Traditional Chinese medicine composition for treating heart failure and preparation method and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicines, and discloses a traditional Chinese medicine composition for treating heart failure, and a preparation method and application thereof. According to mass parts, the traditional Chinese medicine raw materials of the traditional Chinese medicine composition for treating heart failure are as follows: red ginseng 10-22 parts, orange red 5-13 parts and aconite 10-22 parts. The traditional Chinese medicine composition for treating heart failure provided by the application has no cytotoxicity, can improve cell viability, reduce mitochondrial membrane potential, thereby reducing myocardial cell death, protecting myocardial cell mitochondrial function, and can be applied to preparation of a medicine for preventing and / or treating heart failure.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a traditional Chinese medicine composition for treating heart failure, its preparation method and application. Background Technology

[0002] Heart failure (HF) is a complex clinical syndrome caused by initial myocardial damage from various causes, leading to changes in the structure and function of the heart and ultimately resulting in decreased ventricular pumping function. Clinical features of HF include dyspnea, fatigue, decreased exercise tolerance, and pulmonary congestion and peripheral edema caused by fluid retention. It is a common critical condition in clinical practice and is often a major complication of all organic heart diseases of different etiologies.

[0003] Based on his academic thought of "interrelationship of the five internal organs," Professor Deng Tietang believed that the pathogenesis of heart failure is a combination of deficiency in the root and excess in the branch. He emphasized that the location of heart failure is in the heart, but it is not limited to the heart. He proposed the view of "treating heart failure from the perspective of the spleen," believing that the pathogenesis and treatment principles of heart failure can all be addressed by starting with the spleen. The heart belongs to fire, the yang within yang, and governs blood vessels; the spleen belongs to earth, the source of qi and blood production, and governs the transformation and transportation of fluids. When the heart qi or heart yang is deficient, the pumping function is weak, blood circulation is sluggish, the spleen is deficient and unable to transport fluids, fluids accumulate in the chest and heart, and body fluids stagnate to form phlegm. The disease of the child affects the mother, aggravating the deficiency of heart yang qi, and phlegm and fluids combine. In other words, the pathogenesis of heart failure can be summarized as deficiency of heart (yang) qi combined with phlegm and fluid retention. Based on the aforementioned theories and through long-term clinical practice, Professor Deng Tietao developed a traditional Chinese medicine composition with the effects of warming the heart and tonifying qi and removing blood stasis. This composition uses red ginseng as the principal ingredient to replenish the deficiency of the five internal organs' vital energy and strengthen the spleen to control blood stasis; aconite as the assistant ingredient to warm and invigorate the heart's yang, reversing collapse and tonifying fire to assist yang; and tangerine peel and coix seed as adjuvant ingredients to strengthen the spleen and replenish acquired qi. The combined effects of these herbs primarily warm the yang, while also tonifying qi and removing blood stasis, achieving harmony between the internal organs, ensuring sufficient yang qi, unobstructed blood flow, and elimination of edema, thus achieving a comprehensive treatment effect. This composition is mainly formulated into capsules for clinical application and is known as the "Heart-Warming Capsule."

[0004] Warm Heart Capsules have achieved good clinical efficacy. Previous clinical studies have shown that Warm Heart Capsules have a definite curative effect on patients with different types of heart failure, such as heart failure with preserved ejection fraction. The multi-component, multi-target, and multi-pathway effects of Warm Heart Capsules have been proven to have anti-heart failure effects. Summary of the Invention

[0005] The main objective of this invention is to provide a traditional Chinese medicine composition for treating heart failure. Compared to existing heart-warming capsules, the traditional Chinese medicine composition for treating heart failure provided by this invention does not contain coix seed, making the raw materials simpler and the cost lower.

[0006] This invention has demonstrated through cell experiments that neither the Warm Heart Capsule nor the traditional Chinese medicine composition provided in this application is cytotoxic. Both can increase cell viability, reduce mitochondrial membrane potential, thereby reducing cardiomyocyte death and protecting cardiomyocyte mitochondrial function, and there is no statistically significant difference between the two in these effects. Therefore, it can be concluded that the efficacy of the traditional Chinese medicine composition provided in this invention is not significantly different from that of the Warm Heart Capsule.

[0007] According to one aspect of the present invention, a traditional Chinese medicine composition for treating heart failure is provided, wherein the raw materials are, by weight: 10-22 parts of red ginseng, 5-13 parts of tangerine peel and 10-22 parts of aconite.

[0008] In this invention, red ginseng refers to the dried root and rhizome of Panax ginseng CAMey., a plant of the Araliaceae family, after steaming. It has a sweet and slightly bitter taste and is warm in nature. It enters the spleen, lung, heart, and kidney meridians. It greatly replenishes vital energy, restores the pulse and consolidates the body, and benefits qi and stops bleeding.

[0009] Aconite (also known as prepared aconite or black aconite root) is a processed product of the lateral root of *Aconitum carmichaelii* Debx, a plant in the Ranunculaceae family. It has a pungent and sweet taste and is extremely hot in nature. It enters the heart, kidney, and spleen meridians. It restores yang and rescues from collapse, tonifies fire and assists yang, dispels cold and relieves pain.

