Cordyceps sinensis extract, preparation method and use thereof

Abstract

The present application belongs to the field of medicine, and particularly relates to a cordyceps sinensis extract, a preparation method and use of the cordyceps sinensis extract as an immunosuppressive agent in preparation of a medicine for preventing or treating immune-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a Cordyceps sinensis extract, its preparation method, and its use as an immunosuppressant in the prevention or treatment of immune-related diseases. Background Technology

[0002] Cerebrosides (or glycoceramides) belong to the glycosphingolipid class and are mainly found in the animal kingdom, but also in plants and microorganisms. Cerebrosides consist of three parts: a glycosyl group, a fatty acid chain, and a sphingosine chain. Although all compounds in this class are composed of these three parts, they exhibit structural diversity due to variations in the type of sugar, glycosidic bonds, the length and degree of branching of the fatty acid and sphingosine chains, and the number and position of hydroxyl groups and double bonds (J.Nat.Prod.2017,80,6,1734–1741). Different compound structures often result in different types or degrees of pharmacological activity; therefore, cerebroside compounds have been reported to possess varying degrees of antitumor, antiviral, antimicrobial, and neuroprotective activities (Xu Jie, Isolation, Purification, Structural Analysis, and Bioactivity Study of Sea Cucumber Cerebrosides, Doctoral Dissertation, Ocean University of China, 2011). Cerebrosides, composed of hydrophilic glycosidic groups and lipophilic fatty acid and sphingosine chains, are important lipid components of biological membranes, contributing to their integrity. Furthermore, cerebrosides form extensive hydrogen bond networks at the polar and nonpolar interfaces of biological membranes, a crucial factor in the stability of the membrane's layered structure. Therefore, cerebrosides exhibit mild activity and low toxicity. Developing new cerebroside compounds or extracts rich in cerebrosides for the treatment of chronic diseases has become a research goal for scientists.

[0003] Cordyceps sinensis is a dried complex of the stroma of the fungus Cordyceps sinensis (Ber K.) Sacc. (family Clavicipitaceae) parasitizing the larvae of insects in the family Hepialidae (family Hepialidae) and the larval corpse. It possesses various pharmacological effects, including immune regulation, anti-tumor activity, antioxidant activity, and anti-aging. Literature reports that the main chemical components of Cordyceps sinensis include polysaccharides, proteins, sugar alcohols, nucleosides, amino acids, sterols, and organic acids. Extracting new active ingredients from Cordyceps sinensis can provide a scientific basis for the comprehensive quality evaluation of Cordyceps sinensis and the future development and application of related products. Summary of the Invention

[0004] The inventors of this invention extracted the effective components from Cordyceps sinensis and discovered for the first time that the Cordyceps sinensis extract contains cerebrosides. They also conducted immunomodulatory activity studies on the Cordyceps sinensis extract containing cerebrosides and found that it has excellent immunosuppressive activity.

[0005] The purpose of this invention is to provide a type of Cordyceps sinensis extract containing cerebrosides and exhibiting excellent immunosuppressive activity, and the use of this Cordyceps sinensis extract as an immunosuppressant in the preparation of drugs for the prevention or treatment of immune-related diseases.

[0006] On one hand, the present invention provides a Cordyceps sinensis extract, characterized in that the extract contains 0.39%-0.45% of compound 1, 0.10%-0.18% of compound 2, 16%-18% of compound 3, 12%-15% of the total content of compounds 4 and 5, 0.25%-0.45% of compound 6, and 0.40%-0.50% of compound 7; wherein the structures of compounds 1, 2, 3, 4, 5, 6, and 7 are as follows:

[0007]

[0008]

[0009] In some embodiments, the Cordyceps sinensis extract of the present invention contains 0.43% of compound 1, 0.16% of compound 2, 17.79% of compound 3, 13.58% of compounds 4 and 5 in total, 0.38% of compound 6, and 0.46% of compound 7.

[0010] Unless otherwise specified, the content of the compound mentioned in this invention refers to the relative content of the chromatographic peak of the contained compound in HPLC chromatogram 2 relative to the total extract in HPLC chromatogram 2. The method for calculating the relative content of the chromatographic peak of the contained compound relative to the total extract in HPLC chromatogram 2 is the external standard one-point method, and the calculation formula is: relative content of chromatographic peak relative to extract (%) = (sample peak area / reference peak area) * reference concentration * reference purity * volume adjusted / mass of cerebroside extract * 100%. Wherein, the volume adjusted is the volume of cerebroside extract weighed and added to methanol for volume adjustment, and the mass of cerebroside extract is the mass weighed to prepare the test sample. In the specific embodiment of this invention, the reference standard used in this invention is cerebroside A.

[0011] In some embodiments, the Cordyceps sinensis extract of the present invention is subjected to high performance liquid chromatography. Figure 2The image shows six peaks, corresponding to compounds 1, 2, 3, a mixture of compounds 4 and 5, 6, and 7, with retention times of 13.325 min, 13.834 min, 15.006 min, 17.572 min, 19.887 min, and 22.093 min, respectively.

