CN114191391B - Preparation method of myricetin-loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting formation of osteoclast - Google Patents
Preparation method of myricetin-loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting formation of osteoclast Download PDFInfo
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Abstract
The invention provides a preparation method of a myricetin-loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting formation of osteoclasts, belonging to the field of pharmaceutical preparations. Aims to provide a myricetin loaded nano micelle with improved drug loading capacity, so as to improve the solubility and oral bioavailability of myricetin and reduce the bone loss induced by ovariectomy by inhibiting the formation of osteoclasts. The key point of the technical scheme is that the preparation method of the myricetin-loaded nano micelle (hereinafter abbreviated as myricetin micelle) comprises the following steps: cheng Yangmei element bone-targeted micelles were prepared from bone-targeted material AL-P (LLA-CL) -PEG-P (LLA-CL) and myricetin. The prepared myricetin micelle has smaller particle size and higher encapsulation efficiency, enhances the in-vitro solubility and in-vivo bioavailability, remarkably improves the ALP activity of osteoblasts, and enhances the therapeutic effect of resisting castration rat osteoporosis. Provides preliminary information for clinical application of myricetin and other similar water insoluble compounds.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of a myricetin loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting formation of osteoclasts.
Background
Myricetin (3, 5,7,3',4',5' -hexahydroxyflavone) is a polyhydroxy flavonol compound, exists in various natural plants, such as cortex Myricae Rubrae, leaf, tea, onion, and the like, and has resource advantages in research and application of medicines. Research shows that myricetin has wide pharmacological activity, including lowering blood sugar, resisting oxidation, protecting liver, diminishing inflammation, etc. Research has also shown that myricetin has the effects of promoting osteogenesis and inhibiting osteogenesis. At present, myricetin is approved by FDA as an additive to be applied to medicines, foods and health care products, and has great development and application prospects.
Myricetin is yellow needle-like crystal, is easily dissolved in organic solvents such as methanol, ethanol and acetonitrile, is insoluble in water, and contains a plurality of hydroxyl groups in the myricetin structure, so that the myricetin is easy to oxidize and photolyze in the air, is relatively stable under acidic conditions, is relatively poor in stability in neutral and alkaline environments, and is easy to damage. Myricetin is insoluble in water, resulting in poor absorption in the gastrointestinal tract and low bioavailability. Man Na et al have shown that by performing pharmacokinetic studies on a 300mg/kg myricetin suspension by intragastric administration in rats, the maximum absorption concentration in the body of the rats is 0.57g/L, the area under the drug curve is 5.83 mu g/mL, and the oral bioavailability is very low, which limits the development and application space of myricetin to a great extent, so that it is necessary to find a suitable carrier to improve the water solubility and stability in water and bioavailability. At present, various preparation researches such as microemulsion, nanoliposome, micelle, inclusion compound and the like of myricetin have been advanced to some extent. The preparation can greatly improve the water solubility and stability of myricetin, and prolong the residence time of myricetin in vivo so as to improve the bioavailability of myricetin.
Polymeric micelles are ordered aggregates of self-assembled core/shell structures in water from amphiphilic block copolymers. The hydrophobic end of the block copolymer can encapsulate poorly soluble drugs, while the hydrophilic end increases the solubility of the system in water. In addition, the block copolymer is modified to achieve the aim of targeted drug delivery. Polyethylene glycol (PEG), a water-soluble, biocompatible and non-immunogenic nonionic polymer, is widely used in the field of drug delivery system research. The active targeting drug carrier can be constructed by using the modifiable group on PEG as a bridging arm between the targeting ligand molecule and the drug (or drug carrier). The alendronate sodium (ALN) belongs to a third-generation bisphosphonate medicine, has strong bone affinity and strong affinity with hydroxyapatite, can be deposited on bones, inhibits the activity of osteoclasts, promotes the apoptosis of the osteoclasts, can effectively reduce the bone absorption and slow down the bone loss, improves the bone metabolism, has a simple structure, can be chemically modified, and is widely used as a bone targeting ligand in a drug delivery system. P (LLA-CL) is a polymer of poly-L-lactic acid (PLLA) and poly-epsilon-caprolactone (PCL) and is currently used in tissue engineering and drug delivery systems due to its high biocompatibility.
Osteoporosis is a systemic metabolic bone disease, characterized by reduced bone mass per unit volume of bone tissue, degenerative changes in bone tissue microstructure, reduced bone strength, increased fragility of bone, and susceptibility to fracture, severely affecting the quality of life of patients, and being classified by world health organization as one of three major geriatric diseases. Currently, the drugs used clinically for treating osteoporosis are mainly classified into two types, bone resorption promoters (e.g., estrogens) and bone formation promoters (e.g., statins). However, the medicine has the defects of insignificant effect, proper alleviation of osteoporosis symptoms, large dosage, certain toxic and side effects on other non-targeted organs and the like. Therefore, the search for a natural, safe and effective drug with better targeting effect has important significance. The research proves that the flavonoid small molecular compound has a similar chemical structure with estrogen, can effectively restore the bone mass of the osteoporosis animal, and is more suitable for long-term use than the traditional therapeutic medicine.
