CN1594267A - Diterpenoid monomer and its preparation method and application for preparing anti-cancer drugs - Google Patents
Diterpenoid monomer and its preparation method and application for preparing anti-cancer drugs Download PDFInfo
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- CN1594267A CN1594267A CN 200410041150 CN200410041150A CN1594267A CN 1594267 A CN1594267 A CN 1594267A CN 200410041150 CN200410041150 CN 200410041150 CN 200410041150 A CN200410041150 A CN 200410041150A CN 1594267 A CN1594267 A CN 1594267A
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Abstract
Disclosed is an anti-cancer medicament ent-kauran-19-al-17-oic acid which is prepared by the steps of, (1) disintegrating the aired round lichee stems, extracting with ethanol, condensing into extract, (2) extracting with chloroform, column chromatography, purging and column chromatography again, and purging to obtain the end product.
Description
One, technical field
The present invention relates to a kind of cancer therapy drug and method for making thereof, specifically is a kind of anti-cancer active matter kaurane type diterpene compound monomer of raw material extraction and preparation method thereof with slick and sly sweetsop.
Two, background technology
Cancer is the formidable enemy of harm humans health.The annual New Development cases of cancer of China has 1,600,000 people approximately, that dies from cancer every year has 1,300,000 people approximately, rise year by year because of the dead number of cancer in the whole nation, China is the zone occurred frequently of cancers such as liver cancer, cancer of the stomach, the esophageal carcinoma, mammary cancer, lung cancer, and cancer mortality is high.
Still there are not the most of cancer patientss of effective cured substance at present.Though chemotherapeutics has certain curative effect to cancer, toxic side effect is big, and Chinese patent medicine is generally lower to the curative effect of cancer, is 3.5% as the former cancer kitchen range remission rate to lung, cancer of the stomach of ginseng lotus glue, and soft hard oral liquid is 6.0% to the cancer kitchen range remission rate of liver cancer, and curative effect is all undesirable.
The inventor has studied the preparation of the ethanol extraction of the root skin of slick and sly sweetsop (Annona glabra Linn) or bark or branches and leaves or seed or their mixture, shows that through animal experiment zoografting sarcoma S180 and liver cancer H22 are all had significant vivo antitumor result of treatment.On the basis of the above, the inventor extracts anticancer monomeric compound (activeconstituents) again from medicinal extract.
Three, summary of the invention
1, goal of the invention: the purpose of this invention is to provide a kind of high-efficiency low-toxicity, the wide anticancer monomeric compound of anticancer spectrum, is a kind of kaurane type diterpene compound monomer of extracting of raw material and in the application aspect the medicine of preparation treatment liver cancer, cancer of the stomach with slick and sly sweetsop specifically.
2, technical scheme: the inventor has done further research on the basis of existing technology again, finds that its anticancer activeconstituents mainly is a kaurane type diterpene compound monomer of the present invention in the medicinal extract of slick and sly sweetsop cauline leaf.And also there is not bibliographical information kaurane type diterpene compound ent-kauran-19-al-17-oic acid to be used for treatment for cancer such as anti-liver cancer, cancer of the stomach at present through data consultation.A kind of kaurane type diterpene compound monomer that the present invention extracts from slick and sly sweetsop (Annona glabra Linn), it is ent-kauran-19-al-17-oic acid, its molecular formula is C
20H
30O
3, chemical structural formula is as follows:
The preparation method of a kind of kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid the steps include:
A, slick and sly sweetsop stem that will be air-dry are pulverized the back and are extracted with the ethanol 2000ml-4000ml heating of 60%-95%, reclaim ethanol and then extracting solution concentrated, medicinal extract;
B, medicinal extract is used chloroform extraction again, chloroform extracted solution;
C, chloroform extracted solution being carried out silica gel (100-200 order) column chromatography, is eluent A wash-out with 2000ml-4000ml sherwood oil-chloroform (2: 1), obtains extract;
D, the extract of gained among the step C is carried out silica gel (200-300 order) column chromatography again, with 2000ml-4000ml sherwood oil-ethyl acetate (100: 2) is eluent B, obtains the 4th batch of compound under the wash-out and is kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid of the present invention.It can use the re-crystallizing in ethyl acetate purifying.
