Artificial venenum bufonis composition and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an artificial toad venom composition and application thereof, and the core component of the composition comprises one or more of toad cardiotonic steroids, toad tryptamine or toad polypeptides. The invention designs a key raw material, a formula and a preparation method of artificial venenum bufonis, and particularly develops a method for effectively obtaining toad cardiotonic steroid components from plants. Pharmacodynamic evaluation proves that the artificial venenum bufonis composition prepared by the method has the effect equivalent to or close to that of natural venenum bufonis, and has the value for preparing natural venenum bufonis substitutes. According to the invention, sustainable resource guarantee is provided for Chinese patent medicines containing venenum bufonis, and support is provided for wild toad resources and ecological protection in China. The invention also comprises the design and development of a new medicine taking the artificial venenum bufonis composition or the raw materials thereof as components.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to an artificial toad venom composition and its uses. Specifically, the present invention relates to the key raw materials of artificial toad venom or its substitutes, their preparation methods, the formula of toad venom substitutes, and the uses of toad venom substitutes and their key raw materials in the development of pharmaceutical industrial raw materials and pharmaceutical products. Background Art

[0002] Toad venom, a traditional precious Chinese medicine in China, is the processed and dried secretion of the parotid gland and skin gland of animals of the genus Bufo, such as Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider. It has the effects of clearing heat and detoxifying, reducing swelling and alleviating pain, and inducing resuscitation and opening the orifices, and is a key raw material for many large varieties of traditional Chinese medicines. However, the supply of natural toad venom resources has been decreasing year by year.

[0003] In order to alleviate the resource tension, Bufo bufo gargarizans has been artificially farmed, but various problems have prevented the breakthrough of the key farming technologies. Generally speaking, the farming cost is high, the efficiency is low, and the cycle is long. At present, the research and development of artificial toad venom substitutes is very necessary.

[0004] The inventors of the present invention have been engaged in toad venom research for a long time. Under the sponsorship of the National Natural Science Foundation of China, namely "Research on the material basis and mechanism of the compatibility and toxicity reduction of bezoar and toad venom", No.: 3901894, in 2010, "The impact of pollutants in contact with toad skin on the quality of toad venom and its molecular mechanism, 81102762, in 2012", "Research on the macromolecular active substance basis of toad venom based on the biological affinity between polypeptides and tumor cells, 81673563, in 2017", "Research on the structure-activity-toxicity relationship and molecular mechanism of toad venom peptides in anti-tumor based on polypeptide group and biological affinity, 82073975, in 2021", "Research on the molecular mechanism of toad venom in inhibiting the activity of skin inflammatory fibroblasts to improve atopic dermatitis, 82204715, in 2022", "Research on the antidepressant mechanism of toad venom in upregulating PCBP1 to inhibit ferroptosis of hippocampal neurons, 82304743, in 2023", they have deeply explored the active substance basis and safety of toad venom.

[0005] On the basis of these works, the present invention further designs a toad venom substitute with "similar chemical characteristics, similar efficacy, and higher safety", and provides an artificial toad venom composition and its uses. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art, and provide an equivalent substitute for toad venom that is easy to produce and prepare industrially, has stable quality and low price, and discloses its main raw material components, formula, and uses in the preparation of drugs.

[0007] The solution adopted by the present invention to solve its technical problems is as follows:

[0008] In a first aspect, the present invention provides an artificial bufotalin composition, and the core components of the composition include one or more of the following: bufadienolides, bufotenines or bufotoxins.

[0009] Preferably, the bufadienolides, the bufotenines and the bufotoxins are natural products, natural product analogs, natural product modifiers, or the bufotenines and the bufotoxins are prepared by chemical synthesis or synthetic biology techniques.

[0010] As an alternative, in the above artificial bufotalin composition, the core components of the composition consist of bufadienolides, bufotenines and bufotoxins, and the masses of the bufadienolides, the bufotenines and the bufotoxins respectively account for 0.01 - 70%, 0.01 - 80%, 0.01 - 80% of the total mass of the artificial bufotalin composition.

[0011] Preferably, the masses of the bufadienolides, the bufotenines and the bufotoxins respectively account for 10 - 30%, 10 - 50%, 10 - 50% of the total mass of the artificial bufotalin composition.

[0012] More preferably, the masses of the bufadienolides, the bufotenines and the bufotoxins respectively account for 10 - 20%, 20 - 40%, 20 - 40% of the total mass of the artificial bufotalin composition.

[0013] As an alternative, in the above artificial bufotalin composition, the composition further includes auxiliary components, and the auxiliary components include one or more of the following: proteins, amino acids, cholesterol, nucleic acids or organic acids. Conventional pharmaceutically acceptable excipients having functions such as filling, binding, lubricating, dispersing, sustained release, coating, encapsulation, disintegration, promoting penetration, emulsifying, solubilizing, etc.

[0014] Preferably, the auxiliary components consist of cholesterol - like substances, protein - like substances, organic acid - like substances, amino acid - like substances, nucleoside - like substances, and their masses respectively account for 0.01 - 20%, 0.01 - 30%, 0.01 - 20%, 0.01 - 20%, 0.01 - 20% of the total mass of the artificial bufotalin composition.

[0015] As an alternative, in the above-mentioned artificial bufotalin composition, the bufadienolides are cardenolides and their aglycones, bufadienolides and their aglycones, bufadienolide analogues or their chemical modifications. The bufadienolides are of animal origin or plant origin. The animal origin includes amphibians of the families Bufonidae and Ranidae. The plant origin includes: the original plants containing hellebrigenin glycosides and scillarenin glycosides, Convallaria majalis, Allamanda schottii, Asclepias curassavica, Adonis amurensis, various Rohdea plants, and plants of the genus Helleborus in the family Ranunculaceae, natural plants of the genus Urginea in the family Liliaceae, bulbous succulent plants of the genus Bowiea in the family Liliaceae, the genus Urginea in the family Hyacinthaceae, the family Crassulaceae, the family Scrophulariaceae (genus Digitalis), the family Apocynaceae (genus Thevetia, genus Strophanthus), the family Asclepiadaceae (genus Periploca, genus Asclepias), the family Liliaceae (genus Convallaria, genus Rohdea), the family Brassicaceae (genus Cardamine), the family Ranunculaceae (genus Adonis), the South African plant Drimia robusta, Urginea Steinh.

[0016] As an alternative, in the above-mentioned artificial bufotalin composition, the bufadienolides are bufadienolide sterenes, and the bufadienolide sterenes include free bufadienolide sterene components or conjugated bufadienolide sterene components.

[0017] Preferably, the bufadienolides include free bufadienolide components selected from one or more of the following: Resibufogenin, bufalin, resibufagin, Resibufaginol, desacetylcinobufagin, Gamabufotalin, Desacetylbufotalin, 1β-hydroxylbufalin, Telocinobufagin, bufotalinin, arenobufagin, hellebrigenin, dehydrated cinobufagin, cinobufagin, bufotalin, 19-oxo-cinobufagin, 3β,14β,16β-trihydroxy-5α-bufadien-20,22-diene, 3β,14β-dihydroxy-5α-bufadien-20,22-diene, cinobufaginol, cinobufotalin, 19-oxo-cinobufagin, 19-oxo-cinobufotalin, Desacetylcinobufagin, 14β-hydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufadien-20,22-diene, 3β,14β,16β-trihydroxy-5α-bufadien-20,22-diene or 3β,14β-dihydroxy-5α-bufadien-20,22-diene.

