Combined Erzhi pill anti-osteoporosis pill based on hydroxyapatite and preparation method thereof

Abstract

The invention relates to the related technical field of medical drugs, in particular to an anti-osteoporosis pill based on hydroxyapatite combined Erzhi pill and a preparation method thereof, the pill comprises a nano-hydroxyapatite carrier (Ca / P = 1.67: 1, and the particle size is 20-100nm), Erzhi pill active components (the compatibility of ligustrum lucidum ait total glycoside and eclipta ethyl acetate part is 1: 1) and pharmaceutical auxiliary materials, through ionic bond / hydrogen bond loading, the drug loading ratio is (5-20): 1. The preparation method comprises the following steps: synthesizing nano-hydroxyapatite by a hydrothermal method, extracting active components of Erzhi pills by using ethanol, adsorbing and carrying medicines by using a buffer solution with the pH value of 7.4, and tabletting and forming. According to the anti-osteoporosis pill based on the hydroxyapatite combined Erzhi pill and the preparation method of the anti-osteoporosis pill, the synergistic anti-osteoporosis effect is achieved by up-regulating bone morphogenetic genes (osterix, OCN), inhibiting osteoclast genes (c-Src, cathepsin K) and regulating an LCN2 / Sema3A pathway, the targeting property is high, the bioavailability is high, and the anti-osteoporosis pill is suitable for treating postmenopausal and senile osteoporosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical drugs, in particular to an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pill and a preparation method thereof. BACKGROUND

[0002] Osteoporosis is a systemic bone disease characterized by bone loss and bone microstructure destruction. Current clinical treatment drugs mainly include anti-bone resorption drugs (such as bisphosphonates) and bone formation promoting drugs (such as parathyroid hormone analogues), but existing therapies generally have problems such as single target, large side effects (such as jaw necrosis, osteosarcoma risk) and long-term use. Traditional Chinese medicine compound Erzhi pill (female privet fruit + eclipta) shows a multi-target synergistic advantage in regulating bone remodeling. Its active components (such as specnuezhenide, wedelolactone) can improve the bone microenvironment by regulating the neural-immune-endocrine network (such as LCN2, Semaphorin signaling pathway), but there are technical bottlenecks such as low oral bioavailability (<15%) and poor bone targeting, which makes it difficult to maintain therapeutic concentration of effective components at the bone defect site. As the main inorganic component of natural bone, nano-hydroxyapatite (nHA) has excellent biocompatibility and osteoinductivity, and has been widely used in bone repair field. However, existing drug delivery systems mainly use simple chemical drug loading, which cannot achieve the synergistic controlled release of multi-components of traditional Chinese medicine, and lack of systematic regulation of key pathways of bone metabolism. Therefore, developing an anti-osteoporosis drug based on nano-hydroxyapatite carrier combined with active components of Erzhi pill to remodel bone homeostasis through precise bone targeting delivery and multi-target synergistic effect has become a key direction to break through the limitations of existing technology.

[0003] At present, the research on Erzhi pill for anti-osteoporosis mainly focuses on the component splitting of Erzhi pill. The pharmacodynamic study of Erzhi pill whole prescription or single herb extract on animal models of kidney-yin deficiency osteoporosis, glucocorticoid stimulation or surgical castration, and cell models is carried out to observe the influence on neural-immune-endocrine related indicators, bone histomorphometry, bone biomechanics, and bone biochemical indicators. The splitting research of Erzhi pill reveals the regulation of Erzhi pill on the neural-immune-endocrine microenvironment of bone remodeling, bone formation, and bone resorption, but the compatibility mechanism of Erzhi pill is still unclear. The reduction research of traditional Chinese medicine reveals its pharmacodynamic material basis to some extent, but the compatibility mechanism of different traditional Chinese medicines in traditional Chinese medicine compound is still unclear. Animal models can evaluate the overall efficacy of Erzhi pill in regulating bone remodeling, but due to the limitation of sample usage, only the compatibility of extract and high content, easily accessible components can be studied. SUMMARY

[0004] The application aims to provide an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pill and a preparation method, so as to solve the problems of single target of anti-osteoporosis drugs, large side effects, low oral bioavailability of active components of Erzhi pill and poor bone targeting in the prior art.

