阅读说明：本技术 一种山奈苷在缓解糖皮质激素副作用方面的应用 (Application of kaempferitrin in relieving side effects of glucocorticoid ) 是由 谢保城 陈世春 何瑞荣 丁少波 于 2021-01-11 设计创作，主要内容包括：本发明涉及天然化合物在缓解糖皮质激素副作用方面的应用技术领域,具体涉及一种山奈苷在缓解糖皮质激素副作用方面的应用。山奈苷或其药学上可接受的盐、酯或前药在制备缓解糖皮质激素副作用方面的药物中的用途,山奈苷能显著缓解糖皮质激素所致的成骨分化抑制作用,促进地塞米松诱导MC3T3-E1细胞的成骨分化。该实验结果发现了本化合物在减轻糖皮质激素类药物副作用方面的分子作用机制,对于开发新的药物具有重要的临床意义。(The invention relates to the technical field of application of natural compounds in relieving glucocorticoid side effects, in particular to application of kaempferitrin in relieving glucocorticoid side effects. The kaempferide or pharmaceutically acceptable salt, ester or prodrug thereof can be used for preparing the medicine for relieving the side effect of glucocorticoid, and the kaempferide can obviously relieve the osteogenic differentiation inhibition effect caused by the glucocorticoid and promote dexamethasone to induce the osteogenic differentiation of MC3T3-E1 cells. The experiment result discovers a molecular action mechanism of the compound in the aspect of reducing the side effect of glucocorticoid medicaments, and has important clinical significance for developing new medicaments.)

1. The use of a compound of formula I or a pharmaceutically acceptable salt, ester or prodrug thereof in the manufacture of a medicament for alleviating a side effect of a glucocorticoid:

2. use according to claim 1, for alleviating the glucocorticoid side effects by modulating at least one selected from the group consisting of mRNA expression of Runx2, mRNA expression of OPN, mRNA expression of OPG, mRNA expression of bgalap, protein expression of Runx2, protein expression of OPN, protein expression of OPG, and protein expression of bgalap, or modulating alkaline phosphatase activity of osteoblasts, or modulating mineralization of osteoblasts.

3. Use according to claim 1 or 2, the glucocorticoid side effect being selected from the group consisting of: obesity and cushing's syndrome, induced infection, aggravated infection, substance metabolism disorder, water and salt metabolism disorder, cardiovascular system complications, water and sodium retention, hyperlipidemia, hypertension, atherosclerosis, nervousness, neurological abnormality, insomnia, catabolism of skeletal muscle, osteoporosis, bone fracture, cell cycle block of osteoblast lineage cells, promotion of differentiation of osteoclast cells, promotion of activation of osteoclast cells, induction of apoptosis of osteoblast lineage cells, and inhibition of osteogenic differentiation.

4. The application of the compound shown in the formula I in preparing a medicament for treating diseases related to Runx2 expression inhibition, OPN expression inhibition, OPG expression inhibition, Bglap expression inhibition, osteoblast alkaline phosphatase activity inhibition or osteoblast mineralized nodule formation inhibition caused by glucocorticoid.

5. Use according to claim 4, wherein the glucocorticoid is selected from the group consisting of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone or dexamethasone.

6. The use according to claim 4, wherein the disease is selected from at least one of obesity and Cushing's syndrome, induced infection, aggravated infection, substance metabolism disorder, water and salt metabolism disorder, cardiovascular system complications, water and sodium retention, hyperlipidemia, hypertension, atherosclerosis, nervousness, nerve abnormality, insomnia, catabolism of skeletal muscle, osteoporosis, bone fracture, cell cycle arrest of osteoblast lineage cells, promotion of differentiation of osteoclasts, promotion of activation of osteoclasts, induction of apoptosis of osteoblast lineage cells, and inhibition of osteogenic differentiation.

7. Use of a compound of formula I in the preparation of an agent for modulating Runx2 expression.

8. Use of a compound of formula I for the preparation of an agent for modulating OPN expression.

9. Use of a compound of formula I for the preparation of an agent for modulating OPG expression.

10. Use of a compound of formula I in the preparation of an agent for modulating bghap expression.

Technical Field

The invention relates to the technical field of application of natural compounds in relieving glucocorticoid side effects, in particular to application of kaempferitrin in relieving glucocorticoid side effects.

Background

Glucocorticoid (GC) is an extremely important regulatory molecule in the body, plays an important role in regulating development, growth, metabolism, immune function and the like of the body, is the most important regulatory hormone for stress response of the body, and is also the most widely and effectively anti-inflammatory and immunosuppressant in clinic. Glucocorticoids are often preferred in emergency or critical situations. The clinically common glucocorticoid medicaments comprise prednisone, methylprednisolone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone and the like. Has antiinflammatory, antitoxic, antiallergic, antishock, nonspecific immunity inhibiting and antipyretic effects, and can prevent and stop immune inflammatory reaction and pathological immune reaction, and is almost effective for allergic diseases of any type.

