CN106214777B - anti-HIV-1 virus effective part of potentilla chinensis in northwest China, preparation method and application - Google Patents

anti-HIV-1 virus effective part of potentilla chinensis in northwest China, preparation method and application Download PDF

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CN106214777B
CN106214777B CN201610760889.2A CN201610760889A CN106214777B CN 106214777 B CN106214777 B CN 106214777B CN 201610760889 A CN201610760889 A CN 201610760889A CN 106214777 B CN106214777 B CN 106214777B
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林鹏程
胡秦
高世宝
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Qinghai Nationalities University
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Abstract

An anti-HIV-1 virus effective part of potentilla chinensis in northwest China, a preparation method and application thereof, belonging to the technical field of plant extracts. Is n-butanol fraction of Potentilla chinensis Benth or water phase extraction fraction of Potentilla chinensis Benth. Crushing potentilla anserine, adding 65-95% ethanol solution by volume percentage for extraction, and concentrating and drying the obtained extract to obtain an extract; dispersing the extract in distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing the solvent of the obtained n-butanol extract to obtain n-butanol fraction of Potentilla chinensis in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis. The anti-HIV-1 virus effective part of the potentilla anserine in northwest of China is applied to preparing anti-HIV-1 virus medicines, and is suitable for being developed into anti-AIDS new medicines.

Description

anti-HIV-1 virus effective part of potentilla chinensis in northwest China, preparation method and application
Technical Field
An anti-HIV-1 virus effective part of potentilla chinensis in northwest China, a preparation method and application thereof, belonging to the technical field of plant extracts.
Background
Northwest cinquefoil (cornum salesovianum (Stepn.) ash. et Gr.) is a perennial subalbusta plant of the roseceae (Rosaceae) cinquefoil (cornum palustre L.) [ see: china plant matter editing Committee of China academy of sciences, China higher plant [ M ]. Beijing: scientific press 2003 (6) ], which is a traditional medicinal plant. It is mainly grown on hillside forest edges, under forests and river valley wet grasslands with elevation 2800-. Is distributed in the southwest, northwest and northeast China. The northern temperate zone is widely distributed, and also in north america, europe to japan, and asia to himalaya [ see: GBIF (Global biological Information facility). Comarum salesovianum (Stephan) Ash. & Graebn,2015. www.gbif.org/species/3020536.). The Mongolian academy of sciences found that it had significant anti-inflammatory activity [ see: ondinuya G, Banzragchrarav O, Murata T, et al.Antibacterially active phenolic lipid derivatives from Comarum salesovianum (Steph.) Ashers. et Gr. [ J ]. Phytochemistry Letters, 2015, 13: 360-; one japanese patent suggests that it has a staphylococcal inhibitory effect and can prevent acne and allergic dermatitis [ see: y, Nobuo, S, Hiroyuki, JPO, 2011, JP 2011001329-A ].
