CN100450490C - Use of caudatin-3-O-beta-D-cymaroside as medicament for treating tumour - Google Patents
Use of caudatin-3-O-beta-D-cymaroside as medicament for treating tumour Download PDFInfo
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- CN100450490C CN100450490C CNB2007100200328A CN200710020032A CN100450490C CN 100450490 C CN100450490 C CN 100450490C CN B2007100200328 A CNB2007100200328 A CN B2007100200328A CN 200710020032 A CN200710020032 A CN 200710020032A CN 100450490 C CN100450490 C CN 100450490C
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Abstract
The invention relates to a method for preparing the caudatin-3-O-beta-D-cymarose glycoside as the anti-tumor drug, while the caudatin-3-O-beta-D-cymarose glycoside can be extracted from the plant especially the drug plant white polygonum. The external test has proved the restrain functions of caudatin-3-O-beta-D-cymarose glycoside on the human tumor cells, while it can induce the death of tumor cells, and the test has proved that it can be used to treat the transplant liver cancer of mouse.
Description
Technical field
The present invention relates to natural medicine field, be specifically related to a kind of chemical compound Caudatin .-3-O-β-D-Apocynum cannabinum glucosides as the application in the medicine for treating tumor thing.
Background technology
Tumor has become one of the highest disease kind of sickness rate and fatality rate in the west, the M ﹠ M of China's malignant tumor also has the trend that increases year by year, and every dead 6 philtrums just have 1 to be the cancer patient, have 1 family relevant with cancer stricken in per 200 families.If cardiovascular and cerebrovascular disease is separately added up, present malignant tumor has become first of the human cause of death.Medical circle is when seeking and using cancer therapy drug, find that many chemical anticarcinogenic drugs often involve normal cell when acting on target cell, and the most of kinds of the chemicals that is used for the treatment of tumor clinically all have mutagenesis genetoxic in various degree, have increased the probability that patient suffers from second kind of tumor when treating tumor for this reason; As if but the genetoxic of plant amedica is not too obvious.Therefore, should be following main trend to the research and development of anti-tumor botanical, anti-tumor botanical has vast market prospect.
Radix Cynanchi Auriculati is one of motherland's Chinese medicine, and the medicine source is mainly from the tuber of asclepiadaceae Cynanchum plant cynanchum auriculatum Royle (Cynanchumauriculatum Royle ex Wight).Radix Cynanchi Auriculati is considered as the anti-old treasure of health preserving in history as tonification Chinese medicine by ancient Chinese medicine doctor, effects such as the liver benefiting that nourishes blood, the benefit of reinforcing the kidney essence, strengthening the tendons and bones, pitch-black beard and hair and life lengthening.
Modern pharmacological research shows, Radix Cynanchi Auriculati can significantly improve body specificity and nonspecific immunity, removing oxygen-derived free radicals, the liver protecting, heart tonifying, reduction serum cholesterol, suppress effects such as monoamine oxidase-B enzymatic activity, antitumor in the brain; The little malicious scope of the oral genus of radix cynanchi bungei total glucoside, no mutagenic action.
In further research to Radix Cynanchi Auriculati, research worker is therefrom separated and has been obtained number of C-21 steroid, as the new glycosides A of Radix Cynanchi Auriculati, B, its chemical constitution is: Caudatin .-3-O-β-D-Glucopyranyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside; Caudatin .-3-O-β-D-Glucopyranyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-digoxigenin pyrans glycosyl-(1 → 4)-β-D-Apocynum cannabinum pyranoside, these two monomers have shown certain anti-tumor activity, these two chemical compounds of in-vitro pharmacological experiments are 64～74% (" Acta Pharmaceutica Sinicas " 2006,35 (6): 431-437) to the suppression ratio of some tumor.
CN1342654A has also reported several C-21 steroids that separation obtains from Radix Cynanchi Auriculati, and they also have certain anti-tumor activity.Several Caudatin .s in this patent-3-O-β-D-Glucopyranyl-(1 → 4)-β-D-Apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-external antagonism Human Prostate Cancer Cells of D-Apocynum cannabinum pyranoside compounds PC
3, human lung carcinoma cell PAA, human cervical carcinoma cell Hela, colorectal cancer cells Hcc-8693 IC50 (μ g/ml) be about 26～102.In the test of radix cynanchi bungei total glucoside anti-tumor in vivo, the tumour inhibiting rate to the S180 tumor when oral dose is 20mg/kg, 40mg/kg, 80mg/kg distinguishes 35.1%, 39.7%, 47.7%.To the EC ascites tumor under these three dosage is that tumour inhibiting rate distinguishes 22.4%, 29.2%, 44.3%.
