A traditional Chinese medicine composition, a traditional Chinese medicine extract and applications thereof

Abstract

This invention relates to the field of traditional Chinese medicine (TCM) technology, and more particularly to a TCM composition, a TCM extract, and their applications. The TCM composition of this invention is characterized by comprising a first component, a second component, and a third component; the first component is *Prunella vulgaris* and *Lophatherum gracile*; the second component is *Platycodon grandiflorus* and *Glycyrrhiza uralensis*; the third component is *Prunus mume*; the content of the first component in the TCM composition is 50-60 wt%; the mass ratio of the first component to the second component is 1.5-2.5:1; and the mass ratio of *Prunella vulgaris* and *Lophatherum gracile* in the first component is 1:1.5-2.5. The TCM composition of this invention achieves good anti-inflammatory and nodule-dispersing effects through the synergistic combination of its components. While ensuring the synergistic anti-inflammatory and nodule-dispersing effects, it simplifies the formulation, facilitates industrial production and component control, and improves overall sensory quality and drinking compliance, providing a more suitable oral beverage dosage form for this anti-inflammatory and nodule-dispersing active ingredient.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition, a traditional Chinese medicine extract, and their applications. Background Technology

[0002] Acute pharyngitis (AP) refers to an acute inflammation of the pharyngeal mucosa, submucosa, and lymphoid tissue. It generally has a rapid onset, with clinical manifestations including a foreign body sensation in the throat, pain, burning sensation, and speech difficulties. AP has a distinct seasonality, being prevalent in winter and spring. As a very common upper respiratory tract infection, it is widespread globally. Antibiotic overuse is prevalent in clinical treatment, but antibiotics are ineffective against most viral acute pharyngitis cases and can lead to intestinal flora imbalance and the proliferation of drug-resistant bacteria. Traditional Chinese medicine (TCM) compound formulas, with their multi-target mechanism of action, offer an effective approach to reducing antibiotic dependence. However, most TCM preparations currently used to improve acute pharyngitis are traditional decoctions, which suffer from technical drawbacks such as bitter taste, poor patient compliance, cumbersome preparation process, low extraction rate of active ingredients, unstable efficacy, and inconvenience in portability, severely restricting their clinical application and promotion.

[0003] Existing herbal tea products primarily focus on basic health maintenance, lacking targeted formulas for acute pharyngitis with symptoms of excessive heat in the lungs and stomach. They fail to combine the principles of traditional Chinese medicine (TCM) diagnosis and treatment with the convenience and palatability of herbal tea, thus failing to meet patients' needs for safe, effective, and portable adjunctive treatment products. Therefore, developing a compound herbal tea that addresses the shortcomings of traditional Chinese medicine decoctions and is suitable for the prevention and control of acute pharyngitis has become an urgent technical challenge. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, the present invention provides a traditional Chinese medicine composition with good anti-inflammatory effects.

[0005] Another objective of this invention is to provide a traditional Chinese medicine extract.

[0006] Another object of the present invention is to provide an application of a traditional Chinese medicine composition or extract in the preparation of plant-based beverages.

[0007] Another object of the present invention is to provide a plant-based beverage.

[0008] Another object of the present invention is to provide the application of the traditional Chinese medicine composition or the traditional Chinese medicine extract in the preparation of a drug with anti-inflammatory and nodule-dispersing effects.

[0009] Based on this, the present invention has the following technical solution: In a first aspect, the present invention provides a traditional Chinese medicine composition comprising a first component, a second component, and a third component; the first component is Prunella vulgaris and Lophatherum gracile; the second component is Platycodon grandiflorus and Glycyrrhiza uralensis; and the third component is Prunus mume. In the traditional Chinese medicine composition, the content of the first component is 50-60 wt%; The mass ratio of the first component to the second component is 1.5~2.5:1; In the first component, the mass ratio of Prunella vulgaris to Lophatherum gracile is 1:1.5~2.5.

[0010] In this invention, the traditional Chinese medicine composition mainly clears heat and detoxifies, reduces swelling and soothes the throat, and also promotes the production of body fluids and moistens dryness. It is suitable for acute pharyngitis with excessive heat in the lungs and stomach, clinically manifested as red, swollen and painful throat, difficulty swallowing, hoarseness, thick yellow phlegm, fever, dry mouth with thirst, scanty dark urine, red tongue with yellow coating, rapid pulse and other symptoms.

[0011] In the traditional Chinese medicine composition of this invention, the principal ingredient is Prunella vulgaris, which clears heat and purges fire, disperses nodules and reduces swelling. It is a "key medicine for clearing heat and dispersing nodules," capable of clearing heat toxins from the throat and dissipating sore throat and lymph node nodules. Lophatherum gracile clears heat and relieves irritability, promotes urination and guides heat downward. Lophatherum gracile is light and rises, clearing heat from the heart and stomach, guiding heat toxins out through urination, and synergistically enhancing the heat-clearing effect with Prunella vulgaris.

[0012] The assistant herb, Platycodon grandiflorus, promotes lung function, soothes the throat, and eliminates phlegm and pus. Platycodon grandiflorus acts as a "carrier," transporting the medicine upwards, opening up lung qi to soothe the throat, and also eliminating phlegm and pus, relieving sore throat, hoarseness, and sticky, difficult-to-expectorate phlegm. Licorice root clears heat and detoxifies; its sweet and mild nature harmonizes the cold properties of Prunella vulgaris and Lophatherum gracile, enhancing the throat-soothing effect when combined with Platycodon grandiflorus. The adjuvant herb, Prunic acid, astringes yin, generates fluids, and moistens the throat, relieving dryness and burning pain.

[0013] Current technology generally considers nodules to be formed by the interplay of four factors: qi stagnation, phlegm accumulation, blood stasis, and toxin accumulation. Therefore, when using basic medicinal ingredients such as Prunella vulgaris, it is generally believed that the following three types of drugs must be added to achieve the effect of dispersing nodules: Softening and expectorating herbs: such as Fritillaria thunbergii, oyster shell, kelp, and seaweed. It is believed that without such seafood or expectorating herbs, it is impossible to break down hard masses.

[0014] Blood-activating and stasis-removing herbs: such as Sparganium rhizome, Curcuma rhizome, Salvia miltiorrhiza root, and Paeonia lactiflora root. It is believed that "nodules are always caused by blood stasis," and without activating blood circulation, nodules will not disappear. Liver-soothing and qi-regulating herbs: such as Bupleurum, Cyperus, and Citrus reticulata peel. It is believed that the smooth flow of qi will dissipate stagnation, which is a prerequisite for dispersing nodules. In existing anti-inflammatory and nodule-dispersing drugs, it is often necessary to combine multiple auxiliary herbs to achieve the ideal anti-inflammatory and nodule-dispersing effects. This application achieves equivalent or better efficacy by optimizing the compatibility ratio and synergistic effect, while reducing the conventionally added drugs.

[0015] In some specific embodiments, the mass ratio of Platycodon grandiflorus to Glycyrrhiza uralensis in the second component mentioned in this invention is 1:1.5~2.5. Adjusting the mass ratio of Platycodon grandiflorus to Glycyrrhiza uralensis can provide the content of glycyrrhizin and glycyrrhizic acid in the active ingredients of the compound.

[0016] In some more specific exemplary embodiments, the formulation of the traditional Chinese medicine composition mentioned in this invention is detailed as follows: The formula includes the following components in parts by weight: 16-24 parts of Prunella vulgaris; 36-44 parts of Lophatherum gracile; 6-14 parts of Platycodon grandiflorus; 16-24 parts of Glycyrrhiza uralensis; and 16-24 parts of Prunus mume.

[0017] Among them, the amount of Prunella vulgaris can be 16 parts, 18 parts, 20 parts, 22 parts, 24 parts, or any range of values. Lophatherum gracile can be a range of values ​​consisting of 36, 38, 40, 42, or 44 parts, or any other combination thereof. Platycodon grandiflorus can be a range of values, such as 36, 38, 40, 42, or 44 parts, or any other range of values. Licorice can be in point values ​​of 6 parts, 8 parts, 10 parts, 12 parts, 14 parts, or any range of values. The amount of dried plum can be 16, 18, 20, 22, 24, or any range of values.

