Traditional Chinese medicine compound for treating viral myocarditis

Abstract

This invention provides a traditional Chinese medicine compound for treating viral myocarditis, belonging to the field of traditional Chinese medicine pharmaceutical technology. The compound consists of Forsythia suspensa, Fagopyrum dibotrys, Pseudostellaria heterophylla, Rhodiola rosea, Citrus medica, Allium macrostemon, Poria cocos, Gynostemma pentaphyllum, Salvia miltiorrhiza, Dalbergia odorifera, Cynanchum paniculatum, and Valeriana. This traditional Chinese medicine compound exhibits significant protective effects against viral myocarditis, specifically by significantly reducing damage to H9c2 cardiomyocytes caused by CVB3 virus. Its mechanism of action is closely related to downregulating the expression of inflammatory factors TNF-α and IL-6 and inhibiting the inflammatory response. Simultaneously, within the experimental concentration range, the compound shows no toxicity to cardiomyocytes, demonstrating good biocompatibility. Furthermore, this compound can significantly reduce myocardial tissue damage in a viral myocarditis model mouse by inhibiting the in vivo inflammatory response and regulating oxidative stress imbalance, providing a safe and effective new treatment option for the clinical treatment of viral myocarditis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine manufacturing technology, specifically to a traditional Chinese medicine compound for treating viral myocarditis. Background Technology

[0002] Viral myocarditis (VMC) is a localized or diffuse degeneration and necrosis of cardiomyocytes caused by viral infection. Its pathogenesis is mainly related to direct viral damage to cardiomyocytes and subsequent immune-mediated myocardial injury. In the acute phase, the disease often presents with nonspecific systemic symptoms such as fever, myalgia, respiratory symptoms, or gastroenteritis, frequently accompanied by one or more atypical symptoms such as chest pain, palpitations, fatigue, or exertional dyspnea. With the increasing prevalence of viral infections, the incidence of viral myocarditis is rising annually, especially among adolescents and young adults, and it has become one of the common and challenging cardiovascular diseases in clinical practice.

[0003] Currently, there is no specific treatment for viral myocarditis in clinical practice. Treatment mainly focuses on symptomatic and supportive care, including: ① Antiviral therapy, commonly using drugs such as ribavirin and acyclovir. However, these drugs have limited effectiveness in clearing the virus from myocardial tissue, and long-term use can easily lead to drug resistance and may also cause adverse reactions such as gastrointestinal reactions and liver and kidney damage; ② Immunomodulatory therapy, such as the use of immunosuppressants like glucocorticoids. Although these can suppress excessive immune responses to some extent, they may reduce the body's overall immunity and increase the risk of secondary infections, leading to considerable controversy in clinical application; ③ Symptomatic treatment, such as antiarrhythmic drugs, myocardial nutrition, and correction of heart failure. These can only relieve symptoms and cannot fundamentally repair damaged myocardium, block disease progression, or improve long-term prognosis. Overall, existing Western medicine methods are still insufficient in blocking viral replication, regulating the host immune response, and preventing long-term sequelae such as myocardial fibrosis, making it difficult to meet the clinical demand for safe and effective treatment plans.

[0004] In Traditional Chinese Medicine (TCM), viral myocarditis is classified under the category of "Wenbing" (warm disease) based on its pathogenesis. Some physicians, however, categorize it according to different clinical symptoms, such as "Xiongbi" (chest pain), "Xinshui" (heart water), "Zhanchong" (palpitation), and "Xinji" (heart palpitation). Severe and critical cases may be diagnosed as "Jue Luo" (collapse). TCM generally believes its etiology is related to external wind-heat-dampness toxins or epidemic pathogens. The disease location is in the heart, but it is also related to the liver, lungs, spleen, and kidneys. The overall pathogenesis is a deficiency of vital energy and invasion of pathogenic factors into the heart, often manifesting as a complex syndrome of deficiency and excess. Based on thousands of years of clinical practice, TCM has developed a unique antiviral theoretical system and treatment plans. Some TCM compound formulas or prepared medicines (such as modified formulas based on Shengmai San and Zhigancao Tang) have shown certain advantages in improving patient symptoms and quality of life. However, existing TCM treatment options still have significant shortcomings: First, most compound prescriptions lack specific targeting, focusing primarily on a single treatment principle. Their overall understanding and synergistic intervention regarding the complex pathogenesis of the intertwined "toxicity, blood stasis, and deficiency" is insufficient, making it difficult to achieve systemic regulation across multiple targets and pathways. Second, existing compound prescriptions often lack systematic pharmacological experimental verification, and their mechanisms of action remain unclear. Therefore, there is an urgent need in this field for a scientifically formulated TCM compound prescription with a clear mechanism of action, definite efficacy, and high safety. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the shortcomings of the existing technology, the present invention provides a traditional Chinese medicine compound for the treatment of viral myocarditis.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a traditional Chinese medicine compound for treating viral myocarditis, which is composed of the following raw materials in parts by weight: Forsythia suspensa 8-28 parts, Fagopyrum dibotrys 5-25 parts, Codonopsis pilosula 3-20 parts, Rhodiola rosea 5-25 parts, Citronella foenum-graecum 3-22 parts, Allium macrostemon 3-22 parts, Poria cocos 5-25 parts, Gynostemma pentaphyllum 5-25 parts, Salvia miltiorrhiza 8-28 parts, Dalbergia odorifera 5-25 parts, Cynanchum paniculatum 5-25 parts, and Valerian 8-28 parts.

