CN114469898A

CN114469898A - Preparation method of nanocapsule for improving oral bioavailability of ginkgetin aglycone

Info

Publication number: CN114469898A
Application number: CN202210071508.5A
Authority: CN
Inventors: 陆苑; 何艳; 张帅; 宋忠军; 刘�文; 李勇军; 李琴
Original assignee: Affiliated Hospital of Guizhou Medical University
Current assignee: Affiliated Hospital of Guizhou Medical University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-05-13
Anticipated expiration: 2042-01-21
Also published as: CN114469898B

Abstract

The invention discloses a preparation method of a nanocapsule for improving oral bioavailability of ginkgetin aglycone, which comprises the following steps: s1, accurately weighing PLGA-PEG, lecithin and castor oil, adding ethanol, acetonitrile, acetone and GA, and vortex mixing until the organic phase is completely dissolved; s2, accurately weighing Tween80 and Chen' S water to prepare a water phase; and S3, uniformly stirring the water phase on a magnetic stirrer at the rotating speed of 1500rpm, dropwise adding the organic phase, continuously stirring for more than 4 hours to completely volatilize the organic solution, and performing constant volume to obtain polymer Nanocapsules (NCs) wrapping the active ingredient. The preparation method of the nanocapsule for improving the oral bioavailability of the ginkgetin aglycone has the advantages of high drug loading, capability of improving the stability of GA in an aqueous solution and long circulation in vivo, and finally realizes the remarkable improvement of the oral bioavailability of quercetin, kaempferol and isorhamnetin in the GA.

Description

Preparation method of nanocapsule for improving oral bioavailability of ginkgetin aglycone

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of a nanocapsule for improving oral bioavailability of ginkgetin aglycone.

Background

Ginkgo flavone aglycone (GA) is a novel Ginkgo extract, mainly comprises 3 flavone aglycones with the following chemical structures, namely quercetin (46.61%), kaempferol (44.23%) and isorhamnetin (4.77%), and total flavone aglycone is 95.61%.

The research reports show that the potency of the flavonoid aglycone for eliminating the free radicals of the human body is 7 times that of the flavonoid glycoside. Quercetin, kaempferol and isorhamnetin in GA have wide pharmacological activity, such as resisting tumor, protecting heart and cerebral vessels, and can be widely used for preventing and treating senile dementia, coronary heart disease, hypertension, angina pectoris, cerebral hypofunction and atherosclerosis. However, GA has poor drug properties, and the reasons for this are poor water solubility, poor stability (easily oxidized polyhydroxy structure) and poor pharmacokinetic properties in vivo (low oral bioavailability and extremely short half-life) of quercetin, kaempferol and isorhamnetin, which are important factors limiting the deep development and utilization of GA.

Quercetin, kaempferol and isorhamnetin are all polyhydroxy structures, have poor chemical stability, are very easy to be oxidized, and are almost insoluble in water (all are less than 1 mu g/mL). The three have obvious first pass effect, can be rapidly metabolized by drug metabolizing enzymes (particularly CYP3A4) in the small intestine in the process of small intestine absorption, and can be rapidly metabolized by liver drug metabolizing enzymes after being absorbed into the liver, and finally the biological half-life period is extremely short. In addition, the three are substrates of the small intestine P-gp, and the oral absorption process is easily influenced by various factors, so that the bioavailability is extremely low. Therefore, there is a need to develop a high-end product of ginkgo biloba flavonoid aglycone to improve GA stability, oral bioavailability and prolong the half-life in vivo.

Disclosure of Invention

The invention aims to provide a preparation method of nanocapsules for improving oral bioavailability of ginkgetin aglycone, which has high drug loading, can improve stability of GA in aqueous solution and can realize long circulation in vivo, and finally realizes remarkable improvement of oral bioavailability of quercetin, kaempferol and isorhamnetin in GA.