[0012] In some embodiments, the analytical conditions for high-performance liquid chromatography (HPLC) according to the present invention are as follows: chromatographic column: Agilent Zorbax Eclipse Plus C18 (4.6 mm × 150 mm, 5 μm); mobile phase: 10 mM ammonium acetate aqueous solution (A) - methanol (B); flow rate: 1 mL / min; gradient elution: 0–40 min, 93% B–95% B, followed by a 10 min run; column temperature: 30 °C; detection wavelength: 205 nm; injection volume: 10 μL.

[0013] On the other hand, the present invention provides a method for preparing Cordyceps sinensis extract, comprising the following steps:

[0014] (1) Cordyceps sinensis was extracted in 85%-95% methanol solution to obtain an extract;

[0015] (2) Load the extract from step (1) into a solid phase extraction column and elute with 95% methanol and 98%–100% methanol in sequence. Collect the eluent obtained by eluting with 98%–100% methanol.

[0016] In some embodiments, the eluent contains compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7; wherein the structures of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7 are shown below:

[0017]

[0018]

[0019] In some embodiments, in the preparation method of Cordyceps sinensis extract of the present invention, the ratio of Cordyceps sinensis to 85%-95% methanol solution in step (1) is 1:8 to 1:12.

[0020] The material-to-liquid ratio represents the ratio of the mass of the material used to the volume of the solution used, where the unit of measurement for the material is g and the unit of measurement for the solution is mL. When the material-to-liquid ratio is 1:8, it means that 1 g of material is used to produce 8 mL of solution.

[0021] In some implementations, the ratio of Cordyceps sinensis to 85%-95% methanol solution in step (1) is 1:10.

[0022] In some implementations, the methanol solution in step (1) is a 90% methanol solution.

[0023] In some implementations, the extraction is a reflux extraction.

[0024] In some implementations, the extraction time is 2 to 3 hours.

[0025] In some implementations, the extract is the supernatant obtained after centrifuging the extracted mixture.

[0026] In some specific implementations, the centrifugal speed is 5000 r / min.

[0027] In some embodiments, the preparation method of Cordyceps sinensis extract according to the present invention uses ODS packing material in step (2) of the solid phase extraction column.

[0028] In some implementations, step (2) is: loading the extract from step (1) onto a solid-phase extraction column packed with ODS, and eluting sequentially with 95% methanol and 100% methanol, and collecting the eluent obtained by eluting with 100% methanol.

[0029] In some embodiments, the eluent obtained by elution with 100% methanol is further concentrated.

[0030] In some embodiments, the method for preparing the Cordyceps sinensis extract of the present invention includes the following steps:

[0031] (1) Cordyceps sinensis is extracted by reflux in 90% methanol solution for 2-3 hours, centrifuged, and the supernatant is obtained. The ratio of Cordyceps sinensis to 90% methanol is 1:8 to 1:12. Optionally, the ratio of Cordyceps sinensis to 90% methanol is 1:10.

[0032] (2) Load the supernatant from step (1) into an ODS solid phase extraction column and elute with 95% methanol and 100% methanol in sequence, and collect the eluent obtained by elution with 100% methanol.

[0033] On the other hand, the present invention provides a Cordyceps sinensis extract prepared by the method described in the present invention.

[0034] On the other hand, the present invention provides the use of the Cordyceps sinensis extract described in the present invention or the Cordyceps sinensis extract prepared by the method described in the present invention in the prevention or treatment of immune-related diseases.

[0035] In some implementations, the immune-related disease is organ transplant rejection or an autoimmune disease, wherein the autoimmune disease is lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, complications from organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia, or lymphoma.

[0036] Compared with the prior art, the positive effects of the present invention are:

[0037] This patent is the first to extract a novel cerebroside extract from Cordyceps sinensis, and this extract exhibits excellent immunosuppressive activity, demonstrating immunosuppressive activity against Con A-induced proliferation of mouse splenic lymphocytes, with an IC50 value of [missing information]. 50 The value was 4.6 μg / mL.

[0038] The foregoing description only outlines certain aspects of the invention, but is not limited to these aspects. These and other aspects will be described in more detail below.

[0039] Detailed Description of the Invention

[0040] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0041] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0042] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0043] The term "treatment" as used in this invention refers to both therapeutic and preventative treatment. For example, therapeutic treatment includes reducing or improving the progression, severity, and / or duration of immune-mediated symptoms, or improving one or more symptoms (specifically, one or more identifiable symptoms) of immune-mediated symptoms by administering one or more therapies (e.g., one or more therapeutic agents, such as the compounds and compositions of this invention). In certain embodiments, therapeutic treatment includes improving at least one measurable physical parameter of immune-mediated symptoms. In other embodiments, therapeutic treatment includes suppressing the progression of immune-mediated symptoms physically by stabilizing identifiable symptoms or physiologically by stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes reducing or stabilizing the severity of immune-mediated disease. The medicaments of this invention can be used in the community to treat individuals already suffering from immune diseases to reduce the severity of symptoms and the number of days they are ill.

[0044] The compositions, formulations, and administration methods of the present invention

[0045] This invention provides a pharmaceutical composition comprising the extract described herein. The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, and optionally, other therapeutic and / or preventative ingredients. In some embodiments, the pharmaceutical composition comprises an effective amount of at least one pharmaceutically acceptable excipient.

