Cordycepin-steviosin compound as well as preparation method and application thereof

Abstract

The invention discloses a cordycepin-steviosin compound as well as a preparation method and application thereof. The cordycepin-steviosin compound is prepared by mixing cordycepin, steviosin and an organic solvent, dissolving and then drying. The adenosine deaminase degradability of the cordycepin is improved, the cordycepin is endowed with a slow release characteristic, the half-life period of the cordycepin is prolonged, the bioavailability of the cordycepin is improved, and wide application of the cordycepin in the fields of medicines, cosmetics, feeds or health foods is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and pharmaceutical intermediate compound modification, and particularly relates to a cordycepin-stevioside complex and a preparation method and application thereof. Background Art

[0002] Cordycepin, also known as 3′-deoxyadenosine (TDA), is a nucleoside antibiotic isolated from Cordyceps militari. Currently, cordycepin is primarily extracted from cultivated fruiting bodies of Cordyceps militaris or synthesized chemically using adenosine as a starting material. Cordycepin exhibits a wide range of biological and pharmacological activities, including anti-tumor, anti-leukemia, immunomodulatory, antibacterial, anti-inflammatory, antiviral, hypoglycemic, hypolipidemic, and anti-aging effects.

[0003] The chemical structure of cordycepin is shown in Formula I, and the molecular formula is C 10 H 13 N5O3.

[0004]

[0005] However, after intravenous injection, this nucleoside antibiotic is rapidly deaminated by adenosine deaminase (ADA) in the blood, metabolizing it to the inactive metabolite 3'-deoxyhypoxanthine. (J. Agric. Food Chem. 2010, 58, 4638-4643) Cordycepin has an elimination half-life of 1.6 minutes when injected into the tail vein of rats. Co-injection with a nucleoside deaminase inhibitor prolongs the elimination half-life to 23.8 minutes.

[0006] Domestic and foreign scholars have been trying to use various technologies to extend the elimination half-life of cordycepin in vivo and improve its bioavailability. At present, there are two main strategies to try to improve its bioavailability: (1) Use it in combination with ADA inhibitors to protect cordycepin from enzyme metabolism by inhibiting nucleoside deaminase, such as combining it with pentostatin (a potent and irreversible nucleoside deaminase inhibitor with Ki = 2.5 pM). This method can indeed improve the bioavailability of cordycepin and enhance the therapeutic effect, but ADA inhibitors show serious toxicity, such as causing growth retardation, increasing lung and spleen masses, causing lymphotoxicity, thrombocytopenia, severe gastrointestinal toxicity, bone marrow toxicity, etc. (Agarwal, 1980; Rodman et al., 1997). (2) Use a new drug delivery system, such as CN104473874A discloses a cordycepin proliposome, which can increase the bioavailability of cordycepin by 1.4 times compared with cordycepin, but it needs to be administered by injection, which seriously limits the use of future products. CN117462498A discloses a cordycepin liposome multi-responsive microsphere. In vitro tests confirm that the microsphere has multiple responsiveness to temperature, magnetism, pH, and ionic strength, but no in vivo research data are provided, nor can it be confirmed that the system can protect cordycepin from nucleoside deaminase metabolism in vivo, and whether it can improve the bioavailability of cordycepin is uncertain. CN110538203A discloses a cordycepin liposome complex, but only provides an in vitro preparation method and no in vivo and in vitro evaluation of the efficacy. CN116172974B discloses cordycepin self-assembling nanoparticles, and conducts in vitro cell-level tests and enzyme degradation tests, but no in vivo test data are provided. CN107281109A discloses cordycepin carboxymethyl chitosan nanoparticles, also providing only in vitro data. Nucleoside deaminases are expressed in various tissues, organs, and cells in the human body. Therefore, any technology aimed at addressing the enzymatic metabolism of cordycepin in vivo cannot avoid in vivo verification in animals or humans.

[0007] With the continuous deepening of research on the active substances of traditional Chinese medicine, active ingredients of traditional Chinese medicine such as terpenes, alkaloids, flavonoids, quinones, and polysaccharides have shown potential as drug delivery nanocarriers (Research Progress of Active Ingredients of Traditional Chinese Medicine as Drug Delivery Nanocarriers [J]. Chinese Herbal Medicine, 2024, 55(16): 5678-5691.)

