CN106265786A - Willow herb AntiHIV1 RT activity 1 virus effective site and preparation method and application - Google Patents
Willow herb AntiHIV1 RT activity 1 virus effective site and preparation method and application Download PDFInfo
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- CN106265786A CN106265786A CN201610780007.9A CN201610780007A CN106265786A CN 106265786 A CN106265786 A CN 106265786A CN 201610780007 A CN201610780007 A CN 201610780007A CN 106265786 A CN106265786 A CN 106265786A
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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)
- A61K36/185—Magnoliopsida (dicotyledons)
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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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- 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
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
Abstract
Willow herb AntiHIV1 RT activity 1 virus effective site and preparation method and application, belong to plant extract technical field.Position is extracted selected from willow herb n-butanol portion or willow herb aqueous phase.Willow herb herb is pulverized, adds concentration of volume percent 65% ~ 95% ethanol solution and extract, gained extracting solution is concentrated, is dried, obtain extractum；Extractum is dispersed in distilled water to obtain extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid the most successively, gained butanol extraction liquid is flung to solvent, obtains willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.The willow herb AntiHIV1 RT activity 1 virus effective site application in terms of preparing AntiHIV1 RT activity 1 virus drugs of the present invention, it is adaptable to develop into anti-AIDS new drug.
Description
Technical field
The present invention relates to willow herb anti-HIV-1 virus effective site and preparation method and application, belong to plant extract technical field.
Background technology
Willow herb (Epilobium angustifolium L.) be Oenotheraceae (Onagraceae) willow herb belong to perennial slightly
Strong herbaceous plant, is Tibetan medicine's tradition medicinal plants.It is mainly born in hillside border, sylvan life and the river valley of height above sea level 2800 ~ 4250 meters
Wet meadow.It is distributed in Southwestern China, northwest, North China to northeast.North temperate zone blazons, and North America, Europe to Japan, Asia Minor are extremely
The ground such as Himalaya also have.Willow herb herb bitter in the mouth, nontoxic, there are detumescencing diuresis, stimulating milk secretion, the function of intestine moistening.Cure mainly hypogalactia, gas
Impractical swollen.Lattice erg Sang Zhaxi records it in " practical Tibetan medicine name storehouse " and has clearing heat secreting bile, antidiarrheal, insecticidal function.
Acquired immune deficiency syndrome (AIDS) i.e. acquired immune deficiency syndrome (AIDS) (is translated again: acquired immune deficiency syndrome).Nowadays acquired immune deficiency syndrome (AIDS) is own
Become serious harm human survival and the public health of development and social problem in the world.The popular warp of China's acquired immune deficiency syndrome (AIDS)
Crossing afferent phase, diffusion period, oneself enters the quick rise period at present, is in the whole nation low popular popular with some areas and specific crowd height
And the situation deposited.
Acquired immune deficiency syndrome (AIDS) is a kind of great infectious disease of hazardness, aids infection virus (inhibition of HIV) cause.HIV is one
Planting the virus that can attack human immune system, i.e. " HIV (human immunodeficiency virus) ", it destroys the siberian crabapple of human body after invading human body
System, makes human body that the multiple infection being difficult to cure and tumor to occur, ultimately results in death, confirmed that HIV is divided into amphitypy: HIV-1
Type and HIV-2 type.HIV-1 type is Retroviridae lentivirus, and its viral RNA genes group must be through in advance by single stranded RNA
Reverse transcription is cDNA, and after forming RNA-DNA heterozygote, the RNA complementary with DNA is fallen in hydrolysis, then closes with the cDNA of strand for template
Becoming double-stranded DNA, such that it is able to integrate in the middle of human host's DNA genome, this process is catalyzed by reverse transcriptase (RT enzyme).RT enzyme function
Various, it is constituted heterodimer by two protein monomers of P66 and P51, and wherein P66 is the main functional parts of enzymatic activity；Remove
Have outside reverse transcriptase activity, also there is DNA polymerase activity and RNase enzymatic activity.The molecular mechanism of process of reverse-transcription is also
Rather complicated: the initial primers of process of reverse-transcription is not from virus self synthesis, but in utilizing host cell
TRNALye-3, after combining with the primer binding site (PBS) of viral RNA, more efficiently starts process of reverse-transcription.Secondly, reverse
Record enzyme is because lacking general 3'-5' exonuclease activity, it is impossible to complete proofreading function, replicates fidelity poor, is the variation of inhibition of HIV height
One of major reason of property.Human immunodeficiency virus type 1 (i.e. HIV-1 type) reverse transcriptase (RT) is always important resisting
The target spot of HIV, for developing the medicine for the treatment of acquired immune deficiency syndrome (AIDS).Protease is in HIV (human immunodeficiency virus) gene code
Planting special Radix Asparagi acyl protease, its effect is by protein cleavage produced by gene and gene expression, becomes active disease
Poison structural protein and enzyme, be the key substance of inhibition of HIV duplication.It is multiple that hiv protease inhibitor mainly acts on HIV (human immunodeficiency virus)
The final stage of system, owing to protease is suppressed, being allowed to form DNA from the cd4 cell core infected can not assemble and discharge, and leads
Cause amyloid protein precursor to crack and form mature virion.
