Cordyceps sinensis extract and preparation method thereof

Abstract

The invention provides a cordyceps sinensis extract and a preparation method thereof, the cordyceps sinensis extract is a water-soluble cordyceps sinensis extract, and the molecular weight of the cordyceps sinensis extract is less than 10kDa. The cordyceps sinensis extract disclosed by the invention has a good liver and kidney protection function.

Description

Technical Field

[0001] The present application relates to the field of traditional Chinese medicine, and in particular to a Cordyceps extract and a preparation method thereof. Background Art

[0002] Cordyceps sinensis is a complex of the fruiting bodies and larval corpses of the ergot fungus Cordyceps sinensis (Berk.) Sacc. parasitized on the larvae of the bat moth family insects. It is a precious Chinese medicinal material (hereinafter referred to as Cordyceps). Due to the special growth environment of natural Cordyceps and its high price, it cannot meet the market demand. Therefore, people use modern biological fermentation technology to cultivate Cordyceps mycelium to replace natural Cordyceps. In 1982, the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences isolated more than a dozen strains from Cordyceps sinensis samples collected in Diqing Tibetan Autonomous Prefecture, Yunnan Province, and cultured and morphologically studied 4 of them. After identification, they were named Paecilonyces hepialid chen & Dai. In 1982, the Institute of Materia Medica, Chinese Academy of Medical Sciences isolated the Cs-4 strain from fresh Cordyceps sinensis collected in Hualong County, Qinghai Province, which was also identified as Paecilonyces hepialid chen & Dai. Fermented Cordyceps powder Cs-4, hereinafter referred to as Cs-4, is the dry powder of mycelium obtained by fermenting the isolated strain of Paecilomyces cereus Cs-4. It is used as a raw material to make medicines that have been successfully launched on the market. The names of the medicines are Jinshuibao Capsules and Jinshuibao Tablets.

[0003] At present, the research on the active ingredients of Cordyceps mainly focuses on its fat-soluble components and water-soluble macromolecular polysaccharides, while there is little research on the water-soluble low-molecular weight parts. Summary of the invention

[0004] The present application obtains a water-soluble low molecular weight Cordyceps extract by defatting, water extraction, alcohol precipitation and ultrafiltration, and evaluates its efficacy in animal models of acute kidney injury and chronic kidney disease. The Cordyceps extract of the present application has excellent kidney and liver protective effects, showing application prospects in the treatment and prevention of acute kidney injury and / or chronic kidney disease.

[0005] In one aspect, the present application provides a Cordyceps extract, which is a water-soluble Cordyceps extract, and the molecular weight of the Cordyceps extract is less than 10 kDa.

[0006] In certain embodiments, the cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

[0007] In certain embodiments, the Cordyceps is fermented Cordyceps Cs-4.

[0008] In certain embodiments, the fermented Cordyceps includes fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0009] In certain embodiments, the cordyceps is fermented cordyceps powder Cs-4.

[0010] In certain embodiments, the Cordyceps extract is non-lipid soluble.

[0011] In certain embodiments, the total sugar content in the Cordyceps extract is ≥ 5% w / w.

[0012] In certain embodiments, the polypeptide content in the Cordyceps extract is ≥ 20% w / w.

[0013] In certain embodiments, the nucleoside content in the Cordyceps extract is ≥ 2% w / w.

[0014] On the other hand, the present application provides a method for preparing a Cordyceps extract, which comprises the following steps: subjecting the Cordyceps to defatting, water extraction, alcohol precipitation and ultrafiltration processes to obtain a water-soluble extract; wherein the ultrafiltration process obtains a filtrate with a molecular weight of less than 10 kDa.

[0015] In certain embodiments, the method further comprises concentrating and drying the filtrate obtained in the ultrafiltration process.

[0016] In certain embodiments, the degreasing comprises: organic solvent extraction degreasing, supercritical CO2 extraction degreasing.

[0017] In certain embodiments, the defatting is an organic solvent extraction, wherein the organic solvent is selected from: petroleum ether, n-hexane, ethyl acetate, dichloromethane, ethanol and / or methanol.

[0018] In certain embodiments, the cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

[0019] In certain embodiments, the Cordyceps is fermented Cordyceps Cs-4.

[0020] In certain embodiments, the fermented Cordyceps includes fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0021] In certain embodiments, the cordyceps is fermented cordyceps powder Cs-4.

[0022] In certain embodiments, the method comprises: defatting the fermented Cordyceps powder Cs-4 with ethanol, discarding the ethanol extract to obtain a defatted residue; extracting the residue with water and refluxing it, precipitating the obtained extract with ethanol, ultrafiltration of the obtained precipitate to obtain a filtrate with a molecular weight of less than 10 kDa, concentrating and drying to obtain a Cordyceps extract.

[0023] In certain embodiments, the method comprises the following steps:

[0024] 1) Degreasing: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling with 5 to 20 times (by mass) of 60% to 100% v / v ethanol, and the extraction is maintained at a slight boiling temperature for 0.5 to 2 hours, and the alcohol extract is discarded. After degreasing 1 to 3 times, the powder is used for later use;

[0025] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 5 to 20 times the volume of water, reflux and extract 1 to 3 times, each time for 0.5 to 2 hours, combine the obtained water extracts and concentrate to a relative density of 1.08, cool to room temperature, add 95% v / v ethanol to an alcohol concentration of 60 to 80% v / v, stand overnight, discard the supernatant, and dry the precipitate for later use;

[0026] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0027] In certain embodiments, the method comprises the following steps:

[0028] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78° C.) with about 10 times the amount (mass) of 85% v / v ethanol, and the extraction is maintained at a slight boiling temperature for about 1 hour, and the alcohol extract is discarded. The defatted powder is then used for later use;

[0029] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add about 10 times the amount (volume) of water, reflux and extract twice, each time for about 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 70% v / v, let it stand overnight, discard the supernatant, and dry the precipitate for later use;

[0030] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0031] In another aspect, the present application provides a composition comprising the Cordyceps extract described in the present application.

[0032] In certain embodiments, the composition further comprises one or more physiologically acceptable carriers, excipients, and diluents.

[0033] In certain embodiments, the formulation of the composition is selected from the group consisting of tablets, suspensions, granules, pills, powders, pellets, syrups, mixtures, dews, effervescents, pastes, emulsions, teas, powders, injections, gels, patches, plasters, creams, ointments, lotions, suppositories, capsules, and injections.

[0034] In certain embodiments, the composition is a pharmaceutical composition and / or a health food composition.

[0035] On the other hand, the present application provides use of the Cordyceps extract described in the present application or the composition described in the present application in the preparation of a medicament for preventing and / or treating kidney disease.

[0036] In certain embodiments, the kidney disease comprises acute kidney injury and / or chronic kidney disease.

[0037] On the other hand, the present application provides use of the Cordyceps extract described in the present application or the composition described in the present application in preparing health food. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly describe the features and advantages of the present invention, the following is a brief description of the accompanying drawings:

[0039] Figure 1 The UPLC ultraviolet chromatogram (UV 254 nm) of the Cordyceps extract of the present application is shown.

[0040] Figure 2 The result graphs showing the efficacy evaluation of the Cordyceps extract of the present application in improving acute kidney injury are shown; A: serum urea nitrogen; B: serum creatinine; C: kidney coefficient; D: liver coefficient.

