CN108785290B

CN108785290B - Application of effective component of chalcone of resina draconis in preparing medicine

Info

Publication number: CN108785290B
Application number: CN201810358407.XA
Authority: CN
Inventors: 刘予豪; 周驰; 何伟; 徐家科; 邓章荣; 王刚; 王海彬; 张庆文
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-04-18
Filing date: 2018-04-18
Publication date: 2021-03-16
Anticipated expiration: 2038-04-18
Also published as: CN108785290A

Abstract

The invention relates to the field of medicines, in particular to dragon blood and medical application of active ingredients of the dragon blood. The invention provides application of dragon blood and chalcone active ingredients thereof in preparation of medicines or health-care products for inhibiting osteoclast formation or osteoclast bone absorption. The application of at least one of loureirin A, loureirin B, loureirin C, loureirin D, cochinchinenin A or cochinchinenin C in preparing medicine or health product for inhibiting osteoclast formation or osteoclast bone absorption is provided. The prepared medicine can be used for treating and preventing osteolytic diseases. The dragon's blood has small side effect and no toxicity, and can be taken for a long time for preventing and treating administration.

Description

Application of effective component of chalcone of resina draconis in preparing medicine

Technical Field

The invention relates to the field of medicines, in particular to dragon blood and medical application of active ingredients of the dragon blood.

Background

Normal bone tissue in the human body is dynamic and constantly changed tissue, and the shape is reshaped mainly by the bone reconstruction function of osteoblasts and the bone resorption function of osteoclasts. When the number of osteoclasts is increased or the bone absorption function is enhanced in vivo, the bone homeostasis is broken, so that various osteolytic related diseases such as osteoporosis and the like are induced, mainly manifested by the microstructure destruction of bone tissues, and the reduction of bone density leads to the reduction of bone strength, increase of brittleness and the like.

The sanguis Draxonis is extracted from daemonorops draco of Palmaceae or dracaena cochinchinensis of dracaena of Liliaceae [ national standard: WS3-082(Z-016) -99(Z) ], a commonly used medicine in traditional Chinese medicine clinical, is a blood-activating saint medicine in the reputation of Shen nong Ben Cao Jing, and is considered to have various physiological activities such as antioxidation, antibiosis, anti-inflammation, antithrombotic and the like in modern pharmacological research. The research reports that the compound can be used for treating hypertrophic scars, diabetes or cardiovascular diseases or has the effect of relieving pain. The dragon's blood has several chemical components, including loureirin A, loureirin B, loureirin C, loureirin D, flag leaf loureirin A and flag leaf loureirin C, etc. all belong to chalcone compounds.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the medical application of the effective components of the dragon blood and the chalcone.

In order to achieve the purpose, the invention adopts the technical scheme that: the research of the inventor finds that the chalcone active ingredients in the dragon blood can inhibit the formation of osteoclasts or inhibit the bone absorption function, so that the application of the chalcone active ingredients in the dragon blood in preparing the medicines or health-care products for inhibiting the formation of osteoclasts or inhibiting the bone absorption of osteoclasts is provided.

In a preferred embodiment, the effective component is at least one of loureirin A, loureirin B, loureirin C, loureirin D, loureirin A and loureirin C.

The structural formulas of the loureirin A, the loureirin B, the loureirin C, the loureirin D, the loureirin A and the loureirin C are as follows:

loureirin A,

B of loureirin,

Loureirin C,

Loureirin D,

Dracaena cochinchinensis A,

Flag leaf lourerin C.

The inventor conducts pharmacodynamic study on the compound, and the result shows that the loureirin A, the loureirin B, the loureirin C, the loureirin D, the loureirin A and the loureirin C can inhibit osteoclast formation or osteoclast bone resorption.

In a preferred embodiment, said lourerin A, lourerin B, lourerin C, lourerin D, glaucin A and glaucin C inhibits osteoclastogenesis or inhibits osteoclastic bone resorption by inhibiting or attenuating the action of RANKL.

In a preferred embodiment, the lourerin A, lourerin B, lourerin C, lourerin D, sisal leaf lourerin A and sisal leaf lourerin C can inhibit osteoclast formation or inhibit osteoclast bone resorption by inhibiting or weakening the expression or active oxygen production of TRAcP gene, V-ATPase-D2 gene, CTSK gene, MMP9 gene, Calcitonin Receptor gene, NFATc1 protein, V-ATPase-D2 protein or cathepsin K protein.

The invention also provides application of the dragon blood in preparing a medicament or a health-care product for inhibiting osteoclast formation or osteoclast bone absorption.