[0005] To achieve the above-mentioned object, the application provides the following technical scheme: an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pill, comprising the following components: Nano-hydroxyapatite carrier material, with a calcium-phosphorus molar ratio of 1.67:1, a particle size of 20-100 nm and a specific surface area of 60-120 m² / g; Active components of Erzhi pill, composed of total ginsenoside parts of Ligustri Lucidi Fructus and ethyl acetate parts of Ecliptae Prostratae Radix, with a medicinal material weight ratio of 1:1; Pharmaceutically acceptable excipients; The mass ratio of the nano-hydroxyapatite to the active components of Erzhi pill is (5-20):1, and the nano-hydroxyapatite surface is loaded with the active components of Erzhi pill through ionic bonds and hydrogen bonds.

[0006] Preferably, the total ginsenoside parts of Ligustri Lucidi Fructus contain ≥15% specnuezhenide and ≥8% rhodioside, and the ethyl acetate parts of Ecliptae Prostratae Radix contain ≥5% wedelolactone and ≥3% hoslundia glycoside A.

[0007] Preferably, the nano-hydroxyapatite is a rod-shaped crystal structure, and the surface is modified with sodium carboxymethyl cellulose, with a modification amount of 0.5-2% of the total weight of the carrier.

[0008] The application further provides a preparation method of the anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pill, and the specific steps are as follows: (1) Preparation of nano-hydroxyapatite: mix Ca(NO3)2•4H2O and (NH4)2HPO4 solutions according to a calcium-phosphorus molar ratio of 1.67:1, adjust the pH to 10, and react in a water bath at 80-100℃ for 2-6 hours, then centrifuge and wash until neutral, and freeze-dry; (2) Extraction of active components of Erzhi pill: extract Ligustri Lucidi Fructus with 70% ethanol by reflux, purify the total ginsenoside parts with a macroporous resin, extract Ecliptae Prostratae Radix with 95% ethanol, and obtain the active parts by ethyl acetate extraction; (3) Preparation of drug-loaded complex: dissolve the active components of Erzhi pill in a pH 7.4 phosphate buffer, mix with nano-hydroxyapatite according to a mass ratio of 1:(5-20), oscillate for 12 hours at 37℃, and freeze-dry; (4) Pill forming: mix the drug-loaded complex with microcrystalline cellulose and cross-linked polyvinylpyrrolidone according to a ratio of 8:1:1, and press to obtain tablets.

[0009] Preferably, the adsorption conditions in step (3) are: initial drug concentration 1-5 mg / mL, solid-liquid ratio 1:10-1:20 (g / mL), and oscillation speed 120 rpm.

[0010] Preferably, the anti-osteoporosis pill for use in the preparation of a drug for treating osteoporosis plays a synergistic role through the following mechanisms: up-regulating the expression of bone formation marker genes osterix and osteocalcin; down-regulating the expression of osteoclast marker genes c-Src and cathepsin K; regulating the expression levels of nerve-immune-endocrine network factors LCN2 and Sema3A.

[0011] Preferably, the anti-osteoporosis pill for use in the preparation of a drug for treating osteoporosis, wherein the osteoporosis includes postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, or senile osteoporosis.

[0012] Preferably, the excipient includes a disintegrant, sodium croscarmellose, which is used in an amount of 2-5% of the total weight of the pill.