[0018] In some other embodiments, the traditional Chinese medicine composition mentioned in this invention further includes a fourth component, which is dandelion and honeysuckle, and the mass ratio of the first component to the fourth component is 1:1.0~1.5.

[0019] Similarly, in terms of anti-inflammatory and nodule-dispersing effects, existing technologies often consider the anti-inflammatory properties of honeysuckle and dandelion to be limited, and usually require the addition of: Heat-clearing and detoxifying herbs include: Forsythia suspensa, Scutellaria baicalensis, Isatis indigotica, Isatis tinctoria, and Belamcanda chinensis. These herbs are believed to enhance heat-clearing and detoxifying effects, targeting symptoms of excessive heat and toxins.

[0020] Heat-clearing and bowel-regulating herbs include rhubarb and gardenia. It is believed that heat is expelled through the stool.

[0021] Yin-nourishing and dryness-moistening herbs include: Ophiopogon japonicus, Scrophularia ningpoensis, and Rehmannia glutinosa. This is believed to prevent bitter and cold herbs from damaging yin.

[0022] In traditional Chinese medicine theory and existing patent literature, the above-mentioned combination of drugs is considered "indispensable." Chronic nodules are hard in texture, and only "heavy-sedating and phlegm-reducing" herbs such as fritillaria and oyster shell can loosen their foundation. Inflammation is often accompanied by systemic immune responses and microcirculatory disorders. Without the addition of blood-activating drugs, local metabolism cannot be improved, making it difficult for the active ingredients of the drugs to reach the core inflammatory lesion. Without soothing the liver and regulating qi, nodules will "grow back while disappearing."

[0023] This application achieves comparable or better anti-inflammatory and nodule-dispersing effects by reducing the number of conventionally added drugs. The core of this application lies in the fact that the seven medicinal materials (dandelion, honeysuckle, prunella vulgaris, lophatherum gracile, platycodon grandiflorus, licorice, and dried plum) form a synergistic and synergistic organic whole in a specific ratio. The medicinal materials promote and restrain each other, thereby maximizing the efficacy.

[0024] The optimal pairing logic of monarch, minister, assistant, and envoy: Principal herbs: Clearing heat and detoxifying, dispersing nodules and reducing swelling: Dandelion and honeysuckle are the principal herbs. Dandelion excels at clearing heat and detoxifying, dispersing nodules and reducing swelling, and is especially good at treating mastitis and scrofula; honeysuckle is sweet and cold, clearing heat and detoxifying, and is good at clearing heat from the lungs and stomach, and has a strong ability to disperse carbuncles and swelling. When the two are used together, their effects of clearing heat and detoxifying, and dispersing nodules are significantly enhanced.

[0025] Assistant herbs: Prunella vulgaris and Lophatherum gracile are the assistant herbs, assisting the principal herb in clearing heat and specifically targeting nodules. Prunella vulgaris is a key herb for dispersing nodules, effectively clearing liver fire and dispersing stagnation, especially effective in treating scrofula and phlegm nodules; Lophatherum gracile clears heat, drains fire, promotes urination, and guides heat downwards. These two herbs assist the principal herb in enhancing its heat-clearing and nodule-dispersing effects, while Lophatherum gracile, through its diuretic properties, helps to expel heat from below.

[0026] Adjuvant herbs: Platycodon grandiflorus is used as an adjuvant herb to clear the lungs, soothe the throat, resolve phlegm, and drain pus. It can guide the medicine upwards to the upper burner and also resolve phlegm and dissipate nodules. When combined with Prunella vulgaris and Taraxacum mongolicum, it simultaneously resolves phlegm and dissipates nodules, enhancing the therapeutic effect on inflammatory nodules.

[0027] Guiding herbs: harmonizing all herbs, astringent and yin-tonifying: Licorice and dried plum are used together as guiding herbs. Licorice harmonizes all herbs, clears heat and detoxifies, and relieves spasms and pain; dried plum is astringent and generates fluids, which can prevent bitter and cold herbs from damaging yin, and its astringent properties help to disperse stagnation. When used together, the two harmonize the properties of the herbs, so that the whole formula clears heat without damaging yin and disperses stagnation without depleting qi.

[0028] In some preferred exemplary embodiments, the ratio of dandelion, honeysuckle and prunella vulgaris in the traditional Chinese medicine composition mentioned in this invention is 4:3:2. Dandelion and honeysuckle clear heat and detoxify, while dandelion and prunella vulgaris reduce swelling and dissipate nodules. The combination of the three herbs emphasizes both clearing heat and dissipating nodules.

[0029] The ratio of Platycodon grandiflorus to Glycyrrhiza uralensis is 1:2: Platycodon grandiflorus clears the lungs and resolves phlegm, while Glycyrrhiza uralensis harmonizes and detoxifies. The combination of the two enhances the effects of resolving phlegm, draining pus, relieving sore throat and pain.

[0030] Equal parts of dried plum and licorice are combined to maximize the effect of "sour and sweet nourishing yin" and effectively curb the side effects of bitter and cold medicines.

[0031] The efficacy of traditional Chinese medicine compound prescriptions depends not only on the types of ingredients but also on the proportional relationship between them. This invention optimizes the proportions, ensuring that each step is interconnected and mutually reinforcing, thus achieving excellent anti-inflammatory and nodule-dispersing effects while reducing the amount of conventionally added drugs.

[0032] In some more specific exemplary embodiments, the specific formulation of the traditional Chinese medicine composition mentioned in this invention is further preferred as follows: Prunella vulgaris 16-24 parts; Lophatherum gracile 36-44 parts; Platycodon grandiflorus 6-14 parts; Glycyrrhiza uralensis 16-24 parts; Prunus mume 16-24 parts; Taraxacum mongolicum 32-48 parts; Lonicera japonica 24-36 parts.

[0033] Secondly, the present invention also specifically provides a traditional Chinese medicine extract, which is extracted according to the following method: S1: The traditional Chinese medicine composition is dried at 55~70℃ to obtain a raw material with a water content of 3~8wt%; S2: Crush the raw material obtained in S1, soak it in water, and heat it for extraction. The material-to-liquid ratio for extraction is 1:10~50, the extraction temperature is 60~100℃, and the extraction time is 20~60min.

[0034] In some specific embodiments, the extraction method of the traditional Chinese medicine extract protected by the present invention is preferably as follows: the material-to-liquid ratio is 1:20~40 (g / mL), the extraction temperature is 70~90℃, and the extraction time is 30~50min.

[0035] In the extraction process of the traditional Chinese medicine extract mentioned in this invention, by optimizing the material-liquid ratio, extraction temperature and extraction time, a suitable concentration gradient can be maintained, cell membrane permeability can be enhanced, and molecular diffusion and dissolution balance can be controlled, so that active ingredients such as flavonoids and saponins can be fully and stably dissolved, avoiding degradation and waste. At the same time, it can also inhibit the excessive dissolution of bitter, astringent and off-flavor substances, reduce burnt taste and viscosity, and make the extract more mellow in flavor and significantly improve palatability, thus solving the problems of poor taste and low utilization rate of effective ingredients in traditional decoctions.

[0036] In some specific embodiments, the extraction method of the traditional Chinese medicine extract mentioned in this invention, expressed in g / mL, may have a material-to-liquid ratio of 1:20, 1:25, 1:30, 1:35, 1:40, or any other arbitrary range; the extraction temperature may be 70℃, 80℃, 90℃, or any other arbitrary range; and the extraction time may be 30min, 35min, 40min, 50min, or any other arbitrary range.

[0037] According to the preparation method provided by the present invention, S2 is followed by S3, which includes: filtering the extract, centrifuging the filtrate, and concentrating the supernatant under reduced pressure to a specific gravity of 1.05 to 1.15.

