Use of Houttuynia cordata-associated flavonoids in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections

Abstract

This invention relates to the use of Houttuynia cordata-related flavonoids in the preparation of drugs for the prevention and treatment of viral-bacterial co-infections, specifically to the novel use of total Houttuynia cordata flavonoids, quercetin, and quercetin in the preparation of drugs for interstitial pneumonia caused by viral-bacterial co-infections. Total Houttuynia cordata flavonoids are extracted from Houttuynia cordata. Compared with existing technologies, the total Houttuynia cordata flavonoids, quercetin, and quercetin of this invention have significant therapeutic effects on pneumonia caused by viral-bacterial co-infections.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the use of a type of houttuynia cordata-related flavonoid in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections. Background Technology

[0002] Severe viral-induced lung infections are a key factor threatening human life and health, and influenza virus is one of the important factors causing infectious respiratory diseases and a major cause of death worldwide. However, the high mortality rate caused by influenza virus (H1N1) infection is often closely related to secondary bacterial infections during influenza virus infection (Hussell T, Wissinger E et al. Bacterial complications during pandemic influenza infection. Future Microbiology, 2009, 4:269-272.). For example, the 1918 "Spanish flu" was the most severe influenza pandemic in history, with most deaths due to co-infection with influenza virus and bacteria; during the 2009 H1N1 influenza pandemic, 25%-50% of hospitalized patients had viral infection with bacterial pneumonia, significantly increasing their risk of developing the disease. Among the several bacteria that cause pneumonia, *Streptococcus pneumoniae*, *Staphylococcus aureus*, and *Haemophilus influenzae* have been reported to induce secondary infections following influenza virus infection (Centers for Disease Control and Prevention, 2009. Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1). Morbidity and Mortality Weekly Report, 2009, 58:1071-1074.). Recently, *Staphylococcus aureus* has increasingly been recognized as an important influenza-related pathogen in pneumonia cases occurring in many countries and regions worldwide. Simultaneously, the emergence of drug-resistant mutant strains, such as methicillin-resistant clones USA300 and USA400 (MRSA), has further complicated the treatment of viral-bacterial co-infection pneumonia. Current clinical treatment strategies for pneumonia caused by viral and bacterial co-infection include the use of antiviral drugs and antibiotics, vaccine prevention, and supportive care. However, the high mutation rate of viruses, bacterial resistance, and the accompanying overactive immune response in the lungs have limited effectiveness in these clinical prevention and treatment methods. Therefore, it is imperative to find new drug treatment options for severe pneumonia caused by viral and bacterial co-infection.