Not seeing in the existing document has the therapeutic activity report of such monomer to hepatocarcinoma, and another different with above-mentioned monomer structure in addition monomers: the pharmacologically active of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides does not appear in the newspapers.
Summary of the invention
The invention discloses a kind of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides and be used for anti-tumor application, described Caudatin .-3-O-β-D-cymarose can separate from Radix Cynanchi Auriculati and obtains, and structural formula is as follows:
Pharmacological testing proves that Caudatin .-3-O-β-D-Apocynum cannabinum glucosides has stronger anti-tumor activity, especially hepatocarcinoma is had very strong inhibition effect.In-vitro pharmacological experiments proves, the IC of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides antagonism tumor
50Be 16.64 μ mol/L, anti-tumor in vivo evidence, the inhibitory rate to 34.57% of its oral 20mg/kg dosage group.
Description of drawings
Fig. 1 is the apoptosis figure of the cell of blank group
Fig. 2 is the apoptosis figure of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 120umol/L group
Fig. 3 is the apoptosis figure of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 60umol/L group
The specific embodiment
Embodiment 1
The preparation of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides
Cynanchum auriculatum Royle dried root 15kg, with alcohol reflux 3 times, each 2 hours, merge extractive liquid,, decompression and solvent recovery, ethanol extraction, this extract with aqueous dispersion after, defat with petroleum ether 3 times, chloroform extraction 3 times, the reclaim under reduced pressure chloroform gets chloroform extract.The chloroform extract silica gel column chromatography, carry out gradient elution with chloroform-methanol, the thin layer chromatography inspection is shown, chloroform: methanol (95: 5) exhibition layer, the Rf0.5 flow point merges, and uses RP-18 normal pressure column chromatographic isolation and purification then, methanol: water (68: 32) eluting, obtain product Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 4.5 grams, content is greater than 85%.
Caudatin .-3-O-β-unformed the powder of D-Apocynum cannabinum glucosides white, mp:152-155 ℃.UV
max WeOH?nm：221。ESI-MSn/z：633.4[M-H]
-，657.3[M+Na]
+。Molecular formula C
35H
51O
10,
1H-NMR (C
5D
5N): δ 0.94,0.96 (each 3H, d, J=4.55Hz, 5 ', 6 '-CH
3), 1.40 (3H, S, 18-CH
3), 1.98 (3H, s, 19-CH
3), 2.27 (3H, s, 7 '-CH
3), 2.46 (3H, s, 21-CH
3), 3.26 (1H, m, 7-CH), 3.87 (1H, m, 3 α-H), 5.17 (1H, dd, J=9.4Hz, 1 " H), 5.20 (1H, dd, J=11.45,4.2Hz, 12 α-H), 5.33 (1H, brs, 6-CH), 5.86 (1H, s, 2 '-CH).
13C-NMR：39.2(C-1)，29.9(C-2)，77.5(C-3)，39.0(C-4)，139.4(C-5)，119.2(C-6)，33.9(C-7)，74.4(C-8)，44.6(C-9)，37.4(C-10)，25.14(C-11)，72.7(C-12)，58.0(C-13)，89.5(C-14)，34.8(C-15)，32.9(C-16)，92.4(C-17)，10.6(C-18)，18.3(C-19)，209.3(C-20)，27.5(C-21)，165.9(C-1′)，114.2(C-2′)，165.2(C-3′)，38.1(C-4′)，21.0(C-5′)，20.9(C-6′)，16.5(C-7′)，96.3(C-1″)，36.1(C-2″)，78.9(C-3″)，74.2(C-4″)，70.9(C-5″)，19.1(C-6″)，58.0(C-7″)。
Embodiment 2
Pharmaceutical preparation
Get Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 100g, disperse with an amount of water for injection, add lactose 200g, mixing adds the 100mg magnesium stearate, and add water and make soft material in right amount, drying, the granule tabletting is made 1000.