[0010] Preferably, the traditional Chinese medicine compound is composed of the following raw materials in parts by weight: 12-23 parts of Forsythia suspensa, 10-20 parts of Fagopyrum dibotrys, 5-15 parts of Codonopsis pilosula, 10-20 parts of Rhodiola rosea, 8-17 parts of Citronella foenum-graecum, 8-17 parts of Allium macrostemon, 10-20 parts of Poria cocos, 10-20 parts of Gynostemma pentaphyllum, 12-23 parts of Salvia miltiorrhiza, 10-20 parts of Dalbergia odorifera, 10-20 parts of Cynanchum paniculatum, and 12-23 parts of Valerian.

[0011] Preferably, the traditional Chinese medicine compound is composed of the following raw materials in parts by weight: 18 parts forsythia, 15 parts buckwheat, 10 parts codonopsis, 15 parts rhodiola, 12 parts citron, 12 parts allium macrostemon, 15 parts poria cocos, 15 parts sedge, 18 parts salsa, 15 parts dalbergia odorifera, 15 parts cynanchum paniculatum, and 18 parts valerian.

[0012] This invention provides the application of the aforementioned traditional Chinese medicine compound in the preparation of a medicament for treating and / or improving viral myocarditis.

[0013] The pharmacological effects of each herb in the herbal compound of this invention are as follows:

[0014] Forsythia: The dried fruit of Forsythia suspensa, a plant in the Oleaceae family. It tastes bitter and is slightly cold in nature, entering the lung, heart, and small intestine meridians. It has the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and dispersing wind-heat. It is used for carbuncles, scrofula, mastitis, erysipelas, wind-heat colds, the initial stage of febrile diseases, febrile diseases entering the nutritive level, high fever with thirst, delirium with rashes, and painful urination due to heat.

[0015] Golden buckwheat: The dried rhizome of *Fagopyrum cymosum*, a plant in the Polygonaceae family. It has a slightly pungent and astringent taste, is cool in nature, and enters the lung meridian; it has the effects of clearing heat and detoxifying, draining pus and removing blood stasis. It is used for lung abscess with purulent sputum, lung heat with cough and wheezing, and tonsillitis with swelling and pain.

[0016] Codonopsis pilosula: The dried tuberous root of Codonopsis pilosula, a plant in the Caryophyllaceae family. It has a sweet and slightly bitter taste, is neutral in nature, and enters the spleen and lung meridians; it has the effects of invigorating qi and strengthening the spleen, promoting body fluids and moistening the lungs. It is used for spleen deficiency and fatigue, loss of appetite, weakness after illness, qi and yin deficiency, spontaneous sweating and thirst, and dry cough due to lung dryness.

[0017] Rhodiola rosea: The dried root and rhizome of Rhodiola rosea, a plant in the Crassulaceae family. It has a sweet and bitter taste, is neutral in nature, and enters the lung and heart meridians; it has the effects of invigorating qi and blood circulation, clearing the meridians and relieving asthma. It is used for qi deficiency and blood stasis, chest pain, hemiplegia due to stroke, and fatigue with shortness of breath.

[0018] Citron: The dried, ripe fruit of *Citrus medica* or *Citrus medica*, plants of the Rutaceae family. It has a pungent, bitter, and sour taste, is warm in nature, and enters the liver, spleen, and lung meridians; it has the effects of soothing the liver and regulating qi, relieving chest congestion, and resolving phlegm. It is used for liver and stomach qi stagnation, chest and rib pain, abdominal distension, vomiting, belching, and cough with excessive phlegm.

[0019] Allium macrostemon: The dried bulb of Allium macrostemon or Allium chinense, a plant in the Liliaceae family. It has a pungent and bitter taste, and is warm in nature. It enters the heart, lung, stomach, and large intestine meridians; it has the effects of promoting yang and dispersing stagnation, regulating qi and relieving stagnation. It is used for chest pain, abdominal distension and pain, and tenesmus after diarrhea.

[0020] Poria cocos: The dried sclerotium of the fungus Poria cocos (family Polyporaceae). It has a sweet and bland taste, and is neutral in nature. It enters the heart, lung, spleen, and kidney meridians; it has the effects of promoting diuresis and eliminating dampness, strengthening the spleen, and calming the mind. It is used for edema with scanty urine, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, loose stools and diarrhea, restlessness, palpitations, and insomnia.

[0021] Dragon Beard Grass: The whole herb of *Juncus effusus*, a plant in the Juncaceae family. It has a sweet and bland taste, is cold in nature, and enters the Heart and Small Intestine meridians; it has the effects of promoting urination, relieving strangury, clearing heat, and calming the mind. It is used for painful urination, strangury due to heat, edema due to nephritis, dizziness, toothache, epistaxis, sore throat, irritability, insomnia, diabetes, and nocturnal emission.

[0022] Sal seeds: Dried, mature seeds of *Aesculus hippocastanum*, *Aesculus zeylanus*, or *Aesculus tianshiense*, all belonging to the Aesculaceae family. They are sweet in taste and warm in nature, entering the liver and stomach meridians; they have the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain. They are used for liver and stomach qi stagnation, chest and abdominal distension, and stomach pain.