Since the application of glucocorticoid drugs to the treatment of rheumatoid arthritis was first reported in the journal of the rhematic diseases in 1949 by p.hench, e.kendal and t.reichstein, glucocorticoid drugs are widely used in clinical treatments such as inflammation, autoimmune diseases and organ transplantation due to their excellent anti-inflammatory activity and immunosuppressive action. The main causes of the clinical glucocorticoid therapy at present comprise inflammatory diseases, rheumatic diseases (such as rheumatic arthritis, connective tissue diseases and lupus erythematosus), respiratory diseases (asthma and chronic obstructive pulmonary disease) and the like. Although glucocorticoids have good pharmacological activity and are widely used clinically, their side effects after long-term use do not vary considerably.

The side effects of glucocorticoid drugs are mainly manifested in the following aspects: (1) induction or exacerbation of infection; (2) the mass application of glucocorticoid for a long time can cause the disturbance of the mass metabolism and the water salt metabolism; (3) complications of cardiovascular system, long-term application of glucocorticoid can cause sodium, water retention and blood lipid increase, and can induce hypertension and atherosclerosis; (4) neuropsychiatric effects, glucocorticoids can cause many forms of behavioral abnormalities, such as nervousness, agitation, insomnia, affective changes, and the like; (5) inducing glucocorticoid-induced osteoporosis or bone fracture, wherein long-term application of glucocorticoid induces cell cycle arrest and inhibits proliferation of osteoblast lineage cells; inducing apoptosis in cells of the osteogenic lineage; the resulting bone loss can increase the release of a nuclear factor-K beta receptor activator factor ligand (RANKL), promote the differentiation and activation of osteoclasts, inhibit the expression of Osteoprotegerin (OPG), and reduce the proportion of OPG/RANKL, so that the number of osteoclasts is increased, and the damage of bone microstructure is accelerated; inhibiting osteogenic differentiation and function of cells; (6) causing obesity and cushing's syndrome.

Runx2 is Runt-related transcription factor 2, belonging to the Runt-related transcription factor family. Runx2 is a transcription factor specific to osteogenic differentiation, and is capable of regulating the transcription of many genes and up-regulating SP⁷Expression of various bone matrix protein genes such as OPN and OCN. Meanwhile, the gene is also an important regulatory factor for differentiation from mesenchymal cells to osteoblasts, when a osteoblast Runx2 gene is deleted, the differentiation of the osteoblast Runx2 gene is inhibited, researchers use a Runx2 gene knockout mouse to research the function of Runx2 in osteogenic differentiation, and find that the Runx2 gene knockout mouse loses the capability of effectively differentiating osteoblasts and the ossification phenomenon in periosteum and cartilage disappears. The results show that the Runx2 deletion can inhibit the osteogenic differentiation of osteoblasts so as to cause the body to generate ossification disorder.

Osteopontin (OPN) is synthesized and secreted by osteoblasts, osteoclasts, and osteocytes in bone tissue, and is involved in cell migration, adhesion, proliferation, bone remodeling and mineralization, signal transduction, and the like. In physiological conditions, bone formation and bone resorption are in dynamic equilibrium, and when this equilibrium is broken, diseases associated with bone metabolism may occur. In recent years, a plurality of researches find that OPN has irreplaceable position in bone metabolism and can be an important link for causing osteoporosis. The research finds that OPN has a complex dual regulation mechanism on osteoblasts: on one hand, OPN can transform osteoblasts to a mature state, and is one of the markers of osteoblast differentiation and maturation; on the other hand, OPN can promote the expression up-regulation of OPG/RANKL and the proliferation of osteoblasts.

Osteocalcin (OCN), also called Bone Gla Protein, or Bone gamma-carboxyglutamic acid Protein (Bone gamma-carboxyglutamic acid bound Protein, BGLAP or BGP), or Bone-Dependent Vitamin K Protein (Bone Vitamin K Dependent Protein), is a structural Protein specifically synthesized and secreted by osteoblasts, odontoblasts and hypertrophic chondrocytes, and belongs to one of the components constituting the Bone matrix. Glutamate residues in osteocalcin molecules are converted to gamma-carboxyglutamic acid (Gla) residues by vitamin K dependent post-translational modifications. The gamma-carboxylated osteocalcin molecule contains 3 Gla residues. Active vitamin D promotes the biosynthesis of gamma-carboxylated osteocalcin in osteoblasts. Gamma-carboxylated osteocalcin, called active osteocalcin, is capable of binding calcium through Gla residues. Research proves that osteocalcin can maintain normal mineralization rate of bones, inhibit formation of abnormal Hydroxyapatite (HA) crystals, and is a sensitive and reliable index for reflecting metabolic changes of bones of organisms. Since a fraction of osteocalcin molecules are secreted into the blood, serum osteocalcin levels are used as an index for bone formation and bone metabolic disorders.