[0046] As described in this invention, the pharmaceutical compositions or pharmaceutically acceptable compositions of this invention further comprise pharmaceutically acceptable excipients, including any solvent, diluent, liquid excipient, dispersant, suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, or lubricant, etc., suitable for the specific target dosage form, as applied in this invention. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D.B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, discloses various carriers used in formulating pharmaceutically acceptable compositions and their known preparation methods. Any other conventional carrier media and their uses are also within the scope of this invention, except for conventional carrier media incompatible with the compounds of this invention, such as those that produce adverse biological effects or interact harmfully with any other component in the pharmaceutically acceptable composition.

[0047] The extracts or compositions of the present invention can be administered by any suitable means, and can be given to humans or other animals orally, rectally, parenterally, intracerebrospinal, vaginally, intraperitoneally, topically (as in powder, ointment or drops), or orally as a mouth or nasal spray, depending on the severity of the infection being treated.

[0048] The extract used in the methods of the present invention can be formulated into unit dosage forms. The term "unit dosage form" refers to a physical discrete unit suitable as a unit dose for a treated patient, each unit containing a predetermined amount of extract calculated to produce the intended therapeutic effect, optionally combined with a suitable pharmaceutical adjuvant. Unit dosage forms can be used as a single daily dose or as a multiple daily dose (e.g., about 1-4 or more times daily). When using multiple daily doses, the unit dosage form for each dose can be the same or different.

[0049] Uses of the extracts and compositions thereof of the present invention

[0050] The extracts and pharmaceutical compositions provided by this invention can be used as immunosuppressants to prepare drugs for the prevention, treatment or alleviation of immune-related diseases in patients. Preferably, the immune-related diseases are organ transplant rejection reactions or autoimmune diseases. Preferably, the autoimmune diseases are lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, complications caused by organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia or lymphoma.

[0051] This invention provides a method for treating, preventing, or alleviating immune-related diseases in patients, the method comprising administering to the patient a therapeutically effective amount of the extract or pharmaceutical composition thereof described in this invention. Furthermore, the above-described compound or pharmaceutical composition thereof provided by this invention can be administered in combination with other therapies or therapeutic agents. Administration can be simultaneous, sequential, or at time intervals. The other therapies or therapeutic agents are selected from chemotherapeutic agents or antiproliferative agents, anti-inflammatory drugs, immunomodulators or immunosuppressants, neurotrophic factors, active agents for treating cardiovascular diseases, active agents for treating diabetes, and active agents for treating autoimmune diseases.

[0052] The dosage of the extract or pharmaceutical composition required to achieve therapeutic, preventive, or delaying effects generally depends on the specific extract or pharmaceutical composition being administered, the patient, the specific disease or condition and its severity, the route of administration, and the frequency, and needs to be determined by the attending physician based on the specific circumstances. For example, when administering the extract or pharmaceutical composition provided by this invention via intravenous route, it can be administered once a week or even at longer intervals.

[0053] The extracts and pharmaceutical compositions of the present invention, in addition to their therapeutic benefits for humans, can also be used in veterinary treatment of mammals, including pets, introduced breeds, and farm animals. Other examples of animals include horses, dogs, and cats. Herein, the compounds of the present invention include their pharmaceutically acceptable derivatives. Attached Figure Description

[0054] Figure 1 and Figure 2 This is the chromatographic peak identification chromatogram of the Cordyceps sinensis cerebroside extract in Example 1, wherein, Figure 1 Chromatogram of reference substance Cerebroside A (peak 3); Figure 2 The chromatogram of the cordyceps sinensis cerebroside extract shows peaks 1-7, which represent 7 cerebroside components.

[0055] Figure 3 The images show chromatograms of Cordyceps sinensis cerebroside extracts under different extraction solvent conditions in Example 2. A: Chromatogram of Cordyceps sinensis cerebroside extract under 90% methanol extraction solvent; B: Chromatogram of Cordyceps sinensis cerebroside extract under 95% ethanol extraction solvent; Peaks 1-7: 7 cerebroside components.

[0056] Figure 4 , Figure 5 , Figure 6 , Figure 7 The chromatograms are for the eluent of Cordyceps sinensis cerebroside fraction under different eluent conditions in Example 2. Figure 4 , Figure 5 , Figure 6 , Figure 7 The following are chromatograms of Cordyceps sinensis cerebroside fractions under elution conditions of 90% methanol, 95% methanol, 98% methanol, and 100% methanol; a to e are chromatograms of the elution solutions of column volumes 1 to 5; peaks 1 to 7 represent the 7 cerebroside components. Detailed Implementation

[0057] The embodiments of the present invention are described in detail below. It should be noted that the described embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0058] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention, and other methods for preparing the compounds of the present invention are considered to be within the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0059] 1. Source of medicinal materials

[0060] Cordyceps sinensis comes from Yichang Shancheng Shuidu Cordyceps sinensis Co., Ltd. in Hubei Province.