[0023] Dalbergia odorifera: The dried heartwood of the trunk and roots of the Dalbergia odorifera plant, belonging to the legume family. It has a pungent taste and warm properties, and enters the liver and spleen meridians; it has the effects of resolving blood stasis and stopping bleeding, regulating qi and relieving pain. It is used for hematemesis, epistaxis, traumatic bleeding, liver stagnation and hypochondriac pain, chest pain, traumatic injuries, vomiting and abdominal pain.

[0024] Baiwei: The dried root and rhizome of *Cynanchum atratum* or *Cynanchum viniferum*, both belonging to the Asclepiadaceae family. It tastes bitter and salty, is cold in nature, and enters the stomach, liver, and kidney meridians. It has the effects of clearing heat and cooling the blood, promoting urination and relieving strangury, and detoxifying and healing sores. It is used for fever due to febrile diseases, fever due to yin deficiency, steaming bone fever, postpartum fever due to blood deficiency, strangury due to heat, hematuria, carbuncles, and boils.

[0025] Valerian: The root and rhizome of Valeriana and Valeriana niger, both belonging to the Valerianaceae family. It has a pungent and sweet taste, is warm in nature, and enters the heart and liver meridians; it has the effects of calming the mind, regulating qi, and relieving pain. It is used for neurasthenia, insomnia, hysteria, epilepsy, abdominal distension and pain, lower back and leg pain, and traumatic injuries.

[0026] (III) Beneficial Effects

[0027] This invention provides a traditional Chinese medicine compound for treating viral myocarditis. The compound consists of Forsythia suspensa, Fagopyrum dibotrys, Codonopsis pilosula, Rhodiola rosea, Citrus aurantium, Allium macrostemon, Poria cocos, Gynostemma pentaphyllum, Salvia miltiorrhiza, Dalbergia odorifera, Cynanchum paniculatum, and Valeriana. Its treatment principle is to detoxify, clear the heart, invigorate qi, and unblock the meridians. It closely addresses the core pathogenesis of viral myocarditis, namely "heat toxin stagnation and blood stasis obstructing the heart meridians." The specific analysis is as follows: Forsythia suspensa and Fagopyrum dibotrys are the principal herbs. Forsythia suspensa effectively penetrates heat to the exterior, clears the heart and drains fire, and detoxifies and disperses nodules. Fagopyrum dibotrys specializes in clearing heat and detoxifying, promoting blood circulation and eliminating carbuncles, and dispelling wind and dampness, possessing both detoxifying and blood-stasis-removing properties. The two herbs, one for the exterior and one for the interior, one penetrating and one clearing, directly target the core problem of heat toxin and blood stasis in viral myocarditis, working together to detoxify, remove blood stasis, and unblock the heart meridians. The formula uses four herbs as assistant herbs: Codonopsis pilosula, Rhodiola rosea, Citronella arvense, and Allium macrostemon. Codonopsis pilosula tonifies Qi and nourishes Yin, strengthens the spleen and moistens the lungs; Rhodiola rosea tonifies Qi and invigorates blood, unblocks meridians and relieves asthma; Citronella arvense soothes the liver and relieves depression, regulates Qi and relieves chest congestion; Allium macrostemon invigorates Yang, disperses stagnation, and promotes Qi circulation. The combination of these four herbs not only assists the principal herbs in strengthening the clearing and resolving effects, but also addresses the underlying deficiency of Qi and Yin, as well as the Qi stagnation due to Qi stagnation. The formula also uses five herbs as adjuvant herbs: Poria cocos, Gynostemma pentaphyllum, Spatholobus suberectus, Dalbergia odorifera, and Cynanchum paniculatum. Poria cocos invigorates the spleen and eliminates dampness, calms the mind and soothes the nerves; Gynostemma pentaphyllum clears heat, promotes diuresis, cools the blood and calms the nerves; Spatholobus suberectus soothes the liver and regulates Qi, relieves chest congestion and pain; Dalbergia odorifera invigorates blood, disperses stagnation, regulates Qi and relieves pain; Cynanchum paniculatum clears heat, cools the blood, nourishes Yin and relieves irritability. The combination of these five herbs not only precisely treats concurrent symptoms such as dampness, residual heat, and liver stagnation, but also harmonizes the properties of all the herbs in the formula, ensuring that clearing and resolving does not harm the body's vital energy, and tonifying does not hinder the elimination of pathogenic factors. Valerian is used as the guiding herb. It is good at calming the mind and soothing the nerves, regulating qi and relieving pain. It also has the triple functions of guiding the herbs to the meridians, calming the mind and relieving palpitations, and harmonizing the properties of the herbs. It can guide the herbs to act precisely on the chest and heart area, and can directly relieve the core symptoms of palpitations and insomnia in viral myocarditis. At the same time, it harmonizes the cold and hot properties of the herbs in the whole formula.

[0028] This invention systematically verified the protective effect and mechanism of action of the traditional Chinese medicine compound against viral myocarditis through cell and animal experiments, specifically as follows:

[0029] (1) This compound can significantly reduce the damage of H9c2 cardiomyocytes caused by CVB3 virus. Its mechanism of action is closely related to the downregulation of the expression of inflammatory factors TNF-α and IL-6 and the inhibition of inflammatory response. At the same time, the compound has no toxic effect on cardiomyocytes within the experimental concentration range, showing good biosafety.

