CN107011170B - Fucoxanthin derivative and preparation method and application thereof - Google Patents
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Abstract
The invention relates to five fucoxanthin derivatives, wherein the derivatives are prepared by taking fucoxanthin as a raw material, adding a reducing agent for reaction, and concentrating to obtain a fucoxanthin derivative crude extract; separating fucoxanthin derivative crude extract by silica gel column chromatography, separating into different components, and purifying by semi-preparative high performance liquid chromatography. The fucoxanthin derivative prepared by the invention has a strong effect of inhibiting tumor cell proliferation, and has a good clinical application prospect.
Description
Technical Field
The invention relates to a compound and a preparation method thereof, in particular to a fucoxanthin derivative, a preparation method and an application thereof, and especially relates to an application of the fucoxanthin derivative in preparing a medicament for treating cancers.
Background
Fucoxanthin (fucoxanthin), also known as fucoxanthin, is a natural carotenoid extracted from edible brown algae such as Undaria pinnatifida (Laminariaceae) and Laminaria japonica (Laminaria japonica Aresch), and has strong biological activity because both ends of its rigid all-trans long chain have a chemically active 5, 6-epoxy unsaturated allene bond structure, which is different from other carotenoid molecules. In recent years, various biological activities of the compound have been proved, and some potential activities are actively researched by scientists, so that the compound is one of the main focus points of research and development of marine medicines at present.
Traditional Chinese medicines or natural medicines show diversity and complexity in compound types and structures, are naturally-formed 'combined chemical sample libraries', and become important sources for finding lead compounds in the research and development of new medicines. China has rich Chinese herbal medicine resources, and lead compounds extracted and separated from the Chinese herbal medicine resources are structurally modified, so that the pharmacological activity is enhanced, the toxic and side effects are reduced, the pharmacokinetic properties are improved, and the method is an effective way for realizing modernization and innovation of new traditional Chinese medicines.
At present, few reports on fucoxanthin derivatives are reported at home and abroad. Fucoxanthin is one of the currently known fucoxanthin derivatives, is a metabolite of the fucoxanthin derivative without an acetyl group, has a structure similar to that of fucoxanthin, and has strong biological activity. Recent studies have shown that fucoxanthin can be converted into fucoxanthin derivatives for use in mice orally administered with fucoxanthin. Research of Hayato Maeda and the like also finds that fucoxanthin derivatives as fucoxanthin metabolites in 3T3-L1 cell lines can achieve the purpose of inhibiting differentiation from 3T3-L1 pre-adipogenic cell lines to adipocyte lines by reducing PPAR gamma (peroxisome proliferator activated receptor), thereby achieving the effect of losing weight. At present, no report of batch preparation research of fucoxanthin derivatives is found at home and abroad, and high-value development and application of fucoxanthin derivatives cannot be performed, so that a preparation method of high-purity fucoxanthin derivatives is needed to be invented, the biological activity of the fucoxanthin derivatives is researched, and an important basis is provided for development and application of functional products of the derivatives.
Disclosure of Invention
The invention aims to provide a series of fucoxanthin derivatives, and a preparation method and application thereof.
Specifically, the invention relates to a fucoxanthin derivative or a salt thereof, which is characterized by having a structure shown in the following formula (I) -formula (V):
the salts of the derivatives described in the present invention include, but are not limited to, inorganic acid salts such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid and hydrobromic acid, or salts of organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valeric acid.
The invention provides a pharmaceutical composition, which comprises the fucoxanthin derivative or the salt thereof as an active ingredient and can be used as a pharmaceutical adjuvant.
The medicinal auxiliary materials can be conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
Preferably, the dosage form is injection, capsule, tablet, powder, oral liquid.
The third aspect of the invention relates to the application of the fucoxanthin derivative or the salt thereof in preparing a medicament for treating cancer.
