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

The preparation method and anti-dengue virus application of banana leaf extract Download PDF

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CN110123921A
CN110123921A CN201910478232.0A CN201910478232A CN110123921A CN 110123921 A CN110123921 A CN 110123921A CN 201910478232 A CN201910478232 A CN 201910478232A CN 110123921 A CN110123921 A CN 110123921A
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banana leaf
banana
dengue
preparation
leaf extract
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CN110123921B (en
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邓家刚
侯小涛
郝二伟
李聪
杜正彩
秦建峰
韦玮
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Guangxi University of Chinese Medicine
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Guangxi University of Chinese Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Communicable Diseases (AREA)
  • Organic Chemistry (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
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  • Microbiology (AREA)
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  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)

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

The preparation method and anti-dengue virus application of banana leaf extract
Technical field
The invention belongs to dengue fever virus treatment technology field more particularly to a kind of preparation method of banana leaf extract and Anti-dengue virus application.
Background technique
Virus is one kind of nature microorganism, is divided into DNA virus and two kinds of RNA virus by nucleic acid type, in nature It is widely distributed in boundary, can bacterial infection, fungi, plant, animal and people, Chang Yinqi host morbidity.People caused by virus takes a disease Sick very much, dengue fever (Dengue fever) is one of which.
Dengue fever be caused by 1~5 type dengue virus (Dengue virus, DENV) and by Aedes aegypti or lineae ablicantes she The acute mosquito matchmaker infectious disease that mosquito bite is propagated.The popular disease is often in fulminant, and disease incidence is high, but case fatality rate is lower, serious to suffer from Person shows as vascular permeability increases, Hypovolemia and clotting mechanism are abnormal etc., and patient with severe symptoms can lead to death.The disease is mainly sent out Raw in whole world subtropical and tropical zones, DF epidemic situation is in rising trend in recent years, and WHO statistics is shown, there are about 25 in the whole world The threat that~40 hundred million people are infected by DENV estimates that morbidity is up to 0.5~100,000,000 every year, the raw DENV recessiveness sense of nearly 300,000,000 human hair in the whole world Dye.Existing 128 countries of DF epidemic situation occur for the whole world at present, and DF is in Africa, America, Eastern Mediterranean, Southeast Asia and Western Pacific 100 Multiple countries are in endemic conditions, even more serious with America, Southeast Asia and West Pacific region, with going deep into for international exchange, DF has become the serious public health problem in the whole world.Since 1978, China is as caused by Imported cases when DF outbreak of epidemic There is generation, the provinces such as Guangdong, Guangxi, Yunnan, Fujian and Henan report that the DF as caused by Imported cases is broken out, and seriously threatens me The healthy living of the state people.
It, can only be with clinically there are no definite effectively cause of disease therapeutic agent currently, dengue fever there is no vaccine for prevention Based on symptomatic treatment, therefore studying anti-dengue virus drug is the emphasis to work at present.
Banana (Musa nana Lour.) is the perennial large-scale unifacial leaf herbaceous plant of Musaceae Musa, widely distributed In 30 ° of tropical and subtropical region below of north and south latitude.Banana category high heat fruit, pulp rich in carbohydrate, protein, Fat, dietary fiber, various trace elements and vitamin, the fourth-largest grain in the world to be only second to rice, wheat and corn Crop.About 136 countries and regions produce banana, and main producing region concentrates on the states such as India, Philippine, China, Brazil.In recent years Come, World banana harvest area and yield are in ever-increasing trend, are also generated while harvesting a large amount of banana products several The agricultural wastes such as the banana stem leaves of equivalent.It is urgently solved for this purpose, how to be recycled to banana stem leaves and have become one Project certainly.Banana stem leaves volume is big, thus it is high to collect labor intensity；Water content is high, can neither directly burn and increase transport Cost；But itself biomass with higher, nutriment is abundant, possesses huge potentiality to be exploited.Current most perfume (or spice) Any of several broadleaf plants stalk, blade etc. are used as feed, fertilizer, or for producing the products such as low-grade paper, coarse cloth, packing rope, further then may be used Fermentation and biogas production or alcohol.And the exploitation of its medical value is then rarely reported, and is especially used for antiviral research then not See relevant report.
Summary of the invention
The technical problem to be solved in the present invention is to provide the preparation methods and anti-dengue virus of a kind of banana leaf extract Using, sufficiently to excavate the pharmaceutical value of waste Banana Leaf, for treatment dengue fever open up Chinese herbal medicine approach.
In order to solve the above technical problems, the invention adopts the following technical scheme:
Banana leaf extract is preparing the application in anti-dengue virus drug.
Dengue fever virus is dengue 2-type virus.
Banana leaf extract is the ethanol extract of Banana Leaf.
The preparation method of banana leaf extract, takes Banana Leaf that EtOH Sonicate is added to extract, and filters, and filters, filtrate revolving recycling After solvent to concentration liquid, it is centrifuged to dry cream to obtain the final product.
Banana Leaf is Banana Leaf green blade position fresh goods.
Ethyl alcohol is the ethyl alcohol of volumetric concentration 95%, and additional amount is 8 times of Banana Leaf weight.
Ultrasonic extraction 2h, revolving temperature are 40 DEG C.
Aiming at the problem that effective exploitation is not yet received in current agricultural waste material Banana Leaf value, inventor is through furtheing investigate It was found that the ethanol extract of Banana Leaf has certain inhibition dengue virus effect.Accordingly, inventor has also set up Banana Leaf second The corresponding extracting method of alcohol extracting thing.Experiment shows that Banana Leaf ethanol extract has anti-dengue virus effect, to Dengue disease Malicious 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 is further to open The medical applications for warding off Banana Leaf provide strong foundation and reliable guarantee, and banana resource utilization can be improved, and reduce the wasting of resources； Also Chinese herbal medicine approach is opened for treatment dengue fever, to reduce drug production cost.
