CN113940943A - Application of icariside I - Google Patents

Application of icariside I Download PDF

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Publication number
CN113940943A
CN113940943A CN202111211629.7A CN202111211629A CN113940943A CN 113940943 A CN113940943 A CN 113940943A CN 202111211629 A CN202111211629 A CN 202111211629A CN 113940943 A CN113940943 A CN 113940943A
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icariside
epimedium extract
fermented
pichia pastoris
epimedium
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Inventor
李慧灵
黄佳俊
陈川
张利民
罗冬章
林育成
周金林
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Golden Health Biotechnology Co ltd
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Golden Health Biotechnology Co ltd
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Priority to CN202111211629.7A priority Critical patent/CN113940943A/en
Priority to PCT/CN2021/135559 priority patent/WO2023065478A1/en
Publication of CN113940943A publication Critical patent/CN113940943A/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

Abstract

The invention relates to application of icariside I. Experiments show that icariside I can obviously improve the bone density of mice, promote the formation of osteoblasts and inhibit the formation of osteoclasts, so that the icariside I can be applied to the preparation of medicaments or health-care products for preventing and/or treating osteoporosis.

Description

Application of icariside I
Technical Field
The invention relates to the field of medicines, in particular to application of icariside I.
Background
Osteoporosis is a disease characterized by a decrease in bone mass, a change in the microstructure of bone tissue and an increased risk of fracture. The inhibition of osteoblast proliferation and the reduction of osteoblast differentiation are important causes of osteoporosis. The clinical manifestations of osteoporosis are soreness of the waist, pain in the back, weakness of limbs and pain in bones, which are highly regarded by the society and medical field because of high morbidity and great harm in the middle-aged and elderly people. At present, the osteoporosis is mainly treated by taking estrogen, calcium, active vitamin D, calcitonin, fluoride and the like as common medicines, and although the osteoporosis has a certain curative effect, the osteoporosis has the defects of great adverse reaction, incapability of enduring for a long time by patients and the like. As the population life increases and the number of elderly people increases, the risk of osteoporosis becomes more serious. Therefore, finding a medicine for effectively treating osteoporosis is one of the subjects of traditional Chinese medicines in the medical field at present. The traditional Chinese medicine holds that kidney stores essence, mainly takes bones to produce marrow, marrow is stored in bone cavities to nourish bones, the growth and development of bones depend on the nourishing of kidney qi and promote the clinical and experimental results of the traditional Chinese medicine to prove that the traditional Chinese medicine has obvious curative effect on treating primary osteoporosis, and the epimedium is a common traditional Chinese medicine for clinically treating osteoporosis or promoting fracture healing in the traditional Chinese medicine. At present, the Chinese medicine preparation for treating osteoporosis by using the traditional Chinese medicine epimedium is Xianlinggubao. However, long-term use of Xianlinggubao can also cause liver damage. Therefore, the search for a Chinese medicinal preparation with small side effect and obvious curative effect is a difficult problem to be solved urgently in the medical field.
On the other hand, icariside I (Icariside I) is a trace flavonoid glycoside compound in traditional Chinese medicine epimedium, few pharmacological activity research reports about icariside I are reported at present, and epimedium medicaments can increase cardiovascular and cerebrovascular blood flow, promote hematopoietic function, immunologic function and bone metabolism, and also have the effects of tonifying kidney, strengthening yang, resisting aging and the like. Icariside I is a major in vivo metabolite of icariin and has been studied only rarely against osteoporosis.
Disclosure of Invention
The invention aims to solve the technical problem of providing a new application of icariside I, namely an application in preparing a medicament or a health-care product for preventing and/or treating osteoporosis.
The medicament can be prepared into any dosage form, and is exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but is not limited thereto.
Specifically, when the medicine or the health-care product is used for treating or preventing osteoporosis, the daily dosage of icariside I is 5-50mg/kg & BW (body weight).
In one embodiment of the invention, the icariside I is obtained by fermenting an epimedium extract by pichia pastoris engineering bacteria GS 115-KA; the content of icariside I in the fermented epimedium extract is more than or equal to 1 percent; wherein the Pichia pastoris engineering bacteria GS115-KA is a rhamnosidase TpeRhha-K579A gene mutant. The specific amino acid sequence and nucleotide sequence of the rhamnosidase TpeRhha-K579A mutant refer to the preamble application CN113136378B of the applicant.
