Application of tetrapanax papyriferus water extract, mastitis medicine and functional supplement

Abstract

The invention provides an application of at least one of a ricepaperplant pith water extract and phytosphingosine in a medicine for relieving and / or treating mastitis, and also provides a mastitis medicine containing the ricepaperplant pith water extract and phytosphingosine and a functional supplement. In the medicine for relieving and / or treating the mastitis, the tetrapanax papyriferus water extract and the phytosphingosine show obvious anti-inflammatory and antibacterial activity, have the medicine effect of relieving and treating the mastitis and are high in safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an application of a water extract of Tongcao, a mastitis medicine and a functional supplement. BACKGROUND

[0002] Accumulation of milk and bacterial invasion are the main causes of mastitis, which is a common disease in lactating women.

[0003] At present, the treatment of mastitis mainly relies on two means: one is supportive treatment, including massage, cold compress, etc.; the other is drug treatment, such as using antibiotics to inhibit bacterial infection, or using analgesics to relieve pain of patients. However, drug treatment is not perfect. On the one hand, some mastitis patients are prone to drug resistance after using antibiotics, thereby affecting the treatment effect; on the other hand, drug treatment also has safety problems, such as side effects and potential effects on infants, which makes many patients choose to stop breastfeeding during the treatment period.

[0004] Therefore, it is an urgent need to find a safer and more effective drug for the treatment of mastitis. SUMMARY

[0005] The purpose of the embodiments of the present application is to overcome the above-mentioned shortcomings of the prior art, and to provide an application of at least one of a water extract of Tongcao and phytosphingosine, a mastitis medicine and a functional supplement, so as to solve the technical problems of unsatisfactory safety and effectiveness of the existing drug treatment of mastitis.

[0006] In order to achieve the above-mentioned application purposes, the first aspect of the embodiments of the present application provides an application of at least one of a water extract of Tongcao and phytosphingosine, specifically an application of at least one of a water extract of Tongcao and phytosphingosine in a medicine for relieving and / or treating mastitis.

[0007] Since the water extract of Tongcao and phytosphingosine show obvious anti-inflammatory and antibacterial activities, they have the pharmacodynamic effect of relieving and treating mastitis and good safety. Therefore, the water extract of Tongcao and phytosphingosine can be used as a pharmacologically active component for relieving or treating mastitis, and can be applied in a medicine for relieving or treating mastitis.

[0008] In some embodiments, the water extract of Tongcao comprises phytosphingosine, or a mixture of phytosphingosine and golden amide alcohol ester.

[0009] In some embodiments, the water extract of Tongcao further comprises at least one of sucrose-6-phosphate, L-pyroglutamic acid, adenosine, dehydrophytosphingosine, trihydroxyoleic acid and 9,10-dihydroxy-12-octadecenoic acid.

[0010] In some embodiments, the water extract of Tong- cao includes the phytosphingosine, the golden amide alcohol ester, the sucrose-6-phosphate, the L-pyroglutamic acid, the adenosine, the dehydrophytosphingosine, the trihydroxyoleic acid, and the 9,10-dihydroxy-12-octadecenoic acid.

[0011] In some embodiments, the water extract of Tong- cao is a water extract of the raw Tong- cao after soaking treatment, which is soaked in an aqueous extraction solution and treated at 50-100°C for 30 min-2h.

[0012] In some embodiments, the water extract of Tong- cao is a water extract of the raw Tong- cao after soaking treatment, which is soaked in an aqueous extraction solution and treated at 50-100°C for 30-60 min.

[0013] In some embodiments, the effective dose of the water extract of Tong- cao is 0.25-2g / mL.

[0014] In some embodiments, the effective dose of the phytosphingosine is 0.03-10mMol.

[0015] In a second aspect, the present application provides a mastitis medicine. The mastitis medicine of the present application includes a pharmaceutically acceptable excipient and an active ingredient, and the active ingredient includes at least one of the water extract of Tong- cao and the phytosphingosine.

[0016] Since the mastitis medicine of the present application contains at least one of the water extract of Tong- cao and the phytosphingosine, it can be used as an active ingredient for relieving or treating mastitis, so that the mastitis medicine of the present application can effectively relieve or treat mastitis, and is safe.

[0017] In a third aspect, the present application provides a functional supplement. The functional supplement of the present application includes at least one of the water extract of Tong- cao and the phytosphingosine.

[0018] Since the functional supplement of the present application contains at least one of the water extract of Tong- cao and the phytosphingosine, it can be used as an active ingredient for preventing and relieving mastitis, so that the functional supplement of the present application can effectively prevent and relieve mastitis, and is safe. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 This is the basic peak chromatogram (BPC) of the water extract of Thunbergia scoparia in Example 1 of the present application by UPLC-Orbitrap-MS; wherein, Figure A is the basic peak chromatogram in positive ion mode, and Figure B is the basic peak chromatogram in negative ion mode;

[0021] Figure 2 This is a bar graph showing the cytotoxicity of the water extract of Tongcao to HuMEC cells determined by the MTT assay in Example 3 of the present application;

[0022] Figure 3 This is a bar graph showing the inhibitory effect of the water extract of Tongcao on HuMEC cells infected with Staphylococcus aureus in Example 4 of the present application, wherein Figure a is a bar graph showing the inhibitory effect of the water extract of MT (2 mg / ml) on HuMEC cells 2 hours after HuMEC cells were infected with Staphylococcus aureus (S. aureus); Figure b is a bar graph showing the inhibitory effect at the end of the infection period (24 hours);

[0023] Figure 4 This is a bar graph showing the inhibitory effect of the water extract of Tongcao on inflammatory cytokines (TNF-α and IL-6) in HuMEC cells infected with Staphylococcus aureus using an enzyme-linked immunosorbent assay (ELISA) in Example 5 of the present application; wherein, Figure a is a bar graph of the TNF-α secretion content of the MT water extract 2 hours after HuMEC infection with Staphylococcus aureus; Figure b is a bar graph of the IL-6 secretion content of the MT water extract 2 hours after HuMEC infection with Staphylococcus aureus; Figure c is a bar graph of the TNF-α secretion content of the MT water extract 24 hours after HuMEC infection with Staphylococcus aureus; Figure d is a bar graph of the IL-6 secretion content of the MT water extract 24 hours after HuMEC infection with Staphylococcus aureus;

