CN110105422B - Uncaria indole diterpenoid alkaloid and preparation method and application thereof - Google Patents
Uncaria indole diterpenoid alkaloid and preparation method and application thereof Download PDFInfo
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- CN110105422B CN110105422B CN201910508550.7A CN201910508550A CN110105422B CN 110105422 B CN110105422 B CN 110105422B CN 201910508550 A CN201910508550 A CN 201910508550A CN 110105422 B CN110105422 B CN 110105422B
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- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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Abstract
The invention relates to an uncaria indoxyl diterpenoid alkaloid and a preparation method and application thereof, wherein the uncaria indoxyl diterpenoid alkaloid has a structure shown in a formula I:
Description
Technical Field
The invention belongs to the field of natural plant extraction, and particularly relates to uncaria indole diterpenoid alkaloids and a preparation method and application thereof.
Background
Uncaria rhynchophylla (Miq.) Jacks is a plant of Uncaria of Rubiaceae (Rubiaceae), which is one of the main basic source plants of traditional Chinese medicine Uncaria rhynchophylla, and the medicinal part is stem branch with hook, and has effects of calming wind, arresting convulsion, clearing heat and calming liver, etc. Various indole alkaloids (mainly uncarine) are separated from uncaria. The uncaria indole diterpenoid alkaloid compound with a novel structure is prepared by ethanol reflux extraction, silica gel column chromatography and high performance liquid chromatography, and the cytotoxic activity of the uncaria indole diterpenoid alkaloid compound is tested.
Disclosure of Invention
The invention provides uncaria indole diterpenoid alkaloids or pharmaceutically acceptable salts thereof, which are characterized in that the uncaria indole diterpenoid alkaloids have a structure shown in a formula I:
another embodiment of the present invention provides a method for preparing the uncaria indole diterpenoid alkaloids, which is characterized by comprising the following steps:
(1) pulverizing dried stem and branch of ramulus Uncariae cum uncis, extracting with 95% ethanol under reflux for 10-12 hr, filtering, and concentrating the filtrate to obtain extract;
(2) and (2) carrying out chromatographic separation on the extract obtained in the step (1) to obtain the uncaria indole diterpenoid alkaloid.
The pulverization in the step (1) is preferably 20-100 meshes, and the dosage of 95% ethanol is preferably 8-10L per kg of dried stem and branch of uncaria with hooks;
the chromatographic separation in the step (2) is preferably combined with the preparation of silica gel column chromatography and high performance liquid chromatography; specifically, the extract is firstly subjected to silica gel column chromatography, the stationary phase is 200-mesh and 300-mesh silica gel, and the mobile phase adopts stonePerforming gradient elution by using oleyl ether-ethyl acetate according to the ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, collecting 3 column volumes in each gradient, combining fractions obtained by gradient elution according to the ratio of 90:10 and 80:20, concentrating, and preparing the uncaria indole diterpenoid alkaloid by using a high performance liquid chromatography; the chromatographic conditions adopted by the high performance liquid chromatography preparation are as follows: the chromatographic column is Agilent eclipse XDB-C18, 9.4X 250mm,5 μm, the flow rate is 1.5mL/min, and the mobile phase is MeOH H2O＝60:40。
Another embodiment of the invention provides an application of ramulus uncariae cum uncis with hooks in preparing the uncaria indolone diterpenoid alkaloid.
Another embodiment of the invention provides an application of the uncaria indole diterpenoid alkaloid or the pharmaceutically acceptable salt thereof in preparing an antitumor drug.
Another embodiment of the present invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises the above uncaria indole diterpenoid alkaloid or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition may also comprise other antineoplastic agents. The pharmaceutical composition may further comprise pharmaceutically acceptable adjuvants.
The 95% ethanol refers to an ethanol solution with a volume fraction of 95%.
Drawings
FIG. 1 is a compound of formula I1H NMR(CDCl3600MHz) graph;
FIG. 2 is a compound of formula I1H NMRδH7.9-7.0 magnification;
FIG. 3 is a compound of formula I1H NMRδH3.3-0.9 magnification;
FIG. 4 is a compound of formula I13C NMR(CDCl3150MHz) diagram;
FIG. 5 is a MS plot of a compound of formula I.
Detailed Description
In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.
Example 1
(1) Pulverizing dried stem and branch (100g) of ramulus Uncariae cum uncis with hook to 20-100 mesh, extracting with 95% ethanol (800mL) under reflux for 10 hr, filtering, and concentrating the filtrate to obtain extract;
(2) subjecting the extract obtained in step (1) to silica gel column chromatography with a stationary phase of 200-300 mesh silica gel, gradient elution with petroleum ether-ethyl acetate at a ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100 for a mobile phase, collecting 3 column volumes for each gradient, mixing fractions obtained by gradient elution at a ratio of 90:10 and 80:20, concentrating, and subjecting to high performance liquid chromatography (the chromatographic conditions are that the chromatographic column is Agilent Eclipse XDB-C18,9.4 × 250mm,5 μm, the flow rate is 1.5mL/min, and the mobile phase is MeOH: H2O60: 40) to obtain a white solid (38mg), namely the uncaria indole diterpenoid alkaloid with the structure of the formula I.
