WO2023065478A1 - Use of icariside i - Google Patents

Use of icariside i Download PDF

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WO2023065478A1
WO2023065478A1 PCT/CN2021/135559 CN2021135559W WO2023065478A1 WO 2023065478 A1 WO2023065478 A1 WO 2023065478A1 CN 2021135559 W CN2021135559 W CN 2021135559W WO 2023065478 A1 WO2023065478 A1 WO 2023065478A1
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icariside
fermented
epimedium extract
epimedium
extract
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PCT/CN2021/135559
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French (fr)
Chinese (zh)
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李慧灵
黄佳俊
陈川
张利民
罗冬章
林育成
周金林
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广东金骏康生物技术有限公司
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Publication of WO2023065478A1 publication Critical patent/WO2023065478A1/en

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • 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)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A61K36/296—Epimedium
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Definitions

  • the invention relates to the field of medicine, in particular to the application of icariside I.
  • Osteoporosis is a disease characterized by decreased bone mass, changes in the microstructure of bone tissue, and an increased risk of fracture. Osteoblast proliferation inhibition and reduced differentiation are important causes of osteoporosis.
  • the clinical manifestations of osteoporosis are low back pain, limb weakness, and bone pain. Because of its high incidence and great harm in middle-aged and elderly people, it has been highly valued by the society and the medical field.
  • the treatment of osteoporosis mainly uses estrogen, calcium, active vitamin D, calcitonin and fluoride as commonly used drugs. Although it has a certain effect, it has the disadvantages of large adverse reactions and patients cannot tolerate it for a long time. .
  • icariside I is a trace flavonoid glycoside compound in the Chinese herbal medicine Epimedium.
  • epimedium drugs can increase the blood flow of cardiovascular and cerebrovascular, promote hematopoietic function, immune function and bone metabolism, and also have the functions of nourishing kidney and strengthening yang, anti-aging and so on.
  • Icariside I as the main metabolite of icariin, has little research on the anti-osteoporosis.
  • the technical problem to be solved by the present invention is to provide a new application of icariside I, i.e. the application in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis.
  • the medicine can be made into any dosage form, exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but not limited thereto.
  • the daily dosage of icariside I is 5-50 mg/kg ⁇ BW (body weight).
  • the icariside I is obtained by fermenting the epimedium extract with Pichia pastoris engineering bacteria GS115-KA; the icariside I in the fermented epimedium extract The content is greater than or equal to 1%; wherein, the Pichia pastoris engineering strain GS115-KA is a rhamnosidase TpeRha-K579A gene mutant.
  • the specific amino acid sequence and nucleotide sequence of the rhamnosidase TpeRha-K579A mutant refer to the applicant's previous application CN113136378B.
  • the preparation method of Pichia pastoris engineered bacteria GS115-KA is:
  • the preparation method of fermented Epimedium extract is:
  • icariside I is obtained by enzymatically hydrolyzing the epimedium extract with rhamnosidase TpeRha-K579A enzyme, and enzymatically hydrolyzing the content of icariside I in the epimedium extract The content is greater than or equal to 1%.
  • this application also discloses the use of fermented Epimedium extract in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis; wherein, the content of icariside I in the fermented Epimedium extract The content is greater than or equal to 1%.
  • this application also discloses the use of the enzymatically hydrolyzed Epimedium extract in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis; wherein, the enzymatically hydrolyzed Epimedium extract contains The content of I is greater than or equal to 1%.
  • the medicine can be made into any dosage form, exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but not limited thereto.
  • the daily dosage of icariside I is 5-50 mg/kg BW (body weight).
  • the present invention shows that icariside I can be used for treating osteoporosis through pharmacological experiments. Specifically, the results of in vitro experiments show that icariside I can significantly promote the proliferation of osteoblasts, and the results of in vivo experiments show that icariside I can significantly increase the bone density of mice and promote the formation of osteoblasts. Inhibits osteoclast formation. Moreover, it has been proved by in vivo experiments that when the dose ranges from 5-50 mg/kg ⁇ BW, it has the best therapeutic effect, and at high doses (200 mg/kg ⁇ BW), there is no therapeutic effect. In addition, the present invention uses the fermented or enzymatically hydrolyzed Epimedium extract to add pharmaceutically acceptable carriers to prepare various preparations. The preparations have low toxic and side effects and make up for the disadvantages of long-term use of the commercially available drugs.
  • Fig. 1 is the impact diagram of each test group on mouse liver weight in embodiment 2;
  • Fig. 2 is the liver slice figure of Sham group mouse in embodiment 2;
  • Fig. 3 is the liver section figure of OVX group mouse in embodiment 2;
  • Fig. 4 is the liver slice figure of ALN group mouse in embodiment 2;
  • Fig. 5 is the L group mouse liver slice figure among the embodiment 2;
  • Fig. 6 is the liver section figure of group M mice in embodiment 2;
  • Fig. 7 is the liver section figure of group H mice in embodiment 2;
  • Fig. 8 is the figure of influence of each test group on total bone density in embodiment 2;
  • Fig. 9 is the impact diagram of each test group on cortical bone density in embodiment 2.
  • Fig. 10 is the impact diagram of each test group on the bone density content of cancellous bone in embodiment 2;
  • Fig. 11 is the impact diagram of each test group on mouse osteocalcin in embodiment 2;
  • Fig. 12 is the figure of the influence of each test group on the activity of ALP in rBMSCs cells in embodiment 3;
  • Fig. 13 is the impact diagram of each test group on ALP mRNA expression in rBMSCs cells in embodiment 3;
  • Fig. 14 is a diagram showing the influence of each test group on the expression of Run ⁇ 2 mRNA in rBMSCs cells in Example 3.