[0030] (2) This compound can improve the general survival status and increase the survival rate of viral myocarditis model mice; reduce the levels of myocardial injury markers CK-MB and cTnI; inhibit the release of serum pro-inflammatory factors TNF-α, IL-6 and IL-1β; reduce the content of oxidative stress products MDA and ROS; and increase the activity of antioxidant enzyme SOD.

[0031] In summary, the traditional Chinese medicine compound of this invention exhibits a clear antiviral effect against myocardial damage. Its mechanism involves multiple pathways of regulation, including anti-inflammatory and antioxidant effects. Furthermore, the combination of the entire formula has the advantage of synergistic effect, providing a safe and effective new treatment option for the clinical treatment of viral myocarditis. Attached Figure Description

[0032] Figure 1 Figure A shows the survival status of mice in each group; Note: Figure A compares the survival rates of mice in each group. * P<0.05, ** P<0.01); Figure B shows the survival curves of mice in each group.

[0033] Figure 2 Comparison of serum inflammatory factors and oxidative stress markers in mice of different groups; Note: Compared with the blank control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0035] Example 1

[0036] A traditional Chinese medicine compound for treating viral myocarditis, comprising the following parts by weight: Forsythia suspensa 18 parts, Fagopyrum dibotrys 15 parts, Codonopsis pilosula 10 parts, Rhodiola rosea 15 parts, Citronella 12 parts, Allium macrostemon 12 parts, Poria cocos 15 parts, Gynostemma pentaphyllum 15 parts, Salvia miltiorrhiza 18 parts, Dalbergia odorifera 15 parts, Cynanchum paniculatum 15 parts, and Valerian 18 parts.

[0037] Example 2

[0038] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: 8 parts forsythia, 5 parts buckwheat, 3 parts codonopsis, 5 parts rhodiola, 3 parts citron, 3 parts allium macrostemon, 5 parts poria cocos, 5 parts sage, 8 parts salvia miltiorrhiza, 5 parts dalbergia odorifera, 5 parts cynanchum paniculatum, and 8 parts valerian.

[0039] Example 3

[0040] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: Forsythia suspensa 28 parts, Fagopyrum cymosum 25 parts, Codonopsis pilosula 20 parts, Rhodiola rosea 25 parts, Citronella cuspidatum 22 parts, Allium macrostemon 22 parts, Poria cocos 25 parts, Gynostemma pentaphyllum 25 parts, Salvia miltiorrhiza 28 parts, Dalbergia odorifera 25 parts, Cynanchum paniculatum 25 parts, and Valerian 28 parts.

[0041] Example 4

[0042] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: Forsythia 20 parts, Fagopyrum 18 parts, Codonopsis pilosula 12 parts, Rhodiola rosea 15 parts, Citron 14 parts, Allium macrostemon 16 parts, Poria cocos 17 parts, Gynostemma pentaphyllum 17 parts, Salvia miltiorrhiza 20 parts, Dalbergia odorifera 18 parts, Cynanchum paniculatum 18 parts, and Valerian 20 parts.

[0043] Example 5

[0044] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: 10 parts forsythia, 7 parts buckwheat, 5 parts codonopsis, 7 parts rhodiola, 5 parts citron, 5 parts allium macrostemon, 7 parts poria cocos, 7 parts spatholobus suberectus, 10 parts salsa, 7 parts dalbergia odorifera, 7 parts cynanchum paniculatum, and 10 parts valerian.

[0045] Example 6

[0046] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: Forsythia 25 parts, Fagopyrum 23 parts, Codonopsis pilosula 18 parts, Rhodiola rosea 22 parts, Citron 20 parts, Allium macrostemon 20 parts, Poria cocos 23 parts, Gynostemma pentaphyllum 22 parts, Salvia miltiorrhiza 25 parts, Dalbergia odorifera 22 parts, Cynanchum paniculatum 23 parts, and Valerian 25 parts.

[0047] Example 7

[0048] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: Forsythia 23 parts, Fagopyrum 20 parts, Codonopsis pilosula 15 parts, Rhodiola rosea 18 parts, Citron 17 parts, Allium macrostemon 18 parts, Poria cocos 20 parts, Gynostemma pentaphyllum 20 parts, Salvia miltiorrhiza 21 parts, Dalbergia odorifera 20 parts, Cynanchum paniculatum 20 parts, and Valerian 22 parts.

[0049] Example 8

[0050] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following parts by weight: 15 parts forsythia, 10 parts buckwheat, 8 parts codonopsis, 11 parts rhodiola, 9 parts citron, 8 parts allium macrostemon, 8 parts poria cocos, 8 parts sedge, 15 parts salsa, 10 parts dalbergia odorifera, 12 parts cynanchum paniculatum, and 15 parts valerian.

[0051] Experimental Example 1

[0052] 1. Materials

[0053] 1.1 Cell lines and virus strains

[0054] The H9c2 rat cardiomyocyte line was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0055] Coxsackievirus B3 (CVB3, Nancy strain) was revitalized and amplified in HEp-2 cells before use.

