratic elution, collect 4-5 column volume wash-out position, concentrated;
4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure Compound I from water-methanol wash-out position.
For solving the problems of the technologies described above, present invention also offers a kind of diterpene-kind compound, the chemical structural formula of described diterpene-kind compound is as follows:
For solving the problems of the technologies described above, invention further provides a kind of pharmacological activity testing method of diterpene-kind compound as described in aforementioned any one, comprising the steps:
A. take the logarithm vegetative period tumor cell culture in 96 well culture plates, every hole 100 μ L, containing 1000-1200 tumour cell; Next day, administration group adds containing different concns compound, and 4 dosage groups established by every medicine, and often group establishes 3 parallel plates;
B. control group adds solvent isopyknic with described diterpene-kind compound, puts 5%CO 2in 37 DEG C of cultivations in incubator, nutrient solution is discarded after 4d, every hole adds 200 μ L 0.2%MTT solution, 37 DEG C of insulation 4h, abandoning supernatant, every hole adds DMSO 150 μ L and dissolves first hairpin particle, after slight concussion, by microplate reader, at reference wavelength 450nm, under determined wavelength 570nm condition, measure optical density(OD) OD value;
C. with the cell strain of solvent control process for control group, using taxol as positive control medicine, measuring result following formulae discovery drug on tumor cell and Normocellular inhibiting rate:
D. LOGIT method computerized compound IC is used by gained cell inhibitory rate 50numerical value.
For solving the problem, the present invention reoffer a kind of as described in aforementioned any one diterpene-kind compound preparing the application in antitumor drug.
It is colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7 that described diterpene-kind compound prepares the tumour described in the application in antitumor drug.
For solving the problems of the technologies described above, the present invention separately provides a kind of application of diterpene-kind compound in the activity detecting antitumor drug as described in aforementioned any one.
Described tumour is preferably colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
For solving the problems of the technologies described above, the present invention has reoffered a kind of application of diterpene-kind compound in screening antineoplastic drugs as described in aforementioned any one.
Described tumour is preferably colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
The technique effect that the present invention is useful is, diterpene-kind compound of the present invention has very strong restraining effect to colon cancer cell (HT-29, HCT-8), liver cancer cell (Bcl-7402), nonsmall-cell lung cancer (A549) and/or mammary cancer (MCF-7), is wherein better than positive drug contrast to the restraining effect of colon cancer cell (HT-29, HCT-8), liver cancer cell (Bcl-7402) and mammary cancer (MCF-7); On the other hand, the restraining effect of Compound I to normal cell (Human umbilical vein endothelial cells VEC) will be weaker than positive drug, shows that the compounds of this invention I has antitumor action, and has certain selectivity.
Accompanying drawing explanation
Fig. 1 is representative Grayanane type diterpene compound structure iron in prior art;
Fig. 2 is representative kalmanol type diterpene compound structure iron in prior art;
Fig. 3 is the chemical structural formula of compound described in the embodiment of the present invention 1.
Embodiment
Describe embodiments of the present invention in detail below with reference to embodiment, to the present invention, how utilisation technology means solve technical problem whereby, and the implementation procedure reaching technique effect can fully understand and implement according to this.
One embodiment of the invention provides a kind of diterpene-kind compound, and its preparation method comprises:
A. by Rhododendron molle methylene dichloride one or many degreasing, filter, collect filter residue;
B. Rhododendron molle alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
C. added water by alcohol extract, after petroleum ether extraction again degreasing, with extraction into ethyl acetate, the acetic acid ethyl acetate extract of acquisition, again with saturated sodium bicarbonate aqueous solution extraction, is got ethyl acetate phase evaporate to dryness, is obtained acetic acid ethyl ester extract crude product;
D. the crude product of acquisition is carried out column chromatography separating purification, obtain the diterpene-kind compound after purifying.
The detailed step of described step D can comprise:
1. crude product is carried out column chromatography on silica gel, use ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol eluate;
2. get ethyl acetate-ethanol eluate and carry out silica gel column chromatography again, use methylene chloride-methanol isocratic elution, collect 6-8 column volume wash-out position, concentrated;
3. get step 2. enriched material carry out reversed-phase silica gel column chromatography, use water-methanol isocratic elution, collect 4-5 column volume wash-out position, concentrated;
4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure Compound I from water-methanol wash-out position.