[0061] 2. Experimental apparatus

[0062] EYELA rotary evaporator N-1001 (Rikakikai, Tokyo); Dionex analytical and preparative high-performance liquid chromatographs (Thermo Fisher Scientific, USA); Agilent Zorbax Eclipse Plus C18 analytical column (4.6×150mm, 5μm) (Phenomenex, USA); Agilent Extend C18 (2.1mm×50mm, 3.5μm) (Phenomenex, USA); ESI source low-resolution mass spectrometer (Bruker Daltonics, USA); HERA cell 150i cell culture incubator (Thermo Fisher Scientific, USA); Model 680 multi-functional microplate reader (Bio-Rad Laboratories, USA); electronic balance (Mettler Toledo, model: ME204E); constant temperature water bath (Jintan Tianhong Experimental Instrument Factory, model: HH-S28); high-speed refrigerated centrifuge (Thermo Fisher Scientific, model: ST40R); Milli-Q Advantage A10 ultrapure water system (Merck Millipore); JASCO V-550 UV spectrometer (Jasco International, Japan); JASCO FT / IR-480plus infrared spectrometer (Jasco International, Japan); JASCO P-1020 polarimeter (Jasco International, Japan); Bruker AV-400 / 600MHz NMR spectrometer (Bruker BioSpin, Switzerland); APCI ion source low-resolution mass spectrometer (Bruker Daltonics, USA); Waters Synapt G2 TOF high-resolution mass spectrometer (Waters Corporation, USA); 1260-6130 high-performance liquid chromatography-mass spectrometry system with G1315DAD detector (Agilent Technologies, USA). 1260 high-performance liquid chromatograph (Agilent Technologies, USA).

[0063] Phenomenex Gemini C 18Analytical (4.6 × 250 mm, 5 μm), semi-preparative (10.0 × 250 mm, 5 μm), and preparative (20.0 × 250 mm, 5 μm) columns (Phenomenex, USA); Phenomenex Biphenyl analytical (4.6 × 250 mm, 5 μm) and semi-preparative (10.0 × 250 mm, 5 μm) columns (Phenomenex, USA).

[0064] 3. Experimental Materials

[0065] Chromatographic grade methanol was purchased from Shandong Yuwang Industrial Co., Ltd.; chromatographic grade ammonium acetate was purchased from Sigma-Aldrich Chemical Co., Ltd. (USA); anhydrous methanol (analytical grade, Xilong Scientific Co., Ltd.), methanol (chromatographic grade, Thermo Fisher Scientific Co., Ltd.), and ultrapure water were used. ODS-AQ-HG reversed-phase silica gel packing material was purchased from YMC Co., Ltd.; deuterated pyridine was purchased from Sigma-Aldrich AG (Germany); and silica gel was purchased from Qingdao Ocean Chemical Co., Ltd.

[0066] Male BABL / C mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.; 96-well plates were purchased from Corning (Shanghai) Management Co., Ltd.; Con A, Hank's solution, and erythrocyte lysis buffer were purchased from Sigma-Aldrich Chemical Company, USA; and Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies, Japan.

[0067] The following abbreviations are used throughout the instruction manual:

[0068] t R Retention time

[0069] Pyr-d5: Deuterated pyridine

[0070] δ H Chemical shift of hydrogen

[0071] δ C : Carbon compound displacement

[0072] Unless otherwise specified, the detection wavelength for chromatographic analysis in this invention is 205 nm, and the column temperature is 30 °C.

[0073] Example 1: Preparation and Chemical Composition Analysis of Cordyceps sinensis Cerebroside Extract

[0074] 1.1 Preparation of Cordyceps sinensis cerebroside extract

[0075] Take 10g of Cordyceps sinensis and place it in a 250mL round-bottom flask. Add 100mL of 90% methanol and reflux for 3 hours. Centrifuge (5000r / min). The obtained supernatant (about 70-80mL) is loaded in batches onto an ODS column (inner diameter 2.8cm * height 3.0cm). (The ODS column needs to be activated with pure methanol beforehand. After packing the column, equilibrate with 90% methanol before loading the supernatant. In this experiment, the mass of ODS is about 10g.) Then, elute with 95% methanol for 10 BV (BV, column volume, 1 BV is about 10mL), discard the eluent, and elute with 100% methanol for 5 BV. Collect the eluent, evaporate to dryness, and obtain 3.4mg of Cordyceps sinensis cerebroside extract, with a yield of 0.38%. The calculation formula is: Yield = mass of cerebroside extract / mass of sample (dried product).

[0076] 1.2 Chemical composition analysis of Cordyceps sinensis cerebroside extract

[0077] 1.2.1 Preparation of test sample

[0078] Take 3.4 mg of the above cerebroside extract, add 500 μL of methanol to reconstitute, centrifuge at 10000 rpm, and collect the supernatant for later use.

[0079] 1.2.2 Preparation of reference standard

[0080] Weigh 14.10 mg (purity 90.7%) of reference standard cerebroside A (laboratory preparation, stored at Jinan University) and add methanol to prepare a concentration of 2.82 mg / mL for HPLC-UV-ESI / MS analysis.