[0024] Figure 5 This is a protein electrophoresis diagram and a bar graph of the occludin protein density measurement value normalized with β-actin in the experiment of the effect of MT water extract on Staphylococcus aureus (S. aureus, SA) on the blood-milk barrier of HuMEC cells in Example 6 of the present application; wherein, Figure a is a representative electrophoresis band of the occludin protein, and Figure b is a bar graph of the density measurement value of the occludin protein normalized with β-actin;

[0025] Figure 6 This is a bar graph showing the cytotoxicity of Phy to HuMEC cells determined using the MTT assay in Example 7 of the present application;

[0026] Figure 7Bar chart of the inhibitory effect of Phy on S. aureus infected HuMEC cells in Example 8 of the present application, wherein a chart is a bar chart of the inhibitory effect of Phy (10 mg / ml) on HuMEC cells 2 hours after the cells were infected with S. aureus; b chart is a bar chart of the inhibitory effect at the end of the infection period (24 hours);

[0027] Figure 8 Bar chart of the inhibitory effect of Phy on inflammatory cytokine (TNF-α) in S. aureus infected HuMEC cells using enzyme-linked immunosorbent assay (ELISA) in Example 9 of the present application; wherein a chart is a bar chart of the TNF-α secretion content of Phy 2 hours after the cells were infected with S. aureus; b chart is a bar chart of the TNF-α secretion content of Phy 24 hours after the cells were infected with S. aureus. DETAILED DESCRIPTION

[0028] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and not intended to limit the present application.

[0029] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0030] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0031] It should be understood that in various embodiments of the present application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0032] The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the description of the embodiments of the present application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0033] The weight of the related components mentioned in the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the embodiments of the present application is proportionally enlarged or reduced, it is within the scope disclosed in the embodiments of the present application. Specifically, the mass mentioned in the embodiments of the present application can be μg, mg, g, kg, and other mass units commonly known in the chemical industry.

[0034] Mastitis is a common inflammation of the mammary gland, mainly affecting women during lactation, especially women within 26 weeks after childbirth, with an incidence of about 25%. Mastitis is mainly manifested as redness and swelling of the mammary gland tissue, accompanied by pain and fever, which seriously affects the lactation experience of women and the nutritional intake of infants, and becomes one of the main obstacles to pure breast feeding. The occurrence of mastitis is mainly due to the accumulation of milk and the invasion of bacteria. Among them, the invasion of bacteria may be caused by factors such as nipple damage, infant oral inflammation, poor hygiene, and incomplete milk. These factors make mastitis a common condition for women during lactation.

[0035] At present, the treatment of mastitis mainly relies on supportive treatment (such as massage, cold compress, etc.) and drug treatment (such as antibiotics and analgesics). Among them, drug treatment mainly uses antibiotics to inhibit bacterial infection and uses analgesics to relieve pain in patients. However, the existing drug treatment has obvious deficiencies, mainly manifested in the following aspects:

[0036] Firstly, drug treatment may produce drug resistance. For example, long-term use of antibiotics may cause some mastitis patients to develop drug resistance, which means that the therapeutic effect of the drug on bacteria will gradually weaken, or even completely fail. This not only affects the treatment effect of mastitis, but also may increase the difficulty of treatment and the course of the disease. Secondly, drug treatment has safety problems. Some mastitis treatment drugs may have side effects, such as intestinal flora disorder, liver and kidney function damage, etc., which may further damage the health of patients. In addition, since most mastitis patients are lactating women, drugs may also have potential effects on infants through breast milk. According to statistics, mastitis is one of the main reasons for the failure of breast feeding, which makes many patients choose to stop breast feeding during the treatment period, thereby affecting the healthy growth of infants.

[0037] In order to improve the safety and effectiveness of treatment, especially for lactating women, in recent years, the application of Chinese herbal medicine in the treatment of mastitis has gradually been valued, including Australia, Italy and China. Many countries have shown that the use of Chinese herbal medicine for the treatment of mastitis in lactating mothers has increased significantly.

[0038] Tongcao (Latin name: Medulla Tetrapanacis, MT for short), also known as Tongtuo wood, white Tongcao, etc., is the stem pith of the plant Tongtuo wood in the family Araliaceae, distributed in the Yellow River Basin of China. This Chinese herbal medicine occupies a place in the theory of traditional Chinese medicine and has various medicinal effects. According to the public records, the existing known main medicinal effects of Tongcao include clearing heat and diuresis, promoting lactation, etc. In traditional Chinese medicine practice, Tongcao is used to promote urination, lactation and treat edema. It can be used alone or as a component of herbal formulations.

[0039] Although some studies have shown that herbal formulations containing Tongcao may be beneficial to lactating mothers, so far there is no public report on the research of Tongcao crude drug and its extracts in the quality of mastitis. Through research, it is found and verified that Tongcao water extract and its active ingredients contained therein both have the effect of inhibiting or treating mastitis. Based on the research, the following scheme of the embodiments of the present application is proposed.

[0040] [Application of Tongcao water extract and active ingredients thereof]

[0041] In one aspect, the present application provides the application of at least one of Tongcao water extract and phytosphingosine in a drug for relieving and / or treating mastitis.

[0042] In the application of Tongcao water extract and phytosphingosine in the present application, Tongcao water extract means that Tongcao crude drug is treated by water extraction, and the slurry or dry matter obtained by collecting the water extract is collected. The application of Tongcao water extract and phytosphingosine in the drug for relieving and / or treating mastitis should be understood that Tongcao water extract and phytosphingosine are active components or one of the active components of the drug for relieving and / or treating mastitis, which can have the effect of relieving and / or treating mastitis. Phytosphingosine is specifically a compound represented by the following formula (1).

[0043] Due to research and verification, the water extract of Tongcao and phytosphingosine can significantly inhibit bacterial internalization, reduce the secretion of pro-inflammatory cytokines (TNF-α and IL-6) in infected human mammary epithelial cells and inhibit the expression of Occludin protein, and are non-toxic, as shown in the experimental results of Examples 3 to 9 below. Therefore, the water extract of Tongcao and phytosphingosine show obvious anti-inflammatory and antibacterial activity, and have the pharmacodynamic effect of alleviating and treating mastitis, and are highly safe. Therefore, the water extract of Tongcao and phytosphingosine can be used as active pharmaceutical ingredients for alleviating or treating mastitis, and can be used in the application of alleviating and mastitis drugs for the preparation of drugs for alleviating or treating mastitis.

