CN110448623B

CN110448623B - Preparation method of banana leaf extract and application of banana leaf extract in resisting chikungunya virus

Info

Publication number: CN110448623B
Application number: CN201910478724.XA
Authority: CN
Inventors: 邓家刚; 郝二伟; 侯小涛; 李聪; 杜正彩; 秦建峰; 韦玮
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2022-03-01
Anticipated expiration: 2039-06-03
Also published as: CN110448623A

Abstract

The invention discloses that the ethanol extract of banana leaves has a certain function of inhibiting chikungunya virus. Accordingly, the inventor also establishes a corresponding extraction method of the ethanol extract of the banana leaves. Experiments show that the ethanol extract of banana leaves has the effect of resisting chikungunya virus, and the chikungunya virus IC₅₀It was 8.73. mu.g/ml. Therefore, the banana leaves have potential value of the medicine resisting the chikungunya fever. Therefore, the method provides powerful basis and reliable guarantee for further opening up the medical application of the banana leaves, can improve the utilization rate of banana resources, and reduces the resource waste; also opens up a Chinese herbal medicine approach for treating chikungunya fever so as to reduce the production cost of the medicine.

Description

Preparation method of banana leaf extract and application of banana leaf extract in resisting chikungunya virus

Technical Field

The invention belongs to the technical field of chikungunya virus treatment, and particularly relates to a preparation method of a banana leaf extract and application of the banana leaf extract in resisting chikungunya virus.

Background

The virus is one of natural microorganisms, is divided into DNA virus and RNA virus according to nucleic acid types, is widely distributed in nature, can infect bacteria, fungi, plants, animals and human, and often causes host morbidity. Many human diseases are caused by viruses, and Chikungunya Fever (Chikungunya Fever) is one of them.

Chikungunya fever is a mosquito-borne infectious disease caused by Chikungunya virus (CHIKV), and is mainly characterized by fever, headache, myalgia, rash, and arthralgia. Joint pain in some patients persists for years after other symptoms disappear, with severe symptoms remaining in the flexion position, initially prevalent in tropical and subtropical regions of africa, and continuing to spread to southern asia, southeast asia, indian islands, and the americas. Over the past decade, the number of chikungunya fever outbreaks has increased, with an ever-expanding prevalence that has emerged in over 100 countries and regions worldwide, causing approximately 100 million infections annually worldwide.

At present, no vaccine can be used for prevention of chikungunya fever, and no exact effective pathogenic treatment medicine exists clinically, and only symptomatic treatment is taken as the main treatment, so that the research on anti-chikungunya virus medicines is the key point of the current work.

Bananas (Musa nana Lour.) are perennial, large monocotyledonous herbaceous plants of the genus Musa of the family Musaceae, and are widely distributed in tropical and subtropical regions below 30 ° latitude north and south. Bananas belong to high-calorie waterfruits, and the pulp of the bananas is rich in carbohydrates, proteins, fat, dietary fibers, various trace elements and vitamins and is the fourth-generation grain crop next to rice, wheat and corn in the world. There are about 136 countries and regions producing bananas, and the main producing areas are concentrated in india, philippines, china, brazil and other countries. In recent years, the harvest area and yield of bananas are on a growing trend all over the world, and agricultural wastes such as banana stems and leaves with almost the same quantity are generated while a large amount of banana products are harvested. Therefore, how to recycle banana stems and leaves becomes an urgent problem to be solved. The volume of banana stems and leaves is large, so the labor intensity of collection is high; the water content is high, so that the fuel can not be directly combusted, and the transportation cost is increased; but the biomass-rich fertilizer has high biomass and rich nutrient substances, and has great development potential. At present, most banana stems, leaves and the like are used as feed and fertilizer, or used for producing low-grade paper, coarse cloth, packaging ropes and other products, and further can be fermented to produce methane or alcohol. The development of the medicinal value of the compound is rarely reported, and particularly, the compound is not reported in antiviral research.

Disclosure of Invention

The invention aims to provide a preparation method of banana leaf extract and application of the banana leaf extract in resisting chikungunya fever virus, so as to fully explore the medical value of the waste banana leaves and open up a Chinese herbal medicine approach for treating chikungunya fever.

In order to solve the technical problems, the invention adopts the following technical scheme:

application of banana leaf extract in preparing medicine for resisting chikungunya fever virus is provided.

The banana leaf extract is ethanol extract of banana leaf.