Glucocorticoid excess results in increased bone fragility, a high risk factor for patients to develop fractures. Glucocorticoid-induced osteoporosis occurs at a high rate in various types of osteoporosis, and is not easily found by most patients, with latent symptoms. Long-term glucocorticoid treatment of patients leads to a decrease in bone mineral density and the risk of fractures, and high risk groups should begin bone protection therapy as early as possible. At present, bisphosphonates are the most widely clinically used anti-osteoporosis drugs. Research shows that the bisphosphonates type anti-osteoporosis medicine can effectively improve the bone density of lumbar vertebrae, neck of femur and total hip, and reduce the risk of vertebral fracture. The american college of rheumatology updated the glucocorticoid-induced osteoporosis guidelines in 2017. The guidelines strongly recommend teriparatide, raloxifene, denosumab and bisphosphonates as first line drugs for glucocorticoid-induced osteoporosis. However, studies have found that if osteoporosis is treated with bisphosphonates for a long period of time, the risk of atypical femoral fractures is increased, and patients are advised to take for 3 or more years, the patient's condition needs to be evaluated to determine if continued medication is needed. If the teriparatide is used, daily subcutaneous injection is needed, cold storage is needed, and the compliance of patients is reduced. Meanwhile, the medicine has high cost and more side effects such as dizziness, depression, headache and the like, and particularly, patients with renal calculus or patients with increased baseline level of parathyroid hormone need to be cautiously and closely observed. Therefore, the continuous development of the medicament for treating glucocorticoid-induced osteoporosis, which is low in price, safe and effective, is particularly important.

Kaempferide is a naturally occurring flavonoid, is mainly extracted from camphor tree and Chinese redbud leaves, and has the functions of antioxidation, antibiosis, anti-inflammation and blood sugar reduction. Whether the kaempferitrin has the effect of resisting Glucocorticoid Induced Osteoporosis (GIOP) is not found, and no relevant research report is found. The kaempferide serving as a natural nontoxic chemical with rich resources has extremely high medicinal value and wide application prospect.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide the application of kaempferitrin in relieving the side effect of glucocorticoid.

The purpose of the invention is realized by the following technical scheme: the application of kaempferide in relieving side effects of glucocorticoid is disclosed, wherein the kaempferide has a chemical formula shown in formula I, and a compound shown in formula I or a pharmaceutically acceptable salt, ester or prodrug thereof is applied to preparation of a medicament for relieving side effects of glucocorticoid:

in one aspect, the alleviating glucocorticoid side effects is achieved by modulating at least one selected from the group consisting of mRNA expression of Runx2, mRNA expression of OPN, mRNA expression of OPG, mRNA expression of bgalap, protein expression of Runx2, protein expression of OPN, protein expression of OPG, and protein expression of bgalap, or modulating alkaline phosphatase activity of osteoblasts, or modulating mineralization of osteoblasts. Preferably, the alleviating glucocorticoid side effects is achieved by promoting at least one selected from mRNA expression of Runx2, mRNA expression of OPN, mRNA expression of OPG, mRNA expression of bgalap, protein expression of Runx2, protein expression of OPN, protein expression of OPG, and protein expression of bgalap, or promoting alkaline phosphatase activity of osteoblasts, or promoting mineralization of osteoblasts.

In one aspect, the glucocorticoid side effect is selected from the group consisting of: obesity and cushing's syndrome, induced infection, aggravated infection, substance metabolism disorder, water and salt metabolism disorder, cardiovascular system complications, water and sodium retention, hyperlipidemia, hypertension, atherosclerosis, nervousness, neurological abnormality, insomnia, catabolism of skeletal muscle, osteoporosis, bone fracture, cell cycle block of osteoblast lineage cells, promotion of differentiation of osteoclast cells, promotion of activation of osteoclast cells, induction of apoptosis of osteoblast lineage cells, and inhibition of osteogenic differentiation. Preferably, the glucocorticoid side effect is selected from the group consisting of: at least one of catabolism of skeletal muscle, osteoporosis, bone fracture, cell cycle arrest of osteoblast lineage cells, promotion of differentiation of osteoclasts, promotion of activation of osteoclasts, induction of apoptosis of osteoblast lineage cells, and inhibition of osteogenic differentiation.