[0013] Compared with the prior art, the present application has the following beneficial effects: 1. Through the innovative combination of nano-hydroxyapatite and the active components of Erzhi Pill, the present application has achieved a breakthrough in the field of anti-osteoporosis treatment. In terms of treatment mechanism, the present application has creatively combined the bone repair function of nano-hydroxyapatite with the multi-target regulation advantage of Erzhi Pill. The calcium and phosphorus elements provided by nano-hydroxyapatite directly promote bone matrix mineralization, while the active components of Erzhi Pill (including specnuezhenide, salidroside in total ligans of Ligustrum lucidum Ait, and artemisia capillaris lactone, hirsutanol A, etc. in the ethyl acetate fraction of Herba Ecliptae) loaded thereon synergistically act on multiple key links of bone remodeling: significantly up-regulating the expression of bone formation marker genes (osterix, osteocalcin, ALP) to promote osteogenic differentiation; effectively inhibiting the activity of osteoclast marker genes (c-Src, c-fos, cathepsin K) to reduce bone resorption; and further regulating key factors of nerve-immune-endocrine network such as LCN2, Semaphorin 3A / 4D / 7A, etc. to comprehensively improve the bone microenvironment, forming a three-dimensional treatment mode of "material supply-drug regulation-microenvironment remodeling", and breaking through the limitation of single target drug mechanism.

[0014] 2. By optimizing the preparation process of nano-hydroxyapatite (Ca / P molar ratio 1.67:1, particle size 20-100 nm, specific surface area 60-120 m² / g) and surface carboxymethyl cellulose sodium modification (0.5-2%), multiple breakthrough effects are achieved: first, the high affinity of nano-hydroxyapatite with bone matrix improves the targeting enrichment efficiency of drugs in bone defect site by 3-5 times; second, through the ion bond / hydrogen bond interaction between active components and carriers, a stable drug delivery system (drug loading capacity 15-25%) is constructed, realizing the controlled release of two to pill components, maintaining effective blood drug concentration for more than 24 hours; third, the established bone marrow mesenchymal stem cell-osteoclast precursor-dorsal root ganglion neuron three-dimensional co-culture evaluation system provides a precise in vitro evaluation method for the bone targeting and sustained release characteristics of the drug delivery system. These technical innovations fundamentally solve the key technical bottlenecks of traditional Erzhi pills, such as low oral bioavailability (<15%), poor bone targeting, and short action time. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present application will be described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0016] The present application provides a technical solution: an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pills, comprising the following components: Nano-hydroxyapatite carrier material, with a calcium-phosphorus molar ratio of 1.67:1, a particle size of 20-100 nm, and a specific surface area of 60-120 m² / g; Active components of Erzhi pills, composed of total ginsenoside fraction of Ligustrum lucidum and acetic acid ethyl ester fraction of Eclipta prostrata L. at a crude drug weight ratio of 1:1; Pharmaceutically acceptable excipients; Wherein the mass ratio of nano-hydroxyapatite to active components of Erzhi pills is (5-20):1, and the active components of Erzhi pills are loaded on the surface of nano-hydroxyapatite through ionic bonds and hydrogen bonds.

[0017] Further, the total ginsenoside fraction of Ligustrum lucidum contains specnuezhenoside ≥15% and rhodioside ≥8%, and the acetic acid ethyl ester fraction of Eclipta prostrata L. contains onirol ≥5% and helenyn A ≥3%.

[0018] Further, the nano-hydroxyapatite is a rod-shaped crystal structure, and the surface is modified with carboxymethyl cellulose sodium, with a modification amount of 0.5-2% of the total weight of the carrier.

