CN115400166A - Polygonum cuspidatum nanoparticle and preparation method thereof and application of polygonum cuspidatum nanoparticle in medicines for preventing and treating enterovirus and hand-foot-and-mouth disease - Google Patents
Polygonum cuspidatum nanoparticle and preparation method thereof and application of polygonum cuspidatum nanoparticle in medicines for preventing and treating enterovirus and hand-foot-and-mouth disease Download PDFInfo
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Abstract
The invention discloses giant knotweed rhizome nanoparticles, a preparation method thereof and application thereof in medicines for preventing and treating enterovirus and hand-foot-and-mouth disease. In the application, in some embodiments, when the concentration of the polygonum cuspidatum nanoparticles is 2.0mg/ml, the inhibition rates of the polygonum cuspidatum nanoparticles on EV71 and CAVl6 reach 90.91% and 63.77% respectively, and the half Effective Concentration (EC) of the polygonum cuspidatum nanoparticles on EV71 is achieved 50 ) 0.46mg/ml, and the extract EC of Polygonum cuspidatum 50 It was 0.4mg/ml. Further animal experiments prove that the death protection rates of 4mg/kg/d and 1mg/kg/d giant knotweed rhizome nanoparticles on EV71 virus infected ICR mice respectively reach 83.33% and 25.0%. The giant knotweed rhizome nanoparticles have obvious antiviral effect and can be used for treating hand-foot-and-mouth disease virus infectionThe new traditional Chinese medicine.
Description
Technical Field
The invention relates to the technical field of medicines for preventing and treating hand-foot-and-mouth diseases caused by enteroviruses or enteroviruses.
Background
Hand-foot-mouth disease (HFMD) is a common infectious disease of children caused by various enteroviruses, and the incidence rate of the disease is highest in children aged 5 years and younger. Most HFMD (high frequency multiplex disease) patients are mainly characterized by fever and rash or herpes at parts such as hands, feet, oral cavity, buttocks and the like, but a few patients can suffer from encephalitis, meningitis, myelitis, peripheral neuritis, acute flaccid paralysis, myocarditis and the like, and individual severe children have fast disease progress and die due to neurogenic pulmonary edema and exhaustion of cardiopulmonary function. HFMD has various transmission ways, and is common to recessive infection people, the main peak of disease occurrence is summer, the secondary peak is autumn and winter, and the social panic is caused by outbreak and epidemic in people gathering places such as kindergartens, schools and the like.
The major pathogens of HFMD are more than 20, with Enterovirus (Enterovirus, EV) type 71 (EV 71) infection in about 80% of cases, and secondarily more commonly coxsackievirus type 16 (CoxAl 6). In the prior art, effective vaccines for preventing the generation of HFMD are still lacked, and specific antiviral drug treatment on viruses such as EV71 and the like is also not needed. Therefore, the search for new antiviral drugs, particularly drugs against major viruses such as EV71, is a problem to be solved urgently in the prior art.
Polygonum cuspidatum (Polygonum cuspidatum) is a herbaceous plant in Polygonum of Polygonaceae. The medicine is slightly bitter and cold, and has effects of promoting diuresis, eliminating jaundice, clearing heat and detoxicating, removing blood stasis, relieving pain, relieving cough and eliminating phlegm etc. The giant knotweed rhizome extract has been proved to have the activity of inhibiting human AIDS virus, human cytomegalovirus, coxsackie virus B3 and herpes virus. It also has anti-inflammatory and anti-tumor biological activity. However, the effect of polygonum cuspidatum on enterovirus type 71 and coxsackievirus type A16 is not found and reported in the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention firstly provides the application of the polygonum cuspidatum nanoparticles in the prevention and treatment medicines for enterovirus 71 type, coxsackievirus A16 type and hand-foot-and-mouth diseases caused by the enterovirus 71 type, the coxsackievirus A16 type and the hand-foot-and-mouth diseases caused by the enterovirus 71 type and the coxsackievirus A16 type.
