CN114344268B - Naringin nanoparticle with polyhydroxyethyl methacrylate as carrier, and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of high-molecular pharmaceutical preparations, in particular to naringin nanoparticles with poly (2-hydroxyethyl methacrylate) (PHEMA) as a carrier, a preparation method thereof and anti-inflammatory characteristic research. The invention discloses a drug-loaded nanoparticle which is prepared by coating and loading a hydrophobic drug by using PHEMA as a carrier, and specifically comprises the following components: firstly, ultrasonically distributing PHEMA and Naringin (NA) in a penicillin bottle filled with water, uniformly mixing a medicament and a carrier, adding a rotor and zirconia beads, and magnetically stirring; and then sucking out the suspension, diluting the residual liquid medicine on the zirconium beads by water for several times, and combining the liquid medicines to a constant volume to obtain the finished product. The nano drug-loaded particles disclosed by the invention are simple in preparation process, high in drug loading capacity and good in slow release effect; experiments show that the NA-Nps can effectively improve the anti-inflammatory effect, can inhibit cough and reduce the toxic and side effects of the medicine, and has great market application and popularization prospects.

Description

Naringin nanoparticle with poly (hydroxyethyl methacrylate) as carrier, and preparation method and application thereof

Technical Field

The invention relates to the field of polymer pharmaceutical preparations, relates to a nano pharmaceutical preparation, and in particular relates to a naringin nano particle taking poly (hydroxyethyl methacrylate) as a carrier, and a preparation method and application thereof.

Background

The water solubility and fat solubility of the naringin are poor, the application of the naringin in the pharmaceutical industry and the food industry is greatly limited, and the bioavailability is low. At present, the research on new formulations of naringin is mostly in the early basic test stage, but the research on the extraction process of naringin and the in vitro release and other aspects are well progressed, and the problem of low absolute bioavailability of common solid tablets is solved. Therefore, the naringin preparation with high bioavailability, stability, effectiveness and safe application is obtained by utilizing a new technology, using new auxiliary materials, designing a scientific and reasonable prescription and implementing a stable and feasible preparation process, and is the main research direction of people.

Although naringin shows good biological activity in vitro tests, the solubility in water and the absorption rate in human bodies are low, so that the bioavailability of oral naringin is extremely low, which greatly limits the development and utilization of the oral naringin. Therefore, researchers can improve the bioavailability of naringin by means of preparing naringin cyclodextrin inclusion compounds, lecithin compounds, nano particles, liposomes and the like.

Nanotechnology refers to the technology of manufacturing or processing macromolecular substances into substances with nanometer-scale particle size by using single atoms or molecules, and also includes some nanocapsules made of high molecular materials and used for embedding medicines. The preparation using nanotechnology can have various properties, including sustained release, change of the half-life of the drug in vivo, prolongation of the action time of the drug, targeted drug delivery to specific organs, reduction of the drug dosage, alleviation or elimination of toxic and side effects, improvement of the stability of the drug, increase of the drug permeability, increase of the drug transdermal absorption rate, improvement of the drug solubility, and the like.

Poly (2-hydroxyethyl methacrylate) (PHEMA) is prepared by free radical polymerization of hydroxyethyl methacrylate monomer, and is a common high molecular hydrogel material, and PHEMA has controllable permeability, water content, mechanical property and transparency. Has good biocompatibility, and is widely applied to the preparation of soft contact lenses, soft tissue reinforcing materials and supporting materials in bioreactors due to high water content and high permeability. PHEMA also has good viscoelastic behavior, thermal properties, dielectric properties, and the like. With the intensive research on PHEMA hydrogel, PHEMA is also used in other medical fields such as orthopedics, cosmetic surgery, drug release, hemostatic materials, artificial stents, and the like. In order to improve the water solubility of naringin and improve the bioavailability of naringin, the research adopts a high molecular preparation method, hydrophilic polyhydroxyethyl methacrylate is used as a carrier to prepare naringin nanoparticles, and the cough relieving and anti-inflammatory effects of the naringin nano preparation are explored while the water solubility of the naringin is improved.

