WO2023143221A1 - Composition for preventing or treating osteoporosis, preparation thereof and use thereof - Google Patents

Composition for preventing or treating osteoporosis, preparation thereof and use thereof Download PDF

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WO2023143221A1
WO2023143221A1 PCT/CN2023/072505 CN2023072505W WO2023143221A1 WO 2023143221 A1 WO2023143221 A1 WO 2023143221A1 CN 2023072505 W CN2023072505 W CN 2023072505W WO 2023143221 A1 WO2023143221 A1 WO 2023143221A1
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composition
extract
preparation
gpx4
osteoporosis
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PCT/CN2023/072505
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French (fr)
Chinese (zh)
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何蓉蓉
李怡芳
栗原博
张琼谊
江曼亚
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暨南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of medicine, and in particular relates to a composition for preventing or treating osteoporosis and its preparation and application.
  • Osteoporosis is a systemic bone metabolic disease, and its main pathological features are decreased bone mineral density (BMD), impaired bone microstructure/mineralization, and increased fracture risk.
  • BMD bone mineral density
  • impaired bone microstructure/mineralization impaired bone microstructure/mineralization
  • increased fracture risk Modern society is an aging society. With the aging of the population and the extension of life expectancy, the incidence of osteoporosis and osteoporotic fractures continues to rise. Osteoporosis has become a global health problem. According to incomplete statistics, more than 200 million people worldwide suffer from osteoporosis. In addition, the epidemiological survey data of the National Health Commission in 2018 showed that the prevalence of osteoporosis among people over 65 years old in my country was as high as 32%. Fragility fractures of the spine, hip, and wrist caused by osteoporosis seriously affect the quality of life of patients and bring huge economic and medical burdens to the society. Therefore, the drug treatment of osteoporosis has always been an important area of concern for drug researchers
  • osteoporosis research on drugs for the treatment of osteoporosis is mainly based on two strategies.
  • One is to increase the differentiation of osteoblasts and promote the development of drugs for the purpose of bone formation, which aims to supplement the missing bone tissue by increasing osteoblasts and bone cells , to increase bone mass; the other is developed for the purpose of promoting osteoclast apoptosis and inhibiting osteoclast activation, aiming at reducing bone resorption, alleviating the continuous loss of bone mass, and improving osteoporosis and related symptoms.
  • the clinical drugs for the treatment of osteoporosis are through the above two mechanisms.
  • the commercially available drugs are mainly calcium preparations, bisphosphonates, estrogens, androgens, and vitamin D3.
  • Oxidative stress produces a large number of reactive oxygen species (ROS), on the one hand, increases the proliferation and resorption activity of osteoclasts, and promotes bone resorption; on the other hand, ROS promotes the apoptosis of osteoblasts and inhibits the formation of osteoblasts. Differentiate function, reduce bone formation, eventually lead to or exacerbate osteoporosis. Therefore, finding drugs to improve the body's oxidative stress state, inhibit ROS generation or scavenge ROS has gradually become a new focus, new trend and new trend in the research and development of osteoporosis drugs.
  • ROS reactive oxygen species
  • GPX4 and Alox15 play an important role in oxidative stress, especially phospholipid peroxidation, and vitamin E can effectively reduce the level of phospholipid peroxidation in cells and the body, and improve the state of oxidative stress in the body.
  • vitamin E can effectively reduce the level of phospholipid peroxidation in cells and the body, and improve the state of oxidative stress in the body.
  • GPX4 agonist, Alox15 inhibitor and vitamin E can more effectively scavenge ROS, especially lipid ROS, improve the body's oxidative stress state, and correct the function of osteoclasts and osteoblasts.
  • Alox15 is closely related to osteoporosis, the low expression of Alox15 gene can effectively increase the bone density value of mice, and Alox15 inhibitor can effectively improve the degree of osteoporosis in ovariectomized mice.
  • the present invention provides a method for preventing or treating osteoporosis, more specifically, a composition for preventing or treating osteoporosis, its preparation and application.
  • the present invention provides a composition for preventing or treating osteoporosis, the composition comprising a GPX4 agonist and an Alox-15 inhibitor and vitamin E, wherein the GPX4 agonist Enhance the activity of endogenous antioxidant enzyme GPX4, enhance the clearance of lipid peroxides in the body, so
  • the above-mentioned Alox-15 inhibitor inhibits the activity of endogenous lipoxygenase 15, reduces the production of lipid peroxide, and at the same time supplements the small molecule reducing agent vitamin E to directly remove the accumulation of lipid peroxidation, and the three play a synergistic role .
  • the weight ratio of the GPX4 agonist, the Alox-15 inhibitor, and the vitamin E is 0.5-5:1-5:0.5-5.
  • the weight ratio of the GPX4 agonist, the Alox-15 inhibitor, and the vitamin E is 1:4:1.
  • the GPX4 agonist is selected from one or more of 6-gingerol, 8-gingerol, 10-gingerol, and PKUMDL-LC-101-D04;
  • the Alox-15 inhibitor is selected from one or more of quercetin, ferulic acid, baicalein, hesperetin, nobiletin, PD146176, orange flavone, and ML351.
  • the GPX4 agonist is selected from 6-gingerol, 8-gingerol or 10-gingerol;
  • the Alox-15 inhibitor is selected from quercetin, ferulic acid, baicalein, hesperetin or Sichuan Dermoside.
  • the GPX4 agonist is selected from 8-gingerol; the Alox-15 inhibitor is selected from quercetin.
  • the composition comprises ginger extract (GPX4 agonist), seabuckthorn extract (Alox-15 inhibitor) and wheat germ extract (vitamin E source), and the ginger
  • the weight ratio of the extract, the seabuckthorn extract, and the wheat germ extract is 2:5:3.
  • the ginger extract includes water extraction or organic solvent extraction from ginger and dried ginger, and the organic solvent includes lower alcohol ethanol and ethyl acetate;
  • the sea buckthorn extract It includes extracting from seabuckthorn fruit and seabuckthorn leaves;
  • the wheat germ extract is obtained from wheat germ through absolute ethanol extraction.
  • the extraction of the composition includes hot water, or a solvent in which one or two or more of water, lower alcohols such as methanol and ethanol, and polar solvents such as acetone are arbitrarily mixed for extraction.
  • water and ethanol as the extraction solvent as much as possible from the perspective of safety.
  • the present invention provides a preparation for preventing or treating osteoporosis, said preparation comprising the composition described in the first aspect above, and a suitable carrier or adjuvant.
  • the preparation is an oral dosage form.
  • the oral dosage form is selected from tablets, capsules, oral liquids, enteric-coated tablets, granules, syrups, dripping pills, honeyed pills, powders, mixtures, decoctions, liquors, dews, tinctures or tongue Lower the lozenge.
  • the dosage form may also be an external dosage form.
  • external use include but are not limited to ointments, suppositories, hard ointments, liniments, eye drops, oils, creams, aerosols, and sprays.
  • composition provided by the invention can also be added into food and health food products as powder for oral administration.
  • the composition can also be combined with other raw materials to produce certain beverage products, such as juice drinks, sports drinks and the like.
  • the present invention provides the use of the composition described in the first aspect above or the preparation described in the second aspect above in the preparation of products for preventing or treating osteoporosis.
  • the product is medicine, health product or functional food.
  • the osteoporosis includes osteoporosis-related complications, related diseases and related clinical symptoms.
  • the present invention provides the application of the above-mentioned composition in osteoporosis, of course, it also has a good effect on bone-related diseases such as osteopenia, fragility fractures and traumatic fractures related to the inhibition of osteoblast differentiation activity or the increase of osteoclast activity. To improve the effect.
  • the present invention has the following beneficial effects:
  • the formulation of the composition for preventing or treating osteoporosis provided by the present invention has been screened, and when it plays the role of inhibiting the proliferation of osteoclasts and promoting the differentiation of osteoblasts, it is not played by one or several components. It is the result of the synergistic effect of each component, especially under the condition of a specific weight ratio, the synergistic effect is the best, so that it can obviously improve osteoporosis in application.
  • test materials used in the following examples are commercially available products unless otherwise specified.
  • Bone marrow mononuclear cells were isolated from the femur and tibia of a 1-month-old mouse (purchased from Guangdong Provincial Medical Experimental Animal Center), spread evenly in a 24-well plate, with 1 ⁇ 105 cells per well, and after the cells adhered to the wall, use The ⁇ -MEM medium containing 25ng/ml M-CSF, 25ng/ml RANKL cytokines and 10% fetal bovine serum was cultured. At the same time, except the normal control group, other groups were added with different pharmaceutical compositions, every 3 days Change medium once. After 6 days, the data of osteoclasts in each group were calculated by using TRAP staining.
  • the positive number of osteoclasts was defined as: TRAP-positive cells with more than 3 nuclei.
  • E A , E B , and E C are the inhibition rates of A drug group, B drug group, C drug group, and three-drug combination group, respectively.
  • q ⁇ 1 means that the combination of the three drugs produces an antagonistic effect;
  • q>1 means that the combination of the three drugs produces a synergistic effect;
  • the composition can significantly inhibit the formation of osteoclasts in vitro, and its inhibition rate can be as high as 86.67%, while the inhibitory effects of 8-gingerol, quercetin and vitamin E at the same dose are all low In the composition, and when the ratio of the composition is 1:4:1, it has the best inhibitory effect on osteoclasts.
  • the above results demonstrate from the side that the effect of the composition provided by the present invention on inhibiting the activation of osteoclasts is not due to one or several components, but the result of the synergistic and joint action of each component.
  • Table 1 The number and differentiation inhibition rate of osteoclasts Note: The data are expressed as mean ⁇ SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0. Compared with normal group, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001.
  • Example 2 Effect of composition on bone mineral density and trabecular bone in ovariectomized mice
  • mice Female C57BL/6 mice, were purchased from Guangdong Medical Experimental Animal Center. The mice were kept at a temperature of 23 ⁇ 2°C, a humidity of 55 ⁇ 5%, a ventilation frequency of 12-15 times/hour, and a lighting time of 12 hours/day (7:00-19:00 light), 5/breeding cage ( 235 ⁇ 325 ⁇ 170H mm), feeding conditions: solid feed (Guangdong Medical Experimental Animal Center), free diet. When the mice were 3 months old, the mice were randomly divided into a sham operation group, a model group and an administration group. Mice were fasted for 12 hours before surgery water. Mice were anesthetized using isoflurane, then the skin was washed and sterilized with 75% ethanol.
  • mice in each group were operated by ventral incision. Except the sham operation group, the ovaries were not removed, and the mice in the other groups were subjected to bilateral ovariectomy. All operated mice were intraperitoneally injected with penicillin solution. After the operation, the operated animals were placed in an incubator and transferred to a clean breeding cage to recover for 1 week after the animals woke up. During this period, penicillin solution was injected intraperitoneally every other day to avoid wound infection of the mice.
  • mice in each administration group were given the corresponding dose of drugs once a day for 2 consecutive months. After 12 hours of the last administration, the mice were anesthetized, and the right hind limbs of the mice were taken for scanning Bone mineral density (BMD) and trabecular number.
  • BMD Bone mineral density
  • the experimental results are shown in Table 2. Compared with the mice in the sham operation group, the bone mineral density and the number of bone trabeculae in the model group were significantly reduced. After administration of 8-gingerol, quercetin or vitamin E alone, the bone mineral density value and trabecular number of ovariectomized mice were improved, and after the combination was given, the bone mineral density value and trabecular bone The improvement effect of the number is more significant, and the combined use of 8-gingerol, quercetin and vitamin E is better than the combined use of 6-gingerol, quercetin and vitamin E.
  • Table 2 BMD and Trabecular Number of Ovariectomized Mice Note: The data are expressed as mean ⁇ SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0. Compared with the sham operation group, *** P ⁇ 0.001; compared with the model group, # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001.
  • Example 3 Effect of the composition on the antioxidant capacity of bone tissue in ovariectomized mice
  • the antioxidant capacity of mouse bone tissue was determined by oxidative radical absorbance capacity (Oxygen Radical Absorbance Capacity, ORAC) test.
  • Bone tissue sample pretreatment In Example 2, the left hind limb of the mouse was taken, and the muscle and connective tissue were stripped Finally, grind in liquid nitrogen and collect into a centrifuge tube, add perchloric acid solution (3%) to prepare 2% bone tissue homogenate, centrifuge at 10000r/min for 15min at 4°C, and take the supernatant as the ORAC assay. sample solution.
  • ORAC experiment process Add 20 ⁇ L bone tissue homogenate supernatant and 20 ⁇ L phosphate buffer saline of different groups of mice to the 96-well plate, then add 20 ⁇ L sodium fluorescein, and finally add 140 ⁇ L AAPH, and quickly place the 96-well plate in The assay was started in a fluorescent microplate reader with temperature control at 37°C. A point was measured every 2 minutes for a total of 2 hours. The ORAC value was calculated using the protection integral area corresponding to 1 ⁇ mol/L Trolox on the fluorescence decay curve as the standard control.
  • Example 4 Effect of composition on bone density of ovariectomized mice
  • mice The animal female C57BL/6 mouse used in this example was purchased from Guangdong Medical Experimental Animals Heart. The mice were raised at a temperature of 23 ⁇ 2°C, a humidity of 55 ⁇ 5%, a ventilation frequency of 12-15 times/hour, and a lighting time of 12 hours/day (7:00 ⁇ 19:00 light), 5 mice/cage ( 235 ⁇ 325 ⁇ 170H mm), feeding conditions: solid feed (Guangdong Medical Experimental Animal Center), free diet. When the mice were 3 months old, the mice were randomly divided into a sham operation group, a model group and an administration group. Mice were fasted for 12 hours before surgery, without food or water.
  • mice were anesthetized using isoflurane, then the skin was washed and sterilized with 75% ethanol.
  • the mice in each group were operated by ventral incision. Except the sham operation group, the ovaries were not removed, and the mice in the other groups were subjected to bilateral ovariectomy. All operated mice were intraperitoneally injected with penicillin solution. After the operation, the operated animals were placed in an incubator and transferred to a clean breeding cage to recover for 1 week after the animals woke up. During this period, penicillin solution was injected intraperitoneally every other day to avoid wound infection of the mice.
  • mice in each administration group were given the corresponding dose of drugs once a day for 2 consecutive months. After 12 hours of the last administration, the mice were anesthetized, and the right hind limbs of the mice were taken for scanning Bone mineral density (BMD) and trabecular number.
  • BMD Bone mineral density
  • Ginger extract, sea buckthorn extract and wheat germ extract can effectively increase the BMD value of ovariectomized mice, and when ginger extract, sea buckthorn extract and wheat germ extract are used in combination, there is no effect on ovarian
  • the BMD improvement effect of mice is more significant, and the effect is better when the weight ratio of ginger extract, seabuckthorn extract and wheat germ extract is 2:5:3.
  • Table 4 Effects of compositions of the present invention on BMD of ovariectomized mice Note: The data are expressed as mean ⁇ SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0. Compared with the sham operation group, *** P ⁇ 0.001; compared with the model group, # P ⁇ 0.05, ## P ⁇ 0.01.