Another embodiment of the present invention provides a kind of diterpene-kind compound, and the chemical structural formula of described diterpene-kind compound is as follows:
Further embodiment of this invention provides a kind of pharmacological activity testing method of diterpene-kind compound as described in aforementioned any one, comprises the steps:
A. take the logarithm vegetative period tumor cell culture in 96 well culture plates, every hole 100 μ L, containing 1000-1200 tumour cell; Next day, administration group adds containing different concns compound, and 4 dosage groups established by every medicine, and often group establishes 3 parallel plates;
B. control group adds solvent isopyknic with described diterpene-kind compound, puts 5%CO 2in 37 DEG C of cultivations in incubator, nutrient solution is discarded after 4d, every hole adds 200 μ L 0.2%MTT solution, 37 DEG C of insulation 4h, abandoning supernatant, every hole adds DMSO 150 μ L and dissolves first hairpin particle, after slight concussion, by microplate reader, at reference wavelength 450nm, under determined wavelength 570nm condition, measure optical density(OD) OD value;
C. with the cell strain of solvent control process for control group, using taxol as positive control medicine, measuring result following formulae discovery drug on tumor cell and Normocellular inhibiting rate:
D. LOGIT method computerized compound IC is used by gained cell inhibitory rate 50numerical value.
For solving the problem, the present invention reoffer a kind of as described in aforementioned any one diterpene-kind compound preparing the application in antitumor drug.
It is colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7 that described diterpene-kind compound prepares the tumour described in the application in antitumor drug.
Another embodiment of the present invention provides a kind of application of diterpene-kind compound in the activity detecting antitumor drug as described in aforementioned any one.
Described tumour is preferably colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
Yet another embodiment of the invention provides a kind of application of diterpene-kind compound in screening antineoplastic drugs as described in aforementioned any one.
Described tumour is preferably colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bcl-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
Another embodiment of the present invention provides a kind of new compound I with antitumor action that separation and purification goes out from Rhododendron molle.
Another embodiment of the present invention provides a kind of preparation method of separation and purification Compound I from Rhododendron molle.
One more embodiment of the present invention provides Compound I and is preparing the application in antitumor drug.
According to one embodiment of the invention, provide the compound with the following chemical structure formula I:
There is provided a kind of preparation method of separation and purification Compound I from Rhododendron molle according to another embodiment of the present invention, its preparation process comprises:
A. by Rhododendron molle dichloromethane extraction one or many degreasing, filter, collect filter residue;
B. Rhododendron molle alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
C. added water by alcohol extract, after petroleum ether extraction again degreasing, with extraction into ethyl acetate, the acetic acid ethyl acetate extract of acquisition, again with saturated sodium bicarbonate aqueous solution extraction, is got ethyl acetate phase evaporate to dryness, is obtained acetic acid ethyl ester extract (crude product);
D. the crude product of acquisition is carried out column chromatography separating purification, obtain pure Compound I.
The detailed step of above-mentioned steps D comprises as follows: 1. crude product is carried out column chromatography on silica gel, uses ethyl acetate-ethanol gradient elution, obtains ethyl acetate-ethanol (volume ratio 30: 1) eluate; 2. get ethyl acetate-ethanol (volume ratio 30: 1) eluate and carry out silica gel column chromatography again, with methylene chloride-methanol (15: 1) isocratic elution, collect 6-8 column volume wash-out position, concentrated; 3. get step 2. enriched material carry out reversed-phase silica gel column chromatography, with water-methanol (55: 45) isocratic elution, collect 4-5 column volume wash-out position, concentrated; 4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure Compound I from water-methanol (volume ratio 60: 40) wash-out position.