[0038] Thirdly, the present invention provides an application of the traditional Chinese medicine composition or the traditional Chinese medicine extract in the preparation of plant-based beverages.

[0039] Fourthly, the present invention also specifically provides a plant-based beverage containing the above-mentioned traditional Chinese medicine composition or extract for anti-inflammatory and nodule-dispersing purposes and excipients, wherein the excipients include one or more of steviol glycosides, zero-calorie sugar, and N,2,3-trimethyl-2-isopropylbutyramide (WS-23).

[0040] The Xia Zhu Qing extract obtained after the extraction process has a thin taste and lacks sweetness. It can be formulated with certain excipients to better meet consumer taste preferences. With the increasing health awareness of global consumers and the deepening trend of "sugar reduction" in the international food industry, the market demand for natural sweeteners has increased significantly. Steviol glycosides (Stv) are diterpenoid glycoside compounds isolated and purified from the leaves of the Stevia rebaudiana Bertoni plant. Due to their high sweetness and low calorie content, they are now widely used in the food and herbal tea industries. Vanilla-flavored zero-calorie sugar is a complex syrup with a refreshing and crisp overall taste. WS-23 (N,2,3-trimethyl-2-isopropylbutyramide) is a synthetic cooling agent. Compared with traditional menthol, WS-23 has no burning, numbness, or irritating side effects, and its taste is more rounded and refreshing.

[0041] This invention overcomes the technical problems of poor palatability and difficulty in formulating beverages caused by the inherent bitterness and pungent odor of traditional Chinese medicine extracts by combining sweeteners and cooling agents to improve flavor. Simultaneously, through the synergistic combination of these excipients, the taste and cooling sensation of the beverage are significantly improved without affecting the stability of the active ingredients, enhancing overall sensory quality and drinking compliance, and providing a more suitable oral beverage formulation for this anti-inflammatory and nodule-dispersing active ingredient.

[0042] In a specific embodiment, the composition of the excipients in the plant beverage mentioned in this invention is as follows: by weight, 0.008~0.012 parts of steviol glycosides; 0.8~1.2 parts of zero-calorie sugar; and 0.0016~0.0024 parts of N,2,3-trimethyl-2-isopropylbutyramide.

[0043] In some specific embodiments, the preferred mass ratio of steviol glycosides to zero-calorie sugar is 1:50~150, more preferably 1:100.

[0044] In some more specific exemplary preferred embodiments, the plant-based beverages mentioned in this invention may be components in the following weight proportions: 16-24 parts of Prunella vulgaris; 36-44 parts of Lophatherum gracile; 6-14 parts of Platycodon grandiflorus; 16-24 parts of Glycyrrhiza uralensis; 16-24 parts of Prunus mume; 0.008-0.012 parts of Stevioside; 0.8-1.2 parts of Zero-Calorie Sugar; 0.0016-0.0024 parts of WS-23 (N,2,3-trimethyl-2-isopropylbutyramide); 100 parts of Water.

[0045] In some more specific exemplary preferred embodiments, the plant beverage mentioned in this invention may also consist of the following components in parts by weight: 16-24 parts of Prunella vulgaris; 36-44 parts of Lophatherum gracile; 6-14 parts of Platycodon grandiflorus; 16-24 parts of Glycyrrhiza uralensis; 16-24 parts of Prunus mume; 32-48 parts of Taraxacum mongolicum; 24-36 parts of Lonicera japonica; 0.008-0.012 parts of Stevioside; 0.8-1.2 parts of zero-calorie sugar; 0.0016-0.0024 parts of WS-23 (N,2,3-trimethyl-2-isopropylbutyramide); and 100 parts of water.

[0046] In the above-mentioned herbal beverage formula of the present invention, a simple traditional Chinese medicine composition is used to achieve a synergistic effect of anti-inflammatory and nodule-dispersing effects, while the excipient system is combined to specifically correct the taste. Among them, the sweetener steviol glycoside can initially mask the bitterness, and the use of zero-calorie sugar can not only make up for the bitterness of steviol glycoside and enhance the sweetness, but also form a compatibility with the taste of traditional Chinese medicine, avoiding the sweetness from being out of place with the taste of traditional Chinese medicine. At the same time, it does not react with the active ingredients of traditional Chinese medicine and does not affect the anti-inflammatory and nodule-dispersing effects.

[0047] N,2,3-Trimethyl-2-isopropylbutyramide is a non-irritating cooling agent that can alleviate the bitterness and spiciness of traditional Chinese medicine, enhance the refreshing taste of beverages, and work synergistically with sweeteners to create a sweet but not cloying, refreshing but not harsh taste. At the same time, its addition will not affect the stability of the active ingredients of traditional Chinese medicine.

[0048] The aforementioned plant-based beverages can not only meet the public's demand for portable functional drinks, but also achieve daily prevention and control of respiratory diseases, effectively saving medical expenses and optimizing the allocation of health resources.

[0049] In a specific embodiment of the present invention, the preparation method of the mentioned plant-based beverage is as follows: The excipients and concentrated extracts of traditional Chinese medicine are mixed and homogenized, then bottled, degassed, sterilized, and cooled to prepare the beverage.

[0050] The concentrated Chinese herbal extract can be obtained by extracting and concentrating the Chinese herbal composition protected by this invention, or it can be a Chinese herbal extract protected by this invention.

[0051] Fifthly, in a specific embodiment, the present invention also provides the application of the traditional Chinese medicine composition or the traditional Chinese medicine extract in the preparation of a drug with anti-inflammatory and nodule-dispersing effects.

[0052] The traditional Chinese medicine composition of this invention can alleviate oxidative stress by inhibiting the generation of reactive oxygen species (ROS) and nitric oxide (NO), reducing the number and aggregation of neutrophils at the site of injury, and decreasing the relative expression levels of pro-inflammatory factors TNF-α, IL-6, IL-8, IL-1β, and key genes of the NF-κB pathway, BCL2L1, NF-κB, MAPK, and JNK mRNA. This inhibits the anti-inflammatory effect of the NF-κB / MAPK signaling pathway, thus exhibiting good anti-inflammatory and nodule-dispersing effects. It can be applied to treat inflammatory diseases such as acute pharyngitis.

[0053] Based on this, the technical solution of the present invention has the following beneficial effects: This invention provides a traditional Chinese medicine composition, with Prunella vulgaris and Lophatherum gracile as the principal herbs. Prunella vulgaris clears heat and purges fire, disperses nodules and reduces swelling, while Lophatherum gracile clears heat and purges fire, disperses nodules and reduces swelling, thus synergistically enhancing the heat-clearing effect. At the same time, the sweet and mild nature of Glycyrrhiza uralensis harmonizes the cold medicinal properties of Prunella vulgaris and Lophatherum gracile. When used with Platycodon grandiflorus, it enhances the throat-soothing effect. Prunic acid is added to astringe yin, promote fluid production and moisten the throat, which can relieve dryness and burning pain in the throat, and achieve good anti-inflammatory and nodule-dispersing effects.

[0054] The traditional Chinese medicine composition provided by this invention is simple and reasonable, overcoming the drawbacks of existing anti-inflammatory and nodule-dispersing traditional Chinese medicine compositions that are cumbersome and have redundant components. While ensuring synergistic anti-inflammatory and nodule-dispersing effects, the simplified composition facilitates industrial production and component control. Furthermore, this invention overcomes the technical problems of poor palatability and difficulty in formulating beverages due to the inherent bitterness and irritating odor of traditional Chinese medicine extracts by compounding sweeteners and cooling agents, thus improving overall sensory quality and drinking compliance. This provides a more suitable oral beverage formulation for the anti-inflammatory and nodule-dispersing active ingredient. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0056] Figure 1 This invention provides the effect of different experimental groups on the ROS generation rate in LPS-induced zebrafish. - x±s, n=12), where a is the experimental flowchart; b is the fluorescence intensity map of zebrafish in different experimental groups; c is the effect of different experimental groups on LPS-induced ROS generation rate in zebrafish, ##P<0.01 compared with the blank group; compared with the model group, P<0.01.