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Abstract

A composition for preventing or treating osteoporosis, a preparation thereof, and use thereof in preventing or treating osteoporosis. The composition is mainly composed of a GPX4 agonist and a lipoxygenase 15(Alox-15) inhibitor, and cooperates with vitamin E to exert a synergistic effect. The GPX4 agonist comprises one or more of 6-gingerol, 8-gingerol, 10-gingerol and PKUMDL-LC-101-D04; the Alox-15 inhibitor comprises one or more of quercetin, ferulic acid, baicalein, hesperetin, nobiletin, PD146176, sinensetin and ML351; the composition comprises a ginger extract, a sea-buckthorn extract, and a wheat germ extract.

Description

一种预防或治疗骨质疏松症的组合物及其制剂和用途A composition for preventing or treating osteoporosis and its preparation and use
相关申请的交叉引用Cross References to Related Applications
本申请要求2022年01月29日提交的中国申请号202210112526.3的权益。所述申请号202210112526.3据此全文以引用方式并入本文。This application claims the benefit of Chinese application number 202210112526.3 filed on January 29, 2022. Said application number 202210112526.3 is hereby incorporated herein by reference in its entirety.
技术领域technical field
本发明属于医药领域，具体涉及一种预防或治疗骨质疏松症的组合物及其制备和用途。The invention belongs to the field of medicine, and in particular relates to a composition for preventing or treating osteoporosis and its preparation and application.
背景技术Background technique
骨质疏松症(osteoporosis,OP)是一种全身性的骨骼代谢疾病，其主要病理特征是骨密度值(bone mineral density,BMD)降低、骨骼微结构/矿化受损及骨折风险增加。现代社会是一个老龄化社会，随着人口老龄化和寿命的延长，骨质疏松和骨质疏松性骨折发生率不断攀升。骨质疏松已成为全球性的健康问题，据不完全统计，全球范围内有超过2亿人患有骨质疏松。此外，国家卫生健康委2018年的流行病学调查数据显示，我国65岁以上人群的骨质疏松患病率高达32％。骨质疏松引起的脊柱、髋部、腕部等部位的脆性骨折严重影响患者的生活质量，为社会带来巨大的经济及医疗负担。因此，骨质疏松症的药物治疗一直是药物研究者关注的重要领域。Osteoporosis (OP) is a systemic bone metabolic disease, and its main pathological features are decreased bone mineral density (BMD), impaired bone microstructure/mineralization, and increased fracture risk. Modern society is an aging society. With the aging of the population and the extension of life expectancy, the incidence of osteoporosis and osteoporotic fractures continues to rise. Osteoporosis has become a global health problem. According to incomplete statistics, more than 200 million people worldwide suffer from osteoporosis. In addition, the epidemiological survey data of the National Health Commission in 2018 showed that the prevalence of osteoporosis among people over 65 years old in my country was as high as 32%. Fragility fractures of the spine, hip, and wrist caused by osteoporosis seriously affect the quality of life of patients and bring huge economic and medical burdens to the society. Therefore, the drug treatment of osteoporosis has always been an important area of concern for drug researchers.
目前，在治疗骨质疏松药物研究方面，主要基于两种策略，一种以增加成骨细胞分化，促进骨形成为目的药物开发，旨在通过增加成骨细胞和骨细胞以补充缺失的骨组织，增加骨量；另一种则通过促进破骨细胞凋亡、抑制破骨细胞的活化为目的的药物开发，旨在通过降低骨吸收，缓解骨量持续丢失，改善骨质疏松及相关症状。目前，临床上骨质疏松的治疗药物正是通过以上两种机 制实现，市售药物主要为钙制剂、双磷酸盐类、雌激素类、雄激素类、维生素D3类等。但是，这些药物长期使用副作用较多，如长期使用双磷酸盐类药物对肾脏、肝脏和胃肠道等组织器官产生毒副作用；长期使用甲状旁腺激素类药物会增加骨肉瘤的发生风险。因此，研发安全性高、副作用低的、适合长期服用的用于预防、改善和治疗的骨质疏松的食品、保健食品和医药用品及组合物是非常必要且迫切的。At present, research on drugs for the treatment of osteoporosis is mainly based on two strategies. One is to increase the differentiation of osteoblasts and promote the development of drugs for the purpose of bone formation, which aims to supplement the missing bone tissue by increasing osteoblasts and bone cells , to increase bone mass; the other is developed for the purpose of promoting osteoclast apoptosis and inhibiting osteoclast activation, aiming at reducing bone resorption, alleviating the continuous loss of bone mass, and improving osteoporosis and related symptoms. At present, the clinical drugs for the treatment of osteoporosis are through the above two mechanisms. The commercially available drugs are mainly calcium preparations, bisphosphonates, estrogens, androgens, and vitamin D3. However, long-term use of these drugs has many side effects, such as long-term use of bisphosphonates can cause toxic side effects on tissues and organs such as the kidney, liver, and gastrointestinal tract; long-term use of parathyroid hormone drugs will increase the risk of osteosarcoma. Therefore, it is very necessary and urgent to develop foods, health foods, medical supplies and compositions for preventing, improving and treating osteoporosis that have high safety, low side effects and are suitable for long-term use.
近年来，氧化应激作为骨质疏松症的重要危险因子越来越受到学者和药物研发者的关注。氧化应激产生大量的活性氧自由基(reactive oxygen species,ROS)一方面增加破骨细胞的增殖和吸收活性，促进骨吸收；另一方面ROS促进成骨细胞的凋亡和抑制成骨细胞的分化功能，减少骨形成，最终导致或加剧骨质疏松。因此，寻找改善机体氧化应激状态，抑制ROS产生或清除ROS的药物也逐渐成为骨质疏松症药物研发和开发的新热点、新趋势和新潮流。In recent years, oxidative stress, as an important risk factor for osteoporosis, has attracted more and more attention from scholars and drug developers. Oxidative stress produces a large number of reactive oxygen species (ROS), on the one hand, increases the proliferation and resorption activity of osteoclasts, and promotes bone resorption; on the other hand, ROS promotes the apoptosis of osteoblasts and inhibits the formation of osteoblasts. Differentiate function, reduce bone formation, eventually lead to or exacerbate osteoporosis. Therefore, finding drugs to improve the body's oxidative stress state, inhibit ROS generation or scavenge ROS has gradually become a new focus, new trend and new trend in the research and development of osteoporosis drugs.