[0081] 1.2.3 High Performance Liquid Chromatography Analysis

[0082] Chromatographic column: Agilent Zorbax Eclipse Plus C18 (4.6 mm × 150 mm, 5 μm); mobile phase: 10 mM ammonium acetate aqueous solution (A) - methanol (B); flow rate: 1 mL / min; gradient elution: 0–40 min, 93% B–95% B, followed by a 10 min run; column temperature: 30 °C; detection wavelength: 205 nm; injection volume: 10 μL.

[0083] Mass spectrometry conditions: Electrospray ionization source, drying gas (nitrogen) flow rate 9 L / min; drying gas temperature 350 °C; nebulization pressure 50 psi; capillary voltage 3500 V; scanning mode: negative ion mode, SIM scan mode; collision-induced dissociation voltage 70 V; ion scan range: 100-1000 m / z. Retention times and corresponding mass-to-charge ratios of screened ions: Channel 1: 0 min 711, 725, 727, 739, 753.

[0084] Calculation formula (external standard single-point method): Chromatographic peak relative content of extract (%) = (sample peak area / reference peak area) * reference concentration * reference purity * final volume / mass of cerebroside extract * 100%. (Final volume refers to the volume of methanol added to the weighed cerebroside extract, and the mass of the cerebroside extract is the mass weighed to prepare the test sample.)

[0085] The relative contents and mass spectrometry data of the seven cerebroside components of Cordyceps sinensis in the cerebroside extract obtained by the above analytical methods are shown in Table 1. Additionally... Figure 1 and Figure 2 The image shows the corresponding compounds for each chromatographic peak of the Cordyceps sinensis cerebroside extract. Figure 2 Peak 1 corresponds to compound 1, peak 2 corresponds to compound 2, peak 3 corresponds to compound 3, peaks 4 & 5 correspond to compounds 4 and 5, peak 6 corresponds to compound 6, and peak 7 corresponds to compound 7.

[0086] Table 1. Relative content of cerebroside components in Cordyceps sinensis extract.

[0087] Element Retention time (min) Peak area Relative content (%) Mass-to-charge ratio (m / z) Peak 1 13.325 168.6 0.43 711 Peak 2 13.834 61.7 0.16 711 Peak 3 15.006 7043.9 17.79 725 Peak 4 & 5 17.572 5377.6 13.58 727&739 Peak 6 19.887 152.3 0.38 711 Peak 7 22.093 180.6 0.46 753 Reference 14.924 13202.2 - - Total content 32.7884 -

[0088] 1.2.4 Isolation and identification of monomeric compounds 1, 2, 3, 4, 5, 6, and 7 from the cordycepin extract

[0089] The Cordyceps sinensis cerebroside extract (1.4 g) prepared according to method 1.1 was dissolved in methanol and analyzed by preparative high-performance liquid chromatography (HPLC). Separation was performed using a medium-low pressure ODS column (3.1 × 20.0 cm) (YMC Corporation, Japan). The mobile phase was eluted with a methanol-water gradient of 85%–100% (v / v) for 3 hours. The eluent was collected in graduated centrifuge tubes, approximately 30 mL per tube, and every 15 tubes were combined to form a single fraction, yielding seven sub-fractions: F7.1, F7.2, F7.3, F7.4, F7.5, F7.6, and F7.7. HPLC analysis revealed that sub-fractions 7.1, 7.2, and 7.3 did not contain any compounds. Fraction F7.4 (10.8 mg) was separated using a semi-preparative chromatographic column, Phenomenex Biphenyl (10.0 × 250 mm, 5 μm), with isocratic elution using 85% methanol-water (v / v) as the mobile phase at a flow rate of 3 mL / min to obtain compound 1 (t). R :43.8 min, 3.5 mg). Fraction F7.5 (93.8 mg) was separated using a semi-preparative chromatographic column Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 92% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 2 (t R:51.6 min, 4.1 mg). Fraction F7.6 (523.4 mg) was separated using a preparative chromatographic column Phenomenex Gemini C18 (20.0 × 250 mm, 5 μm) with isocratic elution of 93% methanol-water (v / v) at a flow rate of 8 mL / min to obtain fraction F7.6.1 (t R :50.0~55.0min), F7.6.2(t R :60.0~65.0min), F7.6.3(t R Fractions F7.6.1 (tR: 66.0–70.0 min) and F7.6.4 (tR: 74.0–78.0 min) were separated using a semi-preparative chromatographic column, Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm), with isocratic elution using 93% methanol-water (v / v) as the mobile phase at a flow rate of 3 mL / min to obtain compound 3 (tR: 66.0–70.0 min). R :50.9 min, 319.9 mg). Fraction F7.6.2 (20.7 mg) was separated using a semi-preparative chromatographic column Phenomenex Biphenyl (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 87% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 4 (t R :31.6min, 9.3mg) and compound 5 (t R :29.3 min, 8.7 mg). Fraction F7.6.3 (8.8 mg) was separated using a semi-preparative chromatographic column Phenomenex Biphenyl (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 87% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 6 (t R :29.2 min, 2.5 mg). Fraction F7.6.4 (15.6 mg) was separated using a semi-preparative chromatographic column Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 93% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 7 (t R (71.1 min, 12.9 mg).