[0010] Citrus peel is the dried outer pericarp of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It has a pungent and bitter taste, and is warm in nature. It enters the lung and spleen meridians. It regulates qi, relieves chest congestion, dries dampness, and resolves phlegm.

[0011] In some embodiments, the traditional Chinese medicine composition provided by the present invention, by weight, comprises the following raw materials: 22 parts red ginseng, 13 parts tangerine peel, and 22 parts aconite.

[0012] In some embodiments, the dosage form of the traditional Chinese medicine composition provided by the present invention can be conventional drug dosage forms such as decoction, tablet, capsule, powder, oral liquid, pill, ointment, freeze-dried powder or granules.

[0013] According to another aspect of the present invention, a method for preparing the above-mentioned traditional Chinese medicine composition for treating heart failure is also provided:

[0014] When the dosage form of the traditional Chinese medicine composition is a decoction, the preparation method includes the following steps:

[0015] Weigh out red ginseng, tangerine peel, and aconite according to the formula ratio, add water in a volume of 6 to 8 times the total weight of the three raw materials and soak for 20 to 60 minutes. Then decoct 1 to 3 times, each time for 45 to 90 minutes. Combine the decoctions, filter the combined decoctions, remove the residue, and you will get the decoction.

[0016] When the dosage form of the traditional Chinese medicine composition is lyophilized powder, the preparation method includes the following steps:

[0017] The decoction prepared above was concentrated and subjected to alcohol precipitation to obtain an alcohol-precipitated medicinal solution.

[0018] Remove the ethanol from the alcohol-precipitated solution to obtain solution A;

[0019] Drug solution A was freeze-dried to obtain freeze-dried powder.

[0020] When the dosage form of the traditional Chinese medicine composition is other drug dosage forms, the preparation method includes the following steps:

[0021] Weigh out the red ginseng, tangerine peel, and aconite according to the specified ratio;

[0022] Take Aconitum carmichaelii, add 6-8 times its weight of water and decoct 1-3 times, each time for 45-90 minutes. Combine the decoctions, concentrate and precipitate the combined decoction with alcohol to obtain an alcohol-precipitated decoction. Remove the ethanol from the alcohol-precipitated decoction to obtain decoction B. Concentrate decoction B into a thick Aconitum carmichaelii extract with a water content of 15-20%; or, concentrate decoction B into a thick Aconitum carmichaelii extract with a water content of 15-20%, and then dry the thick Aconitum carmichaelii extract to a water content of 3-8% to obtain a dry Aconitum carmichaelii extract.

[0023] Red ginseng and tangerine peel are pulverized into fine powder, sieved, and then mixed with aconite extract or aconite dry extract. Pharmaceutically acceptable excipients are then added and prepared into conventional pharmaceutical preparations such as tablets, capsules, powders, oral liquids, pills, ointments, or granules according to existing pharmaceutical preparation processes.

[0024] In some embodiments, the method for freeze-drying drug solution A may include the following steps: first, pre-freezing drug solution A at -80°C for 12-24 hours; then, vacuuming at -30°C for 48-72 hours; and finally, drying at 30°C for 24-48 hours to obtain freeze-dried powder.

[0025] In some embodiments, pharmaceutically acceptable excipients may be at least one of sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorbents, surfactants, and lubricants.

[0026] The excipients selected in this invention can be conventional excipients in the field of traditional Chinese medicine manufacturing. For example, the sustained-release agent and excipient can be water; the filler can be starch or sucrose; the binder can be cellulose derivatives, alginate, gelatin, or polyvinylpyrrolidone; the humectant can be glycerin; the disintegrant can be agar, calcium carbonate, or sodium bicarbonate; the absorption promoter can be a quaternary ammonium compound; the adsorbent carrier can be kaolin or soap clay; the surfactant can be cetyl alcohol; and the lubricant can be talc, calcium / magnesium stearate, or polyethylene glycol. Other excipients such as flavoring agents and sweeteners can also be added.

[0027] This invention demonstrates through cell experiments that the traditional Chinese medicine composition provided by this invention for treating heart failure has comparable efficacy to existing warming capsules and can be used to prepare drugs for the prevention and / or treatment of heart failure. Attached Figure Description

[0028] Figure 1 Total ion chromatogram of the contents of the heartwarming capsules;

[0029] Figure 2 Total ion chromatogram of serum containing the Warm Heart Capsule;

[0030] Figure 3 This is the total ion chromatogram of the traditional Chinese medicine composition of the present invention analyzed by mass spectrometry;

[0031] Figure 4 The study demonstrated the toxic effects of Warm Heart Capsule lyophilized powder NX, Warm Heart Capsule-containing serum NX4, and Warm Heart Capsule optimized lyophilized powder NX3 on H9C2 cells.

[0032] Figure 5 The effects of lyophilized Nuoxin Capsule powder NX, Nuoxin Capsule-containing serum NX4, and optimized Nuoxin Capsule lyophilized powder NX3 on the viability of H9C2 cells under hypoxia injury were demonstrated.