GPX4和Alox15在氧化应激，尤其是磷脂过氧化中扮演重要角色，且维生素E能有效的降低细胞和机体磷脂过氧化水平，改善机体氧化应激状态。联合使用GPX4激动剂、Alox15抑制剂和维生素E能更有效的清除ROS，特别是清除脂质ROS，改善机体氧化应激状态，矫正破骨细胞和成骨细胞的功能。此外，Alox15与骨质疏松症密切相关，Alox15基因低表达能有效增加小鼠的骨密度值，且Alox15抑制剂能有效改善去卵巢小鼠的骨质疏松程度。GPX4 and Alox15 play an important role in oxidative stress, especially phospholipid peroxidation, and vitamin E can effectively reduce the level of phospholipid peroxidation in cells and the body, and improve the state of oxidative stress in the body. Combined use of GPX4 agonist, Alox15 inhibitor and vitamin E can more effectively scavenge ROS, especially lipid ROS, improve the body's oxidative stress state, and correct the function of osteoclasts and osteoblasts. In addition, Alox15 is closely related to osteoporosis, the low expression of Alox15 gene can effectively increase the bone density value of mice, and Alox15 inhibitor can effectively improve the degree of osteoporosis in ovariectomized mice.
发明内容Contents of the invention
为了解决上述技术问题，本发明提供了一种预防或治疗骨质疏松症的方法，更具体地，一种预防或治疗骨质疏松症的组合物及其制剂和用途。In order to solve the above technical problems, the present invention provides a method for preventing or treating osteoporosis, more specifically, a composition for preventing or treating osteoporosis, its preparation and application.
具体地，通过以下几个方面的技术方案实现了本发明：Specifically, the present invention is realized through the technical solutions of the following aspects:
在第一个方面中，本发明提供了一种预防或治疗骨质疏松症的组合物，所述组合物包含GPX4激动剂和Alox-15抑制剂和以及维生素E，其中，所述GPX4激动剂增强内源性抗氧化酶GPX4的活性，增强机体脂质过氧化物的清除，所 述Alox-15抑制剂抑制内源性脂氧合酶15的活性，降低脂质过氧化物的产生，同时辅以小分子还原剂维生素E直接清除脂质过氧化的累积，三者协同发挥作用。In a first aspect, the present invention provides a composition for preventing or treating osteoporosis, the composition comprising a GPX4 agonist and an Alox-15 inhibitor and vitamin E, wherein the GPX4 agonist Enhance the activity of endogenous antioxidant enzyme GPX4, enhance the clearance of lipid peroxides in the body, so The above-mentioned Alox-15 inhibitor inhibits the activity of endogenous lipoxygenase 15, reduces the production of lipid peroxide, and at the same time supplements the small molecule reducing agent vitamin E to directly remove the accumulation of lipid peroxidation, and the three play a synergistic role .
作为可选的方式，在上述组合物中，所述GPX4激动剂、所述Alox-15抑制剂、所述维生素E的重量比为0.5-5：1-5：0.5-5。As an optional manner, in the above composition, the weight ratio of the GPX4 agonist, the Alox-15 inhibitor, and the vitamin E is 0.5-5:1-5:0.5-5.
优选地，所述GPX4激动剂、所述Alox-15抑制剂、所述维生素E的重量比为1：4：1。Preferably, the weight ratio of the GPX4 agonist, the Alox-15 inhibitor, and the vitamin E is 1:4:1.
作为可选的方式，在上述组合物中，所述GPX4激动剂选自6-姜酚、8-姜酚、10-姜酚、PKUMDL-LC-101-D04中的一种或多种；所述Alox-15抑制剂选自槲皮素、阿魏酸、黄芩素、桔皮素、川皮苷、PD146176、橙黄酮、ML351中的一种或多种。As an alternative, in the above composition, the GPX4 agonist is selected from one or more of 6-gingerol, 8-gingerol, 10-gingerol, and PKUMDL-LC-101-D04; The Alox-15 inhibitor is selected from one or more of quercetin, ferulic acid, baicalein, hesperetin, nobiletin, PD146176, orange flavone, and ML351.
优选地，所述GPX4激动剂选自6-姜酚、8-姜酚或10-姜酚；所述Alox-15抑制剂选自槲皮素、阿魏酸、黄芩素、桔皮素或川皮苷。Preferably, the GPX4 agonist is selected from 6-gingerol, 8-gingerol or 10-gingerol; the Alox-15 inhibitor is selected from quercetin, ferulic acid, baicalein, hesperetin or Sichuan Dermoside.
更优选地，所述GPX4激动剂选自8-姜酚；所述Alox-15抑制剂选自槲皮素。More preferably, the GPX4 agonist is selected from 8-gingerol; the Alox-15 inhibitor is selected from quercetin.
作为可选的方式，在上述组合物中，所述组合物包含姜提取物(GPX4激动剂)、沙棘提取物(Alox-15抑制剂)和小麦胚芽提取物(维生素E来源)，所述姜提取物、所述沙棘提取物、所述小麦胚芽提取物的重量比为2：5：3。As an optional mode, in the above composition, the composition comprises ginger extract (GPX4 agonist), seabuckthorn extract (Alox-15 inhibitor) and wheat germ extract (vitamin E source), and the ginger The weight ratio of the extract, the seabuckthorn extract, and the wheat germ extract is 2:5:3.
作为可选的方式，在上述组合物中，所述姜提取物包括从生姜和干姜中通过水提或有机溶剂提取，所述有机溶剂包括低级醇乙醇和乙酸乙酯；所述沙棘提取物包括从沙棘果、沙棘叶中提取获得；所述小麦胚芽提取物从小麦胚芽中通过无水乙醇提取获得。As an optional mode, in the above composition, the ginger extract includes water extraction or organic solvent extraction from ginger and dried ginger, and the organic solvent includes lower alcohol ethanol and ethyl acetate; the sea buckthorn extract It includes extracting from seabuckthorn fruit and seabuckthorn leaves; the wheat germ extract is obtained from wheat germ through absolute ethanol extraction.
所述组合物的提取，包括采用热水，或水与甲醇、乙醇等低级醇类、丙酮等极性溶剂中的一种或二种以上任意混合的溶剂进行提取。鉴于组合物是以药剂或食品作为最终产品开发，故从安全性角度考虑提取溶剂尽量选择水和乙醇为宜。 The extraction of the composition includes hot water, or a solvent in which one or two or more of water, lower alcohols such as methanol and ethanol, and polar solvents such as acetone are arbitrarily mixed for extraction. In view of the fact that the composition is developed with medicine or food as the final product, it is advisable to choose water and ethanol as the extraction solvent as much as possible from the perspective of safety.
在第二个方面中，本发明提供了一种预防或治疗骨质疏松症的制剂，所述制剂包含上述第一个方面所述的组合物，以及适宜的载体或辅料。In the second aspect, the present invention provides a preparation for preventing or treating osteoporosis, said preparation comprising the composition described in the first aspect above, and a suitable carrier or adjuvant.
作为可选的方式，在上述制剂中，所述制剂为口服剂型。As an optional mode, in the above preparation, the preparation is an oral dosage form.
优选地，所述口服剂型选自片剂、胶囊剂、口服液、肠溶片、颗粒剂、糖浆剂、滴丸、水蜜丸、散剂、合剂、煎膏剂、酒剂、露剂、酊剂或舌下含片。Preferably, the oral dosage form is selected from tablets, capsules, oral liquids, enteric-coated tablets, granules, syrups, dripping pills, honeyed pills, powders, mixtures, decoctions, liquors, dews, tinctures or tongue Lower the lozenge.
或者，所述剂型也可以是外用剂型。外用常用的方式包括但不限于软膏、栓剂、硬质软膏、搽剂、滴眼液、油剂、乳膏、气雾剂、喷雾剂。Alternatively, the dosage form may also be an external dosage form. Common methods for external use include but are not limited to ointments, suppositories, hard ointments, liniments, eye drops, oils, creams, aerosols, and sprays.
