A phenolic acid compound, preparation method, composition and its application

Abstract

The present invention relates to a phenolic acid compound, a preparation method, a composition and an application thereof, and relates to the technical field of natural medicines. The phenolic acid compound includes a compound represented by formula (I), a medicinal salt or a medicinal esterified derivative. The present invention for the first time isolates this new phenolic acid compound from the leaves of Castanopsis lamontii plants. It is confirmed by in vitro pharmacological experiments that it has good colon cancer inhibitory activity, and the IC<subgt;50< / subgt> value is 7.26 ± 0.6 μM.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and particularly relates to a phenolic acid compound, a preparation method, a composition and an application thereof. Background Art

[0002] A tumor is a new organism formed by the abnormal proliferation of a single cell in a local tissue at the gene level under the action of various carcinogenic factors, because this new organism mostly presents as a mass-like protrusion occupying space, also known as a neoplasm. Colorectal cancer is a common malignant tumor of the digestive tract occurring in the colon, which is prone to occur at the junction of the rectum and the sigmoid colon. The incidence rate is the highest in the age group of 40 - 50 years old, accounting for 10 - 15% of all malignant tumors, ranking the 3rd in the mortality rate of malignant tumors, and the incidence rate and mortality rate have shown an accelerating upward trend in recent years. Epidemiological studies have shown that an unbalanced diet with high content of saturated fat and low content of vegetables and fruits, less exercise leading to abdominal fat accumulation, obesity, and a family history of malignant tumors are the main factors for the occurrence of colorectal cancer.

[0003] Currently, the treatment of colorectal cancer mainly relies on Western medicines and Chinese patent medicines. Commonly used Western medicine chemotherapy drugs mainly include oxaliplatin, irinotecan, capecitabine, 5-fluorouracil, etc. These drugs are all cytotoxic chemotherapy drugs. After long-term use, they can produce side effects such as loss of appetite, stomatitis, gastritis, abdominal pain and diarrhea. In severe cases, it can cause symptoms such as bloody diarrhea or hematochezia, which have a great impact on the quality of life of patients. The main Chinese patent medicines for the treatment of colorectal cancer include Shen Dan San Jie Capsule, Yang Zheng Xiao Ji Capsule, Ping Xiao Capsule, etc. They have significant advantages in the treatment of diseases with a multi-target and multi-pathway mode of action and low adverse reactions. However, most Chinese patent medicines are compound preparations, with disadvantages such as unclear active ingredients, difficult effective control of the production process quality, and unclear mechanism of action. Therefore, it is urgent to develop drugs for the treatment of colorectal cancer with definite curative effects and few toxic and side effects. Discovering drugs or lead compounds with development value from natural products is one of the important ways of drug research and development, and the same is true for the research and development of drugs for colorectal cancer.

[0004] Castanopsis lamontii, also known as Castanopsis fissa, Castanea henryi, black charcoal wood, etc., is mainly used for the treatment of diseases such as diarrhea, hemostasis, gastrointestinal inflammation, and unknown swelling and toxicity. It is an arbor of the genus Castanopsis in the family Fagaceae, up to 25 meters tall, with a breast diameter of up to 1 meter. The bark is rough, reticulated and longitudinally cracked, up to 2 cm thick, and the branches, leaves and inflorescence axes are all glabrous. It is distributed in Fujian, Jiangxi, Hunan, Guizhou, Guangdong, Guangxi and Yunnan provinces. The wood is grayish-yellow to light brownish-yellow, with medium hardness, less likely to crack when dry, and quite resistant to decay. At present, there is no report on the chemical composition research of Castanopsis lamontii. In view of this, the present invention provides a phenolic acid compound, a preparation method, a composition and its application, providing a new drug or lead compound for the treatment of colon cancer and making up for the deficiencies of the existing technology. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a phenolic acid compound, a preparation method, a composition and its application. The purpose is to provide a new drug or lead compound for tumor treatment, especially for the treatment of colon cancer.