Disclosure of Invention
Aiming at the problems of poor water solubility, low in-vitro release degree, low bioavailability and the like of myricetin, the invention provides a preparation method of a myricetin loaded nano micelle for reducing the bone loss induced by ovariectomy by inhibiting the formation of osteoclasts.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the preparation method of the myricetin-loaded nano micelle for reducing the ovariectomy-induced bone loss by inhibiting the formation of osteoclasts comprises a bone targeting material and myricetin; the bone targeting material is synthesized by chemical reaction of P (LLA-CL) 5/5 (4500) -PEG (2000) -P (LLA-CL) 5/5 (4500) and alendronate sodium. Wherein the mass ratio of the bone targeting material to the myricetin is 10:1 to 10:4.
preferably, the method for preparing the myricetin-loaded nano-micelle for reducing ovariectomy-induced bone loss by inhibiting the formation of osteoclasts, wherein the mass ratio of the bone targeting material to the myricetin is 10:2.
preferably, the myricetin purity is 98%.
A method for preparing a myricetin-loaded nanomicelle that reduces ovariectomy-induced bone loss by inhibiting the formation of osteoclasts, comprising the steps of:
(1) Dissolving the dried AL-P (LLA-CL) -PEG-P (LLA-CL) in ultrapure water at a concentration of 10mg/mL;
(2) Dissolving Myricetin (MY) in absolute ethanol with the concentration of 10mg/mL;
(3) Taking a certain amount of prepared MY solution according to a proportion, slowly dripping 20mL of AL-P (LLA-CL) -PEG-P (LLA-CL) water solution with the concentration of 10mg/mL under the ultrasonic condition, and uniformly mixing by ultrasonic;
(4) Removing absolute ethyl alcohol by decompression rotary evaporation, and filtering by using a 0.45 mu m microporous filter membrane to obtain the AL-P (LLA-CL) -PEG-P (LLA-CL) -MY drug-loaded micelle.
The ultrasonic power in the step (3) is preferably 200W, and the ultrasonic time is preferably 20min.
The invention has the beneficial effects that:
the study first devised a method for preparing myricetin-loaded nanomicelle that reduces ovariectomy-induced bone loss by inhibiting the formation of osteoclasts. The micelle has narrower particle size distribution, negative zeta potential and higher encapsulation efficiency. In vitro release and in vivo pharmacokinetics studies in rats show that the solubility of myricetin can be obviously improved after the myricetin is encapsulated in micelles, and the oral bioavailability of myricetin is improved by 3.54 times compared with that of free myricetin. Meanwhile, the myricetin bone targeting micelle has better in-vivo and in-vitro bone targeting, can obviously improve ALP activity of osteoblasts, and also has better treatment effect in the treatment of castration rat osteoporosis. In general, the myricetin bone targeting micelle can remarkably improve the water solubility and bioavailability of myricetin, is a potential carrier for treating castration rat osteoporosis, and provides information for possible clinical application of myricetin.
Drawings
FIG. 1 (a, b) is a thin layer chromatogram of a first step (a) and a second step (b) of synthesizing a bone targeting material; FIG. 1 (c, d) shows nuclear magnetic resonance phosphorus spectrum (c) and hydrogen spectrum (d) of the product after separation and purification; fig. 1 (e, f) shows the particle size distribution (e) of myricetin-targeted micelles and the scanning result (f) by a transmission electron microscope.
FIG. 2 of the present invention is a graph showing in vitro release profile comparison of free myricetin and myricetin micelles in different media: a) In vitro release profile of free myricetin and myricetin micelles in hydrochloric acid solution (pH 1.2); b) In vitro release profile of free myricetin and myricetin micelles in PBS (pH 6.8); c) In vitro release profile of free myricetin and myricetin micelles in PBS (Ph 7.4) and d) binding rate of free myricetin and myricetin micelles to hydroxyapatite (mean±sd, n=3).
FIG. 3a shows the plasma concentration profile of free myricetin and myricetin micelles (mean+ -SD, n=6); fig. 3 (b-e) is imaging results of rats orally administered targeted micelles containing fluorescent dye for 4,8, 12 and 16 hours.
FIG. 4 (a, b) shows the effect of different concentrations of free myricetin and myricetin micelles on osteoblast proliferation rate; FIG. 4 (c, d) is the effect of different concentrations of free myricetin and myricetin micelles on osteoblast ALP viability; FIG. 4e shows the effect of varying concentrations of free myricetin and myricetin micelles on the calcium ion content of osteogenesis.