[0018] In addition, preferably, the combined bufadienolide components are selected from one or more of the following: 3-succinate-arginine ester resibufogenin, 3-succinate-arginine ester bufalin, 3-glutarate-arginine ester resibufogenin, (resibufaginol-3-succinate-arginine ester), 3-adipate-arginine ester resibufogenin stereoisomer, 3-adipate-arginine ester bufalin stereoisomer, 3-pimelate-arginine ester resibufogenin, 3-pimelate-arginine ester bufalin, 3-suberate-arginine ester resibufogenin, 3-succinate-arginine ester cinobufagin, 3-suberate-arginine ester desacetylcinobufagin, 3-succinate-arginine ester cinobufaginol, 3-suberate-arginine ester telocinobufagin, 3-suberate-arginine ester gamabufotalin, 3-adipate-arginine ester cinobufagin, 3-suberate-arginine ester hellebrigeninester), arenobufagin-3-suberate-arginine ester, cinobufagin-3-pimelate-arginine ester, cinobufagin-3-suberate-arginine ester, cinobufaginol-3-pimelate-arginine ester, cinobufaginol-3-suberate-arginine ester, 19-oxo-cinobufotalin-3-suberate-arginine ester, deglucohellebrin, 5β,14β,16β-trihydroxy-19-formyl-3β-[(α-L-rhamnopyranosyl)oxy]bufa-20,22-diene, 14β,16β-dihydroxy-3β-[β-D-glucopyranosyl-(1→4)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-diene, 14β,16β-dihydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-diene, Hellebosapogenin, Hellebrin, 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-diene.

[0019] As an alternative, in the above artificial bufotalin composition, the bufadienolides are bufadienolides of plant origin.

[0020] Preferably, the bufadienolides are derived from plants of the genus Helleborus in the family Ranunculaceae, including: Helleborus thibetanus or its variants, including: Helleborus niger, Helleborus caucasicus, Helleborus orientalis, Helleborus viridis, Helleborus bocconei, Helleborus purpurascens, H. Odbrus, H. abchasicus, H. macranthus, H. torquatus, H. multifidus, H. cyclophyllus, H. odorus, H. croaticus, H. istriacus, H. dumetorum, Helleborus foetidus, Helleborus argutifolius, Helleborus atrorubens, Helleborus corsicus, Helleborus lividus,

[0021] Preferably, the plant-derived bufadienolides can be obtained through solvent extraction, chromatographic enrichment, microbial or enzymatic transformation processes.

[0022] Specifically, the preparation method includes the following steps: The roots and rhizomes of the original plant medicinal materials containing bufadienolide cardiac glycosides are ground into coarse powder, soaked in 10 times the amount of ethanol, water or other solvents, extracted by conventional extraction methods, the extraction solutions are combined, filtered, concentrated and dried, and separated by macroporous resin to obtain the elution part with 40%-100% ethanol.

[0023] More preferably, the bufadienolides are prepared by microbial fermentation or enzymatic transformation for the deglycosylation treatment of cardiac glycosides. Industrially, these microorganisms or enzymes can be modified by known techniques of gene sequence mutation, deletion or editing to obtain stronger substrate binding, anti-denaturation and aglycone conversion abilities.

[0024] Specifically, the preparation method includes the following steps:

[0025] The total extract of bufadienolide cardiac glycosides was loaded onto a macroporous adsorption resin column and successively eluted with pure water and ethanol solutions with volume fractions of 10%, 20%, 40%, 60%, 80%, and 100% in a gradient manner. The eluted components at different positions were collected and recovered under reduced pressure. Detection was carried out according to the following color development method to exclude the alkaloid and flavonoid parts. The eluted parts with 40%-100% ethanol were combined to obtain the total glycosides, and the content of steroidal cardiac glycosides was determined by the vanillin-perchloric acid method. A biocatalytic enzyme was added for aglycone conversion. The mass ratio of the enzyme to the total cardiac glycosides was (0.05:1 to 5:1), the pH of the buffer solution was 4.5, and when the enzymatic hydrolysis temperature was 35°C, the enzymatic hydrolysis time was 6 hours. The enzymatic hydrolysis solution was spray-dried to obtain a dry powder, and 0.5-3 times the organic solvent (such as ethyl acetate, dichloromethane, petroleum ether, etc.) was added for reflux extraction to obtain the bufadienolide part of plant origin. The biocatalytic enzymes include: β-glucosidase, glycosidase C, snailase, pectinase, cellulase, mannanase, cellobiase, protease, galactase, etc.

[0026] Immobilized enzymatic hydrolysis-extraction method: The target plant was extracted with an organic solvent or water, and the extract was concentrated and dried to obtain a total extract. The biocatalyst was immobilized by an ion adsorption method, which is an immobilization method in which the enzyme binds to a mixed resin containing cation and anion exchange groups by electrostatic interaction. This method has mild treatment conditions, does not undergo chemical coupling agent treatment and modification, and maintains a high enzymatic catalytic activity. The mass ratio of the enzyme to the total cardiac glycosides was (0.01:1 to 5:1), the pH of the buffer solution was 4.5, and when the enzymatic hydrolysis temperature was normal temperature or 37°C, it was shaken on a shaker overnight. The advantage of this method is that after the reaction, the immobilized enzyme naturally settles with the ion exchange resin and can be reused after filtration and washing, reducing the enzyme dosage. The enzymatic hydrolysis solution was spray-dried to obtain a dry powder, and 0.5-3 times the organic solvent was added for ultrasonic extraction to obtain the bufadienolide part of plant origin.

[0027] Microbial fermentation enzymatic hydrolysis-extraction method: The microbial fermentation method was used to treat cardiac glycoside plants or plant extracts containing natural bufadienolide lactones. The microorganisms include Trichoderma harzianum, Trichoderma, Aspergillus tubingensis, Aspergillus oryzae, Aspergillus niger, Monascus purpureus, Aspergillus awamori, Fusarium fungi, Curvularia fungi for fermentation, and also include probiotic fermentation, such as Lactobacillus, Enterococcus, Bifidobacterium, Bacillus, yeast, etc. for fermentation. Through the production of carbohydrate-active enzymes, etc., the conversion of bufadienolide cardiac glycosides to aglycones was achieved.

[0028] In a preferred embodiment, a mixed strain of Aspergillus niger, Aspergillus oryzae, and Lactobacillus was used for fermentation, and the solid-state fermentation method was adopted. For example, the original plant containing bufadienolide cardiac glycosides was crushed, moistened with 2 times the amount of water, the fermentation bacterial liquid was added, and fermentation was carried out at 28°C for 20 days, dried, and the aglycone was obtained by ethyl acetate extraction and the total bufadienolide part was obtained by enrichment and purification with macroporous resin.

[0029] More preferably, the bufadienolides of plant origin are selected from one or more of the following: 3β,14β-dihydroxy-5α-bufa-20,22-diene, deglucohellebrin, 3β,14β,16β-trihydroxy-5α-bufa-20,22-diene, bufotalidin or hellebrigenin, desacetylcinobufagin, 19-oxo-cinobufagin, 3β,5β,14β,16β-tetrahydroxy-19-aldehyde-bufa-20,22-diene (16-OH-hellebrigenin), 3β,14β,16β-trihydroxy-5β-bufa-20,22-diene (16-OH-bufalin).

[0030] As an alternative, in the above artificial bufotalin composition, the bufotenines are selected from one or more of the following:

[0031] In the above artificial bufotalin composition, the bufotenines are selected from one or more of the following: 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), 5-MeO-NMT, 5-methoxytryptamine, bufobutanoic Acid, bufobutarginine, bufoserotonin A, bufoserotonin B, bufoserotonin C, bufotenidine, N,N-dimethylserotonin (bufotenine), bufotenine Oxide, bufotenine-O-Sulphate, bufoviridine, N-methyl-5-hydroxytryptamine (n-Methylserotonin), 5-hydroxytryptamine (serotonin), tryptamine, dehydrobufotenine, indole-3-acetaldehyde, 5-hydroxyindole-3-aldehyde, 5-hydroxytryptophol, bufobutarginne or bufopyramide.

