Biflavone compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicinal compounds, and particularly discloses a biflavone compound and a preparation method and application thereof.The preparation method comprises the steps that suaeda salsa is taken and cut into small sections, the small sections are soaked in methyl alcohol, extracting solutions are mixed, and the mixed extracting solutions are concentrated into fluid extract to obtain total extract; the preparation method comprises the following steps: sequentially adding diluted hydrochloric acid to adjust the pH value to 3-5, extracting with ethyl acetate, adjusting the pH value to 11-13 with a sodium hydroxide solution, extracting with ethyl acetate, combining extract liquor, concentrating under reduced pressure to remove ethyl acetate to obtain a crude extract, and sequentially performing ODS column chromatography chopping and silica gel column chromatography elution on the crude extract to obtain the compound. According to the method disclosed by the invention, a novel biflavone compound can be extracted and separated from suaeda salsa, and the compound has anti-gastric cancer and antibacterial activity. The biflavone compound extracted and separated by the invention has proliferation inhibition activity on a human gastric cancer 7901 cell strain. According to the present invention, the biflavone compound obtained through extraction and separation has an inhibition effect on Pseudomonas aeruginosa (Pseudomonas aeruginosa)-ATCC27853.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, in particular to a biflavonoid compound and a preparation method and application thereof. Background Art

[0002] According to patent announcement number: CN202010885479.7, a biflavonoid compound with neuroprotective effect and its extraction and separation method, this technology mainly uses ethanol extraction, n-butanol extraction, silica gel column chromatography, polyamide, and Sephadex-LH-20 to extract and separate biflavonoid compounds with neuroprotective effects from cosmos, providing a certain scientific basis for the treatment of neurological diseases, but it was not found that the biflavonoid compound has anti-gastric cancer and anti-Pseudomonas aeruginosa-ATCC27853 activity.

[0003] According to the patent announcement number: CN201510736739.3, the biflavonoid compounds, their preparation methods and medical uses, this technology mainly uses macroporous adsorption resin, silica gel column chromatography and recrystallization to extract and separate two new biflavonoid compounds with toxic activity to liver cancer cells Hepa-lclc7 from Millettia sparse-leaved, providing a certain scientific basis for cancer treatment, but it was not found that the biflavonoid compounds have activity against Pseudomonas aeruginosa Pseudomonas-aeruginosa-ATCC27853.

[0004] Suaeda salsa-Pall., as an edible medicinal halophyte, commonly known as "sea salsa", was first recorded in the "Compendium of Materia Medica for Famine Relief" and is widely distributed in inland saline-alkali land and coastal saline-alkali tidal flats in northwest my country. It is reported that the seeds and young shoots of this plant can be eaten, and its seeds contain a high-quality edible oil rich in unsaturated fatty acids. Recent studies have found that crude extracts of Suaeda salsa-Pall. have a series of biological activities such as anti-cancer, anti-inflammatory, and hypolipidemic, but no biologically active biflavonoid compounds have been found, and there are few studies on the extraction and separation of biflavonoid substances from natural products. Summary of the invention

[0005] The purpose of the present invention is to provide a biflavonoid compound and a preparation method and application thereof to solve the problems raised by the above-mentioned background technology in view of the defects of the prior art.

[0006] To achieve the above object, the present invention provides the following technical solution: a biflavonoid compound having a structural formula as shown in Formula 1,

[0007]

[0008] A method for preparing a biflavonoid compound, the specific steps are as follows:

[0009] Step 1: Take Suaeda salsa, cut it into small pieces of 4-5 cm, soak it in 80% methanol, combine the extracts, and concentrate it to a fluid extract to obtain a total extract;

[0010] Step 2: adding 0.5 mol / L dilute hydrochloric acid in turn to adjust the pH to 3-5, then extracting with ethyl acetate, and then adjusting the pH to 11-13 with 5 mol / L sodium hydroxide solution, and then extracting with ethyl acetate, combining the extracts, and concentrating under reduced pressure to remove ethyl acetate to obtain a crude extract;

[0011] Step 3: The crude extract is sequentially chopped by ODS column chromatography and eluted by silica gel column chromatography to obtain a compound.