Applicant finds under study for action: the raw material of anti-AIDS drug is the deficientest at present.Existing anti HIV-1 virus medicine
As other kinds of Anti-AIDS Drugs, along with HIV1-RT produces resistance mutation, listed controls
The curative effect of the medicine treating acquired immune deficiency syndrome (AIDS) is restricted.At present, in the urgent need to exploitation a new generation anti-HIV-1 type virus drugs.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the deficiencies in the prior art, it is provided that willow herb anti-HIV-1 virus effectively portion
Position and preparation method and application, this willow herb anti-HIV-1 virus effective site can effectively suppress HIV-1 virus activity, it is possible to is used for preparing
Anti-HIV-1 virus drugs, it is adaptable to develop into anti-AIDS new drug.
The technical solution adopted for the present invention to solve the technical problems is: this willow herb anti-HIV-1 virus effective site, and it is special
Levy and be: extract position selected from willow herb n-butanol portion or willow herb aqueous phase.
Described n-butanol portion contains flavonoid glycoside and phenolic compound；Polysaccharide and organic acid are contained in aqueous phase extraction position
Compound.
The preparation method of this willow herb anti-HIV-1 virus effective site, it is characterised in that comprise the steps: willow herb complete
Grass meal is broken, adds concentration of volume percent 65% ~ 95% ethanol solution and extracts, and is concentrated by gained extracting solution, is dried, obtains extractum；Will
Extractum is dispersed in distilled water to obtain extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol to extract as solvent the most successively
Take extract dispersion liquid, gained butanol extraction liquid is flung to n-butyl alcohol, obtain willow herb n-butanol portion；Remaining extractum after extracting
Dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Described reflux, extract, is ultrasonic assistant reflux, extract, and concrete operations are: the willow herb herb after pulverizing adds
Concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds concentration of volume percent
65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
The application of this willow herb anti-HIV-1 virus effective site, it is characterised in that: in preparing anti-HIV-1 virus drugs
Application.
Described anti-HIV-1 virus drugs is to extract position, with pharmaceutic adjuvant by willow herb n-butanol portion or willow herb aqueous phase
After mixing, the oral formulations made or ejection preparation.
Described ejection preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
Described oral formulations is powder, tablet, granule, capsule, oral solution.
Applicant is described as follows for the present invention:
In preparation method, extractum is crude extract, uses different solvent extraction extract dispersion liquids, refers to successively by petroleum ether
Add extract dispersion liquid extract and separate to obtain petroleum ether extraction liquid, ethyl acetate adds extract dispersion liquid extract and separate acquisition second
Acetoacetic ester extract, n-butyl alcohol is added extract dispersion liquid extract and separate obtain butanol extraction liquid；Take acetic acid ethyl acetate extract
Volatilizing respectively with butanol extraction liquid after removing solvent, gained is i.e. selected from willow herb ethyl acetate extract and willow herb n-butanol portion；
Remaining extract dispersion liquid (solvent is water) after extracting, volatilization is removed solvent and is i.e. obtained willow herb aqueous phase extraction position.Aqueous phase extracts
Position may also be referred to as aqueous phase extract, and aqueous phase extraction position is soluble in water；And relative, petroleum ether, ethyl acetate and positive fourth
Alcohol is organic facies, and petroleum ether part, ethyl acetate extract are corresponding with n-butanol portion is soluble in petroleum ether, ethyl acetate and positive fourth
Alcohol.Wherein, willow herb n-butanol portion contains saponin and phenolic compound；Polysaccharide and organic acid are contained in willow herb aqueous phase extraction position
Compound.Fling to solvent i.e. volatilize removal solvent.Flinging to solvent can use normal heating to make solvent (petroleum ether, ethyl acetate
And n-butyl alcohol) volatilization.Preferably, flinging to solvent and use concentrating under reduced pressure, the method efficiency is high, and be avoided that heat-sensitive ingredients because of
High temperature loses the property of medicine.