[0056] 1.2 Experimental Drugs

[0057] Test substance 1: Forsythia suspensa 18 g, Fagopyrum cymosum 15 g, Codonopsis pilosula 10 g, Rhodiola rosea 15 g, Citronella 12 g, Allium macrostemon 12 g, Poria cocos 15 g, Gynostemma pentaphyllum 15 g, Salvia miltiorrhiza 18 g, Dalbergia odorifera 15 g, Cynanchum paniculatum 15 g, Valerian 18 g.

[0058] Test substance 2: 15 g of buckwheat, 10 g of codonopsis, 15 g of rhodiola rosea, 12 g of citron, 12 g of allium macrostemon, 15 g of poria cocos, 15 g of dalbergia odorifera, 15 g of cynanchum paniculatum, and 18 g of valerian.

[0059] Test substance 3: Forsythia suspensa 18 g, Codonopsis pilosula 10 g, Rhodiola rosea 15 g, Citronella 12 g, Allium macrostemon 12 g, Poria cocos 15 g, Gynostemma pentaphyllum 15 g, Salvia miltiorrhiza 18 g, Valerian 18 g.

[0060] Test substance 4: Forsythia 18 g, Fagopyrum 15 g, Citron 12 g, Allium macrostemon 12 g, Gynostemma pentaphyllum 15 g, Salvia miltiorrhiza 18 g, Dalbergia odorifera 15 g, Cynanchum paniculatum 15 g, Valerian 18 g.

[0061] Test substance 5: Forsythia 18 g, Fagopyrum 15 g, Codonopsis 10 g, Rhodiola 15 g, Poria 15 g, Gynostemma pentaphyllum 15 g, Salvia miltiorrhiza 18 g, Dalbergia odorifera 15 g, Cynanchum paniculatum 15 g.

[0062] Preparation process: Weigh each medicinal material according to the above weight ratio, add 10 times the volume of distilled water, soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 1 hour, filter and collect the filtrate; add 8 times the volume of distilled water to the dregs, simmer over low heat for 40 minutes, filter and collect the second filtrate; combine the two filtrates, concentrate to a crude drug concentration of 1 g / mL using a rotary evaporator, filter through a 0.22 μm filter membrane to remove bacteria to obtain test substances 1-5, aliquot and store at -20℃ for later use, and re-dilute to the required concentration at 37℃ before use.

[0063] 2 Experimental Methods

[0064] 2.1 Cell Culture

[0065] H9c2 cells were cultured in DMEM medium (hereinafter referred to as "DMEM complete medium") containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 U / mL penicillin and 100 μg / mL streptomycin) at 37°C in a cell culture incubator with 5% CO2. When the cell confluence reached 80%-90%, the cells were washed twice with PBS, digested with 0.25% trypsin-EDTA digestion solution, and then passaged in DMEM complete medium. Cells in the logarithmic growth phase were used for subsequent experiments.

[0066] 2.2 Preparation of CVB3 virus and determination of median tissue infection dose

[0067] The median tissue infectious dose (TCID) was determined using a 10-fold serial dilution method. 50 The virus was serially diluted to 10⁻⁶ using serum-free DMEM medium. -1 -10 -10 Ten concentrations were selected and seeded into 96-well plates pre-treated with generic H9c2 cells, with 100 μL seeded per well and three replicates per concentration. Three controls were prepared in serum-free DMEM medium. Cells were cultured at 37°C and 5% CO2 for 2-3 days. Cardiomyocyte pathogenesis was observed daily under an inverted microscope. A positive result was defined as more than 50% of cells exhibiting pathological changes such as shrinkage, detachment, and lysis. TCID was calculated using the Reed-Muench method. 50 10 -4.2 100 TCID was used in the experiment. 50 / The virus infects the hole.

[0068] 2.3 Model Preparation

[0069] H9c2 cells in logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 3 100 μL of the test substance was seeded per well in a 96-well plate. A blank control group, a model control group, and groups 1-5 of the test substance were included, with three replicates per group. The plates were incubated at 37°C and 5% CO2 for 24 h. Except for the blank control group, each of the other groups received 20 μL of CVB3 virus solution (final concentration 100 TCID50). 50 / well), incubate at 37℃ for 2 h, discard the virus solution, and wash the cells twice with PBS buffer. Test groups 1-5 were added to culture medium containing the corresponding traditional Chinese medicine solution (final concentration 50 mg / mL, 100 μL / well), while the blank control group and model control group were added to an equal volume of serum-free DMEM medium, and cultured for another 48 h.

[0070] 2.4 Detection Indicators

[0071] 2.4.1 Cell viability

[0072] After culturing for 48 h, 10 μL of CCK-8 was added to each well, and the cells were incubated in a cell culture incubator at 37℃ and 5% CO2 for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0073] Cell viability (%) = (OD) 给药组 -OD 空白对照组 ) / (OD 模型对照组 -OD 空白对照组 ) × 100%

[0074] 2.4.2 Lactate dehydrogenase

[0075] After culturing for 48 h, the cell culture medium from each well was collected and centrifuged at 3000 r / min for 5 min at 4℃. The supernatant was then collected. Reagents were added sequentially according to the LDH detection kit instructions, and the mixture was incubated at 37℃ for 15 min. After terminating the reaction, the OD value at 450 nm was measured using a microplate reader, and the LDH activity of each group was calculated based on the standard curve.