FIG. 5 of the present invention shows the "calcium nodule" resulting from the alizarin red staining of each group of rats. ( In fig. 5, group a complete medium group (control group); group b 1. Mu.g/L myricetin drug substance group; d group 10 mug/L myricetin bulk drug group; d group 50 mug/L myricetin bulk drug group; group e, 100 mug/L myricetin bulk drug group; f group 1. Mu.g/L myricetin micelle group; group g 10. Mu.g/L myricetin micelle group; group h, 50 μg/L Myricetin micelle group; group i 100. Mu.g/L Myricetin micelle group. )
FIG. 6 of the present invention shows BV/TV, tb.Th, tb.N, tb.Sp, SMI and BMD indices for each group of rats; CT three-dimensional reconstruction results of isolated femur of each group of rats. ( In fig. 6, a blank group; a model group B; a group of raw material medicines; d targeting micelle group. )
FIG. 7 shows the expression of TRAP, BALP, OCN, IL-1. Beta. Factor in plasma of rats of each group.
Figure 7 of the present invention shows the pathology analysis of bone trabeculae of rats in each group.
( In fig. 6-8, a blank; a model group B; a group of raw material medicines; d targeting micelle group. )
Detailed Description
Embodiments of the present invention will now be described in detail with reference to the following examples, which are only illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Synthesis of bone targeting material AL-P (LLA-CL) -PEG-P (LLA-CL):
the P (LLA-CL) 5/5 (4500) -PEG (2000) -P (LLA-CL) 5/5 (4500) compound was ultrasonically dissolved in DMF solution, and CDI (N, N' -carbonyldiimidazole) compound was added while stirring and reacted at room temperature for 5 hours. The detection of the relevant substances was carried out using methylene chloride-methanol (15:1, v/v) as developing reagent. After the completion of the first reaction, a mixed solution of alendronate sodium (AL) and tetrabutylammonium hydroxide (molar ratio=1:2; alendronate sodium, amount of trihydrate: 10mg,0.03mmol; tetrabutylammonium hydroxide aqueous solution: 38.9mg,0.06 mmol) was added to the reaction mixture while stirring, and the mixture was heated at 80℃for 5 hours. With dichloromethane: methanol=15:1 run plate monitors the progress of the reaction. After the reaction was completed, DMF was removed by rotary evaporator (70-80 ℃) and the remaining reaction solution was washed with a small amount of water, the supernatant was removed by centrifugation (excess alendronate sodium and tetrabutylammonium hydroxide were removed), the underlying white emulsion was dissolved in a suitable amount of dichloromethane, and purified by separation with a neutral Al2O3 column (eluent: dichloromethane: methanol=30-15:1, product amount: 18mg, yield: 15.9%). AL-P (LLA-CL) -PEG-P (LLA-CL) was characterized by nuclear magnetic resonance spectroscopy to demonstrate successful bonding.
Fig. 1 (a, b) shows thin layer chromatograms of the first and second step reactions. FIG. 1 (c, d) shows the nuclear magnetic resonance hydrogen spectrum and the phosphorus spectrum of the product after separation and purification. Analysis of the phosphorus spectrum revealed a single peak at-20 ppm, corresponding to phosphorus on ALN; in the hydrogen spectrum, the peaks at δ=1.0-2.5 ppm may be protons on- (CH 2) 3-, -OCCH 2-on CL and-CCH 3 on LLA, the peak at δ=3.7 ppm or so corresponds to the monomethoxy proton peak on PEG, the peak at δ=4.06 ppm or so may correspond to the proton on-CH 2 OOC-methylene in CL, and the peak at δ=5.3 ppm may be attributed to the proton on-cho-on LLA. The resulting product has characteristic peaks of ALN, PEG, PLLA and PCL at the same time, indicating successful synthesis of the copolymer AL-P (LLA-CL) -PEG-P (LLA-CL).
Example 2
Preparation of myricetin-loaded nanomicelles to reduce ovariectomy-induced bone loss by inhibiting the formation of osteoclasts:
the dried AL-P (LLA-CL) -PEG-P (LLA-CL) was dissolved in ultrapure water at a concentration of 10mg/mL. Myricetin (MY) was dissolved in absolute ethanol to prepare a solution with a concentration of 10mg/mL. 1, 2,3 and 4mL of prepared MY are respectively taken and slowly dripped into 20mL of AL-P (LLA-CL) -PEG-P (LLA-CL) water solution with the concentration of 10mg/mL under the ultrasonic condition, and the mixture is uniformly mixed by ultrasonic (the ultrasonic power is 200W and the ultrasonic time is 20 min). Removing absolute ethyl alcohol by reduced pressure rotary evaporation, and filtering by using a 0.45 mu m water-based filter membrane to obtain the AL-P (LLA-CL) -PEG-P (LLA-CL) -MY drug-loaded micelle. The particle size, potential and polydispersity of four groups of micelles are measured respectively, and the four groups of micelles are placed in a normal temperature environment for 7 days, and after 7 days, the particle size of the micelles is measured again, and finally a proper formulation prescription is selected. The results of each set of experiments are shown in table 1.