[0032] As an alternative, in the above artificial bufotalin composition, the bufopeptides are selected from one or more of the following:

[0033] Preferably, the toad polypeptides are selected from one or more of the following: Phe-Asp-Phe-Pro-Gly-Asn-Lys-Ile-Thr, Phe-Pro-Ala-Ser-Ala-Ala-Gly-Lys-G1y-Leu-Gln, Ser-Glu-Trp-Glu-Glu-Ash-Pro-Met(+15.99)-Asn-Lys-Tyr-Val-Ser, Asn-Phe-Thr-Gly-Asp-Ser-Ile-Pro-Cys(+57.02)-Arg, Ser(+42.01)-Asp-Glu-Ile-Ile-His-Asn-Pro-Ala-Val-Pro-Phe, Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys-Glu-Leu, Tyr-Glu-Arg-Pro-Leu-Val-Thr, Leu-Thr-Val-Gly-Pro-Arg-Gly-Pro-Leu-Leu-Val-Gln-Asp, Val-Asp-Leu-Asn-Pro-Ser-Asn-Val-Gly-Trp-Asn-Lys-Thr-Thr-Phe-Ala, Lys-Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys-Glu-Leu-Tyr, Gln-A1a-Glu-Phe-Asp-Lys-Ala-A1a-Glu-Asp-Val-Lys-Lys-Leu-Lys-Thr, Met-Leu-Ala-Asn-Glu-Asp-Tyr-Ala-Ser-Leu-Thr-Lys-Gly-I1e-Gln-Asn-Leu-Asn, Ala-Thr-Pro-Val-Asp-Trp-Lys-Pro-Gly-Asp-Arg-Val-Met-Val, Phe-Phe-Asp-Phe-Pro-Gly-Asn-Lys-Ile-Thr-Ser-Val-Ala-Gly-Val-Tyr-Phe, Ser-Asp-Ile-Ser-Ala-Gly-Lys-Ile-Lys-Gln, Gln-Gly-Leu-Ala-Ala-Gly-Met(+15.99)-Pro-Gly-Tyr-Pro-Val-Leu(-.98) Phe-Asp-Lys-Ala-Ala-Glu-Asp-Val-Lys-Lys-Leu-Lys-Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys, Trp-Gly-Trp-Pro-Ile-Asp-Asp-Ala-Thr-Thr-Glu-Lys-Leu-Ser, Pro-Arg-Gly-Pro-Leu-Leu-Val-Gln-Asp.

[0034] In a second aspect, the present invention provides the use of the artificial bufotalin composition described in the first aspect above in the preparation of raw materials for the pharmaceutical industry, which are used for the equivalent substitution of natural bufotalin.

[0035] In a third aspect, the present invention provides the use of the artificial bufotalin composition described in the first aspect above in the preparation of drugs for treating diseases of the throat, heart, skin, tumor and nerve.

[0036] Preferably, other active ingredients may also be included in the drug to form a compound drug for treating diseases such as heart, tumor and throat in combination with the artificial bufotalin composition of the present invention.

[0037] The present invention has the following beneficial effects compared with the prior art:

[0038] The present invention designs a key raw material, formula and preparation method of artificial bufotalin. In particular, the present invention develops a method for effectively obtaining bufadienolide components from plants for industrial production. Pharmacodynamic evaluation confirms that the artificial bufotalin composition prepared by the method of the present invention has an equivalent or nearly equivalent effect to natural bufotalin and has the value for preparing substitutes for natural bufotalin. The present invention provides a sustainable resource guarantee for Chinese patent medicines containing bufotalin and supports the protection of Chinese wild toad resources and the ecological environment. The present invention also includes the design and development of new drugs with the artificial bufotalin composition or its raw materials as components. Description of the Drawings

[0039] Figure 1 : The TP1-TP19 polypeptide sequences used in the example section.

[0040] Figure 2 : Effects of bufotalin (CS) and its potential substitute (NJ) on formalin-induced pain in mice (n = 8-11, means ± SEM).

[0041] Figure 3 : Effects of bufotalin (CS) and its potential substitute (NJ) on the swelling degree of carrageenan-induced right hind paw in mice (n = 8-11, means ± SD).

[0042] Figure 4 : Effects of bufotalin and its potential substitutes on electrocardiogram changes in myocardial ischemia (100 ms / division). A is the control group; B is the ISO-induced model group; C is the nitroglycerin group; D is the high-dose bufotalin group; E is the low-dose bufotalin group; F is the high-dose substitute NJ group; G is the low-dose substitute NJ group.

[0043] Figure 5 : Comparison of HE staining results of myocardial tissues of rats in each group (n = 6, ×100). Among them, A is the control group; B is the ISO-induced model group; C is the nitroglycerin group; D is the low-dose bufotalin group; E is the high-dose bufotalin group; F is the low-dose substitute group; G is the high-dose substitute group.

[0044] Figure 6 : Effects of bufotalin (CS) and its potential substitute (NJ) on lipopolysaccharide-induced depressive behavior in mice. Among them, compared with the control group, ## P < 0.01, compared with the model group, ** P < 0.01. n = 9.

[0045] Figure 7 : Effects of bufotalin (CS) and its potential substitute (NJ) on mRNA expression of inflammatory factors in hippocampal tissues of lipopolysaccharide-induced mice. Among them, compared with the control group, ## P < 0.01, compared with the model group, ** P < 0.01. n = 5. Detailed implementation manners

[0046] The present invention will be further described below in conjunction with specific embodiments, but these embodiments should not be construed as limiting the present invention. Embodiment 1: Preparation of plant-derived bufotalin substitute raw materials

[0047] 1.1 Method

[0048] 1.1.1 Enrichment of plant-derived bufotalin substances by macroporous resin

[0049] The roots and rhizomes of Helleborus thibetanus Franch. of the Ranunculaceae family are ground into coarse powder (sieved), and extracted by heating under reflux with 10 times the amount of ethanol or water. The extraction solutions are combined and filtered. Concentrated and dried to obtain the total extract. The total extract is loaded onto a macroporous adsorption resin column, and gradient elution is carried out successively with pure water, ethanol solutions with volume fractions of 10%, 20%, 40%, 60%, 80%, and 100%. The elution components of different parts are collected and recovered under reduced pressure. Detection is carried out according to the following color development method, excluding the alkaloid and flavonoid parts. The elution parts with 40%-100% ethanol are combined to obtain the total glycosides, and the content of steroidal cardiac glycosides is determined by the vanillin-perchloric acid method.

[0050] 1.1.2 Enzymatic hydrolysis-extraction method

[0051] Glycosidase C, snail enzyme, and pectinase were added for aglycone conversion, the enzyme / total cardiac glycoside mass ratio was (0.05:1 to 5:1), the buffer pH was 4.5, the enzyme hydrolysis temperature was 35°C, and the enzymatic hydrolysis time was 6 hours. The enzymatic hydrolyzate was spray-dried to obtain dry powder, and 0.5-3 times organic solvent (ethyl acetate, dichloromethane, petroleum ether, etc.) was added for reflux extraction to obtain the plant-derived bufadienolide part.

[0052] 1.1.3 Immobilized enzymatic hydrolysis-extraction method

[0053] Break into the coarsest powder (pass through No. 1 sieve), and extract with ethanol or water by heating and refluxing for 3 times. Combine the extracts and filter. Concentrate and dry under reduced pressure to obtain the total extract, and determine the content of steroidal cardiac glycosides by the vanillin-perchloric acid method. The cellulase is immobilized by ion adsorption method, and the enzyme is immobilized by electrostatic force with a mixed resin containing anion and cation exchange groups. The treatment conditions of this method are mild, and it is not modified by chemical coupling agents, and the enzyme catalytic activity is maintained at a high level. When the mass ratio of enzyme / total cardiac glycosides is (2:1 to 5:1), the pH of the buffer is 4.5, and the enzyme hydrolysis temperature is 55°C, the shaker is shaken, and the enzymolysis is carried out overnight. The advantage of this method is that the immobilized enzyme will naturally settle with the ion exchange resin after the reaction is completed, and it can be reused after filtering and washing, reducing the amount of enzyme. The enzymatic hydrolyzate is spray-dried to obtain dry powder, and 0.5-3 times ethyl acetate is added for ultrasonic extraction to obtain the plant-derived bufadienolide part. β-glucosidase, cellulase, mannosidase, enzyme / total cardiac glycosides mass ratio is 2:1, shake on a shaker, enzymolysis at 45℃ overnight.