[0008] In summary, existing cordycepin preparations or delivery systems have the disadvantages of large side effects and limited effect on improving the stability of cordycepinase. Therefore, there is an urgent clinical need for a high-safety cordycepin product with a simple preparation process, which can change the pharmacokinetic characteristics of cordycepin itself, prolong the duration of action in vivo, and significantly improve its bioavailability. Summary of the Invention

[0009] In order to solve the above technical problems and improve the bioavailability of cordycepin, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a use of stevioside in improving the bioavailability of cordycepin, wherein improving the bioavailability of cordycepin includes increasing the whole blood exposure of cordycepin or extending the half-life of cordycepin.

[0011] Preferably, the application comprises mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying to obtain the cordycepin-stevioside complex.

[0012] Preferably, the cordycepin and stevioside assemble to form a complex through non-covalent interactions.

[0013] Furthermore, the non-covalent interaction includes ionic bonds and / or hydrogen bonds.

[0014] Preferably, the XRPD spectrum of the cordycepin-stevioside complex has characteristic peaks at characteristic diffraction angles (2θ°) of 3.46, 4.16, 6.91, 8.23, 12.27, 12.93, 13.61, 15.87, 16.50, 18.36, 19.71, 22.99, 24.41, and 24.79.

[0015] In a second aspect, the present invention provides a cordycepin-stevioside complex, which is obtained by mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying.

[0016] Preferably, the cordycepin and stevioside assemble to form a complex through non-covalent interactions.

[0017] Furthermore, the non-covalent interaction includes ionic bonds and / or hydrogen bonds.

[0018] Preferably, the XRPD spectrum of the cordycepin-stevioside complex has characteristic peaks at characteristic diffraction angles (2θ°) of 3.46, 4.16, 6.91, 8.23, 12.27, 12.93, 13.61, 15.87, 16.50, 18.36, 19.71, 22.99, 24.41, and 24.79.

[0019] Preferably, the weight ratio of the cordycepin to the stevioside is 1:0.5-5, for example: 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0020] Furthermore, the weight ratio of the cordycepin to the stevioside is 1:1-2.

[0021] In a third aspect, the present invention provides a composition comprising the complex according to the first aspect.

[0022] Preferably, the composition further comprises a carrier, and the structural formula of the carrier is as follows:

[0023] OH-ABC

[0024] Wherein, structure A is a five-membered ring or a six-membered ring, preferably a benzene ring, a five-carbon ring, a six-carbon ring, a five-membered heterocycle, or a six-membered heterocycle, and structure A has 0-4 substituents, and the substituents are OH, CH3OH, or NH2;

[0025] Structure B is a 3-5 carbon chain containing 0-3 double bonds or a heterochain containing 0-2 O or N atoms. Structure B has 0-3 substituents, which are OH, NH2, =O, or CH3OH.

[0026] The C structure is a five-membered ring or a six-membered ring, preferably a benzene ring, a five-carbon ring, a six-carbon ring, a five-membered heterocycle, or a six-membered heterocycle. The C structure has 0-4 substituents, and at least one substituent contains 1 or more O and / or N atoms;

[0027] Preferably, the structure B and the structure C are further cyclized into a cyclic structure via a substituent or a heteroatom.

[0028] Preferably, the weight ratio of cordycepin to carrier in the composition is 1:0.1 to 1:5, for example: 1:0.1, 1:0.5, 1:1, 1:1.25, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0029] Furthermore, the weight ratio of cordycepin to carrier in the composition is 1:0.5 to 1:2.

[0030] Preferably, the carrier is a flavonoid or phenolic glycoside compound,

[0031] Furthermore, the carrier is selected from any one or a combination of two or more of dihydrocinnamidine, cinnamidine, deacetylcinnamidine, salidroside, naringenin, hesperetin and quercetin.

[0032] Preferably, the composition further comprises a polymer,

[0033] Preferably, the weight ratio of cordycepin to polymer in the composition is 1:0.1 to 1:5, for example: 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0034] Furthermore, the weight ratio of cordycepin to polymer in the composition is 1:0.5 to 1:2.

[0035] Preferably, the polymer is selected from any one or a combination of two or more of cellulose polymers, synthetic polymers, polysaccharides and their derivatives, proteins and polypeptides.

[0036] Preferably, the cellulose polymer includes but is not limited to hydroxypropyl methylcellulose (HPMC), low-substituted hydroxypropyl cellulose (L-HPC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), sodium carboxymethylcellulose (CMC-Na) or hydroxyethyl cellulose.

[0037] Preferably, the synthetic polymer includes but is not limited to polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), copovidone (PVP-VA64), polyglutamic acid (PGA), polydopamine (PDA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polylactic-co-glycolic acid (PLGA), sodium polyacrylate, siRNA and derivatives (alkyl chains for improving lipophilicity), mRNA and derivatives (alkyl chains for improving lipophilicity) or antisense oligonucleotides and derivatives (alkyl chains for improving lipophilicity).

