CN102659802A - Preparation method of coumarin lignan compounds and application of coumarin lignan compounds in resisting marine fouling - Google Patents

Abstract

The invention relates to a class of coumarin lignan compounds extracted from Hibiscus (Hibiscus hamabo Sieb.et Zucc.), a plant of the genus Hibiscus of Malvaceae (Malvaceae), and a preparation method thereof, and the compounds Anti-cancer, anti-oxidation and anti-marine fouling applications. The characteristic is that the structural formula of compound A is shown on the right, and the preparation method includes 50-95% alcohol extraction, low polarity solvent extraction, and macroporous resin to obtain the concentrated active site of coumarin lignan compounds. The seaside hibiscus obtained by the extraction process and method of the present invention has high yield and purity of active ingredients, has the activity of inhibiting cancer cells such as HeLa, HepG2, HL-60, and the adhesion activity of bryozoa larvae and barnacle larvae, and has anti-marine fouling It can be used to prepare high-efficiency marine antifouling agent. At the same time, the content of active ingredients is specified. The content of compound A is not less than 3%. It is easy to control the effective dose, and it is safer and more stable.

Description

Preparation method of coumarin lignan compound and its application in anti-marine fouling

1. Technical field

The invention relates to a class of coumarin lignan compounds extracted from Hibiscus (Hibiscus) plants of Malvaceae (Malvaceae) Hibiscus (Hibiscus hamabo Sieb.et Zucc.) and a preparation method thereof, as well as the compounds Anti-cancer, anti-oxidation and anti-marine fouling applications. The seaside hibiscus prepared by the invention has definite content of active ingredients, is easy to control the effective dose, and is safer and more stable when used for preparing anti-cancer, anti-oxidation and anti-marine fouling products.

2. Background technology

There are more than 200 species of Hibiscus plants in Malvaceae distributed in tropical and subtropical regions in the world; in my country, there are 24 species and 16 varieties distributed all over the country. Most of the plants of this genus are used for medicinal purposes, and are effective in treating bruises, eczema of the scrotum, skin ringworm, dysentery and diarrhea, hemorrhoids, heart and nervous system diseases, and various inflammations. The types of monomeric compounds isolated from the flowers, bark, roots and heartwood of Hibiscus plants include lignans, flavonoids, terpenoids, mansonones, cyclic peptides, organic acids, coumarins and coumarin lignans ( The pharmacological effects of these compounds include anti-inflammatory, antibacterial, anti-oxidation, anti-tumor and anti-fertility. Hibiscus hamabo Sieb.et Zucc. is a deciduous shrub or small tree of the Malvaceae genus Hibiscus. In the 1980s, Professor Fan Wentao of Zhejiang Agricultural University collected specimens of the tree and named it It is seaside hibiscus, also known as sea hibiscus, sea pond tree, sea pond tree. There are few studies on sea hibiscus, and most of them focus on introduction and domestication. In order to further explore the intrinsic efficacy of sea hibiscus, make reasonable and sustainable use of resources, and improve the added value of comprehensive utilization, the present invention finds sea sea hibiscus through various activity screening experiments. Active extracts have good anti-cancer, anti-oxidation and anti-marine fouling effects.

3. Contents of the invention

The purpose of the present invention is to extract effective parts with multiple biological activities from hibiscus seaside, find the optimum dose, reduce the dosage and obtain significant effect, so as to provide an active ingredient of hibiscus seaside to prepare anti-cancer medicine and anti-oxidation health food and methods and uses of anti-marine fouling organism larvae settling.

The invention clarifies the composition, properties and application of active ingredients of sea hibiscus, and is characterized in that the content of total phenolic acid active ingredients in the extract is 30-90%, and the content of compound A is not less than 3%.