Preferably dosage form is oral formulations or ejection preparation.Oral formulations and ejection preparation oral formulations and ejection preparation are
Optimal route of administration.Oral formulations described herein is through intestines and stomach drug-delivery preparation, it is preferred that oral formulations is slow-release controlled-release type
Oral formulations.Capsule includes soft capsule and hard capsule.It addition, the preparation of this willow herb anti-HIV-1 virus effective site can also
Preparation for other administration route: such as respiratory tract administration preparation (spray, aerosol, powder spray)；Percutaneous drug delivery preparation (external
Solution, lotion, liniment, ointment, plaster, paste, patch)；Film drug-delivery preparation (eye drop, nasal drop, ophthalmic ointment,
Gargarism, sublingual tablet)；Cavity/canal drug administration preparation (suppository).Ejection preparation can be conventional injection preparation.According to drug delivery system
System is classified, it is preferred that the ejection preparation of the present invention is lipidosome injection, nanoparticle injection or micro-balloon injection；Its
He, the injection of the present invention can also be microcapsule injection, polymer micelle injection, microemulsion or Submicroemulsion injection, Asia
Microgranule injection or gel injection, when the injection of above drug delivery system can extend pharmaceutical carrier circulation in vivo
Between, extend medicine carrying microgranule absorption site the time of staying, control medicine release the initial stage burst effect.Pharmaceutic adjuvant includes
Pharmaceutical carrier and solvent, solubilizing agent, cosolvent, emulsifying agent, suspending agent, clarifier, deflocculant, correctives, coloring agent, anti-
Rotten agent, chemosterilant, adsorbent, filter aid, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, binding agent, moistening
In agent, disintegrating agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material
One or more.
In preparation method: described ultrasonic extraction is to carry out under room temperature.Described concentration and being dried as concentrating under reduced pressure, true
Empty dry, to avoid there is scattering and disappearing of volatile willow herb effective site.Described reflux, extract, refers to for 1 ~ 3 time: add to backflow
Thermal adds the filtering residue after supersound extraction and ethanol solution, heating, reflux, extract, lets cool and filter to obtain an extracting solution and medicine
Slag；In medicinal residues, add ethanol solution, heat, carry out second time reflux, extract, let cool and filter to obtain secondary raffinate and medicinal residues；Close
And repeatedly extracting solution, obtain the extracting solution of reflux, extract,.Preferably, each reflux, extract, 0.5 ~ 2 hour；Every time during reflux, extract,
Added ethanol solution did not had medical material surface 1 ~ 2cm.
Compared with prior art, what the willow herb anti-HIV-1 virus effective site of the present invention and preparation method and application were had has
Benefit effect is:
1, this willow herb anti-HIV-1 virus effective site can effectively suppress HIV-1 virus activity, it is possible to is used for preparing anti-HIV-1
Virus drugs, it is adaptable to develop into anti-AIDS new drug.Applicant is by finding under study for action: only have diuretic in prior art
Eliminating dampness by diuresis, the willow herb of merit of promoting blood flow to regulate menstruation, also have effect of anti-AIDS.Applicant carries out drug effect first in HIV-1 virus
Screening, and determines by numerous studies: willow herb n-butanol portion, willow herb aqueous phase extract position by combine reverse transcriptase protein and
Suppression hiv protease can substantially suppress HIV-1 virus, and cytotoxicity increases with administration concentration and strengthens, it is adaptable to be prepared as
Anti-AIDS new drug.Applicant is finding new effect of willow herb, designs preparation method, by determining effective portion of its optimum
The kind of the enzyme that position and this effective site are suppressed, has paid substantial amounts of creative work in the above process.