The preparation method of the banana leaf extract comprises the steps of adding ethanol into banana leaves, carrying out ultrasonic extraction, filtering, carrying out suction filtration, carrying out rotary evaporation on the filtrate to recover the solvent to a concentrated liquid state, and centrifuging to obtain dry paste.

The banana leaves are fresh products of green leaf parts of the banana leaves.

The ethanol is 95% ethanol with volume concentration, and the addition amount is 8 times of the weight of the banana leaves.

Ultrasonic extraction is carried out for 2 hours, and the rotary evaporation temperature is 40 ℃.

Aiming at the problem that the value of the banana leaves which are waste crops is not effectively developed at present, the inventor finds that the ethanol extract of the banana leaves has a certain function of inhibiting the dengue-chikungunya virus through deep research. Accordingly, the inventor also establishes a corresponding extraction method of the ethanol extract of the banana leaves. Experiments show that the ethanol extract of banana leaves has the effect of resisting chikungunya virus, and the chikungunya virus IC₅₀It was 8.73. mu.g/ml. Therefore, the banana leaves have potential value of the medicine resisting the chikungunya fever. Therefore, the method provides powerful basis and reliable guarantee for further opening up the medical application of the banana leaves, can improve the utilization rate of banana resources, and reduces the resource waste; also opens up a Chinese herbal medicine approach for treating chikungunya fever so as to reduce the production cost of the medicine.

Drawings

FIG. 1 is a graph showing the results of the dengue virus plaque resistance test of ethanol extract of banana leaves according to the present invention.

FIG. 2 is a graph showing the results of the test for resistance of ethanol extract of banana leaves to chikungunya virus plaques.

FIG. 3 is a graph showing the results of the inhibition rate of the ethanol extract of banana leaves against dengue virus and chikungunya virus.

Detailed Description

First, experimental material

1. Banana leaf extract

Taking 100.00g of fresh banana leaf green leaf part, adding 8 times of 95% ethanol, carrying out ultrasonic extraction for 2h, filtering, carrying out suction filtration by using qualitative filter paper, putting the filtrate on a rotary evaporator, recovering the solvent at 40 ℃ until the filtrate is in a concentrated liquid state, and carrying out centrifugal concentration at 40 ℃ to obtain 787.40mg of dry paste for later use.

2. Viral strains

Dengue virus, chikungunya virus.

3. Cell line

Human liver cancer cell Huh-7, human cervical carcinoma cell Hela and milk hamster kidney cell BHK 21.

4. Primary reagents and instruments

DMEM culture medium, RPMI-1640 culture medium, FBS, Trypsin, alamar Blue, cell culture plate, DMSO, crystal violet solution, centrifugal concentrator, rotary evaporator, biological safety cabinet, CO2 incubator, pipette gun and microplate reader.

Second, Experimental methods

1. Drug cytotoxicity assay

(1) Taking a proper amount of Huh-7(Hela) tumor cells in logarithmic growth phase into a 75ml culture bottle, culturing at 37 ℃ until the growth state of the cells is good, digesting the cells with Trypsin according to the ratio of 6.5 multiplied by 10³One/hole (1.3 is multiplied by 10)⁴One/well) was inoculated in a 96-well plate and cultured for 24 hours.

(2) After each extract was dissolved in DMSO, the resulting mixture was prepared into a drug-containing medium with drug concentrations of 100.00. mu.g/ml, 50.00. mu.g/ml, 25.00. mu.g/ml, 12.50. mu.g/ml, and 6.25. mu.g/ml in DMEM medium containing serum, and was used. 0.1% DMSO as solvent control.

(3) Taking out the cells which are cultured for 24 hours, pouring out the culture medium in a 96-well plate, adding a drug-containing culture medium, setting 3 parallels for each concentration of each sample, additionally setting a model group and a solvent control group, adding a drug-free culture medium into the model group, adding a culture medium containing 0.1% DMSO into the solvent control group, setting 3 parallels for each group, and continuously culturing for 48 hours at 37 ℃.

(4) The cells after 48h of culture were removed, the medium was decanted, 100. mu.l (1:10 configuration) of alamar bury solution was added to each well, three wells were filled with the same amount of alamar blue solution as a blank, incubated at 37 ℃ for 2h (degree to complete color change of the model group to pink), and the cells were removed and measured for fluorescence in a microplate reader (excitation wavelength 570nm, emission wavelength 585 nm).