  • This implementation provides a preparation method of fermented Epimedium extract, which includes:
  • the rhamnosidase TpeRha-K579A gene was connected to the plasmid pPIC9K, and the plasmid was transformed into Pichia pastoris GS115 to obtain engineering bacteria GS115-KA;
  • the volume of the fermentation medium is 3.5L
  • the medium formula is: 85% (w/v) H 3 PO 4 26.7ml/L, CaSO 4 0.93g/L, K 2 SO 4 18.2g/L, KOH 4.13g/L, MgSO 4 7H 2 O 14.9g/L, glycerin 40g/L, yeast extract 5g/L, peptone 5g/L, trace element PTM1 4.4ml/L, defoaming oil 15mL, 30mL ammonia water to adjust the pH to 5.0.
  • mice that underwent surgery without ovariectomy were used as the sham operation group (SHAM), and the mouse osteoporosis model made in step (1) was randomly divided into the control group (OVX), the A Lunronate sodium intervention group (ALN), fermented or enzymatically hydrolyzed Epimedium extract (based on 1 dose of icariin) low, middle and high dose groups L, M, and H, a total of 6 groups, with three mice in each group.
  • OVX control group
  • Epimedium extract based on 1 dose of icariin
  • FIG. 1 is a graph showing the influence of each test group on the mouse liver weight by each test group. It can be seen from Figure 1 that the livers of the mice in the Sham group and the ALN group were normal and not affected, the livers of the mice in the model group were affected, and the liver anatomy and morphology of the mice were normal under the three doses of low, medium and high.
  • FIGS 2 to 7 are liver slices of mice in each test group. From the pictures, after taking the fermented icariin extract, the liver of the mice returned to normal, indicating that the fermented icariin extract has low toxicity , or may have the effect of repairing the liver.
  • Figures 8 to 10 are diagrams showing the influence of each test group on bone density. It can be seen from the figure that compared with OVX after 4 weeks of administration, the low-dose group (L) and the middle-dose group (M) have improved bone mineral density, and the low-dose group (L) has the best effect , low dose (L) and middle dose group (M) compared with the drug alendronate sodium group, the effect is better than the alendronate sodium group (ALN), and the high dose group (H) has no improvement effect. It shows that the daily dosage of 5-50 mg/kg ⁇ bw in mice does not improve the bone density in a dose-dependent manner, and the high dosage has the opposite effect.
  • Figure 11 is a diagram of the influence of each test group on mouse osteocalcin. It can be seen from the figure that after 4 weeks of administration, in the blood of mice, the content of osteocalcin in the Sham group is the highest, which is the expression of normal mice. Osteocalcin in the OVX group was the lowest, indicating that the bone loss of the mice was severe after ovariectomy, which also indicated that the osteoporosis model was successfully established.
  • the treatment effect of the L group was the best, strong
  • the treatment effect of M group is not as good as that of L group, but it also has a certain effect, which is better than that of OVX group. It has an effect at a dose of 5-50 mg/kg ⁇ BW, the optimal dose is 5 mg/kg ⁇ BW, and there will be opposite results at high doses. Cannot reach the effect of treating osteoporosis.
  • rBMSCs rat bone marrow mesenchymal stem cells
  • ALP alkaline Phosphatase
  • Control group basic culture medium: DMEM+10mL/dL fetal bovine serum (containing 100U/ml penicillin and streptomycin), fermented Epimedium extract Intervention group: basic culture medium+fermented or enzymolyzed Epimedium Husk extract 1 ⁇ 10 -5 mol/L, 1 ⁇ 10 -6 mol/L, 1 ⁇ 10 -7 mol/L, 1 ⁇ 10 -8 mol/L, 1 ⁇ 10 -9 mol/L, 1 ⁇ 10 -10 mol/L.
  • Figure 12 is a diagram showing the influence of each test group on the ALP activity in rBMSCs cells. It can be seen from the figure that the ALP activity of different concentrations of fermented Epimedium extract intervened in rBMSCs was higher than that of the control group, and at 1 ⁇ 10 -7 mol/L, 1 ⁇ 10 -8 mol/L, 1 ⁇ 10 -9 mol/L, 1 ⁇ 10 -10 mol/L have the most significant effect.
  • Figure 13 is a graph showing the influence of each test group on the expression of ALP mRNA in rBMSCs cells; it can be seen from the graph that the expression level of ALP mRNA significantly increased at a concentration of 1 ⁇ 10 -6 mol/L.
  • Figure 14 is a diagram of the influence of each test group on the expression of Run ⁇ 2 mRNA in rBMSCs cells; it can be seen from the figure that the expression level of Run ⁇ 2 mRNA at a concentration of 1 ⁇ 10 -7 mol/L is much higher than that of the control group.
  • Run ⁇ 2 is a specific transcription factor for osteoblasts and a key regulator of osteoblast differentiation, and is an important transcription factor for activating and initiating the differentiation of BMSCs into osteoblasts and regulating the maturation of osteoblasts during bone development.
  • the fermented Epimedium extract can promote the proliferation of primary osteoblasts. And in the range of 1 ⁇ 10 -6 -1 ⁇ 10 -10 mol/L, it can promote the proliferation of primary osteoblasts very well.
  • the present invention proves that the fermented Epimedium extract has little toxic effect on the host through safety experiments; through in vivo experiments on osteoporosis mice, it is proved that the fermented Epimedium extract can increase the bone density of mice, It can promote proliferation of osteoblasts and inhibit osteoclasts. Moreover, icariside I has an effect on the treatment of osteoporosis at a dose of 5-50 mg/kg ⁇ BW, and a high dose will have the opposite result, and the effect of treating osteoporosis cannot be achieved. Through in vitro experiments, it has been proved that the fermented epimedium extract has a proliferative effect on osteoblasts. According to the above examples, it is proved that icariside I and fermented epimedium extract can be used as medicine for treating osteoporosis.