Disclosure of Invention

In view of the above, the present invention aims to provide a naringin nanoparticle using polyhydroxyethyl methacrylate as a carrier, which has the advantages of simple preparation process, high drug loading rate and good sustained release effect.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for preparing naringin nanoparticles by using polyhydroxyethyl methacrylate as a carrier is disclosed, wherein the naringin nanoparticles are prepared by using PHEMA as a carrier to encapsulate naringin; the method specifically comprises the following steps:

(1) Respectively dissolving PHEMA and naringin in water to obtain a water phase I and a water phase II; uniformly mixing the water phase I and the water phase II under an ultrasonic heating condition to obtain a mixed liquid;

(2) And combining a drug carrier by adopting a medium grinding method, and diluting to finally obtain the naringin nanoparticle solution.

The preparation method disclosed and protected by the invention is simple in process operation and good in repeatability, and the naringenin nanosuspension prepared by the method can realize in-vivo slow release of insoluble drugs, improve in-vivo distribution of the insoluble drugs, enhance the phlegm-resolving effect of the drugs and reduce toxic and side effects.

Exemplarily, referring to the attached figures 1-2 of the specification, the invention performs structural characterization on the naringin nanoparticles prepared by taking polyhydroxyethyl methacrylate as a carrier through particle size distribution and scanning electron microscope test.

Preferably, the above preparation method further comprises freeze-drying: 0.5% whey protein is added into the prepared naringin nano particle solution as a freeze-drying protective agent (the dosage is 1mL naringin nano particle solution and 5mg whey protein) to prolong the storage time of the medicine; wherein the freeze drying time is 12h, and the pressure of the freeze dryer is 0.12mbar under vacuum.

Preferably, the water used in the step (1) is deionized water produced by a Hitech ultra-pure water machine, and the naringin and PHEMA are screened according to a mass ratio of 1: 1;

the concentration of the naringin in the naringin nanoparticle solution is 2.2-2.5mg/mL, the ultrasonic power is 100w, and the ultrasonic temperature is 50 ℃.

Preferably, in the step (2), the grinding medium is zirconia beads with the size of 0.4-0.6mm, the dosage is 10g, the magnetic stirring bar is 2cm, the rotating speed is 300r/min, and the water bath temperature is 50 ℃.

In addition, the residual liquid medicine of the zirconia beads in the step (2) is diluted and subjected to constant volume after ultrasonic oscillation, so that the medicine loss caused by medicine adsorption of the zirconia beads due to grinding loss is reduced, and the medicine content of unit volume is increased to the maximum extent.

The invention also aims to provide the naringin nano-particles prepared by the method.

The naringin nano particle is prepared by taking PHEMA as a carrier to encapsulate hydrophobic drug naringin; wherein the PHEMA is prepared from a polymer of 2-hydroxyethyl methacrylate monomer units.

The nano drug-loaded particles (naringin nano particles) disclosed by the invention are simple in preparation process, high in drug loading capacity and good in slow release effect; experiments show that the NA-NPs can improve the anti-inflammatory effect of the medicine and the cough suppression rate, can improve the weight of a mouse, reduces the toxic and side effects of the medicine, and provides reference and reference for naringin nano preparation and clinical application.

The invention also aims to provide the application of the naringin nano-particles taking the poly (hydroxyethyl methacrylate) as the carrier in the pharmaceutical preparation.

In some application scenes, the application of the naringin nanoparticles in cough relieving and inflammation resisting is also included.

According to the technical scheme, compared with the prior art, the naringin nanoparticle taking the polyhydroxyethyl methacrylate as the carrier, the preparation method and the application thereof provided by the invention have the following excellent effects:

the nano drug-loaded particles (naringin nano particles taking poly (hydroxyethyl methacrylate) as a carrier) disclosed by the invention not only have simple preparation process, but also have high drug-loaded amount and good slow-release effect; experiments show that the NA-NPs can improve the anti-inflammatory effect of the medicine and the cough suppression rate, can improve the weight of mice, reduces the toxic and side effects of the medicine, and has great market application and popularization prospects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the distribution of the average particle size of NA-NPs of the present invention.

