CN106265930A - Herba Lycopodii AntiHIV1 RT activity 1 virus effective site and preparation method and application - Google Patents

Herba Lycopodii AntiHIV1 RT activity 1 virus effective site and preparation method and application Download PDF

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CN106265930A
CN106265930A CN201610761208.4A CN201610761208A CN106265930A CN 106265930 A CN106265930 A CN 106265930A CN 201610761208 A CN201610761208 A CN 201610761208A CN 106265930 A CN106265930 A CN 106265930A
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herba lycopodii
extract
hiv
virus
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芦永昌
胡秦
林鹏程
钱帅
田静
王欢
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Qinghai Nationalities University
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    • 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
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

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Abstract

Herba Lycopodii AntiHIV1 RT activity 1 virus effective site and preparation method and application, belong to plant extract technical field.Selected from Herba Lycopodii ethyl acetate extract or Herba Lycopodii n-butanol portion.Herba Lycopodii is pulverized, adds mass percentage concentration 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, by gained butanol extraction liquid solvent flashing, obtain Herba Lycopodii n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Lycopodii aqueous phase extraction position.The Herba Lycopodii 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

Herba Lycopodii anti-HIV-1 virus effective site and preparation method and application
Technical field
The present invention relates to Herba Lycopodii anti-HIV-1 virus effective site and preparation method and application, belong to plant extract technology neck Territory.
Background technology
Herba Lycopodii (Tinospora sinensis) Menispermaceae Tinospora plant Tinospora sinensis Merr. is many enters with rattan Medicine.[seeing: Luo Dashang. book on Chinese herbal medicine (Chinese) [M] is hidden by China. Nationalities Press, 1997.], it is Tibetan medicine's tradition medicinal plants, hides Medicine name " Le Zhe ".Its hillside being mainly born in height above sea level 900 meters and sylvan life.It is distributed in the Motuo in China Tibet, Bomi and Guangdong, sea The ground such as south, Guangxi and Yunnan.[see: State Administration of Traditional Chinese Medicine's " China's book on Chinese herbal medicine " editorial board. China's book on Chinese herbal medicine (Tibetan medicine volume) [M]. Shanghai science tech publishing house, 2002.12:286]." Jingzhubencao " is recorded: strangle wise man's sweet in the mouth, hardship, puckery, pungent, change sweet in the mouth, Acid, property profit, cool, warm.Control bar complication grand, red, Baconic's disease, rheumatism, grand calentura.Peel of stem is sweet, and meat is bitter, for Zhi Longre good medicine. [see: Supreme Being Ma Erdan increases Peng to be arranged, and Mao Jizu translates. Jingzhubencao [M]. Shanghai science tech publishing house, 2012:329.].
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 is Retroviridae lentivirus, and its viral RNA genes group must be through the most inverse by single stranded RNA It is transcribed into cDNA, after forming RNA-DNA heterozygote, the RNA that hydrolysis is fallen and DNA is complementary, then with the cDNA of strand as templated synthesis 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 is many Sample, it constitutes heterodimer by two protein monomers of P66 and P51, and wherein P66 is the main functional parts of enzymatic activity；Except tool Have outside reverse transcriptase activity, also there is DNA polymerase activity and RNase enzymatic activity.The molecular mechanism of process of reverse-transcription is the most quite For complexity: the initial primers of process of reverse-transcription is not from virus self and synthesizes, but the tRNALye-in utilizing host cell 3, after combining with the primer binding site (PBS) of viral RNA, more efficiently start process of reverse-transcription.Secondly, reverse transcriptase because Lack general 3'-5' exonuclease activity, it is impossible to complete proofreading function, replicate fidelity poor, be the important of inhibition of HIV high variability One of reason.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is always the target spot that important AntiHIV1 RT activity infects, For developing the medicine for the treatment of acquired immune deficiency syndrome (AIDS).Protease is the special Radix Asparagi acyl egg of the one in HIV (human immunodeficiency virus) gene code White enzyme, its effect is by protein cleavage produced by gene and gene expression, becomes active virus structural protein and enzyme, It it is the key substance of inhibition of HIV duplication.Hiv protease inhibitor mainly acts on the final stage that HIV (human immunodeficiency virus) replicates, by Being suppressed in protease, being allowed to form DNA from the cd4 cell core infected can not assemble and discharge, and causes the amyloid protein precursor can not Cracking and formation 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.In this area, Herba Lycopodii primary efficacy is relaxing muscles and tendons and activating QI and blood in the collateral, wind-expelling pain-stopping, but at present Also there is no the correlational study in terms of anti-AIDS.