[0041] Figure 3 The result graphs showing the efficacy evaluation of the Cordyceps extract in Example 4 of the present application in improving chronic kidney disease are shown; A: body weight change curve; B. serum creatinine change curve; C. serum urea nitrogen on the 45th day; D. 12h urine volume on the 45th day.

[0042] Figure 4 The figure shows the results of HE staining of kidney pathology in Example 5 of the present application. DETAILED DESCRIPTION

[0043] Other features and advantages of the present invention will be apparent through the following examples, which should not be construed as limiting. As will be appreciated by those skilled in the art, the content of the present invention enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the present invention.

[0044] Definition of terms

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology will be used.

[0046] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] In the present application, Cordyceps refers to all Cordyceps species, including fermented Cordyceps, Cordyceps sinensis ("caterpillar fungus" or "winter worm summer grass"), Cordyceps militaris (C.militaris), Cordyceps capitata (C.capita), Cordyceps pruinosa (C.pruinosa), Cordyceps ophioglossoides, Paecilomyce shepiali, C.chanhua, etc. In some embodiments, the Cordyceps species is selected from fermented Cordyceps, Cordyceps sinensis, Cordyceps militaris, Cordyceps capitata, Cordyceps ophioglossoides, and Cordyceps ophioglossoides.

[0048] In the present application, the term "fermented Cordyceps powder" refers to the dry powder of mycelium obtained by fermentation culture of a strain isolated from fresh Cordyceps sinensis. "Fermented Cordyceps powder Cs-4" is the powder obtained by fermentation of Paecilonyces hepialid chen & Dai Cs-4 strain.

[0049] In the present application, the term "treatment" of a disease or condition generally refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject. The terms "treatment", "treating", "treat", etc. as used herein generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of preventing a disease or its symptoms completely or partially, and / or therapeutic in terms of partial or complete stabilization or cure of a disease and / or the adverse effects attributable to the disease. The term "treatment" encompasses any treatment of a disease in a mammal (particularly a human being), and includes: (a) preventing a disease and / or symptom from occurring in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed with the disease or symptom; (b) inhibiting a disease and / or symptom, such as slowing down or preventing its development (e.g., preventing the growth of a tumor, slowing down the growth rate of a tumor, preventing the proliferation rate of cancer cells, etc.); or (c) alleviating disease symptoms, i.e., causing a regression of the disease and / or symptoms (e.g., causing a reduction in tumor size, reducing the number of cancer cells present, etc.). Patients in need of treatment include those already suffering from it (eg, patients with cancer, etc.) as well as those in need of prevention (eg, patients with increased susceptibility to cancer, etc.).

[0050] In this application, the term "physiologically acceptable excipient, carrier or diluent" generally refers to an excipient, carrier or diluent that can be administered to a subject with an agent without destroying its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the agent.

[0051] In this application, the term "therapeutically effective amount" generally refers to the amount of the subject compound or composition that will cause the biological, physiological, clinical or medical response of the cell, tissue, organ, system or subject that the researcher, veterinarian, doctor or other clinician is seeking. The term "therapeutically effective amount" includes the amount of the compound or composition that is sufficient to prevent or to some extent treat one or more signs or symptoms of the disease or condition being treated when administered. The therapeutically effective amount can vary according to the compound or composition, the disease and its severity and the age, weight, etc. of the subject to be treated.

[0052] In this application, the terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any animal or cell thereof suitable for the methods herein, whether in vivo, in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0053] In the present application, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0054] In this application, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with reference to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0055] References in the specification to "certain embodiments," "some embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0056] The ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and all intermediate decimal values ​​between the above integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" extending from either end of a range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0057] In this application, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. Those skilled in the art can easily determine the degree of flexibility of a specific variable. As used herein, when used in reference to a specific listed numerical value, the term "about" means that the value can vary by no more than 10% from the listed value. For example, the expression "about 100" includes all values ​​between 90 and 110 (e.g., 91, 92, 93, 94, etc.). DETAILED DESCRIPTION OF THE INVENTION

[0059] In one aspect, the present application provides a Cordyceps extract, which is a water-soluble Cordyceps extract and has a molecular weight of less than 10 kDa.

[0060] In certain embodiments, the cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

[0061] In certain embodiments, the fermented Cordyceps includes fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0062] In certain embodiments, the fermented Cordyceps includes fermented Cordyceps mycelium and / or fermented Cordyceps powder.

[0063] In certain embodiments, the cordyceps is fermented cordyceps powder Cs-4.

[0064] In certain embodiments, the extract is non-lipid soluble.

[0065] In certain embodiments, the total sugar content in the Cordyceps extract is ≥1% w / w. In certain embodiments, the total sugar content in the Cordyceps extract is ≥2% w / w. In certain embodiments, the total sugar content in the Cordyceps extract is ≥3% w / w. In certain embodiments, the total sugar content in the Cordyceps extract is ≥5% w / w. In certain embodiments, the total sugar content in the Cordyceps extract is 2% w / w to 20% w / w, 3% w / w to 15% w / w, or 5% w / w to 15% w / w. For example, the concentration of total sugars per 1g of Cordyceps extract is 50mg to 150mg.

[0066] In the context of the present invention, the term "total sugar" refers to the sum of all sugar substances contained in the Cordyceps extract. The term "total sugar content" refers to the concentration of all sugar substances contained in the Cordyceps extract.

[0067] In certain embodiments, the polypeptide content in the Cordyceps extract is ≥10% w / w. In certain embodiments, the polypeptide content in the Cordyceps extract is ≥15% w / w. In certain embodiments, the polypeptide content in the Cordyceps extract is ≥20% w / w. In certain embodiments, the total sugar content in the Cordyceps extract is 10% w / w to 50% w / w, 15% w / w to 40% w / w, or 20% w / w to 40% w / w. For example, the concentration of polypeptides per 1g of Cordyceps extract is 200mg to 400mg.

[0068] In some embodiments, the content of nucleosides in the Cordyceps extract is ≥1% w / w. In some embodiments, the content of nucleosides in the Cordyceps extract is ≥2% w / w. In some embodiments, the total sugar content in the Cordyceps extract is 1% w / w to 6% w / w or 2% w / w to 5% w / w. For example, the concentration of nucleosides in each 1g of Cordyceps extract is 20mg to 50mg.

[0069] For example, the cordyceps extract is a water-soluble cordyceps extract of fermented cordyceps powder Cs-4, and the molecular weight of the cordyceps extract is less than 10 kDa. For another example, the cordyceps extract is a water-soluble cordyceps extract of fermented cordyceps powder Cs-4, and the molecular weight of the cordyceps extract is less than 10 kDa, and the total sugar content in the cordyceps extract is ≥5% w / w, the polypeptide content is ≥20% w / w, and the nucleoside content is ≥2% w / w.

[0070] For another example, the Cordyceps extract is a water-soluble Cordyceps extract of fermented Cordyceps powder Cs-4, and the molecular weight of the Cordyceps extract is less than 10kDa, and the total sugar content of the Cordyceps extract is 50 to 150 mg / g, the polypeptide content is 200 to 400 mg / g and the nucleoside content is 30 to 50 mg / g.

