A zingerone-a-isoleucine-tryptophan conjugate or salt thereof, and a preparation method and application thereof

Abstract

The application provides a zingerone A-isoleucine-tryptophan conjugate or a salt thereof, and a preparation method and application thereof. The application further improves the solubility of the drug, promotes the absorption of the drug, and can increase the effective permeation coefficient of the drug in the intestinal tract by adopting isoleucine-tryptophan dipeptide to modify zingerone A. Pharmacokinetic experimental data show that by intravenous injection of the conjugate, the action time of zingerone A and the isoleucine-tryptophan dipeptide compound in the body can be significantly prolonged, and the bioavailability of the drug can be effectively improved. The experimental results show that the conjugate has the effects of anti-aging and treating cardiovascular diseases, and can be applied to the research and development of functional foods and medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a gingerone A-isoleucine-tryptophan conjugate or its salt, its preparation method, and its application. Background Technology

[0002] From a biological perspective, aging is a natural process involving the decline of cellular function and the gradual aging of tissues. However, in modern society, environmental pollution, changes in dietary structure, and the accelerated pace of life can all accelerate the aging process. Many Chinese people find themselves experiencing signs of aging, such as decreased physical strength and memory loss, in middle age or even earlier. At the same time, socio-cultural perceptions of aging are subtly changing. In the past, aging was often viewed as a natural and irreversible process, but now, with the continuous emergence of anti-aging technologies and products, more and more people are trying to slow down the aging process through technological means.

[0003] Shogaol A, a unique compound found in ginger, has attracted widespread attention in scientific research in recent years. Studies have found that shogaol A possesses various biological activities, including anti-inflammatory, antioxidant, and anti-tumor effects, which are of great significance for the prevention and treatment of various chronic diseases.

[0004] CN118206452A discloses a gingerol A prodrug compound and its application. The gingerol A prodrug compound exhibits superior anti-aging effects and is highly effective in clearing senescent cells. The gingerol A prodrug compound of this invention can be used to treat patients suffering from age-related diseases or disorders, neuroinflammation, pain, and / or amino acid deficiencies.

[0005] CN117981872A discloses the application of gingerone A in the preparation of foods for preventing cardiovascular diseases and drugs for treating cardiovascular diseases, belonging to the field of biomedical technology. This invention protects the application of gingerone A in the preparation of foods for preventing cardiovascular diseases. This invention, by preparing foods containing gingerone A and feeding these foods to mice, found that it can prevent atherosclerosis, thereby preventing cardiovascular diseases caused by atherosclerosis.

[0006] However, gingerone A is an enone compound, and its structure introduces certain defects that affect its drug efficacy: unmodified gingerone A has poor solubility, permeability, stability and absorption, ultimately resulting in low drug bioavailability and difficulty in achieving effective therapeutic concentrations.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] One objective of this invention is to provide a gingerone A-isoleucine-tryptophan conjugate or a salt thereof. This invention modifies gingerone A with an isoleucine-tryptophan dipeptide. This conjugate effectively improves the drug's solubility, permeability, and stability, promotes the intravenous absorption of gingerone A, and significantly increases its bioavailability. This overcomes the deficiency of gingerone A's low bioavailability, which makes it difficult to achieve effective therapeutic concentrations, thereby significantly enhancing its antioxidant, anti-aging, and cardiovascular disease prevention and / or treatment capabilities.

[0009] The second objective of this invention is to provide a method for preparing gingerone A-isoleucine-tryptophan conjugate or its salt. The preparation method described in this invention is simple to operate, operates under mild conditions, produces high-quality products, and has a high yield.

[0010] The third objective of this invention is to provide an application of gingerone A-isoleucine-tryptophan conjugate in the preparation of anti-aging products.

[0011] The fourth objective of this invention is to provide the use of gingerone A-isoleucine-tryptophan conjugate or its salt in the preparation of products for the prevention and / or treatment of cardiovascular diseases.

[0012] The fifth objective of this invention is to provide the application of the gingerone A-isoleucine-tryptophan conjugate or its salt in the preparation of products for lowering blood pressure.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a gingerone A-isoleucine-tryptophan conjugate or a salt thereof, wherein the structural formula of the gingerone A-isoleucine-tryptophan conjugate is shown in Formula I below:

[0014] Formula I.

[0015] Preferably, the salt is a pharmaceutically acceptable salt of gingerone A-isoleucine-tryptophan conjugate, including acid addition salts and / or base addition salts of gingerone A-isoleucine-tryptophan conjugate.

[0016] Preferably, the acid addition salt is selected from any one or a combination of at least two of the following: hydrochloride, formate, trifluoroacetate, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate, or oxalate, preferably formate and / or trifluoroacetate.

[0017] Preferably, the alkali addition salt is selected from lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, iron salts, ferrous salts, copper salts, zinc salts, diethylamine salts, triethylamine salts, isopropylamine salts, etc. N,N - Any one or a combination of at least two of dimethylacetamide salts or trimethylamine salts.

[0018] In a second aspect, the present invention provides a method for preparing the gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect, the preparation method specifically comprising the following steps: Shogaol A and di(p-nitrobenzene) carbonate are esterified to obtain the active ester of shogaol A; The active ester of gingerone A and the isoleucine-tryptophan dipeptide were condensed to obtain the gingerone A-isoleucine-tryptophan conjugate.