[0044] In some embodiments, after testing and verification, the effective dose of the water extract of Tongcao is 0.25 to 2 g / mL, and can be optionally 0.5 to 2 g / mL. Among them, the effective dose refers to the safe administration or pharmacological dosage range of the water extract of Tongcao as an active ingredient to effectively relieve or treat mastitis. The water extract of Tongcao at this concentration range can effectively play a significant role in inhibiting bacterial internalization, reducing the secretion of proinflammatory cytokines (TNF-α and IL-6) in infected human mammary epithelial cells and inhibiting the expression of Occludin protein, and is non-toxic, showing obvious anti-inflammatory and antibacterial activity and high safety.

[0045] In some embodiments, after testing and verification, the effective dose of phytosphingosine is 0.03 to 10 mmoL, optionally 1 to 10 mmoL. Among them, the effective dose refers to the safe administration of phytosphingosine as an active ingredient that can effectively relieve or treat mastitis or exert its efficacy in the system. Phytosphingosine at this concentration range, such as the above-mentioned Tongcao water extract, can also effectively exert a significant inhibitory effect on bacterial internalization, reduce the secretion of proinflammatory cytokines (TNF-α and IL-6) in infected human mammary epithelial cells, and inhibit the expression of Occludin protein. It is non-toxic and also shows obvious anti-inflammatory and antibacterial activity with high safety.

[0046] [Components of Tongcao water extract]

[0047] On the second aspect, it was found that the water extract of Herba Anemarrhenae has the pharmacological effect of alleviating and treating mastitis. The components contained in the water extract of Herba Anemarrhenae were further analyzed. Among them, the analysis method of the components contained in the water extract of Herba Anemarrhenae can be a conventional method for characterizing the components contained in the composition, such as in the embodiment, which can include chromatography, mass spectrometry, atomic absorption spectrometry detection, nuclear magnetic resonance spectrometry detection method and the like or a combination of two or more methods. In an exemplary embodiment, the types of components contained in the water extract of Herba Anemarrhenae can be analyzed by ultra-high performance liquid chromatography-tandem electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Orbitrap-MS).

[0048] In some embodiments, the water extract of Tong Cao is analyzed to include phytosphingosine, or a mixture of phytosphingosine and aurantiamide acetate.

[0049] Phytosphingosine (Chinese alias: 2-amino-1,3,4-octadecanetriol, English name: Phytosphingosine), molecular formula is C 18 H 39 NO3, the molecular structure formula is shown as follows formula (1):

[0050]

[0051] Aurantiamide acetate (Chinese alias: gray-green aspergillus amide, English name: Aurantiamide acetate), molecular formula is C 27 H 28 N2O4, the molecular structure formula is shown as follows formula (2):

[0052]

[0053] In some embodiments, the water extract of Tong Cao is analyzed to include phytosphingosine, or a mixture of phytosphingosine and aurantiamide acetate.

[0054] Sucrose-6-phosphate (English name: Sucrose-6-phosphate), molecular formula is C 12 H 23 O 14 P, the molecular structure formula is shown as follows formula (3):

[0055]

[0056] L-Pyroglutamic acid (Chinese alias: 5-carboxy pyrrolidone, English name: L-Pyroglutamic acid), molecular formula is C5H7NO3, the molecular structure formula is shown as follows formula (4):

[0057]

[0058] Adenosine (Chinese alias: 6-amino-9-Β-D-ribofuranosyl-9H-purine, English name: Adenosine), molecular formula is C 10 H 13 N5O4, the molecular structure formula is shown as follows formula (5):

[0059]

[0060] Dehydrophytosphingosine, molecular formula is C 18 H 37 O5, the molecular structure formula is shown in the following formula (6):

[0061]

[0062] Trihydroxyoleic acid, molecular formula is C 18 H 34 O5, the molecular structure formula is shown in the following formula (6):

[0063]

[0064] 9,10-Dihydroxy-12-octadecenoic acid, molecular formula is C 18 H 34 O4, the molecular structure formula is shown in the following formula (8):

[0065]

[0066] In the exemplary embodiment, the UPLC-Orbitrap-MS method is used to analyze the types of components contained in the water extract of Tongcao, and the components include those shown in Table 1. Among them, the basic peak chromatogram (BPC) of the water extract of Tongcao under positive ion mode (A) and negative ion mode (B) by UPLC-Orbitrap-MS is shown in Figure 1 Figure 1 and Table 1, the water extract of Tongcao is rich in the above golden amide alcohol ester, dehydrophytosphingosine and phytosphingosine and other components.

[0067] The one or more than two components detected by the above analysis may constitute part or all of the active components contained in the water extract of Tongcao, and the content and type of each component can play a synergistic role in relieving and treating mastitis activity, thereby improving the pharmaceutical effect of the water extract of Tongcao in relieving and treating mastitis. In addition, the types and contents of components contained in the water extract of Tongcao can be adjusted by extraction conditions such as temperature and time, and the above-mentioned effects of the water extract of Tongcao in inhibiting bacterial internalization, reducing the secretion of pro-inflammatory cytokines (TNF-α and IL-6) and inhibiting Occludin protein expression in infected human mammary epithelial cells, and non-toxicity, and improving its anti-inflammatory and antibacterial activity and high safety.

[0068] [Water extraction method of Tongcao water extract]​

[0069] In a third aspect, the water extract of Radix Gentianae Macrophyllae as described above is prepared by water extraction of crude Radix Gentianae Macrophyllae, collecting the water extract, and then concentrating the water extract to obtain a paste or dried material.

[0070] In some embodiments, the water extract of Radix Gentianae Macrophyllae can be prepared by water extraction according to a method comprising the following steps:

[0071] S10: soaking crude Radix Gentianae Macrophyllae in an aqueous extraction solution, heating the mixture to perform at least one extraction, and then performing solid-liquid separation to obtain an extract mixture solution;

[0072] S20: concentrating and drying the extract mixture solution to obtain the water extract of Radix Gentianae Macrophyllae.

[0073] In the method for preparing the water extract of Radix Gentianae Macrophyllae, the soaking and the extraction can both be performed by placing the crude Radix Gentianae Macrophyllae in an aqueous extraction solution. The heating of the mixture during the soaking is performed to allow the extraction to be performed at a certain temperature. The heating temperature of the extraction is adjusted to allow the active ingredients in the crude Radix Gentianae Macrophyllae to be effectively extracted by the extraction solution, so that the water extract of Radix Gentianae Macrophyllae has the activity and safety for relieving and treating mastitis.