The technical scheme of the invention is as follows:
a method for preparing giant knotweed rhizome nanoparticles comprises the following steps:
(1) Crushing giant knotweed rhizome or giant knotweed rhizome concentrate, sieving the crushed giant knotweed rhizome or giant knotweed rhizome concentrate with a sieve of 80 to 100 meshes, performing reflux extraction by using 75 to 85 percent ethanol, drying the obtained precipitate, dissolving the precipitate by using 90 to 95 percent ethanol, centrifuging and drying to obtain giant knotweed powder;
(2) Adding the giant knotweed rhizome powder into deionized water to obtain giant knotweed rhizome slurry;
(3) Grinding the polygonum cuspidatum slurry to be less than 50 microns to obtain ground slurry;
(4) And (3) crushing the grinding slurry at the crushing speed of 1100-1800 r/min for 30-60 min, and drying to obtain the polygonum cuspidatum nanoparticles.
The giant knotweed rhizome nano-particles prepared by the preparation method are spherical particles, and are uniformly dispersed and free of agglomeration when used, and the particle size of the giant knotweed rhizome nano-particles can reach 20-300 nm.
The invention also specifically provides application of the giant knotweed rhizome nanoparticles to medicines for preventing and treating enterovirus 71 (EV 71) and/or coxsackievirus A16 (CAV 16).
According to the application, the application of the giant knotweed rhizome nanoparticles to the medicines for preventing and treating the hand-foot-and-mouth disease caused by the enterovirus 71 and/or the coxsackievirus A16 can be further obtained.
According to some embodiments of the present invention, after using the polygonum cuspidatum nanoparticle, the OD value of the viral antigen is decreased and the inhibition rate of the virus is increased in the cell contents infected by the enterovirus 71 and the coxsackievirus a16, which are related in an amount effective manner, for example, the inhibition rate of the polygonum cuspidatum nanoparticle to EV71 and CAVl6 is 90.91% and 63.77% respectively at 2.0 mg/ml.
According to some embodiments of the present invention, after EV71 infected mice were treated with the nanoparticles of polygonum cuspidatum, the mortality of mice decreased and was in a dose-response relationship, while mice without the nanoparticles of polygonum cuspidatum all died. The giant knotweed rhizome nanoparticles have obvious protective effect on EV71 virus infected mice.
According to some embodiments of the present invention, the cytotoxic experiment result shows that when the polygonum cuspidatum nanoparticles are diluted from 6.4mg/ml to 7 concentrations such as 0.1mg/ml, the cell survival rate is increased from 23.08% to 97.80%, and the cell survival rate and the concentration of the polygonum cuspidatum nanoparticles have a reverse gradient relationship, which indicates that the safety coefficient of the polygonum cuspidatum nanoparticles as the drug is high.
According to some specific embodiments of the present invention, antiviral experimental results show that the polygonum cuspidatum nanoparticles have obvious effects of inhibiting enterovirus 71 (EV 71) and coxsackievirus a16 (CAVl 6) in cells, at 2.0mg/ml, the inhibition rates of EV71 and CAVl6 are 90.91% and 63.77%, respectively, and at low concentration (0.25 mg/ml), the inhibition rates of the two viruses are 4.55% and 7.25%, respectively, which indicates that the inhibition effects of the polygonum cuspidatum nanoparticles on the two viruses have an obvious dose-effect relationship.
According to some embodiments of the present invention, further animal experiments prove that the death protection rate of the two doses of 4mg/kg/d and 1mg/kg/d giant knotweed rhizome nanoparticles on EV71 virus infected ICR mice is 83.33% and 25.0%, respectively. The medicine has antiviral effect in mice, and is expected to become a new traditional Chinese medicine for treating hand-foot-and-mouth disease virus infection.
The invention can prepare the giant knotweed into the nano-grade medicament, develops a new dosage form of the giant knotweed medicament, increases the surface area of medicament particles, enlarges the contact area between the medicament particles and tissues, can increase medicament absorption and bioavailability, improves medicament effect, reduces dosage, reduces side effect and can also enhance the targeting property of the medicament.
Drawings
FIG. 1 is a transmission electron microscope image of the nanoparticles of Polygonum cuspidatum obtained in example 1.