[0019] Preferably, the molar ratio of gingerone A to di(p-nitrobenzene) carbonate is 1:(2~6).

[0020] Preferably, the esterification reaction is carried out in the presence of an organic base.

[0021] Preferably, the organic base is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N -Methylmorpholine or N , N Any one of diisopropylethylamine, preferably N , N -Diisopropylethylamine.

[0022] Preferably, the molar ratio of gingerone A to organic base is 1:(2~6).

[0023] Preferably, the esterification reaction is carried out at a temperature of 0~80℃ and for a time of 0.5~24 h.

[0024] Preferably, the molar ratio of the active ester of gingerone A to the isoleucine-tryptophan dipeptide is 1:(2~6).

[0025] Preferably, a condensing agent and / or an organic base are added during the condensation reaction.

[0026] Preferably, the condensing agent is selected from any one of HATU, HBTU, HCTU, HAPyU, HBPyU, TBTU, TSTU, TNTU, NMM, DCC, EDCI, T3P, CDI or HOBT, with HOBT being the most preferred.

[0027] Preferably, the organic base is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N- Methylmorpholine or N,NAny one of diisopropylethylamine, preferably N,N -Diisopropylethylamine.

[0028] Preferably, the molar ratio of the active ester of gingerone A to the condensing agent is 1:(2~6).

[0029] Preferably, the molar ratio of the active ester of gingerone A to the organic base is 1:(3~10).

[0030] Preferably, the temperature of the condensation reaction is 0~80℃, and the time of the condensation reaction is 0.5~24 h.

[0031] Preferably, the preparation method further includes the following steps: The organic solvent of gingerone A-isoleucine-tryptophan conjugate and an aqueous solution of the salt were mixed, stirred, separated, and the aqueous phase was collected and freeze-dried to obtain gingerone A-isoleucine-tryptophan conjugate salt.

[0032] Thirdly, the present invention provides the use of gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of anti-aging products.

[0033] Fourthly, the present invention provides the use of the gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of products for the prevention and / or treatment of cardiovascular diseases.

[0034] Fifthly, the present invention provides the use of the gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of a product for lowering blood pressure.

[0035] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention improves the drug’s bioavailability by modifying gingerone A with isoleucine-tryptophan dipeptide, thereby increasing the drug’s solubility, promoting drug absorption, increasing the effective permeability coefficient of the drug in the intestine, and prolonging the drug’s action time in the body.

[0036] (2) The present invention further improves the antioxidant and anti-aging ability of the conjugate by modifying gingerone A with isoleucine-tryptophan dipeptide.

[0037] (3) The present invention further improves the efficacy of the conjugate in improving, preventing or treating cardiovascular diseases by modifying gingerone A with isoleucine-tryptophan dipeptide.

[0038] (4) The present invention modifies gingerone A with isoleucine-tryptophan dipeptide to further improve the antihypertensive effect of gingerone A. Attached Figure Description

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 The plasma pharmacokinetic curve of gingerone A-isoleucine-tryptophan conjugate.

[0041] Figure 2 This is a plasma pharmacokinetic curve of gingerone A.

[0042] Figure 3 This is a graph showing the dynamic changes in blood pressure.

[0043] Figure 4 The graph shows the determination of Renin, Ang I, ACE, and Ang II content.

[0044] Figure 5 The graph shows the determination of eNOS, ET-1, CGRP, and NO content.

[0045] Figure 6 The graph shows the determination of β-galactosidase content.

[0046] Figure 7 The graph shows the determination of MDA, T-AOC, CAT, and SOD contents.

[0047] Figure 8 The graph shows the determination of iNOS, IL-4, and TNF-α levels.

[0048] Figure 9 Image of HE staining of the heart.

[0049] Figure 10 Image of kidney stained with hematoxylin and eosin (HE). Detailed Implementation

[0050] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0051] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] In a first aspect, the present invention provides a gingerone A-isoleucine-tryptophan conjugate or a salt thereof, wherein the structural formula of the gingerone A-isoleucine-tryptophan conjugate is shown in Formula I below:

[0054] Formula I.

[0055] In this invention, by modifying gingerone A with an isoleucine-tryptophan dipeptide, the conjugate effectively improves the drug's solubility, permeability, and stability, promotes the intravenous absorption of gingerone A, and significantly increases its bioavailability, overcoming the deficiency of gingerone A's low bioavailability and difficulty in achieving effective therapeutic concentrations. The gingerone A-isoleucine-tryptophan conjugate or its salt described in this invention significantly enhances antioxidant, anti-aging, and cardiovascular disease prevention and / or treatment effects.

[0056] As an optional embodiment of the present invention, the salt is a pharmaceutically acceptable salt of gingerone A-isoleucine-tryptophan conjugate.

[0057] As an optional embodiment of the present invention, the pharmaceutically acceptable salts of the gingerone A-isoleucine-tryptophan conjugate include acid addition salts and / or base addition salts of the gingerone A-isoleucine-tryptophan conjugate.

[0058] As an optional embodiment of the present invention, the acid addition salt is selected from any one or a combination of at least two of the following: hydrochloride, formate, trifluoroacetate, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate, or oxalate.

[0059] As an optional embodiment of the present invention, the acid addition salt is preferably a formate or trifluoroacetate.