[0003] Houttuynia cordata, or the whole herb including roots, is a plant belonging to the Saururaceae family. It is a commonly used traditional Chinese medicine for clearing heat and detoxifying, possessing properties such as clearing heat and detoxifying, promoting diuresis, and eliminating carbuncles and pus. It is used to treat phlegm-heat cough, lung abscess with purulent sputum, and other ailments. Flavonoids are the main active components of Houttuynia cordata, mostly existing in the form of glycosides or aglycones. The total flavonoids of Houttuynia cordata mainly consist of quercetin, quercetin, isoquercetin, rutin, and hyperoside, with quercetin being the common aglycone of the other four flavonoids. Invention patents CN109288914A and CN109908223A disclose the specific use of drugs containing total flavonoids from Houttuynia cordata in the preparation of drugs for the prevention and treatment of influenza A and viral pneumonia. Previous studies have confirmed that quercetin, hyperoside, quercetin, and total flavonoids from Houttuynia cordata have a clear therapeutic effect on influenza A virus-induced pneumonia (Jiayue Tang, Lishuang Zhou et al. Therapeutic effects on H1N1-induced pneumonia in mice and intestinalbacteria biotransformation of four main flavonoids from Houttuynia cordata Thunb. Journal of Pharmaceutical and Biomedical Analysis, 2023, 233:115469). However, the microenvironment of viral-bacterial co-infection is more complex, leading to a faster and more severe course of pneumonia. The effects of the aforementioned Houttuynia cordata-related flavonoids, which are effective against single influenza virus infection, on co-infection are unknown. Similar reports have shown that total flavonoids from Houttuynia cordata have a good therapeutic effect on LPS-induced acute lung injury in mice (Lee JH, Ahn J et al. Flavonoids from theaerial parts of Houttuynia cordata attenuate lung inflammation in mice. Archives of Pharmacal Rerearch, 2015, 7(38): 1304-1311.). However, endotoxin LPS is only a component of the outer wall of Gram-negative bacteria and cannot replace live bacterial infection, let alone explain its effect on Gram-positive bacterial infection, especially the situation of drug-resistant bacterial strains.Current research shows that quercetin, isoquercetin, rutin, and quercetin in Houttuynia cordata-related flavonoids do not have direct anti-Staphylococcus aureus activity, but they do have inhibitory effects on the Staphylococcus aureus virulence factor Coa or adhesion protein Sortase A (Liu Bingrun. Study on the inhibitory effect and mechanism of quercetin on Staphylococcus aureus Sortase A [D]. Jilin University, 2015; Li Bangbang. Study on the inhibitory effect and mechanism of quercetin on Staphylococcus aureus Coa [D]. Jilin University, 2018; Wang Yanan. Inhibitory effect of rutin on Staphylococcus aureus Sortase A). Preliminary study on the inhibitory effect of A [D]. Jilin University, 2013; Gao Zeyuan. Study on the inhibitory effect of isoquercitrin on the coagulase Coa activity of Staphylococcus aureus [D]. Jilin University, 2020.), while the total flavonoids of Houttuynia cordata and Houttuynia cordata extract have only been studied for the treatment of mycoplasma / Klebsiella pneumoniae (Gram-negative bacteria) pneumonia (Li Xiangfeng, Chen Wenxia. Chinese Journal of Immunology. 2020, 36(14):1695-1699; Wang Xiaoxin, Bai Jing, Ren Zhihui, Wu Wei, Feng Xia. Chinese Journal of Nosocomial Infection. 2022, 32(22):3375-3379.), and have not yet involved Gram-positive bacteria, especially drug-resistant strains. In summary, current patents and research only report the effects of Houttuynia cordata-related flavonoids on single infections (single viral or bacterial infections). Since viruses and bacteria are two completely different pathogens, their viral replication and bacterial colonization in a co-infection state are vastly different from single infections, which can easily induce severe pneumonia. The pathological state and immune stress complexity are significantly higher than those of single viral or bacterial infections and LPS-induced acute lung injury. Currently, there are no reports on the efficacy of Houttuynia cordata-related flavonoids in viral and bacterial co-infections and their symptoms, as well as on the preparation of corresponding drugs. Summary of the Invention

[0004] The purpose of this invention is to provide the use of houttuynia cordata-related flavonoids in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections.

[0005] The objective of this invention can be achieved through the following technical solution: the use of houttuynia cordata-related flavonoids in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections, and the use of houttuynia cordata-related flavonoids in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections.

[0006] Preferably, the houttuynia cordata-related flavonoids include one or more of quercetin, quercetin, isoquercetin, rutin, hyperoside, and total houttuynia cordata flavonoids.

[0007] More preferably, the total flavonoids from Houttuynia cordata are obtained by ethanol extraction from Houttuynia cordata.

[0008] More preferably, the total flavonoids from Houttuynia cordata contain quercetin, quercetin, isoquercetin, rutin, and hyperoside.

[0009] More preferably, the total flavonoids of Houttuynia cordata contain the following contents: quercetin 26.21%, hyperoside 12.16%, rutin 5.28%, isoquercetin 3.64%, and quercetin content less than 1%, but the metabolites of the four major flavonoids all contain quercetin.

[0010] More preferably, the houttuynia cordata-related flavonoids include quercetin, quercetin, and total houttuynia cordata flavonoids.

[0011] More preferably, the method for preparing the total flavonoids from Houttuynia cordata includes the following steps:

[0012] After being pulverized, Houttuynia cordata was extracted three times by reflux in an 80°C water bath with 70% ethanol. The extract was collected and concentrated. The ethanol extract was then enriched and purified using macroporous resin (AB-8). After elution with 7 column volumes of 10% ethanol, 5 column volumes of 70% ethanol were added. Finally, 2-3 column volumes of the 70% ethanol eluent were collected, concentrated, and dried to obtain purified total flavonoids from Houttuynia cordata.

[0013] Preferably, the viruses involved in the co-infection include respiratory viruses.