Abstract

Use of Icariside I. Experiments show that Icariside I can significantly improve the bone density of a mouse, promote the formation of osteoblasts and inhibit the formation of osteoclasts, such that Icariside I can be used in the preparation of a drug or health care product for preventing and/or treating osteoporosis.

Description

淫羊藿次苷Ⅰ的用途Uses of Icaritin Ⅰ 技术领域technical field
本发明涉及医药领域，尤其涉及一种淫羊藿次苷Ⅰ的用途。The invention relates to the field of medicine, in particular to the application of icariside I.
背景技术Background technique
骨质疏松症是一种以骨量减少为特征、骨组织显微结构改变和骨折危险程度增加的疾病。成骨细胞的增殖抑制、分化程度降低是造成骨质疏松的重要原因。骨质疏松症的临床表现为腰酸背痛，四肢乏力，骨骼疼痛，因其在中老年人中发病率高、危害大而受到社会和医疗界的高度重视。现阶段骨质疏松症的治疗主要以雌激素、钙剂、活性维生素D、降钙素和氟化物等为常用药物，虽然有一定的疗效，但存在不良反应大及患者不能长期耐受等缺点。随着人口寿命的增长，老年的数量日益增多，骨质疏松这种病的危害就越来越严重。因此，寻找一种有效治疗骨质疏松症的药物是当前医学界中药的课题之一。中医认为，肾藏精，主骨生髓，髓藏于骨腔内，滋养骨骼，骨的生长发育依赖肾气的滋养与推动中医临床和实验证明中医药治疗原发性骨质疏松症疗效显著，淫羊藿为中医临床治疗骨质疏松或促进骨折愈合常用中药。目前有关中药淫羊藿治疗骨质疏松症的药物，国内的中药制剂有仙灵骨葆。但长期服用仙灵骨葆也会导致肝损伤。因此寻求一种副作用小，又有显著疗效的中药制剂是医学界亟待解决的难题。Osteoporosis is a disease characterized by decreased bone mass, changes in the microstructure of bone tissue, and an increased risk of fracture. Osteoblast proliferation inhibition and reduced differentiation are important causes of osteoporosis. The clinical manifestations of osteoporosis are low back pain, limb weakness, and bone pain. Because of its high incidence and great harm in middle-aged and elderly people, it has been highly valued by the society and the medical field. At present, the treatment of osteoporosis mainly uses estrogen, calcium, active vitamin D, calcitonin and fluoride as commonly used drugs. Although it has a certain effect, it has the disadvantages of large adverse reactions and patients cannot tolerate it for a long time. . With the growth of population life expectancy, the number of old people is increasing day by day, and the harm of this disease of osteoporosis is just more and more serious. Therefore, finding a kind of medicine for effectively treating osteoporosis is one of the problems of current medical circle traditional Chinese medicine. Traditional Chinese medicine believes that the kidney stores the essence, governs the bone to produce marrow, and the marrow is stored in the bone cavity to nourish the bone. The growth and development of the bone depends on the nourishment and promotion of kidney qi. Clinical and experimental results have proved that Chinese medicine has a significant effect in treating primary osteoporosis. , Epimedium is a commonly used traditional Chinese medicine for the clinical treatment of osteoporosis or the promotion of fracture healing. At present, there is Xianling Gubao in domestic Chinese medicine preparations for the treatment of osteoporosis with Epimedium. However, long-term use of Xianling Gubao can also cause liver damage. Therefore seeking a kind of side effect is little, the traditional Chinese medicine preparation of significant curative effect is the difficult problem demanding prompt solution of medical circle again.
另一方面，淫羊藿次苷I(Icariside I)是中药材淫羊藿中的一种微量黄酮苷类化合物，目前有关淫羊藿次苷I的药理活性研究报道较少，在已有报道中，淫羊藿类药物能增加心脑血管血流量、促进造血功能、免疫功能及骨代谢，还具有补肾壮阳、抗衰老等功效。淫羊藿次苷I作为淫羊藿苷主要体内代谢产物，在对抗骨质疏松症方面的研究很少。On the other hand, icariside I (Icariside I) is a trace flavonoid glycoside compound in the Chinese herbal medicine Epimedium. At present, there are few reports on the pharmacological activity of icariside I. Among them, epimedium drugs can increase the blood flow of cardiovascular and cerebrovascular, promote hematopoietic function, immune function and bone metabolism, and also have the functions of nourishing kidney and strengthening yang, anti-aging and so on. Icariside I, as the main metabolite of icariin, has little research on the anti-osteoporosis.
发明内容Contents of the invention
本发明所要解决的技术问题在于，提供一种淫羊藿次苷I的新用途，即在 制备预防和/或治疗骨质疏松症的药物或保健品中的用途。The technical problem to be solved by the present invention is to provide a new application of icariside I, i.e. the application in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis.
其中，所述药物可制成任意剂型，示例性的为片剂、胶囊剂、颗粒剂、软胶囊、滴丸剂、糖浆剂或注射剂，但不限于此。Wherein, the medicine can be made into any dosage form, exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but not limited thereto.
具体的，使用该药物或保健品治疗或预防骨质疏松症时，淫羊藿次苷Ⅰ的每日用量为5～50mg/kg·BW(体重)。Specifically, when using the drug or health product to treat or prevent osteoporosis, the daily dosage of icariside I is 5-50 mg/kg·BW (body weight).