In order to realize the purpose, the invention provides a preparation method of a nanocapsule for improving the oral bioavailability of ginkgetin aglycone, which comprises the following steps:

s1, accurately weighing PLGA, lecithin and castor oil, sequentially adding ethanol, acetonitrile, acetone and GA, and vortex mixing until the organic phase is completely dissolved;

s2, accurately weighing Tween80 and Chen' S water to prepare a water phase;

and S3, uniformly stirring the water phase on a magnetic stirrer at the rotating speed of 1500rpm, dropwise adding the organic phase, continuously stirring for more than 4 hours to completely volatilize the organic solution, and performing constant volume to obtain polymer Nanocapsules (NCs) wrapping the active ingredient.

Preferably, PLGA-PEG 12.5mg, lecithin 12.5mg, castor oil 75mg are weighed out accurately, and added with ethanol 1mL, acetonitrile 0.5mL, acetone 1.5mL, GA3mg, and mixed by vortexing until all the organic phase is dissolved. Accurately weighing 8010 mg of Tween and 5mL of Chen's water to prepare a water phase. And uniformly stirring the water phase on a magnetic stirrer at the rotating speed of 1500rpm, dropwise adding the organic phase, continuously stirring for more than 4 hours to completely volatilize the organic solution, and carrying out constant volume to 5mL to obtain polymer Nanocapsules (NCs) wrapping the active ingredient.

Therefore, the nanocapsule preparation method for improving the oral bioavailability of the ginkgetin aglycone is adopted, the nanocapsule prepared by adopting a nano precipitation method and taking PLGA-PEG as a capsule shell and castor oil as a capsule core has the advantages of proper size and potential, stable physicochemical property, high drug loading, capability of improving the stability of GA in an aqueous solution and long circulation in vivo, and finally realizes the remarkable improvement of the oral bioavailability of quercetin, kaempferol and isorhamnetin in GA, so that the improvement of the drug concentration of a target site is realized, and the pharmacological effect of GA can be better exerted.

Polymer Nanocapsules (NCs) are composed of an oil core and a polymer capsule shell wrapped around the periphery. The polymer nanocapsule has the advantages of avoiding light and oxygen, and improving the chemical stability of the medicine; secondly, the drug loading capacity is high, and a drug storage is formed in the oil core by the fat-soluble drug; the purpose of long circulation can be achieved by adding polyethylene glycol (PEG); fourthly, the contact between the medicine and the enzyme or the transporter is reduced, the biological half-life period is prolonged, and the absorption efficiency is improved; the EPR effect of the tumor is utilized, so that the targeting property of the medicine for resisting the tumor can be improved.

Compared with GA dispersible tablet, dripping pill, and microcapsule, the drug delivery system can improve poor solubility of GA, improve GA absorption degree (reduce GA metabolism and P-gp influence in small intestine), and prolong GA half-life period. Therefore, the polymer nanocapsule adopted by the invention can effectively solve the problems of extremely poor water solubility, poor stability and poor in-vivo pharmacokinetic property of GA under the premise of carrying high-dose GA.

Polylactic acid-glycolic acid copolymer (PLGA) is an amphiphilic polymer polymerized by lactic acid and glycolic acid, is a degradable functional polymer organic compound, and has good biocompatibility, no toxicity and good encapsulation and film forming performance. The release speed of the medicine is adjusted by adjusting the molecular weight and the molecular ratio of the PLGA, so that the in vivo process of the medicine is adjusted.

The invention adopts PLGA-PEG polymer material with long circulation function to prepare nanocapsules with high drug-loading capacity and in-vivo long circulation, thereby finally realizing the significant improvement of GA oral bioavailability, improving the drug concentration at the target site, better playing the pharmacological action of GA, and further developing the ginkgo leaf flavonoid aglycone product with extremely high application value.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a line graph of GA stability in simulated gastric fluid (A), simulated intestinal fluid (B), 1% Tween80 in PBS (C);

FIG. 2 is a graph of the cumulative release of GA, PLGA-PEG NCs and PLGA NCs in artificial gastric fluid;

FIG. 3 is a graph of the cumulative release of GA, PLGA-PEG NCs and PLGA NCs in PBS;

FIG. 4 is a graph of the relative amounts of quercetin, kaempferol and isorhamnetin after different periods of time of standing solutions of GA, PLGA NCs, and PLGA-PEG NCs;

FIG. 5 is a C-T plot of rats after intravenous injection of GA, PLGA NCs and PLGA-PEG NCs

FIG. 6 is a C-T plot of rats after oral administration of GA, PLGA NCs and PLGA-PEG NCs

Detailed Description

The technical solution of the present invention is further illustrated by the accompanying drawings and examples.

Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

Preparation of GA-NCs

(1) Reagent and apparatus

Ginkgo flavone Aglycone (GA) is offered by the assistant professor of Yoghover in the Biochemical engineering center of Guizhou province; quercetin (batch number: 1000881-201610, 99.1%), kaempferide (batch number: 110861-201812, 93.8%), isorhamnetin (batch number: 110860-201611, 99.1%) were purchased from China food and drug assay research; castor oil (Z23J9Y64268) was purchased from genealogical bio; soybean lecithin (pharmaceutical adjuvant for oral administration, Jiang Yao Standard F10354702, batch No. 180701) was purchased from Jiangsu Mannesmann Biotech Co., Ltd; PLGA_10k(Mw 10k,LA:GA＝50:50)，PLGA_10k-PEG_2k(PLGA Mw 10k, LA: GA 50:50) from Sienna Rexi Biotechnology Ltd, PD-10 desalting chromatography column (Sephadex G-25medium) from GE USA; 95% ethanol (AR, chemical Co., Ltd., national drug group), acetone (AR, Zunyi high-class chemical and Master chemical Co., Ltd.), acetonitrile (chromatographically pure, Merck, Germany), and water for experiments were all ultrapure water.

Laser particle size analyzer (NanoBrook 90Plus PALS, Brookhaven, USA), Agilent 1290 series ultra high performance liquid system, UPLC-TQS (Watts, USA)

Triple quadrupole tandem mass spectrometer), an electronic balance (RP5002K, university keen precision instruments ltd), an ultrapure water machine (SC082767, mithrawa waters processing unit ltd), a low temperature high speed centrifuge (Allegra 64R, Beckman Coulter, usa).

SD rats, male, with a body weight of (220- & lt250-) g, provided by Tianqin Biotechnology Ltd of Changsha, animal license number SCXK (Xiang) 2019- & lt0014.

(2) Test method

Accurately weighing 12.5mg of PLGA, 12.5mg of lecithin and 75mg of castor oil, sequentially adding 1mL of ethanol, 0.5mL of acetonitrile, 1.5mL of acetone and 3mg of GA, and vortex mixing until the organic phase is completely dissolved. Accurately weighing 8010 mg of Tween and 5mL of Chen's water to prepare a water phase. And uniformly stirring the water phase on a magnetic stirrer at the rotating speed of 1500rpm, dropwise adding the organic phase, continuously stirring for more than 4 hours to completely volatilize the organic solution, and fixing the volume to 5 mL. Subsequently, the NCs particle diameter and PDI value were determined, and the results showed that the NCs particle diameter was 184.69. + -. 4.4nm, PDI was 0.14. + -. 0.04, and the potential was-25.2. + -. 1.87 mV.

Taking 50 mu L of GA-NCs, adding 750 mu L of acetonitrile, continuously adding water to 1mL, centrifuging at 10000rpm for 10min, measuring the peak areas of quercetin, kaempferol and isorhamnetin by HPLC at the wavelength of 373nm, and quantitatively calculating each component to obtain the total drug amount (the drug amount encapsulated and unencapsulated in NCs).

The GA-NCs were separated and purified by passing 2mL of GA-NCs solution (containing GA-NCs carrying GA and GA not carrying GA) through a PD-10 column, discarding 2mL of eluate flowing down from the column, eluting with 3.5mL of water and collecting the eluate (the eluate is turbid and clarified), and diluting the collected eluate with water to 5 mL. Taking 50 mu L of purified GA-NCs solution, adding 750 mu L of acetonitrile, then adding water to a constant volume of 1mL, centrifuging at 10000rpm for 10min, measuring peak areas of various components by HPLC, and calculating the amount of quercetin, kaempferide and isorhamnetin, namely the GA encapsulation amount.

