阅读说明：本技术 二苯甲酮衍生物组合物及其制备方法和用途 (Benzophenone derivative composition, preparation method and application thereof ) 是由 杨光忠 金山 陈玉 徐婧 甘飞 于 2020-06-18 设计创作，主要内容包括：本发明属于药物技术领域,具体提供一种二苯甲酮衍生物组合物的制备方法,包括以下步骤：采用95％乙醇浸渍大叶藤黄果实,过滤,取滤液,减压浓缩,得到大叶藤黄果实乙醇提取物,向大叶藤黄果实乙醇提取物中加入90％甲醇溶解,然后加入石油醚萃取,将萃余液减压浓缩,之后加入10％甲醇溶解,再用醋酸乙酯萃取,减压浓缩,得到大叶藤黄果实醋酸乙酯提取物；利用石油醚-醋酸乙酯梯度洗脱分离大叶藤黄果实醋酸乙酯提取物,得到若干馏段；对得到的馏段用丙酮分别进行反复的重结晶,即得到二苯甲酮衍生物组合物。本发明从大叶藤黄果实中分离纯化得到二苯甲酮衍生物组合物,具有良好的抗癌活性,能够用于制备抗癌药物。(The invention belongs to the technical field of medicines, and particularly provides a preparation method of a benzophenone derivative composition, which comprises the following steps: soaking the fruits of the Garcinia macrophylla in 95% ethanol, filtering, taking filtrate, concentrating under reduced pressure to obtain an ethanol extract of the fruits of the Garcinia macrophylla, adding 90% methanol to the ethanol extract of the fruits of the Garcinia macrophylla for dissolving, then adding petroleum ether for extracting, concentrating raffinate under reduced pressure, then adding 10% methanol for dissolving, extracting with ethyl acetate, and concentrating under reduced pressure to obtain an ethyl acetate extract of the fruits of the Garcinia macrophylla; separating the extract of the fruit ethyl acetate of the Garcinia macrophylla by petroleum ether-ethyl acetate gradient elution to obtain a dry distillation section; and repeatedly recrystallizing the obtained fractions by using acetone respectively to obtain the benzophenone derivative composition. The benzophenone derivative composition obtained by separating and purifying the common fibraurea fruits has good anticancer activity and can be used for preparing anticancer drugs.)

1. A preparation method of a benzophenone derivative composition is characterized by comprising the following steps:

s1, soaking the Garcinia macrophylla fruits by using 95% ethanol, filtering, taking filtrate, concentrating under reduced pressure to obtain an ethanol extract of the Garcinia macrophylla fruits, adding 90% methanol to the ethanol extract of the Garcinia macrophylla fruits for dissolving, then adding petroleum ether for extracting, concentrating raffinate under reduced pressure, adding 10% methanol for dissolving, extracting by using ethyl acetate, and concentrating under reduced pressure to obtain an ethyl acetate extract of the Garcinia macrophylla fruits;

s2, performing gradient elution and separation on the ethyl acetate extract of the Garcinia macrophylla fruit by using petroleum ether-ethyl acetate to obtain a dry distillation section;

and S3, repeatedly recrystallizing the obtained distillation sections by using acetone respectively to obtain the benzophenone derivative composition.

2. The method for preparing a benzophenone derivative composition according to claim 1, wherein one of the obtained fractions is repeatedly recrystallized with acetone to obtain a benzophenone derivative composition a, wherein the benzophenone derivative composition a comprises a compound Xanthochymol and a compound gattefenerone E, the mass ratio of the compound Xanthochymol to the compound gattefenerone E is 1-2: 1, and the compound Xanthochymol has the formula:

the compound Gutterperone E has the structural formula:

3. the method for preparing a benzophenone derivative composition according to claim 1, wherein one of the obtained fractions is repeatedly recrystallized with acetone to obtain a benzophenone derivative composition B, wherein the benzophenone derivative composition B comprises a compound Cycloxanthochymol and a compound Isoxanthochymol, the mass ratio of the compound Cycloxanthochymol to the compound Isoxanthochymol is 1-2: 1, and the structural formula of the compound Cycloxanthochymol is:

the structural formula of the compound Isoxanthochymol is as follows:

4. the method of claim 1, wherein in step S2, the gradient elution is performed by using petroleum ether-ethyl acetate at a volume ratio of 19:1, 9:1, 7:3, 6:4, 5:5, 3:7, 0: 1.