The invention also provides application of the compound shown in the formula I in preparing a medicament for treating diseases related to Runx2 expression inhibition, OPN expression inhibition, OPG expression inhibition, Bglap expression inhibition, alkaline phosphatase activity inhibition of osteoblasts, or osteoblast mineralized nodule formation inhibition caused by glucocorticoid.

In one aspect, the glucocorticoid is selected from prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, or dexamethasone. Preferably, the glucocorticoid is selected from prednisolone, hydrocortisone or dexamethasone.

In one aspect, the disease is selected from at least one of obesity and cushing's syndrome, induced infection, exacerbated infection, substance metabolism disorder, water and salt metabolism disorder, cardiovascular system complications, water and sodium retention, hyperlipidemia, hypertension, atherosclerosis, nervousness, neurological abnormality, insomnia, catabolism of skeletal muscle, osteoporosis, bone fracture, cell cycle arrest of osteoblastic lineage cells, promotion of differentiation of osteoclasts, promotion of activation of osteoclasts, induction of apoptosis of osteoblastic lineage cells, and inhibition of osteogenic differentiation.

The invention also provides application of the compound shown in the formula I in preparing a reagent for regulating Runx2 expression.

The invention also provides the use of a compound of formula I in the preparation of an agent for modulating OPN expression.

The invention also provides the use of a compound of formula I in the preparation of an agent for modulating OPG expression.

The invention also provides application of the compound shown in the formula I in preparing a reagent for regulating Bglap expression.

The inventor of the invention develops kaempferide related experimental research through creative work, finds that kaempferide can obviously relieve the inhibition of the activity and the mineralization of alkaline phosphatase of MC3T3-E1 cells caused by dexamethasone, and further finds that kaempferide can relieve the inhibition of mRNA and protein of osteogenic related Runx2, OPN, Bglap and OPG caused by dexamethasone. The research results are combined to show that the kaempferide is creatively found to be capable of remarkably relieving osteogenic differentiation inhibition caused by glucocorticoid and promoting dexamethasone to induce osteogenic differentiation of MC3T3-E1 cells. The experiment result discovers a molecular action mechanism of the compound in the aspect of reducing the side effect of glucocorticoid medicaments, and has important clinical significance for developing new medicaments.

Drawings

FIG. 1 is a graph of the effect of kaempferitrin of the present invention on the activity of MC3T3-E1 cells (. about.P <0.05vs Control);

figure 2 is the effect of kaempferitrin on the inhibition of MC3T3-E1 cell alkaline phosphatase by dexamethasone (. about.p <0.01), OM: osteogenic induction group, DEX: a dexamethasone group;

figure 3 is the effect of kaempferitrin on dexamethasone-induced inhibition of MC3T3-E1 cell mineralization (. about.p <0.01), OM: osteogenic induction group, DEX: a dexamethasone group;

fig. 4 is the effect of kaempferitrin on osteogenesis-related genes in the inhibition of osteogenic differentiation of MC3T3-E1 cells by dexamethasone (. P <0.05,. P <0.01), OM: osteogenic induction group, DEX: a dexamethasone group;

fig. 5 is the effect of kaempferitrin on osteogenesis-related proteins in the inhibition of MC3T3-E1 cell osteogenic differentiation by dexamethasone (. P <0.05,. P <0.01), OM: osteogenic induction group, DEX: dexamethasone group.

Detailed Description

For the understanding of those skilled in the art, the present invention will be further described with reference to the following examples and drawings, which are not intended to limit the present invention.

The term "pharmaceutically acceptable salts or esters" denotes the relatively non-toxic inorganic and organic acid addition salts or esters, and base addition salts, of the compounds of formula I of the present invention. These salts or esters can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts or esters may be prepared by separately reacting the purified compound in free base form with a suitable organic or inorganic acid and isolating the salt or ester so formed. Exemplary acid addition Salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphylate, methanesulfonate, glucoheptonate, lactobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisate, isothionate, ditoluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinic acid salt laurylsulfonate (quinatelsulfonate), and the like (see, e.g., Berge et al, "Pharmaceutical Salts", j.pharm.sci., 66: 1-9(1977) and Remington's Pharmaceutical Sciences, 17 th edition, Mack Publishing Company, Easton, Pa., 1985, page 1418, hereby incorporated by reference in its entirety). Base addition salts may also be prepared by separately reacting the purified acid form of the compound with a suitable organic or inorganic base and isolating the salt so formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminium salts. Sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine base addition salts are prepared from amines which are sufficiently basic to form stable salts, and preferably include those amines commonly used in medicinal chemistry due to their low toxicity and acceptability for pharmaceutical use, examples of such amines include ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids such as lysine and arginine, dicyclohexylamine, and the like.