[0033] Figure 6 The effects of lyophilized Nuoxin Capsule powder NX, Nuoxin Capsule-containing serum NX4, and optimized Nuoxin Capsule lyophilized powder NX3 on hypoxic-induced H9C2 JC-1 cells were demonstrated.

[0034] Figure 7 Flow cytometry analysis of the effects of Nuanxin Capsule lyophilized powder NX, Nuanxin Capsule-containing serum NX4, and Nuanxin Capsule optimized lyophilized powder NX3 on hypoxic-induced H9C2 JC-1 cells. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0036] Example 1

[0037] The prescription for the freeze-dried powder of traditional Chinese medicine composition used in this embodiment for treating heart failure (hereinafter referred to as "Warm Heart Capsule Optimized Freeze-Dried Powder") is as follows: 22g of red ginseng, 13g of tangerine peel and 22g of aconite.

[0038] The preparation method includes the following steps:

[0039] (1) Weigh out red ginseng, tangerine peel and aconite, mix them together, add pure water at a ratio of 1:8 of the total mass of the three raw materials to pure water, soak for 30 minutes, then decoct and keep boiling for 1 hour. Pour out the decoction, add 8 times the amount of pure water of the total mass of the three raw materials, boil again for 1 hour, remove the dregs, combine the two decoctions, filter, and you will get the Chinese herbal decoction.

[0040] (2) The decoction of traditional Chinese medicine composition was concentrated using a rotary evaporator at 50°C and 50 rpm until the volume was about 1 / 8 of the original volume of the decoction of traditional Chinese medicine composition, and then subjected to alcohol precipitation.

[0041] The alcohol precipitation process includes: cooling the drug solution, slowly adding ethanol while stirring until the alcohol content is 50%, sealing with plastic wrap, leaving at room temperature for 24 hours, filtering with a vacuum filter, discarding the drug solution residue, and obtaining the alcohol-precipitated drug solution.

[0042] (3) The ethanol in the final precipitated solution was further evaporated by rotary evaporator. The resulting solution A was then freeze-dried in a freeze dryer to obtain the freeze-dried powder of the optimized version of the Warm Heart Capsule, with a yield of 20%. The freeze-dried powder was then packaged and stored at -80℃.

[0043] The freeze-drying process includes: first, pre-freezing drug solution A at -80℃ for 12 hours; then, vacuuming at -30℃ for 48 hours; and finally, drying at 30℃ for 24 hours to obtain the final product.

[0044] Example 2

[0045] The prescription for the traditional Chinese medicine composition capsule for treating heart failure in this embodiment is: 22g of red ginseng, 13g of tangerine peel, and 22g of aconite.

[0046] The preparation method includes the following steps:

[0047] (1) First decoction: Weigh the aconite, add water with 6 times the weight of the feed, heat to boiling and keep it simmering for 1 hour, filter, and obtain the first filter residue and the first filtrate.

[0048] (2) Second decoction: Add 8 times the weight of water to the first filter residue, heat to boiling, keep simmering for 1 hour, filter, and obtain the second filter residue and the second filtrate.

[0049] (3) Third decoction: Add 8 times the weight of water to the second filter residue, heat to boiling, keep simmering for 1 hour, filter, and obtain the third filter residue and the third filtrate; combine the three filtrates to obtain the filtrate;

[0050] (4) Concentration: The filtrate is concentrated under reduced pressure to a specific gravity of 1.04 to 1.12 (80°C), ethanol is added to make the alcohol content reach 70%, and the mixture is allowed to stand for 24 to 48 hours. The filtrate is then filtered, the ethanol is recovered, and the filtrate is concentrated to a relative density of not less than 1.091 (80°C) to obtain Aconitum carmichaelii extract.

[0051] (5) Spray drying: Pass the aconite extract through a 100-mesh sieve and then spray dry it. The process parameters for spray drying are set as follows: air inlet temperature 160-180℃, air outlet temperature 95-105℃; collect the powder in time to obtain aconite dry extract powder.

[0052] (6) Sieve and mix: Weigh red ginseng and tangerine peel and grind them into fine powder. After passing through a 100-mesh sieve, mix them evenly with aconite extract powder to obtain a mixture.

[0053] (7) Capsule preparation: The mixture is filled into capsules using a mechanical filling method to obtain capsules.

[0054] Example 1: Composition Analysis of Warm Heart Capsules and Optimized Version of Warm Heart Capsule Freeze-Dried Powder

[0055] I. Experimental Materials

[0056] 1. Heartwarming Capsules

[0057] Provided by the preparation room of Guangdong Provincial Hospital of Traditional Chinese Medicine, the prescription for its contents is: 22g red ginseng, 13g dried tangerine peel, 22 parts aconite, and 43g coix seed; the preparation method of the warm heart capsule includes the following steps:

[0058] (1) First decoction: Weigh aconite and coix seed, add water with 6 times the weight of the ingredients, heat to boiling and keep simmering for 1 hour, filter to obtain the first residue and the first filtrate;

[0059] (2) Second decoction: Add 8 times the weight of water to the first filter residue, heat to boiling, keep simmering for 1 hour, filter, and obtain the second filter residue and the second filtrate.