AIDS is acquired immunodeficiency syndrome (acquired immunodeficiency syndrome). Today, AIDS is becoming a public health and social problem that seriously jeopardizes human survival and development worldwide. The prevalence of AIDS in China is in the situation of coexistence of low prevalence in China and high prevalence in local areas and specific people through the afferent period and the diffusion period and enters the rapid growth period at present. AIDS is a very harmful infectious disease caused by infection with the HIV virus. HIV is a virus that attacks the immune system of the human body, i.e., "human immunodeficiency virus", which destroys the immune system of the human body after invading the human body, causing various infections and tumors that are difficult to cure in the human body, and ultimately leading to death, and it has been confirmed that HIV is divided into two types: HIV-type 1 and HIV-type 2. HIV-1 is a lentivirus of the family Retroviridae, whose viral RNA genome must first be reverse transcribed from single-stranded RNA to cDNA to form an RNA-DNA hybrid, then the RNA complementary to the DNA is hydrolyzed, and the double-stranded DNA is synthesized using the single-stranded cDNA as a template, so that it can be integrated into the human host DNA genome, a process catalyzed by reverse transcriptase (RT enzyme). The RT enzyme has multiple functions, and is composed of two protein monomers of P66 and P51 to form a heterodimer, wherein P66 is a main functional part of the enzyme activity; in addition to reverse transcriptase activity, DNA polymerase activity as well as RNAse enzyme activity. The molecular mechanism of the reverse transcription process is also rather complex: the initial primer for reverse transcription is not synthesized by the virus itself, but the reverse transcription is efficiently initiated by combining with the Primer Binding Site (PBS) of the viral RNA by using tRNALye-3 in the host cell. Secondly, reverse transcriptase can not complete the reading correction function because of lacking 3'-5' exonuclease activity, and the replication fidelity is poor, which is one of the important reasons of high variability of HIV virus. Human immunodeficiency virus type 1 (i.e., HIV-type 1) Reverse Transcriptase (RT) has been an important target against HIV infection for the development of drugs for the treatment of aids. The protease is a specific aspartyl protease in human immunodeficiency virus gene coding, has the function of cracking genes and proteins generated by gene expression into active virus structural proteins and enzymes, and is a key substance for HIV virus replication. HIV protease inhibitors act primarily at the final stages of HIV replication, and since the protease is inhibited, DNA formed from infected CD4 nuclei cannot be aggregated and released, resulting in the inability of the protein precursors to cleave and form mature virions.
The applicant found in the study that: at present, the raw materials of the anti-AIDS drugs are deficient. The curative effect of the existing anti-HIV drugs is limited along with the drug resistance mutation of HIV-1 reverse transcriptase like other types of anti-HIV drugs. At present, the development of a new generation of anti-HIV-1 type virus drugs is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the anti-HIV-1 virus effective part of the Potentilla chinensis Bunge can effectively inhibit the activity of HIV-1 virus, can be used for preparing anti-HIV-1 virus medicaments, and is suitable for being developed into anti-AIDS medicaments.
The technical scheme adopted by the invention for solving the technical problems is as follows: the anti-HIV-1 virus effective part of the potentilla chinensis in northwest China is characterized in that: is selected from n-butanol fraction of Potentilla chinensis Benth or water phase extraction fraction of Potentilla chinensis Benth.
The preparation method of the anti-HIV-1 virus effective part of the potentilla chinensis in northwest is characterized by comprising the following steps: crushing potentilla anserine, adding 65-95% ethanol solution by volume percentage for extraction, and concentrating and drying the obtained extract to obtain an extract; dispersing the extract in distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the obtained n-butanol extract to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
The extraction is ultrasonic-assisted reflux extraction, and the ultrasonic-assisted reflux extraction has the specific operations that: adding the crushed potentilla anserine into 65-95% ethanol solution by volume percentage concentration, performing ultrasonic extraction for 0.5-3 hours, filtering, adding the 65-95% ethanol solution by volume percentage concentration into filter residue, performing reflux extraction for 2-3 times, combining extracting solutions, concentrating and drying to obtain an extract. Dispersing the extract in distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the obtained n-butanol extract to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
The application of the anti-HIV-1 virus effective part of the potentilla chinensis in northwest China is characterized in that: the application in preparing anti-HIV-1 virus medicines.
The anti-HIV-1 virus medicine is prepared by the following steps: mixing n-butanol fraction or water phase extract fraction of Potentilla chinensis Bunge with medicinal adjuvants, and making into oral preparation or injection.
The injection preparation is liposome injection, nanoparticle injection or microsphere injection.
The oral preparation is powder, tablets, granules, capsules and oral solution.
Applicants describe the invention as follows: in the prior art, the common application of the potentilla anserine in northwest China is anti-inflammation and antibacterial, and no relevant report that the potentilla anserine has the effect of resisting HIV-1 virus is found. The applicant has found through a large number of studies that: part of the extract in the potentilla anserine in northwest has the effect of resisting HIV-1 virus, but the efficacy is not obvious, and the specific effective part of the extract cannot be determined. The applicant finds out through research that: by adopting the preparation method, the screened petroleum ether part of the potentilla anserine and the ethyl acetate part of the potentilla anserine have no obvious inhibition effect. The n-butanol part and the water-phase extraction part of the northwest Potentilla chinensis have good anti-HIV-1 virus effects.