Embodiment 3
Anticancer experiment in vitro
(1) mensuration of IC50
Be in the tumor cell of exponential phase with 0.25% trypsinization, be made into the individual cells suspension with the RPMI-1640 culture medium that contains 10% calf serum, with 5000 cell inoculations in every hole in 96 well culture plates.Behind the cell inoculation, at 37 ℃, 5%CO
2And cultivated 24 hours under the saturated humidity condition.Other establishes the blank hole, only adds complete culture medium, does not add cell.Caudatin .-3-O-β-D-Apocynum cannabinum glucosides is made into the concentration of 55 times of dilutions, adds in the corresponding culture hole, its final concentration is respectively 300,60,12,2.4,0.48 μ mol/L.Cell continued in incubator continuous culture 48 hours.Then, every hole adds tetrazolium bromide (MTT, Sigma) solution, the continuation cultivation 4 hours of 10 μ l5mg/ml.The careful suction goes supernatant, every hole to add 150 μ lDMSO, and the Shi Jia Za that vibrates gently dissolves fully.On microplate reader, measure OD value (returning to zero) with the blank hole in 570nm wavelength place.Be calculated as follows inhibition rate of tumor cell, and calculate IC
50
Cell inhibitory rate (%)=(matched group OD value-medicine group OD value)/matched group OD value * 100%
(2) to the vitro inhibition effect of human hepatoma cell strain SMMC-7721
Experimental technique is the same, with 5000 cell inoculations in every hole in 96 well culture plates.Behind the cell inoculation, at 37 ℃, 5%CO
2And cultivated 24 hours under the saturated humidity condition.Other establishes the blank hole, only adds complete culture medium, does not add cell.Caudatin .-3-O-β-D-Apocynum cannabinum glucosides is made into 4 variable concentrations, adds in the corresponding culture hole, its final concentration is respectively 120,60,30,15 μ mol/L.Cell continued in incubator continuous culture 24,48,72 hours.Then, every hole adds the MTT solution of 10 μ l5mg/ml, continues to cultivate 4 hours.The careful suction goes supernatant, every hole to add 150 μ lDMSO, and the Shi Jia Za that vibrates gently dissolves fully.On microplate reader, measure OD value (returning to zero) with the blank hole in 570nm wavelength place.Be calculated as follows inhibition rate of tumor cell, draw growth curve.
Cell inhibitory rate (%)=(matched group OD value-medicine group OD value)/matched group OD value * 100%
(3) to the external evoked apoptotic effect of human hepatoma cell strain SMMC-7721
Experimental technique is the same, with 5000 cell inoculations in every hole in 96 well culture plates.Behind the cell inoculation, at 37 ℃, 5%CO
2And cultivated 24 hours under the saturated humidity condition.Other establishes the blank hole, only adds complete culture medium, does not add cell.Caudatin .-3-O-β-D-Apocynum cannabinum glucosides is made into 2 variable concentrations, adds in the corresponding culture hole, its final concentration is respectively 120,60 μ mol/L.Cell continued in incubator continuous culture 72 hours.Then, collecting cell detects the apoptosis situation with AnnexinV-FITC apoptosis test regent box with flow cytometer.
Result of the test:
Adopt mtt assay to measure the anti tumor activity in vitro of Caudatin .-3-O-β-D-Apocynum cannabinum glucosides to human liver cancer cell, it is 16.64 μ mol/L that the result records its IC50.Simultaneously, to Caudatin .-3-O-β-D-Apocynum cannabinum glucosides under the concentration of 120,60,30,15 μ mol/L, the suppression ratio of different action times measures, experimental result finds that chemical compound Caudatin .-external inhibitory action to human liver cancer cell SMMC-7721 of 3-O-β-D-Apocynum cannabinum glucosides is tangible dose-effect and time-effect relationship.The results are shown in Table 1, table 2.