[0074] Therefore, the method for preparing the water extract of Radix Gentianae Macrophyllae directly uses the aqueous soaking to perform the extraction, so that at least the water-soluble components are extracted, and thus the water extract of Radix Gentianae Macrophyllae contains the active ingredients for relieving or treating mastitis, and has the activity for relieving or treating mastitis. In embodiments, the water extract of Radix Gentianae Macrophyllae prepared by the method for preparing the water extract of Radix Gentianae Macrophyllae contains the components as described above, such as one or more components in Table 1.

[0075] In some embodiments, the soaking in step S10 can be performed by directly placing the crude Radix Gentianae Macrophyllae in the aqueous extraction solution at room temperature. The soaking allows the components in the extraction solution to infiltrate the crude Radix Gentianae Macrophyllae, so that the water-soluble components are partially dissolved, and the active ingredients in the crude Radix Gentianae Macrophyllae can be extracted as much as possible in the subsequent extraction, and the efficiency of the subsequent extraction is improved.

[0076] In embodiments, the soaking time should be sufficient, and in exemplary embodiments, the soaking can be performed overnight. The soaking can be accompanied by mixing, such as stirring, to improve the dissolution of the components and the efficiency of the soaking.

[0077] In some embodiments, the soaking treatment can be a soaking of the raw medicinal herbs in a soaking solution at a temperature of 4-30 °C for a time period of 30 min-16 h. In some embodiments, the soaking treatment can be a soaking of the raw medicinal herbs in a soaking solution at a temperature of 20-37 °C for a time period of 6-16 h. The soaking treatment at the above-mentioned temperature and time period can allow the components of the raw medicinal herbs to be soaked and further pre-extracted, so as to improve the subsequent extraction efficiency and the extraction amount of the water extract of the medicinal herbs.

[0078] In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 50-100 °C for a time period of 30 min-2 h. In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 50-100 °C for a time period of 30-60 min. In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 100 °C for a time period of 30 min.

[0079] By adjusting the soaking treatment temperature and time period within the above-mentioned ranges, the types and contents of the components, particularly the active components, of the water extract of the medicinal herbs can be further adjusted, so as to further improve the activity and efficacy of the water extract of the medicinal herbs in relieving and treating mastitis, and to improve the application of the water extract of the medicinal herbs in the preparation of drugs for relieving and treating mastitis.

[0080] In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 50-100 °C for a time period of 30 min-2 h. In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 50-100 °C for a time period of 30-60 min. In some embodiments, the soaking treatment can be a soaking of the soaking mixture in an extraction solution at a temperature of 100 °C for a time period of 30 min.

[0081] In some embodiments, the soaking solution for the soaking treatment and the extraction solution for the extraction treatment can be mixed at a mass ratio of (1-200): 1, or at a mass ratio of (20-200): 1. In some embodiments, the soaking solution for the soaking treatment and the extraction solution for the extraction treatment can be mixed at a mass ratio of 40: 1. The above-mentioned mass ratio of the solid-liquid mixture can effectively adjust the types and contents of the active components of the water extract of the medicinal herbs, improve the activity and efficacy of the water extract of the medicinal herbs in relieving and treating mastitis, and effectively improve the processing efficiency of the soaking solution and the extraction solution.

[0082] In some embodiments, the soaking solution for the soaking treatment and the extraction solution for the extraction treatment can be mixed at a mass ratio of (1-200): 1, or at a mass ratio of (20-200): 1. In some embodiments, the soaking solution for the soaking treatment and the extraction solution for the extraction treatment can be mixed at a mass ratio of 40: 1. The above-mentioned mass ratio of the solid-liquid mixture can effectively adjust the types and contents of the active components of the water extract of the medicinal herbs, improve the activity and efficacy of the water extract of the medicinal herbs in relieving and treating mastitis, and effectively improve the processing efficiency of the soaking solution and the extraction solution.

[0083] The original medicine of Tongcao is generally the dried stem pith of Tongcao after removing the bark. In some embodiments, the original medicine of Tongcao is pretreated before being soaked. In exemplary embodiments, the pretreatment includes washing with clean water and can also include pulverization to improve the efficiency of soaking and extraction.

[0084] In some embodiments, the solid-liquid separation can be filtration or centrifugation to separate the filtrate from the residue and collect the filtrate, i.e., the extraction solution.

[0085] The concentration treatment in step S20 is to remove the water-containing extraction solution in the extraction mixture solution to increase the concentration of the Tongcao water extract in the extraction mixture solution, so as to facilitate subsequent processing of the Tongcao water extract, such as directly using the concentrated solution as a raw material for preparing a medicine for relieving or treating mastitis, or facilitating subsequent drying of the concentrated solution to obtain a Tongcao water extract solid.

[0086] In some embodiments, the concentration treatment can be heating or vacuum concentration treatment of the extraction mixture solution to at least partially evaporate the water extraction solution. In embodiments, the concentrated solution obtained by the concentration treatment can also be subjected to centrifugation to remove suspended solids and improve the purity and content of active ingredients in the Tongcao water extract. In exemplary embodiments, the concentrated solution obtained by the concentration treatment can be centrifuged at 4,000 revolutions per minute for 10 minutes and filtered with No. 1 filter paper to remove any suspended solids.

[0087] In some embodiments, the drying treatment is to remove the water extraction solution remaining in the extraction mixture solution or the concentrated solution obtained by the concentration treatment to obtain a solid Tongcao water extract.

[0088] In exemplary embodiments, the drying treatment can be freeze-drying treatment to improve the physical and chemical stability of active ingredients in the Tongcao water extract and improve the activity of the Tongcao water extract in relieving or treating mastitis.

[0089] [Medicine for mastitis]

[0090] In a fourth aspect, the Tongcao water extract and the plant sphingosine contained therein have activity in relieving or treating mastitis based on the above embodiments of the present application. The present embodiments also provide a medicine for mastitis. In the medicine for mastitis of the present embodiments, pharmaceutically acceptable adjuvants and active ingredients are included. The active ingredients include at least one of the Tongcao water extract and the plant sphingosine.

[0091] Since the mastitis medicine in the embodiments of the present application contains at least one of the water extract of Tongcao and phytosphingosine, based on the above description of the activity of the water extract of Tongcao and phytosphingosine, the water extract of Tongcao and phytosphingosine can be used as an active component for relieving or treating mastitis, and the mastitis medicine in the embodiments of the present application can effectively relieve or treat mastitis, and has good efficacy and safety.