The detailed step of above-mentioned steps D comprises as follows: 1. crude product is carried out column chromatography on silica gel, uses ethyl acetate-ethanol gradient elution, obtains ethyl acetate-ethanol (volume ratio 30: 1) eluate; 2. get ethyl acetate-ethanol (volume ratio 30: 1) eluate and carry out silica gel column chromatography again, with methylene chloride-methanol (15: 1) isocratic elution, collect 6-8 column volume wash-out position, concentrated; 3. get step 2. enriched material carry out reversed-phase silica gel column chromatography, with water-methanol (55: 45) isocratic elution, collect 4-5 column volume wash-out position, concentrated; 4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure Compound I from water-methanol (volume ratio 60: 40) wash-out position.
According to one more embodiment of the present invention, the compounds of this invention I is provided to prepare the application in antitumor drug.The compounds of this invention I activity is evaluated with Standard pharmacological inspection procedure, and result shows that the compounds of this invention I has remarkable cytotoxicity to tumour cell, is better than positive drug or suitable with positive drug to the inhibition of most tumors cell; More weak to normal cell toxic action, inhibition is lower than positive drug.Activity shown in evaluating based on described Standard pharmacological testing procedures, thus the compounds of this invention I may be used for antitumor field, preferred colorectal carcinoma, liver cancer and mammary cancer.
Here is pharmacological test procedures and the activity data of the compounds of this invention I.
The determination of activity of In Vitro Anti human colon cancer cell (HT-29, HCT-8), liver cancer cell (Bcl-7402), nonsmall-cell lung cancer (A549), breast cancer cell (MCF-7) and Human umbilical vein endothelial cells (VEC):
Beating holes method measures KB and cell strain inhibit activities, test method is as follows: take the logarithm vegetative period cell cultures in 96 well culture plates, every hole 100 μ L (containing 1000-1200 tumour cell), next day, administration group adds containing different concns compound, 4 dosage groups established by every medicine, and often group establishes 3 parallel plates.Control group adds isopyknic solvent with compound, puts 5%CO 2in 37 DEG C of cultivations in incubator, nutrient solution is discarded after 4d, every hole adds 200 μ L 0.2%MTT solution, 37 DEG C of insulation 4h, abandoning supernatant, every hole adds DMSO 150 μ L and dissolves first hairpin particle, after slight concussion, by microplate reader, at reference wavelength 450nm, under determined wavelength 570nm condition, measure optical density(OD) (OD).With the cell strain of solvent control process for control group, using taxol as positive control medicine.Measuring result following formulae discovery drug on tumor cell and Normocellular inhibiting rate:
LOGIT method and then computerized compound IC is used by gained cell inhibitory rate 50numerical value.
Test-results shows, the compounds of this invention I has very strong restraining effect to colon cancer cell (HT-29, HCT-8), liver cancer cell (Bcl-7402), nonsmall-cell lung cancer (A549) and mammary cancer (MCF-7), is wherein better than positive drug contrast to the restraining effect of colon cancer cell (HT-29, HCT-8), liver cancer cell (Bcl-7402) and mammary cancer (MCF-7); On the other hand, the restraining effect of Compound I to normal cell (Human umbilical vein endothelial cells VEC) will be weaker than positive drug, shows that the compounds of this invention I has antitumor action, and has certain selectivity.Test-results is in table 1.
Table 1 the compounds of this invention I is to tumour cell and Normocellular restraining effect (IC 50(μM))
Embodiment 1
Get Rhododendron molle (Hubei, the place of production), with dichloromethane extraction three degreasings, filter, collect filter residue.Filter residue 95% ethanolic soln is extracted three times, filters, collect filtrate, concentrating under reduced pressure obtains ethanol extraction.Ethanol extraction is added water and makes extract aqueous suspensions, after petroleum ether extraction three times again degreasing, with extraction into ethyl acetate three times, the acetic acid ethyl acetate extract obtained concentrates, extract three times with saturated sodium bicarbonate aqueous solution again, get ethyl acetate phase evaporate to dryness, obtain acetic acid ethyl ester extract crude product.
Crude product is carried out column chromatography on silica gel, uses ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol (volume ratio 30: 1) eluate, concentrate to obtain medicinal extract E1.Get medicinal extract E1 and again carry out silica gel column chromatography, with methylene chloride-methanol (15: 1) isocratic elution, collect 6-8 column volume wash-out position, concentrate to obtain medicinal extract E2.Get medicinal extract E2 and carry out reversed-phase silica gel column chromatography, with water-methanol (55: 45) isocratic elution, collect 4-5 column volume wash-out position, concentrate to obtain medicinal extract E3.Get medicinal extract E3 preparative high performance liquid chromatography to be separated, water-methanol gradient elution, obtain pure Compound I from water-methanol (volume ratio 60: 40) wash-out position.