[0071] On the other hand, the present application provides a method for preparing a Cordyceps extract, which comprises the following steps: subjecting the Cordyceps to defatting, water extraction, alcohol precipitation and ultrafiltration processes to obtain a water-soluble extract; wherein the molecular weight of the Cordyceps extract is less than 10 kDa.

[0072] In certain embodiments, the method further comprises concentrating and drying the filtrate obtained in the ultrafiltration process.

[0073] In certain embodiments, the degreasing comprises: organic solvent extraction degreasing, supercritical CO2 extraction degreasing.

[0074] In certain embodiments, the defatting is performed by organic solvent extraction, wherein the organic solvent is selected from: petroleum ether, n-hexane, dichloromethane, chloroform, ethyl acetate, ethanol and / or methanol.

[0075] In certain embodiments, the cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps. When preparing the Cordyceps extract according to the present invention, a mixture of different Cordyceps species may be used, such as a mixed Cordyceps including both Cordyceps sinensis and Cordyceps militaris.

[0076] In certain embodiments, the cordyceps is fermented cordyceps. For example, the cordyceps can be fermented cordyceps Cs-4.

[0077] In certain embodiments, the fermented Cordyceps includes fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0078] In some embodiments, the Cordyceps is fresh. In other embodiments, the Cordyceps extract can be prepared based on dried Cordyceps.

[0079] In some embodiments, for the preparation method of Cordyceps extract, the Cordyceps is ground into powder. Grinding increases the surface / volume ratio of the Cordyceps particles, thereby improving the extraction efficiency. For example, Cordyceps mycelium can be ground in water to directly obtain Cordyceps slurry. For another example, the grinding of dry Cordyceps can be achieved by chopping, cutting or other methods. For example, Cordyceps can be ground into a particle size of 0.001mm to 5mm, 0.001mm to 5mm, 0.01mm to 5mm, 0.05mm to 5mm, 0.1mm to 4mm or 0.5mm to 3mm.

[0080] In some embodiments, the cordyceps is fermented cordyceps powder. For example, the cordyceps is fermented cordyceps powder Cs-4.

[0081] In certain embodiments, the method comprises: defatting the fermented Cordyceps powder Cs-4 with ethanol, discarding the ethanol extract to obtain a defatted residue; extracting the residue with water and refluxing it, precipitating the obtained extract with ethanol, ultrafiltration of the obtained precipitate to obtain a filtrate with a molecular weight of less than 10 kDa, concentrating and drying to obtain a Cordyceps extract.

[0082] In certain embodiments, the method comprises the following steps:

[0083] 1) Degreasing: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (about 78° C.) with 5 to 20 times the amount (mass) of 60 to 100% v / v ethanol, and the extraction is maintained at a slight boiling temperature for 0.5 to 2 hours, and the alcohol extract is discarded. After degreasing 1 to 3 times, the powder is used for later use;

[0084] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 5 to 20 times the amount (volume) of water, reflux extraction 1 to 3 times, each time for 0.5 to 2 hours, combine the obtained water extracts and concentrate to a relative density of 1.08 (about 60° C.), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 60 to 80% v / v, let stand overnight, discard the supernatant, and dry the precipitate for later use;

[0085] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0086] For example, the method may include the following steps:

[0087] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78° C.) with about 10 times the amount (mass) of 85% v / v ethanol, and the mixture is extracted for 1 hour while maintaining a slight boiling temperature. The alcohol extract is discarded, and the defatted powder is used for later use;

[0088] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add about 10 times the volume of water, reflux and extract twice, each time for 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 70% v / v, let it stand overnight, discard the supernatant, and dry the precipitate for later use;

[0089] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0090] For example, the method may include the following steps:

[0091] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78° C.) with about 10 times the amount (mass) of 85% v / v ethanol, and the mixture is extracted for 1 hour while maintaining a slight boiling temperature. The alcohol extract is discarded, and the defatted powder is used for later use;

[0092] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add about 10 times the volume of water, reflux and extract twice, each time for 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 70% v / v, let it stand overnight, discard the supernatant, and dry the precipitate for later use;

[0093] 3) Ultrafiltration: Take the above alcohol precipitate, add water to redissolve, centrifuge, take the supernatant and treat it with 150kDa and 10kDa ultrafiltration columns in sequence, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0094] In another aspect, the present application provides a Cordyceps extract obtainable by the above method.

[0095] For another example, the present invention provides a Cordyceps extract, and the preparation method of the Cordyceps extract comprises:

[0096] The fermented Cordyceps powder Cs-4 is defatted with ethanol, and the ethanol extract is discarded to obtain a defatted residue; the residue is refluxed with water for extraction, the obtained extract is precipitated with ethanol, the obtained precipitate is ultrafiltered to obtain a filtrate with a molecular weight of less than 10kDa, and the filtrate is concentrated and dried to obtain a Cordyceps extract.

[0097] For another example, the present invention provides a Cordyceps extract, and the preparation method of the Cordyceps extract comprises the following steps:

[0098] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (about 78° C.) with 5 to 20 times the amount (mass) of 60 to 100% v / v ethanol, and the extraction is maintained at a slight boiling temperature for 0.5 to 2 hours, and the alcohol extract is discarded. After defatting 1 to 3 times, the powder is used for later use;

[0099] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 5 to 20 times the amount (volume) of water, reflux extraction 1 to 3 times, each time for 0.5 to 2 hours, combine the obtained water extracts and concentrate to a relative density of 1.08 (about 60° C.), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 60 to 80% v / v, let stand overnight, discard the supernatant, and dry the precipitate for later use;

[0100] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0101] For another example, the present invention provides a Cordyceps extract, and the preparation method of the Cordyceps extract comprises the following steps:

[0102] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78° C.) with about 10 times the amount (mass) of 85% v / v ethanol, and the mixture is extracted for 1 hour while maintaining a slight boiling temperature. The alcohol extract is discarded, and the defatted powder is used for later use;

[0103] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 10 times the volume of water and reflux extract twice, each time for 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 70% v / v, let it stand overnight, discard the supernatant, and dry the precipitate for later use;

[0104] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0105] On the other hand, the present application provides a composition comprising the Cordyceps extract described in the present application. The composition can be a medicine, a food product, a beverage, a functional health food, a nutrient, a food additive, a nutritional formula, and a pet food.

[0106] In certain embodiments, the composition further comprises one or more physiologically acceptable carriers, excipients, and diluents.

[0107] In certain embodiments, the formulation of the composition is selected from the group consisting of tablets, suspensions, granules, pills, powders, pellets, syrups, mixtures, dews, effervescents, pastes, emulsions, teas, powders, injections, gels, patches, plasters, creams, ointments, lotions, suppositories, capsules, and injections.

[0108] In certain embodiments, the composition is a pharmaceutical composition and / or a health food composition.

[0109] On the other hand, the present application provides use of the Cordyceps extract described in the present application or the composition described in the present application in the preparation of a medicament for preventing and / or treating kidney disease.

[0110] In certain embodiments, the kidney disease comprises acute kidney injury and / or chronic kidney disease.

[0111] On the other hand, the present application provides use of the Cordyceps extract described in the present application or the composition described in the present application in preparing health food.

[0112] In another aspect, the present application provides a method for preventing and / or treating kidney disease, comprising administering the Cordyceps extract or the composition described in the present application to a subject in need thereof.