[0057] Figure 2 This invention provides the effect of different experimental groups on the NO production rate in zebrafish. - (x±s, n=30); compared with the blank group, ##P<0.01; compared with the model group, P<0.01.

[0058] Figure 3 This invention provides the effects of different experimental groups on CuSO4-induced neutrophil migration and aggregation in zebrafish. - x±s, n=15), where a is the experimental flowchart; b is the distribution map of neutrophils in zebrafish in different experimental groups; c is the analysis of neutrophil aggregation in different experimental groups. Compared with the control group, ##P<0.01; compared with the model group, P<0.01.

[0059] Figure 4 This invention relates to the effects of different experimental groups on the relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNA in zebrafish. - x±s, n=30), where a represents the relative expression of TNF-α mRNA; b represents the relative expression of IL-6 mRNA; c represents the relative expression of IL-1β mRNA; d represents the relative expression of IL-8 mRNA. Compared with the control group, ##P<0.01; compared with the model group, P<0.01, P<0.05.

[0060] Figure 5 This invention describes the effects of different experimental groups on the relative expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA in zebrafish, where a represents the relative expression level of BCL2L1 mRNA; b represents the relative expression level of NF-κB mRNA; c represents the relative expression level of MAPK1 mRNA; and d represents the relative expression level of JNK mRNA. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0062] Unless otherwise specified, all raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0063] Example 1 A traditional Chinese medicine composition comprising a first component, a second component, and a third component, wherein the first component is Prunella vulgaris and Lophatherum gracile, the second component is Platycodon grandiflorus and Glycyrrhiza uralensis, and the third component is Prunus mume. The specific composition is as follows: 1g of Prunella vulgaris, 2g of Lophatherum gracile, 0.5g of Platycodon grandiflorus, 1g of Glycyrrhiza uralensis, and 1g of Prunus mume.

[0064] This embodiment also provides a traditional Chinese medicine extract, which is extracted from the above-mentioned traditional Chinese medicine components through the following process: After washing the selected Prunella vulgaris, Lophatherum gracile, and raw Glycyrrhiza uralensis twice with deionized water, they were dried in a 60℃ forced-air drying oven for 3 hours to obtain raw materials with a moisture content of 5.0%. The Prunella vulgaris, stir-fried Lophatherum gracile, Platycodon grandiflorus, raw Glycyrrhiza uralensis, and Prunus mume were then pulverized using a pulverizer and passed through a 20-mesh sieve.

[0065] Place each component in a round-bottom flask, add pure water and soak for 30 min, then heat and reflux for extraction: material-to-liquid ratio 1:20 (g / mL), extraction temperature 70℃, extraction time 30 min.

[0066] After extraction, the solution was filtered through a double layer of 300-mesh nylon cloth, and the filtrates were combined. The filtrate was centrifuged at 8000 rpm for 10 min, concentrated under reduced pressure, and then freeze-dried to obtain a lyophilized powder.

[0067] In this embodiment, the heating reflux method is simple to operate and has high extraction efficiency. Considering the issue of solvent residue, pure water is used for extraction. To address cost considerations for industrial production, extraction is performed twice.

[0068] For the detection of effective active ingredients, please refer to the following methods: Flavonoid content determination The NaNO2-Al(NO3) colorimetric method was used. Accurately weigh rutin reference standard, dissolve it in methanol, and prepare a solution with a mass concentration of 0.4 mg / mL. Take 0, 1, 2, 3, 4, 5, and 6 mL of the rutin stock solution into 10 mL volumetric flasks, add 0.3 mL of 5 wt% sodium nitrite solution to each, shake well, let stand for 6 min, then add 0.3 mL of 10 wt% aluminum nitrate solution, shake well, let stand for 6 min, then add 4 mL of 4 wt% sodium hydroxide solution, dilute to the mark with methanol to 10 mL, shake well, and let stand for 15 min. Measure the absorbance at 510 nm. Plot a standard curve with rutin mass concentration on the x-axis and absorbance on the y-axis, and calculate the regression equation.

[0069] For sample determination, accurately weigh the herbal extract (lyophilized powder), add methanol, sonicate (100W, 60℃) for 1 hour, centrifuge at 8000 rpm for 10 minutes, and collect the supernatant. Dilute the supernatant to 0.1 mg / mL, and perform subsequent operations as per the standard curve determination. After measuring the absorbance, calculate the flavonoid content in Xia Zhu Qing based on the standard curve and dilution factor.

[0070] Determination of total saponin content The vanillin-sulfuric acid method was used. Accurately weigh glycyrrhizic acid reference standard, dissolve it in methanol, and prepare a solution with a mass concentration of 1 mg / mL. Take 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the glycyrrhizic acid stock solution into 10 mL volumetric flasks, evaporate the solvent in a 70°C water bath, add 0.2 mL of 5% (w / v) vanillin-glacial acetic acid solution, then add 0.8 mL of perchloric acid, shake well, and react in a 55°C water bath for 20 min. Immediately remove and cool to room temperature under running water, add glacial acetic acid to the mark, and measure the absorbance at a wavelength of 589 nm. Plot a standard curve with glycyrrhizic acid concentration (mg / mL) on the x-axis and absorbance values ​​on the y-axis, and calculate the regression equation.

[0071] Examples 2-9 Examples 2-9 provide a traditional Chinese medicine extract, which is extracted using the same traditional Chinese medicine components as in Example 1 through the process shown in Table 1 below.

[0072] The content of active ingredients in the herbal extracts of Examples 1-9 was determined, and the determination method was the same as in Example 1. The results are shown in Table 1 below.

[0073] Table 1. Extraction Process

[0074] The comprehensive weighted score of flavonoid and total saponin content is used as the evaluation index. The comprehensive weighted score Y = flavonoid content × 0.5 + total saponin content × 0.5.

[0075] The test results in Table 1 show that the material-to-liquid ratio, extraction temperature, and extraction time synergistically affect the extraction efficiency of the needle. Failure to meet any of these conditions will affect the full release and dissolution of flavonoids, thus impacting extraction efficiency. However, the effects of the extraction process are not linear. Exceeding certain ranges in the material-to-liquid ratio, extraction temperature, and extraction time can also lead to premature reaching of the dissolution equilibrium, reducing flavonoid extraction efficiency. Furthermore, flavonoids may decompose or oxidize due to thermal instability, resulting in a decrease in overall extraction efficiency. This study found the optimal extraction process to be a material-to-liquid ratio of 1:40 (g / mL), an extraction temperature of 90℃, and an extraction time of 40 min. Under these conditions, the flavonoid content was 0.324 mg / mL, the total saponin content was 0.068 mg / mL, and the overall score Y=0.196. Three experiments were conducted under these conditions, and the average flavonoid content was measured to be (0.333±0.04) mg / mL, the total saponin content was (0.071±0.003) mg / mL, and the comprehensive score Y was (0.202±0.02).

[0076] Example 10 This embodiment provides a traditional Chinese medicine extract, which differs from Embodiment 9 in that: the selected Prunella vulgaris, Lophatherum gracile, and Glycyrrhiza uralensis are washed twice with deionized water, and then dried at room temperature without heating before being directly pulverized.

[0077] Compared with Example 1, this example omits the 60°C forced-air drying for 3 hours step in the pretreatment stage, and instead uses room temperature air drying. The results show that, under the same extraction process parameters (material-to-liquid ratio 1:40, extraction temperature 90°C, extraction time 50 min), the extract obtained in this example has a flavonoid content of 0.312 mg / mL, a total saponin content of 0.065 mg / mL, and a comprehensive score of 0.189, which is only slightly lower than that of Example 9.

[0078] This embodiment demonstrates that the method for preparing the traditional Chinese medicine composition of this application has good process robustness. Even if the heating and drying step in the pretreatment process is omitted, a high extraction rate of flavonoids and total saponins can still be obtained, thereby ensuring its anti-inflammatory efficacy.