[0060] As an optional embodiment of the present invention, the alkali addition salt is selected from lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, iron salts, ferrous salts, copper salts, zinc salts, diethylamine salts, triethylamine salts, isopropylamine salts, etc. N,N - Any one or a combination of at least two of dimethylacetamide salts or trimethylamine salts.

[0061] In a second aspect, the present invention provides a method for preparing the gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect, the preparation method specifically comprising the following steps: Shogaol A and di(p-nitrobenzene) carbonate are esterified to obtain the active ester of shogaol A; The active ester of gingerone A and the isoleucine-tryptophan dipeptide were condensed to obtain the gingerone A-isoleucine-tryptophan conjugate.

[0062] As an optional embodiment of the present invention, the molar ratio of gingerone A and di(p-nitrobenzene) carbonate is 1:(2~6), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0063] As an optional embodiment of the present invention, the esterification reaction is carried out in the presence of an organic base.

[0064] As an optional embodiment of the present invention, the organic base in the esterification reaction is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N -Methylmorpholine or N , N Any one of diisopropylethylamine, preferably N , N -Diisopropylethylamine.

[0065] As an optional embodiment of the present invention, the molar ratio of gingerone A to organic base is 1:(2~6), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0066] As an optional embodiment of the present invention, the temperature of the esterification reaction is 0~80℃, for example, it can be 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc.

[0067] As an optional embodiment of the present invention, the esterification reaction time is 0.5 to 24 h, for example, it can be 0.5 h, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, 22 h, 24 h, etc.

[0068] As an optional embodiment of the present invention, the molar ratio of the active ester of gingerone A to the isoleucine-tryptophan dipeptide is 1:(2~6), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0069] As an optional embodiment of the present invention, a condensing agent and / or organic base may be added during the condensation reaction.

[0070] As an optional embodiment of the present invention, the condensing agent in the condensation reaction process is selected from any one of HATU, HBTU, HCTU, HAPyU, HBPyU, TBTU, TSTU, TNTU, NMM, DCC, EDCI, T3P, CDI or HOBT.

[0071] As an optional embodiment of the present invention, the condensing agent used in the condensation reaction process is preferably HOBT.

[0072] As an optional embodiment of the present invention, the organic base in the condensation reaction process is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N- Methylmorpholine or N,N - Any one of diisopropylethylamine.

[0073] As an optional embodiment of the present invention, the organic base in the condensation reaction process is preferably... N,N -Diisopropylethylamine.

[0074] As an optional embodiment of the present invention, the molar ratio of the active ester of gingerone A to the condensing agent is 1:(2~6), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0075] As an optional embodiment of the present invention, the molar ratio of the active ester of gingerone A to the organic base is 1:(3~10), for example, it can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, etc.

[0076] As an optional embodiment of the present invention, the temperature of the condensation reaction is 0~80℃, for example, it can be 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc.

[0077] As an optional embodiment of the present invention, the condensation reaction time is 0.5~24 h, for example, it can be 0.5h, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, 22 h, 24 h, etc.

[0078] As an optional embodiment of the present invention, the preparation method further includes the following steps: The organic solvent of gingerone A-isoleucine-tryptophan conjugate and an aqueous solution of the salt were mixed, stirred, separated, and the aqueous phase was collected and freeze-dried to obtain gingerone A-isoleucine-tryptophan conjugate salt.

[0079] As an optional embodiment of the present invention, the stirring time is 1 to 5 hours, for example, it can be 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, etc.

[0080] Thirdly, the present invention provides the use of gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of anti-aging products.

[0081] In this invention, the preparation of anti-aging products includes, but is not limited to, anti-aging foods and / or pharmaceuticals.

[0082] Fourthly, the present invention provides the use of the gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of products for the prevention and / or treatment of cardiovascular diseases.

[0083] In this invention, the preparation of products for the prevention and / or treatment of cardiovascular diseases includes, but is not limited to, foods and / or medicines for the prevention and / or treatment of cardiovascular diseases.

[0084] Fifthly, the present invention provides the use of gingerone A-isoleucine-tryptophan conjugate or its salt as described in the first aspect in the preparation of a product for lowering blood pressure.

[0085] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0086] Example 1 This embodiment provides a gingerone A-isoleucine-tryptophan conjugate, the structural formula of which is shown in Formula I below:

[0087] Formula I The synthetic route for the gingerone A-isoleucine-tryptophan conjugate is shown below:

[0088] The specific preparation steps for the gingerone A-isoleucine-tryptophan conjugate are as follows: (a) Preparation of gingerone A-di-p-nitrophenyl dicarbonate: In a round-bottom flask, gingerone A (5.0 g, 14.0 mmol), di(p-nitrobenzene) carbonate (10.6 g, 30.0 mmol), DIPEA (5.4 g, 42.1 mmol), and THF (50 mL) were added. The mixture was stirred at 30 °C for 1 h. TLC showed that the starting material reacted completely. Petroleum ether was added to the reaction solution, and a solid precipitated. The solid was slurried twice with methyl tert-butyl ether to give 7.0 g of product (gingerone A-di(p-nitrobenzene) carbonate), with a yield of 72.6%.

[0089] 1 H NMR (400 MHz, CDCl3) δ 8.33-8.27 (m, 4H), 7.51-7.45 (m, 4H), 7.12(t, J = 8.0 Hz, 2H), 6.88-6.86 (m, 1H), 6.85-6.76 (m, 4H), 6.19-6.10 (m, 1H), 3.88 (d, J = 2.0 Hz, 6H), 2.98-2.91 (m, 2H), 2.89-2.84 (m, 2H), 2.82-2.76 (m,2H), 2.59-2.51 (m, 2H).