[0113] This application provides the following specific implementation scheme:

[0114] Embodiment 1: A Cordyceps extract, wherein the extract is a water-soluble Cordyceps extract, and the molecular weight of the Cordyceps extract is less than 10 kDa.

[0115] Embodiment 2: According to the Cordyceps extract of Embodiment 1, the Cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

[0116] Embodiment 3: According to the Cordyceps extract according to any one of Embodiments 1-2, the Cordyceps is fermented Cordyceps.

[0117] Embodiment 4: According to the Cordyceps extract according to any one of Embodiments 1 to 3, the Cordyceps is fermented Cordyceps Cs-4.

[0118] Embodiment 5: According to any one of embodiments 1-4, the Cordyceps extract comprises fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0119] Embodiment 6: According to the Cordyceps extract according to any one of Embodiments 1 to 5, the Cordyceps is fermented Cordyceps fungus powder Cs-4.

[0120] Embodiment 7: The Cordyceps extract according to any one of Embodiments 1-6, wherein the Cordyceps extract has one or more of the following properties:

[0121] 1) The Cordyceps extract is non-fat soluble;

[0122] 2) The total sugar content in the Cordyceps extract is ≥ 5% w / w;

[0123] 3) The polypeptide content in the Cordyceps extract is ≥ 20% w / w; and

[0124] 4) The nucleoside content in the Cordyceps extract is ≥ 2% w / w.

[0125] Embodiment 8: A method for preparing a Cordyceps extract, comprising the following steps: subjecting the Cordyceps to defatting, water extraction, alcohol precipitation and ultrafiltration processes to obtain a water-soluble extract; wherein the ultrafiltration process obtains a filtrate with a molecular weight of less than 10 kDa.

[0126] Embodiment 9: The preparation method according to Embodiment 8, wherein the method further comprises concentrating and drying the filtrate obtained in the ultrafiltration process.

[0127] Embodiment 10: A preparation method according to any one of embodiments 8-9, wherein the degreasing comprises: organic solvent extraction degreasing, supercritical CO2 extraction degreasing.

[0128] Embodiment 11: The preparation method according to any one of Embodiments 8-10, wherein the defatting is an organic solvent extraction, wherein the organic solvent is selected from: petroleum ether, ethyl acetate, ethanol and / or methanol.

[0129] Embodiment 12: The preparation method according to any one of Embodiments 8-11, wherein the cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

[0130] Embodiment 13: The preparation method according to any one of Embodiments 8-12, wherein the Cordyceps is fermented Cordyceps Cs-4.

[0131] Embodiment 14: The preparation method according to any one of Embodiments 8-13, wherein the fermented Cordyceps comprises fermented Cordyceps mycelium, fermented Cordyceps slurry and / or fermented Cordyceps powder.

[0132] Embodiment 15: A preparation method according to any one of Embodiments 8-14, wherein the cordyceps is fermented cordyceps powder Cs-4.

[0133] Embodiment 16: A preparation method according to any one of Embodiments 8-15, wherein the method comprises: defatting the fermented Cordyceps fungus powder Cs-4 with ethanol, discarding the ethanol extract, and obtaining a defatted residue; extracting the residue by reflux with water, precipitating the obtained extract with ethanol, ultrafiltration of the obtained precipitate, obtaining a filtrate with a molecular weight of less than 10 kDa, and concentrating and drying to obtain a Cordyceps extract.

[0134] Embodiment 17: The preparation method according to any one of embodiments 8-16, wherein the method comprises the following steps:

[0135] 1) Degreasing: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling with 5 to 20 times (by mass) of 60 to 100% v / v ethanol, and the extraction is maintained at a slight boiling temperature for 0.5 to 2 hours, and the alcohol extract is discarded. After degreasing 1 to 3 times, the powder is used for later use;

[0136] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 5 to 20 times the volume of water, reflux and extract 1 to 3 times, each time for 0.5 to 2 hours, combine the obtained water extracts and concentrate to a relative density of 1.08, cool to room temperature, add 95% v / v ethanol to an alcohol concentration of 60 to 80% v / v, stand overnight, discard the supernatant, and dry the precipitate for later use;

[0137] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0138] Embodiment 18: The preparation method according to any one of embodiments 8-17, wherein the method comprises the following steps:

[0139] 1) Defatting: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78° C.) with about 10 times the volume fraction (mass) of 85% v / v ethanol, and the extraction is maintained at a slight boiling temperature for about 1 hour, and the alcohol extract is discarded. The defatted powder is then used for later use;

[0140] 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add about 10 times the amount (volume) of water, reflux and extract twice, each time for about 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% v / v ethanol to an alcohol concentration of about 70% v / v, let it stand overnight, discard the supernatant, and dry the precipitate for later use;

[0141] 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

[0142] Embodiment 19: A composition comprising the Cordyceps extract according to any one of Embodiments 1-7.

[0143] Embodiment 20: The composition according to embodiment 19, wherein the composition further comprises one or more physiologically acceptable carriers, excipients and diluents.

[0144] Embodiment 21: A composition according to any one of embodiments 19-20, wherein the formulation form of the composition is selected from the group consisting of: tablets, suspensions, granules, pills, powders, pellets, syrups, mixtures, dews, effervescents, pastes, emulsions, teas, powders, injections, gels, patches, plasters, creams, ointments, lotions, suppositories, capsules, and injections.

[0145] Embodiment 22: The composition according to any one of Embodiments 19 to 21, which is a pharmaceutical composition and / or a health food composition.

[0146] Embodiment 23: Use of the Cordyceps extract according to any one of Embodiments 1-7 or the composition according to any one of Embodiments 19-22 in the preparation of a medicament for preventing and / or treating kidney disease.

[0147] Embodiment 24: The use according to embodiment 23, wherein the kidney disease comprises acute kidney injury and / or chronic kidney disease.

[0148] Embodiment 25: Use of the Cordyceps extract according to any one of Embodiments 1-7 or the composition according to any one of Embodiments 19-22 in the preparation of health food.

[0149] Example

[0150] Example 1 Preparation of Cordyceps Extract

[0151] 1. Defatting: Fermented Cordyceps sinensis powder Cs-4 is heated to boiling (78°C) with 10 times the volume fraction (mass) of 85% ethanol, and the extraction is maintained at a slight boiling temperature for 1 hour. The alcohol extract is discarded, and the defatted powder is set aside.

[0152] 2. Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 10 times the amount (volume) of water, reflux and extract twice, each time for 1 hour, combine the obtained water extracts and concentrate to a relative density of 1.08 (60°C), cool to room temperature, add 95% ethanol to an alcohol concentration of 70%, let stand overnight, discard the supernatant, precipitate and dry for later use.

[0153] 3. Ultrafiltration: Take the above alcohol precipitate, add water to dissolve, centrifuge, take the supernatant and treat it with 150kDa and 10kDa ultrafiltration columns in sequence, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract with a yield of 5% to 15% (see Table 1 for details). UPLC analysis shows Figure 1 shown.