本发明提供的组合物，也可以粉末添加到食品及保健食品制品中用于口服。组合物也可与其他原料结合制成一定的饮料制品，如果汁饮料、运动饮料等。The composition provided by the invention can also be added into food and health food products as powder for oral administration. The composition can also be combined with other raw materials to produce certain beverage products, such as juice drinks, sports drinks and the like.
在第三个方面中，本发明提供了上述第一个方面所述的组合物或者上述第二个方面所述的制剂在制备预防或治疗骨质疏松症的产品中的用途。In the third aspect, the present invention provides the use of the composition described in the first aspect above or the preparation described in the second aspect above in the preparation of products for preventing or treating osteoporosis.
作为可选的方式，在上述用途中，所述产品是药物、保健品或功能性食品。As an optional mode, in the above use, the product is medicine, health product or functional food.
作为可选的方式，在上述用途中，所述骨质疏松症包括骨质疏松相关并发症、相关疾病以及相关临床症状。As an optional mode, in the above use, the osteoporosis includes osteoporosis-related complications, related diseases and related clinical symptoms.
本发明提供上述组合物在骨质疏松症中的应用，当然对其他成骨细胞分化活性抑制或破骨细胞活性增加相关骨质减少症、脆性骨折和创伤性骨折等骨骼相关疾病也有很好的改善效果用。The present invention provides the application of the above-mentioned composition in osteoporosis, of course, it also has a good effect on bone-related diseases such as osteopenia, fragility fractures and traumatic fractures related to the inhibition of osteoblast differentiation activity or the increase of osteoclast activity. To improve the effect.
本发明相对于现有技术，具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明提供的预防或治疗骨质疏松症的组合物配方是经过筛选的，其在发挥抑制破骨细胞增殖及促进成骨细胞分化活性的作用时并不是某一个或某几个成分所起的作用，而是各组分相互协同共同作用的结果，尤其在特定重量比的条件下协同作用最佳，从而在应用中明显改善骨质疏松。The formulation of the composition for preventing or treating osteoporosis provided by the present invention has been screened, and when it plays the role of inhibiting the proliferation of osteoclasts and promoting the differentiation of osteoblasts, it is not played by one or several components. It is the result of the synergistic effect of each component, especially under the condition of a specific weight ratio, the synergistic effect is the best, so that it can obviously improve osteoporosis in application.
具体实施方式Detailed ways
下面参照具体的实施例对本发明做进一步说明。应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明的范围。 The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, and are not intended to limit the scope of the present invention.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买得到的常规产品。If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased through regular channels.
下面实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售产品。The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available products unless otherwise specified.
实施例1：组合物对破骨细胞活性的影响Example 1: Effect of Composition on Osteoclast Activity
破骨细胞过度增殖和活化会引起骨吸收增多，减少骨骼骨量、降低骨密度、增加骨骼脆性和骨折风险。因此，抑制骨髓单核巨噬细胞分化为破骨细胞有助于预防和治疗骨质疏松症。Excessive proliferation and activation of osteoclasts can cause increased bone resorption, reduce bone mass, reduce bone density, increase bone fragility and fracture risk. Therefore, inhibiting the differentiation of bone marrow mononuclear macrophages into osteoclasts is helpful for the prevention and treatment of osteoporosis.
从1月龄小鼠(购自广东省医学实验动物中心)的股骨和胫骨分离骨髓单核细胞，均匀铺到24孔板中，每孔1×105个细胞，待细胞贴壁后，用含有25ng/ml M-CSF、25ng/ml RANKL的细胞因子和10％胎牛血清的α-MEM培养基进行培养，同时除正常对照组外，其余组别加入不同的药物组合物，每3天更换一次培养基。6天后利用TRAP染色，计算每组中破骨细胞数据。破骨细胞阳性个数定义为：TRAP阳性，且细胞核数目在3个以上的细胞。协同指数采用金正均q值法进行判定，q值由以下公式求得q＝EA+B+C/(EA+EB+EC-EA×EB×EC)。式中EA、EB、EC分别是A药组、B药组、C药组和三药联用组抑制率。q<1说明三药合用后产生拮抗作用；q>1说明三药联用后产生协同作用，q＝1说明三药联用后产生相加作用。Bone marrow mononuclear cells were isolated from the femur and tibia of a 1-month-old mouse (purchased from Guangdong Provincial Medical Experimental Animal Center), spread evenly in a 24-well plate, with 1× 105 cells per well, and after the cells adhered to the wall, use The α-MEM medium containing 25ng/ml M-CSF, 25ng/ml RANKL cytokines and 10% fetal bovine serum was cultured. At the same time, except the normal control group, other groups were added with different pharmaceutical compositions, every 3 days Change medium once. After 6 days, the data of osteoclasts in each group were calculated by using TRAP staining. The positive number of osteoclasts was defined as: TRAP-positive cells with more than 3 nuclei. The synergy index is judged by Kim Jong Kyun q-value method, and the q-value is obtained by the following formula: q=E A+B+C /(E A +E B +E C -E A ×E B ×E C ). In the formula, E A , E B , and E C are the inhibition rates of A drug group, B drug group, C drug group, and three-drug combination group, respectively. q<1 means that the combination of the three drugs produces an antagonistic effect; q>1 means that the combination of the three drugs produces a synergistic effect; q=1 means that the combination of the three drugs produces an additive effect.
实验结果如表1所示，组合物在体外可显著抑制破骨细胞的形成，其抑制率可高达86.67％，而相同剂量下的8-姜酚、槲皮素和维生素E的抑制效果均低于组合物，且当组合物的比例为1:4:1时，其对破骨细胞抑制效果最佳。以上结果从侧面说明了本发明提供的组合物在发挥抑制破骨细胞活化的作用是并不是某一个或某几个成分发挥作用的，而是各组分相互协同、共同作用的结果。The experimental results are shown in Table 1, the composition can significantly inhibit the formation of osteoclasts in vitro, and its inhibition rate can be as high as 86.67%, while the inhibitory effects of 8-gingerol, quercetin and vitamin E at the same dose are all low In the composition, and when the ratio of the composition is 1:4:1, it has the best inhibitory effect on osteoclasts. The above results demonstrate from the side that the effect of the composition provided by the present invention on inhibiting the activation of osteoclasts is not due to one or several components, but the result of the synergistic and joint action of each component.
表1：破骨细胞的数目及分化抑制率

