CN111454241B

CN111454241B - Biisopentenyl flavonoid compound and preparation method and application thereof

Info

Publication number: CN111454241B
Application number: CN202010352994.9A
Authority: CN
Inventors: 贾景明; 龙国清; 胡高升; 王安华
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2020-04-29
Filing date: 2020-04-29
Publication date: 2021-11-12
Anticipated expiration: 2040-04-29
Also published as: CN111454241A

Abstract

The invention belongs to the technical field of medicines, and relates to two kinds of diisopentenyl flavonoid compounds extracted and separated from radix sophorae flavescentis root bark. The compound has the chemical structural formulas of formula I and formula II. The two compounds were prepared by the following method: adding ethanol into the root bark of the sophora flavescens ait, carrying out reflux extraction, adjusting the pH value to acidity, then extracting with ethyl acetate, and purifying the ethyl acetate extract by silica gel column chromatography, polyamide column chromatography, medium-pressure ODS column chromatography and finally preparing ODS high performance liquid chromatography by taking methanol-water solution as a mobile phase to obtain the compounds shown in the formula I and the formula II. The diisopentenyl flavonoid compound or the hydrochloride thereof has obvious antitumor activity and can be used for preparing antitumor drugs.

Description

Biisopentenyl flavonoid compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to two kinds of double isopentenyl flavonoid compounds, and a preparation method and application thereof, and particularly relates to two kinds of double isopentenyl flavonoid compounds extracted and separated from dried radix sophorae flavescentis root bark, a preparation method thereof, and application thereof in preparation of various anticancer drugs.

Background

Cancer is one of the most major diseases threatening human health. The mortality rate is second to cardiovascular and cerebrovascular diseases, the incidence rate tends to rise year by year, chemotherapy as a main means of tumor treatment has good curative effect but often causes serious side effect, so that patients are difficult to insist on treatment, and the drug resistance of chemotherapy drugs in the treatment process becomes one of the problems in the current clinical treatment. Therefore, there is a need for therapeutic methods and drugs with good efficacy and low toxic side effects. The traditional Chinese medicine has the advantages of wide medicine source, low toxicity, long application history and the like, and is a hot spot for research on anti-tumor medicines.

Radix Sophorae Flavescentis is the dry root of Sophora flavescens ait of Sophora of Leguminosae, and its main active ingredients are alkaloids and flavonoids. Many researches find that the flavonoid compound in the sophora flavescens has great advantages in the aspect of pharmacological activity. The main component is isopentenyl flavone, and the pharmacological actions of the isopentenyl flavone are blood sugar reduction, insect killing, bacteria resistance, malaria resistance, tumor resistance, arrhythmia resistance and the like. Many researches show that the isopentenyl group in the kuh-seng flavone can increase the lipid solubility of the medicine, so that the medicine can more easily enter into cells to play multiple active sites of pharmacological activity for the medicine. The two related compounds with the double isopentene group flavonoid and various anticancer activities thereof have not been reported in patents or literatures so far.

Disclosure of Invention

The invention provides two kinds of diisopentenyl flavonoid compounds or hydrochloride thereof.

The structure of the compound of the invention is shown as formula I or II,

the invention also provides a preparation method of the compound I or II, which comprises the following steps:

(1) taking dried radix sophorae flavescentis root bark as a raw material, heating and refluxing the dried radix sophorae flavescentis root bark by using high-concentration ethanol, concentrating the extract to obtain an extract-shaped ethanol extract, suspending the ethanol extract in water to obtain a suspension, and adjusting the pH value to weak acidity by using dilute acid;

(2) sequentially adding ethyl acetate with the volume equal to that of the suspension for extraction for several times, and concentrating the extraction solution to obtain an extract;

(3) subjecting the ethyl acetate extract to silica gel column chromatography, and isocratic eluting with dichloromethane/chloroform-methanol solution at volume ratio of 100:0-0: 100;

(4) performing polyamide column chromatography on the eluate of dichloromethane/trichloromethane-methanol 100:1-100:2, and performing gradient elution by using a methanol/ethanol-water solvent with the volume ratio of 0:100 and 100: 0;

(5) performing gradient elution on the eluate with the volume ratio of methanol/ethanol-water of 75:25-100:0 by medium-pressure ODS column chromatography with methanol-water solvent with the volume ratio of 50:50-100: 0;

(6) methanol-water 70: purifying the eluted part at a ratio of 30-80:20 by preparative ODS high performance liquid chromatography, and taking methanol-water solution as a mobile phase to obtain the compounds of the formula I and the formula II.