[0154] Example 2 Analysis of Cordyceps Extract Components

[0155] 1. Determination of total sugar content:

[0156] Refer to the polysaccharide determination method in SN / T 4260-2015 "Determination of crude polysaccharides in plant-derived foods for export - phenol-sulfuric acid method". This method uses the method of hydrolyzing polysaccharides into monosaccharides under the action of concentrated acid, forming aldehyde derivatives through dehydration condensation, and then combining with phenol for color development, and calculating the total sugar content by measuring the absorbance value after color development. When using this method to determine the polysaccharide content, a monosaccharide must be selected as a reference substance to draw a standard curve.

[0157] Standard curve drawing: Weigh 5 mg of glucose, weigh accurately, place in a 50 ml volumetric flask, add water to dissolve and dilute to obtain a 0.1 mg / ml glucose standard solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml of the standard solution into a 20 ml stoppered test tube, add distilled water to 1.0 ml, add 1.0 ml of the freshly prepared 5 wt% phenol solution and 5.0 ml of concentrated sulfuric acid, shake well, place at room temperature for 10 min, use a vortex oscillator to fully mix the reaction solution, then place the test tube in a boiling water bath to react for 20 min, and measure the absorbance at 490 nm. With the glucose concentration (mg) as the horizontal axis and the absorbance value as the vertical axis, linear regression is performed to obtain the standard curve equation.

[0158] Test sample detection: Weigh 50 mg of Cordyceps extract, accurately weigh, place in a 10 ml volumetric flask, ultrasonically dissolve, add water to the mark, shake well, and obtain. Take 0.2 ml of each of the above prepared solutions in a 20 ml stoppered test tube, add water to 1.0 ml. Make up to 1.0 ml with distilled water, add 1.0 ml of the freshly prepared 5wt% phenol solution and 5.0 ml of concentrated sulfuric acid, shake well, place at room temperature for 10 minutes, use a vortex oscillator to fully mix the reaction solution, then place the test tube in a boiling water bath to react for 20 minutes, measure the absorbance at 490 nm, read the total sugar content (mg / ml) in the test solution from the standard curve, and calculate.

[0159] 2. Determination of peptide content

[0160] This method is based on the peptide bonds contained in the polypeptide molecules in alkaline solution with Cu 2+ Chelation forms a polypeptide-copper complex. This complex reduces the phosphomolybdic acid in the phenol reagent to produce a blue compound. At the same time, under alkaline conditions, the phenol reagent is easily reduced by tyrosine, tryptophan, and cysteine ​​in the polypeptide to produce a blue reaction. Within a certain range, the color depth is proportional to the polypeptide concentration.

[0161] Preparation of standard curve: Take serum albumin (bovine) reference substance, dissolve it in water and make a solution containing 0.2 mg per 1 ml. Accurately measure 0.2, 0.4, 0.6, 0.8, 1.0 ml of reference substance solution, place them in stoppered test tubes, add water to 1.0 ml each, then add 1.0 ml of alkaline copper test solution, mix well, leave at room temperature for 10 minutes, add 3.0 ml of folin phenol test solution to each, mix well immediately, leave at room temperature for 20 minutes, and measure the absorbance at a wavelength of 650 nm according to the UV-visible spectrophotometry (2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 0401), with absorbance as the ordinate and concentration (mg / ml) as the abscissa; at the same time, use the corresponding reagent as the blank. Calculate the linear regression equation based on the concentration of the reference solution and its corresponding absorbance.

[0162] Sample determination: Take 25 mg of the test sample, weigh accurately, place in a 25 ml volumetric flask, add appropriate amount of water, dissolve by ultrasonic, cool to room temperature, adjust to volume, shake well, centrifuge, take the supernatant, and obtain the test solution. Take the test solution, measure the absorbance according to the method under the standard curve, calculate the polypeptide concentration (mg / ml) in the test solution from the linear regression equation, and multiply it by the dilution factor, and calculate to obtain.

[0163] 3. Determination of nucleoside content

[0164] The nucleoside content in the extract was determined by UV spectrophotometry.

[0165] Drawing of a series of standard curves: Take about 3.4 mg of adenosine reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve it, dilute it to the scale, shake it well, and you will get the mother solution of the reference substance. Accurately measure 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 of the reference substance solution and place it in a 20 ml volumetric flask, add water to dilute it to the scale line, shake it well for later use, and prepare a series of standard curve solutions. Take the reference substance solution, use the corresponding reagent as a blank, and measure the absorbance at a wavelength of 260 nm according to the UV-Vis spectrophotometer method (General Rules 0401 of Part IV of the 2020 Chinese Pharmacopoeia), and draw a standard curve with absorbance as the ordinate and concentration (mg / ml) as the abscissa.

[0166] Take 1.0g of the test sample, place it in a 10ml volumetric flask, add water to make up the volume, shake well, filter through a 0.45μm filter membrane, and take the filtrate to obtain the test solution. Take the test solution, measure the absorbance at a wavelength of 260nm according to the UV-visible spectrophotometer method (2020 edition of the Chinese Pharmacopoeia Part IV General Rules 0401), read the content of adenosine in the test solution from the standard curve (mg / ml), and calculate to obtain.

[0167] According to the total sugar, polypeptide and nucleoside content detection method in Example 2, the four batches of Cordyceps extracts in Example 1 were detected as shown in Table 1, with a total sugar content of ≥50 mg / g, a polypeptide content of ≥200 mg / g, and a nucleoside content of ≥20 mg / g.

[0168] Table 1 Yield and content of major components of 4 batches of Cordyceps extracts

[0169]

[0170] Example 3 Evaluation of drug efficacy in LPS-induced acute kidney injury mouse model

[0171] Acute kidney injury (AKI) is a common clinical syndrome in the field of critical care. In some critical care departments, the incidence rate even exceeds 50%, which seriously threatens the life safety of patients. Acute kidney injury is manifested by a rapid decline in renal function and the disruption of the homeostasis of plasma electrolytes, hemodynamics, nutrition and water. The pathogenesis of acute kidney injury is complex, involving renal tubules, microvessels and inflammation, etc. These mechanisms may interact and amplify each other. In addition to dialysis, there is currently no other effective and reliable method to treat acute kidney injury. Lipopolysaccharide (LPS) is the main component of Gram-negative bacterial endotoxin. After entering the body, it can produce sepsis, cause cytokine storm, increased oxidative stress, hypotension, and renal insufficiency, and ultimately lead to a gradual decline in renal function and systemic multiple organ failure, which is also a common cause of death in the onset of sepsis. At present, glucocorticoids are used as a rescue treatment measure in AKI caused by sepsis. Clinically, there is an urgent need for drugs with higher safety and better efficacy to treat AKI. In this example, an acute kidney injury mouse model was constructed under LPS, and the glucocorticoid dexamethasone was used as a positive drug to evaluate the efficacy of Cordyceps extract.

[0172] Test method:

[0173] 40 male Balb / c mice (25±1g) that passed the quarantine were selected and randomly divided into 4 groups according to gender and weight, including blank control group, model control group, positive drug dexamethasone group, and Cordyceps extract group. The mice were fasted for 12 hours before surgery and had free access to water. Except for the control group, the remaining groups were injected with a single intraperitoneal injection of 18mg / kg LPS solution, and the control group was injected with an equal volume of normal saline. After 15 minutes, dexamethasone and Cordyceps extract (batch 3) were given intravenously according to the group.