[0012] As a preferred technical solution of the present invention, the mass volume ratio of Suaeda salsa and methanol in step 1 is: 10kg:80L; the extraction conditions are: soaking in 80v / v% methanol at 25°C for 3 times, each time for 24 hours.

[0013] As a preferred technical solution of the present invention, the specific steps of step 2 are: the total extract is suspended in 5.0L of water, 0.5mol / L of dilute hydrochloric acid is added in sequence to adjust the pH to 3-5, extracted with ethyl acetate 3 times, and then adjusted to 11-13 with 5mol / L sodium hydroxide solution, and the same solvent is extracted 3 times, the extracts are combined, and the ethyl acetate is removed under reduced pressure to obtain a crude extract.

[0014] As a preferred technical solution of the present invention, the specific steps of step 3 are:

[0015] Step 3.1: The crude extract was separated by ODS column chromatography, and methanol-water was used in a volume ratio of 10:90, 20:80, 30:70, 50:50, 70:30, 90:10, and 100:0 for gradient elution to obtain Fr.11 component, which was concentrated and dried under reduced pressure for later use;

[0016] Step 3.2: The obtained component Fr.11 was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol 60:1, 30:1, 20:1, and 10:1 in sequence, and the component Fr.11-3 obtained when the volume ratio of dichloromethane-methanol was 30:1 was reserved;

[0017] Step 3.3: Purify component Fr.11-3 by silica gel column chromatography, eluting with petroleum ether-ethyl acetate 1:1 isocratic to obtain a purified compound.

[0018] Application of a new biflavonoid compound in the preparation of anti-gastric cancer drugs.

[0019] A new biflavonoid compound for the preparation of anti-Pseudomonas aeruginosa

[0020] Application of Pseudomonas-aeruginosa-ATCC27853 in medicine.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1: The method of the present invention can extract and separate a new biflavonoid compound from Suaeda salsa, and the compound has anti-gastric cancer and anti-Pseudomonas aeruginosa ATCC27853 activities.

[0023] 2: The biflavonoid compound extracted and separated by the present invention has proliferation inhibition activity on human gastric cancer 7901 cell line, IC 50 It is -89.04-μM.

[0024] 3: The biflavonoid compound extracted and separated by the present invention has an inhibitory effect on Pseudomonas aeruginosa, IC 50 It is 25μg / mL.

[0025] 4: The biflavonoid compounds extracted and separated by the present invention provide a scientific basis for the research and development of antibiotic drugs.

[0026] 5: The biflavonoid compounds extracted and separated by the present invention provide a scientific basis for the development of Suaeda salsa resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the embodiments or the prior art descriptions and the drawings required for use are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The HRESIMS spectrum of the compound provided by the present invention;

[0029] Figure 2 The compound provided by the present invention 1 H-NMR spectrum;

[0030] Figure 3 The compound provided by the present invention 13 C-NMR spectrum;

[0031] Figure 4 HSQC spectrum of the compound provided by the present invention;

[0032] Figure 5 The HMBC spectrum of the compound provided by the present invention;

[0033] Figure 6 A schematic diagram of HMBC key related signals of the compounds provided by the present invention;

[0034] Figure 7 This is a graph showing the anti-gastric cancer activity of the compound provided by the present invention;

[0035] Figure 8 This is a graph showing the antibacterial activity of the compounds provided by the present invention. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0037] Example 1: Preparation of new compounds:

[0038] The specific steps are as follows:

[0039] Step 1: Take 10.0 kg of Suaeda salsa, cut it into 4-5 cm small pieces with scissors, extract it with 80% v / v% methanol (80L) at room temperature (about 25°C) for 3 times, each time for 24 hours, combine the extracts, and concentrate them under reduced pressure with a rotary evaporator to a fluid extract to obtain a total extract (416.58 g);

[0040] Step 2: The total extract obtained in step 1 was suspended in 5.0 L of water, and dilute hydrochloric acid (0.5 mol / L) was added in turn to adjust the pH to 3-5, extracted with ethyl acetate 3 times, and then adjusted to 11-13 with sodium hydroxide solution (5 mol / L), and the same solvent was extracted 3 times, the extracts were combined, and the organic phase was removed by concentration under reduced pressure to obtain a crude extract (145.66 g);