FIG. 2 is a scanning electron micrograph of NA-NPs according to the present invention.

FIG. 3 is a graph showing in vitro release profiles of NA-NPs of the present invention in PBS.

FIG. 4 is a graph showing the change in body weight of mice with time.

FIG. 5 shows the therapeutic effect of NA-NPs on mouse lung; lung tissue H & E staining analysis.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the specification of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The embodiment of the invention discloses a method for preparing naringin nanoparticles by taking polyhydroxyethyl methacrylate as a carrier, which is simple in preparation process, high in drug-loaded rate of the prepared drug-loaded nanoparticles, good in sustained-release effect, capable of reducing the toxic and side effects of drugs and suitable for popularization and application in the market.

The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.

Example (b): preferred preparation process of NA-NPs

And preparing the NA-NPs by adopting a medium grinding method. Firstly, screening the drug loading ratio, precisely weighing the drug and the carrier according to the mass ratio of naringin to PHEMA of 1:1,2:1 and 3:1, respectively co-dispersing the drug and the carrier in water, adding the mixture into a penicillin bottle with the volume of 1/4 of the bottle, grinding the mixture for 5 hours at room temperature, measuring the particle size of the nanoparticle solution by a Malvern particle sizer/potentiometer, and screening out the optimal drug loading ratio of 2:1.

secondly, according to the mass ratio of naringin to PHEMA of 2:1 precisely weighing the medicine and the carrier, fixing the rotating speed at 300r/min for 5h, and grinding at the water bath temperature of 0, 25, 50 and 80 ℃, wherein the particle size at 50 ℃ is the minimum, so that the NA-NPs are prepared at 50 ℃.

Then, at milling times of 1,2, 3, 4, 5, 6, 7h, NA-NPs were prepared with a loading ratio of 2:1, preparing NA-NPs under different grinding times, gradually reducing the particle size of the prepared naringenin nanosuspension with the increase of the grinding time, gradually increasing PDI after 5h, and selecting to prepare the naringenin nanosuspension by grinding for 5h in consideration of the dispersion uniformity of the preparation.

The influence of the magnetic stirring speed is examined under the conditions of the drug loading ratio 2:1, the grinding temperature of 50 ℃ and the grinding time of 5h and at the rotating speeds of 250, 300, 350 and 400 r/min. The grain diameter/PDI is better when the rotating speed is more than or equal to 300r/min, but the rotating speed is 300r/min in consideration of the service life of the instrument.

In conclusion, the optimal process is to completely dissolve PHEMA 5mg in 1mL of water by adopting a medium grinding method, then ultrasonically distribute naringin 10mg in a cillin bottle filled with 2mL of water, add the PHEMA solution into the cillin bottle, shake the cillin bottle, add a rotor of 2cm and zirconia beads of 0.4-0.6mm of 10g, stir for 5 hours at the speed of 300r/min under the water bath condition of 50 ℃. Sucking out the suspension, diluting the residual liquid medicine of the zirconium beads by pure water in several times, combining the liquid medicines and fixing the volume to 4mL to obtain the NA-NPs taking PHEMA as the carrier.

Wherein the particle size of the product NA-NPs is 181.3nm, the polydispersity index (PDI) is 0.287, and the potential is-31.0 mV.

In the invention, the pure water is diluted in several times so as to recover the residual liquid medicine of the grinding medium to the maximum extent, and the aim of the invention can be achieved without diluting the liquid medicine.

To further verify the excellent effects of the present invention, the inventors also conducted the following experiments:

experiment 1: morphological Observation of NA-NPs

The NA-NPs (1 mg/mL) prepared by the optimal process is diluted by 100 times, dropped on a clean silica gel sheet to be dried, fixed by using conductive adhesive, sprayed with gold for 6min under the conditions of negative pressure and current of 30mA, the voltage is increased to 30mV, and the nano system shape is observed by using a scanning electron microscope, as shown in figure 2. And as can be seen from FIG. 2, the NA-NPs are nanorods, and the length-diameter ratio is 40:1 to 60.

