CN107674057B - Genistein calcium chelate as well as preparation method and application thereof - Google Patents

Genistein calcium chelate as well as preparation method and application thereof Download PDF

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CN107674057B
CN107674057B CN201710863406.6A CN201710863406A CN107674057B CN 107674057 B CN107674057 B CN 107674057B CN 201710863406 A CN201710863406 A CN 201710863406A CN 107674057 B CN107674057 B CN 107674057B
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genistein
calcium
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preparation
chelate
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CN107674057A (en
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杨成雄
毛学荣
汪会洋
李立威
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Jingchu University of Technology
Jingmen Pharmaceutical Industry Technology Research Institute
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Jingmen Pharmaceutical Industry Technology Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A23L33/165—Complexes or chelates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention relates to the technical field of preparation of medical health-care products, in particular to a genistein-calcium chelate and a preparation method and application thereof. The genistein calcium chelate disclosed by the invention has good solubility in solvents such as ethanol, can be prepared into tablets, capsules, oral liquid, injection and the like, has the medical health-care functions of oxidation resistance, tumor resistance and the like of genistein, has the function of supplementing calcium elements to human bodies, can be widely applied to medical products and health-care products, and meets the increasing medical health-care requirements of people.

Description

Genistein calcium chelate as well as preparation method and application thereof
Technical Field
The invention relates to the technical field of preparation of medical health-care products, in particular to a genistein calcium chelate which has the functions of oxidation resistance, tumor resistance and the like and also has the function of calcium supplement and the like, and a preparation method and application thereof.
Background
Genistein (Genistein), also known as Genistein, 5,7, 4' -trihydroxyisoflavone, is an active ingredient mainly extracted from leguminous plants such as soybean, clover, kudzu root, sophora flower, sophora fruit, Genistein and subprostrate sophora, is a natural tyrosine kinase and a soybean metabolite, and is called phytoestrogen. Genistein is one of isoflavone compounds with the highest active function, is a single component, is a non-estrogen compound, has dual regulation effects of estrogen and antiestrogen, and can be widely applied in the fields of pharmacy, health products and the like. The study proves that the genistein has the following effects:
(one) have estrogen and antiestrogen properties;
(II) has an anti-oxidation effect;
(III) the activity of tyrosine protein kinase (PTK) can be inhibited;
(IV) the activity of the topologic purchased enzyme II can be inhibited;
(V) inhibiting cell cycle: stopping the cells at G2/M phase; induction of tumor cell apoptosis: by up-regulating and down-regulating the apoptosis-promoting gene and the corresponding anti-apoptosis gene, the apoptosis of tumor cells is promoted, thereby preventing and limiting the occurrence of tumors, and the anti-apoptosis gene has the effects of inducing programmed cell death, improving the anti-cancer efficacy, inhibiting angiogenesis and the like.
The summary of pharmacological activity of genistein records that genistein has obvious physiological activities of reducing blood fat, resisting cancer, resisting tumor, preventing and treating osteoporosis, resisting oxidation, resisting atherosclerosis, having estrogen-like effect and the like, so that the genistein is widely applied to the fields of pharmacy, health care products and the like. In recent years, genistein has become a hot spot of research of scholars at home and abroad.
Calcium is important to the human body, is an essential element for living things, and shares the name "vital element". Calcium is the main inorganic component of human bone and teeth, and is also an essential element for neurotransmission, muscle contraction, blood coagulation, hormone release, milk secretion and the like. Calcium accounts for about 1.4% of the human body mass and participates in human metabolism. The deficiency or excess of calcium in human body affects growth and health, and especially needs to be supplemented for women after 20 years old.
In addition, modern medical research proves that calcium deficiency can cause physiological disorders of human bodies, and further cause a series of serious diseases, such as hypertension, coronary heart disease, lithangiuria, colorectal cancer, muscular pain, tetany of infants, senile osteoporosis and the like.