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 Herba Lycopodii anti-HIV-1 virus is effectively Position and preparation method and application, this Herba Lycopodii anti-HIV-1 virus effective site can effectively suppress HIV-1 virus activity, it is possible to is used for Prepare 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 Herba Lycopodii anti-HIV-1 virus effective site, its It is characterised by: selected from Herba Lycopodii ethyl acetate extract or Herba Lycopodii n-butanol portion.
The preparation method of this Herba Lycopodii anti-HIV-1 virus effective site, it is characterised in that comprise the steps: width muscle Rattan is pulverized, and adds mass percentage concentration 65% ~ 95% ethanol solution and extracts, and is concentrated by gained extracting solution, is dried, obtains extractum；Will leaching Cream is dispersed in distilled water to obtain extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction the most successively Extract dispersion liquid, flings to solvent respectively, obtains Herba Lycopodii ethyl acetate extract, Herba Lycopodii n-butanol portion.
Described is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: will pulverize After Herba Lycopodii add mass percentage concentration 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filter, filtering residue adds matter Amount percentage concentration 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
The application of this Herba Lycopodii 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 oral formulations or ejection preparation.
Described anti-HIV-1 virus drugs is with medicinal by Herba Lycopodii ethyl acetate extract or Herba Lycopodii n-butanol portion Make after adjuvant mixing.
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 prior art, and Herba Lycopodii has the merit of relaxing muscles and tendons and activating QI and blood in the collateral, wind-expelling pain-stopping. Applicant is found by numerous studies: the ethanol water solution extract in Herba Lycopodii also has the effect of anti-HIV-1 virus, but Its effect is inconspicuous, it is impossible to determine its concrete effective site.Applicant is found by research: use preparation method of the present invention, The Herba Lycopodii ethyl acetate extract that filters out, Herba Lycopodii n-butanol portion, have obvious inhibition to HIV-1 virus.
In preparation method: described ultrasonic extraction is to carry out under room temperature.The temperature of reflux, extract, is alcohol reflux Ordinary temperature, need to be higher than the boiling point of ethanol, preferably 80 DEG C ~ 100 DEG C.Described concentration and being dried as concentrating under reduced pressure, vacuum It is dried, to avoid there is scattering and disappearing of volatile Herba Lycopodii effective site.Described filtration reflux, extract, refer to for 1 ~ 3 time: to Be heated at reflux in device and add the medical material after pulverizing and ethanol solution, heating, reflux, extract, let cool filter extracting solution and Medicinal residues；In medicinal residues, add ethanol solution, heat, carry out second time reflux, extract, let cool and filter to obtain secondary raffinate and medicinal residues； Merge repeatedly extracting solution, obtain the extracting solution of reflux, extract,.Preferably, each reflux, extract, 0.5 ~ 2 hour；Reflux, extract, every time Shi Suojia ethanol solution did not had medical material surface 1cm ~ 2cm.
In preparation method, being Herba Lycopodii herb or Herba Lycopodii rattan part for the Herba Lycopodii pulverized, extractum is thick extraction Thing, uses different solvent extraction extract dispersion liquids, refers to that ethyl acetate adds extract dispersion liquid extract and separate successively and obtains Obtain acetic acid ethyl acetate extract, n-butyl alcohol is added extract dispersion liquid extract and separate acquisition butanol extraction liquid；Take ethyl acetate extraction Taking liquid and butanol extraction liquid volatilizees after removing solvent respectively, gained is Herba Lycopodii ethyl acetate extract and Herba Lycopodii n-butyl alcohol Position.Remaining extract dispersion liquid (solvent is water) after extracting, volatilization is removed solvent and is i.e. obtained Herba Lycopodii aqueous phase extraction position.Water Extraction position may also be referred to as aqueous phase extract mutually, and aqueous phase extraction position is soluble in water；And relative, ethyl acetate and n-butyl alcohol Corresponding be soluble in for organic facies, ethyl acetate extract and n-butanol portion, ethyl acetate and n-butyl alcohol.Wherein, Herba Lycopodii acetic acid Terpenoid is contained at ethyl ester position；Herba Lycopodii n-butanol portion contains saponin and phenolic compound.Fling to solvent i.e. volatilize Except solvent.Flinging to solvent can use normal heating to make solvent (petroleum ether, ethyl acetate and n-butyl alcohol) volatilize.Preferably, wave Going solvent to use concentrating under reduced pressure, the method efficiency is high, and is avoided that heat-sensitive ingredients loses the property of medicine because of high temperature.