[0090] (b) Preparation of gingerone A-isoleucine-tryptophan conjugate: In a round-bottom flask, gingerone A-di-p-nitrophenyl dicarbonate (2.5 g, 3.6 mmol), a dipeptide compound (2.5 g, 7.9 mmol), N,N-diisopropylethylamine (DIPEA, 2.8 g, 21.8 mmol), 1-hydroxybenzotriazole (HOBT, 1.2 g, 9.1 mmol), and DMF (40 mL) were added and stirred at 25 °C for 16 h. After the reaction was complete, water was added for dilution, and a yellow solid precipitated. The solid was washed with plenty of water, dissolved in ethyl acetate, dried over anhydrous Na2SO4, filtered, concentrated, and separated by column chromatography (silica, eluent: ethyl acetate: petroleum ether = 1:1) to give 0.94 g of gingerone A-isoleucine-tryptophan conjugate, with a yield of 25%.

[0091] LCMS (ESI): m / z=1043.7, (M+H) + .

[0092] 1H NMR (400 MHz, DMSO) δ 12.62 (s, 2H), 10.84 (s, 2H), 8.17 (d, J =7.1 Hz, 2H), 7.63 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 7.8 Hz, 2H), 7.32 (d, J =8.0 Hz, 2H), 7.15 (s, 2H), 7.06 (t, J = 7.5 Hz, 2H), 7.00-6.88 (m, 7H), 6.75(t, J = 6.9 Hz, 2H), 6.17 (d, J = 15.9 Hz, 1H), 4.51 (dd, J = 13.2, 7.4 Hz, 2H), 3.95 (t, J = 8.4 Hz, 2H), 3.69 (s, 6H), 3.22-3.02 (m, 4H), 2.96-2.88 (m,2H), 2.82-2.70 (m, 4H), 2.56-2.51 (m, 2H), 1.81-1.68 (m, 2H), 1.57-1.41 (m, 2H), 1.31-1.07 (m, 2H), 0.93-0.75 (m, 12H).

[0093] Example 2 This embodiment provides a formate salt of gingerone A-isoleucine-tryptophan conjugate, which is prepared by the following steps: The gingerone A-isoleucine-tryptophan conjugate was separated by column chromatography (eluting agent: acetonitrile / 0.05% formic acid-water system, C18 reversed-phase column, 40% gradient), and then lyophilized to obtain gingerone A-isoleucine-tryptophan conjugate formate.

[0094] Example 3 This embodiment provides a trifluoroacetate of a gingerone A-isoleucine-tryptophan conjugate, which is prepared by the following steps: The gingerone A-isoleucine-tryptophan conjugate was separated by column chromatography (eluting agent: acetonitrile / 0.05% formic acid-water system, C18 reversed-phase column, 40% gradient), and then lyophilized to obtain gingerone A-isoleucine-tryptophan conjugate trifluoroacetate.

[0095] Example 4 This embodiment provides an isopropylamine salt of a gingerone A-isoleucine-tryptophan conjugate, which is prepared by the following steps: 1 g of gingerone A-isoleucine-tryptophan conjugate was dissolved in 5 mL of methanol, and 0.12 g of isopropylamine was dissolved in 1 mL of water. The organic solvent of gingerone A-isoleucine-tryptophan conjugate and the aqueous solution of the salt were then mixed and stirred at room temperature for 3 h. The mixture was separated, and the aqueous phase was collected and freeze-dried to obtain gingerone A-isoleucine-tryptophan conjugate isopropylamine salt.

[0096] Comparative Example 1 This comparative example provides gingerone A compound, the structural formula of which is shown in GA below:

[0097] GA.

[0098] Test Example 1 Pharmacokinetic test Test samples: gingerone A-isoleucine-tryptophan conjugate (hereinafter referred to as PDC4) provided in Example 1 and gingerone A (hereinafter referred to as GA) provided in Comparative Example 1.

[0099] Test method: 1. Test sample: The solvent for preparing the test sample is 0.1 M phosphate buffer (the pH of the prepared solution is approximately 8), and the prepared solution is a clear liquid.

[0100] 2. Experimental System: 2.1 Laboratory animals: Species & strain: SD rat.

[0101] Laboratory animal source: Shanghai Bikeyi Biotechnology Co., Ltd.

[0102] Age at administration: 6-8 weeks at the start of administration.

[0103] Body weight at administration: 160-181 g at the start of administration.

[0104] Number and sex of animals: 12 males, including 2 spare rats.

[0105] 2.2 Feeding and Management: Animals were housed in transparent resin-plastic cages (400 mm x 240 mm x 200 mm). They were lit by fluorescent lamps, with 12 hours of light per day (07:00 - 19:00) and 12 hours of darkness. Dark periods could be intermittently interrupted as needed for research activities. The ambient temperature and relative humidity in the animal housing were controlled within the ranges of 20-26℃ and 40-70%, respectively, and monitored and recorded daily. The feed was certified rodent feed. The batch number of the feed was recorded in the research log. Drinking water was filtered and sterilized using an ultrapure water system. All animals had free access to water during the experiment. Animals were fasted overnight before administration of medication, for at least 12 hours, and were fed again 4 hours after administration.