The Encapsulation Efficiency (%) and the Loading Efficiency (%) were calculated according to equations 1 and 2, and the results are shown in Table 1. As a result, at a GA dosage of 3mg, the encapsulation efficiency and the drug loading rate of GA-NCs using PLGA-PEG and PLGA as materials were not significantly different, indicating that PEG had no significant effect on EE (%) and LE (%). The GA nanocapsule prepared by the method has the drug loading of 29 percent.

TABLE 1 encapsulation efficiency and drug loading of GA-NCs

(3) Physical stability of GA-NCs in Release media

The particle size and PDI value were measured in triplicate for each group by taking 3.8mL each of artificial gastric juice, artificial intestinal juice, and 10% bovine serum PBS, adding 200. mu.L of GA-NCs (GA3mg, PLGA-PEG) and measuring the particle size and PDI value at different time points, and the results are shown in Table 2. As a result, the GA-NCs were stable in particle size and PDI value in the three release media, and it was presumed that the GA-NCs were excellent in physical stability during oral absorption.

TABLE 2 particle size and PDI values of GA-NCs in gastric juice, intestinal juice, 10% bovine serum PBS

Second, GA-NCs in vitro Release test

(1) Chemical stability of GA in Release media

Preparing 1mg/mL GA DMSO solution, respectively adding 125 μ L into gastric juice, intestinal juice and PBS containing 1% Tween80, diluting to 10mL, placing in 37 deg.C water bath constant temperature oscillator, taking 1mL for content determination at 0h, 12h, 24h and 48h, and making 3 groups of parallel samples for each medium.

As a result, GA was found to undergo color changes to various degrees in different release media at 12h, with the greatest change in the artificial intestinal fluid (the darkest yellow), followed by PBS and gastric juice. The degradation profile of the components of GA in a release medium is shown in FIG. 1, which illustrates that the polyhydroxy structure of GA is poorly stable in solution, is easily oxidized, and is more unstable especially under alkaline conditions.

(2) GA stability test after addition of Vc

In order to obtain the in vitro release curve of GA-NCs, firstly, the stability of quercetin, kaempferol and isorhamnetin needs to be ensured, so that Vc is selected to be added into the solution as an antioxidant. Different concentrations of Vc (0.5% and 1%) were added to the three release media, and after ultrasonic filtration gastric juice and PBS containing 0.5% and 1% Vc were prepared and GA stability was examined.

As a result, quercetin, kaempferide and isorhamnetin are stable in 0.5% and 1% Vc-containing gastric juice and PBS, and the components are not obviously changed in 48 hours, so that the 0.5% Vc-containing gastric juice and PBS are selected as release media to carry out subsequent GA-NCs in-vitro release experiments.

(3) GA-NCs in vitro release Profile

Soaking a 10cm dialysis bag in purified water at 80 ℃ for 30min, respectively taking 0.5mL of GA DMSO solution, PLGA NCs and PLGA-PEG NCs concentrated solution (containing about 1.5mg/mL of total GA), adding into the dialysis bag, sealing, putting the dialysis bag into 40mL of release medium preheated at 37 ℃, preparing 3 groups of parallel samples by the same method, respectively taking 1mL of each sample at 0h, 0.5h, 1h, 2h, 4h, 6h, 12h, 24h, 36h, 48h, 3d, 4d, 5d, 6d and 7d, supplementing the release medium with the same volume to each point, measuring the content of each component by HPLC, and drawing a GA in-vitro cumulative release curve. The results of the cumulative release profiles of the components in the artificial gastric juice are shown in FIG. 2, and the results of the cumulative release profiles of the components in PBS are shown in FIG. 3.