Table 1 prescription screening results
Morphology observation: taking the myricetin micelle prepared in the example 2, diluting with water, dripping the myricetin micelle on a copper mesh coated with a supporting film, airing, dripping 2% phosphotungstic acid solution for dyeing, naturally volatilizing, placing the copper mesh under a transmission electron microscope for observation, diluting the myricetin micelle with water, and forming a sphere under the transmission electron microscope, wherein the myricetin micelle is not adhered to each other and is uniformly dispersed. The particle size of myricetin micelle and the transmission electron microscope result are shown in fig. 1 (e, f).
Determination of in vitro Release: the myricetin micelle and myricetin suspension of example 2 were separately taken and placed in a dialysis bag, and the two ends of the dialysis bag were tied up with cotton threads. The dialysis bags were then placed in conical flasks with 100mL of release medium (pH 7.4, pH6.8PBS buffer and pH1.2 hydrochloric acid buffer), respectively. The conical flask was placed in a constant temperature shaking water bath (37 ℃,100 rpm/min) and 1mL of sample was taken in 1.5mL ep tubes at 0.083,0.25,0.5,0.75,1,1.5,2,3,4,6,8, 10, 12, 24, 36, 48, 60, 72h, respectively, while the same volume of fresh culture broth was added to maintain the sink state. The sample solution was centrifuged at 10000rpm for 10 minutes, 200. Mu.L of the supernatant was collected, and subjected to liquid phase detection to calculate the cumulative release rate of myricetin. The result shows that compared with myricetin, the myricetin micelle can be dissolved out more rapidly and completely, and the in vitro dissolution rate of myricetin can be effectively improved. The results are shown in FIG. 2 (a-c).
Example 3
Targeted research of myricetin micelle
Respectively adding nano-scale hydroxyapatite aqueous solution into two 2mL plastic centrifuge tubes, respectively adding 500uL myricetin bulk drug and preparation solution (1 mg/mL), carrying out ultrasonic treatment for 5min to uniformly mix, standing at room temperature, and detecting at different time points for 5min, 10min, 15min, 30min, 60min and 120 min. Centrifuging (10000 rpm,5 min) each tube, collecting supernatant, filtering with 0.22um filter, performing HPLC detection, repeating three times at each time point, taking average value, and calculating myricetin content; the same procedure was followed except that no aqueous solution of nano-scale hydroxyapatite was added and the release of the formulation was detected at different time points.
The result shows that after 1h, the binding rate of myricetin and hydroxyapatite in the myricetin targeting micelle is higher than 80%, and the binding rate of free myricetin and hydroxyapatite is lower than 20%, so that the myricetin micelle can obviously improve the binding rate of myricetin and hydroxyapatite, and further proves that the in vitro bone targeting of the myricetin targeting micelle is far higher than Yu Youli myricetin. As shown in fig. 2 d.
Binding ratio of myricetin to hydroxyapatite= (amount of myricetin added-amount of free myricetin)/amount of myricetin added
Example 4
Pharmacokinetic study of myricetin micelles
(1) In vivo study
Sprague-Dawley (SD) rats (200+ -20 g, males) were supplied by the university laboratory animal center of Jiangsu. After two weeks of adaptive feeding, 12 rats were randomly divided into two groups (n=6). Prior to the experiment, rats were fasted for 12h (free drinking). Two groups of rats were perfused with 200mg/kg of myricetin drug substance and myricetin micelle, respectively, according to the body weight of the rats, and blood was taken from the orbital veins of the rats using capillaries at different time points (0.08,0.25,0.5,0.75,1,1.5,2,3,4,6,8, 10, 12, 24 h) after administration. Blood was centrifuged (3700 rpm,10 min) and plasma was collected and stored at-20℃for further analysis.
(2) Blood sample processing
Adopts a liquid-liquid extraction method to extract myricetin from rat serum. 200 mu L of rat serum is taken, quantitative internal standard solution (luteolin) is added, after vortex mixing, 600 mu L of diethyl ether is added, after vortex 1min extraction, centrifugation (10000 rpm,5 min) is carried out to obtain supernatant, nitrogen is adopted for drying, after repeated twice, 200 mu L of acetonitrile is used for redissolution, a 0.22 mu m microporous filter membrane is adopted, and the content of myricetin in the supernatant is analyzed by High Performance Liquid Chromatography (HPLC).