Preferably dosage form is oral formulations or ejection preparation.Oral formulations and ejection preparation are optimal route of administration.Described Oral formulations refers to through intestines and stomach drug-delivery preparation, it is preferred that oral formulations is spacetabs type or control release type oral formulations.Wherein, Capsule includes soft capsule and hard capsule.It addition, the preparation of this Herba Lycopodii anti-HIV-1 virus effective site can also be given for other The preparation of medicine approach: such as respiratory tract administration preparation (spray, aerosol, powder spray)；Percutaneous drug delivery preparation (externally used solution agent, 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；Preferably, the injection system of the present invention Agent is lipidosome injection, nanoparticle injection or micro-balloon injection, above injection be respectively adopted liposome, nanoparticle, Microsphere as pharmaceutical carrier, can extend medicine carrying microgranule circulation time in vivo, extend the medicine carrying microgranule stop at absorption site Time, control medicine, at the burst effect at release initial stage, strengthen drug effect.Preferably, receiving as carrier in nanoparticle injection The grain of rice is nanorize colloidal particle (i.e. nano-micelle).Anti-HIV-1 virus drugs is by Herba Lycopodii ethyl acetate extract or width Muscle rattan n-butanol portion is made after mixing with pharmaceutic adjuvant；Pharmaceutic adjuvant includes pharmaceutical carrier, and solvent, solubilizing agent, hydrotropy Agent, emulsifying agent, suspending agent, clarifier, deflocculant, correctives, coloring agent, preservative, chemosterilant, adsorbent, drainage Agent, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, binding agent, wetting agent, disintegrating agent, lubricant, fluidizer, anti-stick One or more in agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material.
Compared with prior art, the Herba Lycopodii anti-HIV-1 virus effective site of the present invention and preparation method and application are had Provide the benefit that:
1, this Herba Lycopodii 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 finds under study for action: be currently used for preparing anti-AIDS drug Raw material the deficientest, and prior art only has the Herba Lycopodii of the merit of relaxing muscles and tendons and activating QI and blood in the collateral, wind-expelling pain-stopping, also there is anti-AIDS Effect.Applicant is by design preparation method, and carries out medicine efficacy screening first on HIV1-RT and protease, and Determined by numerous studies: Herba Lycopodii ethyl acetate extract, Herba Lycopodii n-butanol portion are by combining reverse transcriptase protein and pressing down Hiv protease processed can substantially suppress HIV-1 virus, and cytotoxicity increases with administration concentration and strengthens, it is adaptable to be prepared as resisting Acquired immune deficiency syndrome (AIDS) new drug.Applicant, during solution problem of pinpointing the problems above, has paid substantial amounts of creative work.
2, the preparation method of this Herba Lycopodii anti-HIV-1 virus effective site is extracted conveniently, and extraction efficiency is high.Use 65% ~ 95% ethanol solution uses the method for ultrasonic assistant reflux, extract, can be effectively improved extraction efficiency.Use petroleum ether, acetic acid successively Ethyl ester and n-butyl alcohol are as solvent extraction extract dispersion liquid, it is possible to quickly obtain Herba Lycopodii ethyl acetate extract and the positive fourth of Herba Lycopodii Alcohol position.
3, this Herba Lycopodii anti-HIV-1 virus effective site application in terms of preparing anti-HIV-1 medicines, has expanded suppression The raw material channel of HIV-1 virus drugs, expands the purposes of Herba Lycopodii, makes Herba Lycopodii develop into suppression HIV-1 virus medicine The new raw material of thing, is remarkably improved the added value of Herba Lycopodii.