The present invention relates to the application of a kind of kaurane type diterpene compound monomer in the medicine of preparation treatment liver cancer.
Adopt conventional mtt assay.The SMMC-7721 or HepG2 liver cancer cell in vegetative period that takes the logarithm, adjusting cell concn with complete culture solution is 1 * 10
5/ ml, inoculating cell is in 96 orifice plates (1 * 10
4/ hole), to add the compound final concentration respectively be 200ug/ml, 100ug/ml, 50ug/ml, 10ug/ml to experimental group behind the 24h, other establishes 5-Fu group, no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole (making 3 plates simultaneously), and each concentration is established 9 multiple holes (dividing 3 repetitions).After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of incubator.After 72h was cultivated in dosing, every hole added 5mg/mlMTT reagent 10ul, continued to cultivate 4h, added DMSO100ul again, put oscillator concussion 15min, surveyed every hole OD value with microplate reader (λ 570nm).Inhibiting rate (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.
Experimental result shows, rising with kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid concentration, inhibiting rate to human liver cancer cell also raises, diterpene compound monomer ent-kauran-19-al-17-oic acid all has the obvious suppression effect at the above dosage of 50ug/ml to hepatoma cell growth, wherein the restraining effect of 200ug/ml concentration is the highest, and its inhibiting rate reaches 75.19%.
The present invention relates to the application of a kind of kaurane type diterpene compound monomer in the medicine of preparation treatment cancer of the stomach.
Adopt mtt assay to measure kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid (preparing) to the effect of BGC823 stomach cancer cell growth-inhibiting with dehydrated alcohol.The BGC823 stomach cancer cell of taking the logarithm vegetative period is adjusted to 1 * 10
5/ ml, 0.2ml are inoculated on 96 orifice plates, put 37 ℃, 5%CO
2Abandon original fluid after cultivating 24h, adding the compound final concentration respectively is 200ug/ml, 100ug/ml, 50ug/ml, 10ug/ml, each concentration is established 9 multiple holes (dividing 3 repetitions), and establishes 5-Fu group, no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole (making 3 plates simultaneously) in addition.Supernatant liquor is removed in effect 24h hypsokinesis, and each hole adds MTT0.1ml, inhales behind 37 ℃ of 4h and removes supernatant liquor, adds DMSO0.1ml in each hole, goes up microplate reader 570nm place behind the 30min and surveys the OD value.Inhibiting rate (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.
Experimental result shows, rising with kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid concentration, inhibiting rate to stomach cancer cell also raises, growth has obvious restraining effect to diterpene compound monomer ent-kauran-19-al-17-oic acid to stomach cancer cell at the above dosage of 50ug/ml, wherein the restraining effect of 200ug/ml dosage is the highest, and its inhibiting rate reaches 68.50%.
According to the inventor's research, kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid can be used to prepare the medicine of anti-liver cancer or anti-cancer of the stomach.
3, beneficial effect: kaurane type diterpene compound monomer ent-kauran-19-al-l7-oic acid of the present invention is the anticancer monomeric compound that extracts from the slick and sly sweetsop of plant, toxic side effect is little, anticancer spectrum is wide, and cancer cells is had clearly restraining effect.Kaurane type diterpene compound monomer of the present invention is through anticancer pharmacological evaluation, and the result shows its antitumour activity height.Slick and sly sweetsop kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid is more than 50ug/ml concentration, hepatoma cell growth there is the obvious suppression effect, restraining effect is the highest when 200ug/ml dosage, and its inhibiting rate reaches 75.19%; Growth all has obvious restraining effect to gastric carcinoma cells at the above dosage of 50ug/ml, and wherein the restraining effect of 200ug/ml dosage is the highest, and its inhibiting rate reaches 68.50%.