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a phenolic acid compound, which includes the compound shown in formula (Ⅰ), a medicinal salt or a medicinal esterified derivative.

[0007]

[0008] Among them, the medicinal salt includes sodium salt or potassium salt, etc., and the medicinal esterified derivative includes an esterified derivative obtained by esterification with formic acid, acetic acid, etc. The medicinal salt and the medicinal esterified derivative both have equivalent functions to the phenolic acid compound including the compound shown in formula (Ⅰ).

[0009] The beneficial effects of the present invention are: (1) The present invention first isolates the new phenolic acid compound from the leaves of Castanopsis lamontii plant, with the molecular formula C 13 H 10 O7, named 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid.

[0010] (2) The new phenolic acid compound 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid provided by the present invention has been confirmed by in vitro pharmacological experiments to have good colon cancer inhibitory activity, and its IC 50 value is 7.26 ± 0.6 μM.

[0011] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0012] The second object is to provide a preparation method of phenolic acid compounds, which are isolated and purified from Castanopsis plants.

[0013] Furthermore, it includes the following specific steps:

[0014] Step 1: Preparation of the primary extract: Crush the Castanopsis raw material, add an alcohol-containing aqueous solution, and perform cold extraction at room temperature to obtain an alcohol-containing extract. Concentrate the alcohol-containing extract under reduced pressure to remove alcohol and then filter to obtain the primary extract.

[0015] Step 2: Enrichment of crude components: Purify the primary extract obtained in Step 1 by Sephadex LH-20 gel column chromatography, elute with an alcohol-containing aqueous solution, collect the fraction containing phenolic acid compounds, and concentrate to obtain a crude extract.

[0016] Step 3: Preliminary separation and purification: Purify the crude extract obtained in Step 2 by Diaion HP20SS small particle size macroporous adsorption resin column chromatography, elute with an alcohol-containing aqueous solution, collect the fraction containing phenolic acid compounds, and concentrate to obtain a relatively pure extract.

[0017] Step 4: Re-separation and purification: Purify the relatively pure extract obtained in Step 3 by Sephadex LH-20 gel column chromatography, elute with an alcohol-containing aqueous solution in an equal gradient manner, collect the fraction containing phenolic acid compounds, and concentrate to obtain phenolic acid compounds.

[0018] Furthermore, it includes the following specific steps:

[0019] Step 1: Preparation of the primary extract: Crush the Castanopsis raw material, add an alcohol-containing aqueous solution with a volume fraction of 80-85%, and the mass of the alcohol-containing aqueous solution added is 6-8 times that of the crushed Castanopsis raw material. Perform cold extraction at room temperature for 12-24 hours to obtain an alcohol-containing extract. Concentrate the alcohol-containing extract under reduced pressure to remove alcohol and then filter to obtain the primary extract.

[0020] Step 2: Enrichment of crude components: Purify the primary extract by Sephadex LH-20 gel column chromatography, elute successively with alcohol-containing aqueous solutions with volume fractions of 0%, 20%, 40%, 60%, 80%, and 100%, collect the fraction containing phenolic acid compounds, and concentrate to obtain a crude extract.

[0021] Step 3: Preliminary separation and purification: Purify the crude extract obtained in Step 2 by Diaion HP20SS small particle size macroporous adsorption resin column chromatography, elute successively with alcohol-containing aqueous solutions with volume fractions of 0%, 20%, 40%, 60%, 80%, and 100%, collect the fraction containing phenolic acid compounds, and concentrate to obtain a relatively pure extract.

[0022] Step 4: Further separation and purification: The relatively pure extract obtained in Step 3 is purified by Sephadex LH-20 dextran gel column chromatography, eluted isogradiently with an aqueous alcohol solution containing 95% by volume of alcohol, and the fraction containing phenolic acid compounds is collected and concentrated to obtain phenolic acid compounds.