The preparation method of Hibiscus hibiscus active ingredient of the present invention is specifically:

method 1:

(1) Cut sea hibiscus or branches and leaves into 3-10 cm sections, place them in an extraction kettle, add 3-10 times the volume of crude drug lower alcohols, lower alcohols are C1-C5 alcohols, and their concentration is 50-95% , the extraction temperature is 25-70°C, the filtrate is obtained by filtration, and the extraction is repeated 2-4 times, and the filtrate is combined to obtain a crude extract;

(2) After the extract is concentrated under reduced pressure, it is dissolved in water at 25-70°C, the volume ratio of the extract to water is 1:2-3, and petroleum ether is added by volume to extract at a volume of 1:1-3 to remove lipophilic impurities;

(3) Ethyl acetate is added by volume in a ratio of 1:1 to 3 for extraction, and concentrated under reduced pressure to a solid without organic solvent smell to obtain the active ingredient with a yield of 0.8 to 3% by weight.

Method 2:

(1) Cut sea hibiscus or branches and leaves into 3-10 cm sections, place them in an extraction kettle, add 3-10 times the volume of crude drug lower alcohols, lower alcohols are C1-C5 alcohols, and their concentration is 50-95% , the extraction temperature is 25-70°C, the filtrate is obtained by filtration, and the extraction is repeated 2-4 times, and the filtrate is combined to obtain a crude extract;

(2) After the crude extract is concentrated to 1/3 of its original volume, it is adsorbed by a macroporous adsorption resin, and water-soluble impurities, such as sugar, are washed away with 1 times the column volume of water, and then washed away with 20% ethanol of 2 times the column volume For large polar substances, use 30% to 70% ethanol for gradient elution, and collect the eluate;

(3) Concentrating the eluent under reduced pressure to a solid without ethanol odor to obtain the active ingredient site, with a yield of 0.2% to 2% by weight.

The macroporous adsorption resin used in the above-mentioned method 2 is D101 or HP-20 or AB-8; The alcohol used in the above-mentioned method 1,2,3 is anhydrous or water-containing alcohol, and used sherwood oil, ethyl acetate are analytically pure.

The active extract that obtains according to method 1, 2 is carried out normal pressure silica gel column chromatography, with chloroform-acetone, chloroform-methanol, ethyl acetate-methanol, ethyl acetate-acetone solvent system as eluent, from volume ratio 100 : 0 to 0: 100 for gradient elution, use thin layer chromatography to trace the combined components, and the components that can be developed with a chloroform-methanol solvent system with a volume ratio of 7: 3 on thin layer chromatography are combined with a volume ratio The chloroform-methanol solvent system of 8: 2 to 7: 3 carries out gradient elution, collects the components with Rf value of 0.3-0.5 when developing with volume ratio 7: 3 chloroform-methanol solvent system on thin layer chromatography and merges respectively, The crude product of Compound A was obtained, and the final product was obtained after recrystallization.

The present invention utilizes HPLC method to measure the content of compound A:

Instruments and reagents

High performance liquid chromatography Agilent 1100 series, quaternary pump, autosampler, detector: Aglient 1100UV, detection at 254nm, chromatographic column: Aglient Zorbax SB-C ₁₈ column (250×4.6mm, 5.0μm), methanol is Chromatographically pure, acetic acid is analytically pure.

Chromatographic conditions

Mobile phase: 0min, methyl alcohol: 0.2% acetic acid solution maintenance ratio (20: 80), 1-20min, mobile phase ratio is changed to (40: 60) by (20: 80) linear shape, 21-50min, mobile phase ratio is by ( 40:60) was changed to (80:20), the column temperature was 30°C, and the flow rate was 0.8mL/min.

Preparation of reference solution

Accurately weigh the compound A reference substance dried overnight with phosphorus pentoxide, and add methanol to make a solution containing 0.05 mg per 1 mL, as the control solution.

The seaside hibiscus active ingredient prepared by the above method has remarkable effects on anticancer, antioxidation and anti-marine fouling. The active part of seaside hibiscus obtained by the method of the invention has simple method, low production cost, high active ingredient content in the product, stable quality and easy control.