[0092] In some embodiments, the pharmaceutically acceptable adjuvant contained in the mastitis medicine in the embodiments of the present application can be a dosage form adjuvant component for giving the mastitis medicine in the embodiments of the present application different dosage forms or a protective adjuvant component for improving the activity of the active component. In embodiments, the dosage form adjuvant component can include a tablet adjuvant component, a capsule adjuvant component, a powder adjuvant component, an oral liquid adjuvant component, etc., and at this time, the mastitis medicine in the embodiments of the present application can be a tablet, a capsule, a powder, an oral medicine, etc. The different adjuvant components can effectively give the mastitis medicine in the embodiments of the present application different dosage forms, facilitate the patient to carry and take the medicine, and improve the stability of the activity of the active component including at least one of the water extract of Tongcao and phytosphingosine, and the components can play a pharmaceutical effect. Moreover, by selecting the adjuvant component, targeted or site-specific release and controlled release rate of the active component including at least one of the water extract of Tongcao and phytosphingosine can also be achieved.

[0093] In some embodiments, in the mastitis medicine in the embodiments of the present application, the content of at least one of the water extract of Tongcao and phytosphingosine can be flexibly controlled according to the dosage form and the administration mode, so that the total dose taken by the patient each time reaches the effective dose of at least one of the water extract of Tongcao and phytosphingosine.

[0094] The water extract of Tongcao and its extraction method and application in relieving and / or treating mastitis medicine are illustrated by a plurality of specific embodiments below. The water extract of Tongcao in each of the embodiments below is denoted as MT water extract.

[0095] [Functional supplement]

[0096] In a fifth aspect, based on the above description that the water extract of Tongcao and the component including phytosphingosine contained therein in the embodiments of the present application have the activity of relieving or treating mastitis, the embodiments of the present application also provide a functional supplement. In the functional supplement in the embodiments of the present application, it contains an active component. The active component includes at least one of the water extract of Tongcao and phytosphingosine.

[0097] Since the functional supplement in the embodiments of the present application contains at least one of the water extract of Tongcao and phytosphingosine, it can be used as an active component for preventing and relieving mastitis, and the functional supplement in the embodiments of the present application can effectively prevent and relieve mastitis, and is safe.

[0098] In some embodiments, the functional supplement of the present application can be in the form of a functional health product, a food functional additive, etc.

[0099] The activity and application of the water extract of Tongcao and phytosphingosine are demonstrated and proved by a plurality of specific embodiments as follows.

[0100] Embodiment 1:

[0101] This embodiment provides a water extract of Tongcao and an extraction method thereof. The extraction method of the MT water extract comprises the following steps:

[0102] S1: The Tongcao crude drug (from Guangxi, provided by a local supplier) was verified for authenticity according to the macroscopic and microscopic characteristics outlined in the Pharmacopoeia of the People's Republic of China, and was confirmed as Tongcao crude drug;

[0103] S2: 23 grams of the confirmed Tongcao crude drug (stem pith of Tongcao) was sliced and soaked in 920 milliliters of double distilled water (ddH2O) (1:40) overnight; after the soaking treatment, the soaked mixture was boiled for 30 minutes for extraction treatment, and filtration treatment was performed to obtain filtrate and residue; the residue was extracted again with 1:20 ddH2O (w / v) for 30 minutes, and filtration treatment was performed again to obtain filtrate and residue; the filtrates obtained by the two filtration treatments were mixed together to obtain an extraction mixture solution;

[0104] S3: After the extraction mixture solution was concentrated to about 200 milliliters, the concentrated solution was centrifuged at 4000 revolutions per minute for 10 minutes, and filtered with No. 1 filter paper to remove all suspended solids; finally, the filtrate was freeze-dried for 24 to 48 hours to obtain MT water extract dry powder, which was stored at 4°C in the dark and used as a backup. Before using the sample, the MT water extract dry powder was first dissolved in ddH2O, and then sterilized by a needle filter (0.22 microns).

[0105] It was detected that the yield of the Tongcao water extract was 1.27 ± 0.122% (mean ± standard deviation).

[0106] Embodiment 2:

[0107] In this embodiment, the MT water extract extracted in Embodiment 1 was analyzed for components by ultra-high performance liquid chromatography-electrostatic field orbitrap mass spectrometry (UPLC-Orbitrap-MS):

[0108] An aliquot of 0.02 g of the lyophilized powder of the MT water extract prepared in Example 1 was accurately weighed into a 2 mL vial, and 500 μL of 70% methanol containing internal standards (1 ppm cholic acid-2,2,4,4-d4 and L-tryptophan-(indole-d5)) was added. The sample was sonicated for 30 minutes at room temperature, and then centrifuged at 14,000 g for 10 minutes. The supernatant was filtered through a 0.22 μm PTFE syringe filter before analysis.

[0109] Orbitrap-MS analysis of the lyophilized powder of the MT water extract was performed using an UltiMate 3000 UHPLC system coupled to a Thermo Scientific Orbitrap IQ-X LC / MS (Thermo Fisher Scientific, Massachusetts, USA). Separation was performed using a Waters ACQUITY UPLC HSS T3 column (2.1 mm x 100 mm, 1.8 μm) and a HSS T3 pre-column (2.1 mm x 5 mm, 1.8 μm) at a temperature of 40 °C. The mobile phase was a mixture of water (A) and acetonitrile (B), both containing 0.1% (v / v) formic acid, using a linear gradient elution with the following conditions: initial 5% B, 0-1 min, 5% B; 1-1.5 min, 5-35% B; 1.5-3 min, 35-50% B; 3-6.5 min, 50-55% B; 6.5-8.5 min, 55-95% B; 8.5-10.5 min, 95% B; 10.5-11 min, 95-5% B; 11-14 min, 5% B, with an injection volume of 3 μL and a flow rate of 0.30 mL / min. The sample chamber temperature was set at 4 °C, and ESI-MS spectra were obtained in both positive and negative ion modes. The ESI parameters were as follows: spray voltage, 3500 V for ESI+ and 2300 V for ESI-; sheath gas 35 arbitrary units; auxiliary gas 10 arbitrary units; scan gas 3 arbitrary units. The general instrument parameters were set as follows: ion transfer tube temperature 300 °C; gasifier temperature 320 °C. For full scan MS, the mass range was set to 70-1200 m / z with a resolution of 120,000.