In one embodiment of the invention, the preparation method of pichia pastoris engineering bacteria GS115-KA is as follows:
(1) the rhamnosidase TpeRhha-K579A gene is connected to a plasmid (pPIC9K) to obtain a recombinant plasmid;
(2) and (3) transforming the recombinant plasmid into a Pichia pastoris GS115 cell, and carrying out induced expression on the strain to obtain the rhamnosidase TpeRhha-K579A mutant.
In one embodiment of the invention, the preparation method of the fermented epimedium extract comprises the following steps:
(1) providing a fermentation medium;
(2) inoculating the pichia pastoris engineering strain GS115-KA into the fermentation culture medium, fermenting for 2-5 days at 20-40 ℃, then feeding 10-20 mL/(L.h) of methanol, inducing enzyme production for 80-150 h, and separating the rhamnosidase TpeRha-K579A mutant to obtain fermentation liquor;
(3) mixing 1-3L fermentation liquor with 1.5-3 kg of epimedium extract and 60-100L of water, and then reacting at 50-60 ℃ for 10-20 h to obtain the fermented epimedium extract.
In one embodiment of the invention, icariside I is obtained by carrying out enzymolysis on epimedium extract by using rhamnosidase TpeRhha-K579A enzyme, and the content of icariside I in the enzymolyzed epimedium extract is more than or equal to 1%.
Correspondingly, the application also discloses the application of the fermented epimedium extract in preparing the medicine or health-care product for preventing and/or treating osteoporosis; wherein the content of icariside I in the fermented epimedium extract is more than or equal to 1 percent.
Correspondingly, the application also discloses application of the enzymolysis epimedium extract in preparing a medicine or health-care product for preventing and/or treating osteoporosis; wherein, the content of icariside I in the enzymolysis epimedium extract is more than or equal to 1 percent.
The medicament can be prepared into any dosage form, and is exemplified by tablets, capsules, granules, soft capsules, dripping pills, syrups or injections, but is not limited thereto.
Specifically, when the medicine or the health-care product is used for treating or preventing osteoporosis, the daily dosage of icariside I is 5-50mg/kg BW (body weight).
The implementation of the invention has the following beneficial effects:
pharmacological experiments show that icariside I can be used for treating osteoporosis. In particular, in vitro experimental results show that icariside I has obvious proliferation promoting effect on osteoblasts, and in vivo experimental results show that icariside I can obviously improve the bone density of mice, promote the formation of osteoblasts and inhibit the formation of osteoclasts. In addition, in vivo experiments prove that the medicine has the best treatment effect when the dosage is in the range of 5-50mg/kg & BW, and has no treatment effect at high dosage (200mg/kg & BW). In addition, the invention utilizes the fermented or enzymolyzed epimedium extract to be added with a pharmaceutically acceptable carrier to prepare various preparations, the preparation has low toxic and side effects, and overcomes the defect that the market needs to take the preparation for a long time, and the toxic and side effects are larger.
Drawings
FIG. 1 is a graph showing the effect of the test groups of example 2 on the weight of mouse liver;
FIG. 2 is a liver slice of the Sham group mice in example 2;
FIG. 3 is a liver slice of OVX group mice in example 2;
FIG. 4 is a liver slice of ALN group mice in example 2;
FIG. 5 is a liver slice of group L mice in example 2;
FIG. 6 is a liver slice of M groups of mice in example 2;
FIG. 7 is a liver slice of group H mice in example 2;
FIG. 8 is a graph of the effect of each test group on total bone density in example 2;
FIG. 9 is a graph of the effect of the test groups of example 2 on cortical bone density;
FIG. 10 is a graph of the effect of the various experimental groups of example 2 on cancellous bone density content;
FIG. 11 is a graph showing the effect of osteocalcin in mice on each test group in example 2;
FIG. 12 is a graph of the effect of the panels in example 3 on ALP activity in rBMSCs cells;
FIG. 13 is a graph of the effect of the panels in example 3 on ALP mRNA expression in rBMSCs cells;
FIG. 14 is a graph of the effect of each panel on Runx 2mRNA expression in rBMSCs cells in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings. It is only noted that the invention is intended to be limited to the specific forms set forth herein, including any reference to the drawings, as well as any other specific forms of embodiments of the invention.
Example 1
The present implementation provides a preparation method of a fermented epimedium extract, which comprises the following steps:
(1) the gene of rhamnosidase TpeRhha-K579A is connected to a plasmid pPIC9K, and the plasmid is transformed into Pichia pastoris GS115 to obtain engineering bacteria GS 115-KA;