The preparation method comprises pulverizing the whole herb of Potentilla chinensis Bunge in northwest of China. The ultrasonic extraction is carried out at normal temperature. Filtration and reflux extraction refer to: after ultrasonic extraction, filtering to obtain an ultrasonic extracting solution and filter residues, adding an ethanol solution with the mass percent concentration of 65-95% into the filter residues for reflux extraction to obtain a reflux extracting solution, and combining the ultrasonic extracting solution and the reflux extracting solution. The specific operation of reflux extraction is as follows: adding the pulverized medicinal materials and ethanol solution into a reflux heating device, heating, reflux extracting, cooling, and filtering to obtain primary extractive solution and residue; adding ethanol solution into the residue, heating, performing secondary reflux extraction, cooling, and filtering to obtain secondary extractive solution and residue (reflux extraction for 2 times); mixing the extractive solutions to obtain extractive solution. Preferably, the reflux extraction is carried out for 0.5-2 hours each time; the ethanol solution added during each reflux extraction submerges the surface of the medicinal material by 1-2 cm. The reflux extraction temperature is the conventional temperature of ethanol reflux extraction, and is higher than the boiling point of ethanol, preferably 80-100 ℃.
In the preparation method, the extract is a crude extract, and different solvents are adopted to extract dispersion liquid, namely petroleum ether is added into the extract dispersion liquid for extraction and separation to obtain petroleum ether extract, ethyl acetate is added into the extract dispersion liquid for extraction and separation to obtain ethyl acetate extract, and n-butanol is added into the extract dispersion liquid for extraction and separation to obtain n-butanol extract; volatilizing the n-butanol extract to remove the solvent to obtain n-butanol fraction of Potentilla chinensis in northwest of China; volatilizing the residual extract dispersion (solvent is water) after extraction to remove the solvent to obtain the water-phase extraction part of the northwest Potentilla chinensis. The aqueous phase extraction part can also be called aqueous phase extract, and the aqueous phase extraction part is easily dissolved in water; in contrast, the petroleum ether fraction, the ethyl acetate fraction, and the n-butanol fraction refer to a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract, respectively, and are easily soluble in petroleum ether, ethyl acetate, and n-butanol, respectively. Wherein the n-butanol fraction of Potentilla chinensis Bunge comprises glycosides and phenolic compounds; the water phase extraction part of Potentilla chinensis Bunge comprises polysaccharide and organic acid compounds. And volatilizing the solvent to remove the solvent. The solvent (petroleum ether, ethyl acetate and n-butanol) can be volatilized by heating under normal pressure. Preferably, the solvent is volatilized and concentrated under reduced pressure, the method has high efficiency, and the heat-sensitive components can be prevented from losing the property of the medicine due to high temperature.
For the application, the applicant has found through research that: oral and injectable formulations are the preferred routes of administration. The oral preparation is a gastrointestinal administration preparation, and preferably, the oral preparation is a sustained-release and controlled-release oral preparation. Capsules include soft and hard capsules. In addition, the preparation of the anti-HIV-1 virus effective part of the potentilla anserine in northwest China can also be prepared by other administration routes: such as respiratory tract administration preparations (spray, aerosol, powder spray); dermal preparations (external solutions, lotions, liniments, ointments, plasters, pastes, patches); film administration preparations (eye drops, nose drops, ophthalmic ointment, gargle, sublingual tablet); the preparation (suppository) is administered via cavity. The injection preparation can be a conventional injection preparation. The injection preparation is classified according to drug delivery system, preferably, the injection preparation is liposome injection, nanoparticle injection or microsphere injection; in addition, the injection of the invention can also be a microcapsule injection, a polymer micelle injection, a microemulsion injection, a submicron emulsion injection, a submicron injection or a gel injection, and the injection of the drug delivery system can prolong the circulation time of a drug carrier in vivo, prolong the retention time of drug-carrying particles at an absorption part and control the burst effect of the drug at the initial release stage. The pharmaceutic adjuvant comprises a medicine carrier, and also comprises at least one of a solvent, a solubilizer, a cosolvent, an emulsifier, a suspending agent, a clarifying agent, a deflocculant, a flavoring agent, a coloring agent, a preservative, a chemical sterilizing agent, an adsorbent, a filter aid, an antioxidant, a pH regulator, an isotonic regulator, a diluent, an adhesive, a wetting agent, a disintegrating agent, a lubricant, a glidant, an anti-adhesion agent, a slow-release agent, a controlled-release agent, a coating material, a film-forming material and a capsule material.