4. Description of drawings:

Figure 1. Schematic diagram of the structure of compound A

Fig. 2, process flow diagram of the present invention

Figure 3. ¹ H-NMR of Compound A

Figure 4. ¹³ C-NMR of Compound A

Figure 5. ESI-MS of Compound A

Figure 6. HPLC (254nm) detection chart of the active part of hibiscus seashore

5. Specific implementation methods:

Example 1

Using method 1, cut 5 kg of fresh branches and leaves of hibiscus seaside into 10 cm sections, add 95% ethanol with 3 times the volume of the crude drug, extract 3 times at room temperature (25° C.), 7 days each time, and combine the crude extracts by suction filtration. Concentrate the filtrate by rotary evaporation under reduced pressure at 30°C, disperse the obtained extract in water, the extract volume/water volume is 1:2.5, first add petroleum ether at a volume of 1:1 for extraction and degreasing, and then add Ethyl acetate was extracted three times, and the ester extract obtained after recovering ethyl acetate under reduced pressure was dissolved in 1 L of distilled water, and the water-soluble part was filtered with suction, concentrated under reduced pressure, and dried to obtain the active ingredient of hibiscus seaside.

Example 2:

Using method 1, cut 1 kg of dried branches and leaves of hibiscus seaside into 5 cm sections, add 80% ethanol with 6 times the volume of the crude drug, reflux extraction in a water bath at 70° C. for 2 hours each time, and combine the crude extracts by suction filtration. Concentrate the filtrate by rotary evaporation under reduced pressure at 70°C, and dissolve the obtained extract in hot water. The volume of the extract/water volume is 1:2. Ethyl acetate was added in volume and extracted 5 times, and the ester extract obtained after recovering the ethyl acetate under reduced pressure was dissolved in 1 L of distilled water, the water-soluble part was filtered with suction, concentrated under reduced pressure, and dried to obtain the active ingredient of sea hibiscus.

Example 3

Using method 1, cut 1 kg of dried branches and leaves of hibiscus seaside into 3 cm sections, add 50% ethanol 10 times the volume of the crude drug, extract in a water bath at 50°C for 4 times, each time for 2 hours, and combine the crude extracts by suction filtration. Concentrate the filtrate by rotary evaporation under reduced pressure at 50°C, dissolve the obtained extract in hot water at 50°C, the volume of the extract/water volume is 1:1, first add petroleum ether to extract and degrease it according to the volume of 1:1, and then press 1 : 2 volumes were added ethyl acetate and extracted 5 times, the ester extract obtained after reclaiming the ethyl acetate under reduced pressure was dissolved in 1L of distilled water, the water-soluble part was filtered by suction, concentrated under reduced pressure, and dried to obtain the Hibiscus seaside active ingredient.

Example 4

Using method 2, take 5 kg of fresh branches and leaves of hibiscus seaside and cut them into 5 cm sections, soak them in 95% ethanol with 3 times the volume of the crude drug at room temperature (25° C.) for 7 days, filter to obtain the crude extract, and soak them repeatedly for 3 times. Combine the filtrates. Concentrate the filtrate to 1/3 of its original volume by rotary evaporation under reduced pressure at 40°C, pass the concentrated solution through the macroporous adsorption resin D101, so that the active ingredients in the liquid are adsorbed on the resin, discard the effluent, and then use 1 times the column volume Rinse the resin column with distilled water, discard the effluent, then rinse the resin column with 20% ethanol of 2 times the column volume, discard the effluent, continue to carry out gradient elution with 30%, 50%, 70%, and 90% ethanol, and collect 50 %, 70% ethanol eluate, and concentrate under reduced pressure to obtain the active ingredient of sea hibiscus.