Wherein the cancer includes but is not limited to malignant melanoma, pancreatic cancer, thyroid undifferentiated cancer, metastatic malignant bone tumor, leukemia, lymph cancer, osteoma, chondrosarcoma, prostate cancer, esophageal cancer, gastric cancer, liver cancer, gallbladder cancer, rectal cancer, carcinoma of large intestine, colon cancer, lung cancer, nasopharyngeal carcinoma, nervous system cancer, breast cancer, ovarian cancer, cervical cancer, and uterine cancer.
The fourth aspect of the present invention provides a method for preparing the fucoxanthin derivative, which comprises the following steps:
1) fucoxanthin or a fucoxanthin-containing extract is used as a raw material;
2) dissolving the raw materials by using a solvent, and adding a certain proportion of reducing agent for reduction reaction to obtain a reaction solution containing the fucoxanthin derivatives;
3) concentrating the reaction liquid to dryness, and performing primary separation by adopting silica gel column chromatography to obtain components containing different fucoxanthin derivatives;
4) concentrating each part of separated liquid to be dry, adding mobile phase for dissolving, and separating and purifying by adopting semi-preparative/preparative liquid phase chromatography to obtain a prepared liquid phase purified liquid;
5) respectively concentrating the liquid phase purified solutions prepared from the above parts, drying the concentrated solution to obtain fucoxanthin derivatives, and performing structure analysis to determine the compounds represented by formula (I), formula (II), formula (III), formula (IV) and formula (V).
Preferably, the solvent in step 2) is an organic solvent, preferably methanol, ethanol, tetrahydrofuran.
Preferably, the weight ratio of the raw material to the reducing agent in step 2) is 50:1 to 1: 1. can be selected from 50:1, 40:1, 30:1, 20:1,10:1, 5:1 and 1: 1.
Preferably, the reducing agent is one or a combination of several of sodium borohydride, lithium aluminum hydride, stannous chloride, oxalic acid, potassium borohydride, ferrous sulfate, sodium sulfite and the like; among them, lithium aluminum hydride is most preferable.
Preferably, the reaction temperature in step 2) is 20-50 deg.C, and can be selected from 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C; the reaction time is 0.5-48h, and can be selected from 0.5h, 1h, 2h, 4h, 8h, 10h, 12h, 16h, 20h, 24h and 48 h.
Preferably, the silica gel column chromatography in step 3) is used for preliminary separation, and the silica gel column can be selected from normal phase silica gel and reverse phase silica gel.
Preferably, when normal phase silica gel column chromatography is adopted in step 3), the eluent may be petroleum ether-ethyl acetate or petroleum ether-acetone, and the ratio of the eluent is 20: 1-1: 1, when the reversed phase silica gel column chromatography is adopted, the eluent can be selected from methanol-water or ethanol-water, and the ratio is 7: 3 to 10: 0.
preferably, the mobile phase in the step 4) is three system solvents of methanol-water, acetonitrile-water and ethanol-water, and a solvent in which the three system solvents are mixed in any proportion.
Preferably, in the preparation method of the fucoxanthin derivative, the step 4) is that the fucoxanthin derivative series derivative components obtained in the step 3) are dissolved by using a mobile phase, transferred into a semi-preparative/preparative high performance liquid chromatography sample injection bottle, the semi-preparative/preparative high performance liquid chromatography is started for separation and purification, and the fucoxanthin derivative purified liquid is automatically collected by triggering a fraction collector through an ultraviolet online detection signal.
Preferably, the semi-preparative/preparative high performance liquid chromatography of step 4) uses a C8 column or a C18 column as packing.
Preferably, the semi-preparative/preparative high performance liquid chromatography of step 4) employs a semi-preparative/preparative column having a diameter of 10 to 50 mm.
Preferably, the semi-preparative high performance liquid chromatography of step 4) is carried out using a sample size of 100. mu.L/time to 20 mL/time.
Preferably, the semi-preparative high performance liquid chromatography described in step 4) uses a mobile phase flow rate of 5-200mL/min, preferably 5, 6, 10, 20, 28, 50, 80, 100, 120, 150, 180, 200 mL/min.