Four, embodiment
Embodiment 1: the extracting method of slick and sly sweetsop kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid
1. slick and sly sweetsop plant sample picks up from Hainan, behind natural air drying, pulverize, the slick and sly sweetsop stem 3.5kg of pulverizing, the alcohol 3000ml refluxing extraction with 95%, reclaim concentrate behind the ethanol medicinal extract 300g;
2. medicinal extract is used chloroform extraction again, obtain chloroform extracted solution;
3. with chloroform extracted solution, (the 100-200 order 2kg) carries out column chromatography, is eluent A with 3000ml sherwood oil-chloroform (2: 1), obtains the extract part through silica gel;
4. extract recycle silicon glue (200-300 order) 300g is carried out column chromatography, with 3000ml petroleum ether-ethyl acetate (100: 2) is that eluent B carries out wash-out, every 50ml elutriant is that a unit accepts respectively, elutriant is made the silica gel thin sheet chromatography respectively, heat colour developing in 3 minutes down at 105 ℃ behind the fragrant oxalaldehyde sulphuric acid soln of thin plate, merge identical elutriant, the compound under the 4th batch of wash-out is kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid of the present invention.
Embodiment 2: the structure of kaurane type diterpene compound monomer ent-kauran-19-al-17-oic acid is identified:
Ent-kauran-19-al-17-oic acid is a white powder, is soluble in organic solvents such as chloroform, ethyl acetate, fusing point 199-203 ℃ (CHCI
3).
1HNMR prompting ent-kauran-19-al-17-oic acid is the kaurane type diterpene compound monomer.
Molecular formula C
20H
30O
3, its chemical structural formula is as follows:
Embodiment 3: the antihepatocarcinoma effect of diterpene compound monomer ent-kauran-19-al-17-oic acid
1, cell cultures and compound:
Human hepatoma cell strain SMMC-7721 or HepG2 routine are incubated in the RPM11640 nutrient solution that contains 10% deactivation calf serum, 100U/ml penicillin and 100ug/ml Streptomycin sulphate, put saturated humidity, 37 ℃ 5%CO
2Incubator is cultivated.Monomeric compound is the slick and sly sweetsop diterpene compound monomer ent-kauran-19-al-17-oic acid that identifies through chemical structure.
2, experimental technique:
Adopt conventional mtt assay.The SMMC-7721 or HepG2 liver cancer cell in vegetative period that takes the logarithm, adjusting cell concn with complete culture solution is 1 * 10
5/ ml, inoculating cell is in 96 orifice plates (1 * 10
4/ hole), to add diterpene compound ent-kauran-19-al-17-oic acid final concentration respectively be 200ug/ml, 100ug/ml, 50ug/ml, 10ug/ml to experimental group behind the 24h, other establishes 5-Fu group, no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole (making 3 plates simultaneously), and each concentration is established 9 multiple holes (dividing 3 repetitions).After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of incubator.After 72h was cultivated in dosing, every hole added 5mg/mlMTT reagent 10ul, continued to cultivate 4h, added DMSO100ul again, put oscillator concussion 15min, surveyed every hole OD value with microplate reader (λ 570nm).Inhibiting rate (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.
3, experimental result
Result such as table 1, table 2 show, rising with the diterpene compound monomer concentration, inhibiting rate to human liver cancer cell also raises, and diterpene compound monomer ent-kauran-19-al-17-oic acid all has obvious restraining effect at the above dosage of 50ug/ml to hepatoma cell growth.