Accompanying drawing explanation
The HIV-1 R3A wild virus infection suppression ratio of Fig. 1 Herba Lycopodii ethyl acetate extract.
The HIV-1 R3A wild virus infection suppression ratio of Fig. 2 Herba Lycopodii n-butanol portion..
Fig. 3 Herba Lycopodii ethyl acetate extract pseudovirus single cycle infection.
Fig. 4 Herba Lycopodii n-butanol portion pseudovirus single cycle infection.
Fig. 5 Herba Lycopodii ethyl acetate extract CCK8 method cytotoxicity.
Fig. 6 Herba Lycopodii n-butanol portion CCK8 method cytotoxicity.
Fig. 7 affects comparison diagram to HIV-1 suppression reverse transcriptase activity.
The impact on HIV-1 proteinase activity of Fig. 8 vitro detection.
Detailed description of the invention
Embodiment 1 ~ 3 is the Herba Lycopodii anti-HIV-1 virus effective site of the present invention and preparation method and the specific embodiment party of application Formula, wherein embodiment 1 is most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize Herba Lycopodii, uses mass percentage concentration 75% ~ 85% ethanol solution ultrasound wave Extracting 1 ~ 2 hour, filter to obtain ultrasonic extract and filtering residue, filtering residue adds the backflow of mass percentage concentration 75% ~ 85% ethanol solution Extract 2 times, each 0.5 ~ 1 hour, obtain reflux extracting liquid, merge ultrasonic extract and reflux extracting liquid, concentrate drying must soak Cream；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as molten the most successively Agent extraction extract dispersion liquid, obtains the extract of each solvent, respectively acetic acid ethyl acetate extract and butanol extraction liquid is flung to solvent, Obtain Herba Lycopodii ethyl acetate extract and Herba Lycopodii n-butanol portion.
Embodiment 2
Preparation method, comprises the steps: to pulverize Herba Lycopodii, uses mass percentage concentration 85% ~ 95% ethanol solution ultrasound wave Extracting 2 ~ 3 hours, filter to obtain ultrasonic extract and filtering residue, filtering residue adds the backflow of mass percentage concentration 85% ~ 95% ethanol solution Extract 3 times, each 1 ~ 1.5 hour, obtain reflux extracting liquid, merge ultrasonic extract and reflux extracting liquid, concentrate drying must soak Cream；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as molten the most successively Agent extraction extract dispersion liquid, obtains the extract of each solvent, respectively acetic acid ethyl acetate extract and butanol extraction liquid is flung to solvent, Obtain Herba Lycopodii ethyl acetate extract and Herba Lycopodii n-butanol portion.
Embodiment 3
Preparation method, comprises the steps: to pulverize Herba Lycopodii, uses mass percentage concentration 65% ~ 75% ethanol solution ultrasound wave Extracting 0.5 ~ 1 hour, filter to obtain ultrasonic extract and filtering residue, filtering residue adds mass percentage concentration 65% ~ 75% ethanol solution and returns Stream extracts 1 time, 1.5 ~ 2 hours used times, obtains reflux extracting liquid, merges ultrasonic extract and reflux extracting liquid, concentrate drying obtain Extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol conduct the most successively Solvent extraction extract dispersion liquid, obtains the extract of each solvent, flings to molten by acetic acid ethyl acetate extract and butanol extraction liquid respectively Agent, obtains Herba Lycopodii ethyl acetate extract and Herba Lycopodii n-butanol portion.
Performance test
The Herba Lycopodii ethyl acetate extract (being numbered L1) obtained with embodiment 1 and Herba Lycopodii n-butanol portion (being numbered L3) are made For test medicine, test as follows.
One, HIV-1 R3A wild virus infection experiment.