In addition, the related data introduces that the absorption of organic calcium ions in human body is better than that of inorganic calcium ions, so that the current calcium supplement products are all expected to be prepared into organic calcium ion preparations so as to improve the in vivo absorption rate. However, few reports on such organic calcium ion preparations are available in the literature.
Disclosure of Invention
The invention aims to provide genistein-calcium chelate, a preparation method and application thereof to improve the problems. The genistein calcium chelate provided by the invention has the functions of oxidation resistance, tumor resistance and the like, and also has the effect of calcium supplement and the like. In addition, the preparation method is simple to operate, mild in reaction conditions and low in raw material cost, and is a novel method for preparing the genistein chelate.
The invention is realized by the following technical scheme:
genistein calcium chelate, or a pharmaceutically acceptable salt, solvate or hydrate thereof, represented by the following formula I:
Figure BDA0001415562870000021
according to the present invention, when the genistein calcium chelate represented by formula I is a hydrate, it may be a compound containing 5 crystal waters.
The invention also provides a preparation method of the genistein calcium chelate or the pharmaceutically acceptable salt, solvate or hydrate thereof, which comprises the following steps:
1) preparing a genistein solution;
2) preparing a solution containing calcium ions;
3) reacting the solution obtained in the step 1) and the solution obtained in the step 2) to obtain the genistein calcium chelate.
According to the invention, in the step 1), the solvent used for preparing the genistein solution is not particularly limited, and the genistein can be dissolved, and can be an organic solvent; when the organic solvent is miscible with water, the solvent can also be a mixed solvent formed by the organic solvent and water;
the organic solvent may be selected from one, two or more of alcohol solvents, sulfone solvents, halogenated hydrocarbon solvents, ester solvents, ether solvents, ketone solvents, and the like;
for example, the alcoholic solvent may be selected from methanol, ethanol, propanol, isopropanol, butanol, pentanol, decanol, n-dodecanol, cyclopentanol, cyclohexanol, benzyl alcohol, phenethyl alcohol;
the sulfone solvent can be selected from dimethyl sulfoxide, dimethyl sulfone, sulfolane and 2, 4-dimethyl sulfolane;
the halogenated hydrocarbon solvent can be selected from dichloromethane, trichloromethane and carbon tetrachloride;
the ester solvent can be selected from ethyl acetate and methyl acetate;
the ether solvent can be selected from diethyl ether, methyl ethyl ether, methyl tert-butyl ether, dipropyl ether, dibutyl ether, 1, 4-dioxane, tetrahydrofuran;
the ketone solvent can be selected from acetone, methyl ethyl ketone;
the mass percentage of the organic solvent in the mixed solvent may be 40-99%, for example, 60% ethanol, 60% tetrahydrofuran or 50% methanol solvent is used to prepare genistein solution.
According to the invention, in the step 1), a catalyst can be added in the process of preparing the genistein solution; the catalyst can be one or more of potassium hydroxide, sodium carbonate, sodium bicarbonate and potassium carbonate, and the molar ratio of the used amount of the catalyst to the genistein can be (0.0001-0.5): 1, and preferably (0.001-0.2): 1.
According to the invention, in step 1), the solvent is preferably subjected to a heat treatment during the preparation of the genistein solution, preferably at a temperature of 0-90 ℃, more preferably at a temperature of 10-70 ℃, for example at a temperature of 50-70 ℃.
According to the present invention, in the step 2), the solvent used for preparing the solution containing calcium ions is not particularly limited, and the calcium source may be dissolved, and for example, one, two or more selected from organic solvents such as alcohol solvents, sulfone solvents, halogenated hydrocarbon solvents, ketone solvents, and the like, wherein the alcohol solvents, sulfone solvents, halogenated hydrocarbon solvents, and ketone solvents have the definitions as described above.
As an illustrative example, the solvent is selected from one or more of ethanol, dichloromethane, methanol, dimethyl sulfoxide, acetone.