[0019] The preparation method of the anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pills is as follows: (1) Preparation of nano-hydroxyapatite: Ca(NO3)2•4H2O and (NH4)2HPO4 solution were mixed at a calcium-to-phosphorus molar ratio of 1.67:1, the pH was adjusted to 10, and the mixture was reacted in a water bath at 80-100℃ for 2-6 hours. After centrifugation and washing until neutral, the mixture was freeze-dried. (2) Extraction of active components of Erzhi Pill: Ligustrum lucidum was extracted by reflux with 70% ethanol and purified by macroporous resin to obtain total glycoside fraction; Eclipta prostrata was extracted by 95% ethanol and extracted with ethyl acetate to obtain active fraction. (3) Preparation of drug-loaded complex: The active component of Erzhi pill was dissolved in pH 7.4 phosphate buffer, mixed with nano hydroxyapatite at a mass ratio of 1:(5-20), and adsorbed by shaking at 37℃ for 12 hours, and then freeze-dried; (4) Pill forming: The drug-loaded complex is mixed with microcrystalline cellulose and cross-linked polyvinylpyrrolidone in a ratio of 8:1:1 and then compressed into tablets.

[0020] Furthermore, the adsorption conditions in step (3) are: initial drug concentration of 1-5 mg / mL, solid-liquid ratio of 1:10-1:20 (g / mL), and shaking speed of 120 rpm.

[0021] The application of anti-osteoporosis pills in the preparation of drugs for treating osteoporosis exerts a synergistic effect through the following mechanisms: Upregulates the expression of bone formation marker genes osterix and osteocalcin; Downregulates the expression of osteoclast marker genes c-Src and cathepsin K; It regulates the expression levels of neuro-immune-endocrine network factors LCN2 and Sema3A.

[0022] Furthermore, osteoporosis includes postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, or senile osteoporosis.

[0023] Furthermore, the excipients include the disintegrant croscarmellose sodium, which is used in an amount of 2-5% of the total weight of the pill.

[0024] Example 1: Preparation and efficacy evaluation of nano-hydroxyapatite combined with Erzhi Pill for osteoporosis prevention. 1. Preparation of nano-hydroxyapatite Raw material ratio: Mix Ca(NO3)2·4H2O (0.5 mol / L) and (NH4)2HPO4 (0.3 mol / L) solutions at a calcium-to-phosphorus molar ratio of 1.67:1.

[0025] Reaction conditions: Adjust pH to 10, react in a 90℃ water bath for 4 hours, centrifuge and wash until neutral, then freeze dry.

[0026] Surface modification: Dry nano-hydroxyapatite was dispersed in 1% sodium carboxymethyl cellulose (CMC-Na) solution, stirred for 2 hours, washed by centrifugation, and freeze-dried to obtain surface-modified nano-hydroxyapatite (modification amount 1% of the total weight of the carrier).

[0027] 2. Extraction of active components of Ligustri Lucidi Fructus pills Total glycoside fraction of Ligustri Lucidi Fructus: Take the coarse powder of Ligustri Lucidi Fructus, extract with 70% ethanol by refluxing for 3 times (1 hour each time), combine the extract, concentrate under reduced pressure, and then pass through a D101 macroporous resin column. Elute with water and then with 50% ethanol. Collect the eluate, concentrate and dry to obtain the total glycoside fraction of Ligustri Lucidi Fructus (specnuezhenide content 18%, salidroside content 10%).

[0028] Ethanolic extract of Eclipta prostrata: Take the coarse powder of Eclipta prostrata, extract with 95% ethanol by refluxing for 3 times (1 hour each time), combine the extract, concentrate under reduced pressure, and then extract with petroleum ether and ethyl acetate in sequence. Collect the ethyl acetate fraction, concentrate and dry to obtain the ethyl acetate fraction of Eclipta prostrata (inulafulvotomentosa lactone content 6%, ecliptine A content 4%).

[0029] 3. Preparation of drug-loaded complex Drug loading ratio: Mix the total glycoside fraction of Ligustri Lucidi Fructus and the ethyl acetate fraction of Eclipta prostrata at a crude drug weight ratio of 1:1, dissolve in pH 7.4 phosphate buffer (initial concentration 3 mg / mL), and mix with nano-hydroxyapatite at a mass ratio of 1:10.

[0030] Adsorption conditions: Adsorb at 37°C for 12 hours with shaking (shaking speed 120 rpm), centrifuge, and freeze-dry to obtain the drug-loaded complex (drug loading amount 22%).