In the preparation method, the raw materials are mixed,

the concentration of the ethanol in the step (1) is as follows: 65 to 100 percent.

The reflux extraction times are 2-3 times, and the extraction time is 1.5-2 hours each time.

The diluted acid is: dilute HCl or dilute H₂SO₄。

The PH is as follows: 2-5.

The preparation method of the ODS high performance liquid chromatography mobile phase methanol-water solution in the step (6) is characterized in that the appropriate volume ratio is 75: 25-90:10.

The compound of formula i is a yellow oil. By¹³C-NMR data show that the compound has 25 carbon signals, wherein 10 carbon signals are two isopentenyl signals. Delta_C21.6 and delta_C20.1 is the methylene carbon signal, δ_C25.5, 25.6, 17.6 and 17.7 are methyl signals on the isopentenyl group. Furthermore, by¹³The 15 carbon signals in the C-NMR data, 197.0, 161.6, 158.5, 157.6, 157.5, 129.2, 128.0, 115.1, 107.9, 101.9, 95.2, 78.1 and 42.0 indicate the presence of the flavanone structural fragment. Signal delta from another 10 carbons_C21.6, 122.8, 130.4, 25.5, 17.7, 20.1, 122.7, 130.2, 25.6 and 17.6 indicate that there are two isopentenyl groups. On the basis of the above reaction solution, the reaction solution is prepared from (600MHz, DMSO-d)₆) The spectral data gave 4 aromatic proton signals, delta_H 7.30(2H,d,J＝8.4Hz)，δ_H6.78(2H, d, J ═ 8.4Hz) concluded that this compound has an AA 'BB' coupling system. In addition, from¹No unimodal olefinic proton signal was observed in H-NMR, indicating that the flavone has all substitutions on the A ring. Analysis of data by means of the aboveAnd comparing with related documents to obtain the structure of the compound of the formula I.

The compound of formula II is a yellow oil. The compound of formula II has an increased delta compared to the compound of formula I_C146.7(C-2) and 135.5(C-3) carbon signals and δ_H9.34(5-OH) proton signal, indicating that the parent nucleus of the compound of formula II is flavonol, the rest signals are substantially the same as those of formula I.

Through the analysis, the compounds shown in the formula I and the formula II are finally determined to be the flavone with two diisopropenyl groups.

Table 1: process for preparing compounds of formula I¹H-NMR(600MHz,DMSO-d₆) And¹³C-NMR(150MHz,DMSO-d₆) Data of

1H-NMR(600MHz)and 13C-NMR(150MHz)data for compound I in DMSO-d6

Table 2: of compounds of formula II¹H-NMR(600MHz,DMSO-d₆) And¹³C-NMR(150MHz,DMSO-d₆) Data of

1H-NMR(600MHz)and 13C-NMR(150MHz)data for compound II in DMSO-d6

The invention further provides a pharmaceutical composition which comprises one or two of the diisopentenyl flavonoid compounds or the hydrochloride thereof and a pharmaceutically acceptable carrier or excipient.

The diisopentenyl flavone compound or the hydrochloride or the pharmaceutical composition thereof has good anticancer activity on various cancer cells, and can be used for preparing anticancer drugs.

The cancer is liver cancer, gastric cancer, lung cancer, cervical cancer and breast cancer.

Detailed Description

Example 1: preparation of compounds of formula I and formula II:

taking 4.5kg of sophora flavescens ait root bark dry medicinal material as a raw material, heating and refluxing the raw material by absolute ethyl alcohol for 3 times, each time for 2 hours, heating and concentrating to obtain an extractum-shaped ethanol extract, suspending the ethanol extract in water, diluting concentrated hydrochloric acid to 0.5mol/L, adjusting the PH of the ethanol extract suspension to 3, adding ethyl acetate with the volume equal to that of the suspension for extraction for 4 times, and concentrating the extraction solution to obtain an extract. Subjecting the ethyl acetate extract to silica gel column chromatography, performing gradient elution by using dichloromethane-methanol solution with the volume ratio of 100:0-0:100, subjecting combined eluates with the dichloromethane-methanol volume ratio of 100:1 and 50:1 to polyamide column chromatography, performing gradient elution by using ethanol-water solvent with the volume ratio of 0:100-100:0, combining eluates with the ethanol-water volume ratio of 75:25, 90:10 and 100:0, performing gradient elution by using medium-pressure ODS column chromatography with the methanol-water volume ratio of 65:50-100:0 for 5 hours, combining 70:30-85:15 parts, concentrating, and performing ODS liquid chromatography by using methanol-water solution with the volume ratio of 75:25 as a mobile phase to obtain the compound shown in the formula I. The compound of the formula II is prepared by taking methanol-water solution with the volume ratio of 70:30 as a mobile phase through preparative ODS liquid chromatography.