注：数据以平均值±SD表示，数据统计使用SPSS25.0的one-way ANOVA分析，
与正常组比较，*P<0.05，**P<0.01，***P<0.001。Table 1: The number and differentiation inhibition rate of osteoclasts

Note: The data are expressed as mean ± SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0.
Compared with normal group, * P<0.05, ** P<0.01, *** P<0.001.
实施例2：组合物对去卵巢小鼠骨密度和骨小梁的影响Example 2: Effect of composition on bone mineral density and trabecular bone in ovariectomized mice
本实施例中的使用的动物雌性C57BL/6小鼠，购自广东省医学实验动物中心。小鼠饲养在温度23±2℃，湿度55±5％，换气频率为12-15次/小时，照明时间12小时/天(7:00-19:00光照)，5只/饲养笼(235×325×170H mm)，饲育条件：固体饲料(广东省医学实验动物中心)，自由饮食。待小鼠为3月龄时，将小鼠随机分为假手术组、模型组及给药组。小鼠手术前12小时，禁食不禁 水。使用异氟烷麻醉小鼠，然后用75％乙醇清洗皮肤并消毒。各组小鼠均采用腹侧切口进行手术，除假手术组小鼠不摘除卵巢外，其余各组小鼠均进行双侧卵巢切除，所有手术小鼠均腹腔注射青霉素溶液。手术完成后，将手术动物放置恒温箱中待动物清醒后转移到干净的饲养笼中恢复1周，期间隔天腹腔注射青霉素溶液以免小鼠伤口感染。Animals used in this example, female C57BL/6 mice, were purchased from Guangdong Medical Experimental Animal Center. The mice were kept at a temperature of 23±2°C, a humidity of 55±5%, a ventilation frequency of 12-15 times/hour, and a lighting time of 12 hours/day (7:00-19:00 light), 5/breeding cage ( 235×325×170H mm), feeding conditions: solid feed (Guangdong Medical Experimental Animal Center), free diet. When the mice were 3 months old, the mice were randomly divided into a sham operation group, a model group and an administration group. Mice were fasted for 12 hours before surgery water. Mice were anesthetized using isoflurane, then the skin was washed and sterilized with 75% ethanol. The mice in each group were operated by ventral incision. Except the sham operation group, the ovaries were not removed, and the mice in the other groups were subjected to bilateral ovariectomy. All operated mice were intraperitoneally injected with penicillin solution. After the operation, the operated animals were placed in an incubator and transferred to a clean breeding cage to recover for 1 week after the animals woke up. During this period, penicillin solution was injected intraperitoneally every other day to avoid wound infection of the mice.
除假手术组和模型组小鼠外，各给药组小鼠均灌胃相应剂量的药物，每天一次，连续2个月，末次给药12小时后，麻醉小鼠，取小鼠右后肢扫描骨密度值(BMD)与骨小梁数目。Except for the mice in the sham operation group and the model group, the mice in each administration group were given the corresponding dose of drugs once a day for 2 consecutive months. After 12 hours of the last administration, the mice were anesthetized, and the right hind limbs of the mice were taken for scanning Bone mineral density (BMD) and trabecular number.
实验结果如表2所示，与假手术组小鼠相比，模型组小鼠的骨密度和骨小梁数目显著降低。单独给予8-姜酚、槲皮素或维生素E后，去卵巢小鼠的骨密度值和骨小梁数目有所改善，而给予组合物之后，去卵巢小鼠的骨密度值和骨小梁数目的改善效果更显著，且8-姜酚、槲皮素和维生素E联合使用的效果优于6-姜酚、槲皮素和维生素E联合使用。The experimental results are shown in Table 2. Compared with the mice in the sham operation group, the bone mineral density and the number of bone trabeculae in the model group were significantly reduced. After administration of 8-gingerol, quercetin or vitamin E alone, the bone mineral density value and trabecular number of ovariectomized mice were improved, and after the combination was given, the bone mineral density value and trabecular bone The improvement effect of the number is more significant, and the combined use of 8-gingerol, quercetin and vitamin E is better than the combined use of 6-gingerol, quercetin and vitamin E.
表2：去卵巢小鼠的BMD和骨小梁数目

注：数据以平均值±SD表示，数据统计使用SPSS25.0的one-way ANOVA分析，
与假手术组比较，***P<0.001；与模型组比较，#P<0.05，##P<0.01,###P<0.001。Table 2: BMD and Trabecular Number of Ovariectomized Mice

Note: The data are expressed as mean ± SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0.
Compared with the sham operation group, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.
实施例3：组合物对去卵巢小鼠骨组织抗氧化能力的影响Example 3: Effect of the composition on the antioxidant capacity of bone tissue in ovariectomized mice
小鼠骨组织的抗氧化能力通过氧化自由基吸收能力(Oxygen Radical Absorbance Capacity,ORAC)实验测定。The antioxidant capacity of mouse bone tissue was determined by oxidative radical absorbance capacity (Oxygen Radical Absorbance Capacity, ORAC) test.
骨组织样品预处理：在实施例2中，取小鼠左后肢，剥离肌肉和结缔组织 后，在液氮中研磨后收集至离心管，加入高氯酸溶液(3％)制备2％的骨组织匀浆液，4℃条件下以10000r/min离心15min，取上清液作为测定ORAC的样品溶液。Bone tissue sample pretreatment: In Example 2, the left hind limb of the mouse was taken, and the muscle and connective tissue were stripped Finally, grind in liquid nitrogen and collect into a centrifuge tube, add perchloric acid solution (3%) to prepare 2% bone tissue homogenate, centrifuge at 10000r/min for 15min at 4°C, and take the supernatant as the ORAC assay. sample solution.
ORAC实验过程：在96孔板中加入不同组别小鼠的20μL骨组织匀浆上清液和20μL磷酸盐缓冲液，再加入20μL荧光素钠，最后添加140μL AAPH后迅速将96孔板置于控温37℃的荧光酶标仪中开始测定。每2min测定一个点，共测定2h。ORAC值以1μmol/L的Trolox在荧光衰减曲线上对应的保护积分面积作为标准对照计算。ORAC experiment process: Add 20 μL bone tissue homogenate supernatant and 20 μL phosphate buffer saline of different groups of mice to the 96-well plate, then add 20 μL sodium fluorescein, and finally add 140 μL AAPH, and quickly place the 96-well plate in The assay was started in a fluorescent microplate reader with temperature control at 37°C. A point was measured every 2 minutes for a total of 2 hours. The ORAC value was calculated using the protection integral area corresponding to 1 μmol/L Trolox on the fluorescence decay curve as the standard control.
结果如表3所示，与模型组相比，8-姜酚、槲皮素和维生素E均能明显升高去卵巢小鼠骨组织的ORAC值，降低MDA水平，增强其抗氧化能力。此外，联用组1和联用组2能更显著的增加去卵巢小鼠的抗氧化能力，且8-姜酚、槲皮素和维生素E联用的抗氧化效果更优。The results are shown in Table 3. Compared with the model group, 8-gingerol, quercetin and vitamin E could significantly increase the ORAC value of bone tissue in ovariectomized mice, reduce the level of MDA, and enhance its antioxidant capacity. In addition, combination group 1 and combination group 2 can significantly increase the antioxidant capacity of ovariectomized mice, and the combination of 8-gingerol, quercetin and vitamin E has a better antioxidant effect.
表3：去卵巢小鼠骨组织的抗氧化能力