[0031] 4. Pill forming Auxiliary material ratio: Mix the drug-loaded complex, microcrystalline cellulose, and cross-linked polyvinylpyrrolidone at a ratio of 8:1:1, and add 2% cross-linked sodium carboxymethyl cellulose as a disintegrating agent.

[0032] Tabletting process: Use a rotary tablet press to press into pills with a diameter of 8 mm and a weight of 200 mg.

[0033] 5. Efficacy evaluation In vitro experiment: Osteogenic activity: Co-culture the drug-loaded complex with bone marrow mesenchymal stem cells (BMSCs), and after 7 days, detect ALP activity (3.2 times higher than the blank group) and osteocalcin (OCN) expression (up-regulated by 2.8 times).

[0034] Osteoclast inhibition: Co-culture with RAW264.7 cells, and after 5 days, TRAP activity is reduced by 65% compared with the blank group, and cathepsin K gene expression is down-regulated by 70%.

[0035] Animal experiments: Postmenopausal osteoporosis model: ovariectomized rats were orally administered the pill (50 mg / kg) daily, and after 8 weeks, Micro-CT showed that bone mineral density (BMD) increased by 35%, trabecular bone number (Tb.N) increased by 40%, serum Sema3A level increased by 2-fold, and LCN2 level decreased by 50%.

[0036] Example 2: Preparation of pills with different drug loading ratios and comparison of sustained release performance 1. Drug loading ratio optimization Group design: Nanohydroxyapatite and active ingredient of Erzhi pill were prepared into drug-loaded complexes with mass ratios of 5:1, 10:1, and 20:1, respectively.

[0037] Drug loading determination: HPLC detection showed that the drug loading was 15%, 22%, and 18%, respectively, with the highest drug loading efficiency in the 10:1 group.

[0038] 2. In vitro release experiment Conditions: Release in simulated intestinal fluid (pH 6.8) at 37°C with shaking.

[0039] Results: 5:1 group: The release rate reached 90% in 12 hours, with obvious burst effect.

[0040] 10:1 group: The cumulative release was 80% in 24 hours, showing sustained release characteristics.

[0041] 20:1 group: The release was only 60% in 24 hours, and the release was not complete.

[0042] 3. Conclusion: The 10:1 drug loading ratio has high drug loading and ideal sustained release performance, and is the optimal combination.

[0043] Example 3: Effect of surface modification on bone targeting 1. Comparison of modification methods Groups: Group A: Unmodified nanohydroxyapatite.

[0044] Group B: CMC-Na modification (1%).

[0045] Group C: Polyethylene glycol (PEG) modification.

[0046] Bone targeting evaluation: The drug-loaded complex was labeled with fluorescence, and was injected intravenously into an osteoporosis mouse model. The fluorescence intensity of the bone tissue was detected 24 hours later: The bone targeting enrichment efficiency of group B was 4 times higher than that of group A, and was significantly higher than that of group C (P<0.01).

[0047] 2. Mechanism analysis: CMC-Na modification enhances electrostatic adsorption of nanoparticles to bone matrix collagen, promoting specific deposition at bone defect sites.

[0048] Example 4: Compatibility component synergistic verification 1. Component split experiment Group: Single use of total ginseng ginseng.

[0049] Single use of St. John's wort ethyl acetate fraction.

[0050] Both 1:1 compatibility.

[0051] Results: The promoting effect of the compatibility group on BMSCs osteogenic differentiation (3.2-fold increase in ALP activity) was significantly better than that of the single use group (1.8-fold for ginseng, 1.5-fold for St. John's wort), confirming the synergistic effect.

[0052] Example 5: Preclinical safety evaluation 1. Acute toxicity test: no death was observed in rats after single oral administration of 5000 mg / kg for 14 days, and no abnormalities were found in the pathological examination of major organs. 2. Long-term toxicity test: normal blood biochemical indicators and bone tissue morphology were observed after daily administration of 100 mg / kg for 6 months, confirming safety.

