CN110123921A - The preparation method and anti-dengue virus application of banana leaf extract - Google Patents

Abstract

The invention discloses the ethanol extracts of Banana Leaf to have certain inhibition dengue virus effect.Accordingly, inventor has also set up the corresponding extracting method of Banana Leaf ethanol extract.Experiment shows that Banana Leaf ethanol extract has anti-dengue virus effect, to dengue virus IC₅₀For 3.79 μ g/ml.As it can be seen that Banana Leaf has the potential value of anti-dengue drug.Therefore, the present invention provides strong foundation and reliable guarantee further to open up the medical applications of Banana Leaf, and banana resource utilization can be improved, and reduces the wasting of resources；Also Chinese herbal medicine approach is opened for treatment dengue fever, to reduce drug production cost.

Description

Preparation method of banana leaf extract and anti-dengue virus application

technical field

The invention belongs to the technical field of dengue fever virus treatment, and in particular relates to a preparation method of banana leaf extract and an anti-dengue virus application.

Background technique

Viruses are a kind of microorganisms in nature. They are divided into DNA viruses and RNA viruses according to the type of nucleic acid. They are widely distributed in nature and can infect bacteria, fungi, plants, animals and humans, and often cause disease in the host. People suffer from many diseases caused by viruses, and dengue fever (Dengue fever) is one of them.

Dengue fever is an acute mosquito-borne infectious disease caused by dengue virus (DENV) types 1-5 and transmitted through the bite of Aedes aegypti or Aedes albopictus. The prevalence of the disease is often fulminant, with a high morbidity rate, but a low case fatality rate. Severe cases show increased vascular permeability, hypovolemia, and abnormal coagulation mechanisms. Severe cases can lead to death. The disease mainly occurs in tropical and subtropical regions of the world. In recent years, the DF epidemic has been on the rise. According to WHO statistics, about 2.5 to 4 billion people in the world are threatened by DENV infection. Nearly 300 million people have DENV recessive infection. At present, DF epidemics have occurred in 128 countries around the world. DF is endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, Southeast Asia and the Western Pacific, and it is more serious in the Americas, Southeast Asia and the Western Pacific. In-depth, DF has become a serious global public health problem. Since 1978, outbreaks of DF caused by imported cases have occurred frequently in my country. Guangdong, Guangxi, Yunnan, Fujian, Henan and other provinces have reported DF outbreaks caused by imported cases, which seriously threaten the healthy life of our people.

At present, there is no vaccine available for prevention of dengue fever, and there is no exact and effective pathogenic treatment drug in clinical practice, and only symptomatic treatment is given priority to. Therefore, research on anti-dengue virus drugs is the focus of current work.

Banana (Musa nana Lour.) is a perennial large-scale monocotyledonous herbaceous plant of the genus Musa in the Musa family. It is widely distributed in tropical and subtropical regions below 30° north and south latitude. Banana is a high-calorie fruit. The pulp is rich in carbohydrates, protein, fat, dietary fiber, various trace elements and vitamins. It is the fourth largest food crop in the world after rice, wheat and corn. About 136 countries and regions produce bananas, and the main production areas are concentrated in India, the Philippines, China, Brazil and other countries. In recent years, the harvested area and output of bananas in the world have shown a continuous growth trend. While harvesting a large number of banana products, almost the same amount of agricultural waste such as banana stems and leaves is produced. For this reason, how to recycle banana stems and leaves has become an urgent problem to be solved. Banana stems and leaves are bulky, so the collection labor intensity is high; the water content is high, which can neither be burned directly nor increase the transportation cost; but it has high biomass and rich nutrients, and has huge development potential. At present, most of the banana stems and leaves are used as feed, fertilizer, or used to produce low-grade paper, coarse cloth, packaging rope and other products, and further can be fermented to produce biogas or alcohol. However, there are few reports on the development of its medicinal value, especially for antiviral research.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of banana leaf extract and anti-dengue fever virus application, so as to fully explore the medical value of waste banana leaves and open up a way of Chinese herbal medicine for the treatment of dengue fever.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

Application of banana leaf extract in preparation of anti-dengue virus medicine.

Dengue virus is dengue type 2 virus.

Banana leaf extract is the ethanol extract of banana leaves.