在本发明的一个实施例之中，所述淫羊藿次苷Ⅰ由毕赤酵母工程菌GS115-KA对淫羊藿提取物发酵而得；发酵淫羊藿提取物中淫羊藿次苷Ⅰ的含量大于等于1％；其中，毕赤酵母工程菌GS115-KA为鼠李糖苷酶TpeRha-K579A基因突变体。其中，鼠李糖苷酶TpeRha-K579A突变体的具体氨基酸序列、核苷酸序列参申请人的前序申请CN113136378B。In one embodiment of the present invention, the icariside I is obtained by fermenting the epimedium extract with Pichia pastoris engineering bacteria GS115-KA; the icariside I in the fermented epimedium extract The content is greater than or equal to 1%; wherein, the Pichia pastoris engineering strain GS115-KA is a rhamnosidase TpeRha-K579A gene mutant. Wherein, the specific amino acid sequence and nucleotide sequence of the rhamnosidase TpeRha-K579A mutant refer to the applicant's previous application CN113136378B.
在本发明的一个实施例之中，毕赤酵母工程菌GS115-KA的制备方法为：In one embodiment of the present invention, the preparation method of Pichia pastoris engineered bacteria GS115-KA is:
(1)将鼠李糖苷酶TpeRha-K579A基因连接到质粒(pPIC9K)中，得到重组质粒；(1) Linking the rhamnosidase TpeRha-K579A gene into a plasmid (pPIC9K) to obtain a recombinant plasmid;
(2)将所述重组质粒转化至毕赤酵母GS115细胞中，该菌株诱导表达，得到鼠李糖苷酶TpeRha-K579A突变体。(2) The recombinant plasmid was transformed into Pichia pastoris GS115 cells, and the strain was induced to express to obtain the rhamnosidase TpeRha-K579A mutant.
在本发明的一个实施例中，发酵淫羊藿提取物的制备方法为：In one embodiment of the present invention, the preparation method of fermented Epimedium extract is:
(1)提供发酵培养基；(1) Provide fermentation medium;
(2)将所述毕赤酵母工程菌GS115-KA接种至所述发酵培养基中，20～40℃发酵2～5d，然后流加10～20mL/(L·h)的甲醇，诱导产酶80～150h，再将所述鼠李糖苷酶TpeRha-K579A突变体分离，得到发酵液；(2) Inoculate the Pichia pastoris engineered strain GS115-KA into the fermentation medium, ferment at 20-40°C for 2-5 days, and then add 10-20 mL/(L·h) of methanol to induce enzyme production 80 to 150 hours, then separating the rhamnosidase TpeRha-K579A mutant to obtain a fermentation broth;
(3)将1～3L发酵液与1.5～3kg淫羊藿提取物、60～100L水混合，然后在50～60℃下反应10～20h，得到发酵淫羊藿提取物。(3) Mix 1-3L of fermented liquid with 1.5-3kg of Epimedium extract and 60-100L of water, then react at 50-60°C for 10-20h to obtain fermented Epimedium extract.
在本发明的一个实施例中，淫羊藿次苷Ⅰ由鼠李糖苷酶TpeRha-K579A酶对淫羊藿提取物酶解而得，酶解淫羊藿提取物中淫羊藿次苷Ⅰ的含量大于等于1％。In one embodiment of the present invention, icariside I is obtained by enzymatically hydrolyzing the epimedium extract with rhamnosidase TpeRha-K579A enzyme, and enzymatically hydrolyzing the content of icariside I in the epimedium extract The content is greater than or equal to 1%.
相应的，本申请还公开了发酵淫羊藿提取物在制备预防和/或治疗骨质疏松症的药物或保健品中的用途；其中，发酵淫羊藿提取物中淫羊藿次苷Ⅰ的含量大于等于1％。Correspondingly, this application also discloses the use of fermented Epimedium extract in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis; wherein, the content of icariside I in the fermented Epimedium extract The content is greater than or equal to 1%.
相应的，本申请还公开了酶解淫羊藿提取物在制备预防和/或治疗骨质疏松症的药物或保健品中的用途；其中，酶解淫羊藿提取物中淫羊藿次苷Ⅰ的含量 大于等于1％。Correspondingly, this application also discloses the use of the enzymatically hydrolyzed Epimedium extract in the preparation of medicines or health products for the prevention and/or treatment of osteoporosis; wherein, the enzymatically hydrolyzed Epimedium extract contains The content of I is greater than or equal to 1%.
其中，所述药物可制成任意剂型，示例性的为片剂、胶囊剂、颗粒剂、软胶囊、滴丸剂、糖浆剂或注射剂，但不限于此。Wherein, the medicine can be made into any dosage form, exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but not limited thereto.
具体的，使用该药物或保健品治疗或预防骨质疏松症时，淫羊藿次苷Ⅰ每日用量为5～50mg/kg BW(体重)。Specifically, when using the drug or health product to treat or prevent osteoporosis, the daily dosage of icariside I is 5-50 mg/kg BW (body weight).
实施本发明，具有如下有益效果：Implement the present invention, have following beneficial effect:
本发明通过药理实验表明淫羊藿次苷I可用于治疗骨质疏松症。具体的，体外实验结果显示，淫羊藿次苷I对成骨细胞具有明显的促进增殖作用，体内实验结果显示，淫羊藿次苷I可显著提高小鼠骨密度，促进成骨细胞形成，抑制破骨细胞的形成。并且，通过体内实验证明，当剂量范围在5-50mg/kg·BW具有最好的治疗效果，高剂量下(200mg/kg·BW)，无治疗效果。另外，本发明利用发酵的或酶解的淫羊藿提取物添加药学上可接受的载体制备成各种制剂，该制剂毒副作用低，弥补了市售要长期服用毒副作用较大的弊端。The present invention shows that icariside I can be used for treating osteoporosis through pharmacological experiments. Specifically, the results of in vitro experiments show that icariside I can significantly promote the proliferation of osteoblasts, and the results of in vivo experiments show that icariside I can significantly increase the bone density of mice and promote the formation of osteoblasts. Inhibits osteoclast formation. Moreover, it has been proved by in vivo experiments that when the dose ranges from 5-50 mg/kg·BW, it has the best therapeutic effect, and at high doses (200 mg/kg·BW), there is no therapeutic effect. In addition, the present invention uses the fermented or enzymatically hydrolyzed Epimedium extract to add pharmaceutically acceptable carriers to prepare various preparations. The preparations have low toxic and side effects and make up for the disadvantages of long-term use of the commercially available drugs.
