Application of dodder amine and / or 19 alpha-madecassic acid

Abstract

The invention relates to the field of detection of active ingredients of traditional Chinese medicines, in particular to application of dodder amine and / or 19 alpha-madecassic acid. According to the application disclosed by the invention, the osteoporosis resisting effect of the cuscutamine, the 19 alpha-hydroxyasitic acid in a mouse MC3T3-E1 cell and the osteoclast differentiation effect of the cuscutamine, the 19 alpha-hydroxyasitic acid in a mouse macrophage RAW 264.7 cell induced by sRANKL are evaluated by using osteoblast differentiation and mineralization. Experimental results show that in an osteoblast or osteoclast activity evaluation system, the cuscutamine, the 19 alpha-hydroxysidic acid has the anti-osteoporosis activity, and in addition, the cuscutamine, the 19 alpha-hydroxysidic acid has the anti-osteoporosis activity. Semen cuscutae amine and 19 alpha-madecassic acid can be used as anti-osteoporosis quality markers of the eight-component kidney tonifying capsules.

Description

Technical Field

[0001] The invention relates to the field of detection of active ingredients of traditional Chinese medicines, and in particular to the application of dodderamine and / or 19α-hydroxyasiatic acid. Background Art

[0002] Traditional Chinese medicine plays an important role in the prevention and treatment of diseases, and therefore has received increasing attention and recognition worldwide. The quality of traditional Chinese medicine directly affects the clinical efficacy and restricts the development of modernization of traditional Chinese medicine. However, the complexity of chemical composition and the ambiguity of pharmacological substances are huge obstacles to the quality control of traditional Chinese medicine. In the process of strategy exploration, Liu et al. proposed the concept of quality markers. This concept focuses on the measurability, specificity, transferability, effectiveness and traceability of quality markers of traditional Chinese medicine. The characterization of quality markers involves the integration of multiple properties, including component analysis, content determination, pharmacokinetics, network pharmacology, etc. There are a variety of plant secondary metabolites in traditional Chinese medicine, and components with different structures may have different biological activities. Therefore, the quality markers of traditional Chinese medicine need to consider the different structural types of different components, which is of great significance for the quality control of traditional Chinese medicine based on traditional efficacy. In addition, network pharmacology is increasingly being used to reveal the mechanism of action between active ingredients and disease therapeutic targets. At present, the use of network pharmacology to screen quality markers is mainly based on the degree of association between a single compound and a disease target, and the mode of action of multiple components of traditional Chinese medicine covering multiple targets in the form of a combination to exert pharmacological effects is not prominent. In addition, the metabolism of TCM ingredients in the body will lead to changes in their biological activity, and the in vivo process of TCM ingredients is also particularly important for determining the quality markers of TCM. It is well known that TCM prescriptions have the characteristics of multi-component, multi-target, and overall regulation, but there are currently few reports on mining TCM quality markers from a holistic perspective. Therefore, this study proposed a digital and holistic strategy to mine the quality markers of absorbable ingredients in Bazi Bushen Capsule in the form of different compositions to reflect the structural diversity and diversity of biological pathways.

[0003] Bazi Bushen Capsule (BZBS) is a traditional Chinese medicine compound, which is composed of 16 single herbs, including Cuscuta, Lycium, Schisandra, Cnidium, Rosa Laevigata, Rubus, Toosendan, Allium, Epimedium, Morinda, Cistanche, Rehmannia, Cyathula, Ginseng, Pilose Antler, and Hippocampus. It is clinically used to prevent and treat symptoms such as soreness of waist and knees, dizziness, tinnitus, and fatigue caused by insufficient kidney yang. Recent pharmacological studies have shown that Bazi Bushen Capsule can significantly improve the bone morphology of aged mice induced by D-galactose and NaNO2, and increase the mineral content in their bone tissue. In addition, Bazi Bushen Capsule can also improve the bone microstructure of the distal tibia in ovariectomized osteoporosis rats. A large number of literature reports show that a variety of single herbs in Bazi Bushen Capsule, such as Cuscuta, Lycium barbarum, Cnidium monnieri, Epimedium, Ginseng, Cistanche deserticola and Rehmannia root, can improve the bone microstructure parameters of ovariectomized osteoporosis model rats / mice. In summary, Bazi Bushen Capsule has a significant anti-osteoporosis effect. Bazi Bushen Capsule has a definite efficacy, but the quality control research of Bazi Bushen Capsule is seriously insufficient. Therefore, it is urgent to find the quality markers of Bazi Bushen Capsule to improve its quality control level.