[0079] Example 11 A traditional Chinese medicine composition includes a first component, a second component, a third component, and a fourth component. The first component is Prunella vulgaris and Lophatherum gracile, the second component is Platycodon grandiflorus and Glycyrrhiza uralensis, the third component is Prunus mume, and the fourth component is Taraxacum mongolicum and Lonicera japonica. The specific composition is as follows: 1g of Prunella vulgaris, 2g of Lophatherum gracile, 0.5g of Platycodon grandiflorus, 1g of Glycyrrhiza uralensis, 1g of Prunus mume, 2g of Taraxacum mongolicum, and 1.5g of Lonicera japonica.

[0080] This embodiment also provides a traditional Chinese medicine extract, the extraction process of which is the same as that in Example 9.

[0081] The extract obtained in this example contained 0.423 mg / mL of flavonoids and 0.078 mg / mL of total saponins, with a comprehensive score of 0.25. Compared with Example 1, the contents of flavonoids and total saponins were increased. The results indicate that the addition of dandelion and honeysuckle significantly increased the content of flavonoid and saponin active ingredients in the extract.

[0082] Example 12 A plant-based beverage containing the herbal extract and excipients prepared in Example 11 is prepared as follows: The concentrate obtained in Example 11 was mixed with excipients and homogenized. Then, the mixture was filled, degassed, sterilized, and cooled in sequence to prepare the finished beverage product. The excipients are shown in Table 2.

[0083] Examples 13-20 A plant-based beverage contains the herbal extract and excipients prepared in Example 9. The preparation method is the same as in Example 12, except that the excipients are added according to the formula shown in Table 2.

[0084] Table 2.

[0085] In Table 2, the content of steviol glycoside A is the percentage added based on the quality of the Chinese herbal extract.

[0086] For the herbal beverages prepared in Examples 13-20, a sensory evaluation table (Table 3) was developed based on the sensory evaluation requirements of "Herbal Tea" (GB / T31326—2014) and the characteristics of Xia Zhu Qing Liang tea. Ten professionals trained in sensory evaluation, with a male-to-female ratio of 1:1, were selected to evaluate the Xia Zhu Qing Liang tea. The scoring criteria are shown in Table 3.

[0087] Table 3. Sensory Evaluation Table of Summer Bamboo Cooling Tea

[0088] The scoring results are shown in Table 2. Table 2 shows that the addition of sweeteners and cooling agents needs to achieve a synergistic balance to achieve better sensory quality. Appropriate amounts of the sweetener steviol glycosides can improve the sweetness of plant-based beverages, and the appropriate addition of vanilla-flavored zero-calorie sugar can enrich the taste. The cooling agent WS-23 can give Xia Zhu Qing a refreshing taste, but the overall compatibility of the excipient system must also be considered to avoid an overly strong mint flavor that could negatively affect the taste.

[0089] The herbal beverage obtained by this invention is refreshing and soothing to the throat, with a moderate sweetness and a unique, light bamboo leaf aroma. Its optimal sensory score can reach 94 points. The herbal beverage of this invention is light yellow with a uniform and bright color; it has a unique, light bamboo leaf aroma; it has a moderate sweetness, a long-lasting cooling sensation, and a slightly sweet aftertaste, without the bitterness of traditional herbal teas.

[0090] Comparative Example 1 This comparative example provides a method for preparing a traditional Chinese medicine composition for anti-inflammatory purposes. The difference between this method and Example 1 is that the traditional Chinese medicine raw materials also contain 1.0g of Forsythia suspensa and 1.0g of Ophiopogon japonicus.

[0091] Results: The extract obtained in this comparative example contained 0.353 mg / mL of flavonoids and 0.078 mg / mL of total saponins, with a comprehensive score of 0.216, which is basically equivalent to that of the present invention.

[0092] This comparative example demonstrates that the heat-clearing and detoxifying drugs and yin-nourishing drugs, which are considered essential for anti-inflammatory effects in the prior art, are not necessary in the summer bamboo cooling tea combination of this application. The present invention achieves a comparable anti-inflammatory effect even without the aforementioned components, demonstrating the simplicity and cost advantage of the technical solution of this application.

[0093] Comparative Example 2 This comparative example provides a method for preparing a traditional Chinese medicine composition for anti-inflammatory purposes. The difference between this method and Example 1 is that the traditional Chinese medicine raw materials also contain 1.0g of Salvia miltiorrhiza and 1.0g of Bupleurum chinense.

[0094] The extract obtained in this comparative example had a flavonoid content of 0.340 mg / mL, a total saponin content of 0.079 mg / mL, and a comprehensive score of 0.209, which is basically equivalent to that of the present invention.

[0095] This comparative example demonstrates that the blood-activating and stasis-removing drugs and liver-soothing and qi-regulating drugs, which are considered essential for nodules in the prior art, are not necessary in the Xia Zhu Qing Liang Tea combination of this application. Example 1, even without omitting the above-mentioned components, still achieves a considerable anti-inflammatory effect, reflecting the simplicity and cost advantage of the technical solution of this application.

[0096] Test case I. Evaluation and testing of the anti-inflammatory activity of the traditional Chinese medicine composition provided in Example 9: 1 Method 1.1 Zebrafish breeding and embryo collection and culture The day before the experiment, sexually mature zebrafish were placed in a spawning tank at a female-to-male ratio of 1:1 and separated by a transparent partition. The partition was removed at 9:00 AM the following morning, and the fertilized eggs of the zebrafish were collected at 11:00 AM. The collected fertilized eggs were incubated in embryo culture medium, and poorly developed and dead eggs were removed daily, and the embryo culture water was changed.

[0097] 1.2 Grouping and Drug Administration in Zebrafish Experiments The zebrafish experimental groups were: blank group, model group (10 μg / mL LPS (lipopolysaccharide) and 20 μM CuSO4), dexamethasone group (DSMS group: 10 μg / mL), XZQ-L, XZQ-M, and XZQ-H groups (100, 200, and 300 μg / mL, respectively), and throat-clearing and pharyngeal-soothing granules group (QHLYKL group: 200 μg / mL). Each group was given the corresponding drug.

[0098] The XZQ-L, XZQ-M, and XZQ-H groups are freeze-dried powders of traditional Chinese medicine extracts, without added excipients, and are prepared using embryo culture medium.

[0099] 1.3 Screening of non-toxic doses of Xia Zhuqing Five concentrations of Xia Zhu Qing (100, 300, 400, 500, and 600 μg / mL) were selected. AB-strain zebrafish with normal development at 7-9 hpf (embryonic stage) after fertilization were randomly selected and transferred to 6-well plates using pipettes, with 30 juvenile fish per group. The embryo culture medium was carefully discarded, and 5 mL of Xia Zhu Qing at different concentrations was added to each well (except for the normal group) for soaking and treatment. After treatment, the embryos were placed in a 28.5℃ incubator, and the culture medium was changed every 24 hours, with dead embryos removed promptly. Embryo survival was observed and recorded from 0 to 72 hours, and the non-toxic concentration at 72 hours was selected for subsequent experiments.

[0100] 1.4 Establishment and drug administration of a zebrafish inflammation model 1.4.1 LPS Model Zebrafish embryos with normal development at 7-9 hpf (embryonic stage) after fertilization were selected and grouped into three parallel groups according to "1.2". Except for the control group and the model group, each group was given 5 mL of the corresponding drug solution. After 2 hours, the solution was removed, and all groups except the control group were given 5 mL of 10 μg / mL LPS solution and incubated for 72 hours. The development of the zebrafish embryos in each group was observed, and the drug solution was changed every 24 hours.

[0101] 1.4.2 CuSO4 Model Normally developed 3dpf zebrafish juveniles of the AB strain or Tg(lyz:EGFP) were selected and grouped into three parallel groups according to "1.2". After removing the embryo culture water, except for the normal group and the model group, all other groups were immersed in 5 mL of the corresponding drug solution. After 2 h, the drug solution was removed, and except for the normal group, 5 mL of 20 μM CuSO4 solution was added and incubated for 1 h.