Detailed description of the invention
Fig. 1 is the ethanol extract anti-dengue virus plaque assays result figure of Banana Leaf of the present invention.
Fig. 2 is the anti-chikungunya virus plaque assays result figure of ethanol extract of Banana Leaf of the present invention.
Fig. 3 is the ethanol extract of Banana Leaf of the present invention to dengue virus and chikungunya virus inhibiting rate result figure.
Specific embodiment
One, experimental material
1. banana leaf extract
Banana Leaf green blade position fresh goods 100.00g is taken, adds 8 times of 95% ethyl alcohol of amount, ultrasonic extraction 2h is qualitative after filtering Filter paper filters, and filtrate is taken to be placed in 40 DEG C of recycling designs on Rotary Evaporators, until being placed in 40 DEG C of centrifugal concentratings after concentration liquid, obtaining 787.40mg dry cream is spare.
2. Strain
Dengue virus, chikungunya virus.
3. cell strain
Human liver cancer cell Huh-7, human cervical carcinoma cell Hela, newborn hamster kidney cell BHK21.
4. main agents and instrument
DMEM culture medium, RPMI-1640 culture medium, FBS, Trypsin, alamar Blue, tissue culture plate, DMSO, knot Crystalviolet solution, centrifuge concentrator, Rotary Evaporators, Biohazard Safety Equipment, CO2 incubator, liquid-transfering gun, microplate reader.
Two, experimental method
1. drug cytotoxicity is tested
(1) in right amount into 75ml culture bottle, 37 DEG C are cultivated to cell Huh-7 (Hela) tumour cell of logarithmic growth phase Growth conditions are good, and 6.5 × 10 are pressed after Trypsin vitellophag3A/hole (1.3 × 104A/hole) cell concentration be inoculated in 96 holes In plate, continue culture for 24 hours, it is spare.
(2) after dissolving each extract with DMSO, being configured to drug concentration with the DMEM culture medium containing serum is The pastille culture medium of 100.00 μ g/ml, 50.00 μ g/ml, 25.00 μ g/ml, 12.50 μ g/ml, 6.25 μ g/ml, it is spare.0.1% DMSO is as solvent control.
(3) cell cultivated for 24 hours is taken out, drug containing culture medium, each sample is added in the culture medium in 96 orifice plates that incline The each concentration of product sets 3 in parallel, separately sets model group and solvent control group, and not pastille culture medium, solvent control group is added in model group The culture medium containing 0.1%DMSO is added, is all provided with 3 in parallel, continues 37 DEG C of culture 48h.
(4) cell after taking out culture 48h, incline culture medium, and the alamar bule of 100 μ l (1:10 configuration) is added Solution separately takes three holes that same amount of alamar blue solution is added as blank group, and 37 DEG C of incubation 2h are (with model group color Variation is pink degree of being completely), fluorescence is detected in microplate reader (excitation wavelength 570nm, launch wavelength 585nm) after taking-up Value.
Calculation formula are as follows: (drug containing group fluorescence angle value average value-blank group fluorescence angle value average value)/(model group fluorescence Angle value average value-blank group fluorescence angle value average value) × 100%
2. antiviral pharmacodynamic test
(1) it takes culture to Huh-7 (Hela) cell of logarithmic growth phase, 5 × 10 is pressed after digestion4A/hole (7 × 104A/ Hole) amount add in 24 orifice plates, culture for 24 hours.
(2) 24 orifice plates cultivated for 24 hours, the culture medium in each hole of inclining are taken out.37 DEG C of water-bath defrosting DENV-2 (CHIKV) virus 30s~1min is configured to containing virus be 5 × 10 with DMEM culture medium5PFU/ml(7×105PFU/ml) dilute Liquid is released, 100 μ l viral dilutions, which are added, toward every hole makes the viral units 5 × 10 for being inoculated in each hole in 24 orifice plates4PFU(7× 104PFU), 37 DEG C of incubation 1h, after taking-up plus PBS is cleaned twice.Then it is administered, the reference of banana leaf extract administration concentration Safe concentration (Huh-7/Hela cell survival rate >=80% after administration) in cytotoxicity experiment, as 6.25 μ g/ml, 12.50μg/ml,25.00μg/ml,50.00μg/ml.If three parallel holes, using 0.1%DMSO as solvent control group, every hole is given Medicine body accumulates 1ml.Culture medium suction in each hole is filled in the centrifuge tube of 1.5ml after culture and marks by 37 DEG C of culture 48h After note, -80 DEG C of preservations.
(3) the BHK21 cell in logarithmic growth phase is taken, 5 × 10 are pressed after digestion4The amount in a/hole adds in 24 orifice plates, often A each concentration of flow point needs to be inoculated with 6 holes, and culture is for 24 hours.It is taken out after culture, topples over culture medium.- 80 DEG C are taken out under (2) item What is saved contains Virus Sample, and each sample is both needed to by 10 times of gradient dilutions into 6 concentration, and respectively 10-1、10-2、10-3、10-4、 10-5、10-6, being added respectively by the dosage in 100 holes μ l/ after each dilution containing viral dilution is thin to cultured BHK21 In born of the same parents, 37 DEG C of incubation 1h are added PBS cleaning twice, add the hole culture medium (1%CMC+2%FBS) 1ml/ later after taking-up. 37 DEG C of culture 6d (4d).Culture medium is removed after taking-up, and crystal violet solution, shaking table shaking overnight is added.Decrystallize purple solution, clearly It is dried after water cleaning, carries out plaque counting.
(4) 24 orifice plates dried are taken, are visually counted, record plaque quantity is the corresponding plaque in hole between 1~20 Number, is compared (total plaque number=naked eyes plaque number × corresponding dilute with the logarithm of total plaque number of control group by administration group Release multiple × 10), it is drug at this with the mean value of control group plaque number logarithm-administration group plaque number logarithm mean value >=1 There is significant antivirus action, if the mean value of 1 >=control group plaque number logarithm-administration group plaque number logarithm is equal under concentration Value >=0.5 item indicates that drug has antiviral effect under the concentration, while calculating IC50 value according to inhibiting rate.
Three, experimental result
As shown in Figure 1 to Figure 3, there is disease-resistant toxic effect when banana leaf extract administration concentration is greater than or equal to 12.50 μ g/ml Fruit, administration concentration can completely inhibit virus multiplication when being 50.00 μ g/ml, and Banana Leaf is to dengue virus IC50For 3.79 μ g/ml, To chikungunya virus IC50For 8.73 μ g/ml.