The term "pharmaceutically acceptable prodrug" as used herein means a prodrug of a compound that can be used in accordance with the present invention, and, where possible, the zwitterionic form of the compound of the present invention, which prodrug, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for the intended use. The term "prodrug" means a compound that is rapidly transformed in vivo, for example by hydrolysis in blood, to yield the parent compound of the above formula. Functional groups that can be rapidly converted by metabolic cleavage in vivo form a class of groups that react with the carboxyl groups of the compounds of the present invention. The functional group includes, but is not limited to, groups such as alkanoyl (e.g., acetyl, propionyl, butyryl, etc.), unsubstituted and substituted aroyl (e.g., benzoyl and substituted benzoyl), alkoxycarbonyl such as ethoxycarbonyl), trialkylsilyl (e.g., trimethyl and triethylsilyl), monoesters with dicarboxylic acids (e.g., succinyl), and the like. Since the metabolically cleavable group of the compounds useful in the present invention is readily cleaved in vivo, compounds bearing such a group may act as prodrugs. Compounds carrying metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of the improved solubility and/or absorption rate imparted to the parent compound by the presence of the metabolically cleavable group. The following documents provide a thorough discussion of prodrugs: design of Prodrugs, H.Bundgaard eds, Elsevier (1985); methods in Enzymology, K.Widder et al, Academic Press, 42, 309 & 396 (1985); a Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H.Bundgaard, Chapter 5; "Design and Applications of produgs" pp.113-191 (1991); advanced Drug Delivery Reviews, h.bundgard, 8, pages 1-38 (1992); journal of Pharmaceutical Sciences, 77: 285 (1988); nakeya et al, chem.pharm.bull, 32: 692 (1984); higuchi et al, "Pro-drugs as Novel Delivery Systems", volume 14 of a.c.s.symposium Series, and Bioreversible carrier in Drug Design, Edward b.roche editors, American Pharmaceutical Association and Pergamon Press (1987), which are incorporated herein by reference in their entirety.

Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention.

The term "therapeutically effective amount" is meant to describe an amount of a compound of the present invention that is effective to produce the desired therapeutic effect. Such amounts typically vary depending on several factors, and the variations are within a range that can be determined and calculated by one of ordinary skill given the description provided herein. These factors include, but are not limited to: the particular individual and its age, weight, height, general physical condition and medical history, the particular compound used, the carrier in which the compound is formulated, the route of administration of the selected compound, and the nature and severity of the condition being treated.

The term "pharmaceutical composition" means a composition comprising a compound of formula (I) and, depending on the mode of administration and on the nature of the dosage form, at least one pharmaceutically acceptable ingredient selected from the group consisting of: carriers, diluents, adjuvants, excipients or excipients, for example preservatives, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example sugars, sodium chloride and the like are preferably included. Prolonged absorption of the injectable form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents or excipients include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (e.g. olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, sodium citrate, calcium carbonate, dicalcium phosphate. Examples of disintegrants include starch, alginic acid and some complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycols.

The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable dosage form" means a dosage form of the compounds of the present invention, including, for example, tablets, troches, powders, elixirs, syrups, liquid preparations (including suspensions, sprays, inhalation tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories), and liquid preparations for injection, including liposomal preparations. Formulation techniques and formulations are generally found in Remington's Pharmaceutical Science, Mack Publishing co., Easton, PA, latest edition.

The compound of the present invention can be produced by various known methods, and is not particularly limited.

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Examples

Proliferation assay of MC3T3-E1 cells

1.1 drugs and reagents

MC3T3-E1 Cell line (Cell bank, national academy of sciences), Australian Fetal bovine serum (Fetal bovine serum, FBS) (Mun Hai bioengineering Co., Ltd., Lanzhou), alpha-MEM medium (Vc-free) (GIBCO, USA), Dexamethasone (Dexamethane, Dex) (Sigma, USA), beta-Sodium glycerophosphate (Sodium beta-glycerophosphophosphate, beta-GP) (Sigma, USA), ascorbic acid (vitamin C, Vc) (Sigma, USA), NBT/BCIP staining reagent (Sigma, USA), CCK-8 detection Kit (Cell Counting Kit-8, CCK-8) (Japan Co., Ltd.), alkaline phosphatase activity detection Kit (Bilyunnan Biotech Co., Ltd.), Cetylpyridinium Chloride (Cetylpyridinium Chloride, CPC), BCA protein concentration determination Kit (Bilyunnan Biotech Co., Ltd.), MgCl Biotechnology Ltd.), and Chlam₂·6H₂O (west longus chemical plant, guangdong Shantou city), absolute ethanol (mao chemical reagent plant, Tianjin city), alizarin red (Sigma, USA), dimethyl sulfoxide (DMSO), kaempferide (Kdmanst Biotech, Inc.).