[0014] More preferably, the respiratory virus includes influenza A virus and various subtypes of influenza B virus.

[0015] More preferably, the influenza A virus includes H1N1 and H3N2.

[0016] Preferably, the bacteria involved in the co-infection include clinical respiratory infection strains.

[0017] More preferably, the clinical respiratory infection strains include methicillin-resistant Staphylococcus aureus (MRSA), non-drug-resistant Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, and Pseudomonas aeruginosa.

[0018] Preferably, houttuynia cordata-related flavonoids are used to prepare drugs for the prevention and treatment of H1N1-MRSA co-infection.

[0019] Preferably, houttuynia cordata-related flavonoids are used to prepare drugs for the prevention and treatment of respiratory infections caused by viral and bacterial co-infection.

[0020] More preferably, the respiratory infection includes pneumonia and bronchitis.

[0021] More preferably, the respiratory infection is pneumonia.

[0022] More preferably, the pneumonia is interstitial pneumonia.

[0023] Preferably, the drug for preventing and treating viral and bacterial co-infection is an oral or injectable drug.

[0024] Preferably, the antiviral and bacterial co-infection drug does not need to be mixed with other ingredients.

[0025] Preferably, the dosage of the drug for preventing and treating viral and bacterial co-infection is 100 mg / kg.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. This invention provides a novel application of houttuynia cordata-related flavonoids, an important active ingredient in the traditional Chinese medicine houttuynia cordata, in the prevention and treatment of viral and bacterial co-infections;

[0028] 2. This invention provides a novel use of total flavonoids, quercetin, and quercetin from Houttuynia cordata in the preparation of drugs for the prevention and treatment of interstitial pneumonia caused by viral and bacterial co-infection;

[0029] 3. The total flavonoids, quercetin, and quercetin in the Houttuynia cordata-related flavonoids of this invention can inhibit viral and bacterial co-infection and significantly improve pneumonia induced by them;

[0030] 4. This invention demonstrates, through a mouse pneumonia model induced by viral and bacterial co-infection, that total flavonoids, quercetin, and quercetin from Houttuynia cordata have significant therapeutic effects on pneumonia caused by co-infection, including: significantly improving the survival rate and total survival days of mice, reducing the lung index and lung lesions in experimental mice, and simultaneously reducing the viral load and bacterial colonization in lung tissue. Attached Figure Description

[0031] Figure 1 The present invention investigates the effect of Houttuynia cordata-related flavonoids on the rate of change in body weight in mice co-infected with viruses and bacteria.

[0032] Figure 2 This invention investigates the effect of Houttuynia cordata-related flavonoids on the survival rate of mice co-infected with viruses and bacteria.

[0033] Figure 3 This invention investigates the effect of Houttuynia cordata-related flavonoids on the survival days of mice co-infected with viruses and bacteria.

[0034] Figure 4 The present invention investigates the effects of Houttuynia cordata-related flavonoids on the pathological state of the lungs in mice co-infected with viruses and bacteria.

[0035] Figure 5 The present invention investigates the effect of Houttuynia cordata-related flavonoids on lung imaging in mice co-infected with viruses and bacteria.

[0036] Figure 6 The present invention investigates the effect of Houttuynia cordata-related flavonoids on viral load in the lungs of mice co-infected with viruses and bacteria.

[0037] Figure 7The present invention investigates the effect of Houttuynia cordata-related flavonoids on bacterial colonization in the lungs of mice co-infected with viruses and bacteria.

[0038] In the picture: ** Compared with the model group, P<0.01. *** Compared with the model group, P < 0.001. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0040] In the following examples, the total flavonoids of Houttuynia cordata were isolated from Houttuynia cordata extract, and quercetin, isoquercetin, rutin, hyperoside, and quercetin (purity HPLC>98%) were all purchased from Chengdu Kloma Biotechnology Co., Ltd.