附图说明Description of drawings
图1是实施例2中各试验组对小鼠肝脏重量的影响图；Fig. 1 is the impact diagram of each test group on mouse liver weight in embodiment 2;
图2是实施例2中Sham组小鼠肝脏切片图；Fig. 2 is the liver slice figure of Sham group mouse in embodiment 2;
图3是实施例2中OVX组小鼠肝脏切片图；Fig. 3 is the liver section figure of OVX group mouse in embodiment 2;
图4是实施例2中ALN组小鼠肝脏切片图；Fig. 4 is the liver slice figure of ALN group mouse in embodiment 2;
图5是实施例2中L组小鼠肝脏切片图；Fig. 5 is the L group mouse liver slice figure among the embodiment 2;
图6是实施例2中M组小鼠肝脏切片图；Fig. 6 is the liver section figure of group M mice in embodiment 2;
图7是实施例2中H组小鼠肝脏切片图；Fig. 7 is the liver section figure of group H mice in embodiment 2;
图8是实施例2中各试验组对总骨密度的影响图；Fig. 8 is the figure of influence of each test group on total bone density in embodiment 2;
图9是实施例2中各试验组对皮质骨骨密度的影响图；Fig. 9 is the impact diagram of each test group on cortical bone density in embodiment 2;
图10是实施例2中各试验组对松质骨骨密度含量的影响图；Fig. 10 is the impact diagram of each test group on the bone density content of cancellous bone in embodiment 2;
图11是实施例2中各试验组对小鼠骨钙素的影响图；Fig. 11 is the impact diagram of each test group on mouse osteocalcin in embodiment 2;
图12是实施例3中各试验组对rBMSCs细胞中ALP活性的影响图；Fig. 12 is the figure of the influence of each test group on the activity of ALP in rBMSCs cells in embodiment 3;
图13是实施例3中各试验组对rBMSCs细胞中ALP mRNA表达的影响图；Fig. 13 is the impact diagram of each test group on ALP mRNA expression in rBMSCs cells in embodiment 3;
图14是实施例3中各试验组对rBMSCs细胞中Run×2mRNA表达的影响图。Fig. 14 is a diagram showing the influence of each test group on the expression of Run×2 mRNA in rBMSCs cells in Example 3.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。仅此声明，本发明在文中出现或即将出现的上、下、左、右、前、后、内、外等方位用词，仅以本发明的附图为基准，其并不是对本发明的具体限定。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings. It is only stated here that the words for directions such as up, down, left, right, front, back, inside, and outside that appear or will appear in the text of the present invention are only based on the accompanying drawings of the present invention, and are not specific to the present invention. limited.
实施例1Example 1
本实施提供一种发酵淫羊藿提取物的制备方法，其包括：This implementation provides a preparation method of fermented Epimedium extract, which includes:
(1)将鼠李糖苷酶TpeRha-K579A基因连接到质粒pPIC9K上，将该质粒转化到毕赤酵母GS115中，得到工程菌GS115-KA；(1) The rhamnosidase TpeRha-K579A gene was connected to the plasmid pPIC9K, and the plasmid was transformed into Pichia pastoris GS115 to obtain engineering bacteria GS115-KA;
(2)提供发酵培养基；具体的，发酵培养基体积为3.5L，培养基配方为：85％(w/v)H 3PO 4 26.7ml/L，CaSO 40.93g/L，K 2SO 4 18.2g/L，KOH 4.13g/L，MgSO 4·7H 2O 14.9g/L，甘油40g/L，酵母膏5g/L，蛋白胨5g/L，微量元素PTM1 4.4ml/L，消泡油15mL，30mL氨水调pH至5.0。 (2) Provide fermentation medium; specifically, the volume of the fermentation medium is 3.5L, and the medium formula is: 85% (w/v) H 3 PO 4 26.7ml/L, CaSO 4 0.93g/L, K 2 SO 4 18.2g/L, KOH 4.13g/L, MgSO 4 7H 2 O 14.9g/L, glycerin 40g/L, yeast extract 5g/L, peptone 5g/L, trace element PTM1 4.4ml/L, defoaming oil 15mL, 30mL ammonia water to adjust the pH to 5.0.
(3)在发酵培养基中接种5wt％工程菌GS115-KA，并在30℃，200rpm条件下发酵3d，以15ml/L/h的速度流加甲醇进行诱导产酶100h，将菌种分离，得发酵液2L。(3) Inoculate 5wt% engineering bacteria GS115-KA in the fermentation medium, and ferment for 3 days at 30°C under the condition of 200rpm, feed methanol at a speed of 15ml/L/h to induce enzyme production for 100h, and separate the strains, Obtain 2 L of fermented liquid.
(4)取淫羊藿提取物(含20％朝藿定C和10％淫羊藿苷)2kg，80L的纯净水，加入到100L的反应釜，采用缓冲盐调节pH至5，在温度55℃下加入发酵液2L，反应16h后，灭酶，浓缩，干燥，即得发酵淫羊藿提取物。(4) Get 2kg of Epimedium extract (containing 20% epimedin C and 10% icariin), 80L of purified water, join to a 100L reactor, adjust the pH to 5 with buffer salt, at a temperature of 55 Add 2L of fermentation broth at ℃, react for 16 hours, inactivate the enzyme, concentrate, and dry to obtain the fermented Epimedium extract.