The preparation method of the banana leaf extract comprises the following steps: taking banana leaves and adding ethanol for ultrasonic extraction, filtering, suction filtering, rotating the filtrate to recover the solvent to a concentrated liquid state, and centrifuging until the dry paste is obtained.

The banana leaf is a fresh product of the green leaf part of the banana leaf.

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

Ultrasonic extraction was carried out for 2 hours, and the rotary steaming temperature was 40°C.

Aiming at the problem that the value of banana leaves, the agricultural wastes, has not been effectively exploited, the inventors have found through in-depth research that the ethanol extract of banana leaves has a certain inhibitory effect on dengue virus. Accordingly, the inventor has also established a corresponding extraction method of the banana leaf ethanol extract. Experiments show that the ethanol extract of banana leaves has anti-dengue virus effect, and its IC ₅₀ against dengue virus is 3.79 μg/ml. It can be seen that banana leaves have the potential value of anti-dengue drugs. Therefore, the present invention provides a strong basis and reliable guarantee for further developing the medical application of banana leaves, which can improve the utilization rate of banana resources and reduce waste of resources; it also opens up a way of Chinese herbal medicine for the treatment of dengue fever, so as to reduce the cost of drug production.

Description of drawings

Fig. 1 is the result figure of the anti-dengue virus plaque test of the ethanol extract of banana leaf of the present invention.

Fig. 2 is a graph showing the results of the anti-chikungunya virus plaque test of the ethanol extract of banana leaves of the present invention.

Fig. 3 is the result chart of the inhibition rate of dengue virus and chikungunya virus by ethanol extract of banana leaves of the present invention.

Detailed ways

1. Experimental materials

1. Banana Leaf Extract

Take 100.00g fresh product from the green leaf part of banana leaf, add 8 times the amount of 95% ethanol, ultrasonically extract for 2 hours, filter and filter with qualitative filter paper, take the filtrate and put it on a rotary evaporator at 40°C to recover the solvent, until it is concentrated liquid, put it in Concentrate by centrifugation at 40°C to obtain 787.40 mg of dry paste for future use.

2. Virus strains

Dengue virus, Chikungunya virus.

3. Cell lines

Human liver cancer cells Huh-7, human cervical cancer cells Hela, and suckling hamster kidney cells BHK21.

4. Main reagents and instruments

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

2. Experimental method

1. Drug Cytotoxicity Test

(1) Take an appropriate amount of Huh-7 (Hela) tumor cells in the logarithmic growth phase and put them in a 75ml culture flask, culture them at 37°C until the cells grow well, digest the cells with Trypsin at 6.5×10 ³ cells/well (1.3×10 ⁴ cells/well) were inoculated in a 96-well plate, continued to culture for 24 h, and were set aside.

(2) After dissolving each extract with DMSO, use serum-containing DMEM medium to prepare drug concentrations of 100.00 μg/ml, 50.00 μg/ml, 25.00 μg/ml, 12.50 μg/ml, 6.25 μg/ml containing Medicine culture medium, spare. 0.1% DMSO was used as solvent control.

(3) Take out the cells that have been cultured for 24 hours, discard the medium in the 96-well plate, add the medium containing the drug, set 3 parallels for each concentration of each sample, and set up a model group and a solvent control group separately. The drug-containing medium and the medium containing 0.1% DMSO were added to the solvent control group, and three parallel sets were set up, and culture was continued at 37°C for 48h.

(4) Take out the cells cultured for 48 hours, discard the culture medium, add 100 μl (1:10 configuration) of alamar bule solution, add the same amount of alamar blue solution to another three wells as a blank group, and incubate at 37°C for 2 hours ( The color of the model group has completely changed to pink as the degree), and after taking it out, detect the fluorescence value in a microplate reader (excitation wavelength 570nm, emission wavelength 585nm).

The calculation formula is: (the average value of the fluorescence value of the drug-containing group-the average value of the fluorescence value of the blank group)/(the average value of the fluorescence value of the model group-the average value of the fluorescence value of the blank group)×100%

2. Antiviral efficacy test

(1) Huh-7 (Hela) cells cultured to the logarithmic growth phase were taken, digested and added to a 24-well plate at an amount of 5×10 ⁴ cells/well (7×10 ⁴ cells/well), and cultured for 24 hours.