The each 100mg of accurately weighed L1, L3 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, prepares 100mg/ The stock solution of 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 not With dosage 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 wells.Take MT4 Cell (5 × 105/ mL) add HIV-1 R3A virus 1 × 103 TCID50/ mL, 37 DEG C, 5% CO2Cultivate 2h, PBS to wash 3 times, Centrifugal supernatant discarded, adds the thin of 100 these infected by HIV-1 of L after adding complete medium in the 96 each holes added with medicine of orifice plate Born of the same parents (5 × 104/ 100 L), 37 DEG C, 5% CO2Cultivate, the three days later half liquid that changes, keep drug level constant, within the 6th day, collect each Hole takes supernatant, uses ELISA method to measure P24 amount of antigen, and each hole takes supernatant 50 L and joins the TZMbl cell of first 1 day bed board In, 37 DEG C, CO248h is cultivated in incubator.Promega BrightGlo luciferase kit detection luciferase is used to contain Amount, and calculate suppression ratio, refer to Fig. 1 ~ 2.
Two, HIV-1 pseudovirus single cycle infection experiment.
The each 100mg of accurately weighed L1, L3 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, prepares 100mg/ The stock solution of mL.Diluting by the culture medium of serum-free successively during experiment, L1, L3 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 virus control group, adds Enter AZT(zidovudine) organize as positive controls.By TZM-bl cell, (Hela cell expresses CD4, CXCR4 and CCR5, containing Tat The luciferase started and LacZ gene) it is inoculated in 96 orifice plates, after 37 DEG C of incubators cultivate 24 h, supernatant discarded, add pSG3 Pseudovirus supernatant, adds DEAE(final concentration 10 g/mL in viral supernatants), it is simultaneously introduced the medicine of variable concentrations dilution, cultivates After 48h, supernatant discarded, add fixative room temperature and fix 5 min, after PBS, add nitrite ion, 37 DEG C of incubators are cultivated 50 Min, 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 (IC processed50).Result shows that medicine is to HIV-1 Virus has obvious inhibitory action, 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 L1, L3 is in different centrifuge tubes respectively, adds 1mL DMSO and dissolves, prepares 100mg/ The stock solution of 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, 37 DEG C of trainings After 24 h cultivated by foster case, add the medicine of variable concentrations gradient.After continuing to cultivate 48h, use CCK-8 cell proliferation detectable (colleague's chemistry), cultivates 45 min, the absorbance at microplate reader detection 450nm.Calculate extract to normal TZM-bl The suppression ratio of cell, experimental data is fitted by GraphPad Prism 5.0 software, obtains amount effect curve and half poisoning Concentration (TC50).Suppression ratio formula: suppression ratio=[(cell controls group-experimental group)/cell controls group] × 100%, refers to Fig. 5 ~ 6 and table 2.
The cytotoxicity of table 2 Herba Lycopodii 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 L1, L3 extractive part PBS through 0.45 m membrane filtration Being diluted to 200 g/mL, flow velocity 20 L/min, injection rate is 60 L sample introductions, and record sample flows through when target protein combines the phase RU value and the RU value flowing through the BSA protein binding phase, the difference between the two is the combination response value of this sample and target protein, refers to Table 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 2.5ng/mL, and L1, L3 extractive part concentration is 200 g/mL, Experiment sets 2 multiple holes.Experiment adds containing the lysate (20 L/ hole) of 1ng HIV-1 RT in 96 orifice plates, and with medicine 37 DEG C hatch 1h.Solution in 96 orifice bores is transferred in microwell plate, continues 37 DEG C and hatch 1h.Molten in reject microwell plate Liquid, eluent adds 200 L Anti-DIG-POD working solutions, hatches 1h for 37 DEG C after washing 5 times.Reject microwell plate molten Liquid, eluent washes 5 times, and every hole adds 200 L ABTs substrate solution, incubated at room temperature 30 min, uses enzyme Mark instrument detection compound absorbance at 405nm, calculates suppression ratio, refers to Fig. 7 and Biao 4.In Fig. 7,1 is L1, and 2 is L3,3 For NVP(nevirapine).
The table 4 impact on HIV-1 reverse transcriptase activity
Six, the medicine impact on HIV-1 virus activity.
Use the impact on HIV-1 virus activity of the protease detection kit vitro detection medicine.Use Pepstatin A is as positive control, and concentration is 2 × 10-3MM/L, L1, L3 extractive part concentration are 200 g/mL, and experiment sets 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 buffering Liquid, Positive control wells adds protein enzyme solution and buffer, and after incubated at room 15 min, it is molten that every hole adds 10 L protease substrates Liquid, slightly shakes 60s in microplate reader, under room temperature, lucifuge hatches 30 min, light absorption value at detection 340nm/490nm, calculates sample Suppression ratio to reverse transcriptase, refers to Fig. 8 and Biao 5.In Fig. 8,1 is L1, and 2 is L3, and 3 is Popstain A.