注：数据以平均值±SD表示，数据统计使用SPSS25.0的one-way ANOVA分析，
与假手术组比较，***P<0.001；与模型组比较，##P<0.01,###P<0.001。Table 3: Antioxidative capacity of bone tissue from ovariectomized mice

Note: The data are expressed as mean ± SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0.
Compared with the sham operation group, *** P<0.001; compared with the model group, ## P<0.01, ### P<0.001.
实施例4：组合物对去卵巢小鼠骨密度的影响Example 4: Effect of composition on bone density of ovariectomized mice
本实施例中的使用的动物雌性C57BL/6小鼠，购自广东省医学实验动物中 心。小鼠饲养在温度23±2℃，湿度55±5％，换气频率为12-15次/小时，照明时间12小时/天(7:00～19:00光照)，5只/饲养笼(235×325×170H mm)，饲育条件：固体饲料(广东省医学实验动物中心)，自由饮食。待小鼠为3月龄时，将小鼠随机分为假手术组、模型组及给药组。小鼠手术前12小时，禁食不禁水。使用异氟烷麻醉小鼠，然后用75％乙醇清洗皮肤并消毒。各组小鼠均采用腹侧切口进行手术，除假手术组小鼠不摘除卵巢外，其余各组小鼠均进行双侧卵巢切除，所有手术小鼠均腹腔注射青霉素溶液。手术完成后，将手术动物放置恒温箱中待动物清醒后转移到干净的饲养笼中恢复1周，期间隔天腹腔注射青霉素溶液以免小鼠伤口感染。The animal female C57BL/6 mouse used in this example was purchased from Guangdong Medical Experimental Animals Heart. The mice were raised at a temperature of 23±2°C, a humidity of 55±5%, a ventilation frequency of 12-15 times/hour, and a lighting time of 12 hours/day (7:00～19:00 light), 5 mice/cage ( 235×325×170H mm), feeding conditions: solid feed (Guangdong Medical Experimental Animal Center), free diet. When the mice were 3 months old, the mice were randomly divided into a sham operation group, a model group and an administration group. Mice were fasted for 12 hours before surgery, without food or water. Mice were anesthetized using isoflurane, then the skin was washed and sterilized with 75% ethanol. The mice in each group were operated by ventral incision. Except the sham operation group, the ovaries were not removed, and the mice in the other groups were subjected to bilateral ovariectomy. All operated mice were intraperitoneally injected with penicillin solution. After the operation, the operated animals were placed in an incubator and transferred to a clean breeding cage to recover for 1 week after the animals woke up. During this period, penicillin solution was injected intraperitoneally every other day to avoid wound infection of the mice.
除假手术组和模型组小鼠外，各给药组小鼠均灌胃相应剂量的药物，每天一次，连续2个月，末次给药12小时后，麻醉小鼠，取小鼠右后肢扫描骨密度值(BMD)与骨小梁数目。Except for the mice in the sham operation group and the model group, the mice in each administration group were given the corresponding dose of drugs once a day for 2 consecutive months. After 12 hours of the last administration, the mice were anesthetized, and the right hind limbs of the mice were taken for scanning Bone mineral density (BMD) and trabecular number.
实验结果如表4所示，生姜提取物、沙棘提取物和小麦胚芽提取物能有效增加去卵巢小鼠的BMD值，而生姜提取物、沙棘提取物和小麦胚芽提取物联合使用时，对卵巢小鼠的BMD改善效果更加显著，且生姜提取物、沙棘提取物和小麦胚芽提取物的重量比为2:5:3时效果更佳。The experimental results are shown in Table 4. Ginger extract, sea buckthorn extract and wheat germ extract can effectively increase the BMD value of ovariectomized mice, and when ginger extract, sea buckthorn extract and wheat germ extract are used in combination, there is no effect on ovarian The BMD improvement effect of mice is more significant, and the effect is better when the weight ratio of ginger extract, seabuckthorn extract and wheat germ extract is 2:5:3.
表4：本发明组合物对去卵巢小鼠BMD的影响

注：数据以平均值±SD表示，数据统计使用SPSS25.0的one-way ANOVA分析，
与假手术组比较，***P<0.001；与模型组比较，#P<0.05,##P<0.01。 Table 4: Effects of compositions of the present invention on BMD of ovariectomized mice

Note: The data are expressed as mean ± SD, and the data statistics are analyzed using one-way ANOVA of SPSS25.0.
Compared with the sham operation group, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01.
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。 Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (10)

