CN110448623A - The preparation method of banana leaf extract and anti-chikungunya virus application - Google Patents
The preparation method of banana leaf extract and anti-chikungunya virus application Download PDFInfo
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- CN110448623A CN110448623A CN201910478724.XA CN201910478724A CN110448623A CN 110448623 A CN110448623 A CN 110448623A CN 201910478724 A CN201910478724 A CN 201910478724A CN 110448623 A CN110448623 A CN 110448623A
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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)
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- 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
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- 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
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Abstract
The invention discloses the ethanol extracts of Banana Leaf to have certain inhibition chikungunya disease toxic action.Accordingly, inventor has also set up the corresponding extracting method of Banana Leaf ethanol extract.Experiment shows that Banana Leaf ethanol extract has anti-chikungunya virus effect, to chikungunya virus IC50For 8.73 μ g/ml.As it can be seen that Banana Leaf has the potential value of anti-Chikungunya fever 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 Chikungunya fever, to reduce drug production cost.
Description
Technical field
The invention belongs to chikungunya virus treatment technology field more particularly to a kind of preparation methods of banana leaf extract
With anti-chikungunya 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, Chikungunya fever (Chikungunya Fever) is one of which.
Chikungunya fever is a kind of mosquito matchmaker infection caused by chikungunya virus (Chikungunya virus, CHIKV)
Disease is mainly characterized by fever, headache, courbature, fash and arthralgia.After other symptoms disappearance, the joint of some patients
Pain sustainable many years, serious symptom person can only keep bending position, initially be popular in the subtropical and tropical zones in Africa, and
Constantly expand to South Asia, Southeast Asia, Indian Ocean island and America area.In the more than ten years in past, breaking out for Chikungunya fever is secondary
Number increases, and Epidemic Scope constantly expands, and occurs in a countries and regions in the whole world more than 100, cause in global range it is annual about
1000000 people infection.
Currently, Chikungunya fever there is no vaccine for prevention, clinically there are no definite effective cause of disease therapeutic agents, only
Can be based on symptomatic treatment, therefore studying anti-chikungunya disease cytotoxic 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 a kind of preparation method of banana leaf extract and anti-Chikungunya fevers
Virus applications open up Chinese herbal medicine approach sufficiently to excavate the pharmaceutical value of waste Banana Leaf for treatment Chikungunya fever.
In order to solve the above technical problems, the invention adopts the following technical scheme:
Banana leaf extract is preparing the application in anti-Chikungunya fever virus drugs.
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 there is the ethanol extract of Banana Leaf certain inhibition to step on chikungunya virus function.Accordingly, inventor has also set up perfume (or spice)
The corresponding extracting method of any of several broadleaf plants leaf ethanol extract.Experiment shows that Banana Leaf ethanol extract has anti-chikungunya virus effect,
It is to chikungunya virus IC50For 8.73 μ g/ml.As it can be seen that Banana Leaf has the potential value of anti-Chikungunya fever drug.Cause
This, the present invention provides strong foundation and reliable guarantee further to open up the medical applications of Banana Leaf, and banana resource can be improved
Utilization rate reduces the wasting of resources；Also Chinese herbal medicine approach is opened for treatment Chikungunya 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, banana leaf extract administration concentration ginseng
Examine the 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 (6)
1. banana leaf extract is preparing the application in anti-Chikungunya fever virus drugs.
2. applying according to claim 1, it is characterised in that: the banana leaf extract is the ethanol extract of Banana Leaf.
3. 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.
4. the preparation method of banana leaf extract according to claim 3, it is characterised in that: the Banana Leaf is Banana Leaf
Green blade position fresh goods.
5. the preparation method of banana leaf extract according to claim 4, it is characterised in that: the ethyl alcohol is volumetric concentration
95% ethyl alcohol, additional amount are 8 times of Banana Leaf weight.
6. the preparation method of banana leaf extract according to claim 5, it is characterised in that: the ultrasonic extraction 2h, rotation
Steaming temperature is 40 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910478724.XA CN110448623B (en) | 2019-06-03 | 2019-06-03 | Preparation method of banana leaf extract and application of banana leaf extract in resisting chikungunya virus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910478724.XA CN110448623B (en) | 2019-06-03 | 2019-06-03 | Preparation method of banana leaf extract and application of banana leaf extract in resisting chikungunya virus |
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| CN110448623A true CN110448623A (en) | 2019-11-15 |
| CN110448623B CN110448623B (en) | 2022-03-01 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004083526A (en) * | 2002-08-28 | 2004-03-18 | Morinaga Milk Ind Co Ltd | Antiviral composition |
| CN103320476A (en) * | 2012-03-20 | 2013-09-25 | 张爱诚 | Method and technology for high-value comprehensive utilization of banana stems and leaves |
| CN103919939A (en) * | 2014-04-16 | 2014-07-16 | 王祥培 | Application of japanese banana stem and leaf extract to preparation of medicament for treating or preventing diabetes |
-
2019
- 2019-06-03 CN CN201910478724.XA patent/CN110448623B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004083526A (en) * | 2002-08-28 | 2004-03-18 | Morinaga Milk Ind Co Ltd | Antiviral composition |
| CN103320476A (en) * | 2012-03-20 | 2013-09-25 | 张爱诚 | Method and technology for high-value comprehensive utilization of banana stems and leaves |
| CN103919939A (en) * | 2014-04-16 | 2014-07-16 | 王祥培 | Application of japanese banana stem and leaf extract to preparation of medicament for treating or preventing diabetes |
Non-Patent Citations (1)
| 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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