(3) Myricetin micelle in vivo pharmacokinetics study
The main pharmacokinetic parameters such as maximum peak concentration (Cmax), peak time of arrival (Tmax), mean Residence Time (MRT) and area under the concentration-time curve (AUC 0- ≡) were calculated by BAPP2.3 pharmacokinetic software (supplied by the university of chinese pharmacokinetics drug metabolism center). The equation for calculating the relative oral bioavailability (RBA) of a drug is as follows:
wherein AUCt and AUCr represent the areas under the concentration-time curves of myricetin micelles and myricetin suspensions, respectively.
(4) Myricetin micelle in vivo pharmacokinetic analysis
FIG. 3a depicts the plasma drug concentration versus time profile of rats following oral single dose (200 mg/kg) of free myricetin and myricetin micelles, calculated pharmacokinetic variables are shown in Table 2. It was observed that the plasma drug concentration of oral myricetin micelle rats was significantly higher than that of oral free myricetin. In addition, myricetin could not be detected in serum after 24 hours of oral administration of free myricetin to rats, but myricetin could still be detected in vivo after 36 hours of oral administration of myricetin micelle to rats.
Table 2 pharmacokinetic parameters of free myricetin and myricetin micelles after oral administration in rats (n=6)
Two absorption peaks appear on the mean blood concentration-time curves of the free myricetin and myricetin targeted micelles, which may be liver and intestine circulation, gastrointestinal tract movement and the like. The AUC0-36h and Cmax of free myricetin are 15.69+ -0.37 h μg/ml and 1.88+ -0.08 μg/ml, respectively, while the AUC0-36h and Cmax of myricetin micelle are 55.51 + -2.21 h μg/ml and 8.38+ -0.34 μg/ml, respectively. The relative bioavailability of myricetin is improved by 3.54 times. It can be seen that myricetin micelles significantly increase the absorption of myricetin in vivo (p < 0.01); in addition, myricetin can not be detected in serum after the free myricetin is orally taken by the rats for 24 hours, and myricetin can still be detected in vivo after the myricetin micelle is orally taken by the rats for 36 hours, so that the myricetin micelle can prolong the in vivo time of the myricetin.
The pharmacokinetic study shows that the myricetin micelle can obviously improve the oral bioavailability of myricetin, probably because the micelle improves the solubility of myricetin in the gastrointestinal tract, and further improves the absorption availability of myricetin; in addition, the particle size of the micelle is less than 100nm, the micelle is not easy to be cleared by reticuloendothelial cells, and can be adsorbed by endocytosis, so that the intestinal permeability of the medicine is increased, and the bioavailability is increased.
Example 5
Living body imaging study
Myricetin is dissolved in absolute ethanol to prepare a solution with the concentration of 10mg/mL. Next, 2mL of an ethanol solution of myricetin and fluorescein isothiocyanate (FITC, 100. Mu.g/mL) was taken and slowly dropped into an aqueous solution of AL-P (LLA-CL) -PEG-P (LLA-CL) under ultrasonic conditions. Then, absolute ethanol was removed under reduced pressure using a rotary evaporator. Dialysis was performed in dialysis bags (MV 3000D) for 48 hours, with purified water changed every 6 hours. And (5) freeze-drying after dialysis to obtain the targeting micelle containing FITC.
20 SD rats were divided into 4 groups (n=5), and stomach was irrigated with 200mg/kg of FITC-loaded targeted micelles. Rats were sacrificed 4,8, 12, and 16 hours after administration, and the femur was removed for in vivo imaging (excitation wavelength: 495nm, emission wavelength: 519 nm).
The imaging results of rats after oral administration of the fluorochrome-bearing targeting micelle for 4,8, 12, 16 hours are shown in fig. 3 (b-e). From the figure, after 4 hours, the micelle is slowly and intensively distributed at the thigh bone of the rat, which proves that the myricetin micelle has stronger bone targeting property.
Example 6
Research on influence on osteoblast proliferation
1. Cell culture and experimental grouping
Mouse MC3T3-E1 osteoblasts were cultured at 37℃in a 5% CO2, saturated humidity incubator. alpha-MEM medium containing 10% fetal bovine serum, penicillin (100U/mL) and streptomycin (100U/mL) was used, and the medium was changed 1 time for 3 days. After the cells are fused to about 80%, discarding the old culture medium, flushing with PBS for 2 times, replacing with osteogenic induction culture medium, and then adding myricetin raw material medicines with different mass concentrations and myricetin micelles with corresponding concentrations into each culture hole respectively, wherein the culture medium and medicines are replaced every 3 d.