[0023] Furthermore, the Castanopsis lamontii in Step 1 is the leaves of Castanopsis lamontii, and the leaves of Castanopsis lamontii are dry or fresh products. In addition to the leaves of Castanopsis lamontii, other parts can also be used, such as the stems or roots of Castanopsis lamontii.

[0024] Furthermore, the alcohol in the aqueous alcohol solution is one or a mixture of two or more of methanol, ethanol, and propanol.

[0025] The third object is to provide a pharmaceutical composition comprising the phenolic acid compounds as described above.

[0026] The actual dose of the active ingredient (the phenolic acid compounds, pharmaceutical salts, or pharmaceutical ester derivatives of the present invention) in the pharmaceutical composition should be determined according to various relevant factors, including the severity of the tumor to be treated, the administration route, the age, gender, and weight of the patient. Therefore, the above-mentioned dose of the active ingredient should not limit the scope of protection of the present invention in any way.

[0027] Furthermore, it also includes a pharmaceutically acceptable drug excipient or carrier.

[0028] The pharmaceutically acceptable excipient or carrier includes pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients, which depend on the mode of administration and the designed dosage form.

[0029] Furthermore, the dosage form of the pharmaceutical composition is tablets, granules, capsules, nanoparticles, controlled-release preparations, or sustained-release preparations. Any pharmaceutically acceptable dosage form of the pharmaceutical composition is acceptable. The suitable dosage of the pharmaceutical composition can be formulated in various ways according to factors such as the formulation method, the administration mode, the age, weight, gender, morbidity, diet, administration time, administration route, excretion rate, and sensitivity of the patient. Skilled doctors can usually easily determine the formulation and the dosage effective for the desired treatment.

[0030] The fourth object is to provide the application of phenolic acid compounds, using the phenolic acid compounds or pharmaceutical compositions as described above in the preparation of anti-tumor drugs or as lead compounds for anti-tumor drugs. The tumors include colon cancer. Description of the Drawings

[0031] Figure 1of 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid of the present invention 1 1H - NMR (500 MHz, acetone - d6) spectrum;

[0032] Figure 2 of 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid of the present invention 13 13C - NMR (125 MHz, acetone - d6) spectrum;

[0033] Figure 3 is the HSQC spectrum of 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid of the present invention;

[0034] Figure 4 is the HMBC spectrum of 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid of the present invention;

[0035] Figure 5 is the HREIMS spectrum of 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid of the present invention. Detailed implementation mode

[0036] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0037] Example 1

[0038] This example is for the preparation of the phenolic acid 2',3',4',5,6 - pentahydroxy - [1,1'-biphenyl]-2-carboxylic acid, including the following steps:

[0039] 1. Plant source

[0040] The leaf samples of the plant Castanopsis lamontii used for extraction were collected from Hezhou City, Guangxi Zhuang Autonomous Region.

[0041] 2. Extraction and Separation

[0042] The fresh leaves of Castanopsis lamontii were crushed and added to an aqueous ethanol solution with a volume fraction of 80% for extraction at room temperature. The extract was concentrated under reduced pressure to remove ethanol and then filtered to obtain the extract. The extract was purified by chromatography on a Sephadex LH-20 (Sephadex gel column LH-20, purchased from Shanghai Huzhen Industrial Co., Ltd.), and successively washed and purified with aqueous methanol solutions with volume fractions of 0%, 20%, 40%, 60%, 80%, and 100% to remove impurities. The fraction containing 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid was collected and concentrated to obtain its crude extract. The crude extract was purified by chromatography on a Diaion HP20SS small-particle macroporous adsorption resin column (purchased from Mitsubishi, Japan), and eluted successively with aqueous methanol solutions with volume fractions of 0%, 20%, 40%, 60%, 80%, and 100%. The fraction containing the anti-tumor active ingredient 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid was collected and concentrated to obtain a relatively pure extract. The relatively pure extract was purified by chromatography on a Sephadex LH-20 Sephadex gel column, eluted isogradiently with an aqueous ethanol solution with a volume fraction of 95%, and the fraction containing the anti-tumor active ingredient 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid was collected and concentrated to obtain the phenolic acid 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid compound with anti-tumor activity.