[0041] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0042] Example 1

[0043] Effects of Houttuynia cordata-related flavonoids on body weight, survival rate, and survival days in mice co-infected with viruses and bacteria: Ninety 15-16g female C57 mice were randomly divided into three groups according to body weight: a normal control group, a model control group, a group containing total Houttuynia cordata flavonoids-100, quercetin-100, isoquercetin-100, rutin-100, hyperoside-100, and quercetin-100 (all flavonoid dosages were 100 mg / kg), and a positive control group (22.75 mg / kg, oseltamivir phosphate), with 10 mice in each group. All mice were infected with H1N1 virus on day 0 and with MRSA on day 3. Body weight changes were observed over the following 14 days after infection. Mice in the normal control group showed good spirits, agile movement, shiny fur, normal respiration and appetite, and natural weight gain. In the model group mice, after co-infection with the virus and bacteria, the disease progressed rapidly, manifesting as lethargy, difficulty breathing, chills, decreased appetite, and rapid weight loss. The rate of weight change was calculated by dividing the daily weight of the mice by their weight on day 0 after infection. The results showed (…). Figure 1 In the model control group, the body weight of mice reached its lowest point on day 9. Compared with the model control group, the weight loss rate was slowed in the groups containing total flavonoids from Houttuynia cordata, quercetin, isoquercetin, rutin, hyperoside, and quercetin.

[0044] Mice were observed for survival for 14 days after infection, and the results were as follows: Figure 2The results showed that the survival rate of mice in the normal group was 100.0%; the survival rate of the model control group was 0%; the survival rate of the quercetin-100 group was 20.0%; the survival rates of the total flavonoids-100, isoquercetin-100, rutin-100, and hyperoside-100 groups were all 10.0%; and the survival rate of mice in the positive control drug-22.75 group was 30.0%. Meanwhile, the average survival days in the quercetin-100 group and the positive control drug-22.75 group (…) Figure 3 The mean survival days for the Houttuynia cordata total flavonoids-100 group and the isoquercitrin-100 group were 10.2±2.2 and 11.3±2.3, respectively. The mean survival days for the other Houttuynia cordata total flavonoids-100 group, isoquercitrin-100 group, rutin-100 group, hyperoside-100 group, and quercetin-100 group were 8.8±1.9, 8.4±2.1, 9.0±1.8, 9.0±2.3, and 9.1±2.3, respectively. The treated mice showed a trend of prolonged survival compared to the model control group (7.3±0.67 days), but the survival days for quercetin-100 and positive control group (22.75) were significantly different from those in the model group. In conclusion, Houttuynia cordata-related flavonoids all improved the reduction in body weight, decreased survival rate, and reduced mean survival time in mice caused by co-infection.

[0045] Example 2

[0046] The therapeutic effect of houttuynia cordata-associated flavonoids on interstitial pneumonia induced by viral-bacterial co-infection in mice:

[0047] (1) The therapeutic effect of Houttuynia cordata-related flavonoids on viral and bacterial co-infection-induced pneumonia in mice: The method was the same as before. Fifty-four female C57 mice weighing 15-16g were selected and grouped as above, with 6 mice in each group. Samples were collected on the 5th day. Pathological analysis showed that the alveolar outlines of mice in the normal group were clear, with complete alveolar structure, no bleeding, and no inflammation; the alveolar walls of mice in the model control group were significantly thickened, the alveoli were atrophied and deformed, and the blood vessels were dilated, showing a severe pneumonia state. The alveolar structure of mice in the quercetin-100 and positive drug-22.75 groups was intact, with no severe hemorrhage, and the lung inflammation was significantly reduced; the lung immune cell infiltration of mice in the other Houttuynia cordata total flavonoids-100 group, isoquercetin-100 group, rutin-100 group, hyperoside-100 group, and quercetin-100 group was reduced, but there were still vasodilation and other phenomena (see appendix). Figure 4(See table 1). The lung index is the ratio of lung weight to body weight in mice; a higher value indicates more severe inflammatory exudation in the lung tissue. Compared with the normal control group, the lung index and lung lesion score of the model control group mice were significantly increased, suggesting that H1N1-MRSA co-infection led to significant inflammatory lesions in the lungs; 100 mg / kg of total flavonoids from Houttuynia cordata, quercetin, and quercetin significantly reduced the lung index and lung lesion area in mice (as shown in Table 1). If diffuse lung parenchyma, alveolar inflammation, and interstitial fibrosis are observed as the basic pathological lesions in CT images, and the mice of this invention develop pneumonia due to pathogens such as viruses and bacteria, it can be identified as interstitial pneumonia. From the CT images, the lungs of the model group mice showed obvious diffuse infiltrative shadows and interstitial fibrosis. Quercetin and quercetin significantly alleviated the above phenomena, suggesting that 100 mg / kg of total flavonoids from Houttuynia cordata, quercetin, and quercetin can alleviate interstitial pneumonia caused by H1N1-MRSA co-infection (see table 1). Figure 5 Show).