[0102] 1.5 Measurement of neutrophils, reactive oxygen species, and nitric oxide 1.5.1 Reactive Oxygen Detection Dilute DCFH-DA with embryo culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. Take 12 zebrafish juveniles from each group ("1.4.1") and place them in a 24-well plate. Remove the embryo culture medium, add 1 mL of diluted DCFH-DA to each well, and incubate at 28.5℃ in the dark for 30 min. Gently shake the plate periodically to ensure the probe fully contacts the juveniles. After incubation, wash the zebrafish five times with embryo culture medium and then anesthetize them with 0.02% tricaine. Transfer the zebrafish to a culture dish containing 3% sodium methylcellulose aqueous solution, observe and photograph them under a fluorescence microscope, maintaining consistent microscope parameters, and analyze and statistically determine the relative fluorescence intensity using ImageJ software.

[0103] ROS generation rate = (Fluorescence intensity in the drug-treated group / Fluorescence intensity in the blank group) × 100% 1.5.2 NO generation rate Thirty zebrafish juveniles from each group ("1.4.1") were taken, rinsed five times with phosphate buffer, anesthetized with 0.02% tricaine, and then thoroughly ground in a grinder with 150 μL of phosphate buffer and grinding beads. The grinder was run at 70 Hz for 60 seconds, three times. After grinding, the sample was centrifuged at 8000 rpm for 15 min, and the supernatant was collected to obtain the zebrafish sample for testing. The procedure was performed according to the instructions of the zebrafish BCA protein concentration assay kit and the nitric oxide assay kit.

[0104] NO generation rate = NO concentration / BCA concentration × 100% (unit: μmol / mg) 1.5.3 Neutrophil Count Fifteen zebrafish juveniles from each group ("1.4.2") were taken, rinsed with embryo culture medium, and anesthetized with 0.02% tricaine. The zebrafish juveniles were photographed under a fluorescence microscope, and the distribution and number of neutrophils in the lateral line thalamus cell region were observed and counted.

[0105] 1.6 Detection of inflammatory factor mRNA levels using RT-qPCR 1.6.1 Extraction and concentration detection of total RNA from zebrafish Thirty zebrafish from each group ("1.4.2") were selected, washed five times with PBS, homogenized, and centrifuged to collect the supernatant. Total RNA was extracted from each group's samples using an ultrapure RNA extraction kit. RNA concentration was determined by UV absorption spectroscopy: A NanoDrop® ND-2000 was used to determine RNA concentration and purity. Before measurement, the instrument was zeroed with DEPC water used for dissolving RNA. A reading of 1 at 260 nm represented 40 ng / μL RNA. The formula for calculating sample RNA concentration is: A = 260 × 40 ng / μL.

[0106] 1.6.2 RNA reverse transcription into cDNA Step 1: Remove genomic DNA according to the reaction system and procedure in Table 4. Step 2: Prepare the reaction mixture on ice according to the components in Table 5. To ensure the accuracy of the reaction mixture preparation, prepare MasterMix by adding 2 units of reaction number before each reaction, and finally add the product from Step 1.

[0107] Table 4. Genomic DNA Removal Reaction System and Procedure

[0108] Table 5. Reverse transcription reaction system and procedure

[0109] 1.6.3 Real-time PCR reaction Using GAPDH as an internal control, the DNA sequence for target gene amplification was retrieved from the NCBI website. Specific primer sequences are listed in Table 6. The reaction system was prepared according to Table 7, centrifuged with vortexing, and transferred to PCR plates. 1 µl of the corresponding DNA was added, and the plates were carefully sealed with sealing film. The plates were centrifuged with vortexing, and the prepared PCR plates were placed on ice before setting the PCR program. The 96-PCR plates were then placed on a Realtime PCR instrument for PCR. The PCR program was set as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 5 s; 60℃ annealing + extension for 30 s, for a total of 40 cycles. After the reaction, 2- CT method for analyzing the expression level of target genes.

[0110] Table 6. Real-time PCR reaction system

[0111] Table 7. Primer sequence listing

[0112] 1.7 Data Processing All experiments were independently repeated three times, and all data were presented as follows: - x±s. Statistical analysis was performed using Excel 2019 and SPSS 17.0 software. Graphs were generated using OriginLabOriginPro9 software. One-way ANOVA was used to compare data among multiple groups to ensure homogeneity of variance. Least Significant Difference (LSD) t-tests were used for pairwise comparisons. P<0.05 was considered statistically significant.

[0113] 2 Results 2.1 Effects of different concentrations of plant extracts on the survival rate of zebrafish embryos Before further studying the anti-inflammatory effects of plant extracts using zebrafish embryos, their toxicity needs to be evaluated. The effects of different concentrations of *Xia Zhu Qing* on the hatching rate of zebrafish embryos are shown in Table 8. Therefore, based on the results, concentrations of 100, 200, and 300 μg / mL of *Xia Zhu Qing* were selected for subsequent experiments.

[0114] Table 8. Effects of Xia Zhuqing on zebrafish embryo survival rate

[0115] 2.2 ROS generation rate Reactive oxygen species (ROS) are normal byproducts of biological processes. Under normal physiological conditions, biological systems have the ability to scavenge ROS, thereby maintaining the body's redox homeostasis. Excessive ROS production can damage cell structure, trigger oxidative stress, and activate inflammation-related signaling pathways, such as the NF-κB signaling pathway. At the same time, the inflammatory response also promotes the accumulation of ROS.

[0116] Depend on Figure 1 The presence of inconspicuous green fluorescence in the blank group of zebrafish indicates a low level of ROS in zebrafish under normal physiological conditions. LPS induction significantly enhanced fluorescence intensity and greatly increased ROS generation rate (P<0.01), indicating successful model establishment. Compared with the model group, both the XZQ group and the positive control groups (DSMS group and QHLYKL group) significantly reduced fluorescence intensity and ROS generation rate in zebrafish (P<0.01). The DSMS group showed better results than the XZQ and QHLYKL groups, with a ROS generation rate closest to the normal group. The XZQ-L, XZQ-M, and XZQ-H groups showed increasingly better inhibition of ROS generation with increasing dosage, with each XZQ group showing better results than the QHLYKL group. This suggests that Xia Zhuqing may exert its anti-inflammatory effect by inhibiting ROS generation and reducing oxidative stress.

[0117] 2.3 NO generation rate In mammals, three distinct subtypes of NOS have been identified: neuronal nNOS, inducible iNOS, and endothelial eNOS. nNOS and eNOS are primarily expressed in neurons and endothelial cells; these subtypes produce NO via a pulsatile process using low-output Ca2+-dependent enzymes. iNOS is a high-output Ca2+-independent NOS. iNOS can be induced to produce large amounts of NO by endotoxins and cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ). Excess NO can combine with superoxide anions to form peroxynitrite (ONOO). - This leads to oxidative stress, which damages cell membranes, proteins, and DNA, exacerbating inflammation.

[0118] The experiment yielded the BCA standard curve y = 0.8503x + 0.022, RA 2 =0.9902; NO standard curve y=0.008x+0.0007, R2=0.9978. From Figure 4 It was found that the NO content in the blank group of zebrafish was low, and the NO content in zebrafish increased significantly after LPS induction (P<0.01). Compared with the model group, the XZQ-L group had no significant effect on inhibiting NO production (P>0.05), while the XZQ-M group, XZQ-H group, and positive control groups (DSMS group and QHLYKL group) all significantly reduced the NO production rate (P<0.01). Among them, the DSMS group was more effective than the XZQ and QHLYKL groups, and its NO production rate was closest to that of the normal group. The effect of inhibiting NO production rate increased with increasing dosage in the XZQ-L, XZQ-M, and XZQ-H groups, with the XZQ-H group being more effective than the QHLYKL group. This suggests that Xia Zhuqing may reduce the inflammatory response by inhibiting NO production.