The GA release speed is faster than that of each component in PLGA NCs and PLGA-PEG NCs, which shows that the PLGA NCs and the PLGA-PEG NCs have good slow release effect on GA. The release rates of the components in the PLGA NCs and the PLGA-PEG NCs in gastric juice and PBS have no obvious difference, and the release medium does not influence the dissolution rates of the quercetin, the kaempferide and the isorhamnetin. Compared with PLGA-PEG NCs, the PLGA NCs have no obvious difference in the release degree of quercetin, kaempferide and isorhamnetin, which indicates that PEG does not influence the release of each component. According to the peak sequence of each component in a C18 chromatographic column, the fat-soluble sequence of the three components is quercetin < kaempferide < isorhamnetin, and the release rate of each component in NCs in gastric juice and PBS is quercetin > kaempferide > isorhamnetin. The isorhamnetin has the maximum affinity with the fat-soluble castor oil core, so the release speed is the slowest; the affinity of the quercetin and the castor oil core is small, so that the release speed is fastest.

Stability test of GA-NCs

The GA liquid medicine and the purified PLGA NCs and PLGA-PEG NCs solutions are respectively put into a glass bottle, stored at 4 ℃ and sampled for 500 mu L at 0d, 7d, 14d, 21d and 30 d. Wherein 300 mu L of the mixture is put into a centrifugal tube of 1.5mL, freeze-dried and stored at the temperature of minus 20 ℃, after all samples are completely collected, 100 mu L of DMSO, 350 mu L of acetonitrile and 50 mu L of water are added, vortex mixing is carried out uniformly, centrifugation is carried out at 10000r/min for 10min, and the contents of quercetin, kaempferol and isorhamnetin are measured by HPLC. The remaining PLGA NCs and PLGA-PEG NCs solutions were used to determine the particles, PDI values and Zeta potential.

GA. The appearance properties of the PLGA NCs and the PLGA-PEG NCs after being placed for different time can be known, the PLGA NCs and the PLGA-PEG NCs have no obvious change in the placement time of 30d, and the phenomena of aggregation, precipitation, emulsion breaking and the like do not occur. The particle size and PDI values of PLGA NCs and PLGA-PEG NCs after being placed for different time have no obvious change of Zeta potential (Table 3).

TABLE 3 particle diameter, PDI value, Zeta potential of PLGA NCs and PLGA-PEG NCs at different time points

Calculating the residual content of quercetin, kaempferol and isorhamnetin after placing GA, PLGA NCs and PLGA-PEG NCs solution for different time with the content of each component of 0d as 100% (figure 4). The three components in the GA medicament solution are all rapidly degraded, the residual contents of the quercetin, the kaempferol and the isorhamnetin are respectively 18.88 +/-8.65%, 23.61 +/-14.63% and 30.69 +/-2.70% after 14 days, and the residual contents of the three components in the PLGA NCs solution and the PLGA-PEG NCs solution are still more than 90%; at 30 days, the quercetin, kaempferol and isorhamnetin in the GA medicament solution disappear, and the residual contents of the three components in the PLGA NCs and PLGA-PEG NCs solution are still more than 80 percent. It is shown that under the condition of no special protection (such as filling inert gas, avoiding light, adding antioxidant, etc.), the polymers PLGA and PLGA-PEG can isolate light and oxygen, and can raise the chemical stability of quercetin, kaempferol and isorhamnetin. This also suggests that the stability of GA-NCs can be further improved by filling inert GAs, keeping out of the sun, adding antioxidants, etc. at later stage, making it meet the requirements of the commodity.

Fourthly, detecting the oral bioavailability of GA, PLGA-NCs-GA and PLGA-PEG-NCs-GA (1) the analysis conditions of quercetin, kaempferol and isorhamnetin in plasma

Liquid phase conditions: chromatography column Agilent Eclipse Plus C18(2.1 mm. times.50 mm,1.8 μm) column, Guard column Waters Van Guard BEH C18(2.1 mm. times.5 mm,1.7 μm), flow rate 0.25mL/min, column temperature 40 ℃, mobile phase 0.1% formic acid water (A) -0.1% formic acid acetonitrile (B), sample injection volume 1 μ L. The gradient of the mobile phase is 0-0.5min, 10% -10% B; 0.5-1.0min, 10% -30% B; 1.0-4.5min, 30% -90% B; 4.5-5.0min, 90% -90% B; 5.0-6.0min, 90% -10% B.