[0106] 3. Brief description of experimental methods 3.1 Groups and Measurement No randomization was performed. Animal weight was measured before drug administration, and healthy animals of similar weight were selected for the experiment, as shown in Table 1 below. Table 1

[0107] Note: If any unexpected situation occurs during the drug administration process, the control group animals can be used to replace them in a timely manner. After the experiment, the control group animals will be used to collect blank samples.

[0108] Dosage frequency: Single dose.

[0109] 3.2 Sample Collection Approximately 0.2 mL of blood was drawn from the vein. Anticoagulant: EDTA-K2.

[0110] Collection time: Group 1 (intravenous): Blood samples were collected before administration and at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h and 24 h after administration.

[0111] Sample processing: After blood samples are collected, they are placed in labeled centrifuge tubes (pre-cooled with crushed ice) and centrifuged quickly to separate the plasma. Centrifugation conditions: 3500 rpm, 10 minutes, 4°C. Take 50 μL of plasma and add 100 µL of acetonitrile. Vortex mix and store at -40°C or below for later testing (retain the remaining plasma).

[0112] 4. Sample Analysis 4.1 Standard Curve and Quality Control Samples PDC4 Plasma Sample Standard Curve and Quality Control: The standard curve working solution and quality control working solution were prepared using 50% methanol-water (containing 0.5% plasma) as the diluent. 45 µL of blank plasma was mixed with 100 µL of acetonitrile and 5 µL of the working solution to prepare the plasma standard curve and quality control samples. 100 µL of terfenadine 40 ng / mL (acetonitrile) internal standard was added to the prepared standard curve and quality control samples. After vortexing for 1 min, the mixture was centrifuged at 13000 rpm for 10 min at 4 °C. 100 µL of the supernatant was added to 20 µL of 0.1% formic acid-water mixture, vortexed, and then injected for analysis.

[0113] Standard curve and quality control for GA plasma samples: Working solutions for the standard curve and quality control were prepared using 80% methanol-water as the diluent. 45 µL of blank plasma was mixed with 100 µL of acetonitrile and 5 µL of the working solution to prepare the plasma standard curve and quality control samples. 100 µL of internal standard (tolbutamide 1000 ng / mL, acetonitrile) was added to the prepared standard curve and quality control samples. After vortexing for 1 min, the mixture was centrifuged at 13000 rpm for 10 min at 4 °C. 100 µL of the supernatant was added to 20 µL of 0.1% formic acid-water mixture, vortexed, and then injected for analysis.

[0114] 4.2 Standard Curve and Quality Control Samples PDC4 plasma sample pretreatment: After thawing at room temperature, 100 µL of terfenadine 40 ng / mL (acetonitrile) internal standard was added to each pretreated plasma sample. After vortexing for 1 min, the samples were centrifuged at 13,000 rpm for 10 min at 4 °C. 100 µL of the supernatant was added to 20 µL of 0.1% formic acid solution, vortexed, and then injected for analysis.

[0115] Pretreatment of GA plasma samples: After thawing at room temperature, 100 µL of internal standard (tolbutamide 1000 ng / mL, acetonitrile) was added to each pretreated plasma sample. After vortexing for 1 min, the samples were centrifuged at 13000 rpm for 10 min at 4 °C. 100 µL of the supernatant was added to 20 µL of 0.1% formic acid solution, vortexed, and then injected for analysis.

[0116] 5. Data Acquisition and Statistical Analysis: Analyst 1.7.2 and Analyst 1.7.3 software output raw spectral data, concentrations, accuracy, etc. MSDosoft Excel 2007 software calculates the mean, standard deviation, coefficient of variation, etc.

[0117] 6. Test Results: 6.1 General clinical observation: No abnormalities that could affect the experimental results were observed.

[0118] 6.2 Pharmacokinetic Test Results: The results of the plasma standard curve are shown in Table 2 below: Table 2

[0119] The plasma quality control results are shown in Table 3 below: Table 3

[0120] The results of PDC4 blood concentration time in group 1 (intravenous) are shown in Table 4 below: Table 4

[0121] The plasma concentration-time results of group 1 (intravenous) - PDC4-GA are shown in Table 5 below: Table 5

[0122] Furthermore, a plasma drug-time curve was plotted based on the table above. Figure 1 The plasma pharmacokinetic curve of gingerone A-isoleucine-tryptophan conjugate. Figure 2 This is a plasma pharmacokinetic curve of gingerone A.

[0123] The results of plasma concentration-time of pure GA in group 2 are shown in Table 6 below: Table 6

[0124] The main pharmacokinetic parameters of PDC4 and its breakdown products in rat plasma are shown in Table 7 below: Table 7

[0125] The main pharmacokinetic parameters of pure GA in rat plasma are shown in Table 8 below: Table 8

[0126] It should be noted that PDC4 will decompose after intravenous injection or oral administration to obtain the active ingredient PDC4-GA. In Tables 2-5 and Table 7, PDC4 refers to the conjugate in plasma, and PDC4-GA refers to the active ingredient GA that is decomposed from PDC4 after intravenous injection. Tables 6 and 8 refer to the plasma GA concentration parameters and pharmacokinetic parameters after direct intravenous injection of GA from Comparative Example 1.