Preferably, the detector used in the semi-preparative high performance liquid chromatography in the step 4) is an ultraviolet detector or a diode array detector, and the detection wavelength is 400-500nm, preferably 450 nm.
Preferably, after the fucoxanthin derivative purified liquid in the step 5) is subjected to reduced pressure concentration, the concentrated liquid is subjected to freeze drying to obtain five high-purity fucoxanthin derivatives which are respectively represented by formula (I), formula (II), formula (III), formula (IV) and formula (V).
Drawings
FIG. 1 shows a chromatogram of a mixture of fucoxanthin derivatives
FIG. 2 shows a chromatogram representing a compound of formula (I), wherein FIG. 2-1 shows a liquid chromatogram of the compound of formula (I), FIG. 2-2 shows a hydrogen chromatogram of the compound of formula (I), and FIG. 2-3 shows a carbon chromatogram of the compound of formula (I);
FIG. 3 shows a spectrum of a compound of formula (II), wherein FIG. 3-1 shows a liquid chromatogram of a compound of formula (II), FIG. 3-2 shows a hydrogen spectrum of a compound of formula (II), and FIG. 3-3 shows a carbon spectrum of a compound of formula (II);
FIG. 4 shows a spectrum of a compound of formula (III), wherein FIG. 4-1 shows a liquid chromatogram of a compound of formula (III), FIG. 4-2 shows a hydrogen spectrum of a compound of formula (III), and FIG. 4-3 shows a carbon spectrum of a compound of formula (III);
FIG. 5 shows a chromatogram of the compound of formula (IV), wherein FIG. 5-1 shows a liquid chromatogram of the compound of formula (IV), FIG. 5-2 shows a hydrogen chromatogram of the compound of formula (IV), and FIG. 5-3 shows a carbon chromatogram of the compound of formula (IV);
FIG. 6 shows a spectrum of a compound of formula (V), wherein FIG. 6-1 shows a liquid chromatogram of a compound of formula (V), FIG. 6-2 shows a hydrogen spectrum of a compound of formula (V), and FIG. 6-3 shows a carbon spectrum of a compound of formula (V);
Detailed Description
Example 1
1) Preparation of fucoxanthin derivative mixture: dissolving fucoxanthin (purity of 99%) 500mg in methanol 200mL, adding lithium aluminum hydride 100mg, reacting at 37 deg.C for 10 hr, detecting by liquid chromatography that there are multiple fucoxanthin derivative chromatographic peaks, and concentrating to near dry;
2) primary separation of fucoxanthin derivative mixture: separating fucoxanthin derivative concentrate by silica gel column chromatography, eluting with petroleum ether-ethyl acetate (20: 1-2:1) to obtain fucoxanthin derivative silica gel column chromatography isolates 1-A, 1-B, 1-C, 1-D, 1-E, and 1-F, respectively concentrating for use;
3) separation and preparation of fucoxanthin derivatives (I):
a) preparation of raw materials: preparing the 1-A fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: semi-preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the semi-preparative chromatographic column is C8 column (250mm × 10mm), and the mobile phase system is 85%
The flow rate of a mobile phase of the methanol aqueous solution is 3mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 100 mu L of the solution;
obtaining fucoxanthin derivative preparation solution 80mL by running semi-preparative high performance liquid chromatography, concentrating at 30 deg.C under reduced pressure, freeze drying the concentrated solution to obtain fucoxanthin derivative 23mg, and identifying the compound as formula (I).
4) Separating and preparing the fucoxanthin derivative (II):
a) preparation of raw materials: preparing the 1-B fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: semi-preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the semi-preparative chromatographic column is C8 column (250mm × 10mm), and the mobile phase system is 85%
The flow rate of a mobile phase of the methanol aqueous solution is 3mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 100 mu L of the solution;
obtaining 60mL fucoxanthin derivative preparation solution by running semi-preparative high performance liquid chromatography, concentrating under reduced pressure at 30 deg.C, freeze drying the concentrated solution to obtain fucoxanthin derivative 18mg, and identifying the compound as formula (II).