[0119] 2.4 Effects of Xia Zhuqing on neutrophil migration and aggregation in a zebrafish CuSO4 inflammation model Neutrophils have various chemokine receptors on their surface. When tissues are damaged or infected, the site of inflammation releases specific chemokines that bind to these receptors, guiding the neutrophils to chemotactically aggregate at the site of inflammation. As inflammation subsides, inflammatory cells undergo apoptosis and are detected and cleared by phagocytic cells such as macrophages, resulting in a decrease in neutrophils at the site of inflammation. Neutrophils in Tg(lyz:EGFP) transgenic zebrafish are labeled with green fluorescence, allowing for in vivo imaging of neutrophil movement using a fluorescence microscope.

[0120] Depend on Figure 3 It was found that the normal group of zebrafish had a low number of neutrophils in the lateral line thalamus cell region. CuSO4 induction significantly increased the number of neutrophils in this region, and large clusters of neutrophils were observed (P<0.01). Compared with the model group, both the XZQ group and the positive control groups (DSMS group and QHLYKL group) significantly reduced the number of neutrophils in the lateral line thalamus cell region of zebrafish (P<0.01). Among them, the DSMS group was more effective than the XZQ group and the QHLYKL group, and the number of neutrophils in the lateral line thalamus cell region of zebrafish was closest to that of the normal group. With increasing dosage, the number of neutrophils in the XZQ-L, XZQ-M, and XZQ-H groups was closest to that of the normal group, and the XZQ-M and XZQ-H groups were more effective than the QHLYKL group.

[0121] 2.5 Effects of Xia Zhuqing on the expression of TNF-α, IL-6, IL-1β, and IL-8 mRNA in a zebrafish CuSO4 inflammation model TNF-α, IL-6, and IL-1β are common pro-inflammatory cytokines, mainly released by immune cells. Interleukin-8 (IL-8), also known as CXCL8, is a chemokine produced and released by cells such as macrophages, epithelial cells, and endothelial cells under stimuli such as microbial products, injury, and hypoxia. During infection, tissue damage, or other immune responses, pro-inflammatory cytokines and chemokines are rapidly released to help initiate inflammatory responses and recruit immune cells to regulate repair.

[0122] Depend on Figure 4 Compared with the control group, CuSO4 induction significantly increased the relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNA in zebrafish (P<0.01), indicating that CuSO4 successfully induced the inflammatory response. Compared with the model group, the XZQ group and the positive control groups (DSMS group and QHLYKL group) significantly reduced the relative expression levels of IL-6, IL-1β, and IL-8 mRNA (P<0.01). The DSMS group, XZQ-M group, and XZQ-H group significantly reduced the relative expression level of TNF-α (P<0.01), the XZQ-L group significantly reduced the relative expression level of TNF-α (P<0.05), and the QHLYKL group did not significantly reduce the relative expression level of TNF-α (P>0.05). Xia Zhuqing showed significant inhibitory effects on pro-inflammatory and chemokine factors, but the optimal effective doses for different factors varied. IL-8 was optimal at high doses, while TNF-α, IL-6, and IL-1β were optimal at medium doses. The effects of different treatment groups on the relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNA in zebrafish are shown in Table 9.

[0123] Table 9. Effects of different treatment groups on the relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNA in zebrafish ( - (x±s, n=30)

[0124] Note: Compared with the control group, ##P<0.01; compared with the model group, P<0.01, P<0.05.

[0125] 2.6 Effects of Xia Zhuqing on the expression of BCL2L1, NF-κB, MAPK1, and JNK mRNA in zebrafish tissues of a CuSO4 inflammation model BCL2L1 (BCL-XL) is an anti-apoptotic protein in the BCL-2 family, playing a complex role in inflammation by regulating cell survival and death. In mammals, the NF-κB transcription factor family consists of five members: p50, p52, p65 (RelA), c-Rel, and RelB, encoded by NFKB1, NFKB2, RELA, REL, and RELB, respectively. NF-κB is primarily activated through canonical (or classical) and non-canonical (or alternative) pathways. The canonical NF-κB signaling pathway is mainly activated by stimulating pro-inflammatory receptors (such as the TNF receptor superfamily and the Toll-like receptor family (TLRs)) and interleukin cytokine receptors. Activation of the MAPK / JNK pathway promotes the activation of key transcription factors such as NF-κB, driving the expression of inflammatory genes.

[0126] Depend on Figure 5 It was found that, compared with the control group, the relative expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA in zebrafish were significantly increased after CuSO4 induction (P<0.01), indicating that the model was successfully established. Compared with the model group, the relative expression levels of BCL2L1 and NF-κB in the DSMS, XZQ, and QHLYKL groups were extremely significantly decreased (P<0.01). The relative expression levels of MAPK1 and JNK in the DSMS, XZQ-M, and XZQ-H groups were extremely significantly decreased (P<0.01), while the relative expression levels of MAPK1 and JNK in the XZQ-L and QHLYKL groups were significantly decreased (P<0.05).

[0127] Different experimental groups showed varying degrees of inhibitory effects on the expression of BCL2L1, NF-κB, MAPK1, and JNK genes in a CuSO4-induced zebrafish model, as shown in Table 10. Among them, the DSMS group showed the most significant effect in reducing the expression levels of these genes, with highly significant differences (P<0.01), even slightly lower than the normal group. This may be related to differences between individual zebrafish or the immunosuppressive effect of dexamethasone. The QHLYKL group also showed some inhibitory effects, with highly significant differences in reducing the relative expression levels of BCL2L1 and NF-κB mRNA (P<0.01), and significant differences in reducing the relative expression levels of MAPK1 and JNK mRNA (P<0.05). Xia Zhuqing found that the optimal effective doses for different factors varied; high doses were optimal for NF-κB, MAPK1, and JNK, while medium doses were optimal for BCL2L1.

[0128] Table 10. Effects of different experimental groups on the expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA in zebrafish ( - (x±s, n=30)

[0129] Note: Compared with the control group, ##P<0.01; compared with the model group, P<0.01, P<0.05.

[0130] This study systematically evaluated the anti-inflammatory effects and mechanisms of Xia Zhuqing using a zebrafish inflammation model. Through LPS and CuSO4-induced zebrafish inflammation models, combined with cellular-level detection (ROS, NO production rate, and neutrophil migration) and molecular biology techniques (RT-qPCR), the anti-inflammatory mechanism of Xia Zhuqing by regulating inflammatory signaling pathways at multiple targets was revealed.

[0131] Non-toxic dose screening of *Xia Zhu Qing* revealed that *Xia Zhu Qing* had no significant toxicity to embryonic development within the dose range of 100-300 μg / mL. Therefore, *Xia Zhu Qing* at doses of 100, 200, and 300 μg / mL were selected for subsequent experiments.

[0132] A zebrafish inflammation model was established by inducing normal development of 7-9 hpf zebrafish embryos with 10 μg / mL LPS solution for 72 h. Results showed that 10 μg / mL LPS significantly increased the production rates of ROS and NO in zebrafish. All doses of Xia Zhuqing significantly inhibited ROS and NO production in a dose-dependent manner, with the XZQ-H group showing better inhibitory effects on ROS and NO than the positive control QHLYKL group.

[0133] A zebrafish inflammation model was established by inducing normal development (3 days post-flour) in zebrafish with 20 μM CuSO4 for 1 hour. Results showed that 20 μM CuSO4 significantly increased the number and aggregation of neutrophils in the lateral line thalamus region of zebrafish. Different doses of Xia Zhuqing effectively reduced the number and aggregation of neutrophils in the lateral line thalamus region of zebrafish in a dose-dependent manner. The XZQ-M and XZQ-H groups showed better effects than the QHLYKL group.

[0134] The relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 pro-inflammatory factor mRNAs were significantly upregulated in the model group. The relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNAs were significantly downregulated in each dose group of Xia Zhuqing. Among them, the inhibitory effect of Xia Zhuqing on IL-8 expression was best in the XZQ-H group, while the inhibitory effect on TNF-α, IL-6, and IL-1β expression was best in the XZQ-M group.