[0053] Although embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pills, characterized in that, It contains the following components: Nano-hydroxyapatite carrier material with a calcium-to-phosphorus molar ratio of 1.67:1, a particle size of 20-100 nm, and a specific surface area of ​​60-120 m². 2 / g; The active components of Erzhi Pill are composed of total glycosides from Ligustrum lucidum and ethyl acetate from Eclipta prostrata in a 1:1 ratio by weight of the raw materials. Pharmaceutically acceptable excipients; The mass ratio of nano-hydroxyapatite to the active component of the two-stage pellet is (5-20):1, and the active component of the two-stage pellet is loaded on the surface of the nano-hydroxyapatite through ionic bonds and hydrogen bonds.

2. The osteoporosis-resistant pill based on hydroxyapatite combined with Erzhi pills according to claim 1, characterized in that, The total glycosides fraction of Ligustrum lucidum contains ≥15% ligustrin and ≥8% rhodioloside, and the ethyl acetate fraction of Eclipta prostrata contains ≥5% zedoaryl lactone and ≥3% ecliptaside A.

3. The osteoporosis-resistant pill based on hydroxyapatite combined with Erzhi pills according to claim 1, characterized in that, The nano-hydroxyapatite has a rod-shaped crystal structure and its surface is modified with sodium carboxymethyl cellulose, with the modification amount being 0.5-2% of the total weight of the carrier.

4. A method for preparing an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pills, as described in any one of claims 1-3, characterized in that... The specific steps are as follows: (1) Preparation of nano-hydroxyapatite: Ca(NO3)2•4H2O and (NH4)2HPO4 solution were mixed at a calcium-to-phosphorus molar ratio of 1.67:1, the pH was adjusted to 10, and the mixture was reacted in a water bath at 80-100℃ for 2-6 hours. After centrifugation and washing until neutral, the mixture was freeze-dried. (2) Extraction of active components of Erzhi Pill: Ligustrum lucidum was extracted by reflux with 70% ethanol and purified by macroporous resin to obtain total glycoside fraction; Eclipta prostrata was extracted by 95% ethanol and extracted with ethyl acetate to obtain active fraction. (3) Preparation of drug-loaded complex: The active component of Erzhi pill was dissolved in pH 7.4 phosphate buffer, mixed with nano hydroxyapatite at a mass ratio of 1:(5-20), and adsorbed by shaking at 37℃ for 12 hours, and then freeze-dried; (4) Pill forming: The drug-loaded complex is mixed with microcrystalline cellulose and cross-linked polyvinylpyrrolidone in a ratio of 8:1:1 and then compressed into tablets.

5. The method for preparing an anti-osteoporosis pill based on hydroxyapatite combined with Erzhi pills according to claim 4, characterized in that, The adsorption conditions in step (3) are: initial drug concentration of 1-5 mg / mL, solid-liquid ratio of 1:10-1:20 (g / mL), and shaking speed of 120 rpm.

6. The use of the anti-osteoporosis pill according to any one of claims 1-3 in the preparation of a drug for treating osteoporosis, characterized in that, The following mechanisms can be used to achieve synergy: Upregulates the expression of bone formation marker genes osterix and osteocalcin; Downregulates the expression of osteoclast marker genes c-Src and cathepsin K; It regulates the expression levels of neuro-immune-endocrine network factors LCN2 and Sema3A.

7. The application of the anti-osteoporosis pill according to claim 6 in the preparation of a drug for treating osteoporosis, characterized in that, The osteoporosis mentioned includes postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, or senile osteoporosis.

8. The osteoporosis-resistant pill based on hydroxyapatite combined with Erzhi pills according to claim 1, characterized in that, The excipients include the disintegrant croscarmellose sodium, which is used in an amount of 2-5% of the total weight of the pill.