[0054] 1.1.4 Microbial fermentation enzymatic hydrolysis-extraction method

[0055] Use mixed strains of Aspergillus niger, Aspergillus oryzae and Lactobacillus for fermentation. Solid-state fermentation method. Crush the Chinese chopsticks medicinal materials, add 2 times the amount of water to moisten, add fermentation liquid, and ferment for 20 days at 28 degrees. Dry. Extract with ethyl acetate to obtain aglycones, and enrich and purify with macroporous resin to obtain the total sterene part of toad.

[0056] 1.1.5 Determination of total sterol content in toads

[0057] The steroidal saponin components can be identified by colorimetric method, and the total steroid content can be determined by colorimetric method.

[0058] 1.2 Results

[0059] The present invention innovatively provides a method for processing and preparing bufotalins or bufosterenes using Helleborus thibetanus Franch. of the genus Helleborus in Ranunculaceae as the initial raw material. Total glycosides are obtained through organic solvent extraction and macroporous resin enrichment processes, and then bufosterenes are prepared through enzymatic hydrolysis. The structural characteristics of bufotalins or bufosterenes: Hydroxyl groups are mostly located at 3β, 5β, 14β, and 16β; the fusion mode of the A / B ring is mainly cis, the B / C ring is trans-fused, and the C / D ring is cis-fused; the 18th position is a methyl group, the 19th position can be a methyl group, an aldehyde group, or substituted by a hydroxyl group with one less carbon atom: the six-membered unsaturated lactone ring is connected to the 17β position; if there is no hydroxyl group at the 5β position, it can be 5α hydrogen; the 3β hydroxyl group can undergo a dehydration reaction with adjacent hydrogen; the 3β hydroxyl group can be dehydrogenated to form an oxo group.

[0060] The characteristics of bufosterenes were identified by high performance liquid chromatography-mass spectrometry, and eight bufosterene components were selected as characteristic peaks (ATV1 - ATV8), and their molecular formulas, molecular weights, secondary mass spectrometry fragmentation peaks, and identification names are shown in Table 1 below. Their relative content characteristics are shown in Table 1 below. However, due to differences in different regions, harvesting seasons, and storage methods, there will inevitably be a certain range (1 - 10 times) of fluctuations in the ratio of bufosterenes obtained from raw materials of different sources processed according to the processing methods given in this example; if targeted enrichment is carried out, any component in Table 1 can be easily obtained. At the same time, due to differences in secondary metabolic enzymes in Helleborus thibetanus Franch. of different sources, there may also be slight changes in the substituent positions in bufosterenes in some batches of preparations.

[0061] Regardless of which plant containing bufotalins, in the present invention, biotransformation of deglycosylation is required, and this step is the key and characteristic process for preparing artificial bufotalin raw materials. In this example, the activity differences of different biotransformation methods for preparing bufosterene raw materials from Helleborus thibetanus Franch. were compared. The results show that the biotransformation efficiency of the direct microbial fermentation method is lower than that of the direct enzymatic hydrolysis method, and the pharmacological activity of the prepared product is also lower than that of the bufosterene prepared by the enzymatic hydrolysis method. For the enzyme-catalyzed process, the immobilized enzyme technology can reuse the catalytic enzyme and is more economical than the non-immobilized enzymatic hydrolysis method. However, in terms of biotransformation efficiency and IC50 strength, the enzymatic hydrolysis-extraction method is the preferred preparation process among the three methods. The results are shown in Table 2.

[0062] Table 1: Component and ratio characteristics of plant-derived bufosterenes in artificial bufotalin

[0063]

[0064] Table 2: Comparison of the anti-tumor cell activities of plant-derived bufosterenes obtained under different preparation processes (mean ± SD, n = 4)

[0065]

[0066] Example 2: Evaluation of the anti-inflammatory pain activity of the artificial bufotalin substitute composition

[0067] 2.1 Formula

[0068] The formula of the artificial bufotalin substitute composition used in this example is as follows. By mass percentage, the total toad sterenes from Helleborus thibetanus Franch are 20% (the ratio of ATV1 - ATV7 is 4:1:1:1.5:0.5:0.5:0.5), the total toad tryptamines account for 15% (including bufotenine, bufotenidine, bufothionine, and the ratio is 1:4:1), the total toad peptides account for 20% (including equal proportions of TP1, TP2, TP3, TP4, and the specific sequences are shown in Figure 1 ), and other excipients are 35% (cholesterol, starch, albumin).

[0069] 2.2 Experimental method

[0070] Select prequalified ICR mice, weigh them, and randomly divide them into a blank group, a model group, a low-dose bufotalin group (CSL group), a high-dose bufotalin group (CSH group), a low-dose potential substitute group (NJL group), and a high-dose potential substitute group (NJH group) according to body weight. CSL group: gavaged with bufotalin at 30 mg / kg; CSH group: gavaged with bufotalin at 90 mg / kg; NJL group: gavaged with the potential substitute at 30 mg / kg; NJH group: gavaged with the potential substitute at 90 mg / kg; the blank group and the model group were gavaged with an equal amount of normal saline.

[0071] Set the temperature of the hot plate instrument to 55 ± 0.5 °C and stabilize for one hour. After the temperature is stable, measure the pain threshold of the mice. Place the mice in the hot plate instrument, and take the time when the mice first lick their feet as the pain threshold, and record the initial pain threshold. Before drug intervention, adjust the temperature of the hot plate instrument to 55 °C and stabilize for 1 hour. 2.5 hours after administration (2 hours after modeling), place the mice in the hot plate instrument and observe the first licking response time, which is the pain threshold in hot-plate test (HPPT). If the mice show no response within 60 s, take them out immediately and calculate according to 60 s.

[0072] 2.3 Experimental results

[0073] Bufotalin and its potential substitutes can significantly increase the hot plate pain threshold of mice. After carrageenan was given, the hot pain threshold of mice decreased significantly from (27.33 ± 2.43) s before administration to (11.65 ± 3.29) s( ##P < 0.001), indicating that subcutaneous injection of carrageenan in the foot caused pain. The thermal pain threshold before administration in the CSL group was (24.64 ± 2.75) s, and after administration it was (23.25 ± 6.83) s (**P < 0.001). The thermal pain threshold before administration in the CSH group was (25.54 ± 3.07) s, and after administration it was (28.27 ± 8.55) s (**P < 0.001), and the pain threshold change rate was (-0.12 ± 0.34); the thermal pain threshold before administration in the NJL group was (25.04 ± 2.58) s, and after administration it was (24.90 ± 8.55) s (P < 0.001); the thermal pain threshold before administration in the NJH group was (26.93 ± 2.50) s, and after administration it was (29.78 ± 7.93) s (P < 0.001). There was no significant difference in increasing the thermal pain threshold of mice and inhibiting inflammatory foot swelling in mice between equal doses of toad venom and its potential substitutes (P > 0.05), indicating that the two have similar efficacy in this model. The specific results are shown in Table 3.

[0074] Table 3: Effects of toad venom and its potential substitutes on the thermal pain threshold of the right hind foot of mice (n = 8, means ± SD)

[0075]

[0076] Example 3: Evaluation of the analgesic activity of an artificial toad venom substitute composition

[0077] 3.1 Formulation

[0078] The formulation of the artificial toad venom substitute composition used in this example is as follows. By mass percentage, total toad sterols from plant sources are 20% (where the ratio of ATV1 - ATV7 is 4:1:1:1.5:0.5:0.5:0.5), total toad tryptamines account for 15% (including bufotenine, bufotenidine, bufothionine, with a ratio of 1:4:1), total toad peptides account for 20% (including equal proportions of TP5, TP6, TP7, and the specific sequences are shown in Figure 1 ).), and other excipients are 35% (arginine, cholesterol, starch).