[0038] Preferably, the polysaccharide and its derivatives include but are not limited to carboxymethylation, sulfonylation, phosphorylation, acylation or hydroxypropylation, cationic, anionic or other derivatives.

[0039] Furthermore, the polysaccharide and its derivatives are selected from any one or a combination of two or more of carrageenan, guar gum, gum arabic, locust bean gum, konjac gum, agar, gelatin, pectin, gellan gum, hyaluronic acid (HA), aminodextran, chondroitin sulfate, dermatan sulfate, heparin, keratan sulfate, heparan sulfate, sodium alginate, propylene glycol alginate, agar, fucoidan, cyclodextrin and derivatives or chitosan and derivatives (such as acylation, carboxylation, alkylation and quaternization, etc.).

[0040] Preferably, the protein and polypeptide include but are not limited to soy protein, plant protein, bovine serum albumin or casein.

[0041] In a fourth aspect, the present invention provides an enteric-coated preparation comprising the complex described in the second aspect or the composition described in the third aspect.

[0042] Preferably, the enteric-coated preparation does not release or hardly releases in an acidic medium (such as pH 3.0 and below), and releases most or all of the substance in a neutral medium (such as pH 5.0-pH 8.0).

[0043] Preferably, the enteric materials of the enteric preparation include but are not limited to shellac, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (cellulose acetate peptide, CAP), cellulose acetate trimellitate (cellulose acetate trimellitate, CAT), hydroxypropyl methylcellulose phthalate (hydroxypropyl methylcellulose peptide, HPMCP), hydroxypropyl methylcellulose trimellitate (HPMCR), cellulose acetate succinate (CAS), hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate, HPMCAS) or acrylic resins (such as Eudragit).

[0044] Preferably, the enteric-coated preparation contains additives, such as plasticizers, anti-adherents, and opacifiers.

[0045] Preferably, the enteric preparation comprises any one or a combination of two or more of an excipient, a filler, a diluent, a disintegrant, a binder, a lubricant, a glidant, a flavoring agent or a pH regulator.

[0046] Preferably, the dosage form of the enteric-coated preparation includes but is not limited to tablets, capsules, granules, dry suspensions, pills, pellets or coatings.

[0047] In a fifth aspect, the present invention provides a method for preparing a cordycepin-stevioside complex, comprising mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying to obtain the cordycepin-stevioside complex.

[0048] Preferably, the weight ratio of cordycepin to stevioside is 1:0.5-5, for example: 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0049] Furthermore, the weight ratio of the cordycepin to the stevioside is 1:1-2.

[0050] The organic solvent is selected from any one of methanol, ethanol, acetone, tetrahydrofuran, and dichloromethane, or a combination of two or more thereof, preferably ethanol.

[0051] Preferably, the organic solvent further comprises water, and the volume ratio of the water to the organic solvent is 0-1:1.

[0052] In a sixth aspect, the present invention provides the use of the cordycepin-stevioside complex described in the second aspect, the composition described in the third aspect, or the preparation method described in the fifth aspect in the preparation of medicines, cosmetics, feed or health foods.

[0053] Beneficial effects of the present invention:

[0054] The present invention discovers a new function of stevioside. By forming a cordycepin-stevioside complex with cordycepin, the degradation of cordycepin by adenosine deaminase is improved, the cordycepin is given a sustained-release property, the half-life of cordycepin is extended, and the bioavailability of cordycepin is increased, which contributes to the wide application of cordycepin in the fields of medicines, cosmetics, feed or health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Shown is the infrared spectrum of TDA API;

[0056] Figure 2 Shown is the infrared spectrum of stevia;

[0057] Figure 3 Shown is the infrared spectrum of the physical mixture of TDA-stevioside;

[0058] Figure 4 Shown is the infrared spectrum of the TDA-stevioside complex;

[0059] Figure 5 Shown is the XRPD pattern of TDA drug substance;

[0060] Figure 6 Shown is the XRPD pattern of stevioside;

[0061] Figure 7 Shown is the XRPD pattern of the TDA-stevioside physical mixture;

[0062] Figure 8 Shown is the XRPD pattern of TDA-stevioside complex. DETAILED DESCRIPTION

[0063] The technical solutions of the present invention are described clearly and completely below in conjunction with the embodiments and accompanying drawings. The described embodiments are only a portion of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0064] The main reagents in the embodiments of the present invention are as follows:

[0065] Table 1 Reagent names, functions and abbreviations

[0066]

[0067] Note: The synthesis method of cordycepin is referred to the literature (Organic Process Research & Development 2000, 4, 601-605. From Adenosine to 3'-deoxyadenosine: Development and Scale Up).