2, the preparation method of this willow herb anti-HIV-1 virus effective site is extracted convenient, is the side that determines after deliberation of applicant
Method.In preparation method of the present invention, the extraction order of solvent is that applicant determines after deliberation, and order can not overturn replacement, use with
Willow herb n-butanol portion and the willow herb aqueous phase extraction position of upper solvent extraction order gained have preferably suppression HIV-1 virus
Effect.
3, the present invention has expanded the raw material channel of suppression HIV-1 virus, expands the purposes of willow herb, makes willow herb develop into
For suppressing the new raw material of HIV-1 virus drugs, it is remarkably improved the added value of willow herb.
Accompanying drawing explanation
The HIV-1 R3A wild virus infection suppression ratio result of Fig. 1 willow herb n-butanol portion.
The HIV-1 R3A wild virus infection suppression ratio result at Fig. 2 willow herb aqueous phase extraction position..
Fig. 3 willow herb n-butanol portion pseudovirus single cycle infection.
Fig. 4 willow herb aqueous phase extraction position pseudovirus single cycle infection.
Fig. 5 willow herb n-butanol portion CCK8 method cytotoxicity.
Fig. 6 willow herb n-butanol portion and the impact contrast of willow herb aqueous phase extraction position HIV-1 suppression reverse transcriptase activity
Figure.
Fig. 7 willow herb n-butanol portion and willow herb aqueous phase extraction position affect comparison diagram to HIV-1 virus activity.
Detailed description of the invention
Embodiment 1 ~ 3 is willow herb anti-HIV-1 virus effective site and the detailed description of the invention of preparation method, the Qi Zhongshi of the present invention
Execute example 1 for most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 75% ~ 85% ethanol solution to surpass
When sound wave extracts 1 ~ 2, then filtering and obtain ultrasonic extract and filtering residue, filtering residue adds mass percent concentration 75% ~ 85% ethanol
Aqueous solution reflux, extract, 3 times, each 0.5 hour, merges ultrasonic extract and reflux extracting liquid, concentrate drying obtains extractum；Will
Extractum adds distilled water dispersion, obtains extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction the most successively
Extract dispersion liquid, respectively the extract of each solvent, gained butanol extraction liquid is flung to n-butyl alcohol, obtains willow herb n-butyl alcohol portion
Position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Embodiment 2
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 85% ~ 95% ethanol solution to surpass
Sound wave extracts 2 ~ 3 hours, then filters and obtains ultrasonic extract and filtering residue, and filtering residue adds mass percent concentration 85% ~ 95% second
Alcohol-water solution reflux, extract, 2 times, each 1 hour, merges ultrasonic extract and reflux extracting liquid, concentrate drying obtains extractum；Will
Extractum adds distilled water dispersion, obtains extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction the most successively
Extract dispersion liquid, respectively the extract of each solvent, gained butanol extraction liquid is flung to n-butyl alcohol, obtains willow herb n-butyl alcohol portion
Position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Embodiment 3
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 65% ~ 75% ethanol solution to surpass
Sound wave extracts 0.5 ~ 1 hour, then filters and obtains ultrasonic extract and filtering residue, and filtering residue adds mass percent concentration 65% ~ 75%
Ethanol water reflux, extract, 2 times, each 2 hours, merges ultrasonic extract and reflux extracting liquid, concentrate drying obtains extractum；
Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol to extract as solvent the most successively
Take extract dispersion liquid, respectively the extract of each solvent, gained butanol extraction liquid is flung to n-butyl alcohol, obtains willow herb n-butyl alcohol portion
Position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Performance test
The willow herb n-butanol portion (being numbered L14) obtained using embodiment 1 and willow herb aqueous phase extraction position (being numbered L14) as
Test medicine, tests as follows.
One, HIV-1 R3A wild virus infection experiment.