Mass spectrum conditions: electrospray ionization source (ESI); the capillary voltage is 1 kV; the ion source temperature is 150 ℃; the temperature of the solvent gas is 600 ℃; desolventizing gas N₂The flow rate is 1000L/h; reverse blowing N₂The flow rate is 150L/h; collision gas Ar with the flow rate of 0.15 mL/min; the mass spectrum data acquisition and processing software is a MassLynx V4.1 workstation, and the scanning mode is a multi-reactive ion monitoring mode (MRM). The ion pair information is as follows: quercetin [ M-H]^-301.0 → 151.0; kaempferol [ M-H]^-285.0 → 185.0; isorhamnetin [ M-H]^-315.1 → 300.1; puerarin (IS) [ M-H ]]^-415.0 → 295.0. The cone hole voltage of quercetin, kaempferol, isorhamnetin and puerarin is respectively 25V, 30V and 30V, and the collision voltage is respectively 20eV, 30 eV, 20eV and 20 eV.

(2) Test method

Plasma sample processing method

Melting 100 μ L plasma at room temperature, adding 1% formic acid water solution 10 μ L, vortex mixing, adding 20 μ L100 ng/mL puerarin (IS), vortex mixing, adding 400 μ L acetonitrile, vortex mixing for 5min, ultrasonic treating for 10min, centrifuging at 14000 r/min for 10min, collecting supernatant, centrifuging at 37 deg.C and N₂And (5) drying. The residue was reconstituted with 200. mu.L of 50% aqueous methanol, centrifuged at 14000 r/min for 10min and the supernatant was analyzed by UPLC-MS/MS.

Rat grouping, administration method and treatment

And (3) intragastric administration group: 5.0mg of GA mixture was added with 0.5% CMC-Na to prepare a GA suspension of 0.5 mg/mL. Taking a proper amount of PLGA NCs and PEG-PLGA NCs, and preparing NCs solution with GA concentration of 0.5mg/mL by using normal saline.

Group for intravenous administration: and (3) putting 10.0mg of the GA mixture into a 10mL volumetric flask, adding 400 mu L of DMSO, uniformly mixing by vortex, adding 2mL of propylene glycol and ethanol respectively, adding physiological saline to scale, and uniformly mixing to obtain a GA solution (1.0 mg/mL). And preparing a proper amount of prepared PLGA NCs and PEG-PLGA NCs into NCs solution with GA concentration of 1.0mg/mL by using physiological saline.

36 healthy male SD rats were selected and divided into 6 groups (n ═ 6) and administered with GA, PLGA NCs and PLGA-PEG NCs by intravenous injection and intragastric administration, respectively, at a GA dose of 6.64mg/kg for each group. After administration, 0.3mL of blood is taken from orbital venous plexus of rats at 0.08h, 0.17h, 0.33h, 0.50h, 0.75h, 1h, 1.5h, 2h, 3h, 4h and 8h, and is placed in a heparinized EP tube, centrifuged at 5000 r/min for 10min, 100. mu.L of plasma is taken, 10. mu.L of 0.5% Vc aqueous solution is added, and the mixture is placed at-20 ℃ for storage until UPLC-MS/MS analysis.

After the quantitative determination of the samples, t of each group of quercetin, kaempferol and isorhamnetin is calculated by using the NCA model of the pharmacokinetic software WinNonLin 8.2(Phenix, Pharsight company, USA) software_1/2、AUC_0-t、Tmax、Cmax、AUC0-t、AUC_0-∞、V_dThe main pharmacokinetic parameters of/F, Cl/F, MRT and the like. The results are expressed as "mean. + -. standard deviation" (X. + -. SD), and statistical analysis was performed using Spss software, with a t-test for comparison between groups, P<0.05 means statistically significant.