[0004] It is worth noting that Bazibushen Capsule is a complex system composed of 16 single herbs, and its anti-osteoporosis biological activity evaluation has not been reported, which seriously hinders the quality control research of Bazibushen Capsule. Summary of the invention

[0005] In view of this, the present invention provides the use of cuscutamine and / or 19α-hydroxyasiatic acid. Experimental results show that cuscutamine and 19α-hydroxyasiatic acid have anti-osteoporosis activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides the use of cuscutamine and / or 19α-hydroxyasiatic acid in the preparation of a drug for promoting osteoblast differentiation.

[0008] In some specific embodiments of the present invention, the effective concentration of dodderamine is 10 -6 M, the effective concentration of 19α-hydroxyasiatic acid is 10 -6 In some specific embodiments of the present invention, the effective concentration of 19α-hydroxyasiatic acid is 10 -6 and 10 -5 M.

[0009] In a second aspect, the present invention further provides the use of cuscutaneous amine and / or 19α-hydroxyasiatic acid in the preparation of a drug for promoting bone calcification nodules.

[0010] In some specific embodiments of the present invention, the effective concentration of dodderamine is 10 -6 M.

[0011] In a third aspect, the present invention further provides the use of cuscutamine and / or 19α-hydroxyasiatic acid in the preparation of a drug for inhibiting osteoclast differentiation.

[0012] In some specific embodiments of the invention, the inhibiting osteoclast differentiation comprises reducing the number of multinucleated TRAP-positive cells.

[0013] In some specific embodiments of the present invention, the effective concentration of dodderamine is 10 -5 M, the effective concentration of 19α-hydroxyasiatic acid is 10 -7 In some specific embodiments of the present invention, the effective concentration of 19α-hydroxyasiatic acid is 10 -7 and 10 -6 M.

[0014] In a fourth aspect, the present invention further provides the use of cuscutaneous amine and / or 19α-hydroxyasiatic acid in the preparation of anti-osteoporosis drugs.

[0015] In some specific embodiments of the present invention, the anti-osteoporosis comprises:

[0016] (I) Promote osteoblast differentiation;

[0017] (II), promoting bone calcification nodules; and / or

[0018] (III) inhibiting osteoclast differentiation;

[0019] The effective concentration of the dodder amine and / or the 19α-hydroxyasiatic acid is 10 -7 M~10 -5 M.

[0020] In a fifth aspect, the present invention further provides the use of cuscutaneous amine and / or 19α-hydroxyasiatic acid as a quality marker of Bazibushen Capsule in the preparation of an anti-osteoporosis drug.

[0021] The present invention evaluated the anti-osteoporotic effects of cuscutamine and 19α-hydroxyasiatic acid in mouse MC3T3-E1 cells using osteoblast differentiation (ALP) and mineralization (calcified nodules stained with Alizarin red), as well as the osteoclast differentiation effect (TRAP-positive cell number) in sRANKL-induced mouse macrophage RAW 264.7 cells. Figure 2 As shown, cuscutamine (10 -6M),19α-hydroxyasiatic acid(10 -6 and 10 -5 M) can significantly promote ALP activity (P<0.05). Figure 3 A and Figure 3 As shown in B, cuscutamine at 10-6M has a significant effect on promoting the formation of bone calcification nodules (P<0.05). Figure 4 A and Figure 4 As shown in B, cuscutamine (10 -5 M), 19α-hydroxyasiatic acid(10 -7 and 10 -6 M) can significantly reduce the number of multinuclear TRAP-positive cells (P<0.05). The above results show that cuscutamine and 19α-hydroxyasiatic acid have anti-osteoporosis activity in the osteoblast or osteoclast activity evaluation system. Cuscutamine and 19α-hydroxyasiatic acid can be used as quality markers of Bazi Bushen Capsule for anti-osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0023] Figure 1 Figure 2 shows the extracted ion chromatograms of prototype (A / B) and metabolites (C / D) in rat plasma after oral administration of BZBS; wherein, (A) and (B) show the extracted ion chromatograms of prototype compound in rat plasma after oral administration of BZBS; (C) and (D) show the extracted ion chromatograms of metabolites in rat plasma after oral administration of BZBS;