Compared with the prior art, the anti-HIV-1 virus effective part of the potentilla anserine in northwest China, the preparation method and the application have the beneficial effects that:
1. the anti-HIV-1 virus effective part of Potentilla chinensis Bunge can effectively inhibit HIV-1 virus activity, and can be used for preparing anti-HIV-1 virus medicine. The applicant found in the study that: at present, raw materials for preparing anti-AIDS drugs are short, and the northwest Potentilla chinensis which only has anti-inflammatory and antibacterial effects in the prior art also has the effect of inhibiting the activity of HIV-1 viruses, and the drug effect screening is carried out on HIV-1 reverse transcriptase and protease for the first time. The applicant takes HIV-1 virus inhibition as an activity index to carry out pharmacodynamic experiments on the HIV-1 virus resistance, uses a CCK-8 method to carry out cytotoxicity experiments on medicines, and takes the influence on the activities of HIV-1 reverse transcriptase and protease as an entry point to carry out the research on the activity mechanism of the HIV-1 virus resistance. The results show that the n-butanol part of the northwest cinquefoil and the water-phase extraction part of the northwest cinquefoil can obviously inhibit HIV-1 virus by combining reverse transcriptase protein and inhibiting HIV protease, and the cytotoxicity is enhanced along with the increase of the administration concentration. The applicant finds out the new efficacy of the potentilla anserine, designs a preparation method, determines the optimal anti-HIV-1 virus effective part of the potentilla anserine through research, prepares the medicine in various dosage forms, and pays a great deal of creative labor in the process.
2. The preparation method of the anti-HIV-1 virus effective part of the potentilla anserine is convenient to extract and is a method determined by the research of the applicant. The extraction sequence of the solvent in the preparation method is determined by the research of the applicant, the sequence can not be reversed and replaced, and the n-butanol part and the water-phase extraction part of the northwest cinquefoil obtained by adopting the solvent extraction sequence have better anti-HIV-1 virus effects.
3. The invention expands the raw material channel of anti-HIV-1 virus drugs, expands the application of Potentilla chinensis Benth, develops Potentilla chinensis Benth into a new raw material of anti-HIV-1 virus drugs, and can obviously improve the additional value of Potentilla chinensis Benth.
Drawings
FIG. 1P 24 antigen is used for detecting the inhibition rate of n-butanol part of Potentilla chinensis in northwest of China.
FIG. 2P 24 antigen is used to detect the inhibition rate of the water phase extraction part of Potentilla chinensis in northwest of China.
FIG. 3 shows HIV-1 pseudovirus single-cycle infection experiment of n-butanol fraction of Potentilla chinensis in northwest of China.
FIG. 4 shows the single-cycle infection experiment of HIV-1 pseudovirus in the water-phase extracted part of Potentilla chinensis in northwest of China.
FIG. 5 is a CCK8 method cytotoxicity test of n-butanol fraction of Potentilla chinensis in northwest of China.
FIG. 6 is a CCK8 method cytotoxicity test of the water phase extraction part of Potentilla chinensis in northwest of China.
FIG. 7 shows the in vitro binding ability of anti-HIV-1 virus active site of Potentilla chinensis in northwest of China to reverse transcriptase protein.
FIG. 8 shows the effect of anti-HIV-1 virus active site of Potentilla chinensis in northwest of China on the activity of HIV-1 to inhibit reverse transcriptase.
FIG. 9 shows the effect of anti-HIV-1 virus active site of Potentilla chinensis in northwest of China on HIV-1 protease activity.
Detailed Description
Examples 1 to 3 are specific embodiments of the anti-HIV-1 virus active site of Potentilla chinensis in northwest of the world and the preparation method thereof, and example 1 is the most preferred example.