[0068] Example 1. Preparation of cordycepin-stevioside complex

[0069] 1.1 According to the prescription in Table 2, cordycepin and stevioside were added to an ethanol-water solution, stirred and dissolved, and concentrated to dryness under reduced pressure using a rotary evaporator to obtain cordycepin-stevioside complexes F1 to F5.

[0070] Table 2 Cordycepin-stevioside complex prescription

[0071]

[0072] 1.2 Characterization of cordycepin-stevioside complex

[0073] Take the following components and perform infrared spectrum and XRPD pattern determination respectively, the results are shown in Figure 1-4 and Figure 5-8 .

[0074] Control group 1: cordycepin;

[0075] Control group 2: stevia;

[0076] Comparative group 3: a physical mixture of equal mass of cordycepin and stevioside in formulation F2;

[0077] Experimental group 1: cordycepin-stevioside complex F2 powder.

[0078] like Figures 1 to 4 As shown, the characteristic peak 2923cm in the infrared spectrum of cordycepin in comparison group 1 -1 、1892cm -1 、1716cm -1 、1672cm -1 、1374cm -1 、1209cm -1 、1059cm -1 、1006cm -1 The infrared spectrum of the cordycepin-stevioside complex in experimental group 1 exhibited a shift due to non-covalent interactions between the cordycepin and stevioside molecules. However, the infrared spectrum of the physical mixture of cordycepin and stevioside in comparison group 3 exhibited no shift, indicating that simple physical mixing had no effect on the properties of cordycepin.

[0079] like Figures 5 to 8As shown, compared with the physical mixture of cordycepin and cordycepin and stevioside in control group 3, new characteristic peaks appeared in the XRPD spectrum of the cordycepin-stevioside complex in experimental group 1 at characteristic diffraction angles (2θ°) of 3.46, 4.16, 6.91, 8.23, 12.27, 12.93, 13.61, 15.87, 16.50, 18.36, 19.71, 22.99, 24.41, and 24.79, indicating that a cordycepin-stevioside complex at the molecular level was formed inside the cordycepin-stevioside complex through non-covalent interactions.

[0080] Example 2 Preparation of Cordycepin-Containing Composition

[0081] 2.1 According to the prescriptions in Tables 3 and 4, cordycepin, stevioside, a carrier, and a polymer were added to an ethanol-water solution, stirred and dissolved, and concentrated to dryness under reduced pressure using a rotary evaporator to obtain cordycepin-containing compositions Z1 to F14.

[0082] Table 3 Composition of Z1-Z7 prescriptions

[0083]

[0084]

[0085] Table 4: Composition of Z8-Z14 prescriptions

[0086]

[0087] 2.2 According to the prescription in Table 5, the cordycepin-stevioside complex F2, the carrier and the high molecular weight polymer were uniformly mixed to prepare cordycepin-containing compositions Z15 to Z21.

[0088] Table 5: Composition of Z15-Z21 prescriptions

[0089]

[0090] Example 3 Preparation of Enteric-coated Capsules Containing Cordycepin Composition

[0091] According to the prescription in Table 6, the cordycepin-stevioside complex F1 and the carrier HJTG were weighed and mixed uniformly to prepare a cordycepin composition. The other materials in the prescription were then added and mixed uniformly. The enteric-coated empty capsules were filled according to the theoretical filling amount to prepare enteric-coated capsules C1 of the cordycepin composition.

[0092] Table 6 Composition of enteric-coated capsules

[0093]

[0094] Example 4 Preparation of Enteric-coated Tablets Containing Cordycepin Composition

[0095] According to the recipe in Table 7, cordycepin-stevioside complex F2 and carrier CYCG were weighed and mixed uniformly to prepare a cordycepin composition. Silicified microcrystalline cellulose, 1 / 2 the recipe amount of crospovidone, colloidal silicon dioxide, and magnesium stearate were then weighed in sequence, mixed uniformly, and dry granulated. The remaining crospovidone, colloidal silicon dioxide, and magnesium stearate were added, mixed uniformly, and tablets were compressed to a hardness of 30-180 N to prepare the core tablets of the cordycepin composition. A coating solution was prepared using Opadry enteric premixed coating powder, and enteric coating was applied at a weight gain of 5%-30% to prepare enteric-coated tablets T1 of the cordycepin composition.