Embodiment 2
Get Rhododendron molle 20Kg (Hubei, the place of production), with dichloromethane extraction three degreasings (60L × 3), filter, collect filter residue.Extracted three times (60L × 3) by filter residue 95% ethanolic soln, filter, collect filtrate, concentrating under reduced pressure obtains ethanol extraction (1.2Kg).The 10L that added water by ethanol extraction makes extract aqueous suspensions, with after petroleum ether extraction three times again degreasing (10L × 3), with extraction into ethyl acetate three times (10L × 3), the acetic acid ethyl acetate extract obtained is concentrated into about 10L and extracts three times (10L × 3) with saturated sodium bicarbonate aqueous solution, get ethyl acetate phase evaporate to dryness, obtain acetic acid ethyl ester extract (crude product, about 200g).
Crude product (about 200g) is carried out column chromatography on silica gel (600g), uses ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol (volume ratio 30: 1) eluate, concentrate to obtain medicinal extract E1.Get medicinal extract E1 and again carry out silica gel column chromatography, with methylene chloride-methanol (15: 1) isocratic elution, collect 6-8 column volume wash-out position, concentrate to obtain medicinal extract E2.Get medicinal extract E2 and carry out reversed-phase silica gel column chromatography, with water-methanol (55: 45) isocratic elution, collect 4-5 column volume wash-out position, concentrate to obtain medicinal extract E3.Get medicinal extract E3 preparative high performance liquid chromatography to be separated, water-methanol gradient elution, obtain pure Compound I (110mg) from water-methanol (volume ratio 60: 40) wash-out position.
Embodiment 3
Get Rhododendron molle 20Kg (Hubei, the place of production), with dichloromethane extraction three degreasings (60L × 3), filter, collect filter residue.Extracted three times (60L × 3) by filter residue 95% ethanolic soln, filter, collect filtrate, concentrating under reduced pressure obtains ethanol extraction (1.0 ~ 1.4Kg).The 10L that added water by ethanol extraction makes extract aqueous suspensions, with after petroleum ether extraction three times again degreasing (10L × 3), with extraction into ethyl acetate three times (10L × 3), the acetic acid ethyl acetate extract obtained is concentrated into about 10L and extracts three times (10L × 3) with saturated sodium bicarbonate aqueous solution, get ethyl acetate phase evaporate to dryness, obtain acetic acid ethyl ester extract (crude product, 180 ~ 220g).
Crude product (180 ~ 220g) is carried out column chromatography on silica gel (600g), uses ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol (volume ratio 30: 1) eluate, concentrate to obtain medicinal extract E1.Get medicinal extract E1 and again carry out silica gel column chromatography, with methylene chloride-methanol (15: 1) isocratic elution, collect 6-8 column volume wash-out position, concentrate to obtain medicinal extract E2.Get medicinal extract E2 and carry out reversed-phase silica gel column chromatography, with water-methanol (55: 45) isocratic elution, collect 4-5 column volume wash-out position, concentrate to obtain medicinal extract E3.Get medicinal extract E3 preparative high performance liquid chromatography to be separated, water-methanol gradient elution, obtain pure Compound I (105 ~ 115mg) from water-methanol (volume ratio 60: 40) wash-out position.