[0127] As shown in the table above, the gingerone A-isoleucine-tryptophan conjugate provided in Example 1 of this invention exhibits excellent pharmacokinetic properties when administered intravenously. In contrast, the gingerone A provided in Comparative Example 1, after intravenous administration, is metabolized more rapidly in vivo, with faster absorption and elimination, a shorter half-life, and greater fluctuations in dosage and blood drug concentration. This indicates that modifying gingerone A with isoleucine-tryptophan dipeptide results in more rapid absorption, a significantly earlier peak blood drug concentration, slower elimination, higher blood drug concentration, a longer half-life, and the ability to maintain a stable and effective blood drug concentration for a longer period, while also improving bioavailability.

[0128] Test Example 2 Blood pressure lowering effect test Test sample: gingerone A-isoleucine-tryptophan conjugate (hereinafter referred to as PDC4) provided in Example 1.

[0129] Test method: (1) Laboratory animals: Thirty-six male SHR rats, aged 3 months, and six male WKY rats, aged 3 months, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. This study was approved by the Animal Welfare and Ethics Committee of the Guangdong Academy of Biotechnology (IACUC2021147).

[0130] (2) Instruments and equipment: Electronic balance ML204 (Mettler, Switzerland), small animal blood pressure monitor BP-2010A (Softron, Japan), multi-wavelength microplate reader Multiskan Go (Thermo, USA), rotary microtome (Leica, RM2235, Germany), fully automated pathology slide scanner GT 450 (Leica, Germany), small benchtop high-speed centrifuge Micro 21R (Thermo, USA), ultra-low temperature freezer 920-ULTS (Thermo, USA).

[0131] (3) Reagents and consumables: Test substances: gingerone A (GA), PDC4 (dipeptide + gingerone A complex); positive control: enalapril maleate tablets (National Drug Approval Number H32026567); DMSO (Sigma), PEG300 (Sigma), Tween 80 (Sigma); paraformaldehyde; physiological saline; antioxidant index detection kits (MDA, T-AOC, CAT, SOD, etc.) were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.; enzyme activity test kits (RASS pathway indicators and inflammatory factors, etc.) were purchased from Nanjing Enzyme Immunoassay Co., Ltd. and Wuhan Huamei Biotechnology Co., Ltd., respectively.

[0132] Consumables: 1.5 / 15 / 50 mL centrifuge tubes, 16 G gavage device, pipette, 1 mL syringe.

[0133] (4) Experimental grouping All test substances were prepared into gavage solutions of the required concentration using a uniform solvent (1.735% DMSO, 30% PEG300, 5.0% Tween 80, and 63.265% physiological saline), with an administration volume of 2.0 mL / 200 g body weight. Grouping information is detailed in Table 9.

[0134] Table 9

[0135] (5) Blood pressure measurement: Blood pressure in rats was measured noninvasively using the tail artery volume clamping method (also known as the tail-clamp method). Blood pressure measurements for all groups were scheduled at fixed times daily, 6-9 hours after drug administration. Blood pressure was measured at 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the start of the experiment. Before measurement, rats were placed in a 37°C incubator for 10 minutes to allow them to rest. Five stable readings were taken, and the average value was calculated as the final blood pressure of the rat at that time point.

[0136] (6) Evaluation of blood pressure lowering effect: Indicators related to the renin-angiotensin system (RAS) pathway were measured using ELISA, including Renin (MM-0343R1, ELISA), Ang I (MM-0169R1, ELISA), ACE (MM-0212R1, ELISA), Ang II (MM-0547R1, ELISA), eNOS (MM-0381R1, ELISA), ET-1 (MM-0490R1, ELISA), CGRP (MM-0490R1, ELISA), and NO (S3090, Beyotime). Samples and standards were added sequentially to pre-coated antibody-impregnated microplates. After incubation and washing, HRP-labeled detection antibodies were added, followed by incubation and thorough washing. The substrate TMB was used for color development; TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity is positively correlated with the content of the target substance in the sample. The absorbance is measured at a wavelength of 450 nm using an ELISA reader, and the concentration of the target substance is calculated based on the standard curve.

[0137] The specific test results are as follows: Figure 3 As shown: like Figure 3 As shown, blood pressure was measured at 3, 5, 1, 2, 3, 4, 5, 6, 7, and 8 weeks after the start of the gavage experiment. The results showed that the WKY control group maintained a consistently lower blood pressure level, while the placebo control group (SHR) maintained a consistently higher blood pressure level. The positive control group (enalapril maleate tablets) showed a blood pressure reduction, indicating that this trial is feasible. Regarding the antihypertensive effect, the low-concentration and medium-concentration PDC4 groups had no antihypertensive effect, while the high-concentration PDC4 group showed a significant effect around 9 hours after gavage, while GA had no antihypertensive effect. These results indicate that high-concentration PDC4 has an observable antihypertensive effect, while GA has no antihypertensive effect.

[0138] Test Example 3 Anti-aging effect test Test sample: gingerone A-isoleucine-tryptophan conjugate (hereinafter referred to as PDC4) provided in Example 1.

[0139] Test method: (1) Evaluation of anti-aging effects: Anti-aging related indicators were measured using the ELISA method, including aging biomarkers and inflammatory factors.

[0140] Aging biomarkers were determined using a β-galactosidase assay kit (70978, Sigma). β-galactosidase in the sample was reacted with a specific substrate (o-nitrophenyl-β-D-galactoside, ONPG). The substrate was hydrolyzed by β-galactosidase to produce yellow o-nitrophenol (ONP). Absorbance was measured at 570 nm, and its intensity was directly proportional to the β-galactosidase activity in the sample.

