A traditional Chinese medicine probiotic composition, a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine probiotic composition and a preparation method and application thereof, and belongs to the technical field of biological medicines. The technical problem to be solved is that the existing traditional Chinese medicine compound or probiotic has limited synergistic effect when applied alone in intestinal protection and immune regulation, and the storage stability of the compound of lactobacillus plantarum and traditional Chinese medicine ingredients is poor and the viable count of the lactobacillus plantarum is prone to decrease. The technical solution is that lactobacillus plantarum is compounded with the extract of angelica sinensis, rosa davurica, lonicera japonica, gynura japonica and arctium lappa to prepare a traditional Chinese medicine probiotic composition, the synergistic effect of the plant medicine and the probiotic is utilized to improve the intestinal protection and immune regulation effect, and the storage stability of the lactobacillus plantarum is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine technology, specifically relating to a traditional Chinese medicine probiotic composition, its preparation method, and its application. Background Technology

[0002] As the largest immune organ in the human body, the gut microbiota homeostasis is closely related to the body's immune function. Traditional Chinese medicine (TCM) compound formulas are rich in polysaccharides, flavonoids, saponins, and other active components, which can regulate immunity by modulating the intestinal barrier, balancing the gut microbiota, and regulating immune-related signaling pathways. Probiotics can colonize the gut, promote the activation of intestinal mucosal immune cells, and regulate cytokine secretion. The combined use of these two has enormous potential for synergistic immune regulation through multiple pathways, making it an important direction for the research and development of functional health foods. Combining TCM compound formulas with probiotics to create functional foods that are easy to consume daily can meet the public's demand for immune support products.

[0003] Relevant literature retrieved: Patent document CN111109596A (publication date May 8, 2020) discloses a health product for enhancing immunity and its preparation method. The health product is composed of freeze-dried royal jelly powder, Polygonatum extract, Astragalus extract, American ginseng, prebiotics and excipients. The prebiotics can be replaced by probiotic powder containing Bifidobacterium and Lactobacillus. The health product has antioxidant, anti-fatigue, immunity-enhancing and gastrointestinal-regulating effects.

[0004] Patent document CN118497046A (publication date August 16, 2024) discloses a probiotic agent for enhancing intestinal immunity and its application. The probiotic agent includes two strains: Lactobacillus rhamnosus and Bifidobacterium longum. The two strains have potential interactions and can cooperate with each other to synergistically enhance the efficacy of enhancing intestinal immunity.

[0005] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Existing technologies (such as CN111109596A) mainly adopt the approach of combining Chinese medicine extracts with prebiotics, but do not directly combine the active ingredients of Chinese medicine with live probiotics, nor do they study the impact of the active components of Chinese medicine on the activity of live probiotic cells.

[0006] Current research on probiotic compound formulations (such as CN118497046A) mainly focuses on the synergy between strains, without involving the role of traditional Chinese medicine systems in the storage stability of probiotics.

[0007] In summary, in existing technologies, traditional Chinese medicine and probiotics often function individually, and the synergistic effect of their combined system is limited.

[0008] The inventors discovered in their research that if traditional Chinese medicine extracts are directly combined with live Lactobacillus plantarum, factors such as the acidic components in the extracts and the water activity of the system can cause a decrease in the activity of live Lactobacillus plantarum during storage, resulting in a significant reduction in the number of live bacteria. Existing technologies struggle to preserve the active ingredients of traditional Chinese medicine while simultaneously ensuring the storage stability of Lactobacillus plantarum. Summary of the Invention

[0009] The purpose of this invention is to provide: A traditional Chinese medicine probiotic composition and related technologies are proposed to address the technical problems of limited synergistic effects when existing traditional Chinese medicine compound formulas or probiotics are used alone for intestinal protection and immune regulation, as well as the poor storage stability and easy decline of live bacteria count after combining Lactobacillus plantarum with traditional Chinese medicine ingredients, or the technical problems of their combination.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Definitions of standard chemical terms can be found in the reference "Chinese Pharmacopoeia (2025 Edition)".

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as water activity, viable bacterial count, cell proliferation activity, phagocytic activity assays, and nitric oxide (NO) secretion level assays, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0016] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in this invention shall, in all cases, be understood to be modified by the word "about". When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10%, preferably within 5%, of the specified value or range.