The impact on HIV-1 virus activity of table 5 vitro detection
Embodiment 4
The method for preparing tablet thereof of Herba Lycopodii effective site is: Herba Lycopodii n-butanol portion 300mg and starch 100mg mixing, adds matter The gelatinized corn starch 40mg of amount percent concentration 10% makes soft material, and sieve to obtain wet granular, is dried to obtain dry granule, granulate, adds magnesium stearate 4mg mixing, tabletting, to obtain final product.
Embodiment 5
Pellet capsule is composed of the following raw materials by weight: Herba Lycopodii ethyl acetate extract 30 parts, 5 parts of lecithin, Bile Salts 5 Part, microcrystalline Cellulose 30 parts；
The preparation method of pellet capsule: by proportioning by Herba Lycopodii ethyl acetate extract, lecithin, Bile Salts and microcrystalline cellulose Element is mixed, and pours ethanol water into and stirs evenly acquisition soft material, is poured into by soft material in extruder and extrudes, through round as a ball acquisition granule, extrusion 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, filled by micropill Load in capsule shells, to obtain final product.
Embodiment 6
The preparation method of lipidosome injection: 1) under nitrogen protection, by 50g cholesterol succinate, 250g distearyl phosphorus Acyl ethanolamine, 40g soybean lecithin, 50g poloxamer-188 and 10g Herba Lycopodii n-butanol portion are dissolved in 1500ml body Long-pending ratio is in the ethanol of 1:1 and the organic solvent of n-butyl alcohol, and stirring makes it dissolve acquisition suspension；Suspension is dense by decompression Organic solvent is flung in contracting, it is thus achieved that immobilized artificial membrane；
2) under nitrogen protection, adding 8000ml pH in immobilized artificial membrane is the phosphate buffered solution of 6.8, and stirring makes immobilized artificial membrane Eluting abundant swelling hydration, through 0.22 m filtering with microporous membrane, obtain the liposome of Herba Lycopodii n-butanol portion；
3) aseptically, in the liposome of Herba Lycopodii n-butanol portion, add 100g trehalose, stir, ultrasonic Ripple processes 0.5 ~ 1 hour, injects and uses water constant volume, through 0.22 m filtering with microporous membrane, fill, obtains Herba Lycopodii n-butanol portion Lipidosome injection.
Embodiment 7
The preparation method of nanoparticle injection: take 25g Herba Lycopodii ethyl acetate extract and 100g poloxamer188 3000ml Anhydrous alcohol solution, adds the ethanol solution of 30ml 1mol/L zinc chloride, and stirring mixing, ultrasonic Treatment makes it in 0.5 ~ 1 hour Dissolve, it is thus achieved that mixed liquor；Mixed liquor concentrating under reduced pressure is flung to solvent, puts into-20 DEG C of refrigerator freezings 2 hours；Taking-up injects use Water constant volume, ultrasonic Treatment 0.5 ~ 1 hour, through 0.22 m filtering with microporous membrane, fill, obtain Herba Lycopodii ethyl acetate extract Nanoparticle injection.
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 (8)

1. Herba Lycopodii anti-HIV-1 virus effective site, it is characterised in that: selected from the positive fourth of Herba Lycopodii ethyl acetate extract or Herba Lycopodii Alcohol position.
2. the preparation method of the Herba Lycopodii anti-HIV-1 virus effective site described in claim 1, it is characterised in that include following Step: pulverized by Herba Lycopodii, adds mass percentage concentration 65% ~ 95% ethanol solution and extracts, and is concentrated by gained extracting solution, is dried, Obtain extractum；Extractum is dispersed in distilled water to obtain extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol to make the most successively For solvent extraction extract dispersion liquid, fling to solvent respectively, obtain Herba Lycopodii ethyl acetate extract, Herba Lycopodii n-butanol portion.