实施例2Example 2
发酵淫羊藿提取物对骨质疏松小鼠的影响体内实验Effects of Fermented Epimedium Extract on Osteoporosis Mice in Vivo Experiment
(1)造模：将小鼠卵巢切除进行模型构建。(1) Modeling: Ovariectomized mice were used for model construction.
(2)分组：以做了手术未切除卵巢的小鼠为假手术组(SHAM)，将步骤(1)造好的小鼠骨质疏松模型随机分为造模后对照组(OVX)、阿伦磷酸钠干预组(ALN)、发酵或酶解的淫羊藿提取物(以淫羊藿次苷1剂量计)低中高剂量组L、M、H共6组，每组三只小鼠。(2) Grouping: The mice that underwent surgery without ovariectomy were used as the sham operation group (SHAM), and the mouse osteoporosis model made in step (1) was randomly divided into the control group (OVX), the A Lunronate sodium intervention group (ALN), fermented or enzymatically hydrolyzed Epimedium extract (based on 1 dose of icariin) low, middle and high dose groups L, M, and H, a total of 6 groups, with three mice in each group.
(3)给药：采用灌胃方式给药，其中Sham组、OVX组给予0.2mL/kg·BW玉米油；ALN组给予90mg/kg·BW阿伦磷酸钠，L组为低剂量组，给予5mg/kg·BW的淫羊藿次苷I；M组为中剂量组，给予50mg/kg·BW的淫羊藿次苷I；H组为高剂量组，给予200mg/kg·BW的淫羊藿次苷I，连续给药4周。(3) Administration: administration by intragastric administration, in which Sham group and OVX group were given 0.2mL/kg·BW corn oil; ALN group was given 90mg/kg·BW alendronate sodium; 5 mg/kg·BW of icariin I; M group was the middle dose group, given 50 mg/kg·BW of icariin I; H group was the high dose group, given 200 mg/kg·BW of epimedium Huociside I was administered continuously for 4 weeks.
(4)观察与检测：给药期间，每日观察小鼠食欲、排便、尿液、活动等情况；给药4周后对活体小鼠骨密度和小鼠血清，以及小鼠的肝脏进行检测。(4) Observation and detection: During the administration period, observe the mice's appetite, defecation, urine, activity, etc. every day; after 4 weeks of administration, detect the bone density of the living mice, mouse serum, and the liver of the mice .
结果如图1-11所示。其中，图1是各试验组对各试验组对小鼠肝脏重量的影响图。从图1可以看出，Sham组和ALN组小鼠的肝脏正常，没有受到影响，造模组小鼠的肝脏受到影响，而在低中高三种剂量下，小鼠的肝脏解剖形态学正常。The result is shown in Figure 1-11. Wherein, FIG. 1 is a graph showing the influence of each test group on the mouse liver weight by each test group. It can be seen from Figure 1 that the livers of the mice in the Sham group and the ALN group were normal and not affected, the livers of the mice in the model group were affected, and the liver anatomy and morphology of the mice were normal under the three doses of low, medium and high.
图2～图7是各试验组小鼠的肝脏切片图，从图中来看，在服用发酵淫羊藿苷提取物后，小鼠的肝脏恢复正常，说明发酵淫羊藿提取物毒性较低，或有可能具有修复肝脏的作用。Figures 2 to 7 are liver slices of mice in each test group. From the pictures, after taking the fermented icariin extract, the liver of the mice returned to normal, indicating that the fermented icariin extract has low toxicity , or may have the effect of repairing the liver.
图8～图10是个试验组对骨密度的影响图。从图中可以看出，小鼠给药4周后与OVX相比低剂量组(L)和中剂量组(M)对骨密度都有所提升，且低剂量组(L)的效果最好，低剂量(L)和中剂量组(M)和药物阿伦磷酸钠组相比，效果要比阿伦磷酸钠组(ALN)好，高剂量组(H)则没有改善效果。说明小鼠每天的给药剂量在5-50mg/kg·bw，对骨密度的改善不是剂量依赖性的，高剂量下起到相反的作用。Figures 8 to 10 are diagrams showing the influence of each test group on bone density. It can be seen from the figure that compared with OVX after 4 weeks of administration, the low-dose group (L) and the middle-dose group (M) have improved bone mineral density, and the low-dose group (L) has the best effect , low dose (L) and middle dose group (M) compared with the drug alendronate sodium group, the effect is better than the alendronate sodium group (ALN), and the high dose group (H) has no improvement effect. It shows that the daily dosage of 5-50 mg/kg·bw in mice does not improve the bone density in a dose-dependent manner, and the high dosage has the opposite effect.
图11是各试验组对小鼠骨钙素的影响图，从图中可以看出，在给药4周后，小鼠血液中，Sham组骨钙素含量最高，表现的是正常小鼠的骨质情况，OVX组的骨钙素最低，说明通过卵巢切除造摸后，小鼠的骨流失严重，也表明骨质疏松模型造摸成功，给药后，L组的治疗效果最好，强于阳性对照ALN组，而M组的治疗效果不及L组，但也有一定的效果，比OVX组好，H组表现出了负面的效果，说明，淫羊藿次苷Ⅰ对骨质疏松的治疗在5-50mg/kg·BW的剂量下具有作用，最优的剂量为5mg/kg·BW，高剂量下会有相反的结果。达不到治疗骨质疏松的作用。Figure 11 is a diagram of the influence of each test group on mouse osteocalcin. It can be seen from the figure that after 4 weeks of administration, in the blood of mice, the content of osteocalcin in the Sham group is the highest, which is the expression of normal mice. Osteocalcin in the OVX group was the lowest, indicating that the bone loss of the mice was severe after ovariectomy, which also indicated that the osteoporosis model was successfully established. After administration, the treatment effect of the L group was the best, strong In the positive control ALN group, the treatment effect of M group is not as good as that of L group, but it also has a certain effect, which is better than that of OVX group. It has an effect at a dose of 5-50 mg/kg·BW, the optimal dose is 5 mg/kg·BW, and there will be opposite results at high doses. Cannot reach the effect of treating osteoporosis.