[0079] 3.2 Experimental method

[0080] ICR mice were randomly divided into a model group, a morphine hydrochloride group, a low-dose bufotoxin group (CSL group), a high-dose bufotoxin group (CSH group), a low-dose potential substitute group (NJL group), and a high-dose potential substitute group (NJH group). Morphine hydrochloride was intraperitoneally injected at 2 mg / kg; CSL group: Bufotoxin was intragastrically administered at 30 mg / kg; CSH group: Bufotoxin was intragastrically administered at 90 mg / kg; NJL group: The potential substitute was intragastrically administered at 30 mg / kg; NJH group: The potential substitute was intragastrically administered at 90 mg / kg; The model group was given normal saline by gavage. 30 minutes after administration, 20 μL of 2.5% formalin was subcutaneously injected once into the right hind paw of the mice.

[0081] 3.3 Experimental results

[0082] Formalin-induced pain was obvious and rapid. The model mice quickly showed the behavior of lifting the foot and licking the foot. The time of lifting the foot and licking the foot reached (183.33 ± 30.57) s within 5 minutes after modeling. However, compared with the model group, after administration of the positive drug morphine hydrochloride, high and low doses of bufotoxin, and high and low doses of potential substitutes, the time of lifting the foot and licking the foot was significantly shortened within 0-5 minutes after modeling. The low-dose bufotoxin group was (147.90 ± 32.44) s (P < 0.05), and the high-dose bufotoxin group was (110.89 ± 30.16) s (P < 0.001). The low-dose potential substitute group was (140.45 ± 21.18) s (P < 0.01), and the high-dose potential substitute group was (128.75 ± 23.03) s (P < 0.001); within 40 minutes after modeling, the duration of lifting the foot / licking the foot of the mice in the administration group was significantly shortened, showing an analgesic effect. The potential substitute group was basically nearly as effective as the bufotoxin group in shortening the time of lifting the foot and licking the foot of the mice. The specific results are as Figure 2 shown.

[0083] Example 4: Anti-inflammatory equivalence activity of artificial bufotoxin substitute composition

[0084] 4.1 Formula

[0085] The formula of the artificial bufotoxin substitute composition used in this example is as follows. By mass percentage, total toad sterols from plants are 30% (the ratio of ATV1-ATV7 is 3:0.5:3:2:0.5:0.5:0.5), total toad tryptamines account for 15% (including bufotenine, bufotenidine, N-methyl serotonin, with a ratio of 0.5:2:1), total toad peptides account for 20% (including equal proportions of TP8, TP9, TP10, and the specific sequences are shown in Figure 1 ).), and conventional pharmaceutical excipients are 35% (arginine, cholesterol, starch, albumin).

[0086] 4.2 Experimental method

[0087] Preselected qualified ICR mice were weighed and randomly divided into a blank group, a model group, a low-dose bufotoxin group (CSL group), a high-dose bufotoxin group (CSH group), a low-dose potential substitute group (NJL group), and a high-dose potential substitute group (NJH group) according to body weight. CSL group: gavaged with 30 mg / kg bufotoxin; CSH group: gavaged with 90 mg / kg bufotoxin; NJL group: gavaged with 30 mg / kg potential substitute; NJH group: gavaged with 90 mg / kg potential substitute; the blank group and the model group were gavaged with an equal amount of normal saline.

[0088] 30 minutes after administration, except for the blank group, 30 μL of 1% carrageenan was subcutaneously injected into the right hind paw of each mouse in the other groups for a single dose. Cut and weigh the same positions on the right hind paw (experimental paw) and left hind paw (control paw) of the mouse. The swelling rate of the paw given carrageenan = (weight of the paw given carrageenan - weight of the paw not given carrageenan) / weight of the paw not given carrageenan × 100%. The inhibition rate of the drug on paw swelling = (degree of paw swelling given normal saline - degree of paw swelling given the drug) / degree of paw swelling given normal saline × 100%.

[0089] 4.3 Experimental results

[0090] After injecting carrageenan, the paws of the mice were significantly swollen, with a swelling degree of (41.14 ± 19.17) mg and a swelling rate of (25.63 ± 10.88)%, which was significantly more swollen than the normal paw (P < 0.001); the paw swelling degree of the mice in the drug administration groups was significantly inhibited. In the CSL group, the swelling degree was (13.80 ± 6.27) mg and the swelling rate was (8.70 ± 4.21)% (P < 0.01), and the swelling inhibition rate reached 66.06%; in the CSH group, the swelling degree was (10.82 ± 3.99) mg and the swelling rate was (6.52 ± 2.41)% (P < 0.01), and the swelling inhibition rate reached 74.58%; in the NJL group, the swelling degree was (17.83 ± 2.68) mg and the swelling rate was (11.07 ± 1.82)%, with a significantly better inhibitory effect than the model group (P < 0.01), and the swelling inhibition rate reached 56.83%; in the NJH group, the swelling degree was (13.71 ± 3.95) mg and the swelling rate was (8.62 ± 2.30)% (P < 0.001), and the swelling inhibition rate reached 66.38%. It shows that there is no significant difference in the effects of equal-dose bufotoxin and the substitute, and the two are equivalent in this model. The specific results are as Figure 3 shown.

[0091] Example 5: Equivalent activity of artificial bufotoxin substitute composition against myocardial ischemia

[0092] 5.1 Formula

[0093] The formula of the artificial bufotalin substitute composition used in this embodiment is as follows. By mass percentage, total toad sterene from plant sources is 40% (where the ratio of ATV1 - ATV7 is 8:0.5:0.5:0.7:0.2:0.2:0.2), total toad tryptamine accounts for 20% (including bufotenine, N-methyl serotonin, dehydrobufotenine, with a ratio of 0.5:1:1), total toad peptides account for 20% (including equal proportions of TP11, TP12, TP13, specific sequences can be seen in Figure 1 ), and excipients are 20% (cholesterol, starch, albumin, acrylic resin).

[0094] 5.2 Experimental methods

[0095] The experimental SD rats were divided into a normal saline group, an isoproterenol group, a nitroglycerin treatment group, a bufotalin treatment group (low and high doses), and a potential substitute treatment group (low and high doses), with 6 rats in each group. Needle electrodes were inserted subcutaneously into the four limbs and connected to a BL-420N biological signal acquisition and analysis system. After stabilizing for 5 min, the electrocardiogram before blank modeling was recorded to evaluate the degree of acute myocardial ischemia injury and the drug efficacy. Positive determination of acute myocardial ischemia on electrocardiogram: ST segment horizontal shift, upward or downward shift ≥ 0.1 mv; T wave tall and upright, exceeding 1 / 2 of the R wave in the same lead. ELISA method was used to measure the contents of lactate dehydrogenase (LDH), cardiac troponin I (cTnI), and creatine kinase isoenzyme MB (CK-MB) in heart tissues.

[0096] 5.3 Experimental results

[0097] Electrocardiogram monitoring showed that in the model group, the ST segment showed progressive upward arching elevation, with a shift value ≥ 0.1 mv. The T wave was upright, tall and pointed, exceeding 1 / 2 of the R wave in the same lead, indicating acute myocardial ischemic changes (P < 0.01). After intragastric administration of bufotalin and the substitute, it was able to reduce the amplitude of the ST wave and the T wave / R wave ratio, but the relationship with the dose was not obvious. There was no significant difference between the bufotalin group and the substitute group (P > 0.05), indicating that the two had similar drug effects on this index. The specific results are as shown in Figure 4 and Table 4.

[0098] Table 4; Effects of bufotalin and the substitute on the T wave / R wave ratio of electrocardiogram in myocardial ischemia (mean ± SD, n = 6)

[0099]

[0100] H-E staining showed that the myocardial cells in the normal group were arranged neatly. In the isoproterenol-induced model group, obvious myocardial tissue disorder and a large number of inflammatory cell infiltrations were observed, and fatty degeneration was also present. In the toad venom and substitute treatment groups, the inflammatory cell infiltration in myocardial cells was significantly reduced, and the edema decreased, showing a certain dose-dependence. There was no significant difference between the substitute group and the toad venom group. The specific results are as Figure 5 shown.