[0060] (3) Third decoction: Add 8 times the weight of water to the second filter residue, heat to boiling, keep simmering for 1 hour, filter, and obtain the third filter residue and the third filtrate; combine the three filtrates to obtain the filtrate;

[0061] (4) Concentration: The filtrate is concentrated under reduced pressure to a specific gravity of 1.04 to 1.12 (80°C), ethanol is added to make the alcohol content reach 70%, and the mixture is allowed to stand for 24 to 48 hours. The mixture is then filtered, the ethanol is recovered from the filtrate and concentrated to a relative density of not less than 1.091 (80°C) to obtain a thick extract.

[0062] (5) Spray drying: Pass the thick extract through a 100-mesh sieve and then spray dry it. The process parameters for spray drying are set as follows: air inlet temperature 160-180℃, air outlet temperature 95-105℃; collect the powder in time to obtain dry extract powder.

[0063] (6) Sieve and mix: Weigh red ginseng and tangerine peel and grind them into fine powder. After passing through a 100-mesh sieve, mix them evenly with the dry extract powder to obtain a mixture.

[0064] (7) Capsule making: The mixture is filled into capsules using a mechanical filling method to obtain the heartwarming capsules.

[0065] 2. Warm Heart Capsule Optimized Freeze-Dried Powder

[0066] The optimized freeze-dried powder of the heartwarming capsule was prepared using Example 1.

[0067] II. Experimental Methods

[0068] (I) Extraction and preparation of serum containing the warming capsule

[0069] 1. Preparation of Warm Heart Capsule Solution: Weigh 2.34g of the powdered contents of Warm Heart Capsule, add 100mg of sodium carboxymethyl cellulose, mix thoroughly, add ultrapure water to make up to 20mL, and store at 4℃ protected from light for later use.

[0070] 2. Grouping and administration of C57B / 6 mice

[0071] (1) SPF-grade C57B / 6 mice were purchased from the Guangdong Provincial Medical Experimental Animal Center and fed in a standardized manner by the Laboratory Center of Guangdong Academy of Traditional Chinese Medicine. After one week of feeding, they were randomly divided into two groups: a blank serum group and a warm heart capsule administration group, with 3 mice in each group.

[0072] (2) The dosage for mice was 120 mg / kg.

[0073] (3) Before gavage, the mice were weighed and the gavage volume was 1 mL / 150 g. The mice in the drug-containing serum group were given the warm heart capsule solution by gavage according to the above calculated dose, while the blank serum group was given an equal volume of physiological saline. The mice were gavaged once a day for 7 consecutive days.

[0074] 3. Collection and preparation of mouse serum

[0075] The mice were fasted for 12 hours before blood collection. Anesthesia was performed using 1.5% sodium pentobarbital, with the dosage determined based on the mice's weight. Blood was collected from the abdominal aorta. After standing at room temperature for 2 hours, the blood was centrifuged at 2500g for 10 minutes at 4°C. The supernatant serum was collected under aseptic conditions, filtered once through a 0.22μm microporous membrane, and inactivated by water bath at 56°C for 30 minutes. The serum was then placed in 1.5mL EP tubes, sealed, and stored at -80°C.

[0076] (II) Composition analysis of serum and aqueous solution containing Warm Heart Capsules, and aqueous solution of optimized freeze-dried powder of Warm Heart Capsules

[0077] 1. Sample preparation

[0078] (1) Serum sample pretreatment: Take 1 mL of drug-containing serum and precipitate the protein with 3 times the amount of ice-cold acetonitrile. Vortex for 1 min, centrifuge at 14000 rpm for 10 min at 4℃, and dry the supernatant under nitrogen at room temperature. Redissolve the residue with 2 mL of ultrapure water, centrifuge, and then pass the supernatant through an SPE column (Waters Oasis HLB, 6 cc). Before use, activate the SPE column with 6 mL of methanol and then equilibrate it with 6 mL of ultrapure water. Then add 2 mL of sample and elute with 5% methanol and pure methanol for 3 times the sample volume. Collect the pure methanol elution fraction, dry it under nitrogen at room temperature, and redissolve the residue with 200 μL of methanol. Centrifuge at 14000 r / min at 4℃ for 15 min, and then inject 2 μL of the supernatant for LC-MS / MS analysis.

[0079] (2) Preparation of the aqueous solution of the warm heart capsule: Weigh 0.5g of the contents of the warm heart capsule, add 4mL of ultrapure water, vortex thoroughly and mix well, and then filter overnight in the dark. Then filter it in a sterile environment in a laminar flow hood using a 0.22μm microporous filter. The working solution concentration is 125mg / mL. Accurately pipette 1mL of the working solution and dilute it with 50% methanol to a mass concentration of 100μg / mL. Centrifuge (14000r / min, 4℃) for 15min. Take the supernatant and filter it through a 0.22μm microporous membrane for LC-MS / MS analysis.