Based on the concentration of myricetin added, the experiment set 9 groups in total: group a complete medium group (control group); group B1 μg/L myricetin drug substance group; group C10 μg/L myricetin drug substance group; group D, 50 μg/L myricetin drug substance group; group E, 100 mug/L myricetin bulk drug group; group F, 1. Mu.g/L myricetin micelle group; group G10. Mu.g/L myricetin micelle group; group H, 50. Mu.g/L myricetin micelle group; group I100. Mu.g/L myricetin micelle group.
MTT method for detecting osteoblast proliferation
MC3T3-E1 cells were seeded at a density of 1X 104 per well in 27-well plates, 9 groups of 3 duplicate wells per column were set, and cultured in an incubator. And adding A, B, C, D, E, F groups of corresponding mass concentrations into the 6 rows of holes respectively, changing the liquid for 1 time for 3d, performing MTT detection after co-culturing for 3 and 6d respectively, and detecting the absorbance (A) value at the 490nm wavelength by adopting an enzyme-labeled instrument.
As can be seen from FIGS. 4 (a, b), both myricetin micelles and free myricetin promote osteoblast proliferation, while the proliferation rate of myricetin micelles is significantly higher than that of free MY (p < 0.01). Meanwhile, the research shows that the Al-P (LLA-CL) -PEG-P (LLA-CL) material has no cytotoxicity.
Increment rate = (drug group absorbance value-control group absorbance value)/control group absorbance value 100%
3. Alkaline phosphatase Activity assay
MC3T3-E1 cells were inoculated into 27-well plates at a density of 1X 105 per well, 9 groups were set, 3 multiplex wells were set per group, 3d was changed 1 time, and after 4,7d co-culture, alkaline phosphatase assay was performed. The culture solution is discarded, 1% PBS is rinsed for 3 times, 0.1% Triton X-100 is added to lyse the cells, a refrigerator is used for overnight at 4 ℃, the cells are observed to be completely lysed under a microscope, each hole of cells is blown, the lysate is collected, centrifugation is carried out at 1200r/min, the supernatant is collected and operated according to the instructions of an alkaline phosphatase test box, A value at the wavelength of 520nm is detected by a colorimetry enzyme-labeling instrument, and enzyme activity is calculated according to a protein-standardized Sigma calibration curve.
After 4 days of administration, the ALP activity of osteoblasts can be enhanced by the myricetin bulk drugs and myricetin micelles with different concentrations, and the myricetin has concentration dependence; however, for the myricetin bulk drug, the effective concentration is required to be more than 1 mug/ml, and the ALP activity of the myricetin micelle group is obviously high in Yu Yangmei element bulk drug group (p is less than 0.01 and p is less than 0.05). After 7 days of administration, ALP activity of the myricetin bulk drug group and the myricetin micelle group forming bone cells is further enhanced (p is less than 0.01 and p is less than 0.05), but ALP activity of the myricetin targeting micelle group is still obviously higher than that of the bulk drug group (p is less than 0.01 and p is less than 0.05). Demonstrating that myricetin micelles can promote the formation of osteoblasts and increase ALP activity. The results are shown in FIG. 4 (c, d)
4. Alizarin red staining and quantification
MC3T3-E1 cells were inoculated into 27-well plates at a density of 5X 104 per well, 9 groups were set, 3 duplicate wells per group were changed 1 time for 3d, and alizarin red staining was performed after co-culture for 14 d. Removing culture solution, rinsing with PBS for 3 times, fixing with 40g/L paraformaldehyde for 30min, removing the fixing solution, rinsing with distilled water for 3 times, completely sucking water completely, slowly adding alizarin red S staining solution, staining for 30min, removing alizarin red staining solution, rinsing with distilled water for 3 times, adding appropriate amount of distilled water into each hole to prevent drying in the hole, observing under a microscope, and photographing. After photographing, the alizarin red dye is decolorized by using 10% cetylpyridinium chloride (dissolved in 10mmol/L sodium phosphate) for 30min at room temperature, and the calcium ion concentration of the sample is calculated by using a standard calcium ion concentration curve by using a calcium content detection kit (colorimetric method).
The osteoinductive process is such that calcium ions precipitate in the form of calcium salts, i.e. "calcium nodules", which are commonly identified by alizarin red staining. Alizarin red can react with calcium ions in a color development manner to generate a dark red compound. From fig. 5, we can see that the red compound of myricetin micelle group is obviously more than myricetin bulk drug, and the effect of myricetin targeting micelle to promote osteoblast formation is better. In addition, it can be seen from the quantitative results of calcium ions (fig. 4 e), although both the myricetin drug substance and myricetin micelle can increase the content of calcium ions, the effect of myricetin micelle is better than that of drug substance.