[0017] As used herein, the term "room temperature" refers to ambient temperature, ranging from about 10°C to about 30°C. In some embodiments, "room temperature" refers to a temperature ranging from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature ranging from about 25°C to about 30°C; and in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, etc.

[0018] As used in this article, "solid-liquid separation" refers to the process of separating a mixture containing solid particles and liquid components into independent solid and liquid phases using physical or mechanical methods. This process can remove solid impurities, cell debris, precipitates, microbial aggregates, and other solid substances from the mixture to obtain a clear liquid phase or collect the target solid product. It is a commonly used separation and purification method in cell culture, sample pretreatment, and reagent preparation. Centrifugation, filtration, sedimentation, and pressure filtration are all conventional solid-liquid separation methods in this field.

[0019] The term "concentration" as used in this article refers to the process of removing solvents (such as water) from materials using physical or chemical methods, thereby increasing the concentration of effective components and reducing their volume. Conventional concentration methods in this field, such as vacuum concentration, spray concentration, etc., can be used.

[0020] The term "drying" as used in this article refers to the conventional operation of removing moisture from liquid mixtures or solid components to obtain dried products, including but not limited to vacuum freeze drying, low-temperature drying, spray drying, and hot air drying.

[0021] The term "dry extract" as used in this article refers to the extract obtained after the Chinese herbal raw materials are extracted and filtered to remove the residue; the extract is then concentrated and dried to remove most of the solvent (water, ethanol, etc.), resulting in a solid paste / powder extract with a water content of about 5%.

[0022] Angelica sinensis is a plant belonging to the Apiaceae family. Angelica sinensis (Oliv.) Diels The dried roots. Harvested in late autumn, the fibrous roots and soil are removed, and after the moisture has evaporated slightly, they are bundled into small bunches, placed on a shed, and slowly dried with smoke.

[0023] Rosa laevigata is a plant belonging to the Rosaceae family. Rosa laevigata Michx. The dried, ripe fruit. Harvest in October or November when the fruit turns red, dry it, and remove the thorns.

[0024] Honeysuckle is a plant of the Caprifoliaceae family, Lonicera japonica. Japanese honeysuckle Dried flower buds or newly opened flowers of Thunb. Harvested in early summer before the flowers open and dried.

[0025] Centella asiatica is a plant belonging to the Apiaceae family. Centella asiatica (L.) Urb. The dried whole herb. Harvested in summer and autumn, the soil and sand are removed, and then it is sun-dried.

[0026] Burdock seeds are from the plant *Arctium lappa*, belonging to the Asteraceae family. Arctium lappa L. The dried, ripe fruit. The fruit clusters are harvested in autumn when the fruit is ripe, dried in the sun, the fruit is threshed off, impurities are removed, and then dried again.

[0027] Lactobacillus plantarum, also known as Lactobacillus plantarum ( Lactiplantibacillus plantarum It is widely found in fermented vegetables, fermented dairy products, and the human gut, for example, it can be derived from dehydrated fermented milk or the feces of healthy infants.

[0028] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides: a traditional Chinese medicine probiotic composition, comprising Angelica sinensis, Rosa laevigata, Lonicera japonica, Centella asiatica, Arctium lappa and Lactobacillus plantarum.

[0029] In this invention, *Lactobacillus plantarum* is used as the core of immune regulation to reshape the intestinal immune microecology; *Angelica sinensis* and *Rosa laevigata* strengthen the body's resistance and regulate the body's basic immunity; *Lonicera japonica* clears heat, detoxifies, and reduces inflammation; *Centella asiatica* and *Arctium lappa* clear turbidity, detoxify, and promote intestinal barrier repair; by combining herbal medicines with probiotics, multi-pathway synergy is achieved, thereby enhancing the body's immune capacity.

[0030] In some embodiments, the herbal probiotic composition comprises, by weight, 11-25 parts Angelica sinensis, 7-21 parts Rosa laevigata, 6-16 parts Lonicera japonica, 2-10 parts Centella asiatica, 6-14 parts Arctium lappa, and 0.5-3 parts Lactobacillus plantarum.

[0031] In some embodiments, the amount of Angelica sinensis in the herbal probiotic composition may be selected as 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, or any range derived thereof, by weight.

[0032] In some embodiments, the amount of Rosa laevigata in the herbal probiotic composition may be selected as 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, or any range derived thereof, by weight.