[0174] Animals in each group were weighed and killed 24 hours after administration, and the kidneys and livers of mice were taken and weighed to calculate the organ coefficient. 0.4 ml of blood was collected from the heart, centrifuged at 4500 r / min for 10 min, and then serum was collected, separated, and stored at -20°C. The kit was used to detect serum creatinine (Scr) and urea nitrogen (BUN).

[0175] The experimental data were expressed as Mean±SD. The data were statistically analyzed using IBM SPSS Statistics 21, and the data of each indicator were statistically analyzed using One-way ANOVA. The data between the two groups were considered to be significantly different when p<0.05.

[0176] Test results:

[0177] Serum urea nitrogen (BUN) and creatinine (Scr) are two important indicators of renal function. Serum biochemical indicators are shown in Table 2 and Figure 2 As shown in the figure, compared with the normal control group, the serum BUN and Scr of the mice in the model group increased significantly to 3.87±1.21mmol / L and 36.30±24.58μmol / L, and showed statistical differences (p<0.01), indicating that the model was successfully established. After administration of the positive drug dexamethasone 10mg / kg group, the serum BUN and Scr levels of the mice decreased to 1.92±1.00mmol / L and 11.75±6.24μmol / L, respectively, compared with the model group, showing statistical differences (p<0.01); after administration of Cordyceps extract, the serum BUN content of the mice decreased to 2.72±0.41mmol / L, and compared with the model group, the serum BUN of the mice in the administration group showed statistical differences (p<0.05); the serum Scr content of the mice in the administration group decreased to 14.58±7.79μmol / L, and compared with the model group, the dexamethasone group and the Cordyceps extract group showed statistical differences (p<0.05).

[0178] Table 2 Effects of Cordyceps extract on serum urea nitrogen and creatinine in mice with LPS-induced acute renal injury ( n=10)

[0179]

[0180] Note: Compared with the control group ++ P≤0.01; compared with the model group *P≤0.05, **P≤0.01.

[0181] The organ coefficient can objectively reflect the function of the relevant organs and is one of the important biological indicators of experimental animals. Figure 2 As shown in the figure, compared with the control group, the kidney coefficient and liver coefficient of the mice in the model group were 1.88±0.33 and 6.12±0.45g / 100g, respectively, and the kidney coefficient and liver coefficient of the mice in the model group showed statistical differences (p<0.05, p<0.01); compared with the model group, the kidney coefficient and liver coefficient of the mice in the dexamethasone group were equivalent to those in the model group, and no improvement was shown; the kidney coefficient of the mice in the Cordyceps extract group decreased to 1.56±0.20g / 100g, which showed statistical differences compared with the model group (p<0.05); the liver coefficient of the mice in the Cordyceps extract group decreased to 5.66±0.71g / 100g, which showed a downward trend compared with the model group. This shows that Cordyceps extract can significantly inhibit the increase of the kidney and liver coefficients of mice caused by LPS, and its effect is better than that of the positive drug dexamethasone.

[0182] Table 3 Effects of Cordyceps extract on organ coefficients of mice with acute renal injury induced by LPS ( n=10)

[0183]

[0184] Note: Compared with the normal group ## P≤0.01, # P≤0.05; compared with the model group *P≤0.05, **P≤0.01.

[0185] In summary, in this embodiment, mice developed abnormal renal function after LPS injection, and serum urea nitrogen and creatinine levels increased. Cordyceps extract at a dose of 20 mg / kg can significantly reduce serum urea nitrogen and creatinine levels, and the effect is equivalent to that of the positive drug dexamethasone. In terms of organ coefficient indicators, Cordyceps extract also has a significant improvement effect on kidney coefficient, and the effect is better than that of the positive drug dexamethasone.

[0186] Example 4 Evaluation of drug efficacy in a rat model of chronic kidney disease induced by 5 / 6 nephrectomy

[0187] Chronic kidney disease (CKD) is a progressive disease accompanied by multiple complications (cardiovascular disease, metabolic acidosis and anemia, etc.), with high morbidity and mortality in the population. The main methods for establishing animal models of chronic kidney disease are Platt method (5 / 6 nephrectomy) and Yokozawa method (adenine induction). 5 / 6 nephrectomy directly leads to a large reduction in nephrons, high workload of residual nephrons, and hypertension (or non-hypertension). These characteristics are similar to the symptoms of patients with clinical nephrectomy, CKD hypertension syndrome, and solitary kidney. The pathological changes are mainly glomerular sclerosis and tubular fibrosis, which is a good model for studying CKD. Empagliflozin is an oral highly selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, which has been approved by the European Union for the treatment of chronic kidney disease. Sulodexide is an antithrombotic drug, and animal experiments have shown that it has a renal protective effect. In this example, a 5 / 6 nephrectomy-induced chronic kidney disease rat model was used, with body weight, serum creatinine, serum urea nitrogen, body weight, urine volume, etc. as indicators, and marketed drugs empagliflozin and sulodexide as positive control drugs, to investigate the protective effect of the Cordyceps extract on chronic kidney disease rats after multiple oral administration.

[0188] Test method:

[0189] 60 Wistar rats that passed the quarantine were selected, male, 200±10g, and the rats were fasted for 12h before surgery and had free access to water. Except for the control group, the rats were anesthetized with isoflurane, shaved, and skinned, and disinfected. The two forelimbs were fixed, and the prone position was used. The left 2 / 3 kidney was removed. A longitudinal incision was made 1-1.5 cm from the costovertebral angle, about 2 cm long, and the muscle layer was cut open. The kidney was pulled out of the incision, the fat capsule was opened, and the kidney capsule was peeled off from bottom to top. Silk thread was tied to the upper pole 1 / 3 and the lower pole 1 / 3 of the kidney respectively, and the kidney was cut off with silk thread. The cross section was hemostatic with sterile gauze. After observing hemostasis, the residual kidney returned to the abdominal cavity, and 5,000 units of penicillin were dripped into the abdominal cavity. The muscle layer and skin were sutured respectively, and routine disinfection was performed. Return to the cage for rest after surgery. After 5-7 days, the right kidney was removed, and the previous method was the same. The kidney was separated, the renal pedicle was ligated with thick thread, the kidney was cut off, sutured, and disinfected. 30 days after surgery, the positive drugs and the test substances were administered according to the groups, and the administration was continued for 45 days. The positive drugs empagliflozin (1 mg / kg) and sulodexide (50 mg / kg) were converted according to the human clinical dose, and the cordyceps extract dosage (265 mg / kg) was converted according to the cordyceps dosage (4000 mg / kg) and the yield of the cordyceps extract of this batch (6.63%).

[0190] Before the end of the experiment, the animals were placed in metabolic cages to drink water freely, and urine was collected and the urine output was counted. The animals in each group were killed 45 days after administration, and 0.4 ml of blood was collected from the heart. After centrifugation at 4500r / min for 10 minutes, serum was drawn, separated, and stored at -20°C. After the animals were killed, the liver and kidney were taken, half of which was frozen at -80°C and the other half was stored in 10% formaldehyde solution. The kit was used to detect serum creatinine and urea nitrogen.

[0191] The experimental data were expressed as Mean±SD. The data were statistically analyzed using IBM SPSS Statistics 21, and the data of each indicator were statistically analyzed using One-way ANOVA. The data between the two groups were considered to be significantly different when p<0.05.