[0110] The identification results are shown in Table 1 and Fig. 1, wherein the base peak chromatogram of the MT water extract sample is shown in Fig. 2, and the compounds identified by UPLC-Orbitrap-MS are summarized in Table 1 below. According to the identification results shown in Table 1 and Fig. 1, a total of 8 compounds were identified in the MT water extract. These compounds are mainly organic acids and fatty acids. Figure 1 The identification results are shown in Table 1 and Fig. 1, wherein the base peak chromatogram of the MT water extract sample is shown in Fig. 2, and the compounds identified by UPLC-Orbitrap-MS are summarized in Table 1 below. According to the identification results shown in Table 1 and Fig. 1, a total of 8 compounds were identified in the MT water extract. These compounds are mainly organic acids and fatty acids. Figure 1 The identification results are shown in Table 1 and Fig. 1, wherein the base peak chromatogram of the MT water extract sample is shown in Fig. 2, and the compounds identified by UPLC-Orbitrap-MS are summarized in Table 1 below. According to the identification results shown in Table 1 and Fig. 1, a total of 8 compounds were identified in the MT water extract. These compounds are mainly organic acids and fatty acids. Figure 1 The identification results are shown in Table 1 and Fig. 1, wherein the base peak chromatogram of the MT water extract sample is shown in Fig. 2, and the compounds identified by UPLC-Orbitrap-MS are summarized in Table 1 below. According to the identification results shown in Table 1 and Fig. 1, a total of 8 compounds were identified in the MT water extract. These compounds are mainly organic acids and fatty acids.

[0111] Table 1

[0112]

[0113] Example 3:

[0114] MT water extract cytotoxicity experiment on human mammary epithelial cells (HuMEC) cells.

[0115] 1. Dissolve the MT water extract dry powder extracted in Example 1 in ddH2O to prepare solutions with concentrations of 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, and 2 mg / ml, and then sterilize them for use;

[0116] 2. Add HuMEC cells (10,000 cells / ml) to 100 microliters of MT water extract solutions with different concentrations (0 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, and 2 mg / ml) for mixing treatment, and after 24 hours of incubation, remove the culture medium and replace it with 100 microliters of methylthiazolyl tetrazolium (MTT) solution (Gibco, Grand Island, NY, USA) with a final concentration of 0.5 mg / ml. After incubation at 37°C for 2 hours, discard the MTT solution in each well and add 100 microliters of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals. Use a microplate reader (Germany BMG LABTECH CLARIOstar) to measure the absorbance of the samples at 570 nanometers with 650 nanometers as the reference wavelength. The cell survival rate (%) is calculated based on the following formula: sample absorbance / control absorbance x 100%.

[0117] 3. Experimental results:

[0118] The results of the MT water extract cytotoxicity experiment on HuMEC cells determined by MTT method are shown inFigure 2 As shown in Figure 2 It can be seen that the MT water extract did not show significant cytotoxicity at concentrations of 0.25 mg / ml to 2 mg / ml. Thus, it can be seen that the MT water extract has little toxicity to HuMEC cells and is highly safe.

[0119] Example 4:

[0120] Inhibition experiment of MT water extract on S. aureus infected HuMEC cells.

[0121] 1. Dissolve the MT water extract dry powder extracted in Example 1 in ddH2O to prepare solutions with concentrations of 0 mg / ml and 2 mg / ml, and then sterilize them separately for standby use;

[0122] 2. At 2 hours and 24 hours after the infection of HuMEC cells with S. aureus, add the MT water extract solutions (0 mg / ml and 2 mg / ml) to determine the inhibitory effect of the MT water extract on S. aureus infected HuMEC cells by colony forming unit plate counting method.

[0123] 3. Experimental results:

[0124] The results of the inhibitory effect of the MT water extract on S. aureus infected HuMEC cells are shown in Figure 3 , wherein, Figure 3 Figure a is a column chart of the inhibitory effect of the MT water extract (2 mg / ml) on HuMEC cells at 2 hours after the infection of the HuMEC cells with S. aureus (the number of S. aureus internalization, n = 5); and Figure b is a column chart of the inhibitory effect at the end of the infection period (24 hours) (the inhibitory effect on S. aureus, n = 7). Figure 3 The data in the table represent mean ± standard error: the differences between groups were compared using Student's t test. *+p < 0.01 compared with the SA group.

[0125] As shown in Figure 3 Figure a, at 2 hours after the infection, the MT water extract can play a role by preventing the internalization of bacteria into cells or reducing the number of bacteria in cells. As shown in Figure 3 Figure b, at the end of the infection period (24 hours), the inhibitory effect of the MT water extract is more obvious, thus proving that the MT water extract has a sustained inhibitory effect on bacterial growth.

[0126] As shown in Figure 3It can be seen that the MT water extract can effectively reduce the internalization of S. aureus and inhibit its growth on HuMEC cells, thus indicating that the MT water extract has a significant bactericidal or inhibitory effect on S. aureus.

[0127] Example 5:

[0128] This example is an experiment on the effect of MT water extract on the secretion of TNF-a and IL-6 inflammatory cytokines in HuMEC cells infected with S. aureus (SA).

[0129] 1. Dissolve the dry powder of the MT water extract extracted in Example 1 in ddH2O to prepare solutions with concentrations of 0 mg / ml and 2 mg / ml, and then sterilize them for use;

[0130] 2. At 2 hours and 24 hours after the infection of HuMEC cells with S. aureus, add the MT water extract solutions (0 mg / ml and 2 mg / ml), and use a commercial kit (PeproTech, USA) to perform a sandwich enzyme-linked immunosorbent assay (ELISA) for TNF-a and IL-6 according to the manufacturer's instructions.

[0131] 3. Experimental results:

[0132] The results of the use of enzyme-linked immunosorbent assay (ELISA) to detect the inhibitory effect of MT water extract on inflammatory cytokines (TNF-a and IL-6) in HuMEC cells infected with S. aureus are shown in Figure 4 The a graph in Figure 4 is a column chart of the secretion content of TNF-a in HuMEC infected with S. aureus for 2 hours by MT water extract; the b graph is a column chart of the secretion content of IL-6 in HuMEC infected with S. aureus for 2 hours by MT water extract; the c graph is a column chart of the secretion content of TNF-a in HuMEC infected with S. aureus for 24 hours by MT water extract; and the d graph is a column chart of the secretion content of IL-6 in HuMEC infected with S. aureus for 24 hours by MT water extract. The data are represented as mean ± standard error: n = 5 (a graph and b graph), n = 7 (c graph), and n = 6 (d graph). One-way analysis of variance (ANOVA) was used for comparison of differences between groups, followed by Tukey's multiple comparison test. *+P < 0.01 and ww-P < 0.001 indicate comparison with the control group, #P < 0.05, ##P < 0.01, and ###P < 0.001 indicate comparison with the SA group.