The each 100mg of accurately weighed L13, L14 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, preparation
The stock solution of 100mg/mL.During experiment successively with the culture medium of serum-free be diluted to series concentration be 100 g/mL, 33.3 g/
ML, 11.1 g/mL, 3.7 g/mL, 1.2 g/mL, for the administration group of variable concentrations.Experiment sets virus control group, cell controls
Group and medicine various dose group.Administration group adds the drug solution of 100 L variable concentrations in the 96 every holes of orifice plate, and every concentration sets three
Multiple hole.Take MT4 cell (5 × 105/ mL) add HIV-1 R3A virus 1 × 103TCID50/ mL, 37 DEG C, 5% CO2Cultivate 2 h,
PBS washs 3 times, centrifugal supernatant discarded, adds this sense of 100 L after adding complete medium in the 96 each holes added with medicine of orifice plate
The cell (5 × 10 of dye HIV-14/ 100 L), 37 DEG C, 5% CO2Cultivate, the three days later half liquid that changes, keep drug level constant, the
Within six days, collecting each hole and take supernatant, use ELISA method to measure P24 amount of antigen, each hole takes supernatant 50 L and joins first 1 day bed board
In TZMbl cell, 37 DEG C, CO248 h are cultivated in incubator.Use the detection of Promega BrightGlo luciferase kit
Luciferase content, and calculate suppression ratio, refer to Fig. 1 ~ 2.
Two, HIV-1 pseudovirus single cycle infection experiment.
The each 100mg of accurately weighed L13, L14 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, preparation
The stock solution of 100mg/mL.Diluting by the culture medium of serum-free successively during experiment, L13, L14 are 50 g/mL, 16.67 g/
ML, 5.56 g/mL, 1.85 g/mL, 0.62 g/mL, for the administration group of variable concentrations, only adding pseudovirus not dosing group is disease
Poison matched group, adds AZT(zidovudine) organize as positive controls.By TZM-bl cell (Hela cell express CD4, CXCR4 and
CCR5, the luciferase started containing Tat and LacZ gene) it is inoculated in 96 orifice plates, after 37 DEG C of incubators cultivate 24 h, discard
Clearly, add pSG3 pseudovirus supernatant, viral supernatants add DEAE(final concentration 10 g/mL), it is simultaneously introduced variable concentrations dilution
Medicine, after cultivating 48 h, supernatant discarded, add fixative room temperature fix 5min, after PBS add nitrite ion, 37 DEG C of trainings
Supporting and cultivate 50min in case, PBS, under the microscope counting locus coeruleus number, according to suppression ratio formula: suppression ratio=[(virus control
Group-experimental group)/virus control group] × 100%, it is calculated amount effect curve and half suppresses concentration processed (IC50).Result table
Bright medicine has obvious inhibitory action to HIV-1 virus, refers to Fig. 3 ~ 4 and table 1.
The half of table 1 sample suppresses concentration IC50 processed
。
Three, medicine is in the CCK-8 cell toxicity test of cellular level.
The each 100mg of accurately weighed L13, L14 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, preparation
The stock solution of 100mg/mL.During experiment successively with the culture medium of serum-free be diluted to series concentration be 250 g/mL, 125 g/mL,
62.5 g/mL, 31.25 g/mL, 15.63 g/mL, for the administration group of variable concentrations.TZM-bl cell is inoculated in 96 orifice plates,
After 37 DEG C of incubators cultivate 24 h, add the medicine of variable concentrations gradient.After continuing to cultivate 48 h, use CCK-8 cell proliferation
Detectable (colleague's chemistry), cultivates 45min, the absorbance at microplate reader detection 450nm.Calculate extract to normally
The suppression ratio of TZM-bl cell, experimental data is fitted by GraphPad Prism 5.0 software, obtain amount effect curve and
Median toxic concentration (TC50).Suppression ratio formula: suppression ratio=[(cell controls group-experimental group)/cell controls group] ×
100%, refer to Fig. 5 and Biao 2.
The cytotoxicity of table 2 willow herb anti-HIV-1 virus effective site
。
Four, medicine and reverse transcriptase protein vitro binding assay.
Use BIAcore 3000 bio-molecular interaction instrument, use reverse transcriptase protein and CM5 chip coupling,
Use bovine serum albumin (BSA) as negative control.By L13, L14 extractive part with through 0.45 μm membrane filtration
PBS is diluted to 200 μ g/mL, flow velocity 20 L/min, and injection rate is 60 L sample introductions, and record sample flows through target protein and combines the phase
Time RU value with flow through the RU value of BSA protein binding phase, the difference between the two is the combination response value of this sample and target protein,
Refer to Fig. 5 and Biao 3.