5) Separation and preparation of fucoxanthin derivative (III):
a) preparation of raw materials: preparing a 1-C fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: semi-preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the semi-preparative chromatographic column is a C8 column (250mm multiplied by 10mm), the mobile phase system is 85% methanol water solution, the flow rate of the mobile phase is 3mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 100 mu L of the solution;
obtaining fucoxanthin derivative preparation solution 80mL by running semi-preparative high performance liquid chromatography, concentrating at 30 deg.C under reduced pressure, freeze drying the concentrated solution to obtain fucoxanthin derivative 23mg, and identifying the compound as formula (III).
6) Separating and preparing fucoxanthin derivatives (IV) and (V):
a) preparation of raw materials: preparing the 1-B fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: semi-preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the semi-preparative chromatographic column is a C8 column (250mm multiplied by 10mm), the mobile phase system is 90% methanol water solution, the flow rate of the mobile phase is 3mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 100 mu L of the solution;
by operating semi-preparative high performance liquid chromatography, respectively obtaining fucoxanthin derivatives (IV) and (V) preparation solutions, concentrating at 30 deg.C under reduced pressure, freeze drying the concentrated solution to obtain fucoxanthin derivatives (IV) and (V) 25mg and 29mg, respectively, and determining the compounds as formula (IV) and (V) by structure identification.
Example 2
1) Preparation of fucoxanthin derivative mixture: dissolving fucoxanthin (purity of 90%) 1000mg in methanol 500mL, adding lithium aluminum hydride 150mg, reacting at 37 deg.C for 10h, detecting by liquid chromatography that there are multiple fucoxanthin derivative chromatographic peaks, and concentrating to near dry;
2) primary separation of fucoxanthin derivative mixture: separating fucoxanthin derivative concentrate by reverse phase silica gel column chromatography with eluent of 60%, 70%, 80%, 90%, and 100% methanol water solution, gradient eluting, collecting fucoxanthin derivative reverse phase column chromatography isolates 2-A, 2-B, 2-C, 2-D, and 2-E, and concentrating respectively;
3) separating and preparing fucoxanthin derivatives (I), (II) and (IV):
a) preparation of raw materials: preparing the 2-C fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the semi-preparative chromatographic column is a C18 column (250mm multiplied by 20mm), the mobile phase system is 80% methanol water solution, the flow rate of the mobile phase is 10mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 500 mu L of the solution;
obtaining fucoxanthin derivatives (I), (II) and (IV) by running preparative high performance liquid chromatography, wherein the preparation solutions are 210mL, 250mL and 170mL respectively, concentrating under reduced pressure at 30 ℃, freeze-drying the concentrated solution to obtain the fucoxanthin derivatives 23mg, 17mg and 38mg respectively, and the structural identification confirms that the structural formulas of the compounds are respectively as follows: (I), (II), (IV).
4) Separating and preparing the fucoxanthin derivative (V):
a) preparation of raw materials: preparing the 2-D fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: semi-preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the preparative chromatographic column is a C18 column (250mm multiplied by 20mm), the mobile phase system is 85% methanol water solution, the flow rate of the mobile phase is 10mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 500 mu L of the solution;
obtaining 300mL fucoxanthin derivative preparation solution by running semi-preparative high performance liquid chromatography, concentrating under reduced pressure at 30 deg.C, freeze drying the concentrated solution to obtain fucoxanthin derivative 78mg, and identifying the compound as formula (V).
5) Separation and preparation of fucoxanthin derivative (III):
a) preparation of raw materials: preparing a 2-E fucoxanthin derivative crude product into a solution of 5 mg/mL;
b) the instrument comprises the following steps: preparative high performance liquid chromatography;
c) chromatographic conditions are as follows: the preparative chromatographic column is a C18 column (250mm multiplied by 20mm), the mobile phase system is 90% methanol water solution, the flow rate of the mobile phase is 10mL/min, and the detection wavelength is 450 nm;
d) sample introduction volume: 500 mu L of the solution;
obtaining fucoxanthin derivative preparation solution 330mL by running semi-preparative high performance liquid chromatography, concentrating at 30 deg.C under reduced pressure, freeze drying the concentrated solution to obtain fucoxanthin derivative 33mg, and identifying the compound as formula (III).