Detailed Description
The present invention is described in detail with reference to the following examples, but it should be understood that the examples are only for illustrative purposes and should not be construed as limiting the scope of the present invention. All reasonable variations and combinations that fall within the spirit of the invention are intended to be within the scope of the invention.
Example 1
The giant knotweed rhizome nano-particle is prepared by the following steps:
(1) Crushing purchased giant knotweed rhizome decoction pieces, sieving the decoction pieces with a sieve of 80-100 meshes, weighing 100g, carrying out reflux extraction for 3 times and 1 h/time by using 75-85% ethanol, recovering an extract, adding water for dissolving for 5 times, filtering, drying to obtain a precipitate, dissolving by using 95% ethanol, centrifuging, and drying to obtain giant knotweed rhizome powder;
(2) Taking 50g of giant knotweed powder, adding the giant knotweed powder into deionized water according to the solid-to-liquid ratio of 1 (g/ml), and repeatedly stirring the mixture into a uniform slurry;
(3) Adding rhizoma Polygoni Cuspidati homogenate into a stirring mill, and repeatedly grinding the rhizoma Polygoni Cuspidati homogenate to a particle size of below 50 μm;
(4) Grinding the ground polygonum cuspidatum homogenate (with the concentration of 5 wt%) by using a MICROSOFT (MICROCOMPUTER-assisted reaction) pulverizer, pulverizing for 30-60 min at 1100-1800 r/min, crushing and drying to obtain polygonum cuspidatum nanoparticles.
The morphology of the obtained rhizoma Polygoni Cuspidati nanoparticles was analyzed by transmission electron microscope, and the results are shown in figure 1, which shows that the rhizoma Polygoni Cuspidati nanoparticles are mostly distributed in spherical shape with a size of 20-300 nm. The particle size of the particles is 47-252 nm, the average particle size is 159.5nm, the dispersity is 0.351, and the particle size accords with the size characteristics of the nanoparticles.
Further, the obtained polygonum cuspidatum nanoparticles are prepared according to the antiviral experiment requirements, and the preparation method comprises the following steps:
weighing rhizoma Polygoni Cuspidati nanoparticles 10mg, adding sterile distilled water to dissolve, diluting to 10mL (final concentration of 1 mg/mL), autoclaving at 110 deg.C for 15min at 8 pounds, and storing at 4 deg.C. When cell experiments and animal experiments are carried out, diluting the polygonum cuspidatum nanoparticles to a required concentration by using a DMEM cell maintenance culture medium containing 2% fetal calf serum to obtain a polygonum cuspidatum nanoparticle maintenance liquid. The positive control medicament arbidol is diluted to 0.1g/mL by DMEM for standby, and is diluted by the same method when in use.
Example 2
Considering that viruses are intracellular parasites, evaluation of the antiviral effects of antiviral drugs must be carried out intracellularly, and observation of the cytotoxicity of the drugs should be taken as a prerequisite for the study. This example first observes the cytotoxicity of a drug as follows:
good growth with pancreatin containing 0.02%Human malignant embryonal rhabdomyoma cells (RD cells) were dispersed into a single cell suspension, after which the cells were adjusted to 1X 10 with a cell culture solution containing 10% calf serum 5 Per ml concentration 0.2 ml/well in 96-well plates, 5% CO at 37 ℃ 2 Culturing in an incubator for 24h. After the cells grew into a monolayer, the supernatant of the DMEM culture solution was discarded, and 200. Mu.l of the polygonum cuspidatum nanoparticle maintenance solution obtained in example 1 with different concentrations was replaced. The final concentration of the giant knotweed rhizome nano-particles is respectively 6.4mg/ml, 3.2mg/ml, 1.6mg/ml, 0.8mg/ml, 0.4mg/ml, 0.2mg/ml and 0.1mg/ml. Each concentration was repeated for 4 wells, and the experimental experiment was repeated for 3 times while setting a normal cell control group. After further culturing for 24 hours, cell viability (CC) was examined by MTT method 50 )。
The examples show that under microscope, the CPE characteristics of polygonum cuspidatum nanoparticles on RD cells are: the cells become round, the number of intracellular granules increases, the refractive index becomes poor, and the cells die and fall off from the bottle wall. The OD value is decreased because the number of living cells detected by the MTT method is decreased due to decrease in metabolic activity or death of the cells. And the cell survival rate of the polygonum cuspidatum nanoparticles is reduced along with the increase of the concentration of the polygonum cuspidatum nanoparticles in a certain range. Determining CC of rhizoma Polygoni Cuspidati nanoparticles by observing cell morphology under microscope and determining OD value by MTT method 50 It was 2.53mg/ml.