(2) Take out the 24-well plate that has been cultured for 24 hours, and pour off the medium in each well. Thaw DENV-2 (CHIKV) virus in a water bath at 37°C for 30 seconds to 1 minute, prepare a virus-containing dilution of 5×10 ⁵ PFU/ml (7×10 ⁵ PFU/ml) with DMEM medium, and add 100 μl of virus to each well The dilution solution made the virus unit inoculated in each well of the 24-well plate 5×10 ⁴ PFU (7×10 ⁴ PFU), incubated at 37° C. for 1 hour, took it out, and washed it twice with PBS. Administration is then carried out, and the administration concentration of banana leaf extract refers to the safe concentration in the cytotoxicity experiment (Huh-7/Hela cell survival rate ≥ 80% after administration), which is 6.25 μg/ml, 12.50 μg/ml, 25.00 μg/ml, 50.00μg/ml. Three parallel wells were set up, 0.1% DMSO was used as the solvent control group, and the volume of administration in each well was 1 ml. Cultivate at 37°C for 48h, after the end of the culture, suck out the culture medium in each well and put it into a 1.5ml centrifuge tube for marking, then store at -80°C.

(3) BHK21 cells in the logarithmic growth phase were taken, and after digestion, 5×10 ⁴ cells/well were added to a 24-well plate. Each fraction and concentration needed to inoculate 6 wells, and cultured for 24 hours. After the cultivation, take it out and pour out the culture medium. Take out the virus-containing samples stored at -80°C under item (2), and each sample needs to be diluted into 6 concentrations according to a 10-fold gradient, which are 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^{- 5} , 10 ^-6 , each diluted virus-containing dilution was added to the cultured BHK21 cells at a dose of 100 μl/well, incubated at 37°C for 1 hour, removed and washed twice with PBS, and then added to the culture medium (1% CMC+2% FBS) 1 ml/well. Incubate at 37°C for 6d (4d). After taking it out, pour out the medium, add crystal violet solution, and shake overnight on a shaker. Pour off the crystal violet solution, wash with water and dry, and count the plaques.

(4) Take the dried 24-well plate, count with the naked eye, record the number of plaques corresponding to the wells with 1 to 20 plaques, and perform the logarithm of the total number of plaques in the treatment group and the control group Comparison (total plaque number = naked eye plaque number × corresponding dilution factor × 10), the mean value of the logarithmic value of the plaque number in the control group - the mean value of the logarithmic value of the plaque number in the administration group ≥ 1 means that the drug has the effect at this concentration Significant antiviral effect, if 1 ≥ the mean value of the logarithmic value of the plaque number in the control group - the mean value of the logarithmic value of the plaque number in the treatment group ≥ 0.5, it means that the drug has an antiviral effect at this concentration, and the IC50 value is calculated based on the inhibition rate.

3. Experimental results

As shown in Figures 1 to 3, banana leaf extract has an antiviral effect when the administration concentration is greater than or equal to 12.50 μg/ml, and can completely inhibit virus proliferation when the administration concentration is 50.00 μg/ml. The IC ₅₀ of the virus was 3.79 μg/ml, and the IC ₅₀ of Chikungunya virus was 8.73 μg/ml.

Claims (7)

1. Application of banana leaf extract in the preparation of anti-dengue virus medicine. 2. The application according to claim 1, characterized in that: the dengue virus is dengue type 2 virus. 3. The application according to claim 1, characterized in that: the banana leaf extract is the ethanol extract of banana leaves. 4. A preparation method of banana leaf extract, which is characterized in that: take banana leaves and add ethanol for ultrasonic extraction, filter, suction filter, spin the filtrate to recover the solvent to a concentrated liquid, and then centrifuge to a dry paste. 5. the preparation method of banana leaf extract according to claim 4 is characterized in that: described banana leaf is the fresh product of green leaf part of banana leaf. 6. the preparation method of banana leaf extract according to claim 5 is characterized in that: described ethanol is the ethanol of volume concentration 95%, and add-on is 8 times of banana leaf weight. 7. The method for preparing the banana leaf extract according to claim 6, characterized in that: the ultrasonic extraction takes 2 hours, and the rotary steaming temperature is 40°C.