[0192] Test results:

[0193] As shown in Table 4 and Figure 3 As shown, compared with the control group, the body weight of rats in the model group was significantly reduced (p<0.01). After administration of the drugs, the body weight of the animals increased more than that of the model group. The body weight of the Cordyceps extract increased significantly after administration on the 15th and 45th days, showing statistical differences compared with the model group (p<0.05, p<0.01), which was better than the positive drugs empagliflozin, sulodexide and Cordyceps group.

[0194] Table 4 Effect of Cordyceps extract on body weight of 5 / 6 nephrectomized rats ( n=10)

[0195]

[0196] Note: Comparison between model group and normal control group ## P≤0.01, # P≤0.05; compared with the model control group, *P≤0.05, **P≤0.01. The positive drugs empagliflozin (1 mg / kg) and sulodexide (50 mg / kg) were converted according to the human clinical dose, and the cordyceps extract dosage (265 mg / kg) was converted according to the cordyceps dosage (4000 mg / kg) and the yield of the cordyceps extract of this batch (6.63%).

[0197] As shown in Table 5 and Figure 3 Compared with the control group, the serum creatinine and urea nitrogen of the rats in the model group increased significantly, and showed statistical differences (p<0.01), indicating that the model was successfully established. The test substances were given separately, and the blood Scr of the rats in each group showed different degrees of reduction. Compared with the model group, the Cordyceps extract showed statistical differences at 15 days, 30 days, and 45 days of administration (p<0.05, p<0.01). The Cordyceps extract had a better effect on improving the blood Scr of rats than the positive drug, and the onset time was earlier than the positive drug. Compared with the model group, the Cordyceps extract showed a statistical difference after 45 days of administration (p<0.05), and the effect was better than the positive drugs empagliflozin, sulodexide and the Cordyceps group.

[0198] Table 5 Effects of Cordyceps extract on serum biochemistry in 5 / 6 nephrectomized rats ( n=10)

[0199]

[0200] Note: Compared with the normal control group ## P≤0.01, # P≤0.05; compared with the model control group *P≤0.05, **P≤0.01.

[0201] like Figure 3 As shown, compared with the control group, the urine output of rats in the model group was significantly reduced (p<0.05). After administration, the urine output of each drug group increased. The urine output of empagliflozin and Cordyceps extract increased significantly, showing statistical differences compared with the model group (p<0.05, p<0.01).

[0202] In summary, under the experimental conditions of this embodiment, the efficacy of Cordyceps extract on chronic kidney disease was evaluated from the aspects of body weight changes and blood and urine sample indicators during the chronic kidney disease model test. The experimental results showed that the model group was successfully established. Compared with the model group, the body weight, serum creatinine, serum urea nitrogen and urine volume indicators of the Cordyceps extract group were significantly improved, and the effect and onset time were better than those of the positive drugs empagliflozin, sulodexide and Cordyceps group.

[0203] Example 5 Evaluation of drug efficacy in adenine-induced chronic kidney disease mouse model

[0204] The adenine-induced chronic renal failure modeling method was first reported by Yokozawa in 1986 and is now widely used. The model is characterized by tubular destruction as the main pathological feature, and can be used to study some new drugs that restore tubular function. High concentrations of adenine are converted into 2,8-dihydroxyadenine, which is extremely insoluble in water, through the action of xanthine oxidase. The latter is deposited in the renal tubules, affecting the excretion of nitrogen compounds, and eventually causing renal damage. In addition, a large amount of adenine intake leads to excessive production of oxygen free radicals and lipid peroxidation in the body, which may be an important mechanism for adenine to cause renal damage. Glucocorticoids have a precise and powerful anti-inflammatory and immunosuppressive effect, and are widely used in the clinical treatment of kidney disease, but long-term use is prone to serious side effects. In this embodiment, an animal model of chronic renal injury in mice was established by adenine induction, and the glucocorticoid dexamethasone was used as a positive drug to evaluate the renal protective effect of Cordyceps extract.

[0205] Test method:

[0206] SPF SD rats, male, except for the normal control group, were given adenine (dissolved in saline) 200 mg / kg by gavage once a day for 2 consecutive weeks, followed by 100 mg / kg for the next two weeks, and only given the drug in the last week without modeling. The drug was given from the second week after modeling, and continued for 4 weeks; before drug administration, the modeling group was randomly divided into 8 rats in each group according to the blood BUN value, and 10 rats in the model group: normal control group, model group, Cordyceps extract group, Cordyceps group, and dexamethasone group. Blood was collected from the eye sockets of the rats on the 7th, 14th, 21st, 28th, and 35th days of modeling, centrifuged at 3000 rpm for 10 minutes, and blood samples were taken to test serum biochemical indicators. The rats were placed in metabolic cages, fasted (water intake as usual), urine volume was recorded, urine sample indicators were tested, creatinine clearance rate was calculated, and urine microalbumin / creatinine (UACR) was calculated. At the end of the experiment, the rats were dissected to remove the kidneys, and the renal sebum was separated on ice, weighed, and the organ index was calculated. Then fix with 4% neutral formaldehyde solution, embed in paraffin, stain with hematoxylin-eosin (HE), examine under light microscope, and read the film. The positive drug dexamethasone (0.1 mg / kg) is converted according to the human clinical dose, and the cordyceps extract dosage (205 mg / kg) is converted according to the cordyceps dosage (2500 mg / kg) and the yield of the cordyceps extract of this batch (8.07%).

[0207] Test results:

[0208] The changes in animal body weight are shown in Table 6. From the second week to the end of the experiment, the body weight of the rats in the model group was significantly reduced compared with the normal control group (p<0.05, P<0.01, P<0.001); the body weight of the dexamethasone group was significantly reduced from the second week to the end of the experiment compared with the model group (p<0.001), suggesting that it was related to the side effects of hormones. Compared with the model group, the body weight of the Cordyceps extract and Cs-4 groups showed an upward trend, but it was not significant.

[0209] Table 6 Effect of adenine on the body weight of animals during the experimental period

[0210]

[0211] Note: Comparison between model group and normal control group # P≤0.05, ## P≤0.01, ### P≤0.001; Comparison between each drug group and the model group * P≤0.05, ** P≤0.01, *** P≤0.001; the positive drug dexamethasone (0.1 mg / kg) was converted according to the human clinical dose, and the cordyceps extract dosage (205 mg / kg) was converted according to the cordyceps dosage (2500 mg / kg) and the yield of the cordyceps extract in this batch (8.07%).

[0212] The rat kidney weight and kidney index at the end of the experiment are shown in Table 7. Compared with the normal control group, the model group rat kidney weight increased significantly (P<0.001); compared with the model group, the Cordyceps extract and Cs-4 groups kidney weight decreased significantly (P<0.01), the Cordyceps extract group kidney weight coefficient decreased significantly (P<0.001), the dexamethasone group rat kidney weight decreased significantly (P<0.001), but the kidney index had no statistical difference. The remission rate of the kidney coefficient of the Cordyceps extract and Cs-4 groups was 32.48% and 33.53%, respectively, which was much higher than the remission rate of dexamethasone on the kidney weight coefficient of 2.69%.