[0101] Creatine kinase isoenzyme MB and cardiac troponin cTnI are common biomarkers for myocardial injury and myocardial ischemia. In this experiment, after intraperitoneal injection of isoproterenol, the above-mentioned biomarkers in the model group were significantly increased (P < 0.01). After administration of toad venom and the substitute, the concentrations of the above-mentioned markers were significantly decreased (P < 0.01), indicating that both toad venom and the substitute can improve isoproterenol-induced myocardial injury, and their effects on anti-acute myocardial ischemia are similar, without significant difference. The specific results are shown in Table 5.

[0102] Table 5; Effects of toad venom and artificial toad venom substitute on the levels of cTnI and CK-MB in rat myocardium (mean±SD, n = 6)

[0103]

[0104] Note: The results were statistically analyzed. Compared with the blank group, # P < 0.05, ## P < 0.01; compared with the model group, * P < 0.05, ** P < 0.01. mean±SD, n = 6.

[0105] Example 6; Equivalence evaluation of artificial toad venom composition and natural toad venom against lipopolysaccharide-induced neuroinflammation in mice

[0106] 6.1 Formulation

[0107] The formulation of the artificial toad venom substitute composition used in this example is as follows. By mass percentage, total toad sterene from plant sources is 10% (the ratio of ATV1 - ATV7 is 2∶1∶1∶4∶0.5∶0.5∶0.5), total toad tryptamine accounts for 20% (including bufotenine, N-methyl serotonin, bufothionine, with a ratio of 0.2∶1∶1), total toad peptides account for 20% (including equal proportions of TP14, TP15, TP16, and the specific sequences are shown in Figure 1 ), and excipients account for 20% (cholesterol, starch, soy protein, cellulose).

[0108] 6.2 Experimental methods and grouping

[0109] Sixty-three SPF male ICR mice were randomly divided into a control group, a model group, a fluoxetine group, a low-dose bufalin group (CSL group), a high-dose bufalin group (CSH group), a low-dose potential substitute group (NJL group), and a high-dose potential substitute group (NJH group), with 9 mice in each group. Except for the blank group, each group was given LPS (0.83 mg / kg, intraperitoneal injection) once. Fluoxetine group: Fluoxetine was administered by gavage at a dose of 30 mg / kg; CSL group: Bufalin was administered by gavage at a dose of 30 mg / kg; CSH group: Bufalin was administered by gavage at a dose of 90 mg / kg; NJL group: The substitute was administered by gavage at a dose of 30 mg / kg; NJH group: The substitute was administered by gavage at a dose of 90 mg / kg. The control group and the model group were given an equal volume of normal saline by gavage. After the mouse behavior test, blood was collected from the orbital cavity for the detection of serum IL-1β and TNF-α levels. Subsequently, the mice were decapitated, and the left hippocampus of the brain was isolated for qPCR detection, while the right brain was used for lipidomics.

[0110] 6.3 Detection methods

[0111] 6.3.1 Behavioral indicators

[0112] Tail suspension test: The inside of the tail suspension box is black, with a length, width, and height of 25 cm × 25 cm × 25 cm. A clip is connected to the center of the top with a rope and fixed at 2 cm from the end of the mouse's tail, making the mouse hang upside down. The head is about 3.5 cm from the bottom of the tail suspension box. Observe for 6 min and count the cumulative immobility time in the last 4 min. Forced swimming: The mouse is placed in a circular glass container with a height of 20 cm and a diameter of 12 cm, the water temperature is 25 °C, and the water depth is 10 cm. Observe each mouse for 6 min and count the cumulative immobility time in the last 4 min.

[0113] 6.3.2 Detection of inflammatory factor levels in serum by ELISA

[0114] The blood coagulates naturally at 4 °C for 2 h, and then centrifuged at 4 °C and 4000 r·min -1 for 30 min. The supernatant is collected, and the levels of IL-1β and TNF-α in the serum are detected by ELISA according to the kit instructions.

[0115] 6.3.3 Detection of the expression of inflammatory factors and BDNF mRNA in mouse hippocampal tissue by qPCR

[0116] Total RNA was extracted from mouse hippocampal tissue using the Trizol method, and the RNA concentration was measured using a Nano-Drop ultra-micro ultraviolet spectrophotometer. Samples were prepared and amplified using the Taq Pro Universal SYBR qPCR Master Mix premix. The amplification conditions were pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, annealing / extension at 60 °C for 30 s, for a total of 40 cycles. GAPDH was used as an internal reference, 2 -ΔΔCtMethod for calculating the relative expression level of mRNA

[0117] 6.3.4 High-sensitivity lipidomics analysis of inflammation-related mediators

[0118] The right brain of the mouse was extracted with ethyl acetate and n-hexane (v / v = 1:1, cooled to -20 °C), and 25 μL of AA-d8 solution (1 μg / mL). It was sonicated in an ice-water bath, centrifuged, and the supernatant was collected. The sample was concentrated and redissolved in 500 μL of 90% acetonitrile. Chromatographic analysis was performed on an XBridge C18 column (4.6 mm × 100 mm, 3.5 μm, Waters, USA) using an HPLC system. Mobile phase A was water, acetonitrile, and formic acid (70:30:0.02, v / v / v). Mobile phase B was acetonitrile and isopropanol (50:50, v / v). The flow rate was 0.7 mL / min, and the sample separation was as follows: 0 - 3 min, 0 - 25% B; 3 - 11 min, 25 - 45% B; 11 - 13 min, 45 - 60% B; 13 - 18 min, 60 - 75% B; 18 - 18.5 min, 75 - 90% B; 18.5 - 20 min, 90% B; 20 - 21 min, 90 - 0%; 21 - 25 min, 0%. The injection volume was 5 μL. A QTRAP 5500 mass spectrometer was used, and the anion, multiple reaction monitoring (MRM) mode was adopted. The main instrument parameters were: curtain gas pressure 10 psi, nebulizer gas pressure 30 psi, auxiliary gas pressure 30 psi, spray voltage -4500 V, ionization temperature 525 °C.

[0119] 6.4 Experimental results

[0120] 6.4.1 Effects of bufotoxin and its substitutes on LPS-induced depressive behavior in mice

[0121] As Figure 6 shown, the results indicated that compared with the control group, the cumulative immobility time in the tail suspension test and the cumulative immobility time in the forced swimming test of the model group were significantly increased (P < 0.01), showing depressive-like behavior; after administration of the positive drug fluoxetine, high and low doses of bufotoxin, and high and low doses of potential bufotoxin substitutes, the cumulative immobility time in the tail suspension test and the cumulative immobility time in the forced swimming test were significantly shortened (P < 0.01). There was no significant difference in the comparison of each dose of bufotoxin and each dose of potential bufotoxin substitute (P > 0.05), indicating a certain equivalence between the two in terms of antidepressant effects.

[0122] 6.4.2 Effects of bufotoxin and artificial bufotoxin substitutes on the expression of inflammatory factors TNF-α, IL-6, and BDNF mRNA in the hippocampal tissue of LPS-induced mice

[0123] As Figure 7As shown, the results indicated that, compared with the control group, the relative expressions of inflammatory factors TNF-α and IL-6 mRNA in the hippocampal tissues of mice in the model group were significantly increased (P < 0.01); compared with the model group, the relative expressions of inflammatory factors TNF-α and IL-6 mRNA in the hippocampal tissues of mice in the fluoxetine group, the high- and low-dose bufotoxin groups, and the high- and low-dose potential substitutes groups were significantly decreased (P < 0.01), indicating that bufotoxin and its potential substitutes could reduce the release of inflammatory factors in the hippocampal tissues and had the effect of anti-brain tissue inflammation; there was no significant difference in the comparison of each dose of bufotoxin and each dose of the potential substitute of bufotoxin (P > 0.05); the relative expression of brain-derived neurotrophic factor BDNF in the hippocampal tissues of mice in the high- and low-dose potential substitute groups was significantly increased (P < 0.01), indicating that bufotoxin and its potential substitutes could increase the expression of brain-derived neurotrophic factor BDNF. It was shown that bufotoxin and its substitutes had a certain equivalence in the effect of anti-brain tissue inflammation.