Example 5

Using method 2, take 1 kg of dry branches and leaves of hibiscus seaside and cut them into 10 cm sections, use 80% ethanol with 6 times the volume of the crude drug, heat and reflux at 70°C for 2 hours, and obtain the crude extract by suction filtration, extract repeatedly 4 times, and combine filtrate. Concentrate the filtrate to 1/3 of its original volume by rotary evaporation under reduced pressure at 70°C, pass the concentrated solution through the macroporous adsorption resin HP20, so that the active ingredients in the liquid are adsorbed on the resin, discard the effluent, and then use 1 times the column volume Rinse the resin column with distilled water, discard the effluent, and then carry out gradient elution with 10%, 30%, 50%, 70%, and 95% ethanol of 2 times the column volume, and collect 30%, 50%, and 70% ethanol. liquid, and concentrated under reduced pressure to obtain the active ingredient of hibiscus hibiscus.

Example 6

Using method 2, take 1 kg of dry branches and leaves of hibiscus seaside and cut them into 3 cm sections, heat and extract with 50% ethanol 10 times the volume of the crude drug at 80°C for 2 hours, filter with suction to obtain a crude extract, repeat the extraction twice, and combine the filtrates . Concentrate the filtrate to 1/3 of its original volume by rotary evaporation under reduced pressure at 60°C, pass the concentrated solution through the macroporous adsorption resin AB-8, so that the active ingredients in the liquid are adsorbed on the resin, discard the effluent, and then use 1 times the column volume of distilled water to rinse the resin column, discard the effluent, and then carry out gradient elution with 10%, 30%, 50%, 70%, 80%, and 95% ethanol of 2 times the column volume, and collect 30%, 50%, and The 70% ethanol eluate was concentrated under reduced pressure to obtain the active ingredient of sea hibiscus.

Example 7

Determination of Compound A in Active Components of Hibiscus seashore by HPLC

Instruments and reagents

High-performance liquid chromatography Agilent 1100 series, quaternary pump, autosampler, detector: Aglient 1100UV, detection at 254nm, 365nm, chromatographic column: Aglient Zorbax SB-C ₁₈ column (250×4.6mm, 5.0μm), Methanol was chromatographically pure, and phosphoric acid was analytically pure.

Chromatographic conditions

Mobile phase: 0min, methyl alcohol: 0.2% acetic acid solution maintenance ratio (20: 80), 1-20min, mobile phase ratio is changed to (40: 60) by (20: 80) linear shape, 21-50min, mobile phase ratio is by ( 40:60) was changed to (80:20), the column temperature was 30°C, and the flow rate was 0.8mL/min.

Preparation of reference solution

Accurately weigh the compound A reference substance dried overnight with phosphorus pentoxide, and add methanol to make a solution containing 0.05 mg per 1 mL, as the control solution.

Preparation of the test solution: Take by weighing 0.1 g of the active ingredient extract of Hibiscus hibiscus above in Examples 1, 3, and 5, respectively, into a 50 mL volumetric flask, and dilute to the mark with methanol to be the solution to be tested.

Example 8

In vitro antioxidant activity of different solvent extracts from different parts of Hibiscus seashore

1 Materials, reagents and instruments

1.1 Materials

Seaside hibiscus was collected from Qingzhu Farm, Ninghai County, Zhejiang Province in 2010. It was identified by researcher Yuan Changqi, Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The specimen is now deposited in the Medicinal Plant Research and Development Center, Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.

1.2 Instruments

Infinite M200 microplate reader (TECAN company); rotary evaporator R-210 (Switzerland Buchi company); LIBRORAEL-200 electronic balance (Shimadzu company); HH-S water bath (Zhengzhou Great Wall Technology Industry and Trade Co., Ltd.); KQ -300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); LTJ-12 freeze dryer (manufactured by Beijing Songyuan Huaxing Technology Development Co., Ltd.); TGL-16M centrifuge (Saitexiangyi Company); GZX-9070 MBE Digital blast drying oven (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

1.3 Reagents

1,1-diphenyl-2-trinitrophenylhydrazine free radical (DPPH ) (Solarbio company); gallic acid standard (Sinopharm Chemical Reagent Co., Ltd.); Foline-Phenol (BIOSHARP company); other reagents were For analytical purity.