[0090] The structures and identification data of compounds 1, 2, 3, 4, 5, 6, and 7 are shown below:

[0091] Compound 1:

[0092]

[0093] Compound 1: White powder; UV(MeOH)λ max (logε)203(4.09)nm;IR(KBr)v max 3332,2915,1659,1539,1466,1073cm -1 ;APCI-MS / MS(positive)m / z 712,694,532,294,276; HRESIMS(positive)m / z 712.5357[M+H] + (calcd.for C 40 H 74 NO9,712.5358); 1 H and 13 The C NMR values ​​are shown in Table 2.

[0094] Compound 2:

[0095]

[0096] Compound 2: White powder; UV(MeOH)λ max (logε)202(4.02)nm;IR(KBr)v max 3218,2921,1639,1539,1466,1078cm -1 ;APCI-MS / MS(positive)m / z 712,694,532,280,262; HRESIMS(positive)m / z 712.5354[M+H] + (calcd.for C 40 H 74 NO9,712.5358); 1 H and 13 The C NMR values ​​are shown in Table 2.

[0097] Compound 3:

[0098]

[0099] Compound 3: White powder; APCI-MS / MS (positive) m / z 726,708,546,294,276; HRESIMS (positive) m / z 726,5505 [M+H] + (calcd.for C 41 H 76 NO9,726.5515); 1 H and 13 The C NMR values ​​are shown in Table 3.

[0100] Compound 4:

[0101]

[0102] Compound 4: White powder; UV(MeOH)λ max (logε)202(4.22)nm;IR(KBr)v max 3283,2921,1639,1531,1466,1081,1029,968 cm -1 ; APCI-MS / MS(positive)m / z740,722,560,294,276; HRESIMS(positive)m / z 740.5668[M+H] + (calcd.for C 42 H 78 NO9,740.5671), the molecular formula of compound 4 was determined to be C 42 H 77 NO9; 1 H and 13 The C NMR values ​​are shown in Table 3.

[0103] Compound 5:

[0104]

[0105] Compound 5: White powder; APCI-MS / MS (positive) m / z 728, 710, 548, 294, 276; HRESIMS (positive) m / z 728, 5668 [M+H] + (calcd.For C 41 H 78 NO9,728.5671); 1 H and 13 The C NMR spectra are shown in Table 3. Compound 6:

[0106]

[0107] Compound 6: White powder; UV(MeOH)λ max (logε)202(3.36)nm;IR(KBr)v max 3172,2918,1720,1542,1466,1079cm -1;APCI-MS / MS(positive)m / z 712,694,532,294,276; HRESIMS(positive)m / z 712.5721[M+H] + (calcd.for C 41 H 78 NO8,712.5722); 1 H and 13 CNMR is shown in Table 4.

[0108] Compound 7:

[0109]

[0110] Compound 7: White powder; APCI-MS / MS (positive) m / z 754,736,574,294,276; HRESIMS (positive) m / z 754,5830 [M+H] + (calcd.for C 43 H 80 NO9,754.5828); 1 H and 13 The C NMR values ​​are shown in Table 4.

[0111] Table 2. NMR data assignments for Compound 1 and Compound 2 (600 MHz, in Pyr-d5)

[0112]

[0113]

[0114] * Signals belonging to the same column may be interchanged.

[0115] Table 3. NMR data assignments for compounds 3, 4, and 5.

[0116]

[0117]

[0118] * Signals belonging to the same column may be interchanged; MHz, in Pyr-d5; MHz, in Pyr-d5.

[0119] Table 4. NMR data assignments for compounds 6 and 7

[0120]

[0121]

[0122] * Signals belonging to the same column may be interchanged; MHz, in Pyr-d5; MHz, in Pyr-d5.

[0123] Example 2: Preparation of Cordyceps sinensis cerebroside extract under different conditions

[0124] 2.1 Different extraction solvents

[0125] The sample preparation method was the same as in "1.1", with 95% ethanol and 90% methanol as the extraction solvents. After centrifugation, the extracts were loaded onto an ODS column and eluted directly with 100% methanol. The chromatographic conditions described in "1.2.3" were then followed, and the peak area of ​​chromatographic peak 3 was used as the evaluation index to compare the effectiveness of the two extraction solvents. The results showed that the peak area of ​​chromatographic peak 3 obtained with the 90% methanol extract was 2661.7, while the peak area obtained with the 95% ethanol extract was 1008.4. Therefore, the 90% methanol extraction was more effective. Figure 3 As shown.

[0126] 2.2 Different elution solutions

[0127] The elution effects of 90% methanol, 95% methanol, 98% methanol, and 100% methanol eluents on cerebroside components were compared. Cordyceps sinensis extract was prepared according to the method described in section "1.1". After centrifugation, the supernatant was placed on an ODS column and eluted with 90% methanol, 95% methanol, 98% methanol, and 100% methanol eluents, respectively. The chromatographic conditions described in section "1.2.3" were followed for detection, and the results are as follows: Figure 4 , Figure 5 , Figure 6 , Figure 7 As shown, 90% methanol and 95% methanol cannot elute the target component; 98% methanol can elute the target component, but the target component is still present in 100% methanol (elution with 98% methanol cannot completely elute the target compound). Therefore, the eluent conditions are optimized to use 95% methanol for impurity removal and 98%-100% methanol for elution and enrichment of the target compound.