[0041] Step 3: The crude extract (145.66 g) obtained in step 2 was separated by ODS column chromatography, and methanol-water was used in a volume ratio of 10:90, 20:80, 30:70, 50:50, 70:30, 90:10, and 100:0 for gradient elution, and similar components were combined after TLC spotting to obtain eighteen components of Fr.1-18, which were then concentrated and dried under reduced pressure by a rotary evaporator for later use;

[0042] Step 4: The obtained component Fr.11 (weight) was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol at a volume ratio of 60:1, 30:1, 20:1, and 10:1 in sequence. After TLC spotting, similar components were combined to obtain six components Fr.11-1 to 11-6, among which Fr.11-3 was the component eluted when the volume ratio of dichloromethane-methanol was 30:1;

[0043] Step 5: Purify the component Fr.11-3 by silica gel column chromatography, isocratically eluting with petroleum ether-ethyl acetate in a volume ratio of 1:1 to obtain a new compound as shown in Formula 1 - Compound Name (10.0 mg);

[0044]

[0045] Structural identification: High resolution mass spectrometry (HRESIMS), NMR 1 H-NMR, 13 C-NMR, HSQC and HMBC were used to identify the structure of the new compound obtained in step 5. Figures 1 to 6 ;

[0046] After identification, the quasi-molecular ion peak of the new compound obtained in step 5 was m / z 575.2949 [M+Na + ](The calculated value is C 31 H 20 O 10 Na + , 575.0954), the molecular formula is estimated to be C 31 H 20 O 10 , and its NMR data are shown in Table 1.

[0047] Table 1: Compounds of formula (1) 1 H-NMR and 13 C-NMR data ( 1 H-NMR 500MHZ, 13 C-NMR 125MHZ, inDMSO-d6, δ: ppm, JinHz.)

[0048]

[0049]

[0050] The relevant signals of the HMBC spectrum of the obtained compound are as follows Figure 6 shown.

[0051] The structural formula of the obtained compound is shown below:

[0052]

[0053] The chemical formula is C 31 H 20 O 10 , named: 3',4"',5,5"-tetrahydroxy-4'-methoxy-[C7-OC 7” ]-Biflavonoids.

[0054] Example 2: Anti-gastric cancer activity of the compound:

[0055] Step 1: Prepare gastric cancer 7901 cell suspension and count;

[0056] Step 2: Inoculate the cell suspension in a 96-well plate, approximately 100-μL per well, in triplicate;

[0057] Step 3: Place the culture plate in an incubator for a pre-culture period of 4 h (37 °C, -5% CO ) to allow the cells to adhere;

[0058] Step 4: Add new compounds to each well of the culture plate;

[0059] Step 5: Place the culture plate in an incubator and incubate for 4 h for color development;

[0060] Step 6: Add 10 μL CCK-8 solution to each well and gently shake the culture plate to mix after adding the reagent;

[0061] Step 7: Place the culture plate in an incubator for incubation;

[0062] Step 8: Measure the absorbance (OD) at 450-nm using a microplate reader;

[0063] Step 9: Cytotoxicity (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] - × 100

[0064] A (drug addition): OD value of the wells with cells, CCK-8 solution, and drug solution;

[0065] A (0 drug addition): OD value of the well with cells and CCK-8 solution but no drug solution;

[0066] A (blank): OD value of the well without cells.

[0067] Step 10: Determine the IC of the new biflavonoid compound against human gastric cancer 7901 cell line 50 It is -89.04-μM.

[0068] The new compound 3',4"',5,5"-tetrahydroxy-4'-methoxy-[C7-OC 7” ]-The test results of biflavonoids are as follows Figure 7 The results showed that the compound had proliferation inhibition activity against human gastric cancer cell line 7901, IC 50 The concentration of 1:1 was -89.04 μM, and the concentration was quantitatively correlated. Therefore, the compound has proliferation inhibition activity against human gastric cancer cell line 7901.