[0076] 2.4.3 Inflammatory factors

[0077] The cell culture supernatant after centrifugation was collected, and the secretion levels of TNF-α and IL-6 in each group were detected by enzyme-linked immunosorbent assay (ELISA). The ELISA kit instructions were strictly followed: standards and samples were added, and the mixture was incubated at 37°C for 1 h. After washing, enzyme-labeled secondary antibody was added, and incubation continued for 30 min. Color development was performed for 15 min, and the reaction was terminated with stop solution. The OD value at 450 nm was measured using a microplate reader, and the secretion levels of TNF-α and IL-6 were calculated based on the standard curve.

[0078] 2.5 Statistical Methods

[0079] Data were processed using SPSS 25.0 software, and experimental data are expressed as mean ± standard deviation. This indicates that one-way ANOVA was used for comparisons among multiple groups. P < 0.05 indicates that the difference is statistically significant.

[0080] 3 Results

[0081] 3.1 Effects of different traditional Chinese medicine compound formulas on cardiomyocyte viability

[0082] As shown in Table 1, compared with the blank control group, the cell viability of the model control group decreased significantly to 51.96% (P<0.01), indicating that viral infection caused severe damage to cardiomyocytes; while the cell viability of test group 1-5 was not significantly different from that of the blank control group (P>0.05), indicating that the traditional Chinese medicine compound of the present invention has no obvious toxic effect on cardiomyocytes and can effectively resist the inhibition of cell viability by CVB3 virus.

[0083] 3.2 Effects of different traditional Chinese medicine compound formulas on lactate dehydrogenase activity

[0084] Table 1 shows that, compared with the blank control group, the LDH activity in the cell culture supernatant of the model control group was significantly increased (P<0.01), indicating that viral infection caused damage to the cardiomyocyte membrane. Compared with the model control group, the LDH content in the cell culture supernatant of test substance groups 1-5 was significantly decreased (P<0.01), with the largest decrease in LDH activity in test substance group 1, reaching 43%. These results indicate that the traditional Chinese medicine composition of the present invention can effectively protect the integrity of the cardiomyocyte membrane and alleviate CVB3 virus-induced cardiomyocyte damage.

[0085] 3.3 Effects of different traditional Chinese medicine compound formulas on the levels of inflammatory factors

[0086] As shown in Table 1, compared with the blank control group, the levels of TNF-α and IL-6 in the model control group were significantly increased (P<0.01), indicating that CVB3 virus infection triggered an inflammatory stress response in cells. Compared with the model control group, the levels of TNF-α and IL-6 in the cell culture supernatant of test substance groups 1-5 were decreased to varying degrees (P<0.05, P<0.01), with the most significant decrease in test substance group 1. This indicates that the traditional Chinese medicine compound of the present invention can inhibit the excessive inflammatory response caused by viral infection by downregulating the expression of inflammatory factors, thereby reducing inflammation-mediated cardiomyocyte damage.

[0087] Table 1. Effects of different traditional Chinese medicine compound formulas on cardiomyocyte viability, LDH activity, and inflammatory factor secretion.

[0088] Group Cell viability (%) LDH(U / L) TNF-α (pg / mL) IL-6 (pg / mL) Blank control group 100.00±0.00 273.82±17.78 137.62±14.17 71.93±8.65 Model control group <![CDATA[51.96±7.36 ## ]]> <![CDATA[614.56±28.66 ## ]]> <![CDATA[309.03±17.11 ## ]]> <![CDATA[153.62±10.52 ## <!-- 6 -->]]> Group 1 of test substances 104.89±8.08 <![CDATA[350.11±23.12 ** ]]> <![CDATA[195.11±13.09 ** ]]> <![CDATA[86.71±5.65 ** ]]> Group 2 of test substances 97.55±8.99 <![CDATA[410.21±16.39 ** ]]> <![CDATA[247.25±19.25 ** ]]> <![CDATA[103.04±8.34 ** ]]> Group 3 of test substances 99.17±6.95 <![CDATA[481.58±17.21 ** ]]> 274.20±13.42 131.34±10.69 Group 4 of test substances 102.91±7.65 <![CDATA[501.14±15.39 ** ]]> <![CDATA[213.65±12.79 ** ]]> <![CDATA[108.25±12.15 ** ]]> Group 5 of test substances 102.82±6.90 <![CDATA[458.22±28.07 ** ]]> <![CDATA[260.40±21.08 * ]]> <![CDATA[123.56±11.08 * ]]>

[0089] Note: Compared with the blank control group ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0090] 4. Conclusion

[0091] This experiment used CVB3 virus to infect H9c2 cardiomyocytes to construct a viral myocarditis cell injury model. By detecting cell viability, LDH activity, and inflammatory factor levels, the protective effect of the traditional Chinese medicine compound of this invention on infected cardiomyocytes was evaluated. The following conclusions were drawn: The traditional Chinese medicine compound of this invention can significantly alleviate CVB3 virus-induced cardiomyocyte damage. Its mechanism of action is closely related to the downregulation of inflammatory factors (TNF-α, IL-6) expression, thereby inhibiting the inflammatory response. Furthermore, the traditional Chinese medicine compound at the experimental concentration showed no toxicity to H9c2 cardiomyocytes, demonstrating good biocompatibility. In summary, the traditional Chinese medicine compound of this invention has a significant protective effect against CVB3 infection-induced cardiomyocyte damage, and the complete formula (test substance 1) showed the best overall improvement effect in all indicators, significantly superior to other simplified formulations, verifying the scientific and rational nature of the formula's formulation structure.