(2) providing a fermentation medium; specifically, the volume of the fermentation medium is 3.5L, and the formula of the medium is as follows: 85% (w/v) H3PO4 26.7ml/L，CaSO40.93g/L，K2SO4 18.2g/L，KOH 4.13g/L，MgSO4·7H214.9g/L of O, 40g/L of glycerol, 5g/L of yeast extract, 5g/L of peptone, 14.4mL/L of trace element PTM, 15mL of defoaming oil and 30mL of ammonia water for adjusting the pH to 5.0.
(3) Inoculating 5 wt% engineering bacteria GS115-KA into the fermentation culture medium, fermenting at 30 deg.C and 200rpm for 3d, adding methanol at 15ml/L/h speed to induce enzyme production for 100h, and separating the strain to obtain 2L fermentation liquid.
(4) Taking 2kg of herba Epimedii extract (containing 20% epimedin C and 10% icariin), adding 80L of purified water into a 100L reaction kettle, adjusting pH to 5 with buffer salt, adding 2L of fermentation liquid at 55 deg.C, reacting for 16h, inactivating enzyme, concentrating, and drying to obtain fermented herba Epimedii extract.
Example 2
In vivo experiment of influence of fermented epimedium extract on osteoporosis mice
(1) Molding: mouse ovariectomy was modeled.
(2) Grouping: the mice without ovaries removed after operation are taken as a pseudo-operation group (SHAM), the mouse osteoporosis model manufactured in the step (1) is randomly divided into 6 groups of a post-molding control group (OVX), an Allen sodium phosphate intervention group (ALN) and a fermented or enzymolyzed epimedium extract (with an icariside 1 dosage) low-medium dose group L, M, H, and each group comprises three mice.
(3) Administration: the medicine is administrated by a gastric lavage way, wherein 0.2mL/kg BW corn oil is administrated to a Sham group and an OVX group; ALN group is given 90mg/kg BW Allen sodium phosphate, L group is low dose group and is given 5mg/kg BW icariside I; m group is middle dose group, and 50mg/kg BW icariside I is given; group H was a high dose group, and was given icariside I at 200 mg/kg. BW for 4 weeks.
(4) And (3) observation and detection: during the administration period, the conditions of appetite, defecation, urine, activity and the like of the mice are observed every day; bone density and mouse serum of the living mice, and liver of the mice were examined 4 weeks after the administration.
The results are shown in FIGS. 1 to 11. FIG. 1 is a graph showing the effect of each test group on the weight of mouse liver. As can be seen from fig. 1, the livers of the Sham and ALN groups of mice were normal, not affected, and the livers of the model group of mice were affected, whereas the liver anatomical morphology of the mice was normal at the low, medium and high doses.
FIGS. 2 to 7 are sectional views of the livers of mice in each test group, from which the livers of the mice recovered to normal after administration of the fermented icariin extract, indicating that the fermented icariin extract had low toxicity or may have an effect of repairing the livers.
Fig. 8 to 10 are graphs showing the influence of the individual test groups on bone density. As can be seen from the figure, the low dose group (L) and the medium dose group (M) both increased bone density and showed the best effect compared to OVX at 4 weeks after administration to mice, and the low dose group (L) and the medium dose group (M) showed better effect than the sodium alene phosphate group (ALN) and did not show improved effect compared to the sodium alene phosphate group (H). The mice are shown to be dosed at 5-50 mg/kg-bw each day, the improvement of the bone density is not dose-dependent, and the opposite effect is achieved at high dose.
FIG. 11 is a graph showing the effect of osteocalcin in mice on each test group, wherein after 4 weeks of administration, the content of osteocalcin in Sham group in blood of mice is highest, which shows the bone condition of normal mice, and the content of osteocalcin in OVX group is lowest, which shows that the bone loss of mice is serious after ovariectomy, which also shows that the osteoporosis model is successful, and after administration, the treatment effect of L group is best, which is stronger than that of positive control ALN group, and the treatment effect of M group is not as good as that of L group, but has a certain effect, which is better than that of OVX group, and H group shows negative effect, which shows that icariside I has effect on osteoporosis treatment at 5-50mg/kg BW, the optimal dose is 5mg/kg BW, and the opposite result at high dose. The effect of treating osteoporosis can not be achieved.
Example 3
In vivo experiment of influence of fermented epimedium extract on osteoporosis mice
(1) The method comprises the following steps: adherent screening method for in vitro culture of rBMSCs (rat bone marrow mesenchymal stem cells), drug intervention for osteogenic differentiation of rBMSCs with fermented herba Epimedii extract of different concentrations, ELISA method for detecting ALP (alkaline phosphatase) activity, and RT-Time PCR method for detecting ALP and Runx2 mRNA gene expression.
(2) Experiment grouping
Control group: basic culture solution: DMEM +10mL/dL fetal bovine serum (containing 100U/mL penicillin and streptomycin), fermented epimedium extract dry group: basic culture solution and fermented or enzymolyzed herba Epimedii extract 1 × 10-5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8mol/L、1×10-9mol/L、1×10-10mol/L。
The results are shown in FIGS. 12 to 14.