[0213] Table 7 Study on the effect of adenine on chronic renal failure at the end of the experiment on rat kidney weight and kidney coefficient

[0214]

[0215] Note: Comparison between model group and normal control group # P≤0.05, ## P≤0.01, ### P≤0.001; Comparison between each test group and the model group * P≤0.05, **P≤0.01, *** P≤0.001;

[0216] The serum urea nitrogen (BUN) indexes are shown in Table 8: Compared with the normal control group, the BUN level of the model group increased significantly from the first week to the end of the experiment (P<0.001); compared with the model group, the BUN levels of the cordyceps extract group, the cordyceps group and the dexamethasone group tended to decrease. In the fourth week of the experiment, the BUN levels of the cordyceps group and the cordyceps extract group decreased significantly (P<0.05), and the improvement of the BUN level was greater than that of the positive drug dexamethasone.

[0217] Table 8 Changes in BUN during the experimental period of the study on the effect of adenine on chronic renal failure

[0218]

[0219] Note: Comparison between model group and normal control group # P≤0.05, ## P≤0.01, ### P≤0.001; comparison between test group and model group * P≤0.05, ** P≤0.01, *** P≤0.001;

[0220] Serum creatinine (Scr) indicators are shown in Table 9. From the first week to the end of the experiment, the Scr level in the model group increased significantly (P<0.001). Compared with the model group, the Scr levels in the Cordyceps extract group, Cordyceps group and dexamethasone group showed a downward trend. Among them, from the first week to the end of the experiment, the Scr level in the positive drug dexamethasone group was significantly improved (P<0.05, P<0.01, P<0.001).

[0221] Table 9 Changes of Scr during the experimental period of the effectiveness study on adenine-induced chronic renal failure (± sd)

[0222]

[0223] Note: Comparison between model group and normal control group # P≤0.05, ## P≤0.01, ### P≤0.001; Comparison between each test group and the model group * P≤0.05, ** P≤0.01, *** P≤0.001;

[0224] The concentration of cystatin C in the blood is determined by the glomerular filtration rate and is independent of any external factors, such as gender, age, and diet. It is an ideal endogenous marker that reflects changes in glomerular filtration rate. The serum cystatin C level is shown in Table 10: Compared with the normal control group, the serum cystatin C level in the model group increased significantly from the first week to the end of the experiment (P<0.01, P<0.001); compared with the model group, the serum cystatin C level in each drug-treated group showed a downward trend, among which the improvement effects of the cordyceps extract and dexamethasone groups were comparable, and significantly improved in the third and fifth weeks (P<0.05, P<0.01).

[0225] Table 10 Changes of Cystatin C during the experimental period of the effectiveness study on adenine-induced chronic renal failure

[0226]

[0227]

[0228] Note: Comparison between model group and normal control group # P≤0.05, ## P≤0.01, ### P≤0.001; Comparison between each drug group and the model group * P≤0.05, ** P≤0.01, *** P≤0.001;

[0229] The results of HE staining of kidney pathological sections are as follows Figure 4 The results showed that the structure and morphology of the glomeruli, tubules and renal interstitium in the normal control group were clear, and no obvious abnormalities were found; the renal structure of the model group was disordered, the number of glomeruli was significantly reduced and atrophied, the cyst cavity was significantly dilated, the renal tubules were significantly dilated, protein casts appeared in the renal tubules, and a large number of inflammatory cells infiltrated the renal interstitium. The kidneys of the animals in the drug-treated groups all had the above lesions, but the degree was alleviated to varying degrees. The pathological scores are shown in Table 11. The pathological scores of the model group were significantly increased (P<0.001); compared with the model group, the renal pathological scores of each drug-treated group were significantly decreased (P<0.001), and the improvement effect of Cordyceps and Cordyceps extract on renal pathological scores was equivalent to that of the positive drug dexamethasone.

[0230] Table 11. Effect of the pathological scores of renal pathological sections at the end point of the study on the effectiveness of adenine-induced chronic renal failure

[0231]

[0232] Note: Comparison between model group and normal control group # P≤0.05,## P≤0.01, ### P≤0.001; Comparison between each drug group and the model group * P≤0.05, ** P≤0.01, *** P≤0.001;

[0233] In summary, under the experimental conditions of this embodiment, the efficacy of Cordyceps extract on chronic kidney disease was evaluated from the aspects of weight change, blood sample indicators and pathology during the adenine-induced chronic kidney disease model test. The test results showed that the model group was successfully established. Compared with the model group, the kidney coefficient, serum urea nitrogen, serum cystatin C and pathological indicators in the Cordyceps extract group were significantly improved, and the effect was equivalent to that of Cordyceps and the positive drug dexamethasone.

[0234] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A Cordyceps extract, wherein the Cordyceps extract is a water-soluble Cordyceps extract, and the molecular weight of the Cordyceps extract is less than 10 kDa.

2. The Cordyceps extract according to claim 1, wherein the Cordyceps is selected from one or more of Cordyceps sinensis, Cordyceps militaris and fermented Cordyceps.

3. The Cordyceps extract according to claim 1, wherein the Cordyceps is fermented Cordyceps Cs-4.

4. A method for preparing a Cordyceps extract, comprising the following steps: The cordyceps is subjected to defatting, water extraction, alcohol precipitation and ultrafiltration processes to obtain a water-soluble extract; The molecular weight of the extract is less than 10 kDa.

5. The preparation method according to claim 4, comprising: The fermented Cordyceps sinensis powder Cs-4 is defatted with ethanol, and the ethanol extract is discarded to obtain a defatted residue; The residue is added with water for reflux extraction, the obtained extract is precipitated with ethanol, the obtained precipitate is treated with ultrafiltration to obtain a filtrate with a molecular weight of less than 10 kDa, and the filtrate is concentrated and dried to obtain a Cordyceps extract.

6. The preparation method according to any one of claims 4-5, comprising the following steps: 1) Degreasing: The fermented Cordyceps sinensis powder Cs-4 is heated to boiling with 5 to 20 times (by mass) of 60 to 100% v / v ethanol, and the extraction is maintained at a slight boiling temperature for 0.5 to 2 hours, and the alcohol extract is discarded. After degreasing 1 to 3 times, the powder is used for later use; 2) Extraction and alcohol precipitation: Take the above defatted bacterial powder, add 5 to 20 times the volume of water, reflux and extract 1 to 3 times, each time for 0.5 to 2 hours, combine the obtained water extracts and concentrate to a relative density of 1.08, cool to room temperature, add 95% v / v ethanol to an alcohol concentration of 60 to 80% v / v, stand overnight, discard the supernatant, and dry the precipitate for later use; 3) Ultrafiltration: Take the above alcohol precipitate, add water to dissolve it, centrifuge it, take the supernatant and treat it with a 10kDa ultrafiltration column, concentrate the 10kDa permeate, and dry it to obtain the refined Cordyceps extract.

7. A composition comprising the Cordyceps extract according to any one of claims 1 to 3.

8. The composition of claim 7, further comprising one or more physiologically acceptable carriers, excipients and diluents.

9. Use of the Cordyceps extract according to any one of claims 1 to 3 or the composition according to any one of claims 7 to 8 in the preparation of a medicament for preventing and / or treating kidney disease.

10. Use of the Cordyceps extract according to any one of claims 1 to 3 or the composition according to any one of claims 7 to 8 in preparing health food.

Images