[0133] From Figure 4As shown in Figures a to d, S. aureus significantly induced the secretion of TNF-α and IL-6 by HuMEC cells, while the experimental MT water extract (2 mg / ml) reduced this LPS-induced TNF-α and IL-6 secretion. Since TNF-α and IL-6 are two important proinflammatory cytokines, their secretion levels can reflect the degree of inflammatory response in cells. The secretion of these cytokines was observed 2 hours and 24 hours after infection, and significant differences in TNF-α and IL-6 secretion were found between the MT water extract-treated group and the SA control group. Specifically, compared with the control group, the MT water extract-treated group had significantly lower TNF-α and IL-6 secretion at both 2 hours and 24 hours after infection, and this difference was statistically significant (P < 0.01 or P < 0.001). These results suggest that the MT water extract can inhibit the secretion of TNF-α and IL-6 in HuMEC cells infected with S. aureus, thereby potentially alleviating the inflammatory response.

[0134] Example 6:

[0135] This example experiments were conducted to investigate the effect of MT water extract on the destruction of the blood-milk barrier of HuMEC cells by Staphylococcus aureus (SA).

[0136] 1. The MT water extract powder obtained in Example 1 was dissolved in ddH2O to prepare solutions with concentrations of 0 mg / ml and 2 mg / ml, and then sterilized separately for later use;

[0137] 2. Follow Figure 5 As shown in Figure (a), SA-infected HuMEC cells were pretreated with different concentrations of MT aqueous extract (0 mg / ml and 2 mg / ml) for 2 hours. After incubation, the culture medium was collected. The HuMEC cells in the culture medium were lysed according to existing lysis methods, and the occludin protein was separated by gel electrophoresis. Occludin content was quantified using densitometry.

[0138] 3. Experimental results:

[0139] The experimental results of the effect of MT water extract on the destruction of the blood-milk barrier of HuMEC cells by Staphylococcus aureus (SA) are as follows Figure 6 As shown. Among them, Figure 6Panel a shows a representative band of occludin protein, and panel b shows a bar graph of occludin protein density measurements normalized to β-actin. Data are expressed as mean ± standard error (SD); n = 3. Intergroup differences were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the control group, and ### indicates p < 0.001 compared with the SA group.

[0140] The blood-milk barrier (BMB) is a crucial line of defense in breast tissue. Its integrity is crucial for maintaining normal mammary gland function and preventing pathogen invasion. When Staphylococcus aureus infects the mammary gland, it disrupts the BMB, leading to impaired mammary gland function and an inflammatory response. Changes in occludin expression can reflect the stability and strength of the BMB in HuMEC cells.

[0141] Depend on Figure 5 As shown in Figure b, MT water extract can protect the blood-milk barrier of HuMEC cells from being destroyed by Staphylococcus aureus, and enhance the barrier function through the expression of the protein Occludin. Moreover, there is a significant difference in the expression level of Occludin between the MT water extract-treated group and the control group and SA group, indicating that MT water extract can effectively reverse the damage caused by Staphylococcus aureus to the Occludin protein.

[0142] Example 7:

[0143] This example is a cytotoxicity experiment of phytosphingosine (Phy).

[0144] 1. Dissolve Phy in ddH2O to prepare solutions with concentrations of 0μmoL, 0.03μmoL, 0.1μmoL, 1μmoL, 3μmoL, and 10μmoL, and then sterilize them separately for later use;

[0145] 2. HuMEC cells (10,000 cells / ml) were added to 100 μl of different concentrations of MT water extract solution (0 μmoL, 0.03 μmoL, 0.1 μmoL, 1 μmoL, 3 μmoL, 10 μmoL) for mixing treatment, and after 24 hours of incubation, the culture medium was removed and replaced with 100 μl of methyl thiazolyl tetrazolium (MTT) solution (Gibco, Grand Island, NY, USA) with a final concentration of 0.5 mg / ml. After incubation at 37°C for 2 hours, the MTT solution in each well was discarded and 100 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals by gentle shaking. The absorbance of the samples was measured at 570 nm using a microplate reader (BMG LABTECH CLARIOstar, Germany) with 650 nm as the reference wavelength. The cell survival rate (%) was calculated based on the following formula: sample absorbance / control absorbance x 100%.

[0146] 3. Experimental results:

[0147] The results of the Phy HuMEC cell toxicity experiment determined by the MTT method are shown in Figure 6 As can be seen from Figure 6 Phy did not show significant cytotoxicity at concentrations of 0.03 μmoL to 10 μmoL. Thus, it can be seen that Phy has little toxicity to HuMEC cells and is highly safe.

[0148] Example 8:

[0149] This example is an experiment on the inhibition of Phy on HuMEC cells infected with S. aureus (SA).

[0150] 1. Phy was dissolved in ddH2O to prepare solutions with concentrations of 0 μmoL and 10 μmoL, and then sterilized for use;

[0151] 2. Two hours and 24 hours after the HuMEC cells were infected with S. aureus, Phy solutions (0 μmoL, 10 μmoL) were added to determine the inhibitory effect of Phy on HuMEC cells infected with S. aureus by colony forming unit plate counting method.

[0152] 3. Experimental results:

[0153] The results of the inhibition of Phy on HuMEC cells infected with S. aureus are shown in Figure 7 wherein, Figure 7Figure a is a bar graph showing the inhibitory effect of Phy (10 μmoL) on HuMEC cells 2 hours after HuMEC cells were infected with Staphylococcus aureus (S. aureus); Figure b is a bar graph showing the inhibitory effect at the end of the infection period (24 hours). Figure 7 The data are expressed as mean ± standard error (n = 5). The differences among the groups were compared using Student's t test, *+p < 0.01 compared with the SA group.

[0154] Depend on Figure 7 As shown in Figure a, 2 hours after infection, Phy may play a role by preventing bacteria from internalizing into cells or reducing the number of bacteria in cells. Figure 7 As shown in Figure b, the inhibitory effect of Phy was more obvious at the end of the infection cycle (24 hours), which proved that Phy also had a sustained inhibitory effect on bacterial growth.

[0155] Depend on Figure 7 It can be seen that Phy, like the MT water extract, can also effectively reduce the internalization of Staphylococcus aureus and inhibit its growth on HuMEC cells, which shows that Phy also has a significant bactericidal or inhibitory effect on Staphylococcus aureus.

[0156] Example 9:

[0157] This example experiments on the effect of phytosphingosine (Phy) on the secretion of TNF-α inflammatory cytokine in HuMEC cells infected with Staphylococcus aureus (SA).