The external IC50 to human liver cancer cell SMMC-7721 of table 1 Caudatin .-3-O-β-D-Apocynum cannabinum glucosides measures
The table 2 Caudatin .-external inhibitory action of 3-O-β-D-Apocynum cannabinum glucosides to human liver cancer cell SMMC-7721
With AnnexinV-FITC apoptosis test regent box, detect the apoptosis situation with flow cytometer, the result shows, Caudatin .-3-O-β-D-Apocynum cannabinum glucosides is under the concentration of 120,60 μ mol/L, in the time of 48 hours, can induce the apoptosis of human liver cancer cell SMMC-7721 significantly, the matched group apoptosis rate: 1.9%, Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 120umol/L apoptosis rate: 29.8%; Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 60umol/L apoptosis rate: 5.9%.See Fig. 1, Fig. 2, Fig. 3.
Embodiment 4
The anti-tumor in vivo experiment
100 of Kunming mouses, male and female half and half, body weight 18-22 gram, all mices are all in the H of right fore armpit subcutaneous vaccination dilution in 1: 4
22Oncocyte liquid 0.2ml/ Mus.Next day, tumor-bearing mice is divided into 4 groups at random was lotus tumor matched group, fluorouracil (5-FU) chemotherapy group and Caudatin .-3-O-β-D-Apocynum cannabinum glucosides high and low dose group behind inoculated tumour.The administration group is irritated stomach respectively and is given Caudatin .-3-O-β-D-Apocynum cannabinum glucosides 20mg/kg and 10mg/kg, and lotus tumor matched group filling stomach gives the distilled water with volume, the equal successive administration of mice 10 days.Positive drug group mice behind inoculated tumour the 3rd day, every day lumbar injection fluorouracil 25mg/kg, continuous 7 days.After the last administration 24 hours, mice takes off cervical vertebra put to death, and peels off sarcoma, scales/electronic balance weighing.Calculate and respectively organize heavy meansigma methods of mouse tumor and tumour inhibiting rate.
Inoculation liver-cancer solid tumor H22 in the mice body, and the filling stomach gives chemical compound Caudatin .-3-O-β-D-Apocynum cannabinum glucosides, found that this product has significant inhibitory effect to mouse entity tumor H22, show that Caudatin .-3-O-β-D-Apocynum cannabinum glucosides oral administration has obvious inhibitory action to rat liver cancer solid tumor H22, proves that this product has significant anti-tumor in vivo activity.See Table 3.
The influence that table 3 Caudatin .-3-O-β-D-Apocynum cannabinum glucosides is heavy to rat liver cancer H22 tumor (x ± s)
Annotate: compare (t-check) with lotus tumor matched group,
*P＜0.05,
* *P＜0.001.
Claims (1)
1, following formula: compound is used to prepare the purposes of the medicine for the treatment of liver cancer diseases
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| CN104693259B (en) * | 2015-02-14 | 2016-06-15 | 陶雪芬 | A kind of aspirin Caudatin. esters derivative compound and its preparation method and application |
| KR102467971B1 (en) * | 2020-11-16 | 2022-11-16 | 제주대학교 산학협력단 | Composition for anti-cancer comprising caudatin compound |
Citations (2)
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|---|---|---|---|---|
| CN1342654A (en) * | 2000-12-16 | 2002-04-03 | 浙江省医学科学院 | C21 steriod glycoside extracted from white fleece flower root with antineoplastic function |
| CN1660865A (en) * | 2004-12-29 | 2005-08-31 | 中国人民解放军第二军医大学 | Saponin compound, general saponin of cynanchum bunaei and application in medication for treating disease of gstrointestinal tract |
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2007
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342654A (en) * | 2000-12-16 | 2002-04-03 | 浙江省医学科学院 | C21 steriod glycoside extracted from white fleece flower root with antineoplastic function |
| CN1660865A (en) * | 2004-12-29 | 2005-08-31 | 中国人民解放军第二军医大学 | Saponin compound, general saponin of cynanchum bunaei and application in medication for treating disease of gstrointestinal tract |
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| 百首乌中C21甾甙元的分离与结构鉴定. 宋俊梅，陶冠军，汤坚等.无锡轻工大学学报，第21卷第2期. 2002 * |
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Assignee: Jiangsu Sunan Pharmaceutical Industry Co., Ltd. Assignor: Jiangsu Provincial Insititute of Traditional Chinese Medicine Contract record no.: 2011320000998 Denomination of invention: Use of -3-O- beta -D- as a therapeutic drug for oncology Granted publication date: 20090114 License type: Exclusive License Open date: 20070808 Record date: 20110714 |
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