[0024] Figure 2 The figure shows the effects of different test concentrations of dodder ine and 19α-hydroxyasiatic acid on the differentiation activity of MC3T3-E1 cells; Ctrl is the blank control group; 17β-E2 is 17β-estradiol; cuscutamine is dodder ine; 19α-hydroxyasiatic acid is 19α-hydroxyasiatic acid; * indicates that the concentration group is significantly different from the blank control group, and ** indicates that the concentration group is very significantly different from the blank control group;

[0025] Figure 3The results of the effects of different test concentrations of dodder and 19α-hydroxyasiatic acid on the mineralization activity of MC3T3-E1 cells are shown; A is a quantitative bar graph of calcium nodule staining by the alizarin red method; B is a representative graph of calcium nodule staining by the alizarin red method; Ctrl is a blank control group; 17β-E2 is 17β-estradiol; cuscutamine is dodder; 19α-hydroxyasiatic acid is 19α-hydroxyasiatic acid; * indicates that the concentration group is significantly different from the blank control group, and ** indicates that the concentration group is very significantly different from the blank control group;

[0026] Figure 4 The figures show the effects of different test concentrations of dodderine and 19α-hydroxyasiatic acid on the differentiation activity of osteoclasts; A is a quantitative bar graph of TRAP-positive multinuclear osteoclasts; B is a representative graph of TRAP-positive multinuclear osteoclasts; NS is a negative control group; Ctrl is a blank control group; cuscutamine is dodderine; 19α-hydroxyasiatic acid is 19α-hydroxyasiatic acid; * indicates that the concentration group is significantly different from the blank control group; ### indicates that the blank control group is extremely significantly different from the negative control group. DETAILED DESCRIPTION

[0027] The present invention discloses the application of dodderamine and / or 19α-hydroxyasiatic acid. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] The present invention studies the use of eight chemical components, namely, osthole, hyperin, epimedin C, icariin, schisandrin, cuscutamine, magnoflorine, and 19α-hydroxyasiatic acid, in anti-osteoporosis, and uses them as quality markers for anti-osteoporosis of Bazi Bushen Capsule. The experimental results show that cuscutamine and 19α-hydroxyasiatic acid have anti-osteoporosis effects, and cuscutamine and 19α-hydroxyasiatic acid can be used as quality markers for anti-osteoporosis of Bazi Bushen Capsule.

[0029] Materials and methods

[0030] Instruments and reagents:

[0031] Samples were measured on a Waters ACQUITY UPLC I-Class Plus system equipped with an autosampler, online degasser, binary solvent delivery system, and a Waters Xevo G2-XS QTOF with ESI interface.

[0032] Cuscuta, Lycium, Schisandra, Cnidium, Rosa laevigata, Rubus idaeus, Toosendan, Allium, Epimedium, Morinda officinalis, Cistanche, Rehmannia, Cyathula, Ginseng, Pilose antler, Hippocampus and 10 batches of Bazi Bushen Capsules were provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., China.