2 methods

2.1 Preparation of different solvent extracts from different parts of Hibiscus seashore

Weigh respectively 4 parts of seashore Hibiscus root, stem bark and leaf dry powder, each 5g. The samples were ultrasonically extracted with n-hexane, dichloromethane, ethyl acetate, and methanol as solvents, the ratio of solid to liquid was 1:20 (mg/mL), the ultrasonic power was 100W, and the extraction was performed twice, 30min each time, and the extracts were combined. Concentrate under reduced pressure and dry in vacuo to obtain 4 different solvent extracts for future use.

2.2 Determination experiment of total phenol content

Accurately weigh 15mg of gallic acid and place it in a 10mL volumetric flask, dissolve it with distilled water, adjust the volume to the mark, shake well, and prepare a 1.5mg/mL standard solution, transfer it to a brown bottle for later use; prepare 100mg/mL Na _{2 CO} solution. Add gallic acid standard solution 0, 1, 2, 4, 6, 8 μL in sequence in 96-well plate; Make up each sample well to 50 μL, then add 50 μL of Folin-Ciocalteu reagent respectively, shake for 3 minutes, add 50 μL of 100 mg/mL Na ₂ CO ₃ solution and mix well, after standing at room temperature for 5 hours, put the 96-well plate into a microplate reader The absorbance of each sample was measured at a wavelength of 760 nm. Each test was repeated 4 times and the average value was taken. Finally, the total phenol content in each sample was obtained according to the standard curve of gallic acid.

2.3 Determination of antioxidant activity

2.3.1 Scavenging DPPH free radical experiment

Take different solvent extracts of each component, dissolve in DMSO, and prepare a 10 mg/mL mother solution. DPPH was prepared with absolute ethanol to 0.16mmol/L, and placed in a brown bottle for later use. In a 96-well plate, add 100 μL of 0.16 mmoL/L DPPH, 2 μL of sample solution, and 98 μL of distilled water to each well, and the reaction system is 200 μL. After adding the sample, shake at room temperature for 15 minutes, put the 96-well plate into a microplate reader and measure the absorbance (Ai) of the sample at a wavelength of 517nm; take 20 μL DMSO instead of the sample solution to measure the blank absorbance (A0); replace the DPPH solution with 100 μL absolute ethanol Measure the background absorbance (Aj) of the sample; do 4 parallels for each concentration, and take the average value. With Vc as positive control, the clearance rate was calculated according to formula (1).

2.3.2 Scavenging effect on hydroxyl radical (.OH)

Add 2 μL of different solvent extracts of each component with a mass concentration of 10 mg/mL to a clean 96-well plate, make up to 50 μL with distilled water, and then add 50 μL of 6 mmoL/L FeSO ₄ solution, 6 mmol/L 50 μL of H ₂ O ₂ , shake well, let stand for 10 min, then add 50 μL of 6 mmoL/L salicylic acid solution therein, and mix well. After reacting at 37°C for 30 minutes, put the 96-well plate into a microplate reader to measure the absorbance (Ai) at a wavelength of 510nm; replace the sample in the system with DMSO, and measure the blank absorbance (Ao); replace the system with distilled water In the salicylic acid, measured the sample background absorbance value (Aj). Each test was repeated 4 times and the average value was taken. With Vc as the positive control, the clearance rate was calculated according to the formula:

2.3.3 Scavenging effect on superoxide anion radical (O ₂ ^- .) ^[8]

Add 2 μL of each extract with a mass concentration of 10 mg/mL to a clean 96-well plate, then add 50 μL of 0.05 mol/L Tris-HCl buffer solution (pH8.2) in sequence, make up to 190 μL with distilled water, shake well, After preheating in a water bath at 25°C for 20 minutes, add 10 μL of 25 mM pyrogallic acid prepared with 10 mM HCl, mix well and react in an aqueous solution at 25°C for 5 minutes, and measure the absorbance (Ai) at 299 nm; use DMSO Instead of the sample in the system, the blank absorbance (Ao) was measured; 10mM HCl was used to replace the pyrogallic acid in the system, and the background absorbance of the sample (Aj) was measured. Each test was repeated 4 times and the average value was taken. With Vc as the positive control, the formula for calculating the clearance rate is:

3 Results and Analysis

3.1 Determination of total phenol content

In the range of 0 to 80 μg/mL, with the absorbance (y) as the ordinate and the concentration of gallic acid (x) as the abscissa, draw the gallic acid standard curve to obtain the regression equation: y=0.0318x+0.0445 (R=0.9996), The results indicated that there was a good linear relationship between the absorbance value and the concentration of gallic acid in the range of 0-80 μg/mL.

Table 1 Total phenolic content in different solvent extracts from different parts of Hibiscus seashore (mg/g)

Total phenol content (mg/g)=x/c, where x (mg/mL) refers to the total phenol concentration in the sample solution calculated according to the regression equation by the absorbance value, expressed in gallic acid, c (g/mL ) is the sample concentration corresponding to the absorbance value, from which the total phenol content in each sample is calculated, and the results are shown in Table 1.

3.2 Scavenging effect on DPPH free radicals

DPPH is a stable free radical, which is purple in organic solvents and has a large absorption at 517nm wavelength. When antioxidants are added, part of the free radicals are removed, which weakens the absorption intensity at this wavelength, which can be used to To evaluate the antioxidant activity of a substance.

Table 2 The scavenging effect of 100 μg/ml different solvent extracts of different parts of Hibiscus seashore on DPPH free radicals (%)

3.3 Scavenging effect on hydroxyl radical (.OH)

Referring to the Fenton reaction method to establish a reaction system model, the use of H ₂ O ₂ and Fe ²⁺ mixed to generate .OH, but because .OH has a high reactivity, the survival time is short, if salicylic acid is added to the system, it can Effectively captures .OH and produces colored products. The product has a strong absorption at 510nm wavelength. If the analyte with the function of scavenging .OH is added to the reaction system, it will compete with salicylic acid for .OH, so that the amount of colored products is reduced. Using the fixed reaction time method, Add a series of extracts of wheat bran total flavonoids with different concentrations to the reaction system of the same volume, and use distilled water as a reference to compare with the reagent blank solution. By measuring the absorbance A at each concentration at a wavelength of 510nm, it can be detected. The scavenging effect of the test substance on .OH.

Table 3 The scavenging effect of 100 μg/ml different solvent extracts of different parts of Hibiscus seashore on hydroxyl radicals (%)

3.4 Scavenging effect on superoxide anion radical (O ₂ ^- .)

O ₂ ^- . and intermediates are produced by pyrogallol under alkaline conditions. The intermediate product has a characteristic absorption peak at 299nm wavelength. When O ₂ ^- . scavenger is added, the formation of O ₂ ^- . Inhibited, the autoxidation process of pyrogallol is hindered, and the absorption of the solution at 299nm wavelength is weakened. Therefore, by measuring the inhibitory effect of a substance on the autoxidation of pyrogallol, its scavenging effect on superoxide anion radicals can be characterized.

Table 4 The scavenging effect of 100 μg/ml different solvent extracts from different parts of Hibiscus seashore on superoxide anion radicals (%)