[0096] Table 7 Composition of enteric-coated tablets containing cordycepin compositions

[0097]

[0098] Example 5 Preparation of Enteric-coated Granules Containing Cordycepin Composition

[0099] According to the recipe in Table 8, the excipients and cordycepin-containing composition Z11 were weighed, mixed uniformly, and dry granulated. Granules that passed a 10-mesh sieve but not a 20-mesh sieve were screened and coated with a coating solution using Opadry enteric premixed coating powder. Enteric coating was applied to the granules, controlling the weight gain to 15%-50%, to prepare enteric-coated granules G1 of the cordycepin composition.

[0100] Table 8 Composition of Cordycepin-containing Granules

[0101] Prescription G1 Dosage Cordycepin-containing composition Z11 (calculated as TDA) 50 microcrystalline cellulose 100 Croscarmellose sodium 15 Sodium stearyl fumarate 1.0

[0102] Example 6 Preparation of Cordycepin Composition Enteric-Coated Micropellets

[0103] Sucrose pellet cores were added to a fluidized bed, and then an 80% ethanol mixed solution containing the cordycepin composition Z3 was added. The pellets were layered and coated with Opadry enteric premixed coating powder to prepare a coating solution. The pellets were then enteric-coated with a weight gain of 10%-40% to obtain enteric-coated pellets P1 of the cordycepin composition.

[0104] Example 7 Preparation of Enteric-coated Microtablets of Cordycepin Composition

[0105] The total mixed granules prepared in Example 6 were weighed, pressed into microtablets, and enteric-coated to obtain enteric-coated microtablets MT1 of the cordycepin composition.

[0106] Example 8 Preparation of Enteric-coated Capsules of Cordycepin Composition

[0107] The enteric-coated granules of Example 5 were loaded into hollow capsules to obtain enteric-coated capsules C2 (granular type) of the cordycepin composition.

[0108] The enteric-coated micropellets of Example 6 were loaded into hollow capsules to obtain enteric-coated capsules C3 (micropellet type) of the cordycepin composition.

[0109] The enteric-coated micro-tablets of Example 7 were loaded into hollow capsules to obtain enteric-coated capsules C4 (micro-tablet type) of the cordycepin composition.

[0110] Example 9 Preparation of Enteric-Coated Sustained-Release Preparation of Cordycepin Composition

[0111] Table 9 Cordycepin composition enteric-coated sustained-release tablets prescription

[0112] Prescription ER1 Dosage Cordycepin-containing composition Z14 (calculated as TDA) 50 Microcrystalline cellulose (a sustained-release drug) 200 Sodium stearyl fumarate 3.0

[0113] 9.1 According to the prescription in Table 9, Z14, microcrystalline cellulose, and 1 / 2 of the prescribed amount of sodium stearyl fumarate were weighed, mixed evenly, and dry granulated. The remaining sodium stearyl fumarate was then added and mixed evenly. Tablets were compressed and enteric-coated to obtain cordycepin composition enteric-coated sustained-release tablets ER1.

[0114] 9.2 Take the sucrose pellet cores and add them to a fluidized bed. Then, add a mixed solution of 80% ethanol containing the cordycepin composition Z7 and apply the drug by layering. Use Opadry enteric premixed coating powder to prepare a coating solution. Apply enteric coating and control the weight gain to 10-40% to obtain enteric-coated sustained-release micropellets ER2 of the cordycepin composition.

[0115] 9.3 Take the sucrose pellet cores, add them into a fluidized bed, then add a mixed solution of 80% ethanol containing the cordycepin composition Z2, and layer the drugs using the drug layering method to prepare cordycepin composition micropellets.

[0116] The cordycepin composition pellets were coated with Colorcon Surelease to increase their weight by 8% and 20%, respectively, to prepare Surelease-coated 8% weight-gain pellets and Surelease-coated 20% weight-gain pellets.

[0117] Uncoated cordycepin pellets, 8% weight-gain pellets coated with SuLiSi, and 20% weight-gain pellets coated with SuLiSi were mixed evenly in a weight ratio of 2:1:1, and filled into enteric-coated capsules to obtain enteric-coated sustained-release capsules ER3 of the cordycepin composition.

[0118] Example 10 Preparation of Cordycepin Composition Enteric-coated Tablets

[0119] The enteric-coated pellets prepared in Example 6 were added with appropriate amounts of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, and magnesium stearate, and mixed to form enteric-coated tablets of the cordycepin composition.