According to the present invention, in step 2), there is no limitation on the calcium source used for preparing the solution containing calcium ions, and a compound that can be used for preparing a calcium-containing chelate may be commonly used, for example, an inorganic salt containing calcium, such as one or more of calcium chloride, calcium acetate, calcium nitrate, calcium sulfate, and the like.
According to the invention, in the step 3), the molar ratio of the genistein in the genistein solution to the calcium source in the calcium ion-containing solution may be (1-10): 10, preferably (2-7): 10, for example, 2.7:10, 4.2:10 or 6.2: 10.
In step 3), the reaction of genistein with the calcium source may be carried out under heating, and the reaction temperature is preferably 0 to 90 ℃, and more preferably 10 to 70 ℃.
By way of example, the genistein-calcic chelates according to the invention can be prepared, for example, by the following method:
adding a solvent and a catalyst into a three-mouth glass bottle containing genistein, heating and stirring to prepare a genistein solution A for later use;
adding calcium-containing compound into solvent, stirring to dissolve it to obtain solution B;
and thirdly, adding the solution B into a constant-pressure dropping filter funnel, opening a piston, dropwise adding the solution B into the solution A while stirring for reaction, filtering after the reaction is finished, and drying filter residues to obtain the genistein calcium chelate.
The invention also provides the application of the genistein calcium chelate or the pharmaceutically acceptable salt, solvate or hydrate thereof, and the compound can be used for preparing medicines, health-care products and the like;
the medicine can be an antioxidant and antitumor medicine or a calcium supplement medicine;
the health product can be antioxidant, antitumor or calcium supplementing.
The invention has the beneficial effects that:
1) the genistein calcium chelate disclosed by the invention has good solubility in solvents such as ethanol, can be prepared into tablets, capsules, oral liquid, injection and the like, has the medical health-care functions of oxidation resistance, tumor resistance and the like of genistein, has the function of supplementing calcium elements to human bodies, can be widely applied to medical products and health-care products, and meets the increasing medical health-care requirements of people.
2) The preparation method disclosed by the invention is simple to operate, mild in reaction conditions, low in raw material cost, simple in product post-treatment, high in product yield which can reach 75%, and high in purity which can reach 99%, and is a novel method for preparing the genistein calcium chelate.
Drawings
FIG. 1 is a HPLC analysis chart of genistein-calcium chelate prepared in example 1 of the present invention.
FIG. 2 is an X-ray diffraction pattern of genistein-calcium chelate prepared in example 2 of the present invention.
FIG. 3 is an X-ray diffraction pattern of genistein according to the present invention.
Detailed Description
The compounds of the present invention, methods for their preparation and their use are described in further detail in the following examples. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
Adding 50ml of 60% ethanol solvent and 5ml of 5% sodium hydroxide solution into a 250ml three-mouth glass bottle with 1g of genistein, heating and stirring at 50-70 ℃ to prepare genistein ethanol solution A, and adjusting the pH value to 10 to obtain a solution for later use;
② adding 1.5g of calcium chloride into a 250ml beaker filled with 50ml of 60 percent ethanol solvent, stirring to dissolve the calcium chloride to prepare solution B for standby;
thirdly, adding the solution B into a 250ml constant pressure dropping filter funnel, opening a piston, dropwise adding the solution B into the solution A, reacting while stirring at 55 ℃, cooling to room temperature after the reaction is finished, filtering, and drying filter residues in vacuum at 50 ℃ to obtain 2.2g of gray solid dye lignin calcium chelate with the purity of 99.9%.