[0080] (3) Preparation of the lyophilized powder aqueous solution of the optimized version of Nuanxin Capsules: Weigh 0.5g of the lyophilized powder of the optimized version of Nuanxin Capsules, add 4mL of ultrapure water and vortex thoroughly to mix well. After overnight in the dark, filter it using a 0.22μm microporous filter in a sterile environment of a laminar flow hood. The working solution concentration is 125mg / mL. Accurately pipette 1mL of the working solution and dilute it with 50% methanol to a mass concentration of 100μg / mL. Centrifuge (14000r / min, 4℃) for 15min. Take the supernatant and filter it through a 0.22μm microporous membrane for LC-MS / MS analysis.

[0081] 2. Chromatographic-mass spectrometry conditions

[0082] (1) Chromatographic conditions

[0083] ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), mobile phase composition: A = 0.1% formic acid-water, B = 0.1% formic acid-acetonitrile, column temperature 40℃, flow rate 0.3 mL / min, injection volume 2 μL. Gradient elution conditions are shown in Table 1.

[0084] Table 1 Gradient Elution Table

[0085] time 0.1% Formic Acid-Water (%) 0.1% Formic acid-acetonitrile (%) 0 98 2 2 95 5 3 89 11 12 60 40 15 40 60 17 0 100 18 0 100 18.5 98 2 20 98 2

[0086] (2) Mass spectrometry conditions

[0087] The ion source was an electrospray ionization (ESI) source; positive and negative ion modes were used for detection; ion transmission tube temperature: 320℃; sheath gas flow rate: 35 arb; auxiliary gas flow rate: 10 arb; spray voltage: 3.5 kV(+) / 3.0 kV(-); heating temperature: 350℃(+) / 350℃(-). Samples were scanned by primary and secondary mass spectrometry. Primary mass spectrometry used full scan (Full MS) with a resolution of 70,000 m / z and a scan range of 70-1050 m / z; secondary mass spectrometry used data-dependent secondary scan (dd-MS2) with a resolution of 17,500 m / z, and high-energy collision-induced dissociation (HCD) was used for fragmentation at collision energies (NCE): 20, 40, and 60.

[0088] (III) Chemical Composition Analysis

[0089] Data were acquired using LC-MS / MS technology with an ESI ion source in both positive and negative ion Full-MS-dd MS2 modes. Positive and negative ion chromatograms, retention times (RT), characteristic fragment ion analysis, and precise mass determination were performed. Molecular formulas with deviations of less than 10 ppm between theoretical and measured values ​​were selected for secondary spectral analysis to infer their structures and fragmentation processes. These findings were then compared with relevant literature data to confirm the chemical components.

[0090] III. Experimental Results

[0091] (I) Component analysis of Warm Heart Capsules, Warm Heart Capsule-containing Serum, and Optimized Version of Warm Heart Capsule Freeze-dried Powder

[0092] The results are as follows Figures 1-3 , Figure 1 , 2 The images 3 are, in order, the total ion chromatograms of mass spectrometry analysis of Warm Heart Capsules, Warm Heart Capsule-containing serum, and Warm Heart Capsule lyophilized powder.

[0093] The results showed that the Nuanxin Capsule contained 191 components, and after removing common differences with the blank serum control group, the serum containing the Nuanxin Capsule contained 63 components. Among the traditional Chinese medicine substances that exerted the efficacy of both the Nuanxin Capsule and the serum containing the Nuanxin Capsule, five components were found to be overlapping (see Table 2): carmichaeline A, vanillic acid, vanillic acid isomer, yesanchinoside A (24S), and quinquenoside L3. These are mainly alkaloids, flavonoids, and triterpenoid saponins.

[0094] The optimized freeze-dried powder of Warm Heart Capsules contains 176 ingredients. As shown in Table 3, the main ingredients of Warm Heart Capsules and the optimized freeze-dried powder of Warm Heart Capsules are not significantly different.

[0095] Table 2. Identification results of common components in Warm Heart Capsules and their drug-containing serum.

[0096] Element Molecular formula source Heartwarming Capsules Drug-containing serum grade Classification carmichaelineA C22H29NO3 aconite 1655052 1165082 A alkaloids Vanillicacid C8H8O4 Orange-red 5781508 46215 A Flavonoids and flavonoid glycosides Vanillicacidisomer C8H8O4 Orange-red 463431 43700 B Flavonoids and flavonoid glycosides yesanchinosideA(24S) C30H50O5 Red Ginseng 6625810 229160 B Triterpenoid saponins quinquenosideL3 C36H60O9 Red Ginseng 188128 49095 B Triterpenoid saponins

[0097] Table 3. Identification results of common components in Nuanxin Capsules and the freeze-dried powder of the traditional Chinese medicine composition in this application.

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Experimental Example 2: Pharmacological Study of Warm Heart Capsule Lyophilized Powder, Warm Heart Capsule-Containing Serum, and Optimized Warm Heart Capsule Lyophilized Powder on Hypoxia-Induced H9C2 Cardiomyocyte Injury.