Experiment 2: NA-NPs drug loading study

Precisely weighing the NA-NPs prepared by the optimal process, respectively adding 1mL of chromatographic methanol, vortexing for 15min to fully dissolve the loaded drug, centrifuging at a high speed of 13000r/min for 30min, taking supernatant, adding the chromatographic methanol to dilute 20 times, loading the supernatant to detect the mass of the model drug, and calculating according to the following formula:

the drug loading (DLC%) = total mass of loaded drug/total mass of nano particles x 100%,

as a result, DLC% =48.36%

The calculation results show that the NA-NPs have higher drug loading capacity and are suitable for large-dose administration.

Experiment 3: NA-NPs Placement stability investigation

The NA-NPs (2.3 mg/mL) prepared by the optimal process is placed in a sealing mode at room temperature, samples are taken at preset time points of 0, 2, 4, 6, 8, 10, 12 and 14d respectively, the particle size of the NA-NPs is measured by a Malvern Nano-ZS particle sizer at room temperature, and the NA-NPs is measured in parallel for 3 times.

The results of the particle size change are shown in table 1 below:

TABLE 1

Time(d)	Size(nm)	PDI
			0	181.3±6.67	0.28±0.03
2	178.2±2.60	0.28±0.07
			4	173.4±3.05	0.27±0.03
6	177.7±0.6	0.26±0.05
			8	174.3±1.85	0.24±0.03
10	175.6±2.74	0.24±0.01
			14	172.1±4.73	0.24±0.02

As can be seen from the data in Table 1, the NA-NPs have good stability, no significant change in particle size after 14 days of storage, and a decrease in PDI.

Experiment 4: NA-NPs in vitro Release assay

The dialysis method is adopted to carry out an in vitro drug release test, and the specific operation is as follows:

in the experiment, a dialysis bag method is adopted to carry out in-vitro release experiment on the NA-NPs prepared by the optimal process, and meanwhile, the coarse powder of the naringin medicament sold in the market is used as a control. Taking 2mL (containing 4 mg) of the sample, placing the sample in a dialysis bag (with molecular weight cut-off of 8000-14000), placing the bag in 100mL of release medium, stirring at constant temperature of 37 ℃ and 100rpm, taking 1mL of release external liquid at each time point, measuring the content of the drug in the solution by HPLC, simultaneously supplementing the release medium with the same volume as the constant temperature, calculating the cumulative release degree, adopting purified water as the release medium, carrying out parallel experiments on 3 parts of the same sample, and showing the experimental result in figure 3.

NA-NPs are slowly released in PBS (pH 7.4) at a constant speed, and the 120-hour cumulative release rate reaches 80.72%; the naringin original drug is released quickly within 24h and slowly within 24-120 h, and the cumulative release rate within 120h reaches 49.21%. Therefore, after the naringin is prepared into the nanoparticles, the in vitro release rate is obviously improved, and the in vivo bioavailability of the naringin is favorably improved.

Experiment 5: screening of naringenin nanosuspension freeze-drying protective agent

1mL of NA-NPs was placed in a vial, and three groups were added in parallel in a mass ratio of 0.5% P188, 0.5% BSA, 0.5% whey protein, 0.5% mannitol, 0.5% glucose, 0.5% trehalose, 0.5% maltose, 0.5% sucrose, 0.5% galactose, 0.5% xylitol, and lyophilized as a lyoprotectant, and a blank control group was set. And (4) carrying out decompression freeze drying for 12h, manually shaking the freeze-dried powder with 1mL of deionized water for redissolving, observing the redissolving condition, and determining the particle size and PDI after freeze-drying and redissolving.

TABLE 2 NA-NPs different lyoprotectants screening

The particle size and the polydispersity index are used as evaluation indexes, and the results in table 2 show that 0.5% of whey protein is used as a good freeze-drying protective agent for NA-NPs, and freeze-dried powder is in a loose and porous fluffy state with uniform texture, so that the dosage of the freeze-drying protective agent is not increased. Has the advantages of long-term storage and convenient transportation.