[0124] 6.4.3 Effects of Bufotoxin (CS) and Artificial Bufotoxin Substitute (NJ) on Inflammatory Mediators in the Cyclooxygenase (COX) Pathway of Brain Tissue

[0125] In the model group, 10 lipids from the COX pathway were up-regulated. After treatment with the bufotoxin substitute, most lipids were significantly reduced. For example, the content of 20-ethyl PGE 2 increased from (10.09 ± 1.16) ng / g in the control group to (32.34 ± 4.67) ng / g in the model group (P = 0.000), decreased to (9.4 ± 0.7) ng / g in the CSL group (P = 0.000), decreased to (7.36 ± 0.58) ng / g in the CSH group (P = 0.000), decreased to (3.8 ± 0.32) ng / g in the NJL group (P = 0.000), and decreased to (6.02 ± 0.26) ng / g in the NJH group (P = 0.000). And 19(R)PGF1a, 11-deoxy-PGF1α / 1b, δ-12-PGJ2 / PGJ2, PGF1a / 1b, 2,3-dinor-PGE1, etc. After administration of high-dose bufotoxin and its potential substitutes, some lipids were restored to the control level. These lipid metabolites included 20-ethylPGE2α, PGF2β, dhk-PGF1a, etc.

[0126] Example 7: Evaluation of the Equivalence of Artificial Bufotoxin Composition and Natural Bufotoxin in Anti-Tumor Cell Proliferation

[0127] 7.1 Prescription

[0128] The formula of the artificial bufotalin substitute composition used in this example is as follows. By mass percentage, total bufadienolides of plant origin are 30% (where the ratio of ATV1 - ATV7 is 6:1:1:0.3:0.2:0.2:0.2), total bufotenines are 20% (the ratio of bufotenine, bufotenidine, and bufothionine is 0.5:5:0.5), total bufopeptides are 10% (including equal proportions of TP17, TP18, and TP19, the specific sequences are shown in Figure 1 ), and excipients are 40% (cyclodextrin, starch, soy protein, cholesterol).

[0129] 7.2 Experimental methods and grouping

[0130] Preparation of drug-containing serum: Fifteen SD rats were divided into a normal group, a low-dose (50 mg / kg) and high-dose (100 mg / kg) bufotalin group, and a low-dose (50 mg / kg) and high-dose (100 mg / kg) artificial bufotalin group. The above doses of drugs were given by gavage once a day for 3 consecutive days. Blood was collected from the orbital cavity 1 h after the last administration. Serum containing drugs was obtained by centrifugation at 3500 rpm for 20 min. Serum samples of the same group were combined, filtered and sterilized, aliquoted, and stored in a -80 °C refrigerator for later use. A549 and SMMC-7721 cells growing normally were seeded in 96-well plates at 5×10 3 cells / well. Incubated overnight at 37 °C in 5% CO 2 . The culture medium was discarded, and blank serum and drug-containing serum with concentrations of 5% and 10% were added. Incubated at a constant temperature for 72 h. 20 μL of 5.0 mg / mL MTT solution was added to each well in the dark and incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well and shaken for 5 min. The absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The cell proliferation inhibition rate was calculated. The cell proliferation inhibition rate (%) = 1 - (absorbance of the drug-administered well / absorbance of the blank well). By preparing drug-containing serum in vivo, the anti-tumor cell activities of artificial bufotalin and natural bufotalin were compared.

[0131] 7.3 Results

[0132] Drug-containing serum is a classic method for in vitro administration of complex components of traditional Chinese medicine. After oral administration of bufotalin products, their active ingredients are absorbed through the gastrointestinal tract and undergo the first-pass effect in the liver before entering the blood to take effect. In this example, bufotalin and artificial bufotalin of the formula of this scheme were prepared under the above conditions, and drug-containing serum was prepared. At 5% and 10% drug administration concentrations, the proliferation of lung cancer A549 and liver cancer SMMC-7721 cells could be significantly inhibited. Under the condition of 10% drug-containing serum, the inhibition rates of the high-dose groups of bufotalin and artificial bufotalin both exceeded 50%, and there was no significant difference in the drug effects between the two, indicating that they have certain anti-tumor equivalent activities. The specific results are shown in Table 6.

[0133] Table 6: Comparison of the inhibitory rates of the drug-containing sera of rats in the natural bufotalin and artificial bufotalin groups on the proliferation of tumor cells

[0134]

[0135] Although the present invention discloses the key method for obtaining total bufotalins from plants and the use of preparing artificial bufotalin, and uses it as the main raw material to combine bufotenine-like components or bufotoxin-like components and related auxiliary components to prepare artificial bufotalin. The above features have been illustrated and described herein, but those of ordinary skill in the art can think of many combinations of proportional components, modifications, substitutions, changes and equivalents based on this. Therefore, it should be understood that the appended claims are intended to cover all such combinations of analogs and changes that fall within the true spirit of the present invention.

Claims

1. An artificial toad venom composition, characterized in that: The core components of the composition include one or more of the following: toad cardiotonic steroids, toad tryptamines or toad polypeptides. Preferably, the toad cardiotonic steroids, the toad tryptamines and the toad polypeptides are natural products, natural product analogs, natural product modifications, or the toad tryptamines and the toad polypeptides are prepared by chemical synthesis or synthetic biology technology.

2. The artificial toad venom composition according to claim 1, characterized in that: The core components of the composition are composed of toad cardiotonic steroids, toad tryptamines and toad polypeptides, and the weight of the toad cardiotonic steroids, the toad tryptamines and the toad polypeptides account for 0.01-70%, 0.01-80% and 0.01-80% of the total weight of the artificial toad venom composition respectively; Preferably, the mass of the toad cardiotonic steroids, the toad tryptamines and the toad polypeptides account for 10-30%, 10-50% and 10-50% of the total mass of the artificial toad venom composition respectively; More preferably, the masses of the toad cardiotonic steroids, the toad tryptamines and the toad polypeptides account for 10-20%, 20-40% and 20-40% of the total mass of the artificial toad venom composition, respectively.

3. The artificial toad venom composition according to claim 1, characterized in that: The composition further comprises auxiliary components, which include one or more of the following: protein, amino acid, cholesterol, nucleic acid or organic acid; pharmaceutically acceptable conventional excipients with functions of filling, bonding, lubrication, dispersion, sustained release, coating, encapsulation, disintegration, penetration promotion, emulsification, solubilization and the like. Preferably, the auxiliary components are composed of cholesterol, proteins, organic acids, amino acids, and nucleosides, and their mass accounts for 0.01-20%, 0.01-30%, 0.01-20%, 0.01-20%, and 0.01-20% of the total mass of the artificial toad venom composition, respectively.

4. The artificial toad venom composition according to claim 1, characterized in that: The toad cardiotonic steroids are type A cardiac glycosides and their aglycones, type B cardiac glycosides and their aglycones, cardiotonic steroid analogs or their chemical modifications. The toad cardiotonic steroids are of animal or plant origin. The animal origin includes amphibians of the Toad family and the Ranidae family. The plant origin includes plants of the genus Helleborus of the Ranunculaceae family, plants of the genus Onion of the Liliaceae family, succulent plants of the genus Candida of the Liliaceae family, Onion of the genus Hyacinthaceae, Crassulaceae, Scrophulariaceae (Digitalis), Apocynaceae (Nerium spp., Capricorn), Asclepiadaceae (Periploca, Asclepiadaceae), Liliaceae (Convallaria, Dieffenbachia), Cruciferae (Saccharum), and Ranunculaceae (Oleaster).