Example 1
The preparation method comprises the following steps: crushing Potentilla chinensis Bunge, performing ultrasonic extraction with 75-85% ethanol solution by volume percentage concentration for 1-2 times, filtering to obtain ultrasonic extraction solution and filter residue, adding 75-85% ethanol solution by mass percentage concentration into the filter residue, performing reflux extraction for 3 times, each time for 0.5-1 hour to obtain reflux extraction solution, combining the sound wave extraction solution and the reflux extraction solution, concentrating and drying to obtain extract; dispersing the extract with distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the n-butanol extractive solution to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
Example 2
The preparation method comprises the following steps: crushing Potentilla chinensis Bunge, performing ultrasonic extraction for 2-3 hours by adopting an ethanol solution with the volume percentage concentration of 85-95%, filtering to obtain an ultrasonic extraction solution and filter residues, adding the filter residues into the ethanol solution with the mass percentage concentration of 85-95%, performing reflux extraction for 2 times, and performing reflux extraction for 1.5-2 hours each time to obtain a reflux extraction solution, combining the acoustic wave extraction solution and the reflux extraction solution, concentrating and drying to obtain an extract; dispersing the extract with distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the n-butanol extractive solution to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
Example 3
The preparation method comprises the following steps: crushing Potentilla chinensis Bunge, performing ultrasonic extraction for 0.5-1 hour by using 65-75% ethanol solution in volume percentage concentration, filtering to obtain ultrasonic extraction solution and filter residue, adding 75-85% ethanol solution in mass percentage concentration into the filter residue, performing reflux extraction for 3 times, 1-1.5 hours each time to obtain reflux extraction solution, combining the ultrasonic extraction solution and the reflux extraction solution, concentrating and drying to obtain extract; dispersing the extract with distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the n-butanol extractive solution to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
Performance testing
The following experiment was carried out using the n-butanol fraction (labeled B) and the aqueous fraction (labeled C) of Potentilla chinensis (North West China) obtained in example 1 as the test drugs.
I, HIV-1R 3A wild virus infection experiment (namely P24 antigen detection).
Each 100mg of B, C sites was precisely weighed into a different centrifuge tube, and dissolved in 1mL of DMSO to prepare a 100mg/mL stock solution. Serum-free culture media are sequentially used for dilution to series of concentrations of 100 mug/mL, 33.3 mug/mL, 11.1 mug/mL, 3.7 mug/mL and 1.2 mug/mL during experiment, and the series of concentrations are administration groups with different concentrations. The experiment is provided with a virus control group, a cell control group and a group administered with different doses. The drug administration group adds 100 muL of test drug solutions with different concentrations into each well of a 96-well plate, and three wells are arranged for each concentration. MT4 cells (5X 10) were harvested5Per mL) withInto HIV-1R 3A virus 1X 103TCID50/mL，37℃，5% CO2Culturing for 2 h, P; s washing for 3 times, centrifuging to remove supernatant, adding complete culture medium, and adding 100 μ L of the HIV-1 infected cells (5 × 10) to each well of 96-well plate4/100µL），37℃，5% CO2Culturing, changing liquid after three days, keeping the concentration of the drug unchanged, collecting supernatant from each hole on the sixth day, measuring the amount of P24 antigen by adopting an ELISA method, taking 50 mu L of supernatant from each hole, adding the supernatant into the plated TZM-bl cells on the previous 1 day, and culturing at 37 ℃ in CO2Culturing in an incubator for 48 h. Luciferase content was detected using Promega Bright-Glo luciferase kit and inhibition was calculated as detailed in FIGS. 1-2.
II, HIV-1 pseudovirus monocycle infection experiment.