[0120] Example 11 Preparation of Cordycepin Composition Enteric-coated Powder

[0121] The cordycepin composition Z1 to Z21 powders are respectively coated with enteric coatings to obtain enteric-coated cordycepin composition powders.

[0122] Performance test 1 Chemical stability inspection

[0123] The samples were sealed according to Table 10 and placed in a high temperature environment of 50°C. Samples were taken at 0 days, 14 days and 30 days respectively to examine their chemical stability. The results are shown in the table below.

[0124] Table 10 Chemical stability test results

[0125]

[0126]

[0127]

[0128] As shown in Table 10, after all samples were stored in their packaging at 50°C for 30 days, no increase in individual or total impurities was observed in any of the formulations compared to the results of the samples tested at day 0. The cordycepin-stevioside complex, cordycepin-containing compositions, and formulations exhibited excellent chemical stability.

[0129] Performance test 2 acid resistance test

[0130] Prepare pH 1.0 and pH 3.0 dissolution media in 750 mL volumes according to the "Technical Guidelines for Dissolution Testing of Ordinary Solid Oral Preparations." Use a slurry method (capsules can be placed in a sinker) with a paddle speed of 50 rpm and the temperature set at 37°C. Add 50 mg of the active ingredient to each cup of enteric-coated formulation. Samples were taken after 2 hours to determine the active ingredient content in the dissolution medium.

[0131] The experimental results showed that when all enteric-coated preparations were tested in pH 1.0 and pH 3.0 media for 2 hours, no main drug was detected in the dissolution medium, indicating that the enteric-coated preparations of cordycepin compositions all had excellent acid resistance.

[0132] Performance test 3Dissolution curve investigation in pH 6.8 phosphate medium

[0133] The dissolution method used the small cup method, paddle method (capsule plus sinker), 100 rpm, 37°C; pH 6.8 phosphate buffer prepared in accordance with the "Technical Guidelines for Dissolution Testing of Ordinary Oral Solid Preparations"; the medium volume was 100 mL; and the dosage was 50 mg (in terms of TDA).

[0134] The sampling and sample processing method is to sample 5 mL at different times and add 5 mL of blank medium to the dissolution cup. The dissolution liquid is taken out and placed in a 5 mL centrifuge tube. Centrifuge at 14000 rpm and 37°C for 3 min. The TDA content in the cordycepin enteric-coated preparation (see Table 12) is detected by HPLC (see Table 11). The results are shown in Table 12.

[0135] Table 11HPLC process conditions

[0136] Chromatographic columns Agilentporoshell120EC-C184.6×150mm, 2.7μm Mobile phase Water:acetonitrile=92.5:7.5 (V / V); flow rate 0.7mL / min Column temperature (℃) 35℃ Wavelength (nm) 260nm Injection volume 10 μL Std concentration 50 μg / mL

[0137] Table 12 Cumulative dissolution of cordycepin enteric-coated preparation in pH 6.8 medium (%)

[0138]

[0139]

[0140] As can be seen from Table 12, the enteric-coated rapid-release preparations of cordycepin can be completely released in a neutral medium within 60 minutes, and the enteric-coated sustained-release preparations can be almost completely released within 12 hours.

[0141] Performance Test 4 Rat Dosing Experiment

[0142] (1) Experimental animals

[0143] SPF male Sprague-Dawley rats, weighing 220-260 g, were purchased from the China Food and Drug Administration (China Food and Drug Administration) under license number SCXK (Beijing) 2022-0002. They were acclimated for one week under a 12-hour daylight cycle, 22 ± 2°C, and a relative humidity of 55 ± 5%. They were fasted (with free access to water) for at least 10 hours the day before the experiment. Water was withheld for 1 hour before dosing, and food and water were freely available 2 hours after dosing.

[0144] (2) Dosage: The dosages for the API group and the commercially available oral tablets were 80 mg / kg and 40 mg / kg, respectively. The dosage for the prescription was 10 mg / kg. The rat body weight was calculated as 250 g.

[0145] (3) Drug delivery preparation

[0146] Take TDA raw material and fill it into No. 9 gelatin capsules, which is recorded as TDA-API;

[0147] Take the physical mixture of cordycepin and stevioside in the cordycepin-stevioside complex F2 and F2 formulation respectively, weigh appropriate amounts (containing about 5 mg of TDA) and fill them into No. 9 gelatin capsules, respectively, and record them as F2 and F2-PM;

[0148] The cordycepin-containing compositions Z16, Z4, Z5, Z12, and Z19 were respectively loaded into No. 9 gelatin capsules and designated as Z16, Z4, Z5, Z12, and Z19;

[0149] Commercially available tablets: 10 cordycepin tablets (specification: 100 mg) were weighed, the average tablet weight was calculated, and the tablets were ground into fine powder using a mortar. The HPLC content was 40.6%; The average content of cordycepin tablets should be 24.6 mg of powder weighed and placed in a No. 9 gelatin capsule, recorded as WSK.