[0033] In some embodiments, the amount of honeysuckle in the herbal probiotic composition may be selected as 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, or any range derived thereof, by weight.

[0034] In some embodiments, the amount of Centella asiatica in the herbal probiotic composition may be selected as 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any range derived thereof, by weight.

[0035] In some embodiments, the amount of burdock seed in the herbal probiotic composition may be selected as 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, or any range derived thereof, by weight.

[0036] In some embodiments, the amount of Lactobacillus plantarum in the herbal probiotic composition may be selected as 0.5 parts, 1.0 parts, 1.5 parts, 2.0 parts, 2.5 parts, 3.0 parts, or any range derived thereof, by weight.

[0037] In some embodiments, the total viable count of *Lactobacillus plantarum* in the traditional Chinese medicine probiotic composition is (1-10) × 10⁻¹⁰. 10 CFU / g.

[0038] In some embodiments, the herbal probiotic composition comprises, by weight, 15-21 parts of Angelica sinensis, 10-18 parts of Rosa laevigata, 8-14 parts of Lonicera japonica, 4-8 parts of Centella asiatica, 8-12 parts of Arctium lappa, and 1-2 parts of Lactobacillus plantarum.

[0039] In some embodiments, the herbal probiotic composition comprises, by weight, 18 parts Angelica sinensis, 14 parts Rosa laevigata, 12 parts Lonicera japonica, 6 parts Centella asiatica, 10 parts Arctium lappa, and 1.5 parts Lactobacillus plantarum.

[0040] Secondly, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine probiotic composition, comprising the following steps: (1) Angelica alcohol was refluxed for extraction, solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 1; (2) The extracts of Rosa laevigata and Arctium lappa were extracted by reflux extraction with alcohol, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 2; (3) Honeysuckle and Centella asiatica were decocted in water, the solid and liquid were separated, the decoctions were combined and concentrated and dried to obtain dry extract 3; (4) After mixing dry extract 1, dry extract 2 and dry extract 3, add Lactobacillus plantarum and mix well.

[0041] In some embodiments, the alcohol in step (1) is 90%-95% ethanol by volume, for example, the volume fraction of ethanol may be 90%, 91%, 92%, 93%, 94%, 95%, or a range derived therefrom.

[0042] In some embodiments, the alcohol in step (2) is 10%-50% ethanol by volume. For example, the volume fraction of ethanol can be selected as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a range derived therefrom.

[0043] In some implementations, the reflux extraction in steps (1) and (2) is performed with a material-to-liquid mass ratio of 1:8-15, at least once, for 0.5-3 hours each time. For example, the material-to-liquid mass ratio can be selected as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or a range derived therefrom; the number of extractions can be selected as once, twice, three times, four times, or a range derived therefrom; and the extraction time can be selected as 0.5 hours, 1.0 hour, 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, or a range derived therefrom.

[0044] In some implementations, the decoction in step (3) is performed with a material-to-liquid mass ratio of 1:8-20, and at least once. For example, the material-to-liquid mass ratio can be selected as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or a range derived therefrom; the number of times the decoction is performed can be 1, 2, 3, 4, or a range derived therefrom; and the decoction time can be selected as 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, or a range derived therefrom.

[0045] In some embodiments, the preparation method includes the following steps: (1) Angelica sinensis was extracted by reflux with 90-95% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 1; (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted by reflux with 10%-50% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 2; (3) Honeysuckle and Centella asiatica were boiled in water, the solid and liquid were separated, the boiled liquid was combined, concentrated and dried to obtain dry extract 3; (4) After mixing dry extract 1, dry extract 2 and dry extract 3, add Lactobacillus plantarum and mix well.

[0046] Thirdly, the present invention provides the application of the above-mentioned Chinese herbal probiotic composition or the Chinese herbal probiotic composition prepared by the above-mentioned preparation method in the preparation of products that help enhance immunity.

[0047] Fourthly, the present invention provides a product that helps enhance immunity, including the above-mentioned traditional Chinese medicine probiotic composition or the traditional Chinese medicine probiotic composition prepared by the above-mentioned preparation method.

[0048] In some implementations, the product includes health foods, special dietary foods, or dietary supplements.

[0049] In some embodiments, the product is made from the above-described Chinese herbal probiotic composition or the Chinese herbal probiotic composition prepared by the above-described preparation method and acceptable excipients.