Example 9

In vitro anti-tumor activity screening experiment

Human cancer cell lines were selected, and the OD value of each well on the 96-well plate was measured by MTT method, the cell growth inhibition rate was calculated, and the 50% growth inhibitory concentration IC ₅₀ value was calculated by Logit method. MTT method: Collect lung cancer cell line A549, cervical cancer cell line Hela, liver cancer cell line HegG-2, human histiocytic lymphoma cell line U-937, human myeloid leukemia cell line HL-60, Human breast cancer cell line MCF-7 and brain cancer cell line U-251 were cultured with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin in DMEM culture medium, suspended, and 0.25% Trypsin-0.02% EDTA was digested and passaged, seeded in 96-well culture plate, the number of cells per well was 5000/80 μL, placed in a 5% CO ₂ incubator, and incubated at 37°C. Compounds A and B were respectively diluted with 10% serum DMEM culture solution into experimental solutions with concentrations of 25 μg/mL, 50 μg/mL and 100 μg/mL. On the next day, 20 μL of the experimental solution with a concentration of 25 μg/mL was added to the culture plate of the experimental group 1, 20 μL of the experimental solution with a concentration of 50 μg/mL was added to the culture plate of the experimental group 2, and the concentration of 100 μg was added to the culture plate of the experimental group 3. /mL of the experimental solution 20 μL, and make the final concentration in each well reach the test (the concentration of the experimental group is double-diluted). In addition, an equal amount of 10% serum DMEM culture solution was added to the negative control group. After 48 hours, the culture solution in the experimental group and the control group was discarded, and 20 μL (2.5 mg/mL) of MTT solution was added to each well, and the culture was continued for 4 hours. Add 100 μL of DMSO to each well to terminate the reaction, place at 37°C for 20 minutes, use a microplate reader to detect the absorbance A value of each well at 570 nm, calculate the cell growth inhibition rate, and use the following formula to calculate the survival rate of tumor cells after drug treatment: cell survival rate %=(A _{570-dosed group} -A _{690-dosed group} )/(A _{570 control group} -A _{690 control group} )×100%.

Tanble 5.In vitro cytotoxicity of A against Hela, U-937, U-251, HL-60, MCF-7, HepG2 and A549 cell lines (IC ₅₀ μM).

NA＝not active up to 100μM

Example 10

Screening test for anti-adhesive activity against Bryxophora multilocularis larvae

The experiment used a polystyrene 24-aperture plate. Compound A was dissolved in disulfoxide (DMSO) as a test sample, and then added to seawater (FSW) filtered by an autoclave (0.22 μM) to prepare different Concentration (0.25 μg/mL, 0.5 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL). Add 20 swimming larvae and 1 mL of the prepared test sample into each well plate, make 4 parallel wells for each sample, and use FSW seawater added with DMSO as a negative control. Put the 24-well plate equipped with test samples in an incubator at 28° C. for 1 hour, and then observe the experimental results. Calculate by microscope: 1) the number of larvae attached to the plate wall; 2) the number of larvae not attached to the plate wall; 3) the number of dead larvae. Calculate the percentage of the number of larvae attached to the plate wall to the total number of larvae used in the experiment, and then calculate the half-effective inhibitory concentration EC ₅₀ for inhibiting the attachment of larvae by EPA PROBIT ANALYSIS PROGRAM VERSION 1.5 software. The half effective inhibitory concentration (EC _{50 )} of the larvae attaching to B. cerevisiae was 12.3 μg/mL, and the half effective lethal concentration (LC _{50 )} was >100 μg/mL.

Claims (8)

1. The structural formula of coumarin lignan compound A is:

. 2. a preparation method of hibiscus seashore active extract, is characterized in that, comprises the following processing steps: (1) Mince the twigs of the sea hibiscus and place them in an extraction kettle, add 3 to 10 times the volume of lower alcohols of the sea hibiscus twigs for extraction, the extraction temperature is 30 to 70°C, the extraction time is 6 to 12 hours, and the filtrate is obtained by filtration Repeatedly extracting 1 to 4 times according to the above extraction temperature and time, filtering, and combining the filtrates to obtain a crude extract; the lower alcohols are C1-C5 alcohols with a concentration of 50-95%; (2) The crude extract is concentrated under reduced pressure (instrument:

Rotary evaporator R-210, water bath 60°C, Vacuum PumpV-700 decompression pump, 70-100mbar (7-10kPa)), the crude extract concentrated under reduced pressure is dissolved in hot water at 30-70°C, crude The volume ratio of extract and hot water is 1:2-3; then add petroleum ether for extraction according to the volume ratio of 1:1-3 to remove lipophilic impurities; (3) adding ethyl acetate at a volume ratio of 1: 1 to 3 for extraction, extracting 5 times, combining the ethyl acetate extracts, concentrating under reduced pressure to a dry solid without organic solvent smell, to obtain ethyl acetate extract; (4) Add a certain amount of water to the ethyl acetate extract to dissolve (approximately 20 mg of ethyl acetate extract plus 1 mL of water), and the aqueous solution after suction filtration (filtering out water-insoluble solids) is concentrated to dryness to obtain the sea hibiscus active substance , the yield of the active substance is 0.8% to 3% by weight (i.e. the weight (g) of seaside hibiscus ÷ seaside hibiscus branches and leaves (g)×100%), the content of compound A is 1-3%, and the content of compound B is 3% -5%. 3. a preparation method of hibiscus seashore active extract, is characterized in that, comprises the following processing steps: (1) Chopping the branches and leaves of Hibiscus seashore and placing them in an extraction kettle, adding 3 to 10 times the volume of lower alcohols of branches and leaves of Hibiscus seashore for extraction, the extraction temperature is 30 to 70°C, the extraction time is 6 to 12 hours, and the filtrate is obtained by filtering; The above extraction temperature and time are repeatedly extracted for 1-4 times, filtered, and the filtrates are combined to obtain a crude extract; the lower alcohols are C1-C5 alcohols with a concentration of 50-95%; (2) Concentrate the crude extract under reduced pressure (instrument: Rotary evaporator R-210, water bath 60°C, Vacuum Pump V-700 decompression pump, 70-100mbar (7-10kPa)) to 1/3 of the original volume (about 500mL), use macroporous adsorption resin 1Kg for adsorption , wash with 1000mL of water to remove water-soluble impurities, and then use 3000mL of ethanol with a concentration of 30%, 50%, 70%, and 90% respectively for gradient elution, and collect the eluate; (3) The eluent is concentrated under reduced pressure (instrument:

Rotary evaporator R-210, water bath 60°C, Vacuum Pump V-700 decompression pump, 70-100mbar (7-10kPa)) to dry solid without organic solvent odor, to obtain the sea hibiscus active extract; the sea hibiscus active The yield is 0.2% to 2% by weight (i.e. the weight (g) of sea hibiscus active matter ÷ sea hibiscus branches and leaves (g) × 100%), the content of compound A is 3-5%, and the content of compound B is 5-7% . 4. The macroporous adsorption resin used in claim 3 is D101 or HP-20 or AB-8. 5. the preparation method of the described compound of claim 1 is characterized in that comprising the following steps: The active extract that claim 2,3 obtains is carried out normal pressure silica gel column chromatography, with chloroform-acetone, chloroform-methanol, ethyl acetate-methanol, ethyl acetate-acetone solvent system as eluent, from volume ratio 100 : 0 to 0: 100 for gradient elution, use thin layer chromatography to trace the combined components, and the components that can be developed with a chloroform-methanol solvent system with a volume ratio of 7: 3 on thin layer chromatography are combined with a volume ratio The chloroform-methanol solvent system of 8: 2 to 7: 3 carries out gradient elution, collects the components with Rf value of 0.3-0.5 when developing with volume ratio 7: 3 chloroform-methanol solvent system on thin layer chromatography and merges respectively, The crude product of Compound A was obtained, and the final product was obtained after recrystallization. 6. The application of an active extract of hibiscus seashore in the preparation of anti-oxidation and scavenging free radicals. 7. The application of an active extract of sea hibiscus in the preparation of a drug for treating cancer. 8. The application of a seaside hibiscus active extract in anti-marine fouling biological larvae attachment. the

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