实施例3Example 3
发酵淫羊藿提取物对骨质疏松小鼠的影响体内实验Effects of Fermented Epimedium Extract on Osteoporosis Mice in Vivo Experiment
(1)方法：贴壁筛选法体外培养rBMSCs(大鼠骨髓间充质干细胞)，以不同浓度的发酵淫羊藿提取物对rBMSCs的成骨性分化进行药物干预，ELISA法检测ALP(碱性磷酸酶)的活性，以RT-Time PCR法检测ALP和Run×2mRNA基因的表达。(1) Method: rBMSCs (rat bone marrow mesenchymal stem cells) were cultured in vitro by adherent screening method, the osteogenic differentiation of rBMSCs was intervened with different concentrations of fermented Epimedium extract, and ALP (alkaline Phosphatase) activity, the expression of ALP and Run × 2 mRNA gene was detected by RT-Time PCR method.
(2)实验分组(2) Experimental grouping
对照组：基础培养液：DMEM+10mL/dL胎牛血清(含100U/ml青、链霉素)，发酵淫羊藿提取物干预组：基础培养液+发酵后的或酶解后的淫羊藿提取物1×10 -5mol/L、1×10 -6mol/L、1×10 -7mol/L、1×10 -8mol/L、1×10 -9mol/L、1×10 -10mol/L。 Control group: basic culture medium: DMEM+10mL/dL fetal bovine serum (containing 100U/ml penicillin and streptomycin), fermented Epimedium extract Intervention group: basic culture medium+fermented or enzymolyzed Epimedium Husk extract 1×10 -5 mol/L, 1×10 -6 mol/L, 1×10 -7 mol/L, 1×10 -8 mol/L, 1×10 -9 mol/L, 1× 10 -10 mol/L.
结果如图12～14所示。The results are shown in Figures 12-14.
具体的，图12是各试验组对rBMSCs细胞中ALP活性的影响图，从图中可以看出，不同浓度的发酵淫羊藿提取物干预rBMSCs，ALP活性都比对照组高，且在1×10 -7mol/L、1×10 -8mol/L、1×10 -9mol/L、1×10 -10mol/L效果最显著。 Specifically, Figure 12 is a diagram showing the influence of each test group on the ALP activity in rBMSCs cells. It can be seen from the figure that the ALP activity of different concentrations of fermented Epimedium extract intervened in rBMSCs was higher than that of the control group, and at 1× 10 -7 mol/L, 1×10 -8 mol/L, 1×10 -9 mol/L, 1×10 -10 mol/L have the most significant effect.
图13是各试验组对rBMSCs细胞中ALP mRNA表达的影响图；从图中可以看出，在1×10 -6mol/L浓度下ALP mRNA表达水平显著升高。 Figure 13 is a graph showing the influence of each test group on the expression of ALP mRNA in rBMSCs cells; it can be seen from the graph that the expression level of ALP mRNA significantly increased at a concentration of 1×10 -6 mol/L.
图14是各试验组对rBMSCs细胞中Run×2mRNA表达的影响图；从图中可以看出，在1×10 -7mol/L的浓度下Run×2mRNA的表达水平远远高于对照组。其中，Run×2是成骨细胞的特异转录因子和成骨细胞分化的关键调节因子，是骨发育过程中激活与启动BMSCs向成骨细胞分化并调节成骨细胞成熟的重要转录因子。 Figure 14 is a diagram of the influence of each test group on the expression of Run×2 mRNA in rBMSCs cells; it can be seen from the figure that the expression level of Run×2 mRNA at a concentration of 1×10 -7 mol/L is much higher than that of the control group. Among them, Run×2 is a specific transcription factor for osteoblasts and a key regulator of osteoblast differentiation, and is an important transcription factor for activating and initiating the differentiation of BMSCs into osteoblasts and regulating the maturation of osteoblasts during bone development.
综合图12～图14的结果可以看出：发酵淫羊藿提取物能够促进原代成骨细胞的增殖。且在1×10 -6-1×10 -10mol/L的范围内对原代成骨细胞增殖具有很好的促进作用。 Based on the results of Figures 12 to 14, it can be seen that the fermented Epimedium extract can promote the proliferation of primary osteoblasts. And in the range of 1×10 -6 -1×10 -10 mol/L, it can promote the proliferation of primary osteoblasts very well.
综上，本发明通过安全性实验证明发酵后淫羊藿提取物对宿主的毒性影响很小；通过骨质疏松小鼠的体内实验，证明了发酵淫羊藿提取物能够提高小鼠骨密度，对成骨细胞具有促进增殖的作用，对破骨细胞具有抑制的作用。且淫羊藿次苷Ⅰ对骨质疏松的治疗在5-50mg/kg·BW的剂量下具有作用，高剂量下会有相反的结果，达不到治疗骨质疏松的作用。通过体外实验，证明了发酵淫羊藿提取物对成骨细胞具有增殖作用。根据以上实施例证明，淫羊藿次苷Ⅰ、发酵淫羊藿提取物可以作为治疗骨质疏松症的药物使用。In summary, the present invention proves that the fermented Epimedium extract has little toxic effect on the host through safety experiments; through in vivo experiments on osteoporosis mice, it is proved that the fermented Epimedium extract can increase the bone density of mice, It can promote proliferation of osteoblasts and inhibit osteoclasts. Moreover, icariside Ⅰ has an effect on the treatment of osteoporosis at a dose of 5-50 mg/kg·BW, and a high dose will have the opposite result, and the effect of treating osteoporosis cannot be achieved. Through in vitro experiments, it has been proved that the fermented epimedium extract has a proliferative effect on osteoblasts. According to the above examples, it is proved that icariside I and fermented epimedium extract can be used as medicine for treating osteoporosis.