Each 100mg of B, C sites was precisely weighed into a different centrifuge tube, and dissolved in 1mL of DMSO to prepare a 100mg/mL stock solution. According to the experiment, the medicine is sequentially diluted by serum-free culture medium, B, C are 50 mug/mL, 16.67 mug/mL, 5.56 mug/mL, 1.85 mug/mL and 0.62 mug/mL, the medicine is taken as administration groups with different concentrations, only a pseudovirus medicine-free group is added to serve as a virus control group, and an AZT (zidovudine) group is added to serve as a positive control group. Inoculating TZM-bl cells (Hela cells express CD4, CXCR4 and CCR5, and contain Tat-initiated luciferase and LacZ genes) to a 96-well plate, culturing for 24 hours in an incubator at 37 ℃, discarding the supernatant, adding pSG3 pseudovirus supernatant, adding DEAE (final concentration is 10 mu g/mL) in the virus supernatant, adding diluted medicines with different concentrations, culturing for 48 hours, discarding the supernatant, adding a fixing solution, fixing for 5 min at room temperature, adding a developing solution after PBS is washed, culturing for 50min in the incubator at 37 ℃, washing the PBS, counting the number of blue spots under a microscope according to an inhibition ratio formula: inhibition rate = [ (virus control group-experimental group)/virus control group]X 100%, calculating to obtain dose-effect curve and half Inhibition Concentration (IC)50). The results show that the medicine has obvious inhibiting effect on HIV-1 virus, and the details are shown in figures 3-4 and table 1.
TABLE 1 half inhibitory concentration IC50 of anti-HIV-1 virus effective fraction of Potentilla chinensis in northwest of China
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And thirdly, testing the cytotoxicity of the drug at the cellular level by CCK-8.
Precisely weighing B, C parts each 100mg in different centrifuge tubes, and using the effective part of willow herb obtained in example 1 for resisting HIV-1 virus as the tested medicine. Separately, B, C parts (100 mg) were weighed precisely into a centrifuge tube, and dissolved in 1mL of DMSO to prepare a 100mg/mL stock solution. Serum-free culture media are sequentially used for dilution to series of concentrations of 250 mug/mL, 125 mug/mL, 62.5 mug/mL, 31.25 mug/mL and 15.63 mug/mL during experiment, and the series of concentrations are administration groups with different concentrations. TZM-bl cells were seeded on 96-well plates and cultured in an incubator at 37 ℃ for 24h, and then drugs with different concentration gradients were added. After further culturing for 48h, CCK-8 cell proliferation detection reagent (homonymous chemistry) is adopted for culturing for 45 min, and the absorbance value at 450 nm is detected by an enzyme-labeling instrument. The inhibition rate of the extracts on normal TZM-bl cells was calculated and the GraphPad Prism 5.0 software was fitted to the experimental data to obtain dose-effect curves and median toxic concentration (TC 50). Inhibition rate formula: the inhibition rate = [ (cell control group-experimental group)/cell control group ] × 100%, which is detailed in fig. 5-6 and table 2.
TABLE 2 cytotoxicity of anti-HIV-1 virus active fractions of Potentilla chinensis in northwest China
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And fourthly, in-vitro combination experiment of the medicament and the reverse transcriptase protein.
A BIAcore 3000 biomolecule interaction instrument was used, reverse transcriptase protein was coupled to a CM5 chip, and Bovine Serum Albumin (BSA) was used as a negative control. Diluting the B, C part to 200 mug/mL by PBS filtered by a 0.45 mu m filter membrane, carrying out sample injection at the flow rate of 20 mu L/min and the injection amount of 60 mu L, recording the RU value of the sample flowing through the target protein binding period and the RU value of the sample flowing through the BSA protein binding period, wherein the difference between the RU value and the RU value is the binding response value of the sample and the target protein, and detailed data are shown in FIG. 7 and Table 3.
TABLE 3 in vitro binding RU values of anti-HIV-1 virus effective fractions of Potentilla chinensis (northwest China)
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And fifthly, determining the activity of the HIV-1 reverse transcriptase by the medicine.