Table 1 ent-kauran-19-al-17-oic acid is to the restraining effect (n=9) of SMMC-7721 liver cancer cell growth
Drug level (ug/ml) OD value (inhibiting rate (%) of A ± S)
24h 48h 24h 48h
0 0.570±0.017 0.673±0.021
5-Fu 100 0.128±0.012 0.135±0.015 77.54 79.94
Diterpene compound 10 0.440 ± 0.016 0.457 ± 0.019 22.81 32.10
Monomer 50 0.403 ± 0.017 0.330 ± 0.014 29.30 50.97**
100 0.323±0.013 0.267±0.015 43.33* 60.33**
200 0.150±0.010 0.173±0.012 73.68** 74.29**
* P＜0.05, * * P＜0.01 and control group comparison
Table 2 ent-kauran-19-al-17-oic acid is to the restraining effect (n=9) of HepG2 liver cancer cell growth
Drug level (ug/ml) OD value (inhibiting rate (%) of A ± S)
24h 48h 24h 48h
0 0.186±0.011 0.270±0.013
5-Fu 100 0.067±0.010 0.090±0.014 63.98 66.67
Diterpene compound 10 0.183 ± 0.012 0.180 ± 0.015 1.61 32.33
Monomer 50 0.147 ± 0.013 0.153 ± 0.012 20.97 43.03*
100 0.096±0.010 0.133±0.013 48.39** 50.74**
200 0.053±0.009 0.067±0.010 71.51** 75.19**
* P＜0.05, * * P＜0.01 and control group comparison
The anti-cancer of the stomach effect of embodiment 4:ent-kauran-19-al-17-oic acid
1, cell cultures and monomeric compound
People's cancer of the stomach BGC823 cell cultures in the RPM11640 nutrient solution, in add 10% calf serum, cultivate at 37 ℃, 5%CO
2In the incubator.Monomeric compound is the diterpene compound monomer ent-kauran-19-al-17-oic acid that identifies through chemical structure.
2, experimental technique
Adopt conventional mtt assay.The BGC823 stomach cancer cell of taking the logarithm vegetative period is adjusted to 1 * 10
5/ ml, 0.2ml are inoculated on 96 orifice plates, put 37 ℃, 5%CO
2Abandon original fluid after cultivating 24h, adding the compound final concentration respectively is 200ug/ml, 100ug/ml, 50ug/ml, 10ug/ml, each concentration is established 9 multiple holes (dividing 3 repetitions), and establishes 5-Fu group, no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole (making 3 plates simultaneously) in addition.Supernatant liquor is removed in effect 24h hypsokinesis, and each hole adds MTT0.1ml, inhales behind 37 ℃ of 4h and removes supernatant liquor, adds DMSO0.1ml in each hole, goes up microplate reader 570nm place behind the 30min and surveys the OD value.Inhibiting rate (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.
3, experimental result
The result with the rising of diterpene compound monomer concentration, also raises to the inhibiting rate of stomach cancer cell as shown in Table 3, and growth has obvious restraining effect to diterpene compound monomer ent-kauran-19-al-17-oic acid to stomach cancer cell at the above dosage of 50ug/ml.
Table 3 ent-kauran-19-al-17-oic acid is to stomach cancer cell growth-inhibiting effect (n=9)
Drug level (ug/ml) OD value (inhibiting rate (%) of A ± S)
24h 48h 24h 48h
0 0.320±0.019 0.327±0.016
5-Fu 100 0.173±0.012 0.009±0.013 45.94 72.48
Diterpene compound 10 0.241 ± 0.018 0.220 ± 0.017 24.69 32.72
Monomer 50 0.193 ± 0.015 0.193 ± 0.015 39.97* 40.98*
100 0.187±0.010 0.177±0.014 41.56* 45.87*
200 0.117±0.009 0.103±0.010 63.44** 68.50**
* P＜0.05, * * P＜0.01 and control group comparison
Claims (8)
1, a kind of cancer therapy drug kaurane type diterpene compound monomer is characterized in that it is ent-kauran-19-al-17-oic acid, and molecular formula is C
20H
30O
3, its chemical structural formula is as follows:
2, a kind of method for preparing the described ent-kauran-19-al-17-oic acid of claim 1 is characterized in that preparation process is as follows:
A, slick and sly sweetsop stem that will be air-dry are pulverized the back and are extracted with the ethanol heating, reclaim ethanol and then extracting solution concentrated, medicinal extract;
B, medicinal extract, use chloroform extraction again, chloroform extracted solution;
C, chloroform extracted solution being carried out silica gel (100-200 order) column chromatography, is eluent A wash-out with sherwood oil-chloroform, obtains extract;
D, the extract of gained among the step C being carried out silica gel (200-300 order) column chromatography again, is eluent B with sherwood oil-ethyl acetate, obtains the 4th batch of compound under the wash-out and is ent-kauran-19-al-17-oic acid of the present invention.