Table 3 and the external combination RU value of reverse transcriptase
。
Five, the medicine mensuration to HIV-1 reverse transcriptase activity.
Use the impact on HIV-1 reverse transcriptase activity of the reverse transcriptase detection kit vitro detection compound.Nai Wei
Even up (nevirapine, NVP) as positive drug, concentration is 2.5 ng/mL, and L13, L14 extractive part concentration is 200 μ
G/mL, experiment sets 2 multiple holes.Experiment is the addition lysate (20 L/ hole) containing 1 ng HIV-1 RT in 96 orifice plates, and with
Medicine 37 DEG C hatches 1 h.Solution in 96 orifice bores is transferred in microwell plate, continues 37 DEG C and hatch 1h.Reject microwell plate
Interior solution, eluent adds 200 L Anti-DIG-POD working solutions, hatches 1h for 37 DEG C after washing 5 times.Reject microwell plate
Solution, eluent washes 5 times, and every hole adds 200 L ABTs substrate solution, incubated at room temperature 30min, makes
With microplate reader detection compound absorbance at 405nm, calculate suppression ratio, refer to Fig. 6 and Biao 4.In Fig. 6,1 is L13, and 2 are
L14,3 is NVP(nevirapine).
The table 4 impact on HIV-1 reverse transcriptase activity
。
Six, the medicine impact on HIV-1 viral protease activity.
Use the impact on HIV-1 viral protease activity of the protease detection kit vitro detection medicine.Use
Pepstatin A is as positive control, and concentration is 2 × 10-3MML, L13, L14 extractive part concentration are 200 μ g/mL, experiment
If 2 multiple holes.Addition medicine (10 L/ hole) and protein enzyme solution (10 L/ hole) in 384 orifice plates, solvent control group adds
Medicine and buffer, Positive control wells adds protein enzyme solution and buffer, and after incubated at room 15min, every hole adds 10 L eggs
White enzyme substrate solution, slightly shakes 60s in microplate reader, under room temperature, lucifuge hatches 30min, detects light absorption value at 340nm/490nm,
Calculate the sample suppression ratio to reverse transcriptase, refer to Fig. 7 and Biao 5.In Fig. 7,1 is L13, and 2 is L14, and 3 is Popstain A.
The impact on HIV-1 viral protease activity of table 5 vitro detection
。
Embodiment 4
The method for preparing tablet thereof of willow herb effective site is: willow herb n-butanol portion 300mg and starch 100mg mixing, adds quality hundred
The gelatinized corn starch 40mg of proportion by subtraction concentration 10% makes soft material, and sieve to obtain wet granular, is dried to obtain dry granule, granulate, adds magnesium stearate 4 mg
Mixing, tabletting, to obtain final product.
Embodiment 5
The preparation method of electuary: by weight ratio willow herb aqueous phase is extracted 5 parts of position, sucrose 1 part, the mixing of 3 parts of dextrin, adds
Appropriate volume percent concentration 95% ethanol solution 2 ~ 5 parts, stirring while adding, prepare soft material, soft material is dried, crosses 16 mesh sieves, point
Fill and get final product.
Embodiment 6
Pellet capsule is composed of the following raw materials by weight: willow herb n-butanol portion 30 parts, 5 parts of lecithin, Bile Salts 5 parts,
Microcrystalline Cellulose 30 parts；
The preparation method of pellet capsule: willow herb n-butanol portion, lecithin, Bile Salts and microcrystalline Cellulose are mixed by proportioning
All, pour ethanol water into and stir evenly acquisition soft material, soft material is poured in extruder and extrude, through round as a ball acquisition granule, extrude rotating speed
250r/min, round as a ball rotating speed 800r/min, round as a ball time 20min, be dried, cross 24 ~ 30 mesh sieves acquisition micropills, be filled into by micropill
In capsule shells, to obtain final product.
The above, be only presently preferred embodiments of the present invention, is not the restriction that the present invention makees other form, appoints
What those skilled in the art changed possibly also with the technology contents of the disclosure above or be modified as equivalent variations etc.