The influence of the compound on the activity of HIV-1 reverse transcriptase is detected in vitro by using a reverse transcriptase detection kit. Nevirapine (NVP) is used as a positive drug, the concentration is 2.5 ng/mL, the concentration of B, C parts is 200 mug/mL, and 2 multiple wells are set in an experiment. Experiment lysis solution (20 μ L/well) containing 1 ng HIV-1 RT was added to 96-well plates and incubated with drug at 37 ℃ for 1 h. The solution in the wells of the 96-well plate was transferred to the microplate and incubation continued at 37 ℃ for 1 h. Discarding the solution in the microplate, washing with the eluent for 5 times, adding 200 μ L of Anti-DIG-POD working solution, and incubating at 37 ℃ for 1 h. The microplate solution was discarded, the eluate was washed 5 times, 200 μ L ABTs substrate solution was added to each well, incubated at room temperature for 30 min, absorbance value of the compound at 405nm was detected using a microplate reader, and inhibition was calculated, as detailed in fig. 8 and table 4.
TABLE 4 Effect of anti-HIV-1 virus active site of Potentilla chinensis in northwest of the northwest on HIV-1 reverse transcriptase activity
Figure 425041DEST_PATH_IMAGE004
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Sixthly, influence of the medicine on the activity of HIV-1 protease.
The influence of the medicine on the activity of HIV-1 protease is detected in vitro by adopting a protease detection kit. Peptatin A was used as a positive control at a concentration of 2X 10-3The concentration of mML, B and C parts is 200 mug/mL, and 2 multiple holes are arranged in the experiment. Adding a drug (10 muL/well) and a protease solution (10 muL/well) into a 384-well plate, adding the drug and a buffer solution into a solvent control group, adding the protease solution and the buffer solution into a positive control well, incubating at room temperature for 15 min, adding 10 muL protease substrate solution into each well, slightly shaking for 60s in an enzyme-linked immunosorbent assay instrument, incubating at room temperature in a dark place for 30 min, detecting the absorbance value at 340nm/490nm, and calculating the inhibition rate of the sample on the reverse transcriptase, which is detailed in a graph 9 and a table 5.
TABLE 5 in vitro determination of the Effect of anti-HIV-1 Virus active fractions of Potentilla chinensis Benth on the activity of HIV-1 protease
Figure DEST_PATH_IMAGE005
。
Example 4
The preparation method of the tablet of the effective part of the northwest Potentilla chinensis comprises the following steps: mixing water phase extract of Potentilla chinensis (northwest) 300mg and starch 100mg, adding starch paste with volume percentage concentration of 10% 40mg, making into soft material, sieving to obtain wet granule, drying to obtain dry granule, grading, adding magnesium stearate 4 mg, mixing, and tabletting.
Example 5
The preparation method of the granule comprises the following steps: mixing 5 parts of water-phase extraction part of Potentilla chinensis, 1 part of sucrose and 3 parts of dextrin uniformly according to the weight part ratio, adding 2-5 parts of ethanol solution with a proper mass percentage of 95%, stirring while adding to prepare a soft material, drying the soft material, sieving with a 16-mesh sieve, and subpackaging to obtain the Potentilla chinensis extract.
Example 6
The pellet capsule comprises the following raw materials in parts by weight: 30 parts of n-butanol part of northwest Potentilla chinensis, 5 parts of lecithin, 5 parts of sodium taurocholate and 30 parts of microcrystalline cellulose;
the preparation method of the pellet capsule comprises the following steps: uniformly mixing the n-butanol part of the northwest Potentilla chinensis, lecithin, sodium taurocholate and microcrystalline cellulose according to a ratio, pouring an ethanol water solution, uniformly stirring to obtain a soft material, pouring the soft material into an extruder for extrusion, carrying out spheronization to obtain granules, carrying out extrusion at a rotation speed of 250r/min, carrying out spheronization at a rotation speed of 800r/min for 20min, drying, sieving by a 24-30-mesh sieve to obtain pellets, and filling the pellets into a capsule shell to obtain the capsule.