Example 2: pharmacological activity of Compounds of formula I and formula II

Test methods and results

1. MTT method for detecting influence of compound shown in formula I on proliferation of 6 cancer cells A549, H1299 and H460

Collecting 6 kinds of cancer cells HELA, HEP-3B, MCF-7, A549, H1299 and H460 cultured in logarithmic growth phase, wherein the A549 cells are cultured in fresh F12 culture medium containing 10% fetal calf serum, the rest cells are cultured in fresh 1640 culture medium containing 10% fetal calf serum, and the cell density is adjusted to 5 × 10⁴one/mL was inoculated in a 96-well plate. Place the well plate in a 5% CO₂After 24h incubation at 37 ℃ 100. mu.M test compound was added, the medium was aspirated after 48h drug pre-treatment and 200. mu.L of 500. mu.g/ml MTT solution was added to the incubator for a further 4h incubation. Carefully discard MTT, 150. mu.L of DMSO was added to each well. After standing for 10min, the solution was shaken for about 40s to completely dissolve formazan crystals. The 96-well plate was placed in a microplate reader and the OD was measured at 490 nm. And (3) carrying out data processing by using a microplate reader and corresponding software, and calculating the cell inhibition rate by using the average value according to the following formula: percent cytostatic was 1-sample OD mean/blank OD mean x 100%.

2. Results of anticancer Activity of Compounds of formula I and formula II

Through preliminary screening of the influences of the compounds shown in the formula I and the formula II on the proliferation of 6 cancer cells HELA, HEP-3B, MCF-7, A549, H1299 and H460, the two compounds are found to have obvious inhibitory activity on the 6 cancer cells.

The inhibition rates of the compound of formula I on HELA, HEP-3B, MCF-7, A549, H1299 and H460 of 6 cancer cells were 99%, 100% and 100% respectively at a concentration of 100. mu.M; IC of Compounds of formula I on 6 cancer cells₅₀The values were 25.1, 64.5, 9.6, 7.4, 12.3 and 32.2. mu. mol/L, respectively.

The inhibition rates of the compound of formula II on HELA, HEP-3B, MCF-7, A549, H1299 and H460 of 6 cancer cells were 98%, 90%, 97%, 95% and 91%, respectively, at a concentration of 100. mu.M; IC of Compounds of formula II on 6 cancer cells₅₀The values were 32.6, 60.0, 17.3, 20.6, 4.6 and 13.7. mu. mol/L, respectively.

Claims

1. The preparation method of the diisopentenyl flavonoid compound shown in the formula I or II is characterized by comprising the following steps:

(1) taking a sophora flavescens root-bark dry medicinal material as a raw material, heating and refluxing the raw material by 65-100% ethanol, concentrating the raw material to obtain an extract-shaped ethanol extract, suspending the ethanol extract in water to obtain a suspension, and adjusting the pH value to subacidity by using dilute acid;

(3) subjecting the ethyl acetate layer extract to silica gel column chromatography, and performing gradient elution with dichloromethane/chloroform-methanol solution at volume ratio of 100:0-0: 100;

(4) performing polyamide column chromatography on the eluate with the volume ratio of dichloromethane/trichloromethane-methanol of 100:1-100:2, and performing gradient elution by using a methanol/ethanol-water solvent with the volume ratio of 0:100 and 100: 0;

(6) methanol-water 70: purifying the eluted part at a ratio of 30-80:20 by preparative ODS high performance liquid chromatography, and taking methanol-water solution as a mobile phase to obtain a compound shown in a formula I and a compound shown in a formula II;

2. the method according to claim 1, wherein the reflux extraction in the step (1) is performed 2 to 3 times for 1.5 to 2 hours.

3. The method of claim 1, wherein the dilute acid of step (1) is: dilute HCl or dilute H₂SO₄The pH is: 2-5.

4. The method of claim 1, wherein the volume of the ethanol in step (1) is 10-30 times of the weight of the dried Sophorae radix root bark.

5. The method according to claim 1, wherein the mobile phase in the step (6) is methanol-water in a volume ratio of 75:25 to 90: 10.