1.2 MC3T3-E1 cell culture

After recovering MC3T3-E1 cells from a liquid nitrogen tank, MC3T3-E1 cells were continuously cultured in an alpha-MEM medium (not containing Vc) containing 10% fetal bovine serum at 37 ℃ in a 5% CO2 incubator, the medium was changed every 3 days, and when the cells had grown to about 90% of the bottom of the flask, they were digested with 0.25% pancreatic enzyme, centrifuged at 900rpm for 2min, and the cells were collected to perform the following experiment.

1.3 CCK-8 method for detecting influence of kaempferitrin on MC3T3-E1 cell activity

The digested MC3T3-E1 cells were prepared into a uniform single cell suspension in an alpha-MEM complete medium, and the cell density was adjusted to 3X 10 by cell count³Per well; each well was inoculated with 100. mu.l of cell suspension in a 96-well plate, and then Dioxo-dioxide was addedAnd (5) culturing in a carbon incubator. After 24h cells were attached, the cells were grouped as follows: 0 mu M kaempferide glycoside (Control group), 1 mu M kaempferide glycoside group, 5 mu M kaempferide glycoside group, 10 mu M kaempferide glycoside group, 20 mu M kaempferide glycoside group, 40 mu M kaempferide glycoside group, 60 mu M kaempferide glycoside group, 80 mu M kaempferide glycoside group and 100 mu M kaempferide glycoside group. And detecting the cell proliferation condition by using a CCK-8 detection kit 48h and 72h after the administration. Adding 100 mul of CCK-8 working solution, putting the solution into an incubator to continue culturing for 2h, taking out a 96-well cell culture plate after the incubation time is reached, measuring the absorbance of each well at the wavelength of 450nm of an enzyme labeling instrument, and comparing the OD (optical density) values among the groups.

The experimental results are as follows: as shown in FIG. 1, the experimental results show that kaempferitrin has no inhibitory effect on the activity of MC3T3-E1 after being cultured for 48h and 72h within the range of 1-80 μ M.

1.4 alkaline phosphatase staining and Activity detection of cells

Alkaline phosphatase (ALP) is a glycoprotein which is a marker for the early differentiation of osteoblasts, and the activity of ALP is closely related to the osteoblast differentiation of cells. The intensity of ALP activity in the early stages of osteogenic differentiation and functional maturation can be used as an index for evaluating bone formation and bone turnover. However, under the action of the glucocorticoid with a super-physiological quantity, the activity of alkaline phosphatase in the in vitro osteogenic differentiation process is obviously inhibited and the osteogenic differentiation capacity of cells is influenced. In the experiment, alkaline phosphatase staining and alkaline phosphatase activity quantitative detection are adopted to probe whether kaempferide has the effect of relieving the inhibition of the alkaline phosphatase activity of MC3T3-E1 cells caused by dexamethasone.

The experimental method comprises the following steps:

the digested MC3T3-E1 cells were prepared in alpha-MEM complete medium to give a uniform single cell suspension, and the cell density was adjusted to 2.5X 10 by cell counting⁴Per well; a24-well plate was used and 500. mu.l of cell suspension was inoculated per well.

Grouping processing: grouping experiments: the composition comprises a first osteogenic differentiation induction group (a control group), a second dexamethasone model group, a third dexamethasone +5 mu M kaempferitrin group, a fourth dexamethasone +10 mu M kaempferitrin group and a fifth dexamethasone +20 mu M kaempferitrin group. ALP staining: after 7 days of cell culture, ALP staining was performed, the staining procedure was as follows: firstly, absorbing and discarding the original culture medium, and adding 500 mu l of PBS into each hole for washing; absorbing PBS and fixing with 70% ethanol for 15 min; removing the fixing liquid by suction, adding an ALP buffer solution into each hole for balancing for 5min, and repeating twice; fourthly, adding 300 mul of newly prepared NBT/BCIP staining solution into each hole; incubating at 37 ℃ in a dark place for 15-30 min, or until obvious blue particles are generated; sixthly, absorbing and discarding the staining solution, stopping the reaction by using distilled water, and observing and photographing under a microscope.

ALP Activity assay: ALP activity in a sample was measured using an ALP activity kit, absorbance was measured at 405nm, and ALP activity in the sample was calculated according to the definition of the enzyme activity. Mapping and statistical analysis: plotting in Graphpad prism software, one-way analysis of variance.