Specific cytotoxicity results are shown in table 1 below:
TABLE 1 cytotoxic Effect of Polygonum cuspidatum nanoparticles on RD cells (x + -s)
Example 3
The inhibition effect of the giant knotweed rhizome nanoparticles on two hand-foot-and-mouth disease virus infected RD cells is detected through the following processes:
in 96-well culture plates in which a monolayer of RD cells had been grown, obtained in example 2, 50. Mu.l of 100TCID was seeded per well 50 Adsorbing EV71 and CAV16 virus liquid at 37 ℃ for 90min, and discarding virus supernatant. According to the results of cytotoxicity test obtained in example 2, the concentration of half cytotoxicity of the polygonum cuspidatum nanoparticles is within the range of half cytotoxicity0.2ml of the DMEM cell maintenance medium containing the giant knotweed nanoparticles obtained in example 1 was added to each well at different concentrations to give final concentrations of 2.0mg/ml, 1.0mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, and 0.0625mg/ml, and the mixture was allowed to stand at 37 ℃ and 5% CO 2 Culturing, observing CPE every day, and changing fresh medicated maintenance solution every other day. Repeat 4 holes for each concentration, and set normal cell control group and virus control group without drug in the experiment. And after continuously culturing for 48 hours, detecting the inhibition rate of the polygonum cuspidatum nanoparticles on virus-infected cells.
Through observation under a light microscope, the effect of the giant knotweed nanoparticles on the cytopathic effect of the pores is very light compared with that of a virus control group, most cells have normal shapes, and the giant knotweed nanoparticles inhibit the formation of CPE (CPE) caused by EV71 and CAV16 virus infected cells. The change of the OD value of the virus antigen is in a negative relation with the concentration of the giant knotweed rhizome nanoparticles. These results suggest that the polygonum cuspidatum nanoparticles have obvious inhibition effect on two hand-foot-and-mouth disease virus infected cells.
The MTT method is adopted to detect the inhibition effect of the giant knotweed rhizome nanoparticles on EV71 and CAV16 viruses. The antiviral effect of the polygonum cuspidatum nanoparticles on two viruses was recorded according to the virus cytopathic effect (CPE). According to the CPE observation results, when the CPE of the virus control group was 75% or more, the culture supernatant was discarded, 50. Mu.l of MTT solution containing 5mg/ml was added to each well, and the mixture was further incubated at 37 ℃ and 5% 2 After culturing for 2-3 h, washing off MTT supernatant, adding DMSO solution, uniformly mixing 100 mu L of the MTT supernatant in each hole, measuring the OD value of the optical density at the wavelength of 570nm by using an enzyme-labeling instrument after 5-10 minutes, and calculating the inhibition rate of the polygonum cuspidatum nanoparticles to two viruses according to the following formula:
the results are shown in table 2 below:
TABLE 2 inhibition of giant knotweed rhizome nanoparticles on RD cells infected by two hand-foot-and-mouth disease viruses (x + -s)
It can be seen that the giant knotweed rhizome nanoparticles have better inhibition effect on cytopathic effect caused by two hand-foot-and-mouth disease viruses, and present a more obvious dose-effect relationship in the range of 0.5mg/ml to 2.0 mg/ml. And the three action modes of the giant knotweed rhizome nanoparticle have obvious effect on virus inhibition.
We used 0.4mg/ml arbidol as positive drug control, and the inhibition ratio on EV71 was 54.09%. And 6 antiviral effects of the polygonum cuspidatum extract with different concentrations from 0.01mg/ml to 0.32mg/ml and the like are synchronously detected, and the result shows that the polygonum cuspidatum extract EC 50 It was 0.4mg/ml.