[0150] (3) Animal grouping and dosing regimen

[0151] All animals were randomly divided into 13 groups, with 6 animals in each group, and the drugs were administered according to the dosing schedule in Table 13. Specific dosing method: After the rats were anesthetized, they were dissected along the midline of the abdomen, the ileum segment was fixed, and an incision of approximately 0.5 cm was made with surgical scissors. After the No. 9 capsule was implanted, the incision was sutured, the ileum tissue was returned to its place, and then sutured layer by layer.

[0152] Table 13 Oral gavage administration regimen for rats

[0153]

[0154]

[0155] (3) Sampling and sample processing

[0156] At 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours after administration, 0.45 mL of blood was collected from the medial canthus and anticoagulated with sodium heparin. Simultaneously, an appropriate amount of pentostatin (PSTT) solution (35 mg / mL) was added to each blood collection centrifuge tube and gently mixed. Whole blood samples were stored at -20°C.

[0157] 150 μL of whole blood sample containing PSTT was accurately drawn, 10 μL of diphenhydramine (BHLM) internal standard solution (BHLM-600 ng / mL) was added, vortexed for 30 seconds, 440 μL of protein precipitant (MeOH) was added, vortexed for 30 seconds, allowed to stand for 10 minutes, centrifuged at 4°C and 13000 rpm for 10 minutes, 50 μL of supernatant was accurately drawn, 450 μL of deionized water was added, vortexed for 2 minutes, and analyzed by liquid chromatography-mass spectrometry according to Tables 14 and 15 (LLOQ: 10 ng / mL, injection volume 10 μL). Phoenix Win Nonlin 7.0 software was used to calculate the pharmacokinetic parameters (AUC 0-t , Cmax, T 1 / 2 , Tmax) results are shown in Table 16.

[0158] Table 14 TDA content determination method in whole blood

[0159]

[0160]

[0161] Table 15 Elution process

[0162] - 0min 4.5min 6min 10min 11.5min 15min A(％) 80 80 5 5 80 80 B(％) 20 20 95 95 20 20

[0163] Table 16 TDA pharmacokinetic results

[0164]

[0165] Note: -- indicates that the amount is below the minimum limit of quantification and cannot be reported.

[0166] As can be seen from Table 16, when the TDA raw material was encapsulated at a dose of 80 mg / kg body weight, no TDA was detected in the whole blood regardless of oral gavage or ileal administration (Groups A and B).

[0167] Commercially available cordycepin tablets were ground into powder and then encapsulated at a dose of 40 mg / kg body weight. TDA was not detected in the whole blood regardless of oral gavage or ileal administration (Groups F and G).

[0168] F2 cordycepin-stevioside complex was encapsulated and the dosage was 20 mg / kg body weight. 1 / 2 The cordycepin exposure (AUC 0-∞ ) were 576.15±92.5ng·hr / mL and 1365.70±171.9ng·hr / mL (Groups C and D), respectively. The exposure after ileal administration was 2.37 times that of oral gavage. Compared with commercially available preparations, the whole blood exposure and elimination half-life were greatly increased.

[0169] The physical mixture of cordycepin and stevioside in the F2 formulation was encapsulated and administered to the rats via ileal administration. No TDA was detected in the whole blood, indicating that the TJS in the F2 formulation had no inhibitory effect on nucleoside deaminase (Group E).

[0170] The cordycepin composition Z16 is packed in capsules, with a dosage of 20 mg / kg body weight, administered orally or by gavage or ileal administration. 1 / 2 1.4 hours and 2.2 hours respectively, and the cordycepin exposure (AUC 0-∞ ) were 433.97±71.6ng·hr / mL and 1113.62±126.51ng·hr / mL, respectively. The exposure after ileal administration was approximately 2.56 times that of oral gavage administration, and the bioavailability was greatly improved (groups H and I);

[0171] The J, K, L, and M groups were administered with capsules containing cordycepin compositions Z4, Z5, Z12, and Z19, respectively, by gavage. The dosage was 20 mg / kg body weight. Cordycepin was detected in the whole blood, and the whole blood elimination half-life was 1.5-3.3 hr. The whole blood exposure and elimination half-life were significantly increased.