Claims (7)

1. banana leaf extract is preparing the application in anti-dengue virus drug.
2. applying according to claim 1, it is characterised in that: the dengue fever virus is dengue 2-type virus.
3. applying according to claim 1, it is characterised in that: the banana leaf extract is the ethanol extract of Banana Leaf.
4. a kind of preparation method of banana leaf extract, it is characterised in that: it takes Banana Leaf that EtOH Sonicate is added to extract, filters, filter, After filtrate rotates recycling design to concentration liquid, it is centrifuged to dry cream to obtain the final product.
5. the preparation method of banana leaf extract according to claim 4, it is characterised in that: the Banana Leaf is Banana Leaf Green blade position fresh goods.
6. the preparation method of banana leaf extract according to claim 5, it is characterised in that: the ethyl alcohol is volumetric concentration 95% ethyl alcohol, additional amount are 8 times of Banana Leaf weight.
7. the preparation method of banana leaf extract according to claim 6, it is characterised in that: the ultrasonic extraction 2h, rotation Steaming temperature is 40 DEG C.
CN201910478232.0A 2019-06-03 2019-06-03 Preparation method of banana leaf extract and application of banana leaf extract in resisting dengue fever virus Active CN110123921B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004083526A (en) * 2002-08-28 2004-03-18 Morinaga Milk Ind Co Ltd Antiviral composition
CN106336390A (en) * 2016-08-25 2017-01-18 岑健 Method for extracting flavone from banana leaves

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004083526A (en) * 2002-08-28 2004-03-18 Morinaga Milk Ind Co Ltd Antiviral composition
CN106336390A (en) * 2016-08-25 2017-01-18 岑健 Method for extracting flavone from banana leaves

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MICHAEL D. SWANSON ETAL.: "A Lectin Isolated from Bananas Is a Potent Inhibitor of HIV Replication", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 *

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