Example 4
SPF grade ICR suckling mice were purchased per nest and bred with the mother mice. After the virus infection is successful, 12 ICR suckling mice are reserved in each group, less than 12 ICR suckling mice are reserved in each group, and two litters are combined into 1 group, so that the suckling mouse and the suckling mouse are fed in the same litter. Fasting was 2h before the experiment. 58 3-day-old ICR mice were randomized into 5 groups for in vivo experiments, including:
(1) normal animal control group, normal saline is taken orally;
(2) a virus infection group, after virus infection, the medicine is not added for treatment, and normal saline is orally taken;
(3) the high and low dose of the giant knotweed nanoparticle treatment group respectively takes 4mg/kg/d and 1mg/kg/d;
(4) the dosage of the positive control medicament arbidol group is 4mg/kg/d.
Except for normal control group mice, each group was given 15LD 50 The virus solution is orally infected with ICR mice, each of which is 0.05mL, and normal control mice are orally administered with normal saline. The gavage administration is started 24h after infection, the administration is carried out 2 times a day, 0.1mL of each time, and the giant knotweed rhizome nanoparticles are continuously administered for 5d.
The morbidity state of the suckling mice is observed and recorded every day after infection, and indexes such as average survival time, mortality, death protection rate and the like of each group of animals are calculated. Observation was carried out for 12 days, then cervical vertebrae were removed to sacrifice all animals, and histoviral titration was carried out according to a conventional method.
The results of in vivo experiments show that the animals have morbidity symptoms 3 days after being infected with EV71 virus, and the symptoms are manifested by hind limb paralysis, walking difficulty, paralysis of part of mice and death within 5-11 days. Normal control animals did not die. The rats treated by the polygonum cuspidatum nanoparticles and the positive control medicament have tremor and paralysis, but are recovered to be normal after a few days. The mortality rates of animals in the high-dose and low-dose giant knotweed nanoparticle groups are 16.67% and 75.0%, respectively, and the details are shown in the following table 3:
TABLE 3 protective Effect (x + -s) of Polygonum cuspidatum Nanoparticles (NPC) on EV 71-infected ICR mice
Meanwhile, the results in Table 4 show that the virus loads in muscle and brain tissues of mice in different experimental groups are also obviously different in high and low dose giant knotweed nanoparticle groups (4 mg/kg/d and 1 mg/kg/d), wherein the former is obviously lower than the latter and has obvious dose-effect relationship. Differences were also shown compared to untreated virus-infected controls. Muscle and brain tissue compare, the former is higher than the latter.
TABLE 4 Effect of Polygonum cuspidatum Nanoparticles (NPC) on viral titers in tissues of EV 71-infected ICR mice (pfu/ml)
The results of in vivo experiments show that the polygonum cuspidatum nanoparticles have obvious protective effect on EV-71 infected mice.
The above examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the above examples. All technical schemes belonging to the idea of the invention belong to the protection scope of the invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention should also be considered as within the scope of the present invention.
Claims (4)
1. A preparation method of giant knotweed rhizome nanoparticles is characterized by comprising the following steps:
(1) Crushing giant knotweed rhizome or giant knotweed rhizome concentrate, sieving with a 80-100 mesh sieve, performing reflux extraction with 75-85% ethanol, drying the precipitate obtained by extraction, dissolving with 90-95% ethanol, centrifuging, and drying to obtain giant knotweed powder;
(2) Adding the giant knotweed rhizome powder into deionized water to obtain giant knotweed rhizome slurry;
(3) Grinding the polygonum cuspidatum slurry to be less than 50 microns to obtain ground slurry;
(4) And (3) crushing the grinding slurry at the crushing speed of 1100-1800 r/min for 30-60 min, and drying to obtain the polygonum cuspidatum nanoparticles.
2. The giant knotweed rhizome nanoparticle prepared according to the preparation method of claim 1.
3. Application of rhizoma Polygoni Cuspidati nanoparticles in medicine for preventing and treating enterovirus 71 and/or Coxsackie virus A16 is provided.
4. The giant knotweed rhizome nanoparticles are applied to the medicines for preventing and treating the hand-foot-and-mouth disease caused by the enterovirus 71 type and/or the coxsackievirus A16 type.
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