Example 7
Therapeutic effect of myricetin micelle on castration rat osteoporosis
1. Establishing castration rat osteoporosis model
Since the bone metabolic process of rats has many similarities to humans, rats are a common model animal for the study of osteoporosis. The common rat osteoporosis model modeling modes mainly comprise a castration model, a glucocorticoid intervention model, a disuse model and the like, wherein the castration method (OVX) can simulate the high-conversion bone metabolic process caused by the sudden reduction of the estrogen level after the menopause of human beings, and is the optimal animal model recommended by the American FDA for researching the osteoporosis
SD rats were randomly divided into 4 groups, A blank; b, model; c, raw material medicine; d myricetin micelle. B. Group C, D rats were subjected to ovariectomy to construct a castration osteoporosis model. Group B is given with physiological saline, and groups C and D are respectively administered with 200mg/kg myricetin and myricetin micelle by gastric lavage once daily, and subsequent related experiments are carried out 30 days after administration.
micro-CT analysis
Each sample was scanned using Scaner software from Skyscan1174 Micro CT. The rat femur was scanned at a voltage of 50kV and a current of 800. Mu.A with a scan resolution of 12. Mu.m, a field size of 1304X 1024. The bottommost end of a growth plate at the knee joint side of the femur is set as a scanning reference line, 250 continuous slices, namely, a region with the thickness of 3mm is set as a three-dimensional reconstruction region of interest (ROI), three-dimensional image reconstruction is carried out by using N-Recon software, three-dimensional analysis is carried out by using CT-AN software, and bone density (BMD) measurement of the region of interest is carried out. The analysis index includes: bone trabecular volume percent BV/TV (precent bone xolume, BV/TV), bone trabecular thickness (trabecular thickness, tb.th), bone trabecular separation (trabecular separation, tb.sp), bone trabecular number (Trabecular number, th.n), and structural model index SMI.
BV/TV refers to the ratio of the total volume of bone trabeculae to the total volume of the sample in the selected area; tb. Th refers to the average thickness of trabecular bone; sp refers to the average width of the medullary cavity between trabeculae; tb.N refers to the number of trabeculae per mm of distance; SMI is an index reflecting the characteristics of the trabecular plate-like structure or rod-like structure. When osteoporosis occurs, trabeculae change from a plate-like structure to a rod-like structure, and the SMI value becomes large. BMD is often used in clinical diagnosis of osteoporosis, and low levels of BMD are considered as the major risk factor for bone fracture. In quantitative analysis of CT scan results, BV/TV, tb.Th, tb.N and BMD of the model group are obviously reduced compared with a blank group, and Tb.Sp and structural model index SMI are obviously increased, which indicates that bone mass of rats is reduced, bone mass is obviously lost, and further indicates that modeling is successful. After the myricetin bulk drug and the myricetin micelle are subjected to the intervention treatment, various indexes are improved to a certain extent, wherein the myricetin micelle has more obvious treatment effect on osteoporosis, BV/TV is obviously increased, tb.Sp is obviously reduced (p is less than 0.05, and p is less than 0.01). The CT 3D reconstruction results of the isolated femur of each group of rats also show that the trabecular bone structure of the myricetin micelle group is more compact. Therefore, the myricetin micelle has a certain degree of bone targeting capability and has a better treatment effect on osteoporosis. The results are shown in FIG. 6.
3. Physical and chemical index detection
Rats were sacrificed after blood withdrawal and colon and liver were withdrawn for further experiments. And (3) preserving the obtained blood sample at 4 ℃ for 3 hours, centrifuging at low temperature, and preserving the upper-layer pale yellow transparent serum at-20 ℃ for testing. According to the requirements of the kit, the level of the tartaric acid-resistant acid phosphatase (TRAP), bone alkaline phosphatase (B-ALP), interleukin-1 beta (IL-1 beta) and Osteocalcin (OCN) in serum is detected.
TRAP, BALP are common serum biomarkers used to assess skeletal remodeling. BALP generally increases the concentration of phosphorus by decomposing phospholipids, thereby promoting matrix mineralization. As can be seen from FIG. 7, the levels of the bone formation biochemical index BALP and the bone resorption biological index TRAP were significantly increased (p < 0.01) in rats in the model group compared to the blank group. After the administration, the myricetin bulk drug can inhibit the content of the myricetin bulk drug and the myricetin bulk drug to a certain extent, but the effect is not obvious; the myricetin micelle has small influence on BALP (p is less than 0.05), but can obviously reduce the content of TRAP in serum (p is less than 0.01), and the myricetin micelle can regulate and control bone metabolism of the ovariectomized osteoporosis rat mainly by inhibiting bone resorption, namely by inhibiting the formation of osteoclasts, so that bone loss is reduced. It has been reported that osteoporosis can also lead to inflammation, and thus, many cytokines such as IL-1β and the like have a close relationship with osteoporosis. As can be seen from fig. 7, the blank group of the model group also had a significant increase in IL-1 beta ratio, consistent with the study. After the administration treatment, the myricetin bulk drug and myricetin micelle can reduce the level of IL-1 beta to a certain extent, and the difference is statistically significant, but the inhibiting effect of myricetin micelle is more remarkable (p is less than 0.01). Osteocalcin (OCN) is a vitamin K-dependent calcium binding protein secreted by bone cells, is closely related to osteogenic activity, plays an important role in bone calcium metabolism, can reflect the degree of bone formation and bone transformation to a certain extent, and has important value in diagnosing diseases such as abnormal calcium metabolism, osteoporosis and the like. In this study, after modeling, the level of OCN was significantly increased (p < 0.01), and after myricetin treatment, the level was significantly decreased (p <0.01 or p < 0.05), indicating that OCN may participate in and affect the development process of osteoporosis, presumably OCN may decrease the activity of bone cells, affecting the balance of bone formation and bone resorption. In general, myricetin micelle can improve bone metabolism related blood index of OVX rats through formation of osteoclast, and has good therapeutic effect on osteoporosis.