3, preparation method according to claim 2 is characterized in that alcohol concn is 60%-95% in steps A, and consumption is 2000ml-4000ml.
4, preparation method according to claim 2 is characterized in that eluent A sherwood oil-chloroform was with 2: 1 proportionings among the step C, and consumption is 2000ml-4000ml.
5, preparation method according to claim 2 is characterized in that eluent B sherwood oil-ethyl acetate was with 100: 2 proportionings among the step D, and consumption is 2000ml-4000ml.
6, the application of diterpene compound monomer ent-kauran-19-al-17-oic acid in preparation anti-liver cancer of treatment or anti-cancer of the stomach medicine.
7, the application of ent-kauran-19-al-17-oic acid according to claim 6 in preparation anti-liver cancer of treatment or anti-cancer of the stomach medicine is characterized in that ent-kauran-19-al-17-oic acid has obvious restraining effect at the above dosage of 50ug/ml to hepatoma cell growth; Growth all has obvious restraining effect to gastric carcinoma cells at the above dosage of 50ug/ml.
8, the application of ent-kauran-19-al-17-oic acid according to claim 7 in anti-liver cancer of preparation or anti-cancer of the stomach medicine, it is characterized in that ent-kauran-19-al-17-oic acid is when 200ug/ml dosage, restraining effect to hepatoma cell growth is the highest, and its inhibiting rate is 75.19%; When 200ug/ml dosage, the highest to the restraining effect of gastric carcinoma cells growth, its inhibiting rate is 68.50%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900046B (en) * | 2005-07-20 | 2011-03-30 | 北京华医神农医药科技有限公司 | Kaurane diterpine compound and its preparing method and use |
| CN101375936B (en) * | 2007-08-29 | 2012-02-29 | 北京星昊医药股份有限公司 | Anti-tumor compound cherimoya formulation and preparation method thereof |
| CN102584760A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院上海药物研究所 | Enantiomorphous-kaurene diterpene and derivative and preparation method thereof |
| CN115282135A (en) * | 2022-07-26 | 2022-11-04 | 扬州大学 | Application of ent-kaurane diterpenoid DKA in preparation of anti-tumor metastasis drugs or inhibitors |
-
2004
- 2004-07-02 CN CN 200410041150 patent/CN1594267A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900046B (en) * | 2005-07-20 | 2011-03-30 | 北京华医神农医药科技有限公司 | Kaurane diterpine compound and its preparing method and use |
| CN101375936B (en) * | 2007-08-29 | 2012-02-29 | 北京星昊医药股份有限公司 | Anti-tumor compound cherimoya formulation and preparation method thereof |
| CN102584760A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院上海药物研究所 | Enantiomorphous-kaurene diterpene and derivative and preparation method thereof |
| CN102584760B (en) * | 2011-01-13 | 2015-06-17 | 中国科学院上海药物研究所 | Enantiomorphous-kaurene diterpene and derivative and preparation method thereof |
| CN115282135A (en) * | 2022-07-26 | 2022-11-04 | 扬州大学 | Application of ent-kaurane diterpenoid DKA in preparation of anti-tumor metastasis drugs or inhibitors |
| CN115282135B (en) * | 2022-07-26 | 2023-07-21 | 扬州大学 | Application of enantiomer-kaurane diterpenoid DKA in preparation of anti-tumor metastasis drugs or inhibitors |
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