Experiment 6: cough test in mice

50 Kunming mice are weighed, randomly divided into 5 groups according to body constitution, and subjected to adaptive breeding for 5 days, and then the model is made. Except for the blank group, other 4 groups of teachers smoke and smoke to make the mold. When smoking, putting the smoked product into a self-made smoking box, putting a moxibustion box at the upper right corner of the box, igniting 4 cigarettes respectively in the morning and afternoon at 1-10 d, putting the cigarettes into the moxibustion box, covering the moxibustion box with a cover, covering the moxibustion box with a towel to prevent smoke from overflowing, taking out the mouse after 30min, and keeping for 10d for 2 times/d; the concentration of the mixture is 0.4 mg/ml in 11 th, 14 th and 17 th morning ^-1 The LPS of (5) is dripped into the nose according to the proportion of 10 mu L to 10g ^-1 Calculating nasal drop amount by weight; the concentration of the mixture is 30 mg/mL in the afternoon between 11 th and 19 th ^-1 The thyroxine suspension was gavaged, and the gavage dose per mouse was 0.3mL. Mice were treated at night at concentrations of 1 × 10 at 12, 13, 15, 16, 18, 19d ^-4 mol·L ^-1 The capsaicin atomizing induced cough has each atomizing time of 3min,1 times d ^-1 . And (3) performing capsaicin induced cough stimulation experiment on the model-making mice by using a multifunctional induced cough asthma apparatus at the 20 th day of model making, and prompting the success of model making if the mice frequently sneeze, stretch forward the neck, twitch abdominal muscles, open the mouth and other characteristic positions appear and the cough frequency is about 10 times within 3 min.

The administration scheme is as follows: after the molding is successful, randomly dividing the raw materials into a blank group, a normal saline group, a positive medicine group, a naringin original medicine group and a naringin nano-particle group; the same amount of physiological saline is administered to the group of physiological saline for intragastric administration, and 1.2 mg/kg is administered to the positive group ^-1 The Montelukast sodium suspension (suspended in 0.5% CMC-Na) was intragastrically administered, and naringin crude drug 10 ml/kg ^-1 Naringin nano granule 13.33 mg.kg ^-1 The intragastric dose of each group is 0.1 ml.10 g ^-1 Mass, 1 times d ^-1 And 7d of continuous administration.

After the adaptive feeding is finished, measuring the body mass once at 1 st d in the molding period as a basic value, measuring the body mass once at every 10d in the smoking period, measuring the body mass once at every 5d after the smoking period, after the molding is finished, measuring the body mass again at 7 th d after the administration, recording experimental data, and observing the body mass change of each group of mice as shown in figure 4. In the experimental process, the blank control group mice have normal behaviors, smooth and bright hair and sensitive response. With the increase of smoking days, the mice of each model group have the symptoms of mental depression, slow movement, brown hair, easy shedding, heavy breath and the like. During the modeling period, the blank group weight was not affected, and gradually increased and leveled off. Compared with the blank group, the body weight of the mice of the model group and the mice of the administration group are slightly reduced, the growth speed begins to slow down, and the difference has very significant statistical significance (P is less than 0.001). During the administration period, the body weight of the mice in the model group is slowly increased compared with that in the blank group, and the difference has statistical significance (P is less than 0.01); compared with the model group, the weight of the mice of each administration group gradually returns to normal, and the difference has statistical significance (P is less than 0.05). The results show that the weight of the mice is obviously reduced after the model is made, and the naringin can improve the mental activity state of the mice and effectively treat cough after infection.

* Denotes P ≦ 0.05 compared to the blank group and ≦ 0.01 compared to the blank group.

Mouse cough sensitivity assay experiment: collecting 30.54mg/L capsaicin as cough inducing liquid in the morning, adding atomizer of multifunctional cough inducing and asthma inducing instrument, atomizing for 3min, observing for 2min after atomization is stopped, and recording cough time and cough frequency of mouse within 5 min.