[0048] Table 1. Animal experimental groupings, drug dosages, lung index, and lung injury area for each group.

[0049]

[0050]

[0051] * Compared with the model group, P<0.05. ** Compared with the model group, P<0.01. *** Compared with the model group, P < 0.001;

[0052] Lung index (mg / g) = (mouse lung weight / mouse body weight) × 100%.

[0053] (2) Detection of viral load in lung tissue: Lung tissues from each group of mice were mixed with Trizol at a mass:volume ratio of 1:9, homogenized, and treated with chloroform, isopropanol, and 75% ethanol (prepared with DEPC water) to obtain total RNA. cDNA was obtained after reverse transcription, and the copy number of the H1N1 M gene in the lung tissue was quantified using RT-PCR, representing the influenza virus replication load. Compared with the normal group, the mRNA level of the H1N1 M gene in the model group mice was significantly increased, indicating a high viral load in the lungs. 100 mg / kg of total flavonoids, quercetin, hyperoside, and quercetin from Houttuynia cordata significantly reduced the mRNA level of the H1N1 M gene. Other flavonoids, such as isoquercetin and rutin, also partially reduced the viral load in the lungs of co-infected mice. Figure 6 (As shown).

[0054] (3) Bacterial colonization count: Lung tissue from each group of mice was homogenized with physiological saline at a mass:volume ratio of 1:9. The original homogenate, diluted 5 times, and 25 times were inoculated onto agar plates and incubated at 37°C for 24 hours. Colony counts were observed; a higher colony count indicated a higher MRSA bacterial colonization load in the mice. Compared with the normal group, bacterial colonization in the model group was significantly increased. 100 mg / kg quercetin and quercetin significantly reduced the MRSA colonization in the lungs of mice co-infected with viruses and bacteria. Other flavonoids, such as total flavonoids from Houttuynia cordata, isoquercetin, rutin, and hyperoside, also showed a tendency to reduce bacterial colonization in the lungs of co-infected mice. Figure 7 As shown.

[0055] This invention investigates the protective effects of total flavonoids from Houttuynia cordata, quercetin, isoquercetin, rutin, hyperoside, and quercetin against H1N1-MRSA co-infection in an animal model. The main indicators were mouse body weight and mortality, lung pathological assessment, viral load in lung tissue, and bacterial colonization. Results confirmed that compared to the co-infection model group, the active components of Houttuynia cordata—total flavonoids, quercetin, and quercetin—significantly inhibited pneumonia and pathogen replication and colonization.

[0056] The total flavonoids, quercetin, and quercetin of Houttuynia cordata of this invention have been shown in animal experiments to have a good therapeutic effect on pneumonia caused by H1N1-MRSA co-infection in mice. The total flavonoids, quercetin, and quercetin of Houttuynia cordata can be used to prepare drugs for the treatment of viral and bacterial co-infection.

[0057] This invention provides a new use for the active components of Houttuynia cordata, namely total flavonoids, quercetin and quercetin in the Houttuynia cordata-related flavonoids, namely, for the preparation of drugs to prevent and treat interstitial pneumonia caused by viral and bacterial co-infection.

[0058] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of houttuynia cordata-related flavonoids in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections, characterized in that, Houttuynia cordata-related flavonoids were used to prepare drugs for the prevention and treatment of viral and bacterial co-infections. The houttuynia cordata-related flavonoids are total houttuynia cordata flavonoids; the total houttuynia cordata flavonoids include quercetin, quercetin, isoquercetin, rutin and hyperoside; Houttuynia cordata-related flavonoids were used to prepare drugs for the prevention and treatment of respiratory infections caused by H1N1-MRSA co-infection; The respiratory infection was interstitial pneumonia.

2. The use of the houttuynia cordata-related flavonoids according to claim 1 in the preparation of drugs for the prevention and treatment of viral and bacterial co-infections, characterized in that, The total flavonoids from Houttuynia cordata were obtained by ethanol extraction from Houttuynia cordata.

Images