[0033] monotropein,deacetylaspelurosidic acid,rehmannioside D,echinacoside,cyasterone,xanthotoxol,isopimpinellin,xanthotoxin,toosendanin,19α-hydroxyasiatic acid,rubiadin-1-methyl ether,schisandrin,schisandrol B,imperatorin,osthole,schisantherin A,schisantherin B,schisandrin A,cuscutamine,schisandrin B,kaempferol 3-O-rutinoside,kaempferol,isochlorogenicacid C,isoquercitrin were purchased from Chengdu Pusi Biotechnology Co., Ltd. Nystose, ginsenoside Rg2, ginsenoside Rc, ginsenoside Rb2, icariin, epimedin A, epimedin B, epimedin C, icariside I, icariside II, icaritin, betaine, magnoflorine, neochlorogenic acid, chlorogenic acid were purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. Hyperin, gallic acid, elagic acid, ginsenoside Rb1, γ-Glctamyl-S-allylcysteine, were purchased from Chengdu Purifa Technology Development Co., Ltd. Ferulic acid, astragalin, azelaic acid, ginsenoside Re, ginsenoside Ro, ginsenoside Rd were purchased from Chengdu Kroma Co., Ltd. Adenine, 6-hydroxypurine were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Ginsenoside Rg1, quercetin were purchased from Chengdu Efa Biological Co., Ltd. Acteoside was purchased from Anhui Zesheng Technology Co., Ltd. After HPLC identification, the purity of all reference substances was ≥98%.

[0034] Water for mass spectrometry (HPLC grade) was purchased from Beijing Watsons Distilled Water Co., Ltd.; mass spectrometry grade methanol and acetonitrile were purchased from Thermo Fisher Scientific, USA; mass spectrometry grade formic acid was purchased from Sigma Aldrich, USA; other reagents were of analytical grade and purchased from the market.

[0035] In the application of dodderamine and / or 19α-hydroxyasiatic acid provided by the present invention, the raw materials and reagents used can be purchased from the market.

[0036] The present invention will be further described below in conjunction with embodiments:

[0037] Example 1 Experimental Animals and Dosage of Bazi Kidney-tonifying Drug

[0038] Female Sprague-Dawley rats (220 ± 20 g) were obtained from the Guangdong Experimental Animal Center (Guangzhou). The animal experiment protocol was reviewed and approved by the Experimental Animal Ethics Committee of Jinan University (IACUC-20220625-01). All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (Jinan University). Rats were housed in an environmentally controlled animal room (22-25 ° C, relative humidity 50-60%, 12-h light-dark cycle) and were given a normal diet for two weeks before the experiment. Bazi Bushen Capsules were dissolved in sterile water and gavaged at a dose of 0.2 g / mL; 8 rats (Bazi Bushen group) were gavaged for 3 consecutive days at a dose of 1 g / kg / day; the remaining 2 rats (blank group) were gavaged with an equal amount of sterile water.

[0039] Example 2 Sample collection and pre-treatment method

[0040] Whole blood samples were collected at 0.5 hours, 1 hour, 2 hours and 4 hours (four time points) after the last administration of the Ba Zi Bu Shen group in Example 1. After the blood samples were centrifuged at 13,200 rpm (10°C) for 10 minutes, the plasma was separated into a clean test tube to obtain the plasma samples of the Ba Zi Bu Shen group, which were stored at -80°C until drug analysis. Blank plasma samples were obtained from blank group rats in the same way.

[0041] Three times the amount of acetonitrile was added to 2 mL of plasma sample, vortexed and centrifuged at 13200 rpm (10°C) for 10 minutes, and the supernatant was blown dry with nitrogen. The residue was redissolved in 200 μL of methanol, vortexed, and then centrifuged at 13200 rpm (10°C) for 20 minutes.

[0042] Example 3 Instrumental Analysis Conditions

[0043] Liquid phase conditions: Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm), and the column temperature was 35°C. The mobile phase consisted of 0.1% (v / v) formic acid (phase A) and 0.1% (v / v) formic acid in acetonitrile (phase B). Flow rate: 0.4 mL / min; elution gradient: 0-3.5 min, 2%-15% B; 3.5-12.5 min, 15%-36% B; 12.5-19.5 min, 36%-55% B; 19.5-23.5 min, 55%-80% B; 23.5-26 min, 80%-100% B; 26-28 min, 100% B; 28-29 min, 100%-2% B; 29-30 min, 2% B. The injection volume was 2 μL.