5. Benzophenone derivative composition a obtained by the process according to claim 2.

6. The use of the benzophenone derivative composition a prepared by the process of claim 2 in the preparation of a medicament for the treatment of cancer.

7. The use of claim 6, wherein the cancer includes, but is not limited to, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, head and neck squamous cell carcinoma.

8. A benzophenone derivative composition B obtained by the process of claim 3.

9. The use of benzophenone derivative composition B prepared by the process of claim 3 in the preparation of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer includes, but is not limited to, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, head and neck squamous cell carcinoma.

Technical Field

The invention relates to the technical field of medicines, in particular to a benzophenone derivative composition, a preparation method and application thereof.

Background

Cancer refers to malignant tumors originating in epithelial tissues and is the most common type of malignant tumor, and cancer chemotherapy is the most effective method for treating malignant tumors nowadays. Most of the current clinical antitumor drugs are chemically synthesized drugs and plant-derived drugs, plant-derived drugs account for about one third, and the plant-derived drugs comprise plant natural products and modified derivatives thereof, and the plant natural products play an irreplaceable important role in the pharmaceutical development.

Garcinia xanthophylla of Garcinia of Guttiferae, commonly known as "Renfei", "Lingnan inverted fruit", "crooked neck fruit", etc., is a traditional Dai medicine used in folks for clearing heat, expelling parasites, etc. At present, scholars separate a great deal of chemical components from different parts of the garcinia macrophylla and most of the chemical components have biological activity.

Disclosure of Invention

In view of the above, the present invention provides a benzophenone derivative composition capable of treating cancer, wherein an active ingredient in the composition is derived from garcinia macrophylla.

The invention provides a preparation method of a benzophenone derivative composition, which comprises the following steps:

step S1: soaking the fruits of the Garcinia macrophylla in 95% ethanol, filtering, taking filtrate, concentrating under reduced pressure to obtain an ethanol extract of the fruits of the Garcinia macrophylla, adding 90% methanol to the ethanol extract of the fruits of the Garcinia macrophylla for dissolving, then adding petroleum ether for extracting, concentrating raffinate under reduced pressure, then adding 10% methanol for dissolving, extracting with ethyl acetate, and concentrating under reduced pressure to obtain an ethyl acetate extract of the fruits of the Garcinia macrophylla;

step S2: separating the gamboge fruit ethyl acetate extract by adopting normal-phase silica gel column chromatography and utilizing petroleum ether-ethyl acetate gradient elution to obtain ten fractions Fr.1-10;

step S3: and repeatedly recrystallizing the obtained fractions by using acetone respectively to obtain the benzophenone derivative composition.

Further, repeatedly recrystallizing the obtained section Fr.2 with acetone to obtain a benzophenone derivative composition A, wherein the benzophenone derivative composition A comprises a compound Xanthochymol and a compound Gutteferone E, the mass ratio of the compound Xanthochymol to the compound Gutteferone E is 1-2: 1, and the structural formula of the compound Xanthochymol is as follows:

the compound Gutterperone E has the structural formula:

further, repeatedly recrystallizing the obtained section Fr.3 with acetone to obtain a benzophenone derivative composition B, wherein the benzophenone derivative composition B comprises a compound Cyclosantochymol and a compound Isoxanthochymol, the mass ratio of the compound Cyclosantochymol to the compound Isoxanthochymol is 1-2: 1, and the structural formula of the compound Cyclosantochymol is as follows:

the structural formula of the compound Isoxanthochymol is as follows:

further, in step S2, petroleum ether and ethyl acetate were used in a gradient elution at a volume ratio of 19:1, 9:1, 7:3, 6:4, 5:5, 3:7, 0: 1.

The invention also provides a benzophenone derivative composition A prepared by the preparation method, wherein the benzophenone derivative composition A comprises a compound Xanthochymol and a compound Gutteferone E, and the mass ratio of the compound Xanthochymol to the compound Gutteferone E is 1-2: 1.

The invention also provides a benzophenone derivative composition B prepared by the preparation method, wherein the benzophenone derivative composition B comprises a compound Cyclosantochymol and a compound Isoxanthochymol, and the mass ratio of the compound Cyclosantochymol to the compound Isoxanthochymol is 1-2: 1.

The invention also provides application of the benzophenone derivative composition A or the benzophenone derivative composition B in preparation of a medicament for treating cancer.

Further, the benzophenone derivative composition a or the benzophenone derivative composition B treats cancer by inducing apoptosis of cancer cells.