[0043] 3. Identification of New Phenolic Acid Compounds

[0044] Figure 1 is the 1 1H-NMR spectrum of the compound 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid; Figure 2 is the 13 13C-NMR spectrum of the compound 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid; Figure 3 is the HSQC spectrum of the compound 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid; Figure 4It is the HMBC spectrum of 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid; Figure 5 It is the HREIMS spectrum of 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid. 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid is a white amorphous powder, showing a bright spot under 254 nm UV light and presenting a brown spot when reacting with 1% FeCl3-EtOH developer. Figure 5 The HREIMS spectrum shows that its quasi-molecular ion peak is m / z: 279.0440 [M+H] + (Calculated value 279.0499, C 13 H 11 O7), suggesting the molecular formula is C 13 H 10 O7, and the degree of unsaturation is 9. Its 1 1H-NMR (500 MHz, acetone-d6), 13 13C-NMR (125 MHz, acetone-d6) data are shown in Table 1 below.

[0045] Table 1 1 1H-NMR and data of 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid 13 13C-NMR (in acetone-d6)

[0046] No <![CDATA 1 H (ppm)]]> <![CDATA 13 C (ppm)]]> 1 140.9 2 121.1 3 7.24(1H, d, J = 8.6 Hz) 113.4 4 7.05(1H, d, J = 8.6 Hz) 111.1 5 146.5 6 144.7 7 168.3 1' 111.6 2' 148.8 3' 143.4 4' 157.7 5' 7.02(1H, d, J = 8.6 Hz) 109.8 6' 7.36(1H, d, J = 8.6 Hz) 110.9

[0047] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance and other spectroscopic data, see Figures 1 - 5 and Table 1, the structural formula of the compound is deduced and shown as formula (Ⅰ), named 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid.

[0048]

[0049] Example 2:

[0050] This example is for the detection of the in vitro colon cancer inhibitory activity of the phenolic compound 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid.

[0051] The cell line used in the experiment is an internationally common tumor cell line, namely: SW480 (colon cancer cells).

[0052] MTT method for the induction of cancer cell differentiation by 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid samples in vitro. The principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The absorbance value is measured at a wavelength of 492 nm with an enzyme-linked immunosorbent detector, and the change in the number of cancer cells affected by the anticancer drug can be obtained. Once the survival rate of the cells is calculated, the IC 50 is used to judge the sensitivity of the cells to the anticancer drug.

[0053] According to the cell growth rate, tumor cells in the logarithmic growth phase are inoculated into a 96-well plate at 100 μL / well, and allowed to adhere and grow for 24 hours. After aspirating the supernatant, 100 μL / well is added. Six replicate wells are set for each concentration, and a medium control without the addition of the drug solution and a cell-free zeroing well are also set. The tumor cells are cultured in an incubator at 37 °C and 5% CO2 for 24 hours, then 10 μL of MTT is added to each well, and the cells are further cultured in the incubator for 4 hours. After taking out, the supernatant is aspirated, and 150 μL / well of DMSO is added. The OD value is measured at a wavelength of 492 nm with an enzyme-labeled instrument. The experimental result shows that the IC 50 value of the sample against SW480 (colon cancer cells) is: 7.26 ± 0.6 μM.

[0054] It can be seen that the above experimental results show that 2',3',4',5,6-pentahydroxy-[1,1'-biphenyl]-2-carboxylic acid has good inhibitory activity against SW480 (colon cancer cells). Therefore, it is expected to be developed as a new colon cancer treatment drug as a compound or a lead compound.