The preparation method of Herba Lycopodii anti-HIV-1 virus effective site the most according to claim 2, it is characterised in that: described Be extracted as ultrasonic assistant reflux, extract,；Reflux, extract, is that the concrete operations of ultrasonic assistant reflux, extract, are: after pulverizing Herba Lycopodii add mass percentage concentration 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filter, filtering residue adds quality Percentage concentration 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
4. the application of the Herba Lycopodii anti-HIV-1 virus effective site described in claim 1, it is characterised in that: prepare AntiHIV1 RT activity- Application in 1 virus drugs.
The application of Herba Lycopodii anti-HIV-1 virus effective site the most according to claim 4, it is characterised in that: described anti- HIV-1 virus drugs is oral formulations or ejection preparation.
The application of Herba Lycopodii anti-HIV-1 virus effective site the most according to claim 4, it is characterised in that: described anti- HIV-1 virus drugs be mixed with pharmaceutic adjuvant by Herba Lycopodii ethyl acetate extract or Herba Lycopodii n-butanol portion after make.
The application of Herba Lycopodii anti-HIV-1 virus effective site the most according to claim 5, it is characterised in that: described note Penetrating preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
The application of Herba Lycopodii anti-HIV-1 virus effective site the most according to claim 5, it is characterised in that: described mouth Formulation is powder, tablet, granule, capsule, oral solution.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101167771A (en) * 2007-10-26 2008-04-30 中国热带农业科学院热带生物技术研究所 Application of vinca flower and its extraction in preparing medicine for resisting AIDS
CN101376652A (en) * 2008-10-10 2009-03-04 昆明理工大学 Novel triterpenoid schisanlactone H and extracting and separating method thereof
CN101411796A (en) * 2008-12-05 2009-04-22 中国热带农业科学院热带生物技术研究所 Novel use of betelnut and extract thereof in preparing anti-AIDS medicament
CN102204970A (en) * 2011-05-19 2011-10-05 中国人民武装警察部队医学院 Application of silverweed cinquefoil root extract to preparation of anti-AIDS medicament
CN102727551A (en) * 2011-04-07 2012-10-17 上海鑫昊生物科技有限公司 Use of effective part of traditional Chinese medicine in HIV latency-resistant treatment
CN103156907A (en) * 2011-12-09 2013-06-19 天津市国际生物医药联合研究院 Pharmaceutical applications of traditional Chinese medicine Cassia fistula L. fruit and extract thereof
CN104815002A (en) * 2015-05-12 2015-08-05 青海民族大学 Acquisition method and application of tinospora sinensis merr extract
CN104906191A (en) * 2015-02-04 2015-09-16 新疆医科大学 Tinospora sinensis Merr acetylcholinesterase inhibition active extract product, medical purpose of its composition and preparation method thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101167771A (en) * 2007-10-26 2008-04-30 中国热带农业科学院热带生物技术研究所 Application of vinca flower and its extraction in preparing medicine for resisting AIDS
CN101376652A (en) * 2008-10-10 2009-03-04 昆明理工大学 Novel triterpenoid schisanlactone H and extracting and separating method thereof
CN101411796A (en) * 2008-12-05 2009-04-22 中国热带农业科学院热带生物技术研究所 Novel use of betelnut and extract thereof in preparing anti-AIDS medicament
CN102727551A (en) * 2011-04-07 2012-10-17 上海鑫昊生物科技有限公司 Use of effective part of traditional Chinese medicine in HIV latency-resistant treatment
CN102204970A (en) * 2011-05-19 2011-10-05 中国人民武装警察部队医学院 Application of silverweed cinquefoil root extract to preparation of anti-AIDS medicament
CN103156907A (en) * 2011-12-09 2013-06-19 天津市国际生物医药联合研究院 Pharmaceutical applications of traditional Chinese medicine Cassia fistula L. fruit and extract thereof
CN104906191A (en) * 2015-02-04 2015-09-16 新疆医科大学 Tinospora sinensis Merr acetylcholinesterase inhibition active extract product, medical purpose of its composition and preparation method thereof
CN104815002A (en) * 2015-05-12 2015-08-05 青海民族大学 Acquisition method and application of tinospora sinensis merr extract

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
张海霞 等: ""GS-MS分析宽筋藤中不同萃取物成分比较"", 《生物技术世界》 *

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