Further, the benzophenone derivative composition a or benzophenone derivative composition B induces apoptosis of cancer cells by inhibiting JAK/STAT3 signaling.

Further, the benzophenone derivative composition a or benzophenone derivative composition B inhibits JAK/STAT3 signaling by downregulating IL-6 levels and downregulating phosphorylation levels of JAK2 protein and STAT3 protein.

Further, the benzophenone derivative composition a or the benzophenone derivative composition B induces apoptosis of cancer cells through a mitochondrial apoptosis pathway.

Further, the benzophenone derivative composition A or the benzophenone derivative composition B regulates the mitochondrial apoptosis pathway by down-regulating the expression level of survivin protein, Bcl-XL protein, Bcl-2 protein and Mcl-1 protein.

Further, the benzophenone derivative composition A or the benzophenone derivative composition B regulates mitochondrial apoptosis pathways by up-regulating TNF-alpha, activating Caspase-8 and Caspase-3.

Further, the benzophenone derivative composition a or the benzophenone derivative composition B is useful for treating cancer by inducing cell cycle arrest.

Further, the benzophenone derivative composition a or the benzophenone derivative composition B treats cancer by reducing tumor angiogenesis.

Further, the benzophenone derivative composition a or the benzophenone derivative composition B reduces tumor angiogenesis by down-regulating VEGF protein expression levels.

Further, the cancer includes, but is not limited to, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, head and neck squamous cell carcinoma, and the like.

The technical scheme provided by the invention has the beneficial effects that: the benzophenone derivative composition A and the benzophenone derivative composition B are obtained by separating and purifying the common jasminorange fruits, have good anticancer activity, play an anticancer role by inducing apoptosis, triggering cell cycle block and reducing tumor angiogenesis, and can be used for preparing anticancer drugs.

Drawings

FIG. 1 is a flowchart of the preparation of ethyl acetate extract of Garcinia macrophylla fruit according to example 1 of the present invention;

FIG. 2 is a flow chart of the preparation of benzophenone derivative composition according to example 1 of the present invention;

FIG. 3 is a liquid chromatography analysis chart of benzophenone derivative composition A and benzophenone derivative composition B in example 1 of the present invention;

FIG. 4 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on the morphology of tumor tissues in a liver cancer model mouse in experimental example four of the present invention;

FIG. 5 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on the expression level of p-STAT3 and VEGF in tumor tissues of a liver cancer model mouse in experimental example V of the present invention, wherein the ordinate of the bar graph is the optical density value;

FIG. 6 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on MCF-7 apoptosis in six experimental examples of the present invention, in which the ordinate of the flow cytometry analysis graph is PI staining intensity and the abscissa is annexin V-FITC staining intensity;

FIG. 7 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on Caspase-8/3 in MCF-7 cells in experimental example seven of the present invention, wherein the ordinate of the bar graph is relative gray scale values;

FIG. 8 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on Bcl-2 family anti-apoptotic proteins in MCF-7 cells in experimental example eight of the present invention, the ordinate of the bar graph is relative gray scale value;

FIG. 9 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on the JAK/STAT3 signal pathway of MCF-7 cells in experimental example nine of the present invention, and the ordinate of the bar graph is relative gray scale values;

FIG. 10 is a graph showing the effect of benzophenone derivative composition A and benzophenone derivative composition B on the MCF-7 cell cycle in the experimental example of the present invention, wherein the ordinate of the flow cytometry analysis graph is the number of cells and the abscissa is the staining intensity of PI; the histogram ordinate is the percentage of each cycle;

in the figure, Control represents a Control group, Model represents a Model group, CTX represents cyclophosphamide, A represents a benzophenone derivative composition A, and B represents a benzophenone derivative composition B.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the following examples and accompanying drawings.