The calculation formula is as follows: (mean value of fluorescence values for drug-containing group-mean value of fluorescence values for blank group)/(mean value of fluorescence values for model group-mean value of fluorescence values for blank group) × 100%

2. Antiviral efficacy test

(1) Taking Huh-7(Hela) cells cultured to logarithmic growth phase, digesting and then pressing 5 x 10⁴One hole (7 is multiplied by 10)⁴One/well) were added to 24-well plates and cultured for 24 hours.

(2) The 24-well plates that had been cultured for 24h were removed and the medium in each well was decanted. Thawing DENV-2(CHIKV) virus in water bath at 37 ℃ for 30 s-1 min, and preparing the virus-containing 5 multiplied by 10 by using DMEM medium⁵PFU/ml(7×10⁵PFU/ml) was added to each well 100 μ l of virus dilution to make the virus unit of 5 × 10 per well inoculated in 24-well plates⁴PFU (7×10⁴PFU), incubation at 37 ℃ for 1h, taking out and washing twice with PBS. Then the administration is carried out, and the administration concentration of the banana leaf extract refers to the safe concentration in the cytotoxicity test (the Huh-7/Hela cell survival rate is more than or equal to 80 percent after the administration), namely 6.25 mu g/ml, 12.50 mu g/ml, 25.00 mu g/ml and 50.00 mu g/ml. Three parallel wells were set, and a volume of 1ml was administered per well using 0.1% DMSO as a solvent control. Culturing at 37 deg.C for 48h, sucking out the culture medium from each well, and labeling in 1.5ml centrifuge tube, and storing at-80 deg.C.

(3) BHK21 cells were harvested at logarithmic growth phase and digested at 5X 10⁴The amount per well was added to 24 well plates, and 6 wells were inoculated per concentration per fraction and incubated for 24 h. After the culture, the medium was taken out and poured. Taking out the virus-containing samples stored at-80 deg.C under item (2), and diluting each sample to 6 concentrations of 10 times by gradient^-1、10^-2、 10^-3、10^-4、10^-5、10^-6Adding each diluted virus-containing diluent into cultured BHK21 cells according to the dosage of 100 mul/hole, incubating at 37 deg.C for 1h, taking out, adding PBS, washing twice, and adding culture medium (C)1% CMC + 2% FBS)1 ml/well. Cultured at 37 ℃ for 6d (4 d). After being taken out, the culture medium is poured out, the crystal violet solution is added, and the shaking table is shaken overnight. And pouring out the crystal violet solution, cleaning with clear water, wiping dry, and counting the plaques.

(4) Taking a wiped 24-well plate, counting by naked eyes, recording the number of plaques corresponding to the wells with the number of plaques ranging from 1 to 20, comparing the log values of the total number of plaques of the administration group and the control group (the total number of plaques is the number of plaques of the naked eyes multiplied by the corresponding dilution multiple multiplied by 10), taking that the mean value of the log values of the number of plaques of the control group-the mean value of the log values of the number of plaques of the administration group is more than or equal to 1 as the drug has obvious antiviral effect at the concentration, and if the mean value of 1 more than or equal to the mean value of the log values of the number of plaques of the control group-the mean value of the log values of the number of plaques of the administration group is more than or equal to 0.5, the drug has the antiviral effect at the concentration, and meanwhile, calculating the IC50 value according to the inhibition rate.

Third, experimental results

As shown in FIGS. 1 to 3, the banana leaf extract has antiviral effect when administered at a concentration of 12.50. mu.g/ml or more, and completely inhibits virus proliferation when administered at a concentration of 50.00. mu.g/ml, and the banana leaves have dengue virus IC₅₀3.79 μ g/ml, p-chikungunya virus IC₅₀It was 8.73. mu.g/ml.

Claims

1. The application of the banana leaf extract in preparing the medicine for resisting chikungunya fever virus is characterized in that the banana leaf extract is an ethanol extract of banana leaves and is prepared by the following steps: ultrasonic extracting folium Musae with ethanol, filtering, vacuum filtering, rotary evaporating the filtrate to recover solvent to concentrated liquid, and centrifuging to obtain dry extract; the ethanol is 95% ethanol with volume concentration, and the addition amount is 8 times of the weight of the banana leaves; the ultrasonic extraction is carried out for 2 hours, and the rotary evaporation temperature is 40 ℃.