Effect embodiment.But every without departing from technical solution of the present invention content, the technical spirit of the foundation present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made, still falls within the protection domain of technical solution of the present invention.
Claims (7)
1. willow herb anti-HIV-1 virus effective site, it is characterised in that: selected from willow herb n-butanol portion or willow herb aqueous phase extraction portion
Position.
2. the preparation method of the willow herb anti-HIV-1 virus effective site described in claim 1, it is characterised in that include following step
Rapid: willow herb herb is pulverized, add concentration of volume percent 65% ~ 95% ethanol solution and extract, gained extracting solution is concentrated, does
Dry, obtain extractum；Extractum is dispersed in distilled water to obtain extract dispersion liquid, uses petroleum ether, ethyl acetate and positive fourth the most successively
Gained butanol extraction liquid, as solvent extraction extract dispersion liquid, is flung to n-butyl alcohol by alcohol, obtains willow herb n-butanol portion；Will extraction
Rear remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
The preparation method of willow herb anti-HIV-1 virus effective site the most according to claim 2, it is characterised in that: described
Being extracted as ultrasonic assistant reflux, extract, concrete operations are: the willow herb herb after pulverizing add concentration of volume percent 65% ~
95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds the backflow of concentration of volume percent 65% ~ 95% ethanol solution
Extract 1 ~ 3 time, united extraction liquid, to obtain final product.
4. the application of the willow herb anti-HIV-1 virus effective site described in claim 1, it is characterised in that: preparing anti-HIV-1
Application in virus drugs.
The application of willow herb anti-HIV-1 virus effective site the most according to claim 4, it is characterised in that: described anti-HIV-1
Virus drugs is to extract position, after mixing, the oral system made with pharmaceutic adjuvant by willow herb n-butanol portion or willow herb aqueous phase
Agent or ejection preparation.
The application of willow herb anti-HIV-1 virus effective site the most according to claim 5, it is characterised in that: described injection
Preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
The application of willow herb anti-HIV-1 virus effective site the most according to claim 5, it is characterised in that: described is oral
Preparation is powder, tablet, granule, capsule, oral solution.
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| CN (1) | CN106265786A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010024664A1 (en) * | 1999-03-19 | 2001-09-27 | Obukowicz Mark G. | Selective COX-2 inhibition from edible plant extracts |
| US20020182272A1 (en) * | 2001-05-30 | 2002-12-05 | Bruce Halstead | Methods of treatment of HIV-associated conditions |
| US20070036834A1 (en) * | 2001-08-29 | 2007-02-15 | Pauletti Giovanni M | Method for augmentation of intraepithelial and systemic exposure of therapeutic agents having substrate activity for cytochrome P450 enzymes and membrane efflux systems following vaginal and oral cavity administration |
| JP4531637B2 (en) * | 2005-06-15 | 2010-08-25 | 株式会社 ヒロインターナショナル | Anti-menopausal agent comprising a plant-derived extract |
-
2016
- 2016-08-30 CN CN201610780007.9A patent/CN106265786A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010024664A1 (en) * | 1999-03-19 | 2001-09-27 | Obukowicz Mark G. | Selective COX-2 inhibition from edible plant extracts |
| US20040052870A1 (en) * | 2000-12-15 | 2004-03-18 | Obukowicz Mark G. | Selective cox-2 inhibition from edible plant extracts |
| US20020182272A1 (en) * | 2001-05-30 | 2002-12-05 | Bruce Halstead | Methods of treatment of HIV-associated conditions |
| US20070036834A1 (en) * | 2001-08-29 | 2007-02-15 | Pauletti Giovanni M | Method for augmentation of intraepithelial and systemic exposure of therapeutic agents having substrate activity for cytochrome P450 enzymes and membrane efflux systems following vaginal and oral cavity administration |
| JP4531637B2 (en) * | 2005-06-15 | 2010-08-25 | 株式会社 ヒロインターナショナル | Anti-menopausal agent comprising a plant-derived extract |
Non-Patent Citations (1)
| Title |
|---|
| 宋小妹等: "《中药化学成分提取分离与制备》", 30 April 2004, 人民卫生出版社 * |
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