Example 3 fucoxanthin derivative antitumor cell Activity screening
1) Five fucoxanthin derivatives are prepared by 5 concentration gradients
Adding DMSO into five fucoxanthin derivative powders to prepare 20mg/mL of mother liquor, and subpackaging at-20 deg.C for storage. The mother liquor was diluted to 20. mu.g/mL with 1640 medium and was diluted in a gradient of 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, 1.25. mu.g/mL.
2) Culturing H460, A549 and MCF-7 cells according to corresponding cell culture methods;
3) plating H460, A549, MCF-7 cells in logarithmic phase of normal culture, digesting with 0.25% Typsin + 0.02% EDTA, centrifuging at 1500rpm for 5min, counting under counting plate, plating 96-well plate, adding 5 × 10 per well3And (5) placing the cells into an incubator and standing for 24 hours. Adding fucoxanthin derivative solutions with different concentrations respectively.
4) After CCK8 detects that fucoxanthin solutions with different cell activities and different concentrations act for different time points, the culture medium is sucked away, 100 mu L of 1640 detection solution containing 10 mu L of CCK8 is added into each well, a blank control well is arranged, the cells are reacted for 2h in a dark place at 37 ℃, and the OD value of each well is read at 450 nm.
TABLE 1 IC50 values for fucoxanthin derivatives for H460, A549, MCF-7
Claims (6)
1. The application of fucoxanthin derivatives or salts thereof in preparing medicaments for treating lung cancer is disclosed, wherein the structures of the fucoxanthin derivatives are shown as the following formula (I) -formula (V):
2. the use as claimed in claim 1, wherein the fucoxanthin derivative is prepared by a method comprising the steps of:
1) fucoxanthin or a fucoxanthin-containing extract is used as a raw material;
2) dissolving the raw materials by using a solvent, and adding a certain proportion of reducing agent for reduction reaction to obtain a reaction solution containing the fucoxanthin derivatives;
3) concentrating the reaction liquid to dryness, and performing primary separation by adopting silica gel column chromatography to obtain components containing different fucoxanthin derivatives;
4) concentrating each part of separated liquid to be dry, adding mobile phase for dissolving, and separating and purifying by adopting semi-preparative/preparative liquid phase chromatography to obtain a prepared liquid phase purified liquid;
5) respectively concentrating the liquid phase purified solutions prepared from the above parts, drying the concentrated solution to obtain fucoxanthin derivatives, and performing structure analysis to determine the compounds represented by formula (I), formula (II), formula (III) and formula (V).
3. Use according to claim 2, characterized in that the solvent in step 2) is methanol, ethanol, tetrahydrofuran.
4. The use according to claim 2, wherein the weight ratio of the feedstock to the reducing agent in step 2) is 50:1 to 1: 1.
5. the method as claimed in claim 2, wherein the reducing agent in step 2) is one or more of sodium borohydride, lithium aluminum hydride, stannous chloride, oxalic acid, potassium borohydride, ferrous sulfate, sodium sulfite, etc.
6. The use according to claim 2, wherein the reaction temperature in step 2) is 20 to 50 ℃ and the reaction time is 0.5 to 48 hours.
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| CN110092766B (en) * | 2019-04-18 | 2021-01-29 | 自然资源部第三海洋研究所 | Fucoxanthin derivative with anti-inflammatory effect and preparation method thereof |
| CN109876079A (en) * | 2019-04-24 | 2019-06-14 | 上海市皮肤病医院 | The application of Haizao Yuhu Tang and its active constituent fucoxanthine in the drug of preparation treatment liver cancer |
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