[0108] I. Experimental Materials

[0109] 1. Warm Heart Capsules Contain Medicated Serum

[0110] The extraction and preparation methods are the same as in Example 1. When using the medication, the serum containing the Warm Heart Capsule should be taken out of the -80℃ refrigerator in advance and warmed up before use.

[0111] 2. Warm Heart Capsule Freeze-Dried Powder

[0112] The preparation method includes the following steps:

[0113] (1) Take 22g of red ginseng, 13g of dried tangerine peel, 22g of aconite and 43g of coix seed, mix them together and soak them in pure water for 30 minutes at a mass ratio of 1:8 of the total mass of the four ingredients to pure water. Then decoct them and keep boiling for 1 hour. Then pour out the decoction, add 8 times the mass of the three ingredients to pure water, boil again for 1 hour, remove the dregs, combine the two decoctions, filter and get the decoction.

[0114] (2) The decoction was concentrated using a rotary evaporator at 50°C and 50 rpm until the volume was 1 / 8 of the original volume, and then subjected to alcohol precipitation.

[0115] (3) The alcohol precipitation process includes: cooling the drug solution, slowly adding ethanol while stirring until the alcohol content is 50%, sealing with plastic wrap, placing at room temperature for 24 hours, filtering with a vacuum filter, discarding the drug solution residue, and obtaining the alcohol-precipitated drug solution.

[0116] (4) The ethanol-precipitated solution was then evaporated using a rotary evaporator to remove all the ethanol. The resulting solution was then freeze-dried in a freeze dryer. The freeze-drying process included: pre-freezing the solution at -80°C for 12 hours; then vacuum-drying it at -30°C for 48 hours; and finally drying it at 30°C for 24 hours to obtain the freeze-dried powder of the Warm Heart Capsules, with a yield of 20%. The freeze-dried powder was then packaged and stored at -80°C.

[0117] Before use, remove the lyophilized powder from the -80℃ freezer, warm it to room temperature, and then prepare a solution with deionized water to a concentration of 125mg / mL.

[0118] 3. Warm Heart Capsule Optimized Freeze-Dried Powder

[0119] The optimized freeze-dried powder of the heartwarming capsule was prepared using Example 1.

[0120] Before use, remove the lyophilized powder from the -80℃ freezer, warm it to room temperature, and then prepare a solution with deionized water to a concentration of 125mg / mL.

[0121] II. Experimental Methods

[0122] (I) Culture of H9C2 cardiomyocytes and establishment of a hypoxia model

[0123] 1. Cell culture: H9C2 cells are placed in T25 cell culture flasks. Add 4-6 mL of DMEM medium containing 10% FBS to each T25 flask and place it in a normal incubator at 37℃ and 5% CO2. When the cells grow to about 80%-90%, passage them at a ratio of 1:3.

[0124] 2. Cell grouping:

[0125] Cytotoxicity assays: Control group, Nuanxin Capsule lyophilized powder group (NX), Nuanxin Capsule drug-containing serum group (NX4), and Nuanxin Capsule optimized lyophilized powder group (NX3).

[0126] Cell viability assay and mitochondrial membrane potential assay: normal group (Control), hypoxia group (Hypoxia), Nuoxin Capsule lyophilized powder group (NX), Nuoxin Capsule drug-containing serum group (NX4), and Nuoxin Capsule optimized version lyophilized powder group (NX3).

[0127] 3. Establishment of the hypoxia model: When the cell confluence density reached approximately 80% in a 37℃, 5% CO2 incubator, the original culture medium was discarded, and the cells were rinsed twice with 3 mL of PBS per bottle. Except for the normal group, which was replaced with 5 mL of 10% FBS DMEM medium per bottle and continued to be cultured in a normal incubator, the original culture medium for all other groups was replaced with approximately 5 mL of serum-free and glucose-free medium per bottle. The drug-treated groups were given the corresponding drug until the final drug concentration reached 4 mg / mL. Finally, the hypoxia group and the drug-treated group were transferred to a 37℃, 0.1% O2, 5% CO2, 95% N2 three-gas hypoxia incubator for 24 hours of hypoxia.

[0128] (II) Cytotoxicity Detection

[0129] 1. Cell treatment: Prepare a 96-well plate and seed cells at a density of 6000 per well, with 3 replicates per group. Repeat the experiment 3 times. After incubating overnight in a normal incubator, add the corresponding drug to the drug treatment groups (Nuanxin Capsule lyophilized powder group (NX), Nuanxin Capsule drug-containing serum group (NX4), and Nuanxin Capsule optimized version lyophilized powder group (NX3)) to a final drug concentration of 4 mg / mL and continue to incubate for 24 h.

[0130] 2. Cytotoxicity assay: The culture medium was removed from the 96-well plates, and the plates were washed twice with 1×PBS. MTT solution (0.5 mg / mL) was added to each well, and the plates were incubated at 37°C in the dark for 4 hours. Then, the MTT solution was removed, and 100 μL of DMSO solution was added to each well for 10 minutes at room temperature in the dark to dissolve the formazan crystals. Finally, the OD value of each well was recorded at 570 nm using a microplate reader (Thermo Scientific, Multiskan FC, USA), and the percentage of cytotoxicity was calculated.