[0135] The relative expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA were significantly increased in the model group. Each dose of Xia Zhuqing significantly downregulated the relative expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA. Among them, the inhibitory effect of Xia Zhuqing on the expression levels of NF-κB, MAPK1, and JNK was best in the XZQ-H group, while the inhibitory effect on the expression level of BCL2L1 was best in the XZQ-M group.

[0136] In summary, Xia Zhuqing can significantly downregulate pro-inflammatory factors such as TNF-α and IL-1β, and inhibit the expression of NF-κB and MAPK / JNK signaling molecules, suggesting that it may exert its anti-inflammatory effect by blocking the activation of the NF-κB and MAPK pathways.

[0137] Effects of different experimental groups on ROS generation rate in LPS-induced zebrafish ( - (x±s, n=12) see Figure 2 Figure a shows the experimental flowchart; figure b shows the fluorescence intensity in zebrafish from different experimental groups; and figure c shows the effect of different experimental groups on LPS-induced ROS generation rate in zebrafish. Compared with the control group, ##P<0.01; compared with the model group, P<0.01.

[0138] Effects of different experimental groups on NO production rate in zebrafish ( - (x±s, n=30) see Figure 4 Compared with the control group, ##P<0.01; compared with the model group, P<0.01.

[0139] Effects of different experimental groups on CuSO4-induced neutrophil migration and aggregation in zebrafish - (x±s, n=15) see Figure 3 Figure a shows the experimental flowchart; Figure b shows the distribution of neutrophils in zebrafish in different experimental groups; Figure c shows the analysis of neutrophil aggregation in different experimental groups. Compared with the control group, ##P<0.01; compared with the model group, P<0.01.

[0140] Effects of different experimental groups on the relative expression levels of TNF-α, IL-6, IL-1β, and IL-8 mRNA in zebrafish ( - (x±s, n=30) see Figure 4Figure a shows the relative expression of TNF-α mRNA; figure b shows the relative expression of IL-6 mRNA; figure c shows the relative expression of IL-1β mRNA; and figure d shows the relative expression of IL-8 mRNA. Compared with the control group, ##P<0.01; compared with the model group, P<0.01, P<0.05.

[0141] The effects of different experimental groups on the relative expression levels of BCL2L1, NF-κB, MAPK1, and JNK mRNA in zebrafish are shown in the figure. Figure 5 Figure a shows the relative expression level of BCL2L1 mRNA; figure b shows the relative expression level of NF-κB mRNA; figure c shows the relative expression level of MAPK1 mRNA; and figure d shows the relative expression level of JNK mRNA.

[0142] II. The nodule-dispersing effect of the traditional Chinese medicine composition used in the examples was verified.

[0143] 1. Experimental Materials and Modeling Experimental animals: SPF grade rats, half male and half female.

[0144] Model establishment: A rat lung nodule model was induced by intraperitoneal injection of urethane. The modeling period was 8-12 weeks. After CT imaging confirmed the presence of obvious solitary or multiple nodules (≥1 mm in diameter) in the lungs, the rats were grouped.

[0145] Experimental Groups: Normal control group: fed according to routine.

[0146] Model group: After modeling, the model was given an equal volume of physiological saline by gavage.

[0147] Example 1 group: After modeling, the extract prepared in Example 1 (2g / kg) was administered.

[0148] Example 3 group: After modeling, the extract prepared in Example 3 (2g / kg) was administered.

[0149] 2. Observation Indicators Nodule count: The total number of nodules on the surface and cut surfaces of both lungs is counted by visual inspection and microscopic examination.

[0150] Nodule diameter: The average diameter of the largest lung nodule is measured using calipers.

[0151] Inflammatory factor detection: Detect the levels of inflammatory factors (such as TNF-α and IL-6) in serum that are associated with nodule formation.

[0152] 3. Experimental Results Table 11. Comparison of the intervention effects of different groups on rat pulmonary nodules (after 4 weeks of drug administration)

[0153] Note: Compared with Example 1 group P<0.05, indicating a significant difference.

[0154] 4. Results Analysis and Conclusions The experimental results showed that the model group rats exhibited significant nodular hyperplasia in their lungs. While Example 1 could reduce inflammation levels to some extent, its effect on reducing the number and diameter of existing nodules was limited. In contrast, Example 11 significantly reduced the number of lung nodules and compressed the nodule diameter by more than 69.7%, with a nodule dispersion inhibition rate as high as 74.1%.

[0155] The traditional Chinese medicine composition provided in Example 11, through a rigorous formulation ratio and preparation process, exhibits excellent dual effects of dispersing nodules and reducing inflammation in the treatment of pulmonary nodules.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these 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 composition, characterized in that, It includes a first component, a second component, and a third component; the first component is Prunella vulgaris and Lophatherum gracile; The second component consists of Platycodon grandiflorus and Glycyrrhiza uralensis; The third component is dried plum; In the traditional Chinese medicine composition, the content of the first component is 50-60 wt%; The mass ratio of the first component to the second component is 1.5~2.5:1; In the first component, the mass ratio of Prunella vulgaris to Lophatherum gracile is 1:1.5~2.

5.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, In the second component, the mass ratio of Platycodon grandiflorus to Glycyrrhiza uralensis is 1:1.5~2.

5.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The formula includes the following components in parts by weight: 16-24 parts of Prunella vulgaris; 36-44 parts of Lophatherum gracile; 6-14 parts of Platycodon grandiflorus; 16-24 parts of Glycyrrhiza uralensis; and 16-24 parts of Prunus mume.

4. The traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, The traditional Chinese medicine composition further includes a fourth component, which is dandelion and honeysuckle, and the mass ratio of the first component to the fourth component is 1:1.0~1.5; The preferred traditional Chinese medicine composition comprises the following components in parts by weight: 16-24 parts of Prunella vulgaris; 36-44 parts of Lophatherum gracile; 6-14 parts of Platycodon grandiflorus; 16-24 parts of Glycyrrhiza uralensis; 16-24 parts of Prunus mume; 32-48 parts of Taraxacum mongolicum; and 24-36 parts of Lonicera japonica.

5. A traditional Chinese medicine extract, characterized in that, Extract using the following method: S1: The traditional Chinese medicine composition according to any one of claims 1 to 4 is dried at 55 to 70°C to obtain a raw material with a water content of 3 to 8 wt%; S2: Crush the raw material obtained in S1, soak it in water, and heat it for extraction. The material-to-liquid ratio for extraction is 1:10~50, the extraction temperature is 60~100℃, and the extraction time is 20~60min.

6. The herbal extract according to claim 5, characterized in that, The material-to-liquid ratio for extraction is 1:20~40 (g / mL), the extraction temperature is 70~90℃, and the extraction time is 30~50 min.

7. The use of a traditional Chinese medicine composition according to any one of claims 1 to 4 or a traditional Chinese medicine extract according to claim 5 or 6 in the preparation of plant-based beverages.

8. A plant-based beverage, characterized in that, The composition contains the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine extract according to claim 5 or 6 and excipients, wherein the excipients include one or more of steviol glycosides, zero-calorie sugar and N,2,3-trimethyl-2-isopropylbutyramide; Preferably, the amount of steviol glycosides is 0.008 to 0.012 parts by weight; Zero-calorie sugar 0.8~1.2 parts; N,2,3-trimethyl-2-isopropylbutyramide 0.0016~0.0024 parts.

9. The use of a traditional Chinese medicine composition according to any one of claims 1 to 4 or a traditional Chinese medicine extract according to claim 5 or 6 in the preparation of a drug with anti-inflammatory and nodule-dispersing effects.

10. The application according to claim 9, characterized in that, The anti-inflammatory and nodule-dispersing effects described in the application include at least one of the following characteristics: a. Downregulate the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-8; b. Inhibit the gene expression of inflammatory signaling pathways BCL2L1, NF-κB, MAPK1, and JNK.

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