5. The artificial toad venom composition according to claim 4, characterized in that: The toad cardiotonic steroid is bufotalin, and the bufotalin includes free bufotalin components or bound bufotalin components. Preferably, the bufotalin includes free bufotalin components selected from the following one or more: resibufogenin, bufalin, resibufagin, resibufaginol, desacetylcinobufagin, and gamabufotalin, desacetylbufotalin, 1β-hydroxylbufalin, telocinobufagin, bufotalinin, arenobufagin, hellebrigin, dehydrated cinobufagin, cinobufagin), cinobufagin, bufotalin, 19-oxo-cinobufagin, 3β,14β,16β-trihydroxy-5α-bufodor-20,22-diene, 3β,14β-dihydroxy-5α-bufodor-20,22-diene, cinobufaginol, cinobufotalin, 19-oxo-cinobufagin, 19-oxo-cinobufagin o-cinobufotalin), Desacetylcinobufagin, 14β-hydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufastat-20,22-diene, 3β,14β,16β-trihydroxy-5α-bufastat-20,22-diene or 3β,14β-dihydroxy-5α-bufastat-20,22-diene, wherein the combined toad steroid component is selected from one or more of the following: resibufogenin-3-succinate-arginine ester), bufalin-3-succinate-arginine ester, resibufogenin-3-glutarate-arginine ester, (resibufaginol-3-succinate-arginine ester), resibufogenin-3-adipate-arginine esterstereoisomer), bufalin-3-adipate-arginine ester stereoisomer, resibufogenin-3-pimelate-arginine ester, bufalin-3-pimelate-arginine ester, resibufogenin-3-suberate-arginine ester, cinobufagin-3-succinate-arginine ester, desacetylcinobufagin-3-suberate-arginine ester, cinobufaginol-3-succinate-arginine ester), telocinobufagin-3-suberate-arginine ester, gamabufotalin-3-suberate-arginine ester, cinobufagin-3-adipate-arginine ester, hellebrigenin-3-suberate-arginine ester, arenobufagin-3-suberate-arginine ester, cinobufagin-3-pimelate-arginine ester, cinobufagin-3-suberate-arginine ester, cinobufaginol-3-pimelate-arginine ester), cinobufaginol-3-suberate-arginine ester, 19-oxo-cinobufotalin-3-suberate-arginineester), deglucohellebrin, 5β,14β,16β-trihydroxy-19-aldehyde-3β-[(α-L-rhamnopyranosyl)oxy]bufastat-20,22-diene, 14β,16β-dihydroxy-3β-[βD-glucopyranosyl-(1→4)-(β-D-glucopyranosyl)-oxy]-5α-bufastat-20,2 2-diene, 14β,16β-dihydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufasta-20,22-diene, Hellebosapogenin, Hellebrin, 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)-oxy]-5α-bufasta-20,22-diene.

6. The artificial toad venom composition according to claim 4, characterized in that: The toad cardiotonic steroid is a plant-derived toad steroid. Preferably, the toad cardiotonic steroid is derived from Helleborus niger, Helleborus caucasicus, Helleborus orientalis, Helleborus viridis, Helleborus bocconei, Helleborus rubrum, Helleborus rubrum var. purpurascens), scented iron chopsticks H.Odbrus, H.abchasicus, H.macranthus, Turkish iron chopsticks (H.torquatus), pointed iron chopsticks (H.multifidus), round-leaved Christmas rose H.cyclophyllus, H.odorus, Croatian Christmas rose H.croaticus, H.istriacus, H.dumetorum, odorous iron chopsticks (Helleborus foetidus), toothed iron chopsticks (Helleborusargutifolius), dark red iron chopsticks (Helleborus atrorubens), Corsican iron chopsticks (Helleboruscorsicus), lead-colored Christmas rose (Helleborus lividus), preferably, the toad cardiotonic steroid is prepared by deglycosylation of cardiac glycosides by microbial fermentation or enzyme conversion, and more preferably, the plant-derived toad steroid is selected from one or more of the following: 3β,14β-dihydroxy-5α-toad steroid-20,22-diene, deglucohellebrin, 3β,14β,16β-trihydroxy-5α-toad steroid-20,22-diene, hellebrigenin, desacetylcinobufagin, 19-oxo-cinobufagin, 3β,5β,14β,16β-tetrahydroxy-19aldehyde-toad steroid-20,22-diene (16-OH-hellebrigenin), 3β,14β,16β-trihydroxy-5β-toad steroid-20,22-diene (16-OH-bufalin).

7. The artificial toad venom composition according to claim 1, characterized in that: The toad tryptamines are selected from one or more of the following: 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT), 5-MeO-NMT, 5-methoxytryptamine (5-Methoxytryptamine), bufobutanoic acid, bufobutarginine, bufoserotonin A, bufoserotonin B, bufoserotonin C, bufotenidine, N, N-dimethyl bufotenine, oxidized bufotenine, ...serotonin A, bufoserotonin B, bufoserotonin C, bufotenidine, bufotenine, bufotenine, bufotenine Oxide, bufotenine-O-sulphate, bufoviridine, n-Acetylserotonin, n′-Formylserotonin, n-Methylserotonin, o-Methylbufoviridine, serotonin, tryptamine, bufopyramide, N-methylserotonin, bufothionine, dehydrobufotenine, indole-3-acetaldehyde, 5-hydroxyindole-3-aldehyde, 5-Hydroxy Tryptophol, bufobutarginine or bufopyramide.

8. The artificial toad venom composition according to claim 1, characterized in that: The toad polypeptides are selected from one or more of the following: Phe-Asp-Phe-Pro-Gly-Asn-Lys-Ile-Thr、Phe-Pro-Ala-Ser-Ala-Ala-Gly-Lys-Gly-Leu-Gln、Ser-Glu-Trp-Glu-Glu-Asn-Pro-Met(+15.99)-Asn-Lys-Tyr-Val-Ser、Asn-Phe-Thr-Gly-Asp-Ser-Ile-Pro-Cys(+57.02)-Arg、Ser(+42.01)-Asp-Glu-Ile-Ile-His-Asn-Pro-Ala-Val-Pro-Phe、Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys-Glu-Leu、Tyr-Glu-Arg-Pro-Leu-Val-Thr、Leu-Thr-Val-Gly-Pro-Arg-Gly-Pro-Leu-Leu-Val-Gln-Asp、Val-Asp-Leu-Asn-Pro-Ser-Asn-Val-Gly-Trp-Asn-Lys-Thr-Thr-Phe-Ala、Lys-Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys-Glu-Leu-Tyr、Gln-Ala-Glu-Phe-Asp-Lys-Ala-Ala-Glu-Asp-Val-Lys-Lys-Leu-Lys-Thr、Met-Leu-Ala-Asn-Glu-Asp-Tyr-Ala-Ser-Leu-Thr-Lys-Gly-Ile-Gln-Asn-Leu-Asn、Ala-Thr-Pro-Val-Asp-Trp-Lys-Pro-Gly-Asp-Arg-Val-Met-Val、Phe-Phe-Asp-Phe-Pro-Gly-Asn-Lys-Ile-Thr-Ser-Val-Ala-Gly-Val-Tyr-Phe、Ser-Asp-Ile-Ser-Ala-Gly-Lys-Ile-Lys-Gln、Gln-Gly-Leu-Ala-Ala-Gly-Met(+15.99)-Pro-Gly-Tyr-Pro-Val-Leu(-.98)、Phe-Asp-Lys-Ala-Ala-Glu-Asp-Val-Lys-Lys-Leu-Lys-Thr-Lys-Pro-Thr-Asp-Asp-Glu-Leu-Lys、Trp-Gly-Trp-Pro-Ile-Asp-Asp-Ala-Thr-Thr-Glu-Lys-Leu-Ser、Pro-Arg-Gly-Pro-Leu-Leu-Val-Gln-Asp。.

9. Use of the artificial toad venom composition according to any one of claims 1 to 8 in the preparation of raw materials for the pharmaceutical industry, characterized in that: The pharmaceutical industry raw material is used as an equivalent substitute for natural toad venom.

10. Use of the artificial toad venom composition according to any one of claims 1 to 8 in preparing a medicament for treating throat diseases.

11. Use of the artificial toad venom composition according to any one of claims 1 to 8 in preparing a medicament for treating heart diseases.

12. Use of the artificial toad venom composition according to any one of claims 1 to 8 in the preparation of a medicament for treating skin, tumor or neurological diseases.

Images