[0092] Experimental Example 2

[0093] 1. Materials

[0094] 1.1 Laboratory Animals

[0095] Six-week-old SPF-grade male BALB / c mice, weighing 17-21 g, were acclimatized for 7 days (hospital environment: temperature 22-25℃, relative humidity 50%-60%, 12 h / 12 ​​h diurnal rhythm, free access to food and water). After confirming good health, the mice were used for subsequent experiments.

[0096] 1.2 Experimental Drugs

[0097] The ingredients are: Forsythia 18 g, Fagopyrum 15 g, Codonopsis 10 g, Rhodiola 15 g, Citron 12 g, Allium macrostemon 12 g, Poria cocos 15 g, Gynostemma pentaphyllum 15 g, Salvia miltiorrhiza 18 g, Dalbergia odorifera 15 g, Cynanchum paniculatum 15 g, and Valerian 18 g.

[0098] Preparation process: Weigh each medicinal material according to the above weight ratio, add 10 times the volume of distilled water, soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 1 hour, filter and collect the filtrate; add 8 times the volume of distilled water to the dregs, simmer over low heat for 40 minutes, filter and collect the second filtrate; combine the two filtrates, concentrate to a crude drug concentration of 1 g / mL using a rotary evaporator, filter through a 0.22 μm filter membrane for sterilization, dispense into portions, freeze at -20℃ for later use, warm to 37℃ before use, and dilute with physiological saline to three concentrations of 50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively as low, medium, and high dose administration solutions of the traditional Chinese medicine compound of this invention.

[0099] 2 Methods

[0100] 2.1 Model Preparation

[0101] Sixty male BALB / c mice were randomly divided into six groups of ten mice each: a blank control group, a model control group, a low-dose group (50 mg / kg), a medium-dose group (100 mg / kg), a high-dose group (200 mg / kg), and a positive control group (ribavirin). Except for the blank control group, the other five groups of mice were inoculated intraperitoneally with a diluted CVB3 virus solution (concentration 1×10⁻⁶). 6 The positive control group received 0.2 mL of ribavirin per mouse (PFU / mL), while the blank control group received an equal volume of physiological saline. Drug administration began 2 hours after model establishment. The low, medium, and high dose groups of the herbal compound of this invention were administered 20 mL / kg of the corresponding drug solution by gavage. The positive control group received 0.06 g / kg ribavirin, while the blank control group and the model control group received an equal volume of physiological saline by gavage. Administration was once daily for 14 consecutive days.

[0102] 2.2 Detection Indicators

[0103] 2.2.1 General Situation

[0104] During the experiment, the general condition of mice in each group was observed and recorded daily, including food and water intake, mental state, fur condition, and survival status. After the experiment, the survival rate of mice in each group was calculated, and survival curves were plotted.

[0105] 2.2.2 Myocardial Injury Markers

[0106] After the last administration, mice were anesthetized intraperitoneally with 10% chloral hydrate (3 mL / kg) and euthanized by cervical dislocation. Heart tissue was isolated, washed three times with pre-cooled PBS to remove blood and connective tissue, and myocardial tissue was collected. Nine times the volume of pre-cooled PBS buffer was added, and the homogenate was prepared into a 10% myocardial tissue homogenate using a tissue homogenizer in an ice bath. The homogenate was centrifuged at 10,000 r / min for 10 min at 4°C, and the supernatant was collected. The levels of creatine kinase isoenzyme (CK-MB) and cardiac troponin I (cTnI) in myocardial tissue were detected strictly according to the ELISA kit instructions.

[0107] 2.2.3 Detection of immune and oxidative stress indicators

[0108] After the last administration, mice were anesthetized intraperitoneally with 10% chloral hydrate, and 3 mL of blood was collected from the abdominal aorta. After standing at room temperature for 30 min, the blood was centrifuged at 4℃ and 3000 r / min for 15 min to separate the serum. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), as well as malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and superoxide dismutase (SOD) activity in the serum were detected strictly according to the ELISA kit instructions.

[0109] 2.3 Statistical Methods

[0110] Data were processed using SPSS 25.0 software, and experimental data are expressed as mean ± standard deviation. This indicates that one-way ANOVA was used for comparisons among multiple groups. P < 0.05 indicates that the difference is statistically significant.

[0111] 3 Results

[0112] 3.1 Effects of different dosages on the general condition and survival rate of mice

[0113] The mice in the blank control group were in good mental condition, with stable food intake, active spontaneous activity, smooth and shiny fur, and normal behavior. The mice in the model control group, the low, medium and high dose groups of the traditional Chinese medicine compound of this invention and the positive control group all showed varying degrees of significantly reduced food intake, lethargy, fluffy and messy fur, puffing up hair and arching back and curling up after modeling, indicating that the overall condition of the mice was significantly affected after modeling.