[0158] 1. Dissolve Phy in ddH2O to prepare solutions with concentrations of 0 μmoL and 10 μmoL, and then sterilize them separately for later use;

[0159] 2. HuMEC cells were infected with Staphylococcus aureus for 2 hours and 24 hours, and MT aqueous extract solution (0 mg / ml, 10 mg / ml) was added, respectively. TNF-α sandwich enzyme-linked immunosorbent assay (ELISA) was performed at 2 hours and 24 hours, respectively, using a commercial kit (PeproTech, USA) according to the manufacturer's instructions.

[0160] 3. Experimental results:

[0161] The results of enzyme-linked immunosorbent assay (ELISA) were used to detect the inhibitory effect of Phy on inflammatory cytokines (TNF-α) in HuMEC cells infected with Staphylococcus aureus at 2 hours and 24 hours. Figure 8Figure 6. Phy and MT water extract inhibit TNF-a secretion in S. aureus infected HuMECs. Figure 6a and 6b show the TNF-a secretion levels in HuMECs 2 hours and 24 hours post-infection, respectively. Data are expressed as mean ± SEM: n = 5 (a and b), n = 7 (c), n = 6 (d). Data are expressed as mean ± SEM (n = 5). One-way ANOVA was used to compare differences between groups, followed by Tukey’s multiple comparison test. * indicates p < 0.05 compared to control, ** indicates p < 0.01 compared to control, # indicates p < 0.05 compared to SA.

[0162] From Figure 8 As shown in Figure 6a and 6b, S. aureus significantly induced TNF-a secretion in HuMECs, while Phy and MT water extract similarly reduced this SA-induced TNF-a secretion. Since the secretion level of TNF-a can reflect the degree of inflammatory response of the cells, the secretion of this cytokine was observed at 2 hours and 24 hours post-infection, respectively, and it was found that there was a significant difference in the amount of TNF-a secretion between the Phy treatment group and the SA control group. These results show that Phy, like MT water extract, can also inhibit the secretion of TNF-a in S. aureus infected HuMECs, thus possibly reducing the inflammatory response.

[0163] In combination with the above Examples 1 to 8, mammary epithelial cells (MECs) are the front line of defense against infection in the mammary gland, and S. aureus (SA) can invade mammary epithelial cells by internalization, allowing it to colonize and multiply in the mammary gland, which is one of the key processes that exacerbate the development of mastitis. In addition to this, inflammation is another key process in the development of mastitis, and the invasion of SA triggers an immune response in mammary epithelial cells (MRECs) to eliminate SA infection by producing reactive oxygen species (ROS) and secreting pro-inflammatory cytokines such as IL-6 and TNF-a. However, excessive inflammatory response can exacerbate mastitis, aggravate damage to the mammary tissue and disrupt the integrity of the blood-milk barrier.

[0164] From the above Examples 1 to 2, in combination with Figure 1 and Table 1, it can be seen that the water extract of Tong Cao is rich in phytosphingosine and golden amide alcohol ester.

[0165] From the above Examples 3 to 5, in combination with Figure 2 and Figure 4 it can be seen that the water extract of Tong Cao is non-toxic to cells, and has high safety ( Figure 2 ), significantly inhibits bacterial internalization at 2 hours post-infection, and inhibits bacterial growth at 24 hours post-infectionFigure 3 ), while the water extract of Tong Cao significantly reduced the SA-induced inflammatory markers (TNF-a and IL-6) Figure 4 ) at both 2 hours and 24 hours post-infection.

[0166] From the above Examples 7 to 9, in combination with Figure 6 and Figure 8 , it is known that the active ingredient, phyto-sphingosine (Phy), contained in the water extract of Tong Cao, is similar in activity to the water extract of Tong Cao, Phy is non-toxic to cells, has high safety Figure 6 , and can significantly inhibit bacterial internalization at 2 hours post-infection, inhibit bacterial growth at 24 hours post-infection Figure 7 , and significantly reduce the SA-induced inflammatory markers (TNF-a and IL-6) Figure 8 ) at both 2 hours and 24 hours post-infection.

[0167] From the above Example 6, in combination with Figure 5 , it is known that the water extract of Tong Cao can significantly restore the Occludin protein expression inhibited by SA.

[0168] Therefore, it is verified that the water extract of Tong Cao and its active ingredients (such as Phy and AA) can alleviate, treat the symptoms of mastitis by inhibiting the internalization of Staphylococcus aureus (SA), reducing the inflammation caused by SA, and repairing the damaged blood-milk barrier integrity, so that the water extract of Tong Cao and its active ingredients (such as Phy and AA) can be applied in drugs for alleviating and treating mastitis, thereby exerting the efficacy of alleviating and treating mastitis.

[0169] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of at least one of a water extract of Tong- cao and phytosphingosine in a drug for relieving and / or treating mastitis.

2. Use according to claim 1, characterized in that: The water extract of Tong- cao comprises phytosphingosine or a mixture of phytosphingosine and golden amide alcohol ester.

3. Use according to claim 2, characterized in that: The water extract of Tong- cao further comprises at least one of sucrose-6-phosphate, L-pyroglutamic acid, adenosine, dehydrophytosphingosine, trihydroxyoleic acid and 9,10-dihydroxy-12-octadecenoic acid.

4. Use according to claim 3, characterized in that: The water extract of Tong- cao comprises the golden amide alcohol ester, the sucrose-6-phosphate, the L-pyroglutamic acid, the adenosine, the dehydrophytosphingosine, the trihydroxyoleic acid and the 9,10-dihydroxy-12-octadecenoic acid.

5. Use according to any one of claims 1 to 4, characterized in that: The water extract of Tong- cao is obtained by immersing the original Tong- cao after soaking treatment in an aqueous extraction solution and treating at 50-100℃ for 30min-2h.

6. Use according to any one of claims 1 to 4, characterized in that: The water extract of Tong- cao is obtained by immersing the original Tong- cao after soaking treatment in an aqueous extraction solution and treating at 50-100℃ for 30-60min.

7. Use according to any one of claims 1 to 4, characterized in that: The effective dose of the water extract of Tong- cao is 0.25-2g / mL.

8. Use according to any one of claims 1 to 4, characterized in that: The effective dose of the phytosphingosine is 0.03-10mMol.

9. A medicament for mastitis, characterized by comprising: The drug comprises pharmaceutically acceptable adjuvants and active components, the active components comprising at least one of a water extract of Tong- cao and phytosphingosine.

10. A functional supplement, characterized in that: The drug comprises at least one of a water extract of Tong- cao and phytosphingosine.

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