[0055] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A phenolic acid compound, characterized in that, The phenolic acid compound is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, （Ⅰ）。 2. The preparation method of the phenolic acid compound according to claim 1, characterized in that The phenolic acid compounds are separated and purified from the genus Cone of the genus Cone, and the method comprises the following specific steps: Step 1: Preparation of primary extract: crush the antler cone raw material, add an alcohol-containing aqueous solution for cold extraction at room temperature to obtain an alcohol-containing extract, concentrate the alcohol-containing extract under reduced pressure to remove alcohol, and filter to obtain a primary extract; Step 2: Crude component enrichment: The primary extract obtained in step 1 is purified by Sephadex LH-20 dextran gel column chromatography, eluted with an alcohol-containing aqueous solution, and the fractions containing phenolic acid compounds are collected and concentrated to obtain a crude extract; Step 3: Preliminary separation and purification: The crude extract obtained in step 2 is purified by chromatography on a Diaion HP20SS small-particle macroporous adsorption resin column, eluted with an alcohol-containing aqueous solution, and the fractions containing phenolic acid compounds are collected and concentrated to obtain a relatively pure extract; Step 4: Re-isolation and purification: The relatively pure extract obtained in step 3 is purified by Sephadex LH-20 dextran gel column chromatography, eluted with an alcohol-containing aqueous solution in an isocratic manner, and the fractions containing phenolic acid compounds are collected and concentrated to obtain phenolic acid compounds.

3. The preparation method of the phenolic acid compound according to claim 2, wherein The specific steps include: Step 1: Preparation of primary extract: crush the antler cone raw material, add 80-85% alcohol aqueous solution by volume, the mass of the alcohol aqueous solution added is 6-10 times that of the crushed antler cone raw material, and cold-immerse and extract at room temperature for 12-24 hours to obtain an alcoholic extract, and then concentrate the alcoholic extract under reduced pressure to remove alcohol and filter to obtain a primary extract; Step 2: Crude component enrichment: The primary extract was purified by Sephadex LH-20 gel column chromatography, eluted with 0%, 20%, 40%, 60%, 80%, and 100% alcohol aqueous solutions in sequence, and the fractions containing phenolic acid compounds were collected and concentrated to obtain a crude extract; Step 3: Preliminary separation and purification: The crude extract obtained in step 2 was purified by chromatography on a Diaion HP20SS small-particle macroporous adsorption resin column, eluting with aqueous alcohol solutions containing 0%, 20%, 40%, 60%, 80%, and 100% by volume, collecting the fractions containing phenolic acid compounds and concentrating them to obtain a relatively pure extract; Step 4: Further separation and purification: The relatively pure extract obtained in step 3 is purified by Sephadex LH-20 dextran gel column chromatography, eluted with a 95% alcohol aqueous solution in an isocratic gradient, and the fractions containing phenolic acid compounds are collected and concentrated to obtain phenolic acid compounds.

4. The preparation method of the phenolic acid compound according to claim 3, wherein The staghorn cone described in step 1 is a leaf of the staghorn cone.

5. The preparation method of the phenolic acid compound according to claim 3, wherein The alcohol in the alcohol-containing aqueous solution is one of methanol, ethanol, and propanol, or a mixture of two or more thereof.

6. A pharmaceutical composition, characterized in that, The invention comprises the phenolic acid compound as claimed in claim 1.

7. The pharmaceutical composition according to claim 6, wherein Also included are pharmaceutically acceptable excipients and / or carriers.

8. The pharmaceutical composition according to claim 6, wherein The dosage form of the pharmaceutical composition is tablets, granules, capsules, nanometer dosage forms, controlled-release preparations or sustained-release preparations.

9. Application of phenolic acid compounds, characterized in that, The phenolic acid compound according to claim 1 or the pharmaceutical composition according to any one of claims 6 to 8 is used to prepare an anti-tumor drug; the tumor is colon cancer.

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