[0131] (III) Cell viability detection

[0132] 1. Cell treatment: Cells were seeded at a density of 6000 per well in 96-well plates, with 3 replicates per group, and the experiment was repeated 3 times. When the cells reached 80% confluence, the original culture medium was removed from all groups, and the cells were washed twice with 1×PBS. The normal group was then replaced with complete culture medium and cultured in a normal incubator for 24 h. The hypoxia group and the drug-treated group were replaced with glucose-free and serum-free culture medium. In addition, the corresponding drug was added to the drug-treated group to a final drug concentration of 4 mg / mL. Finally, the hypoxia group and the drug-treated group were transferred to a 37°C, 0.1% O2, 5% CO2, 95% N2 triple-gas hypoxia incubator and cultured for 24 h.

[0133] 2. Cell viability assay: After cell treatment, the culture medium was removed from the 96-well plates, and the plates were washed twice with 1×PBS. MTT solution (0.5 mg / mL) was added to each well, and the plates were incubated at 37°C in the dark for 4 hours. Then, the MTT solution was removed, and 100 μL of DMSO solution was added to each well for 10 minutes at room temperature in the dark to dissolve the formazan crystals. Finally, the OD value of each well was recorded at 570 nm using a microplate reader (Thermo Scientific, Multiskan FC, USA), and cell viability was calculated.

[0134] (iv) Mitochondrial membrane potential detection

[0135] 1. Cell processing: The cell processing for mitochondrial membrane potential detection is the same as in "(III) Cell viability detection".

[0136] 2. Mitochondrial Membrane Potential Detection: After cells received different treatments, cell samples were collected following the method described in the "Cytotoxicity Detection" section. Following the instructions of the mitochondrial membrane potential detection kit (Shanghai Beyotime Biotechnology Co., Ltd., China), JC-1 detection solution and JC-1 buffer were prepared in advance and stored on ice. Then, under light-protected conditions, 500 μL of JC-1 staining solution was added to each group of cell samples and incubated at room temperature for 20 minutes. The samples were then centrifuged, the supernatant discarded, and each sample was washed twice with 1 mL of JC-1 buffer, thoroughly mixed, and set aside. Finally, the degree of ΔΨm damage was immediately detected by flow cytometry (Agilent Technologies, NovoCyteQuanton, USA).

[0137] (V) Data Analysis

[0138] All experimental values ​​were statistically analyzed using SPSS 26.0 software. Data conforming to a normal distribution and with homogeneous variances were presented as mean ± standard deviation (x ± s), and one-way ANOVA was used for comparisons among multiple groups. When variances were homogeneous, the LSD test was used for pairwise comparisons between groups; when variances were unequal, Dunnett's T3 test was used for pairwise comparisons between groups. Data not conforming to a normal distribution were presented as median and quartiles (M(P25, P75), and a nonparametric Kruskal-Wallis one-way ANOVA (k samples) was used for comparisons. P < 0.05 indicated statistically significant differences.

[0139] III. Experimental Results

[0140] (I) Effects of different drugs on H9C2 cytotoxicity.

[0141] To investigate the effects of different drugs on H9C2 cell cytotoxicity, the MTT assay was used in this invention. The results showed that the drugs in different groups (Nuanxin Capsule lyophilized powder group (NX), Nuanxin Capsule drug-containing serum group (NX4), and Nuanxin Capsule optimized lyophilized powder group (NX3)) had no cytotoxic effect on H9C2 cells; compared with the Control group, there were no statistically significant differences among the groups (Table 4). Figure 4 ).

[0142] Table 4. Effects of different drugs on hypoxia-induced H9C2 cytotoxicity.

[0143]

[0144]

[0145] Note: The data conforms to a normal distribution. One-way ANOVA was used, and the result of the homogeneity of variance test was 0.644, which satisfies the homogeneity of variance. The LSD test was used for multiple comparisons. P is the p-value between groups, and P' is the p-value compared with the model group.

[0146] (II) Effects of different drugs on the viability of hypoxia-induced H9C2 cells

[0147] This invention employs the MTT assay to investigate the effects of different drug groups (Nuanxin Capsule lyophilized powder group (NX), Nuanxin Capsule drug-containing serum group (NX4), and Nuanxin Capsule optimized lyophilized powder group (NX3)) on cell viability in H9C2 cells damaged by hypoxia. The results show that cell viability was significantly reduced in the Hypoxia group (hypoxia group). Conversely, all drug groups effectively improved cell viability, and there were no statistically significant differences between the drug groups (Table 5). Figure 5 ).

[0148] Table 5. Effects of different drugs on hypoxia-induced H9C2 cell viability.

[0149]

[0150] Note: The data conform to a normal distribution. One-way ANOVA was used, and the homogeneity of variance test result was 265.978, which satisfies the homogeneity of variance requirement. The LSD test was used for multiple comparisons. P is the p-value between groups, and P' is the p-value compared with the model group. Post-hoc comparisons showed that there were no statistically significant differences between the NX and NX4 groups (P = 0.160 > 0.05), the NX and NX3 groups (P = 0.203 > 0.05), and the NX4 and NX3 groups (P = 0.878 > 0.05).