[0128] 2.3 Different feed-to-liquid ratios

[0129] Three 1g portions of Cordyceps sinensis powder were accurately weighed and placed in 50mL Erlenmeyer flasks. 6mL, 10mL, and 12mL of 90% methanol were added at material-to-liquid ratios of 1:6, 1:10, and 1:12, respectively, and the mixture was refluxed for 3 hours. The mixture was centrifuged (5000rpm, 10min) and the supernatant was collected. The supernatant was loaded onto a equilibrated ODS column and eluted with 95% methanol for 10 BV (discarded). Then, it was eluted with 100% methanol for 5 BV. The sample was collected in a constant-weight evaporating dish and evaporated to dryness in a water bath. Afterward, it was dried at 105℃ for 3 hours and cooled in a desiccator for 30 minutes. The weight of the cerebroside extract was quickly and accurately determined. The yield was calculated based on the dried product weight using the formula: Yield = Cerebroside extract mass / Sample mass (dried product). (Sample moisture content calculation method: 2g of sample was tested using a rapid moisture analyzer; the moisture content was 11.8%.)

[0130] The results are shown in Table 5. The yields of Cordyceps sinensis cerebroside extract obtained by material-liquid ratio extraction of 1:6, 1:10 and 1:12 were 0.18%, 0.38% and 0.50% respectively, indicating that a material-liquid ratio between 1:8 and 1:12 is more suitable.

[0131] Table 5. Yield of Cordyceps sinensis cerebroside extract with different material-liquid ratios

[0132]

[0133]

[0134] Example 3: Immunosuppressive Activity of Cordyceps sinensis Cerebroside Extract

[0135] 3.1 Solution Preparation

[0136] Culture medium: 89% culture medium + 10% fetal bovine serum + 1% antibiotics, store at 2-8℃.

[0137] PBS: Dissolve one packet of PBS completely in 1L of pure water, dispense into smaller containers, and sterilize at high temperature.

[0138] 10% CCK-8: Add 1 mL of CCK8 to 9 mL of culture medium to prepare a 10% CCK-8 detection solution. Prepare and use immediately.

[0139] Concanavalin A (ConA): Accurately weigh 5 mg of concanavalin A, add 1 ml of sterile PBS and dissolve thoroughly to prepare a 5 mg / mL stock solution. Aliquot into 50 μL tubes and store at -20°C. When using, thaw and prepare a working solution with complete culture medium to a concentration of 150 μg / mL.

[0140] 3.2 Experimental Methods

[0141] 3.2.1 Cell Acquisition: After exsanguination and bloodletting of BABL / C male mice, the mice were euthanized and soaked in 75% alcohol for 10 min. The spleen was aseptically harvested in a laminar flow hood and placed in a culture dish containing PBS. The spleen surface and interior were rinsed with PBS using a syringe until the spleen turned white. The spleen was gently torn apart with forceps and then gently crushed on a steel mesh using a 5 mL syringe needle to prepare a single-cell suspension. The suspension was filtered through a 200-mesh sieve and centrifuged for 10 min (1000 rpm / min). After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in PBS and centrifuged again for 10 min (1000 rpm / min). This process was repeated once. 2 mL of erythrocyte lysis buffer was added to the cell pellet, mixed, and allowed to stand for 5 min. Then, 8 mL of PBS was added, mixed, and centrifuged at 1000 rpm / min for 5 min. After centrifugation, discard the supernatant, resuspend the cell pellet in PBS, and centrifuge again for 10 min (1000 rpm / min). Repeat this process once more. After centrifugation, discard the supernatant, resuspend the cell pellet in 1 mL of RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody), add 2-3 mL of medium to dilute, and count the new cell type using a cell counter. Adjust the cell concentration to 2*10⁻⁶ cells / mL with complete medium. 6 per mL.

[0142] 3.2.2 Sample preparation: Take an appropriate amount of the Cordyceps sinensis cerebroside extract from Example 1 and dissolve it in DMSO to prepare a stock solution of 25.6 mg / mL. Then dilute it with culture medium to obtain a solution with a concentration of 128 μg / mL. Then dilute it serially with culture medium to obtain sample dilutions with concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL and 4 μg / mL, respectively.

[0143] 3.2.3 Cell viability assay: The prepared cell suspension was added to 96-well plates, and the cell density was 2*102. 5 Cells were added to each well at a concentration of 100 μL. Five replicates were performed for each concentration. The wells were then incubated at 37°C with 5% CO2 for 60 h. Afterward, serum-free medium with 10% CCK-8 was added, and the wells were incubated at 37°C for 4–5 h. The absorbance (A) of each well was measured at 450 nm using a microplate reader. A control group was included: cells + 1640 medium. Results showed that, compared with the control group, the samples at all concentrations (64, 32, 16, 8, 4, 2 μg / mL) showed no cytotoxic effect on spleen lymphocytes.