In a preferred embodiment, the drug or health product for inhibiting osteoclastogenesis or inhibiting bone resorption function is used for osteolytic diseases.

In another preferred example, the osteolytic disease is osteoporosis, Paget's disease of the bone, osteomyelitis, bone cysts, myeloma, bone metastasis, osteoblastoma, non-ossifying fibroids, giant cell tumor of the bone.

The loureirin A, loureirin B, loureirin C, loureirin D, flag leaf loureirin A and flag leaf loureirin C can be extracted from dragon's blood by conventional method in the field, or can be purchased from commercial sources or synthesized by conventional compound synthesis method in the prior art by using commercial raw materials. The compounds of the present invention can be synthesized by one of ordinary skill in the art based on the prior art. The synthesized compound can be further purified by means of column chromatography, high performance liquid chromatography, crystallization or the like.

The medicine prepared by the application can be administrated by oral, intravenous, intramuscular, subcutaneous, intranasal, intrarectal and other routes. Solid carriers such as: starch, lactose, phosphate glycol, microcrystalline cellulose, brown sugar and kaolin, the pharmaceutical dosage form can be tablet, powder, and liquid carrier such as: sterile water, polyethylene glycols, non-ionic surfactants and edible oils (such as corn, peanut and sesame oils) as are appropriate to the nature of the active ingredient and the particular mode of administration desired. Adjuvants conventionally used in the preparation of pharmaceutical compositions may also advantageously be included, such as flavouring agents, colouring agents, preservatives and antioxidants such as vitamin E and vitamin C.

The invention has the beneficial effects that: sanguis Draxonis, loureirin A, loureirin B, loureirin C, loureirin D, cochinchinenin A or cochinchinenin C can inhibit osteoclast formation or osteoclast bone absorption, and can be used for preparing medicine or health product for inhibiting osteoclast formation or osteoclast bone absorption. The dragon's blood has small side effect, and the toxicity of dragon's blood is not reported, so that the dragon's blood can be taken for a long time for preventing and treating administration. The details of the various aspects of the invention are set forth in the examples that follow, and in part will be apparent from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a graph showing the results of an experiment for inhibiting osteoclast differentiation in example 1.

FIG. 2 is a graph of the results of experiments on the inhibition of osteoclast-induced differentiation by LrB at different concentration gradients in example 1.

FIG. 3 is a graph showing the results of experiments on the function of inhibiting bone resorption in example 2.

FIG. 4 is a graph of the results of experiments conducted to inhibit Reactive Oxygen Species (ROS) production.

FIG. 5 is a graph showing the results of the gene expression assay of example 4.

FIG. 6 is a graph showing the results of the protein expression experiment in example 5.

FIG. 7 is a graph showing the results of experiments in the osteoporosis model in castrated mice in example 6.

Detailed Description

The inventor finds out through laboratory research that: the dragon blood chalcone chemical components can be used for preparing medicines or health products with the functions of inhibiting osteoclast formation or inhibiting bone absorption, and can be used for preventing or treating osteolytic diseases.

Example 1 experiment for inhibiting osteoclast differentiation

1.1 purpose: the role of lourerin A (LrA), lourerin B (LrB), lourerin C (LrC), lourerin D (LrD), flag leaf lourerin A (CcA) and flag leaf lourerin C (Ccc) in the process of osteoclast differentiation and toxicity to mouse bone marrow macrophages were investigated.

1.2 materials and reagents

Lourerin A (LrA), lourerin B (LrB), lourerin C (LrC), lourerin D (LrD), flag leaf lourerin A (CcA) and flag leaf lourerin C (Ccc) were purchased from Durman Biotechnology Ltd (City, Sichuan province); alpha-MEM medium, FBS (fetal bovine serum) purchased from gibco (Sydney, Australia); beta-actin antibodies, NFATc1 antibodies, V-ATPase-d2 antibodies, CTSK antibodies were purchased from Santa Cruz Biotechnology, Inc. (Calif., USA); MTS assay kit, ROS (reactive oxygen species) reaction kit, PCR reaction kit purchased from Promega corporation (Sydney, Australia); M-CSF (macrophage colony stimulating factor) reagent, RANKL (nuclear factor kappa beta ligand) reagent were purchased from School of Pathology and Laboratory Medicine, Australian university of Western Australian.