Compound I Structural Identification prepared by above-mentioned 3 embodiments:
Amorphous powder, is soluble in methyl alcohol.HRESIMS shows [M+Na] +for m/z 357.2044, in conjunction with 13c-NMR spectrum can determine that this compound molecule formula is C 20h 30o 4, degree of unsaturation is 6.Infrared (IR) points out hydroxyl to there is (3417em -1). 1h-NMR composes prompting has three methyl to there is (δ H 1.22,1.12,1.12), is connected with quaternary carbon; Two companiesies oxygen methyne (δ H 3.85,4.21); Two alkene hydrogen (δ H 5.08,5.11). 13c-NMR spectrum and DEPT compose prompting 20 carbon by 3 methyl, 6 methylene radical (1 olefinic carbon, δ C 103.8), 5 methynes (2 company's oxygen, δ C 84.8,64.9) and 6 quaternary carbon (3 olefinic carbons, δ C 126.9,157.3,154.6; Two company's oxygen, δ C 86.9,81.9) composition.Four olefinic carbons prove existence two double bonds, remove two degrees of unsaturation, also surplus 4 degrees of unsaturation.Comparative compound I and rhodomollein XXIV 1h-NMR spectrum and 13c-NMR composes, and more than combining, analysis can infer that Compound I is the further dehydrated structure of rhodomollein XXIV.Known according to HMBC spectrum, dehydration location is between C1 and C5.Determined the sterie configuration of Compound I by ROESY spectrum, final confirmation Compound I is rhodomollein XXIV 5 hydroxyls and 1 hydrogen dewatered product.
1h-NMR and 13c-NMR spectral data is in table 2.
Table 2 NMR Assignments data
All above-mentioned this intellecture properties of primary enforcement, not setting restriction this product innovation of other forms of enforcement and/or novel method.Those skilled in the art will utilize this important information, and foregoing is revised, to realize similar implementation status.But all modifications or transformation belong to the right of reservation based on product innovation of the present invention.

Claims (10)

1. a diterpene-kind compound, is characterized in that, preparation method comprises:
A. by Rhododendron molle methylene dichloride one or many degreasing, filter, collect filter residue;
B. Rhododendron molle alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
C. added water by alcohol extract, after petroleum ether extraction again degreasing, with extraction into ethyl acetate, the acetic acid ethyl acetate extract of acquisition, again with saturated sodium bicarbonate aqueous solution extraction, is got ethyl acetate phase evaporate to dryness, is obtained acetic acid ethyl ester extract crude product;
D. the crude product of acquisition is carried out column chromatography separating purification, obtain the diterpene-kind compound after purifying.
2. diterpene-kind compound according to claim 1, is characterized in that, the detailed step of described step D comprises:
1. crude product is carried out column chromatography on silica gel, use ethyl acetate-ethanol gradient elution, obtain ethyl acetate-ethanol eluate;
2. get ethyl acetate-ethanol eluate and carry out silica gel column chromatography again, use methylene chloride-methanol isocratic elution, collect 6-8 column volume wash-out position, concentrated;
3. get step 2. enriched material carry out reversed-phase silica gel column chromatography, use water-methanol isocratic elution, collect 4-5 column volume wash-out position, concentrated;
4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure Compound I from water-methanol wash-out position.
3. a diterpene-kind compound, is characterized in that, the chemical structural formula of described diterpene-kind compound is as follows:
4. the pharmacological activity testing method of diterpene-kind compound according to any one of claims 1 to 3, is characterized in that, comprise the steps:
A. take the logarithm vegetative period tumor cell culture in 96 well culture plates, every hole 100 μ L, containing 1000-1200 tumour cell; Next day, administration group adds containing different concns compound, and 4 dosage groups established by every medicine, and often group establishes 3 parallel plates;
B. control group adds solvent isopyknic with described diterpene-kind compound, puts 5%CO 2in 37 DEG C of cultivations in incubator, nutrient solution is discarded after 4d, every hole adds 200 μ L 0.2%MTT solution, 37 DEG C of insulation 4h, abandoning supernatant, every hole adds DMSO 150 μ L and dissolves first hairpin particle, after slight concussion, by microplate reader, at reference wavelength 450nm, under determined wavelength 570nm condition, measure optical density(OD) OD value;
C. with the cell strain of solvent control process for control group, using taxol as positive control medicine, measuring result following formulae discovery drug on tumor cell and Normocellular inhibiting rate:
D. LOGIT method computerized compound IC is used by gained cell inhibitory rate 50numerical value.
5. one kind according to any one of claims 1 to 3 diterpene-kind compound preparing the application in antitumor drug.
6. diterpene-kind compound prepares the application in antitumor drug according to claim 5, it is characterized in that, described tumour is colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bc1-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
7. the application of diterpene-kind compound in the activity detecting antitumor drug according to any one of claims 1 to 3.