[0144] 3.2.4 Cell proliferation inhibition assay: The prepared cell suspension was added to a 96-well plate, and the cell density was 2*102. 5Cells were added to each well at a volume of 100 μL. Samples were added at 100 μL per well, with five replicates per concentration. The wells were then incubated at 37°C with 5% CO2 for 12 h. After carefully aspirating 10 μL of supernatant, 10 μL of ConA at a concentration of 150 μg / mL (final concentration 7.5 μg / mL) was added to the corresponding group. The wells were then incubated at 37°C with 5% CO2 for 48 h. Finally, serum-free medium with 10% CCK-8 was added, and the wells were incubated at 37°C for 4–5 h. The absorbance (A) of each well was measured at 450 nm using a microplate reader. One negative control group and one model control group were set up: cells + 1640 medium and cells + ConA + 1640 medium, respectively.

[0145] 3.2.5 Data Processing: The proliferation rate (viability) of spleen lymphocytes in the sample was calculated using the following formula:

[0146] Proliferation rate % = [As / Ac] × 100%

[0147] As: Absorbance of sample group (spleen lymphocytes + test sample + CCK-8)

[0148] Ac: Absorbance of control group (splenic lymphocytes + CCK-8)

[0149] The inhibition rate of spleen lymphocyte proliferation by the sample was calculated using the following formula:

[0150] Proliferation inhibition rate % = [(Am-As) / (Am-Ac)] × 100%

[0151] Am: Absorbance of the model group control (splenic lymphocytes + ConA + CCK-8)

[0152] As: Absorbance of sample group (spleen lymphocytes + test sample + ConA + CCK-8)

[0153] Ac: Absorbance of negative control group (splenic lymphocytes + CCK-8)

[0154] 3.3 Experimental Results

[0155] The experimental results are shown in Table 6 below:

[0156] Table 6. Inhibition rate (%) of Cordyceps sinensis cerebroside extract on Con A-induced spleen lymphocyte proliferation.

[0157]

[0158] ***: p < 0.001 compared to the model group; **: p < 0.01 compared to the model group; *: p < 0.05 compared to the model group.

[0159] Table 6 shows that the cordyceps sinensis cerebroside extract exhibits immunosuppressive activity against Con A-induced proliferation of mouse splenic lymphocytes, with an IC50 value of [missing information]. 50 The value was 4.6 μg / mL.

[0160] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "some implementations," "other implementations," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0161] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the concept of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. A Cordyceps sinensis extract, characterized in that, The compound contains 0.39%-0.45% of compound 1, 0.10%-0.18% of compound 2, 16%-18% of compound 3, 12%-15% of compounds 4 and 5 combined, 0.25%-0.45% of compound 6, and 0.40%-0.50% of compound 7; the structures of compounds 1, 2, 3, 4, 5, 6, and 7 are shown below: 、 、 、 、 、 、 ； The preparation method of the Cordyceps sinensis extract includes the following steps: (1) Cordyceps sinensis is refluxed in 85%-95% methanol solution for 2-3 hours to obtain extract; the material-liquid ratio of Cordyceps sinensis and 85%-95% methanol solution in step (1) is 1:8 to 1:

12. (2) The extract from step (1) is loaded into a solid phase extraction column, the solid phase extraction column is packed with ODS packing, and eluted with 95% methanol and 98%~100% methanol in sequence, and the eluent obtained by elution with 98%~100% methanol is collected.

2. The Cordyceps sinensis extract according to claim 1, characterized in that, The content of compound 1 is 0.43%, the content of compound 2 is 0.16%, the content of compound 3 is 17.79%, the total content of compounds 4 and 5 is 13.58%, the content of compound 6 is 0.38%, and the content of compound 7 is 0.46%.

3. The Cordyceps sinensis extract according to claim 1, characterized in that, In step (1), the methanol solution is a 90% methanol solution.

4. The Cordyceps sinensis extract according to claim 1, characterized in that, The extract is the supernatant obtained after centrifugation of the extracted mixture.

5. The Cordyceps sinensis extract according to claim 1, characterized in that, Step (2) is as follows: the extract from step (1) is loaded into a solid phase extraction column of ODS packing and eluted sequentially with 95% methanol and 100% methanol, and the eluent obtained by elution with 100% methanol is collected.

6. The Cordyceps sinensis extract according to claim 1, characterized in that, The eluent obtained by elution with 100% methanol is further concentrated.

7. The Cordyceps sinensis extract according to claim 1, characterized in that, Includes the following steps: (1) Cordyceps sinensis is extracted by reflux in 90% methanol solution for 2-3 hours, centrifuged, and the supernatant is obtained. The ratio of Cordyceps sinensis to 90% methanol is 1:8 to 1:

12. (2) Load the supernatant from step (1) into an ODS solid phase extraction column and elute with 95% methanol and 100% methanol in sequence, and collect the eluent obtained by elution with 100% methanol.

8. The Cordyceps sinensis extract according to claim 7, wherein the ratio of Cordyceps sinensis to 90% methanol is 1:

10.

9. Cordyceps sinensis extract prepared by the method according to any one of claims 1-8.

10. The use of the Cordyceps sinensis extract according to any one of claims 1 or 2, or the Cordyceps sinensis extract according to claim 9, in the preparation of immunosuppressants.

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