(3) Oral bioavailability results analysis

The pharmacokinetic results for each group are shown in tables 4 and 5, and the plasma drug concentration versus time (C-T curves) are shown in FIGS. 5 and 6. The results show that oral administration of PLGA NCs and PLGA-PEG NCs can significantly improve the peak concentration Cmax and oral bioavailability of three components in GA, and no obvious absorption phase exists, which indicates that the absorption process is faster. Compared with GA group, Cmax of quercetin, kaempferol and isorhamnetin is increased by 7.91, 5.07 and 5.31 times after PLGA NCs are orally taken; after PLGA-PEG NCs are orally taken, Cmax of quercetin, kaempferol and isorhamnetin is increased by 6.53, 3.87 and 2.89 times. The oral bioavailability of quercetin, kaempferol and isorhamnetin in GA is low, and is respectively 11.05%, 6.21% and 2.19%. Compared with GA, after PLGA NCs are prepared by GA, the oral bioavailability of quercetin, kaempferol and isorhamnetin is respectively improved by 1.96, 1.44 and 2.32 times; after PLGA-PEG NCs are prepared from GA, the oral bioavailability of quercetin, kaempferol and isorhamnetin is respectively improved by 2.53 times, 1.87 times and 1.81 times. Because the oral administration dose is extremely low (6.64mg/kg), the oral pharmacokinetic parameters are not accurate enough, and the intravenous administration can more simply and directly embody the elimination process of the three components in the GA. As can be seen from the C-T plot of FIG. 5, the components of the PLGA-PEG NCs group metabolically eliminated slower than the PLGA-PEG NCs group, and both were slower than GA. The clearance rates (CL) of quercetin, kaempferol and isorhamnetin of PLGA NCs are respectively 0.02, 0.406 and 0.528 times of that of GA; the CL of PLGA-PEG NCs for quercetin, kaempferol, isorhamnetin is 0.014, 0.94 and 0.75 times that of GA. The results show that PLGA and PLGA-PEG can obviously improve the oral absorption process of GA, can reduce the first pass effect and the in-vivo elimination speed of GA, and improve the systemic circulation time of GA, and the action effect of PLGA-PEG is superior to that of PLGA.

TABLE 4 pharmacokinetic parameters after intravenous injection of GA, PLGA NCs and PLGA-PEG NCs in rats

Remarking: ", indicates P <0.05, P <0.01, P <0.001, respectively, compared to the GA group; "#", "# #", "# # # # # # #" indicates that P <0.05, P <0.01, P <0.001, respectively, in the PLGA-PEG-NCs-GA and PLGA-NCs-GA groups.

TABLE 5 pharmacokinetic parameters after intragastric administration of GA, PLGA NCs and PLGA-PEG NCs in rats

Therefore, the preparation method of the nanocapsule for improving the oral bioavailability of the ginkgetin aglycone has high drug loading, can improve the stability of GA in an aqueous solution and can realize long circulation in vivo, and finally realizes the obvious improvement of the oral bioavailability of quercetin, kaempferol and isorhamnetin in GA.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims

1. A preparation method of nanocapsules for improving oral bioavailability of ginkgetin aglycone is characterized by comprising the following steps:

s2, accurately weighing Tween80 and Chen' S water to prepare a water phase;

and S3, uniformly stirring the water phase on a magnetic stirrer, dropwise adding the organic phase, continuously stirring to completely volatilize the organic solution, and performing constant volume to obtain polymer Nanocapsules (NCs) wrapping the active ingredient.

2. The method of claim 1, wherein the nanocapsule is further configured to enhance oral bioavailability of ginkgetin aglycone by: PLGA-PEG 12.5mg, lecithin 12.5mg, castor oil 75mg were weighed out accurately, and 1mL of ethanol, 0.5mL of acetonitrile, 1.5mL of acetone, and 3mg of GA were added and vortex mixed until the organic phase was completely dissolved. Accurately weighing 8010 mg of Tween and 5mL of Chen's water to prepare a water phase. And uniformly stirring the water phase on a magnetic stirrer at the rotating speed of 1500rpm, dropwise adding the organic phase, continuously stirring for 4 hours to completely volatilize the organic solution, and carrying out constant volume to 5mL to obtain polymer Nanocapsules (NCs) wrapping the active ingredient.