[0172] Cordycepin was injected into the tail vein of rats (according to J. Agric. Food Chem., Vol. 58, No. 8, 2010) at a dose of 10 mg / kg body weight. 1 / 2About 1.6min, in the presence of nucleoside deaminase inhibitors, T 1 / 2 Compared with the injection of cordycepin alone, the T 1 / 2 The T 1 / 2 The extension was 3.6 times to 10.04 times, indicating that the cordycepin-stevioside complex significantly prolonged the elimination half-life of cordycepin in whole blood (P<0.05).

[0173] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Application of stevioside in improving the bioavailability of cordycepin, characterized in that: The improvement of cordycepin bioavailability includes increasing the whole blood exposure of cordycepin and / or extending the half-life of cordycepin.

2. The use according to claim 1, characterized in that The application comprises mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying to obtain the cordycepin-stevioside complex; the cordycepin and stevioside assemble to form the complex through non-covalent interactions; the non-covalent interactions include ionic bonds and / or hydrogen bonds; The XRPD spectrum of the cordycepin-stevioside complex shows characteristic peaks at characteristic diffraction angles (2θ°) of 3.46, 4.16, 6.91, 8.23, 12.27, 12.93, 13.61, 15.87, 16.50, 18.36, 19.71, 22.99, 24.41, and 24.

79.

3. A cordycepin-stevioside complex, characterized in that: Mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying to obtain the cordycepin-stevioside complex; The cordycepin and stevioside assemble to form a complex through non-covalent interactions; the non-covalent interactions include ionic bonds and / or hydrogen bonds; The XRPD spectrum of the cordycepin-stevioside complex shows characteristic peaks at characteristic diffraction angles (2θ°) of 3.46, 4.16, 6.91, 8.23, 12.27, 12.93, 13.61, 15.87, 16.50, 18.36, 19.71, 22.99, 24.41, and 24.

79.

4. The cordycepin-stevioside complex according to claim 3, characterized in that The weight ratio of the cordycepin to the stevioside is 1:0.5-5.

5. A composition, characterized in that The composition comprises the complex according to claim 2 or 3; Preferably, the composition further comprises a carrier, and the structural formula of the carrier is as follows: OH-ABC Wherein, structure A is a five-membered ring or a six-membered ring, preferably a benzene ring, a five-carbon ring, a six-carbon ring, a five-membered heterocycle, or a six-membered heterocycle, and structure A has 0-4 substituents, and the substituents are OH, CH3OH, or NH2; Structure B is a 3-5 carbon chain containing 0-3 double bonds or a heterochain containing 0-2 O or N atoms. Structure B has 0-3 substituents, which are OH, NH2, =O, or CH3OH. The C structure is a five-membered ring or a six-membered ring, preferably a benzene ring, a five-carbon ring, a six-carbon ring, a five-membered heterocycle, or a six-membered heterocycle. The C structure has 0-4 substituents, and at least one substituent contains 1 or more O and / or N atoms; Preferably, the structure B and the structure C are further cyclized into a cyclic structure via a substituent or a heteroatom.

6. The composition according to claim 5, characterized in that The carrier is a flavonoid or phenolic glycoside compound; Preferably, the carrier is selected from any one or a combination of two or more of dihydrocinnamidine, calendulaside, deacetylcalendulaside, salidroside, naringenin, hesperetin and quercetin.

7. The composition according to claim 5, characterized in that The composition further comprises a polymer, Preferably, the polymer is selected from any one or a combination of two or more of cellulose polymers, synthetic polymers, polysaccharides and their derivatives, proteins and polypeptides.

8. An enteric-coated preparation, characterized in that The enteric-coated preparation comprises the complex according to claim 3 or 4 or the composition according to any one of claims 5 to 8; preferably, the dosage form of the enteric-coated preparation is tablets, capsules, granules, dry suspensions, pills, pellets or coatings.

9. A method for preparing a cordycepin-stevioside complex, characterized in that: Mixing and dissolving cordycepin, stevioside and an organic solvent, and then drying to obtain the cordycepin-stevioside complex; The weight ratio of the cordycepin to the stevioside is 1:0.5-5; The organic solvent is selected from any one of methanol, ethanol, acetone, tetrahydrofuran, and dichloromethane, or a combination of two or more thereof.

10. Use of the cordycepin-stevioside complex according to claim 3 or 4, the composition according to any one of claims 5 to 7, or the preparation method according to claim 9 in the preparation of medicines, cosmetics, feeds, or health foods.

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