The embodiment of the invention provides a preparation method of a benzophenone derivative composition, which comprises the following steps:

step S1: soaking the fruits of the Garcinia macrophylla in 95% ethanol, filtering, taking filtrate, concentrating under reduced pressure to obtain an ethanol extract of the fruits of the Garcinia macrophylla, adding 90% methanol to the ethanol extract of the fruits of the Garcinia macrophylla for dissolving, then adding petroleum ether for extracting, concentrating raffinate under reduced pressure, then adding 10% methanol for dissolving, extracting with ethyl acetate, and concentrating under reduced pressure to obtain an ethyl acetate extract of the fruits of the Garcinia macrophylla;

step S2: separating the gamboge fruit ethyl acetate extract by adopting normal-phase silica gel column chromatography and utilizing petroleum ether-ethyl acetate gradient elution to obtain ten fractions Fr.1-10;

step S3: repeatedly recrystallizing the obtained fraction Fr.2 with pure acetone to obtain a benzophenone derivative composition A, wherein the benzophenone derivative composition A is composed of a compound Xanthochymol and a compound Gutteferone E, the mass ratio of the compound Xanthochymol to the compound Gutteferone E is 1-2: 1, and the structural formula of the compound Xanthochymol is as follows:

the compound Gutterperone E has the structural formula:

and repeatedly recrystallizing the obtained section Fr.3 with pure acetone to obtain a benzophenone derivative composition B, wherein the benzophenone derivative composition B is composed of a compound Cyclosantochymol and a compound Isoxanthochymol, the mass ratio of the compound Cyclosantochymol to the compound Isoxanthochymol is 1-2: 1, and the structural formula of the compound Cyclosantochymol is as follows:

the structural formula of the compound Isoxanthochymol is as follows:

the benzophenone derivative composition A and the benzophenone derivative composition B have obvious anticancer activity, and researches show that the benzophenone derivative composition A and the benzophenone derivative composition B play an anticancer role by inducing apoptosis, triggering cell cycle retardation and reducing tumor angiogenesis, and can be used for preparing anticancer drugs.

Animal experiments and cell experiments prove that the benzophenone derivative composition A and the benzophenone derivative composition B can induce apoptosis and reduce tumor angiogenesis by down-regulating JAK/STAT3 signal paths.

The JAK/STAT3 signal pathway is considered to be a link connecting chronic inflammation and cancer, wherein STAT3 is called a signal transduction and transcription activation factor, which performs two functions of signal transduction and transcription activation, transduces upstream signals into cell nucleus and combines with DNA, and then activates a series of target genes to start transcription, thereby playing an important role in apoptosis, immunoregulation and malignant proliferation, and shows abnormally high expression of STAT3 protein in a considerable number of tumor cell lines, which cover esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, head and neck squamous cell carcinoma and the like.

STAT3 protein exhibits abnormal activation in various cancer cell lines, and in a tumor microenvironment, the transduction of JAK/STAT3 signal pathway comprises the following links: the inflammatory factor IL-6 in the tumor microenvironment and a corresponding transmembrane receptor on a cell membrane recognize each other and form a complex, JAK in cytoplasm is gathered near the receptor, adjacent JAK is activated through mutual phosphorylation, the receptor is phosphorylated, meanwhile STAT3 on the receptor acts on a tyrosine residue phosphorylated by the receptor through an SH2 structural domain of the receptor to be activated, and the activated STAT3 is separated from the receptor to form a dimer, is transferred into a cell nucleus from cytoplasm and is combined with a corresponding target gene in the cell nucleus through a DNA binding domain of the receptor, so that transcription of the corresponding target gene is regulated and controlled, and a series of effects are generated.

The occurrence and development of cancer depend on the continuous abnormal activation of STAT3, in cancer cells, the continuous abnormal activation of STAT3 leads to the up-regulation of the expression level of downstream target genes, which comprises anti-apoptosis proteins such as Bcl-2, Bcl-XL, Mcl-1, Survivin and the like, and cell cycle regulatory proteins such as Cyclin D1, c-Myc and the like, thereby greatly promoting the survival and proliferation of tumor cells, in addition, matrix metalloproteases have a destructive effect on the barrier of tumor metastasis by enzymolysis of intercellular matrix and basement membrane, and as the target genes of STAT3, the high expression of MMP-7 also promotes the invasion and metastasis of tumors.

Therefore, the JAK/STAT3 signaling pathway may serve as an important target for cancer therapy.

The benzophenone derivative composition A and the benzophenone derivative composition B can obviously reduce the level of IL-6 in serum and the phosphorylation of JAK2 protein and STAT3 protein in cancer cells, namely, the expression of related target genes at the downstream of the cancer cells is inhibited by down-regulating the over-activation of a JAK/STAT3 signal channel.