Specifically, FIG. 12 is a graph showing the effect of the test groups on ALP activity in rBMSCs cells, and it can be seen that the ALP activity was higher than that of the control group at 1X 10 when the fermented Epimedium extract at different concentrations intervenes in rBMSCs-7mol/L、1×10-8mol/L、1×10-9mol/L、1×10-10The mol/L effect is most remarkable.
FIG. 13 shows the respective experimentsGraph demonstrating the effect on ALP mRNA expression in rBMSCs cells; as can be seen from the figure, the value is 1 × 10-6ALP mRNA expression levels were significantly elevated at mol/L concentrations.
FIG. 14 is a graph of the effect of each panel on Runx 2mRNA expression in rBMSCs cells; as can be seen from the figure, the value is 1 × 10-7The expression level of Runx 2mRNA at the concentration of mol/L is much higher than that of the control group. Among them, Runx2 is a specific transcription factor of osteoblast and a key regulator of osteoblast differentiation, and is an important transcription factor for activating and starting BMSCs to differentiate into osteoblast and regulating osteoblast maturation in the bone development process.
The results of fig. 12 to 14 are combined to show that: the fermented epimedium extract can promote the proliferation of primary osteoblasts. And is at 1X 10-6-1×10-10Has good promoting effect on the proliferation of primary osteoblasts within the mol/L range.
In conclusion, the invention proves that the toxicity of the epimedium extract after fermentation has little influence on the host through a safety experiment; in vivo experiments of osteoporosis mice prove that the fermented epimedium extract can improve the bone density of mice, has the effect of promoting proliferation of osteoblasts and has the effect of inhibiting osteoclasts. And icariside I has an effect on the treatment of osteoporosis at a dose of 5-50mg/kg & BW, and opposite results can be obtained at a high dose, so that the effect on the treatment of osteoporosis cannot be achieved. In vitro experiments prove that the fermented epimedium extract has the proliferation effect on osteoblasts. According to the above examples, icariside I and the fermented epimedium extract can be used as the medicine for treating osteoporosis.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. Application of icariside I in preparing medicine or health product for preventing and/or treating osteoporosis is provided.
2. The use according to claim 1 or 2, wherein the icariside I is used in an amount of 5 to 50 mg/kg-BW per day.
3. The use of claim 1, wherein icariside i is obtained by fermenting an epimedium extract with pichia pastoris engineering bacteria GS 115-KA; the content of icariside I in the fermented epimedium extract is more than or equal to 1 percent; or
The icariside I is obtained by carrying out enzymolysis on an epimedium extract by using TpeRha-K579A enzyme, wherein the content of the icariside I in the enzymolyzed epimedium extract is more than or equal to 1%.
4. The use of claim 3, wherein the Pichia pastoris GS115-KA is Pichia pastoris engineering bacteria integrating a rhamnosidase mutant gene TpeRha-K579A.
5. The use of claim 4, wherein the preparation method of pichia pastoris GS115-KA is as follows:
(1) connecting the rhamnosidase TpeRhha-K579A gene to a plasmid to obtain a recombinant plasmid;
(2) amplifying the recombinant plasmid to obtain a mutation product;
(3) and (3) transforming the mutation product into a pichia pastoris GS115 cell to obtain a pichia pastoris engineering strain GS115-KA, and performing induced expression on the strain to obtain the rhamnosidase TpepRha-K579A mutant.
6. The use as claimed in claim 5, wherein the fermented epimedium extract is prepared by a method comprising:
(1) providing a fermentation medium;
(2) inoculating the pichia pastoris engineering strain GS115-KA into the fermentation culture medium, fermenting for 2-5 days at 20-40 ℃, then feeding 10-20 mL/(L.h) of methanol, inducing enzyme production for 80-150 h, and separating the rhamnosidase TpeRha-K579A gene mutant to obtain fermentation liquor;
(3) mixing 1-3L fermentation liquor with 1.5-3 kg of epimedium extract and 60-100L of water, and then reacting at 50-60 ℃ for 10-20 h to obtain the fermented epimedium extract.
7. The application of the fermented epimedium extract in preparing the medicine or the health-care product for preventing and/or treating osteoporosis is characterized in that the fermented epimedium extract contains icariside I.
8. The application of the enzymolysis epimedium extract in preparing the medicine or the health care product for preventing and/or treating osteoporosis is characterized in that the enzymolysis epimedium extract contains icariside I.
9. The use according to claim 7 or 8, wherein the icariside I is used in an amount of 5 to 50 mg/kg-BW per day.
10. The use according to claim 1 or 7 or 8, wherein the medicament is in the form of tablets, capsules, granules, soft capsules, drop pills, syrups or injections.
CN202111211629.7A 2021-10-18 2021-10-18 Application of icariside I Pending CN113940943A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023065478A1 (en) * 2021-10-18 2023-04-27 广东金骏康生物技术有限公司 Use of icariside i

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