Example 2
Adding 50ml of 60% tetrahydrofuran solvent and 10ml of 5% sodium carbonate solution into a 250ml three-mouth glass bottle with 1.5g of genistein, heating and stirring at 50-70 ℃, adjusting the pH value to 10, and preparing a genistein tetrahydrofuran solution A for later use;
② adding 2.1g of calcium acetate compound into a 250ml beaker filled with 20ml of 60 percent tetrahydrofuran solvent, stirring to dissolve the calcium acetate compound to prepare solution B;
thirdly, a constant pressure dropping filter funnel is taken, the solution B is added, a piston is opened, the solution B is dropwise added into the solution A, the dropwise addition is carried out while stirring reaction is carried out at 55 ℃, the reaction product is cooled to room temperature after the reaction is finished, the filtration is carried out, and the filter residue is dried in vacuum at 50 ℃ to obtain 2.6g of solid dye calcium lignin chelate with the purity of 99.31 percent.
Example 3
Adding 60ml of 50% methanol solvent and 5ml of 5% potassium hydroxide into a 250ml three-mouth glass bottle with 1.5g of genistein, heating and stirring at 50-70 ℃ to prepare a genistein methanol solution A, and adjusting the pH value to 8-11 to prepare a solution for later use;
② adding 2.2g of calcium nitrate compound into a 250ml beaker filled with 30ml of 50 percent methanol solvent, stirring to dissolve the calcium nitrate compound to prepare solution B for standby;
thirdly, a constant pressure dropping filter funnel is taken, the solution B is added, a piston is opened, the solution B is dropwise added into the solution A, the dropwise adding is carried out while stirring for reaction at the temperature of 40-60 ℃, the reaction product is cooled to room temperature after the reaction is finished, the filtration residue is filtered, and the filtration residue is dried in vacuum at the temperature of 50 ℃, so that 2.12g of solid dye lignin calcium chelate with the purity of 99.34 percent is prepared.
Example 4
The compound obtained in example 2 and genistein were subjected to X-ray diffraction characterization, and the results are shown in fig. 2 and 3. By comparing fig. 2 and fig. 3, it can be seen that the first characteristic peak of genistein disappears in fig. 2 and the second characteristic peak of calcium acetate shifts, indicating that the reaction produces a new substance.
The genistein calcium chelates obtained in examples 1-3 were further analyzed for elements, and the results are shown in the following table:
TABLE 1 (unit:% m/m)
Figure BDA0001415562870000071
According to the detection data, the structure of the genistein-calcium chelate prepared in the example is as follows:
Figure BDA0001415562870000072
example 5 genistein-calcium chelate antitumor Activity test
The results of preliminary activity test using the MTT method on the genistein calcium chelate prepared in example 1 of the present invention are as follows.
1) Test for inhibitory Effect on Hela cervical cancer cells
The number of cells in a 96-well plate is 5000 per well, the drug action time after the wall attachment is 24h, and the experimental results are shown in the following tables 2 and 3:
TABLE 2
Figure BDA0001415562870000081
Cisplatin (CDDP) as a positive control
TABLE 3
Figure BDA0001415562870000082
2) Human lung cancer A549 cell activity inhibition assay
The number of cells in a 96-well plate is 5000 per well, the drug action time after the wall attachment is 24h, and the experimental results are shown in the following tables 4 and 5:
TABLE 4
Figure BDA0001415562870000083
Cisplatin (CDDP) as a positive control
TABLE 5
Figure BDA0001415562870000084
The result of the inhibition rate shows that the genistein calcium chelate has better inhibition effect on the growth of Hela cervical carcinoma cells and human lung cancer A549 cells.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (1)

1. The application of the genistein calcium chelate shown as follows in preparing the medicine for resisting cervical cancer or lung cancer:
Figure FDA0002952504680000011
wherein the genistein calcium chelate has an XRD pattern substantially as shown in figure 2.
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刘潇等.染料木黄酮钙配合物的合成、表征及清除自由基和抑菌活性.《化工学报》.2010,第61卷(第11期),第3006-3013页. *
染料木素抑制人胰腺癌细胞株PANC-1细胞增殖机制的研究;马志亮等;《中华细胞与干细胞杂志(电子版)》;20150531;第5卷(第2期);第113-118页 *
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