[0069] Example 3: Antibacterial activity of compounds:

[0070] Step 1: Resuscitate the preserved standard strain of Pseudomonas aeruginosa ATCC27853 on the NA medium plate, pick a single colony, inoculate it into NB medium, and culture it at 37°C with shaking at 200-r / min until the OD value of the bacterial solution is measured by the microplate reader. 600 The value is 0.3.

[0071] Step 2: Add 5% DMSO solution to prepare compound - with an initial concentration of 500-μg / mL, then add NB culture medium to dilute to a 96-well plate to obtain test compounds with concentrations of 100, 50, 25, 12.5, and 6.25 μg / mL.

[0072] Step 3: Add 100 μL of Pseudomonas aeruginosa-ATCC27853 bacterial solution to each well of the 96-well plate, and finally obtain the detection concentration of 50, 25, 12.5, 6.25, and 3.125 μg / mL of the compound. Gentamycin was used as the positive control and 5%-DMSO was used as the negative control. Each concentration was operated in parallel 3 times, and the 96-well plate was cultured at 30°C for 8 hours to obtain IC 50 It is 25μg / mL.

[0073] The new compound 3',4"',5,5"-tetrahydroxy-4'-methoxy-[C7-OC 7” ]-The test results of biflavonoids are as follows Figure 8 As shown, the results show that the compound has growth inhibition activity against Pseudomonas aeruginosa-ATCC27853, and the activity is quantitatively correlated. Therefore, the compound has growth inhibition activity against Pseudomonas aeruginosa-ATCC27853.

[0074] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A biflavonoid compound, characterized in that: The compound has a structural formula as shown in Formula 1, 2. A method for preparing the biflavonoid compound as claimed in claim 1, characterized in that: The specific steps are as follows: Step 1: Take Suaeda salsa, cut it into small pieces of 4-5 cm, soak it in 80% methanol, combine the extracts, and concentrate it to a fluid extract to obtain a total extract; Step 2: adding 0.5 mol / L dilute hydrochloric acid in turn to adjust the pH to 3-5, then extracting with ethyl acetate, and then adjusting the pH to 11-13 with 5 mol / L sodium hydroxide solution, and then extracting with ethyl acetate, combining the extracts, and concentrating under reduced pressure to remove ethyl acetate to obtain a crude extract; Step 3: The crude extract is sequentially chopped by ODS column chromatography and eluted by silica gel column chromatography to obtain a compound.

3. The method for preparing the biflavonoid compound according to claim 1, characterized in that: The mass volume ratio of Suaeda salsa and methanol in step 1 is: 10kg:80L; the extraction conditions are: soaking in 80v / v% methanol at 25°C for 3 times, each time for 24 hours.

4. The method for preparing the biflavonoid compound according to claim 1, characterized in that: The specific steps of step 2 are: the total extract is suspended in 5.0L of water, 0.5mol / L of dilute hydrochloric acid is added in sequence to adjust the pH to 3-5, extracted with ethyl acetate 3 times, and then adjusted to 11-13 with 5mol / L sodium hydroxide solution, extracted with the same solvent 3 times, combined the extracts, and concentrated under reduced pressure to remove ethyl acetate to obtain a crude extract.

5. The method for preparing the biflavonoid compound according to claim 1, characterized in that: The specific steps of step 3 are: Step 3.1: The crude extract was separated by ODS column chromatography, and methanol-water was used in a volume ratio of 10:90, 20:80, 30:70, 50:50, 70:30, 90:10, and 100:0 for gradient elution to obtain Fr.11 component, which was concentrated and dried under reduced pressure for later use; Step 3.2: The obtained component Fr.11 was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol 60:1, 30:1, 20:1, and 10:1 in sequence, and the component Fr.11-3 obtained when the volume ratio of dichloromethane-methanol was 30:1 was reserved; Step 3.3: Purify component Fr.11-3 by silica gel column chromatography, eluting with petroleum ether-ethyl acetate 1:1 isocratic to obtain a purified compound.

6. Use of the biflavonoid compound extracted and separated by the method according to any one of claims 2 to 5 in the preparation of anti-gastric cancer drugs.

7. Use of the biflavonoid compound extracted and separated by the method according to any one of claims 2 to 5 in the preparation of an anti-Pseudomonas aeruginosa-ATCC27853 drug.

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