[0114] Observe the survival status of mice ( Figure 1 In the control group, all mice survived (100% survival rate). In the model control group, only 3 mice survived (30% survival rate, 3 / 10), significantly lower than the control group (P<0.01), indicating that experimental myocardial injury can significantly increase the mortality rate in mice. In the low-dose group of the herbal compound of this invention, 5 mice survived (50% survival rate, 5 / 10); in the medium-dose group, 6 mice survived (60% survival rate, 6 / 10); in the high-dose group, 8 mice survived (80% survival rate, 8 / 10); and in the positive control group, 9 mice survived (90% survival rate, 9 / 10). The survival rate of each dose group of the herbal compound of this invention showed a gradient increasing trend, and the survival rate of the high-dose group was significantly higher than that of the model control group (P<0.01), while there was no statistically significant difference in survival rate between the high-dose group and the positive control group (commonly used myocardial protective drugs group) (P>0.05).

[0115] The above results indicate that the traditional Chinese medicine composition of the present invention can effectively improve the condition of mice with viral myocarditis, significantly increase the survival rate of mice, and the protective effect has a clear dose-dependent effect.

[0116] 3.2 Effects of different dosages on the levels of myocardial injury markers in mice

[0117] As shown in Table 2, compared with the blank control group, the levels of CK-MB and cTnI in the myocardial tissue of mice in the model control group were significantly increased (P<0.01), confirming the successful establishment of the mouse myocardial injury model. Compared with the model control group, the levels of CK-MB and cTnI in the myocardial tissue of mice in each dose group of the traditional Chinese medicine compound of the present invention were significantly decreased. Among them, the high dose group had the most significant downregulation effect on the two injury markers, and its index level was comparable to that of the positive control group, indicating that the traditional Chinese medicine composition of the present invention can effectively reduce the degree of myocardial injury.

[0118] Table 2. Levels of myocardial injury markers in mice of each group

[0119] Group CK-MB (ng / mL) cTnI (ng / mL) Blank control group 38.27±4.93 0.028±0.009 Model control group <![CDATA[136.07±10.03 ## ]]> <![CDATA[0.059±0.008 ## ]]> low-dose group <![CDATA[115.57±11.06 * ]]> 0.052±0.004 medium dose group <![CDATA[89.10±6.44 ** ]]> <![CDATA[0.044±0.003 * ]]> High-dose group <![CDATA[68.52±5.97 ** ]]> <![CDATA[0.038±0.004 ** ]]> Positive control group <![CDATA[53.02±6.25 ** ]]> <![CDATA[0.032±0.005 ** ]]>

[0120] Note: Compared with the blank control group ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0121] 3.3 Effects of different dosages on immune and oxidative stress indicators in mice

[0122] Depend on Figure 2 It was found that, compared with the blank control group, the serum levels of inflammatory factors TNF-α, IL-6, and IL-1β in the model control group mice were significantly increased (P<0.01), the levels of oxidative stress indicators MDA and ROS were significantly increased (P<0.01), and the activity of SOD was significantly decreased (P<0.01), indicating that there was a significant inflammatory response and oxidative stress imbalance in the mice in the viral myocardial injury model. Compared with the model control group, the serum levels of TNF-α, IL-6, and IL-1β in the low, medium, and high dose groups of the traditional Chinese medicine compound of this invention were significantly decreased (P<0.05, P<0.01), the levels of MDA and ROS were decreased (P<0.05, P<0.01), and the activity of SOD was significantly increased (P<0.05, P<0.01). Among them, the high dose group had the most significant inhibitory effect on inflammatory factors and the most significant regulatory effect on oxidative stress, indicating that the traditional Chinese medicine compound of this invention can exert a cardioprotective effect by inhibiting the excessive activation of inflammatory response and regulating the balance of oxidative stress, and this effect has a clear dose-dependent effect.

[0123] 4. Conclusion

[0124] This experiment, using a mouse model of viral myocarditis, demonstrated that the traditional Chinese medicine compound of this invention can significantly reduce myocardial damage, improve overall survival, and increase survival rate in mice by inhibiting inflammatory responses and regulating oxidative stress imbalance. Specifically, the compound significantly improved the general condition of model mice, such as lethargy and reduced food intake, and effectively increased their survival rate; it significantly downregulated the abnormally elevated levels of myocardial injury-specific markers CK-MB and cTnI in the myocardial tissue of model mice; simultaneously, it inhibited the excessive release of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of model mice, reduced the levels of oxidative stress products MDA and ROS, and increased the activity of the antioxidant enzyme SOD. In summary, the traditional Chinese medicine compound of this invention has a clear cardioprotective effect on mouse models of viral myocarditis, providing a safe and effective new treatment option for the clinical treatment of viral myocarditis.

[0125] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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 of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine compound for treating viral myocarditis, characterized in that, The traditional Chinese medicine compound is composed of the following ingredients in parts by weight: 18 parts forsythia, 15 parts buckwheat, 10 parts codonopsis, 15 parts rhodiola, 12 parts citron, 12 parts allium macrostemon, 15 parts poria cocos, 15 parts sedge, 18 parts salsa, 15 parts dalbergia odorifera, 15 parts cynanchum paniculatum, and 18 parts valerian.

2. The application of the traditional Chinese medicine compound as described in claim 1 in the preparation of a drug for treating viral myocarditis.

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