1.3 Experimental methods

A healthy 12-week-old C57BL/6J mouse (SPF grade, available from School of Pathology and Laboratory Medicine laboratories, university of Western Australia) was sacrificed by cervical dislocation after ether inhalation anesthesia. Rapidly taking down femurs and shinbones on two sides of a mouse, removing clean muscle tissues in a culture dish containing an alpha-MEM culture medium, and exposing a marrow cavity; and (5) flushing the bone marrow in the marrow cavity to another clean culture dish by using a 5ml syringe, and blowing and uniformly mixing. Filtering out impurities in the bone marrow suspension by using a filter screen, centrifuging the suspension, and retaining the precipitate; the pellet was resuspended using complete α -MEM medium (containing 10% fetal bovine serum, 1% P/S (penicillin/streptomycin) and 1: 20M-CSF). The resuspended bone marrow was transferred to a T75 flask and cultured in a cell culture incubator with complete medium change on day 2 followed by medium change every other day until C57BL/6J mouse Bone Marrow Macrophages (BMMs) mature (about day 5 growing to cover 95% of the area of the flask).

Freshly extracted C57BL/6J mouse Bone Marrow Macrophages (BMMs) were cultured in T75 flasks containing complete medium (α -MEM medium + 10% FBS + 1% P/S + 5% M-CSF) and seeded into 96-well plates at a rate of 6 × 103 cells per well after the BMMs differentiated to maturity. Next day, 50ng/ul RANKL was added to each well to differentiate osteoclasts, 10uM of lourerin A, lourerin B, lourerin C, lourerin D, lourerin A and lourerin C were added to each experimental group, and the same volume of PBS was added to the control group. The differentiation solution is changed every two days until the osteoclast is differentiated and matured. After osteoclasts were differentiated and matured, they were fixed with 2.5% glutaraldehyde for 15 minutes, followed by TRAcP (tartrate-resistant acid phosphatase) staining, and the number of osteoclasts (number of nuclei. gtoreq.3) was counted for each group under the microscope.

Freshly extracted C57BL/6J mouse Bone Marrow Macrophages (BMMs) were cultured in T75 flasks containing complete medium (α -MEM medium + 10% FBS + 1% P/S + 5% M-CSF) and seeded into 96-well plates at a rate of 6 × 103 cells per well after the BMMs differentiated to maturity. Next day, 50ng/ul RANKL was added to each well to differentiate osteoclasts, 1, 2.5, 5, 10uM of LongxuineB was added to each experimental group, and the same volume of PBS was added to each control group. The differentiation solution is changed every two days until the osteoclast is differentiated and matured. After osteoclasts were differentiated and matured, they were fixed with 2.5% glutaraldehyde for 15 minutes, followed by TRAcP (tartrate-resistant acid phosphatase) staining, and the number of osteoclasts (number of nuclei. gtoreq.3) was counted for each group under the microscope.

Differentiating mature BMMs at 6x10 per well³The individual cell ratios were plated in 96-well plates and the intervention started the following day: the experimental group was added 10uM of lourerin A, lourerin B, lourerin C, lourerin D, flag leaf lourerin A and flag leaf lourerin C to each well, the control group was added with the same volume of PBS, incubated in alpha-MEM medium containing 10% FBS and 1% P/S for 48 hours, then 20ul of MTS reagent was added to each well, incubated in the dark for 2 hours and then measured for 490nm photometric number using ELISA reader (German BMG) to determine MTS absorbance.

1.4 results of the experiment

The results show that the number of osteoclasts in the experimental group is obviously less than that in the control group, and lourerin A, lourerin B, lourerin C, lourerin D, lourerin A and lourerin C all have a certain inhibition effect on the formation of osteoclasts, wherein the inhibition effect of LrB is most obvious (fig. 1C and D). Toxicity test results for each component showed that each component was not toxic to BMMs (fig. 1 b). Meanwhile, the research uses LrB with different concentration gradients to intervene in the induced differentiation process of osteoclast, and the result shows that LrB starts to inhibit the formation of osteoclast at low concentration (2.5uM), and the inhibition effect of 10uM concentration is obvious. (FIG. 2)

Example 2 test of function of inhibiting bone resorption

2.1 purpose: the effect of loureirin A (LrA), loureirin B (LrB), loureirin C (LrC), loureirin D (LrD), flag leaf loureirin A (CcA) and flag leaf loureirin C (Ccc) on bone resorption was studied.

2.2 Experimental materials the same as example 1

2.3 Experimental methods

Differentiating mature BMMs at 1 × 10 per well⁵The individual cells are seeded into collagen plates for osteoclast differentiation until osteoclast formation begins, and then the cells are transferred to hydroxylsOsteoclast induction was continued in ashlar board, and 10uM of loureirin A, loureirin B, loureirin C, loureirin D, loureirin A and loureirin C were added to the experimental groups, while PBS of the same volume was added to the control group. After osteoclasts are differentiated and mature, one half of the wells in the same group of cells are stained with TRAcP, the other half of the cells are rinsed and dried, the number of osteoclasts and the hydroxyapatite absorption condition are respectively observed, and Image J software is used for quantitative analysis.