[0044] Mass spectrometry conditions: mass range 50-1500Da; capillary voltage 3.0kV (ESI + ) and 2.1kV (ESI-); cone voltage was 35V; ion source temperature was 140℃; desolvation temperature was 500℃; cone backflush gas flow rate was 50L / h; desolvation gas flow rate was 1000L / h; low channel collision energy was 6-30eV, and high channel collision energy was 40-90V; when the TIC intensity exceeded 5000intensity / s, it automatically switched to MS / MS mode and switched back to MS scan after five MS / MS scans; the MS and MS / MS survey scan times were both set to 0.2. In one MS scan, the five ions with the highest response were automatically selected for MS / MS. The mass accuracy was calibrated using a leucine-enkephalin solution internal standard (m / z 556.2771[M+H] in positive ion mode) + , m / z 554.2615 [MH]- in negative ion mode). LockSpray TM The flow rate was 5 μL / min and data were collected using MassLynx 4.2 software (Waters, Milford, MA, USA).

[0045] Example 4 In vivo metabolic experimental results in rat plasma

[0046] According to the analytical method described in Example 3, a total of 26 prototype substances (13 lignans, 5 coumarins, 3 alkaloids, 1 triterpene, 1 organic acid and 3 other substances) were detected in the plasma of rats after oral administration of Bazi Bushen Capsule, of which 11 prototype components such as magnoflorine, cuscutamine, 19α-hydroxyasiatic acid, schisandrin, imperatorin, osthole, isopimpinellin, xanthotoxin, schisantherin A, schisantherin B and schisandrin B were confirmed by comparison with the standard. TM The software processed the data and used the mass defect filter (MDF) to identify the metabolites. The structures of several characteristic components were input into Metabolynx as parent components. TM The software contains metabolites such as icaritin, hyperin, rutin, osthole, xanthotoxin, schisandrin, schisantherin A, monotropein, echinacoside, cuscutamine, cyasterone, ginsenosideRg1 and ginsenoside Rb1. The characteristics of 16 metabolites (3 prenylated flavonoids, 2 flavonols, 5 coumarins and 6 lignans) of rutin, hyperin, icaritin, icariside II, osthole and schisantherin A were preliminarily determined by accurate mass, characteristic ion and neutral loss analysis. The main metabolic pathways are oxidation, demethylation, hydrolysis, sulfation and glucuronidation. The extracted ion chromatograms (EIC) of the prototype and metabolites in the plasma of rats after oral administration of Bazi Bushen Capsule are shown in Figure 1 The UPLC-Q-TOF-MS data of the prototype are shown in Table 1. The UPLC-Q-TOF-MS data of the metabolites are shown in Table 2.

[0047] It is noteworthy that epimedin C, icariin, icariside II, icaritin, rutin, and hyperin were not detected in vivo after oral administration of Bazibushen capsules, but interestingly, their glucuronide metabolites (M1-M5) were detected in plasma. Therefore, magnoflorine, cuscutamine, 19α-hydroxyasiatic acid, imperatorin, osthole, isopimpinellin, xanthotoxin, schisandrin, schisantherin A, schisantherin B, schisandrin B, epimedin C, icariin, icariside II, icaritin, rutin, and hyperin were selected as absorbable candidate components.

[0048] Table 1. Characterization of the prototype of Bazi Bushen Capsule in rat plasma after oral administration

[0049]

[0050]

[0051] Table 2. Characteristics of metabolites in rat plasma after oral administration of Bazi Bushen Capsule

[0052]

[0053]

[0054] Example 5 Osteoblast differentiation activity

[0055] Mouse embryonic osteoblast precursor cells MC3T3-E1 cells were purchased from ATCC, USA. The cells were cultured in α-MEM cell culture medium (purchased from Gibco, USA) with 1% P / S (100 units / mL penicillin, 100 mg / mL streptomycin) and 10% FBS (purchased from Gibco, USA) added in 5% CO2, 37°C, and 95% humidity. After the cells were fully grown, the cells were counted using a Beckman Coulter cell counter and then inoculated into a 12-well plate. Each well was added with 1.2×10 40.5 mL of α-MEM cell culture medium (1% P / S and 10% FBS) was added to each cell, and the culture medium was discarded after the cells grew to more than 80% adherent culture, and replaced with α-MEM (1% P / S, 10% FBS) osteogenic differentiation induction medium containing mineralization inducer (10 mM β-sodium glycerophosphate, 50 μg / m ascorbic acid) for culture. The specific drug addition was as follows: 0.5 mL of the above osteogenic differentiation induction medium was added with 0.5 μL of the following solutions: blank solvent (absolute ethanol), 10 μM positive drug 17β-estradiol, 0.1 μM, 1 μM, 10 μM cuscutamine, 0.1 μM, 1 μM, 10 μM 19α-hydroxyasiatic acid, and cultured for 7 days, and the drug-containing induction culture medium was replaced every 2 to 3 days.