1H NMR(CDCl3,600MHz)δ:7.78(1H,s,NH-1),7.46-7.44(1H,m,H-20),7.33-7.30(1H,m,H-23),7.12-7.10(2H,m,H-21,22),3.26-3.22(1H,dd,J＝12.0,2.4Hz,H-9),3.08-3.04(1H,dd,J＝12.0,3.6Hz,H-7),2.82-2.75(1H,m,H-16),2.72-2.68(1H,dd,J＝13.3,6.6Hz,Ha-17),2.38-2.33(1H,dd,J＝13.3,10.9Hz,Hb-17),2.02-1.95(1H,m,Ha-5),1.87-1.77(2H,m,Hb-11,Ha-15),1.72-1.60(4H,m,Ha-14,Ha-10,Hb-5,Hb-15),1.52-1.39(3H,m,H-13,Hb-14,Hb-10),1.22(3H,s,CH3-29),1.21(3H,s,CH3-28),1.16(3H,s,CH3-26),1.15-1.14(1H,m,Ha-11),1.05(3H,s,CH3-25),0.91(3H,s,CH3-30)；13C NMR(CDCl3,125MHz)δ:150.8,140.0,125.1,120.4,119.5,118.4,118.2,111.4,85.7,84.7,72.0,53.0,48.8,46.4,40.0,37.7,36.6,33.9,26.1,25.3,24.6,23.7,22.0,21.9,20.0,14.6,12.7；ESIMSm/z[M+H]+422.61,[M+H]+444.60.
Example 2
The experiment tests the cytotoxicity of the hepatoma cell strain HepG2 according to the MTT methodAnd (4) activity. First, the cells in logarithmic phase are digested with pancreatin, and the cells are diluted uniformly to 2X 104The single cell suspension was inoculated into a 96-well plate and 100. mu.L each, followed by incubation in a cell incubator (isothermal 37 ℃ C., 5% CO)2Conditioned culture), 24h later when the cells had adhered, the compound of formula I was added to give a final concentration of 0.625. mu.g/mL, 1.25. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL, 25. mu.g/mL in this order, and three blank control wells and three positive control wells (positive control cisplatin) were provided, and 48h later the absorbance at 490nm of each well was measured using an automatic ELISA plate reader. Repeating the steps three times, and respectively calculating the inhibition rate and IC of the compound on tumor cells compared with negative control50The value is obtained. The results show that the IC of the compounds of the formula I according to the invention50The value was 14.2. + -. 2.1. mu.g/mL.
Claims (1)
1. A preparation method of uncaria indole diterpenoid alkaloids is characterized by comprising the following steps:
(1) pulverizing dried stem and branch of ramulus Uncariae cum uncis, extracting with 95% ethanol under reflux for 10-12 hr, filtering, and concentrating the filtrate to obtain extract;
(2) carrying out chromatographic separation on the extract obtained in the step (1) to obtain uncaria indole diterpenoid alkaloid;
the pulverization in the step (1) is to pulverize to 20-100 meshes, and the dosage of 95% ethanol used for each kilogram of dried stem branches with hooks of uncaria is 8-10L;
the chromatographic separation in the step (2) is the preparation combination of silica gel column chromatography and high performance liquid chromatography; the specific steps are that the extract is firstly subjected to silica gel column chromatography, the stationary phase is 200-mesh 300-mesh silica gel, the mobile phase adopts petroleum ether-ethyl acetate to carry out gradient elution according to the ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, 3 column volumes are collected in each gradient, fractions obtained by gradient elution according to the ratio of 90:10 and 80:20 are combined and concentrated, and then the uncaria indole diterpene alkaloid is prepared by high performance liquid chromatography; the chromatographic conditions adopted by the high performance liquid chromatography preparation are as follows: the chromatographic column is Agilent Eclipse XDB-C18, 9.4X 250mm,5 μm, the flow rate is 1.5mL/min, and the mobile phase is MeOH H2O＝60:40；
The uncaria indole diterpenoid alkaloid has a structure shown in a formula I:
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5541208A (en) * | 1994-01-24 | 1996-07-30 | Merck & Co., Inc. | Indole diterpene alkaloid compounds |
| US6548535B2 (en) * | 2000-01-18 | 2003-04-15 | Merck & Co., Inc. | Method for treating ocular hypertension |
| WO2003105868A1 (en) * | 2002-06-17 | 2003-12-24 | Merck & Co., Inc. | Novel maxi-k channel blockers, methods of use and process for making the same |
| CN102311436A (en) * | 2011-07-05 | 2012-01-11 | 南京泽朗农业发展有限公司 | Preparation method of isorhynchophylline |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5541208A (en) * | 1994-01-24 | 1996-07-30 | Merck & Co., Inc. | Indole diterpene alkaloid compounds |
| US6548535B2 (en) * | 2000-01-18 | 2003-04-15 | Merck & Co., Inc. | Method for treating ocular hypertension |
| WO2003105868A1 (en) * | 2002-06-17 | 2003-12-24 | Merck & Co., Inc. | Novel maxi-k channel blockers, methods of use and process for making the same |
| CN102311436A (en) * | 2011-07-05 | 2012-01-11 | 南京泽朗农业发展有限公司 | Preparation method of isorhynchophylline |
Non-Patent Citations (1)
| Title |
|---|
| Alkaloids from the endophytic fungus Penicillium brefeldianum and their cytotoxic activities;Na Gao et al.;《Chinese Chemical Letters》;20170302;第28卷;1194-1199 * |
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