2.4 results of the experiment

The results show that the bone absorption area of the bone plate of the experimental group is obviously smaller than that of the control group, and the lourerin A, the lourerin B, the lourerin C, the lourerin D, the lourerin A and the lourerin C all have certain inhibition effects on the bone absorption function of osteoclasts, wherein the inhibition effect of LrB is most obvious. (FIG. 3)

Example 3 inhibition of Reactive Oxygen Species (ROS) production assay

3.1 purpose: the effect of lourerin b (lrb) on the production of Reactive Oxygen Species (ROS) during RANKL-induced osteoclast differentiation was studied.

3.2 Experimental materials similar to example 1

3.3 Experimental methods

Differentiating mature BMMs at 6x10 per well³The individual cell ratios were seeded into 96-well plates. Next day, 50ng/ul RANKL was added to each well to perform osteoclast differentiation, and at the same time, 10uM LrB was added to each experimental group, and the same volume of PBS was added to the control group, and the differentiation solution was changed every two days. Fluorescence staining was performed on day four using 2', 7' -dichlorodihydrofluorescein diacetate (H2DCF) reagent, ROS levels were observed for each group using Nikon confocal microscopy, and fluorescence intensity was analyzed using NIS-Elements Viewer software.

3.4 results of the experiment

The results showed that 10uM of LrB already significantly inhibited the production of reactive oxygen species early in osteoclast formation (third day). (FIG. 4)

Example 4 Gene expression detection assay

4.1 purpose: the effect of LrB on RANKL-induced osteoclastogenesis and bone resorption function-related gene expression was investigated.

4.2 Experimental materials the same as example 1

4.3 Experimental methods

Differentiating mature BMMs at 8 × 10 per well⁴The individual cell ratios were seeded into 6-well plates. Next day, 50ng/ul RANKL was added to each well to perform osteoclast differentiation, 5uM and 10uM LrB were added to the experimental group, and the same volume of PBS was added to the control group, and the medium was changed every two days. After the sixth day (osteoclast differentiation and maturation), cells were lysed using Trizol lysate and RNA was extracted; performing reverse transcription by using Oligo-dT, and then detecting by adopting a SYBR Green method and using a ViiATM7Real-time PCR instrument, wherein the primer sequences of the target genes are respectively as follows:

TRAcP(5'-T GTGGCCATCTTTATGCT-3'；5'-GTCATTTCTTTGGGGCTT-3')，V-ATPase-d2(5'-GTGAGACCTTGGAAGACCTGAA-3'；

5'-GAGAAATGTGCTCAGGGGCT-3')，CTSK(5'-GGGAGAAAAACCTGAAGC-3'；5'-ATTCTGGGGACTCAGAGC-3')，MMP9(5'-CGTGTCTGGAGATTCGACTTGA-3'；

5'-TTGGAAACTCACACGCCAGA-3')，Calcitonin receptor(5'-TGGTTGAGGTTGTGCCCA-3'；5'-CTCGTGGGTTTGCCTCATC-3')。

and analyzing the expression quantity of each target gene by adopting a delta-delta Ct method.

4.4 results of the experiment

The results of researches on interference of RANKL-induced osteoclast formation by LrB with different concentration gradients show that LrB has an inhibitory effect on TRAcP related to osteoclast formation, V-ATPase-d2 and CTSK related to bone resorption function, MMP9, Calcitonin Receptor and other gene expressions and is dose-dependent. (FIG. 5)

EXAMPLE 5 protein expression assay

5.1 purpose: LrB was investigated for its effect on RANKL-induced osteoclastogenesis and bone resorption function-related protein expression.

5.2 materials tested are the same as in example 1.

5.3 Experimental methods

Differentiating mature BMMs at 8 × 10 per well⁴The individual cell ratios were seeded into 6-well plates. Osteoclast induction by 50ng/ul of RANKL added to each well starting the next dayDifferentiation was performed while LrB was added at 10uM to the experimental group and the same volume of PBS was added to the control group, and the medium was changed every two days. After the sixth day (osteoclast differentiation and maturation), cells were lysed using RIPA lysate and protein was extracted. Proteins were separated using SDS-PAGE electrophoresis, membrane-transferred using nitrocellulose membrane, and then NFATc1, V-ATPase-d2, CTSK and β -actin protein antibodies were incubated overnight at 4 ℃ for 2 hours, followed by development using ECL developer, LAS 4000 developer. The images were analyzed using Gel-pro software.