[0056] On the eighth day, discard the old drug-containing induction culture medium, then rinse each well twice with 0.5mL PBS (concentration 0.01M, pH 7.4, the same below), then add 100μL Passive Lysis Buffer cell lysis solution and continue shaking on ice for 30 minutes. First, take 20μL of cell lysate from each well, follow the relevant steps described in the Wako lab assay ALP detection kit (purchased from Wako, Japan), and finally use a microplate reader to detect the OD value at a wavelength of 405nm. At the same time, take another 2μL of cell lysate from each well, follow the instructions of the BCA protein concentration detection kit (purchased from Shanghai Jining Industrial Co., Ltd.), and finally detect the total protein content of each well at 562nm. The alkaline phosphatase ALP activity is measured using the OD ratio: OD per well ALP Value / OD per well BCA The relative activity of alkaline phosphatase, i.e., the average value of the OD ratio of each well × 100% / the OD ratio of the blank solvent control group, was used to represent the activity of ALP. The experiment was repeated at least 3 times for statistical analysis. The experimental data were statistically analyzed using ANOVA test, and p < 0.05 was considered statistically significant.

[0057] Example 6 Osteoblast mineralization activity

[0058] MC3T3-E1 cells were counted using a Beckman Coulter cell counter and then seeded in 12-well plates. Each well was added with 1×10 51.0 mL α-MEM cell culture medium (1% P / S and 10% FBS) was added to each well of the cells. When the cells were attached to the wall and the cells grew to more than 80%, they were replaced with fresh osteogenic differentiation induction medium for induction culture of mineralized calcium nodules. The specific drug addition was as follows: 1 mL of differentiation induction medium was added with 1 μL of the following solutions: blank solvent (anhydrous ethanol), 10 μM positive drug 17β-estradiol, 0.1 μM, 1 μM, 10 μM cuscutamine, 0.1 μM, 1 μM, 10 μM 19α-hydroxyasiatic acid, and co-cultured for about 30 days. Fresh drug-containing induction culture medium was replaced every 2 to 3 days until the mineralized calcium nodules were observed and the culture was terminated. After the culture was terminated, the old culture medium was discarded and gently washed twice with 0.5 mL PBS solution. Subsequently, 300 μL of 4% paraformaldehyde solution was added to each well for fixation for 15 minutes, and then the alizarin red S staining method was used for qualitative staining observation.

[0059] The specific steps of staining are: each well is washed once with 0.5mL PBS, then 0.5mL 1% Alizarin Red S dye solution is added for staining for 10 minutes, and then repeatedly rinsed with double distilled water to wash away the background color, and the cell plate is dried and photographed for qualitative observation and comparison. In order to further quantitatively analyze the formation of mineralized calcium nodules, this experiment uses 500μL of 0.5M HCl-5% SDS solution (pH 3-4) to each well to continuously oscillate and dissolve the mineralized calcium nodules for 30 minutes, and finally 200μL of the dissolved solution is transferred to each well into a 96-well plate, and the wavelength is set to 415nm in the microplate reader to detect the OD value. The promotion rate of mineralized calcium nodules is characterized by the average value of each well OD value × 100% / blank solvent control group OD. The experiment was repeated at least 3 times for statistical analysis. The experimental data were statistically analyzed using the ANOVA test, and p<0.05 was statistically significant.

[0060] Example 7 Osteoclast differentiation activity

[0061] Mouse RAW264.7 cells were obtained from the American Type Culture Collection (ATCC) and routinely cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin in an environment of 37°C, 95% humidity, and 5% CO2. The cells were subcultured every two to three days.