以上所述是发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。The above is the preferred embodiment of the invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered as protection scope of the present invention.

Claims (10)

  1. 淫羊藿次苷Ⅰ在制备预防和/或治疗骨质疏松症的药物或保健品中的用途。Use of icariside I in the preparation of medicines or health products for preventing and/or treating osteoporosis.
  2. 如权利要求1或2所述的用途，其特征在于，所述淫羊藿次苷Ⅰ的每日用量为5～50mg/kg·BW。The use according to claim 1 or 2, characterized in that the daily dosage of said icariside I is 5-50 mg/kg·BW.
  3. 如权利要求1所述的用途，其特征在于，所述淫羊藿次苷Ⅰ由毕赤酵母工程菌GS115-KA对淫羊藿提取物发酵而得；发酵淫羊藿提取物中淫羊藿次苷Ⅰ的含量大于等于1％；或The use as claimed in claim 1, characterized in that, said icariside I is obtained by fermenting the extract of Epimedium by Pichia pastoris engineering bacteria GS115-KA; The content of inosinoside I is greater than or equal to 1%; or
    所述淫羊藿次苷Ⅰ由TpeRha-K579A酶对淫羊藿提取物酶解而得，酶解淫羊藿提取物中淫羊藿次苷Ⅰ的含量大于等于1％。The icariside I is obtained by enzymatically hydrolyzing the epimedium extract with TpeRha-K579A enzyme, and the content of the icariside I in the enzymatically hydrolyzed epimedium extract is greater than or equal to 1%.
  4. 如权利要求3所述的用途，其特征在于，所述毕赤酵母工程菌GS115-KA为整合鼠李糖苷酶突变基因TpeRha-K579A的毕赤酵母工程菌。The use according to claim 3, characterized in that the Pichia engineered bacterium GS115-KA is an engineered Pichia bacterium integrating the rhamnosidase mutant gene TpeRha-K579A.
  5. 如权利要求4所述的用途，其特征在于，所述毕赤酵母工程菌GS115-KA的制备方法为：purposes as claimed in claim 4, is characterized in that, the preparation method of described Pichia pastoris engineered bacterium GS115-KA is:
    (1)将鼠李糖苷酶TpeRha-K579A基因连接到质粒中，得到重组质粒；(1) connecting the rhamnosidase TpeRha-K579A gene into the plasmid to obtain a recombinant plasmid;
    (2)将所述重组质粒扩增，得到突变产物；(2) amplifying the recombinant plasmid to obtain a mutant product;
    (3)将所述突变产物转化至毕赤酵母GS115细胞中，获得毕赤酵母工程菌GS115-KA，该菌株诱导表达，得到鼠李糖苷酶TpeRha-K579A突变体。(3) Transform the mutant product into Pichia pastoris GS115 cells to obtain Pichia pastoris engineered strain GS115-KA, and induce expression of the strain to obtain the rhamnosidase TpeRha-K579A mutant.
  6. 如权利要求5所述的用途，其特征在于，所述发酵淫羊藿提取物的制备方法为：purposes as claimed in claim 5, is characterized in that, the preparation method of described fermented epimedium extract is:
    (1)提供发酵培养基；(1) Provide fermentation medium;
    (2)将所述毕赤酵母工程菌GS115-KA接种至所述发酵培养基中，20～40℃发酵2～5d，然后流加10～20mL/(L·h)的甲醇，诱导产酶80～150h，再将所述鼠李糖苷酶TpeRha-K579A基因突变体分离，得到发酵液；(2) Inoculate the Pichia pastoris engineered strain GS115-KA into the fermentation medium, ferment at 20-40°C for 2-5 days, and then add 10-20 mL/(L·h) of methanol to induce enzyme production 80 to 150 hours, and then isolate the rhamnosidase TpeRha-K579A gene mutant to obtain a fermentation broth;
    (3)将1～3L发酵液与1.5～3kg淫羊藿提取物、60～100L水混合，然后在50～60℃下反应10～20h，得到发酵淫羊藿提取物。(3) Mix 1-3L of fermented liquid with 1.5-3kg of Epimedium extract and 60-100L of water, then react at 50-60°C for 10-20h to obtain fermented Epimedium extract.
  7. 发酵淫羊藿提取物在制备预防和/或治疗骨质疏松症的药物或保健品中的用途，其特征在于，所述发酵淫羊藿提取物含有淫羊藿次苷Ⅰ。The use of fermented epimedium extract in the preparation of medicines or health products for preventing and/or treating osteoporosis is characterized in that the fermented epimedium extract contains icariside I.
  8. 酶解淫羊藿提取物在制备预防和/或治疗骨质疏松症的药物或保健品中的用途，其特征在于，所述酶解淫羊藿提取物中含有淫羊藿次苷Ⅰ。The use of the enzymatically hydrolyzed epimedium extract in the preparation of medicines or health products for preventing and/or treating osteoporosis is characterized in that the enzymolyzed epimedium extract contains icariside I.
  9. 如权利要求7或8所述的用途，其特征在于，所述淫羊藿次苷Ⅰ的每日用量为5～50mg/kg·BW。The use according to claim 7 or 8, characterized in that the daily dosage of said icariside I is 5-50 mg/kg·BW.
  10. 如权利要求1或7或8所述的用途，其特征在于，所述药物为片剂、胶囊剂、颗粒剂、软胶囊、滴丸剂、糖浆剂或注射剂。purposes as claimed in claim 1 or 7 or 8, is characterized in that, described medicine is tablet, capsule, granule, soft capsule, drop pill, syrup or injection.
PCT/CN2021/135559 2021-10-18 2021-12-04 Use of icariside i WO2023065478A1 (en)

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