5.4 results of the experiment

The results showed that LrB significantly inhibited the osteoclast formation-associated NFATc1 protein expression on the third day; the expression of V-ATPase-d2 and CTSK proteins related to bone resorption functions is inhibited at the third day, wherein the inhibition effect is more obvious at the fifth day. (FIG. 6)

Example 6 castrated mouse osteoporosis model

6.1 purpose: LrB Effect on Estrogen deficient mice

6.2 Experimental materials

C57BL/6J female mice, 10 weeks old, were purchased from the animal testing center, university of medicine, Guangzhou, and were otherwise identical to example 1.

6.3 Experimental methods

Animal experiments are carried out in SPF animal houses of animal experiment center of Guangzhou Chinese medicine university, and animal experiment schemes and ethical examination are reported to be approved by the ethical examination committee of animal experiments of Guangzhou Chinese medicine university. 24C 57BL/6J female mice, 10 weeks old, were acclimatized for 1 week and randomized into two groups: model group (OVX)16, Sham group (Sham)8, OVX group performed bilateral dorsal incisions and both ovaries were removed and both fallopian tubes were ligated, Sham group performed bilateral incisions in the same OVX group but without ovaries or tubal ligation. After 3-5 days of adaptive feeding, OVX groups were divided into two groups at random: 8 control (OVX + PBS) groups and 8 experimental (OVX + LrB) groups; mice were concurrently intervened with LrB solution or PBS: group (OVX + LrB) was intraperitoneally injected with LrB solution (4mg/kg), group (OVX + PBS) and group Sham were intraperitoneally injected with PBS of the same volume, 1 time every 2 days, and 20 times in total. After injection was completed (about 6 weeks), three groups of mice were sacrificed by cervical dislocation at the same time, and bilateral femoral bone tissue was obtained. And respectively carrying out micro-CT scanning on each group of thighbones by taking the lower ends of the thighbones as the interested areas so as to observe the change of the microstructure of the bone tissues, reconstructing by using NReco software, carrying out quantitative analysis on the interested areas (the range of 1mm of 0.5mm under epiphysis) by using CTAn software, and carrying out three-dimensional reconstruction on the interested areas by using CTvox software.

6.4 results of the experiment

micro-CT scanning and analysis are carried out by taking the distal femur as an interested area, and the result shows that compared with a Sham operation group (Sham), the trabecular bone volume (BV/TV), the trabecular bone number (Tb.N) and the trabecular bone thickness (Tb.Th) of the model group are obviously reduced, and the trabecular bone dispersion (Tb.Sp) is obviously increased. This shows that the animal modeling of this experiment is successful. Compared with the model group, the LrB group has obviously increased trabecular bone volume and trabecular bone number, and the trabecular bone dispersion is obviously reduced. (FIG. 7b) the three-dimensional reconstruction model of cancellous bone in the region of interest at the distal femur was consistent with the above quantitative analysis results. (FIG. 7a) the above results show that LrB can inhibit the estrogen deficiency from causing osteoporosis to some extent.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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Application of effective component of chalcone of dragon's blood in preparing medicine

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Claims

1. The application of chalcone active ingredients in dragon blood in preparing a medicament for inhibiting osteoclast formation or inhibiting osteoclast bone absorption is disclosed, wherein the active ingredients are at least one of lourerin A, lourerin B, lourerin C, lourerin D, lourerin A and sword-leaf lourerin C.

2. The use according to claim 1, wherein the active ingredient inhibits osteoclastogenesis or inhibits osteoclastic bone resorption by inhibiting or attenuating the action of RANKL.

3. The use according to claim 2, wherein the active ingredient inhibits osteoclastogenesis or inhibits osteoclastic bone resorption by inhibiting or attenuating expression or active oxygen production of a TRAcP gene, a V-ATPase-d2 gene, a CTSK gene, a MMP9 gene, a Calcitonin Receptor gene, a NFATc1 protein, a V-ATPase-d2 protein, or a cathepsin K protein.

4. Use according to any one of claims 1 to 3, wherein the medicament is for the treatment of Paget's disease, osteomyelitis, bone cysts, myeloma, bone metastasis, osteoblastoma, non-ossifying fibroids, giant cell tumors of the bone in osteolytic diseases.

5. Use according to any one of claims 1 to 3, wherein the medicament is in the form of a solid tablet, powder.