[0141] Oxidative stress and inflammatory factor-related indicators were measured using ELISA. Oxidative stress factors included MDA (S0131, Beyotime), T-AOC (S0119, Beyotime), CAT (S0051, Beyotime), and SOD (S0101, Beyotime). Inflammatory factors included iNOS (CSB-E08325r, Huamei), IL-4 (MM-0180R1, ELISA), and TNF-α (MM-0191R1, ELISA). Samples and standards were added sequentially to pre-coated antibody-impregnated microplates. After incubation and washing, biotin- or enzyme-labeled detection antibodies were added, followed by incubation and thorough washing. Incubation with enzyme substrate solution in the dark resulted in the substrate changing color under catalysis. The color intensity was positively correlated with the concentration of the target substance in the sample. The absorbance was measured at a specific wavelength using an ELISA reader, and the concentration of the target substance was calculated based on the standard curve.

[0142] (2) Pathological staining: Heart and kidney tissues were fixed with 4% paraformaldehyde, dehydrated, cleared, and embedded in paraffin, then sectioned to a thickness of 5 μm and adhered to glass slides. Histological evaluation was performed by H&E staining. After dewaxing, the slides were graded rehydrated, stained with hematoxylin for 5 minutes, and then differentiated with hydrochloric acid and ethanol for a few seconds. After rinsing with water for 2 minutes, they were stained with eosin for 1 minute, dehydrated with ethanol, cleared three times with xylene, mounted with neutral resin, and examined under a microscope.

[0143] (3) Statistical analysis: Statistical analysis was performed using GraphPad 8.0 software. Quantitative data are expressed as mean ± standard deviation (±s). Paired t-tests were used to determine whether differences between groups were statistically significant. A p-value < 0.05 was considered statistically significant.

[0144] The specific test results for each project are shown below: The results of the renin-angiotensin system marker test are as follows: Figure 4 , Figure 5 : After the experiment, rat serum was collected to detect relevant indicators in the renin-angiotensin system (RAS) pathway, including Renin, Ang I, ACE, Ang II, eNOS, ET-1, CGRP, and NO. The results showed that compared with the placebo control group: (1) the levels of Renin and Ang I increased significantly only in the positive control group, while there were no significant changes in the other groups. Figure 4 A, Figure 4 B); (2) The levels of ACE and Ang II decreased significantly in the positive drug group and the high concentration group of PDC4, while there were no significant changes in the other groups. Figure 4 C Figure 4D); (3) eNOS content increased significantly in the positive drug group and the high concentration group of PDC4, while no significant change was observed in the other groups. Figure 5 A); (4) Except for the GA group, where there was no significant change, the ET-1 content decreased significantly in all other groups ( Figure 5 B); (6) Except for the low concentration PDC4 group and the GA group, where there was no significant change in NO content, all other groups showed a significant increase ( Figure 5 D). The high-concentration PDC4 group inhibited ACE and ANG-2, achieving a hypotensive effect; at the same time, both could increase eNOS and NO, and reduce ET-1, thus protecting the vascular endothelium.

[0145] The specific test results for β-galactosidase are as follows: Figure 6 As shown: After the experiment, rat serum was collected for the detection of the aging marker β-galactosidase. The results showed that, compared with the placebo control group, β-galactosidase levels were significantly decreased in both the high-concentration PDC4 group and the GA group (p<0.05), with the high-concentration PDC4 group showing a more significant effect than the GA group. Figure 6 PDC4 exhibits certain anti-aging effects.

[0146] The specific test results for antioxidants are as follows: Figure 7 As shown: After the experiment, rat serum was collected for oxidative stress markers, including MDA, T-AOC, CAT, and SOD. The results showed that compared with the placebo control group, MDA levels decreased significantly in all groups except for the low-concentration and medium-concentration PDC4 groups, where no significant changes were observed. Figure 7 A); T-AOC levels increased significantly in all groups ( Figure 7 B); CAT levels showed no significant change, while all other groups showed significant increases ( Figure 7 C); SOD levels showed no significant change in any of the groups ( Figure 7 D). PDC4 showed antioxidant effects.

[0147] The specific test results for the inflammatory factor indicators are as follows: Figure 8 As shown: After the experiment, rat serum was collected for the detection of inflammatory factors, including iNOS, IL-4, and TNF-α. The results showed that compared with the placebo group: (1) the iNOS content did not change significantly in any group ( Figure 8 A); (2) The IL-4 content decreased significantly only in the high concentration group of PDC4, and there was no significant change in the other groups. Figure 8 B); (3) TNF-α levels decreased significantly in all groups, with the PDC4 concentration groups showing more significant effects compared to the GA group. Figure 8 C). PDC4 showed some anti-inflammatory effects.

[0148] The specific pathological test results are as follows: Figure 9 and Figure 10 As shown: After the experiment, heart and kidney tissues from rats were collected for pathological scoring. The results showed that in the normal control group, cardiomyocytes were regularly and neatly arranged, and blood vessels were normal. In the placebo and GA groups, most cardiomyocytes had indistinct boundaries, enlarged and deeply stained nuclei, myofibril dissolution and fibrosis in some areas, cardiomyocyte necrosis or apoptosis, and significant perivascular fibrosis. In the positive control group and PDC4 group, cardiomyocyte fibrosis was significantly improved, and perivascular fibrosis was reduced, with the positive control group showing the most significant improvement. Figure 9 ).