  1. 一种预防或治疗骨质疏松症的组合物，其特征在于：所述组合物包含GPX4激动剂和Alox-15抑制剂和以及维生素E，其中，所述GPX4激动剂增强内源性抗氧化酶GPX4的活性，增强机体脂质过氧化物的清除，所述Alox-15抑制剂抑制内源性脂氧合酶15的活性，降低脂质过氧化物的产生，同时辅以小分子还原剂维生素E直接清除脂质过氧化的累积，三者协同发挥作用。A composition for preventing or treating osteoporosis, characterized in that: the composition comprises a GPX4 agonist and an Alox-15 inhibitor and vitamin E, wherein the GPX4 agonist enhances endogenous antioxidant enzymes The activity of GPX4 enhances the clearance of lipid peroxides in the body. The Alox-15 inhibitor inhibits the activity of endogenous lipoxygenase 15 and reduces the production of lipid peroxides. At the same time, it is supplemented with small molecule reducing agent vitamin E directly removes the accumulation of lipid peroxidation, and the three work synergistically.
  2. 根据权利要求1所述的组合物，其特征在于：所述GPX4激动剂、所述Alox-15抑制剂、所述维生素E的重量比为0.5-5：1-5：0.5-5。The composition according to claim 1, characterized in that: the weight ratio of the GPX4 agonist, the Alox-15 inhibitor, and the vitamin E is 0.5-5:1-5:0.5-5.
  3. 根据权利要求1或权利要求2所述的组合物，其特征在于：所述GPX4激动剂选自6-姜酚、8-姜酚、10-姜酚、PKUMDL-LC-101-D04中的一种或多种；所述Alox-15抑制剂选自槲皮素、阿魏酸、黄芩素、桔皮素、川皮苷、PD146176、橙黄酮、ML351中的一种或多种。The composition according to claim 1 or claim 2, wherein the GPX4 agonist is selected from one of 6-gingerol, 8-gingerol, 10-gingerol, and PKUMDL-LC-101-D04 One or more kinds; the Alox-15 inhibitor is selected from one or more of quercetin, ferulic acid, baicalein, hesperetin, nobiletin, PD146176, orange flavone, and ML351.
  4. 根据权利要求1或权利要求2所述的组合物，其特征在于：所述组合物包含姜提取物、沙棘提取物和小麦胚芽提取物，所述姜提取物、所述沙棘提取物、所述小麦胚芽提取物的重量比为2：5：3。The composition according to claim 1 or claim 2, characterized in that: said composition comprises ginger extract, sea buckthorn extract and wheat germ extract, said ginger extract, said sea buckthorn extract, said The weight ratio of wheat germ extract is 2:5:3.
  5. 根据权利要求4所述的组合物，其特征在于：所述姜提取物包括从生姜和干姜中通过水提或有机溶剂提取，所述有机溶剂包括低级醇乙醇和乙酸乙酯；所述沙棘提取物包括从沙棘果、沙棘叶中提取获得；所述小麦胚芽提取物从小麦胚芽中通过无水乙醇提取获得。Composition according to claim 4, it is characterized in that: described ginger extract comprises from ginger and dried ginger by water extraction or organic solvent extraction, and described organic solvent comprises lower alcohol ethanol and ethyl acetate; The extract includes extracting from seabuckthorn fruits and seabuckthorn leaves; the wheat germ extract is obtained from wheat germ through absolute ethanol extraction.
  6. 一种预防或治疗骨质疏松症的制剂，其特征在于：所述制剂包含权利要求1至5中任一项所述的组合物，以及适宜的载体或辅料。A preparation for preventing or treating osteoporosis, characterized in that the preparation comprises the composition according to any one of claims 1 to 5, and a suitable carrier or adjuvant.
  7. 根据权利要求6所述的制剂，其特征在于：所述制剂为口服剂型或外用剂型，优选地，所述口服剂型选自片剂、胶囊剂、口服液、肠溶片、颗粒剂、糖浆剂、滴丸、水蜜丸、散剂、合剂、煎膏剂、酒剂、露剂、酊剂或舌下含片；所述外用剂型选自软膏、栓剂、硬质软膏、搽剂、滴眼液、油剂、乳膏、气雾剂或喷雾剂。 The preparation according to claim 6, characterized in that: the preparation is an oral dosage form or an external dosage form, preferably, the oral dosage form is selected from tablets, capsules, oral liquids, enteric-coated tablets, granules, and syrups , dripping pills, honeyed water pills, powders, mixtures, decoctions, liquors, dews, tinctures or sublingual tablets; the external dosage forms are selected from ointments, suppositories, hard ointments, liniments, eye drops, oils , cream, aerosol or spray.
  8. 权利要求1至5中任一项所述的组合物或者权利要求6或权利要求7所述的制剂在制备预防或治疗骨质疏松症的产品中的用途。Use of the composition described in any one of claims 1 to 5 or the preparation described in claim 6 or claim 7 in the preparation of products for preventing or treating osteoporosis.
  9. 根据权利要求8所述的用途，其特征在于：所述产品是药物、保健品或功能性食品。The use according to claim 8, characterized in that: the product is medicine, health product or functional food.
  10. 根据权利要求8或权利要求9所述的用途，其特征在于：所述骨质疏松症包括骨质疏松相关并发症、相关疾病以及相关临床症状。 The use according to claim 8 or claim 9, characterized in that: said osteoporosis includes osteoporosis-related complications, related diseases and related clinical symptoms.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2007129634A (en) * 2007-08-02 2009-02-10 Закрытое акционерное общество "Биологические исследовани и системы" (RU) PHARMACEUTICAL COMPOSITION FOR COMPLEX THERAPY OF OSTEOPOROSIS
CN102240388A (en) * 2010-05-11 2011-11-16 廖晖 Ginger extract and preparation method and applications thereof
US20190015384A1 (en) * 2017-07-13 2019-01-17 Summit Innovation Labs LLC Treatment and Prevention of Joint Disorders
CN110934883A (en) * 2019-12-14 2020-03-31 湛江广医医药科技开发有限公司 Naringin preparation for improving male osteoporosis and preventing fracture
CN114225037A (en) * 2022-01-29 2022-03-25 暨南大学 Composition for preventing or treating osteoporosis and preparation and application thereof
CN114344289A (en) * 2022-01-29 2022-04-15 暨南大学 Composition with anti-aging activity and preparation and application thereof

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100408231B1 (en) * 2000-08-14 2003-12-01 한국 한의학 연구원 Flavonoid derivateives for prevention and treatment of osteoporosis
JP2003095971A (en) * 2001-09-27 2003-04-03 National Institute Of Health & Nutrition Therapeutic agent or composition for osteoporosis
US20090304827A1 (en) * 2008-05-08 2009-12-10 Kim Darrick S H L Combinations of Ingredients Having Synergistic Anti-Inflammatory Effects
KR20100009151A (en) * 2008-07-18 2010-01-27 한국화학연구원 Composition containing mixture extract of saururus chinesis and scutellaria baicalensis for prevention or treatment of osteoporosis
JP2012158579A (en) * 2011-02-02 2012-08-23 Shonan Institute For Medical & Preventive Science Food, medical product and animal feed for preventing and ameliorating osteoporosis
FR3005419B1 (en) * 2013-05-13 2015-09-04 Agronomique Inst Nat Rech USE OF A COMBINATION OF TWO COMPOUNDS FOR THE TREATMENT AND / OR PREVENTION OF BONE DISORDERS
CN104721678B (en) * 2014-07-12 2020-11-27 广东医科大学 Application of galangal extract in preparation of anti-osteoporosis medicine
CN106333977B (en) * 2015-07-06 2020-02-21 陕西天奎生物医药科技有限公司 Natural pharmaceutical composition for treating osteoporotic fracture and/or osteoarthritis and application thereof
US20190015448A1 (en) * 2017-07-13 2019-01-17 Summit Innovation Labs LLC Treatment and Prevention of Bone and Joint Disorders
JP2021177705A (en) * 2020-05-11 2021-11-18 辻製油株式会社 Bone metabolism improving composition and method of producing the same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2007129634A (en) * 2007-08-02 2009-02-10 Закрытое акционерное общество "Биологические исследовани и системы" (RU) PHARMACEUTICAL COMPOSITION FOR COMPLEX THERAPY OF OSTEOPOROSIS
CN102240388A (en) * 2010-05-11 2011-11-16 廖晖 Ginger extract and preparation method and applications thereof
US20190015384A1 (en) * 2017-07-13 2019-01-17 Summit Innovation Labs LLC Treatment and Prevention of Joint Disorders
CN110934883A (en) * 2019-12-14 2020-03-31 湛江广医医药科技开发有限公司 Naringin preparation for improving male osteoporosis and preventing fracture
CN114225037A (en) * 2022-01-29 2022-03-25 暨南大学 Composition for preventing or treating osteoporosis and preparation and application thereof
CN114344289A (en) * 2022-01-29 2022-04-15 暨南大学 Composition with anti-aging activity and preparation and application thereof

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