4. Histopathological analysis
Bone was fixed with paraformaldehyde (4%) at 4 ℃ for 24 hours, then washed with PBS pH 7.4. Bone tissue was paraffin embedded, sectioned at 5 μm, HE stained. Histopathological changes were observed under a Nikon microscope.
According to HE dyeing results, the bone trabecular structures of rats in blank groups are uniformly distributed, and the morphology is complete; the bone trabecula structure of the model group rat is obviously thinned, the bone cortex is thinned, the morphological structure is poor, the model group rat has the phenomena of distortion and fracture, and the model group rat is in a typical osteoporosis shape, which indicates that the modeling is successful. After 30 days of treatment by the myricetin and myricetin targeted micelle, the morphological structure of bone tissues can be found to be improved, wherein the treatment effect of the myricetin targeted micelle is more remarkable. The specific expression is that the trabeculae are slightly thin, the arrangement is tidy, the gaps among the trabeculae are larger, but the morphological structure is basically complete, and the micelle has good bone targeting treatment effect and can effectively treat osteoporosis. The results are shown in FIG. 8.
Preparation method of myricetin-loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting formation of osteoclast
The invention designs and successfully prepares the myricetin AL-P (LLA-CL) -PEG-P (LLA-CL) bone targeted micelle for the first time. The micelle has narrower particle size distribution, negative zeta potential and higher encapsulation efficiency. In vitro release and in vivo pharmacokinetics studies in rats show that the solubility of myricetin can be obviously improved after the myricetin is encapsulated in micelles, and the oral bioavailability of myricetin is improved by 3.54 times compared with that of free myricetin. Meanwhile, the myricetin bone cement bundle has better in-vivo and in-vitro bone targeting, can obviously improve ALP activity of osteoblasts, and also has better treatment effect in the treatment of castration rat osteoporosis. In general, the myricetin bone micelle can remarkably improve the water solubility and bioavailability of myricetin, is a potential carrier for treating castrated rat osteoporosis, and provides information for possible clinical application of myricetin.
Claims (3)
1. A method for preparing a myricetin-loaded nanomicelle that reduces ovariectomy-induced bone loss by inhibiting the formation of osteoclasts, characterized by: the myricetin-loaded nano micelle is prepared from a bone targeting material and myricetin; the bone targeting material is synthesized by the reaction of raw materials P (LLA-CL) 5/5 (4500) -PEG (2000) -P (LLA-CL) 5/5 (4500) and alendronate sodium, and the bone targeting material is AL-P (LLA-CL) -PEG-P (LLA-CL); the mass ratio of the bone targeting material to myricetin in the micelle is 10:1-10:4, a step of; the preparation method of the nano micelle comprises the following steps:
(1) Dissolving dried AL-P (LLA-CL) -PEG-P (LLA-CL) in ultrapure water, and dissolving myricetin in absolute ethanol;
(2) Slowly dripping myricetin ethanol solution into the AL-P (LLA-CL) -PEG-P (LLA-CL) water solution in the step (1) according to a proportion, and uniformly mixing;
(3) And removing absolute ethyl alcohol by reduced pressure rotary evaporation, and filtering by using a 0.45 mu m microporous filter membrane to obtain the myricetin-loaded nano micelle capable of reducing the bone loss induced by ovariectomy by inhibiting the formation of osteoclast.
2. The method for preparing a myricetin-loaded nano micelle capable of reducing ovariectomy-induced bone loss by inhibiting the formation of osteoclasts according to claim 1, wherein the myricetin has a purity of 98%.
3. The method for preparing a myricetin-loaded nano micelle for reducing ovariectomy-induced bone loss by inhibiting the formation of osteoclasts according to claim 1, wherein the dropping and mixing steps in the step (2) are performed under ultrasonic conditions.
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