The experimental results are as follows: as shown in FIG. 2, it was found that dexamethasone significantly inhibited the ALP activity of cells at 1 μ M, and the ALP staining became light. Compared with the dexamethasone model group, ALP staining was gradually deepened and ALP activity was gradually restored with the kaempferitrin group. The experimental results show that kaempferitrin can relieve the effect of inhibiting the activity of ALP of MC3T3-E1 cells caused by dexamethasone to a certain extent.

1.5 mineralized nodule staining and quantitative detection

The mineralized nodules mainly appear in the late osteoblast differentiation stage of cells, are hydroxyapatite crystals and amorphous calcium phosphate generated in the osteoblast differentiation process and secreted into extracellular matrix, are combined with osteocalcin, osteopontin, collagen and the like to form hydroxyapatite crystals, and are one of important indexes for judging the late osteoblast differentiation stage. Therefore, alizarin red staining and calcium nodule quantification are adopted in the experiment to study the influence of kaempferitrin on dexamethasone inhibition of MC3T3-E1 cell mineralization.

The experimental method comprises the following steps:

the digested MC3T3-E1 cells are prepared into a uniform single-cell suspension by using an alpha-MEM complete medium, and the cells are inoculated and cultured. Cell count was adjusted to 2.5X 10 cell density⁴Per well; a24-well plate was used and 500. mu.l of cell suspension was inoculated per well.

Grouping processing: grouping experiments: the composition comprises a first osteogenic differentiation induction group (a control group), a second dexamethasone model group, a third dexamethasone +5 mu M kaempferitrin group, a fourth dexamethasone +10 mu M kaempferitrin group and a fifth dexamethasone +20 mu M kaempferitrin group. After inoculation and culture to day 14, cell mineralization was observed using alizarin red staining. Alizarin red staining procedure was as follows: firstly, after cells are inoculated for 14 days, adding 500 mu l of PBS into each hole to wash the cells twice, secondly, absorbing the PBS, fixing the PBS by 70 percent of ethanol for 15min, thirdly, absorbing the fixing liquid, adding 500 mu l of 0.5 percent alizarin red solution (pH is 4.1) into each hole, fourthly, incubating for 15-30 min at 37 ℃ in a dark place, or until orange calcium knots appear, fifthly, absorbing the staining solution, adding distilled water into each hole to stop the reaction, repeating the reaction for two to three times, airing, observing under a microscope and taking pictures.

Calcium nodule staining quantitative analysis: a. adding 500 μ l 10% cetylpyridinium chloride solution into each well, and incubating at room temperature for 30 min; b. placing the mixture in a shaking table, oscillating for 15-30 min to fully dissolve the crystals, absorbing the solution into a 96-hole plate, and measuring the absorbance of each hole at the 562nm wavelength of an enzyme labeling instrument; c. statistical analysis: OD values were compared between groups and analyzed using Spss 17.0 one-way anova. d. Plotting: mapping was performed in Graphpad prism software.

The experimental results are as follows: as shown in FIG. 3, after MC3T3-E1 cells were subjected to group intervention for 14 days, alizarin red staining was found as follows: compared with the induction group, alizarin red staining of the dexamethasone group is obviously lightened, and mineralization is inhibited; adding kaempferitrin group, and gradually deepening alizarin red dyeing. And (3) quantitative results of mineralized nodules are found: compared with the induction group, the formation of mineralized nodules in the dexamethasone group is inhibited, and the formation of mineralized nodules is gradually recovered by adding the kaempferitrin group. The experimental results show that dexamethasone can obviously inhibit the formation of mineralized nodules of MC3T3-E1 cells, and kaempferitrin can relieve the inhibition effect of the mineralized nodules of MC3T3-E1 cells caused by dexamethasone.

1.6 Effect of Kaempferitrin on mRNA expression of Runx2, OPN, OPG and Bglap in MC3T3-E1 cells

And (3) real-time quantitative PCR detection: the digested MC3T3-E1 cells are prepared into a uniform single-cell suspension by using an alpha-MEM complete medium, and the cell count is 10⁵One/well was seeded in 6-well plates.

Grouping experiments: the composition comprises a first osteogenic differentiation induction group (a control group), a second dexamethasone model group, a third dexamethasone +5 mu M kaempferitrin group, a fourth dexamethasone +10 mu M kaempferitrin group and a fifth dexamethasone +20 mu M kaempferitrin group. On day 3, total cellular RNA was extracted with Trizal and reverse transcribed into cDNA using a cDNA synthesis kit. And (3) carrying out RT-PCR reaction on the cDNA by using a Real-Time fluorescent quantitative PCR kit and a 7500Real Time PCR instrument, and detecting the expression conditions of relevant genes Runx2, Bglap, OPN and OPG.