The benzophenone derivative composition A and the benzophenone derivative composition B induce cancer cells to generate apoptosis through a mitochondrial pathway. Mitochondrial apoptotic pathways are triggered if cells produce DNA damage, escape from the original growth environment, or lose the support of relevant growth factors. Mitochondrial Permeability Transition Pore (MPTP) exists between the inner mitochondrial membrane and the outer mitochondrial membrane, under the normal condition, MPTP is periodically opened and closed, positive ions in the membrane gap enter the inside of mitochondria, so that excessive accumulation of the positive ions in the mitochondrial membrane gap is prevented, factors such as cell aging and exogenous injury can trigger continuous opening of MPTP, Bax is activated and is transferred to mitochondria from cytoplasm, so that mitochondrial membrane potential disappears, the inner mitochondrial membrane swells, matrix osmotic pressure is increased, cytochrome C is released into cytoplasm, and is combined with Caspase activating factor Apaf-1 to activate Caspase-9, so that downstream Caspase-3 is activated, and apoptosis is triggered.

In the mitochondrial apoptosis pathway, the change of the permeability of the mitochondrial membrane plays a key role, and the permeability of the mitochondrial membrane is controlled by a Bcl-2 family, so that the Bcl-2 family becomes a leading person of the mitochondrial apoptosis pathway, the Bcl-2 family can be mainly divided into pro-apoptotic genes represented by Bax and Bak, anti-apoptotic genes represented by Bcl-2 and Bcl-XL, and pro-apoptotic genes which play a role by activating the pro-apoptotic genes and inhibiting the anti-apoptotic genes, such as Bim and Bid. Meanwhile, main anti-apoptosis genes Bcl-2, Bcl-XL, Mcl-1 and the like of a Bcl-2 family are target genes regulated and controlled by STAT3, and in a tumor cell, the expression quantity of the anti-apoptosis genes is up-regulated due to the continuous abnormal activation of STAT3, so that the apoptosis of the cell is inhibited, the over-activation of the STAT3 of the tumor cell is inhibited, the expression quantity of the anti-apoptosis genes is down-regulated, the tumor cell can be apoptotic, and the anti-tumor effect is achieved.

The benzophenone derivative composition A and the benzophenone derivative composition B can reduce the expression of Bcl-2, Bcl-XL, Mcl-1 and Survivin proteins in cancer cells, thereby promoting mitochondrial apoptosis.

Transmembrane death receptors such as Fas/CD95, TNFR1, TNFR2, DR3/APO-3 and DR4/5 exist on the cell surface, and all belong to the tumor necrosis factor-alpha (TNF-alpha) receptor family, and the most important of them are Fas and TNFR. For example, extracellular Fas ligand binds specifically to Fas, leading to the activation of Fas, followed by recruitment of Fas-related death structures to form FasL-Fas-FADD complexes with protein (FADD) to activate intracellular Caspase-8, which can either activate the Caspase cascade to directly activate Caspase-3 or cleave cytosolic Bid into tBid and act on mitochondria, allowing them to release cytochrome C into the cytosol, activating Caspase-9 and Caspase-3, resulting in apoptotic effects.

The benzophenone derivative composition A and the benzophenone derivative composition B can improve TNF-alpha in serum and can obviously activate Caspase-8 and Caspase-3 in cancer cells, so that the cancer cells are subjected to apoptosis.

The benzophenone derivative composition A and the benzophenone derivative composition B of the invention can be used for treating cancers by reducing tumor angiogenesis. The generation of tumor microvessels plays an important role in the growth and metastasis of tumors, the long-term infiltration of growth factors such as VEGF, EGF and the like in a tumor microenvironment leads the activation of vascular endothelial cells STAT3, so that the generation of the tumor microvessels is promoted, meanwhile, the VEGF is also an important target gene regulated and controlled by STAT3, the over-activation of STAT3 is inhibited, the expression of the VEGF can be reduced to a certain extent, the generation of the tumor microvessels is reduced, and the progress of the tumors is inhibited.

The benzophenone derivative composition A and the benzophenone derivative composition B can reduce the phosphorylation level of STAT3 and the expression of VEGF in tumor tissues, thereby reducing the angiogenesis of tumors.

The benzophenone derivative composition a and the benzophenone derivative composition B of the present invention treat cancer by inducing cell cycle arrest.

The benzophenone derivative composition A and the benzophenone derivative composition B can induce cancer cells to generate G2/M phase block, thereby playing a role in resisting cancers.

The benzophenone derivative composition A and the benzophenone derivative composition B can be used for treating cancers, including but not limited to esophagus cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, head and neck squamous carcinoma and the like.

The benzophenone derivative composition provided by the present invention is described in detail with reference to the following examples.