[0149] In the normal control group, the glomeruli were of regular size, the renal tubular epithelial cells were intact and neatly arranged, and there were no lesions in the renal interstitium; in the placebo group and GA group, the glomeruli showed mild atrophy, and the tubular epithelial cells showed vacuolar and granular degeneration; in the positive control group and PDC4 group, the glomerular atrophy was improved, and the vacuolar and granular degeneration decreased. Figure 10 ).

[0150] High-concentration PDC4 showed a hypotensive effect in spontaneously hypertensive rats after administration, while GA had no hypotensive effect. The high-concentration PDC4 group inhibited ACE and ANG-2 levels, while increasing eNOS and NO and decreasing ET-1, thus achieving hypotensive effect and protection of vascular endothelium. Both PDC4 and GA can reduce β-galactosidase, but the high-concentration PDC4 group had a more significant effect, and showed a more significant anti-aging effect on inflammatory factors IL-4 and TNF-α. Pathological results showed that PDC4 improved myocardial fibrosis and glomerular atrophy.

[0151] In summary, the novel peptide complex PDC4 exhibits both a blood pressure-lowering effect and the anti-aging effect of its second component, GA.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gingerone A-isoleucine-tryptophan conjugate or a salt thereof, characterized in that, The structural formula of the gingerone A-isoleucine-tryptophan conjugate is shown in Formula I below: Formula I.

2. The gingerone A-isoleucine-tryptophan conjugate or its salt according to claim 1, characterized in that, The salt is a pharmaceutically acceptable salt of gingerone A-isoleucine-tryptophan conjugate, including acid addition salts and / or base addition salts of gingerone A-isoleucine-tryptophan conjugate.

3. The gingerone A-isoleucine-tryptophan conjugate or its salt according to claim 2, characterized in that, The acid addition salt is selected from any one or a combination of at least two of the following: hydrochloride, formate, trifluoroacetate, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate, or oxalate, preferably formate and / or trifluoroacetate.

4. The gingerone A-isoleucine-tryptophan conjugate or its salt according to claim 2, characterized in that, The alkali addition salt is selected from lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, iron salts, ferrous salts, copper salts, zinc salts, diethylamine salts, triethylamine salts, and isopropylamine salts. N,N - Any one or a combination of at least two of dimethylacetamide salts or trimethylamine salts.

5. A method for preparing the gingerone A-isoleucine-tryptophan conjugate or its salt according to any one of claims 1 to 4, characterized in that, The preparation method specifically includes the following steps: Shogaol A and di(p-nitrobenzene) carbonate are esterified to obtain the active ester of shogaol A; The active ester of gingerone A and the isoleucine-tryptophan dipeptide were condensed to obtain the gingerone A-isoleucine-tryptophan conjugate.

6. The method for preparing the gingerone A-isoleucine-tryptophan conjugate or its salt according to claim 5, characterized in that, The molar ratio of gingerone A to di(p-nitrobenzene) carbonate is 1:(2~6); Preferably, the esterification reaction is carried out in the presence of an organic base; Preferably, the organic base is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N -Methylmorpholine or N , N Any one of diisopropylethylamine, preferably N , N -Diisopropylethylamine; Preferably, the molar ratio of gingerone A to organic base is 1:(2~6); Preferably, the esterification reaction is carried out at a temperature of 0~80℃ and for a time of 0.5~24 h. Preferably, the molar ratio of the active ester of gingerone A to the isoleucine-tryptophan dipeptide is 1:(2~6); Preferably, a condensing agent and / or an organic base are added during the condensation reaction. Preferably, the condensing agent is selected from any one of HATU, HBTU, HCTU, HAPyU, HBPyU, TBTU, TSTU, TNTU, NMM, DCC, EDCI, T3P, CDI or HOBT, with HOBT being preferred; Preferably, the organic base is selected from triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, morpholine, etc. N- Methylmorpholine or N,N Any one of diisopropylethylamine, preferably N,N -Diisopropylethylamine; Preferably, the molar ratio of the active ester of gingerone A to the condensing agent is 1:(2~6); Preferably, the molar ratio of the active ester to the organic base of gingerone A is 1:(3~10); Preferably, the temperature of the condensation reaction is 0~80℃, and the time of the condensation reaction is 0.5~24 h.

7. The method for preparing the gingerone A-isoleucine-tryptophan conjugate or its salt according to claim 5, characterized in that, The preparation method further includes the following steps: The organic solvent of gingerone A-isoleucine-tryptophan conjugate and an aqueous solution of the salt were mixed, stirred, separated, and the aqueous phase was collected and freeze-dried to obtain gingerone A-isoleucine-tryptophan conjugate salt.

8. The use of the gingerone A-isoleucine-tryptophan conjugate or its salt according to any one of claims 1 to 4 in the preparation of anti-aging products.

9. The use of the gingerone A-isoleucine-tryptophan conjugate or a salt thereof according to any one of claims 1 to 4 in the preparation of products for the prevention and / or treatment of cardiovascular diseases.

10. The use of the gingerone A-isoleucine-tryptophan conjugate or a salt thereof according to any one of claims 1 to 4 in the preparation of a product for lowering blood pressure.

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