8. diterpene-kind compound is detecting the application in antitumor activity according to claim 7, it is characterized in that, described tumour is colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bc1-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
9. the application of diterpene-kind compound in screening antineoplastic drugs according to any one of claims 1 to 3.
10. the application of diterpene-kind compound in screening antineoplastic drugs according to claim 9, it is characterized in that, described tumour is colon cancer cell line HT-29, colon cancer cell line HCT-8, hepatoma cell strain Bc1-7402, non-small cell lung cancer cell strain A549 and/or breast cancer cell line mcf-7.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104788291A (en) * 2015-03-25 2015-07-22 青岛华仁技术孵化器有限公司 Antitumor drug prepared from diterpenoid compound isolated from Chinese azalea flower, method and use
CN107828663A (en) * 2017-06-05 2018-03-23 海南师范大学 A kind of indoles diterpene-kind compound crystal and its application as antineoplastic
CN109593820A (en) * 2019-01-24 2019-04-09 江西师范大学 The anticancer activity detection method of Chinese azalea extract and its application
CN111759831A (en) * 2019-04-02 2020-10-13 武汉红猎生物科技有限公司 Application of 3-oxo-5 beta-steroid-4-dehydrogenase inhibitor
CN111943845A (en) * 2020-08-20 2020-11-17 济南大学 Two sesquiterpenes with antitumor activity and preparation method and application thereof
CN115974695A (en) * 2022-12-13 2023-04-18 沈阳药科大学 Preparation method and application of Vibsane diterpenoid compounds in coral trees

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030756A (en) * 2010-12-03 2011-04-27 沈阳药科大学 6,7-methylene-dioxy-1,2,3,4-tetrahydroisoquinoline derivative and preparation method and application thereof
CN104788291A (en) * 2015-03-25 2015-07-22 青岛华仁技术孵化器有限公司 Antitumor drug prepared from diterpenoid compound isolated from Chinese azalea flower, method and use

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030756A (en) * 2010-12-03 2011-04-27 沈阳药科大学 6,7-methylene-dioxy-1,2,3,4-tetrahydroisoquinoline derivative and preparation method and application thereof
CN104788291A (en) * 2015-03-25 2015-07-22 青岛华仁技术孵化器有限公司 Antitumor drug prepared from diterpenoid compound isolated from Chinese azalea flower, method and use
CN104788291B (en) * 2015-03-25 2017-04-05 漯河医学高等专科学校 The antitumor drug and method for application of the detached diterpene-kind compound of Flos Rhododendri Mollis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHUAI-ZHEN ZHOU ET AL.: "Diterpenoids from the Flowers of Rhododendron molle", 《JOURNAL OF NATURAL PRODUCTS》 *
张枝润: "两种杜鹃花属植物化学成分及质量标准研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104788291A (en) * 2015-03-25 2015-07-22 青岛华仁技术孵化器有限公司 Antitumor drug prepared from diterpenoid compound isolated from Chinese azalea flower, method and use
CN107828663A (en) * 2017-06-05 2018-03-23 海南师范大学 A kind of indoles diterpene-kind compound crystal and its application as antineoplastic
CN107828663B (en) * 2017-06-05 2021-06-01 海南师范大学 Indole diterpenoid compound crystal and application thereof as antitumor drug
CN109593820A (en) * 2019-01-24 2019-04-09 江西师范大学 The anticancer activity detection method of Chinese azalea extract and its application
CN111759831A (en) * 2019-04-02 2020-10-13 武汉红猎生物科技有限公司 Application of 3-oxo-5 beta-steroid-4-dehydrogenase inhibitor
CN111943845A (en) * 2020-08-20 2020-11-17 济南大学 Two sesquiterpenes with antitumor activity and preparation method and application thereof
CN115974695A (en) * 2022-12-13 2023-04-18 沈阳药科大学 Preparation method and application of Vibsane diterpenoid compounds in coral trees
CN115974695B (en) * 2022-12-13 2024-05-03 沈阳药科大学 Preparation method and application of vibsane diterpenoid compounds in coral tree

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