The target gene primer sequence: upstream of Runx2 is 5 '-gaatgcactacccagccac-3', and downstream is 5 '-tggcaggtacgtgtggtag-3'; OPN upstream "5 '-tccaaagccagcctggaac-3", downstream "5' -tgacctcagaagatgaactc-3"; OPG upstream "5 '-agaagaagactgtttctcaagcact-3", downstream "5' -gcctcactctccggactcag-3"; bglap upstream "5 '-ttctgctcactctgctgacc-3", downstream "5' -gccggagtctgttcactacc-3"; the amplification process of beta-actin upstream 5 '-gccaaccgtgaaaagatgac-3 and downstream 5' -accagaggcatacagggacag-3 comprises the following steps: 95 ℃, 30s, amplification: gene amplification was carried out at 95 ℃ for 5 seconds, 60 ℃ for 34 seconds, and 40 cycles. Use of beta-actin as an internal reference 2^-△△CtThe relative amount of mRNA expression of each gene group was determined by a relative quantitative method.

The experimental results are as follows: as shown in FIG. 4, the mRNA expression of the osteogenesis related genes Runx2, OPN, OPG and Bglap was detected by real-time fluorescent quantitative PCR. The result shows that dexamethasone can obviously inhibit mRNA expression of genes of Runx2, OPN, Bglap and OPG, kaempferitrin can relieve inhibition conditions of the genes of Runx2, OPN, Bglap and OPG caused by dexamethasone to a certain extent, and difference statistical significance is achieved.

1.7 Effect of Kaempferitrin on protein expression of Runx2, OPN, OPG and Bglap of MC3T3-E1 cells

Detection by western blotting: the digested MC3T3-E1 cells were prepared as a homogeneous single cell suspension in alpha-MEM complete medium at 10⁵One/well was seeded in 6-well plates.

Grouping experiments: the kit comprises a first osteogenic differentiation induction group (a control group), a second dexamethasone model group, a third dexamethasone +10 mu M kaempferitrin group and a fourth dexamethasone +20 mu M kaempferitrin group. After induced differentiation for 5 days, the cells were washed twice with PBS, lysed by RIPA method, centrifuged, and total protein was extracted. The BCA method is used for determining the protein concentration, and the Western blot is used for detecting the content of the target protein in the cells. The sample is denatured by heating at 95 ℃ for 5min after adding the loading buffer. Each well was loaded with 30. mu.L of total protein for SDS-PAGE. After electrophoresis, semi-dried protein transfer is carried out on a cut electrotransformation membrane, and after electrotransformation is finished, the electrotransformation membrane is sealed by 5 percent of skimmed milk powder and is kept at 37 ℃ for 2 hours. The primary antibodies were Runx2(Cell Signal Technology,1:1000), OPN (Cell Signal Technology,1:1000), Bglap (Cell Signal Technology,1:1000), OPG (Cell Signal Technology,1:1000), beta-actin (Cell Signal Technology,1:5000), respectively. Incubating at 4 deg.C overnight, after primary antibody incubation, taking out protein band from the primary antibody mixed solution, placing into a container containing 1 × TBST, rapidly shaking and rinsing on a horizontal shaking table for 5min, and discarding and repeating for 3-4 times; placed into pre-prepared secondary antibody dilution (type of incubation secondary antibody selected according to antibody instructions) and incubated with slow shaking at room temperature for 1 h.

Developing and analyzing results: after the PVDF membrane is rinsed, the ECL developing solution which is prepared in situ is added, and the PVDF membrane is placed under a gel imaging system for exposure. And after copying the data, performing gray level analysis on the result by using Image J or other software.

The experimental results are as follows: as shown in FIG. 5, we used Western blotting to detect the expression of the osteogenesis-related proteins Runx2, OPN, Bglap and OPG. Experimental results show that dexamethasone can obviously inhibit the protein expression of Runx2, OPN, Bglap and OPG, kaempferitrin can relieve the protein inhibition of Runx2, OPN, Bglap and OPG caused by dexamethasone to a certain extent, and the difference has statistical significance.

Statistical analysis: statistical analysis is carried out on experimental data by adopting SPSS 20.0 software, single-factor variance analysis is adopted for comparison of differences among groups, and an LSD multiple test method is adopted for pairwise comparison. P <0.05 indicates that the difference is statistically significant.

The above-described embodiments are preferred implementations of the present invention, and the present invention may be implemented in other ways without departing from the spirit of the present invention.