TABLE 3 cough sensitivity in mice

Anti-inflammatory experiments in mice:

collecting peripheral whole blood of each group of mice by eyeball hemospasia, placing collected whole blood sample into low temperature high speed refrigerated centrifuge, setting centrifugation conditions at 4 deg.C and 3000 r.min ^-1 Centrifuging for 20min, collecting supernatant as mouse serum, and freezing at-20 deg.C.The level contents of IL-6 and CRP (C-reactive-protein) lung inflammatory factors in the serum of each group of mice are determined by adopting an ELISA method, and the operation process and the method are carried out strictly according to the instruction of a kit.

TABLE 4 serum IL-6, CRP level content in mice

The result shows that the content of proinflammatory cytokines IL-6 and CRP in the mice in the normal saline group is obviously increased (P is less than 0.05). Compared with the model group, the contents of IL-6 and CRP in the positive medicine group and the naringin nanoparticle group are both obviously reduced (P is less than 0.05).

In addition, comparing the observation results of the lung tissue slices of each group (fig. 5), the lung tissue of the mice in the normal group can show that the mucous membranes of each bronchus and alveolus are intact, the structure is normal, cilia are orderly arranged, inflammatory exudates are not seen in the blood vessels and alveolus of the lung tissue, and congestion is not obvious. The mouse lung tissue of the model group has incomplete bronchus and pulmonary alveolar mucous membrane, so that part of epithelial cells fall off in a tube cavity, cilia are arranged unevenly, inflammatory cell infiltration and congestion can be seen in the tube wall, the smooth muscle of the bronchus is thickened, and the pulmonary alveolar structure is disordered. Compared with the model group, the lung tissues of mice in the positive drug group and the naringin group are reduced in pathological changes and epithelial exfoliation is reduced. Inflammatory cell infiltration in the vessel wall is reduced, bronchial smooth muscle is thinned, and the alveolar structure is still intact.

In conclusion, the NA-NPs prepared by the method disclosed by the invention can effectively inhibit cough after infection, can reduce cough frequency and achieve the effects of relieving cough and resisting inflammation.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (5)

1. A method for preparing naringin nanoparticles by using polyhydroxyethyl methacrylate as a carrier is characterized in that the naringin nanoparticles are prepared by using PHEMA as a carrier to encapsulate naringin; the method specifically comprises the following steps:

(1) Respectively dissolving PHEMA and naringin in water to obtain a water phase I and a water phase II;

(2) Uniformly mixing the water phase I and the water phase II under an ultrasonic condition, and grinding in a water bath to obtain an initial solution;

(3) Diluting the initial solution to a constant volume to obtain the naringin nanoparticle solution;

in the step (1), the adding mass ratio of naringin to PHEMA is 2:1, and the concentration of the naringin in the naringin nano-particles is 2.2-2.7 mg/mL;

in the step (2), the water bath grinding temperature is 25-70 ℃, and the grinding time is 1.0-7.0 h; the water bath grinding medium is zirconia beads with the grain diameter of 0.4-0.6mm, and the grinding speed is 250-400 r/min; the ultrasonic power is 100w, and the ultrasonic temperature is 50 ℃.

2. The method for preparing the naringin nanoparticles by using the polyhydroxyethyl methacrylate as the carrier according to claim 1, which is characterized by further comprising freeze drying; the method comprises the following specific operations: adding whey protein with the mass concentration of 0.5% into the prepared naringin nanoparticle solution, and freezing; wherein the freeze-drying time is 12h, and the freeze-drying pressure is 0.12mbar under vacuum.

3. A naringin nanoparticle prepared by the method of claim 1 or 2, wherein the naringin nanoparticle is prepared by coating a hydrophobic drug naringin with PHEMA as a carrier; wherein the PHEMA is prepared from a polymer of 2-hydroxyethyl methacrylate monomer units.

4. Use of the naringin nanoparticles prepared by the method of claim 1 or 2 or the naringin nanoparticles of claim 3 in the preparation of a pharmaceutical formulation.

5. The use of claim 4, further comprising: application of naringin nanoparticle in preparing antitussive and antiinflammatory medicine is provided.

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