[0062] To investigate the effects of cuscutamine and 19α-hydroxyasiatic acid on cytotoxicity, RAW264.7 cells were seeded into 96-well plates at a density of 3,000 cells / well. After 24 hours, cells were treated with different concentrations of cuscutamine and 19α-hydroxyasiatic acid (10-7 -10 -5 Cells were treated with differentiation medium (0.1% DMSO) or blank solvent (0.1% DMSO) for four days for cytotoxicity experiments, and the medium was changed every two days. After the cells were treated as above, the cytotoxicity was determined using a CCK-8 kit (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. The absorbance at 450 nm was measured on a multifunctional microplate reader (BioTEK Synergy H1 Hybrid Multi-Mode Reader, USA).

[0063] RAW 264.7 cells were seeded at a density of 3000 cells / well in a 96-well plate. -7 -10 -5 M) differentiation medium (αMEM, 10% FBS, containing 30ng / mL M-CSF, 100ng / mL sRANKL) was used to induce the cells for 4 days. The cells were then fixed and stained with tartrate-resistant acid phosphatase (TRAP) using a leukocyte acid-resistant phosphatase kit (sigma, USA). TRAP-positive cells with more than three nuclei were considered mature osteoclasts. The experimental data were statistically analyzed using the ANOVA test, and p<0.05 was statistically significant. All statistical analyses were performed using GraphPad Prism (version 6.0, GraphPad Software, USA).

[0064] Effect example

[0065] The anti-osteoporotic effects of cuscutamine and 19α-hydroxyasiatic acid were evaluated in mouse MC3T3-E1 cells using osteoblast differentiation (ALP) and mineralization (calcified nodules stained with Alizarin red), and in sRANKL-induced osteoclast differentiation (TRAP-positive cell count) in mouse macrophage RAW 264.7 cells. Figure 2 As shown, cuscutamine (10 -6 M),19α-hydroxyasiatic acid(10 -6 and 10 -5 M) can significantly promote ALP activity (P<0.05). Figure 3 A and Figure 3 As shown in B, cuscutamine -6 M had a significant effect on promoting the formation of bone calcification nodules (P<0.05). Figure 4 A and Figure 4As shown in B, cuscutamine (10 -5 M),19α-hydroxyasiatic acid(10 -7 and 10 -6 M) can significantly reduce the number of multinuclear TRAP-positive cells (P<0.05). The above results show that cuscutamine and 19α-hydroxyasiatic acid have anti-osteoporosis activity in the osteoblast or osteoclast activity evaluation system.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of cuscutaamine and / or 19α-hydroxyasiatic acid in the preparation of a drug for promoting osteoblast differentiation.

2. The use according to claim 1, characterized in that The effective concentration of dodder amine is 10 -6 M, the effective concentration of 19α-hydroxyasiatic acid is 10 -6 M.

3. Use of dodderamine and / or 19α-hydroxyasiatic acid in the preparation of drugs for promoting bone calcification nodules.

4. The use according to claim 3, characterized in that The effective concentration of dodder amine is 10 -6 M.

5. Use of cuscutaneous amine and / or 19α-hydroxyasiatic acid in the preparation of drugs for inhibiting osteoclast differentiation.

6. The use according to claim 5, characterized in that The inhibition of osteoclast differentiation includes reducing the number of multinucleated TRAP-positive cells.

7. The use according to claim 5 or 6, characterized in that The effective concentration of dodder amine is 10 -5 M, the effective concentration of 19α-hydroxyasiatic acid is 10 -7 M.

8. Use of cuscutaneous amine and / or 19α-hydroxyasiatic acid in the preparation of anti-osteoporosis drugs.

9. The use according to claim 8, characterized in that The anti-osteoporosis drugs include: (I) Promote osteoblast differentiation; (II), promoting bone calcification nodules; and / or (III) inhibiting osteoclast differentiation; The effective concentration of the dodder amine and / or the 19α-hydroxyasiatic acid is 10 -7 M~10 -5 M.

10. Application of cuscutaneous amine and / or 19α-hydroxyasiatic acid as quality markers of Bazi Bushen Capsule in the preparation of anti-osteoporosis drugs.

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