[0151] (III) Effects of different drugs on hypoxia-induced mitochondrial membrane potential in H9C2 cells

[0152] To determine whether the herbal composition of this invention can reduce hypoxia-induced H9C2 cardiomyocyte damage by protecting against mitochondrial damage, changes in mitochondrial membrane potential (ΔΨm) were detected by JC-1 staining. The results showed that the mitochondrial membrane potential rate in the Hypoxia group (hypoxia group) was significantly increased compared to the Control group (normal group), while the mitochondrial membrane potential rate in all drug groups showed a decreasing trend with statistically significant differences. There were no statistically significant differences in mitochondrial membrane potential rate among the different drug groups (Table 6). Figure 6-7 ).

[0153] Table 6. Effects of different drugs on JC-1 in hypoxia-induced H9C2.

[0154]

[0155]

[0156] Note: Data follow a normal distribution. One-way ANOVA was used, and the homogeneity of variance test result was 112.726, satisfying the homogeneity of variance requirement. LSD test was used for multiple comparisons. P is the p-value between groups, and P' is the p-value compared to the model group. Post-hoc comparisons showed no statistically significant differences among the NX and NX4 groups (P = 0.075 > 0.05), the NX and NX3 groups (P = 0.969 > 0.05), and the NX4 and NX3 groups (P = 0.080 > 0.05).

[0157] In summary, both the traditional Chinese medicine composition for treating heart failure provided in this application and the Nuanxin capsules are non-cytotoxic and can both improve cell viability, reduce mitochondrial membrane potential, thereby reducing cardiomyocyte death and protecting cardiomyocyte mitochondrial function. There is no statistically significant difference in the related effects, indicating that there is no significant difference in efficacy between the traditional Chinese medicine composition for treating heart failure provided in this application and the Nuanxin capsules.

[0158] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for treating heart failure caused by deficiency of heart yang qi combined with phlegm and fluid retention, characterized in that, By weight, the Chinese medicinal materials are: 10-22 parts red ginseng, 5-13 parts tangerine peel, and 10-22 parts aconite.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, By weight, the Chinese medicinal materials are: 22 parts red ginseng, 13 parts tangerine peel, and 22 parts aconite.

3. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The dosage form of the traditional Chinese medicine composition is decoction, tablet, capsule, powder, oral liquid, pill, ointment, freeze-dried powder or granules.

4. The method for preparing the traditional Chinese medicine composition for treating heart failure caused by deficiency of heart yang qi combined with phlegm and water retention according to claim 1 or 2, characterized in that, Includes the following steps: Weigh out red ginseng, tangerine peel, and aconite according to the formula ratio, add water in a volume of 6 to 8 times the total weight of the three raw materials and soak for 20 to 60 minutes. Then decoct 1 to 3 times, each time for 45 to 90 minutes. Combine the decoctions and filter the combined decoction to obtain the soup.

5. The preparation method according to claim 4, characterized in that, It also includes the following steps: The decoction was concentrated and subjected to alcohol precipitation to obtain an alcohol-precipitated medicinal solution. Remove the ethanol from the alcohol-precipitated solution to obtain solution A; Drug solution A was freeze-dried to obtain freeze-dried powder.

6. The method for preparing the traditional Chinese medicine composition for treating heart failure caused by deficiency of heart yang qi combined with phlegm and water retention according to claim 1 or 2, characterized in that, Includes the following steps: Weigh out the red ginseng, tangerine peel, and aconite according to the specified ratio; Take Aconitum carmichaelii, add 6 to 8 times its weight of water and decoct 1 to 3 times, each time for 45 to 90 minutes. Combine the decoctions and concentrate and precipitate the combined decoctions with alcohol to obtain alcohol-precipitated decoction. Remove the ethanol from the alcohol-precipitated solution to obtain solution B; Concentrate the herbal solution B into a thick extract of Aconitum carmichaelii with a water content of 15-20%; or, concentrate the herbal solution B into a thick extract of Aconitum carmichaelii with a water content of 15-20%, and then dry the thick extract of Aconitum carmichaelii to a water content of 3-8% to obtain a dry extract of Aconitum carmichaelii. Red ginseng and tangerine peel are pulverized into fine powder, sieved, and then mixed with aconite extract or aconite dry extract. Pharmaceutically acceptable excipients are then added to prepare a pharmaceutical preparation.

7. The preparation method according to claim 6, characterized in that, The pharmaceutical preparation is a tablet, capsule, powder, oral liquid, pill, ointment or granule.

8. The preparation method according to claim 6 or 7, characterized in that, The pharmaceutically acceptable excipients are at least one of the following: sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, adsorbents, surfactants, and lubricants.

9. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 for treating heart failure caused by deficiency of heart yang qi combined with phlegm and water retention in the preparation of a medicament for preventing and / or treating heart failure caused by deficiency of heart yang qi combined with phlegm and water retention.

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