Example 7
The preparation method of the liposome injection comprises the following steps:
1) under the protection of nitrogen, 50g of cholesterol succinate, 250g of distearoyl phosphatidyl ethanolamine, 40g of soybean lecithin, 50g of poloxamer-188 and 10g of n-butanol part of northwest Potentilla chinensis are dissolved in 1500ml of solution with the volume ratio of 1: 1, stirring and dissolving the mixture in an organic solvent of ethanol and n-butanol to obtain a suspension; concentrating the suspension under reduced pressure to volatilize the organic solvent, and obtaining a phospholipid membrane;
2) adding 8000ml of phosphate buffer solution with the pH value of 6.8 into a phospholipid membrane under the protection of nitrogen, stirring to enable the phospholipid membrane to elute, fully swell and hydrate, and filtering by a microporous filter membrane of 0.22 mu m to obtain liposome at the n-butyl alcohol part of the Potentilla chinensis in North West;
3) under the aseptic condition, adding 100g of trehalose into the liposome at the n-butyl alcohol part of the potentilla chinensis, uniformly stirring, carrying out ultrasonic treatment for 0.5-1 hour, adding injection water to a constant volume, filtering through a microporous membrane of 0.22 mu m, and filling to obtain the liposome injection at the n-butyl alcohol part of the potentilla chinensis.
Example 8
The preparation method of the nanoparticle injection comprises the following steps: dissolving 25g of n-butanol part of Potentilla chinensis in northwest China and 100g of poloxamer 407 in 3000ml of absolute ethyl alcohol, adding 30ml of 1mol/L zinc chloride ethanol solution, stirring and mixing, and performing ultrasonic treatment for 0.5-1 hour to dissolve the zinc chloride ethanol solution to obtain a mixed solution; concentrating the mixed solution under reduced pressure to volatilize the solvent, and freezing in a refrigerator at-20 ℃ for 2 hours; taking out and adding water for injection to a constant volume, carrying out ultrasonic treatment for 0.5-1 hour, filtering through a 0.22 mu m microporous filter membrane, and filling to obtain the nanoparticle injection at the n-butyl alcohol part of the Potentilla chinensis.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (4)

1. The application of the anti-HIV-1 virus effective part of the potentilla chinensis in northwest China is characterized in that: the application in preparing anti-HIV-1 virus medicines;
the anti-HIV-1 virus effective part of Potentilla chinensis Bunge in northwest is selected from n-butanol part of Potentilla chinensis Bunge in northwest or water phase extraction part of Potentilla chinensis Bunge in northwest;
the preparation method of the anti-HIV-1 virus effective part of the potentilla chinensis in northwest China comprises the following steps: crushing potentilla anserine, adding 65-95% ethanol solution by volume percentage for extraction, and concentrating and drying the obtained extract to obtain an extract; dispersing the extract in distilled water to obtain extract dispersion, sequentially extracting the extract dispersion with petroleum ether, ethyl acetate and n-butanol as solvents, and volatilizing n-butanol from the obtained n-butanol extract to obtain n-butanol fraction of Potentilla chinensis Bunge in northwest of China; concentrating and drying the remaining extract dispersion liquid after extraction to obtain the water-phase extraction part of the northwest Potentilla chinensis.
2. The use of the anti-HIV-1 virus effective fraction of Potentilla chinensis in northwest of claim 1, wherein: the anti-HIV-1 virus medicine is prepared by the following steps: mixing n-butanol fraction or water phase extract fraction of Potentilla chinensis Bunge with medicinal adjuvants, and making into oral preparation or injection.
3. The use of the anti-HIV-1 effective site of Potentilla chinensis Bunge in northwest of claim 2, which is characterized in that: the injection preparation is liposome injection, nanoparticle injection or microsphere injection.
4. The use of the anti-HIV-1 effective site of Potentilla chinensis Bunge in northwest of claim 2, which is characterized in that: the oral preparation is powder, tablets, granules, capsules and oral solution.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Antibacterially active phenolic lipid derivatives from Comarum salesovianum (Steph.) Aschers. et Gr.;G. Odontuya etal;《Phytochemistry Letters》;20151231(第13期);360-364，第361页左栏第2段 *
G. Odontuya etal.Antibacterially active phenolic lipid derivatives from Comarum salesovianum (Steph.) Aschers. et Gr..《Phytochemistry Letters》.2015,(第13期),360-364，第361页左栏第2段. *
蕨麻的化学成分及药理活性研究进展;刘志军等;《食品安全质量检测学报》;20150925;第06卷(第09期);3569-3574 *

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