[0050] Preferably, the acceptable excipients are those acceptable for health food products.

[0051] The traditional Chinese medicine probiotic composition of this invention can be prepared into various health foods or clinical preparations using conventional formulation methods in the art. For example, acceptable excipients for health foods can be added to an effective amount of the traditional Chinese medicine probiotic composition of this invention. This invention does not impose any special restrictions on the types of excipients; any types of excipients known to those skilled in the art are acceptable. In this invention, the excipients can be one or more of disintegrants, lubricants, binders, and dispersants, and can be prepared into various oral medications, such as tablets, granules, capsules, powders, pills, or oral liquids.

[0052] For those skilled in the art, knowing the active ingredients, various pharmaceutical products can be easily prepared using conventional formulation methods. At the same time, the excipients used in the preparation are also well known to those skilled in the art, such as sodium carboxymethyl starch, sucrose powder, honey, sesame oil, etc.

[0053] Fifthly, the present invention provides a method for improving the storage stability of Lactobacillus plantarum, comprising the following steps: mixing Lactobacillus plantarum with a traditional Chinese medicine extract to obtain a traditional Chinese medicine probiotic composition, and then storing it in a sealed, light-proof container.

[0054] In some embodiments, the herbal probiotic composition comprises, by weight, 11-25 parts Angelica sinensis, 7-21 parts Rosa laevigata, 6-16 parts Lonicera japonica, 2-10 parts Centella asiatica, 6-14 parts Arctium lappa, and 0.5-3 parts Lactobacillus plantarum.

[0055] In some embodiments, the method for preparing the traditional Chinese medicine extract includes the following steps: (1) Angelica sinensis was extracted by reflux with 90%-95% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 1; (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted by reflux with 10%-50% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 2; (3) Honeysuckle and Centella asiatica were boiled in water, the solid and liquid were separated, the boiled liquid was combined, concentrated and dried to obtain dry extract 3; (4) Mix dry extract 1, dry extract 2 and dry extract 3 evenly.

[0056] In some embodiments, the alcohol in step (1) is 90%-95% ethanol by volume, for example, the volume fraction of ethanol may be 90%, 91%, 92%, 93%, 94%, 95%, or a range derived therefrom.

[0057] In some embodiments, the alcohol in step (2) is 10%-50% ethanol by volume. For example, the volume fraction of ethanol can be selected as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a range derived therefrom.

[0058] In some implementations, the reflux extraction in steps (1) and (2) is performed with a material-to-liquid mass ratio of 1:8-15, and at least one extraction. For example, the material-to-liquid mass ratio can be selected as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or a range derived therefrom; the number of extractions can be selected as 1, 2, 3, 4, or a range derived therefrom.

[0059] In some implementations, the decoction in step (3) is performed with a material-to-liquid mass ratio of 1:8-20, and at least once. For example, the material-to-liquid mass ratio can be selected as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or a range derived therefrom; the number of times the decoction is performed can be once, twice, three times, four times, or a range derived therefrom.

[0060] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred solution: the dosage relationship of the traditional Chinese medicine probiotic composition. This technical solution, while solving the technical problem of "enhancing immunity", further strengthens the above-mentioned technical effects.

[0061] The second preferred option is a method for preparing traditional Chinese medicine extracts. This technical solution, while solving the technical problem of "enhancing immunity," further improves the storage stability of live bacteria and strengthens the intestinal protection effect.

[0062] In this invention, Examples 1-3 at least support the protection scope of "the Chinese herbal probiotic composition, by weight, includes 11-25 parts of Angelica sinensis, 7-21 parts of Rosa laevigata, 6-16 parts of Lonicera japonica, 2-10 parts of Centella asiatica, 6-14 parts of Arctium lappa and 0.5-3 parts of Lactobacillus plantarum".

[0063] The technical feature "the Chinese herbal probiotic composition, by weight, includes 11-25 parts of Angelica sinensis, 7-21 parts of Rosa laevigata, 6-16 parts of Lonicera japonica, 2-10 parts of Centella asiatica, 6-14 parts of Arctium lappa, and 0.5-3 parts of Lactobacillus plantarum" is summarized from the foregoing explanation and / or the corresponding dosages of Angelica sinensis, Rosa laevigata, Lonicera japonica, Centella asiatica, Arctium lappa, and Lactobacillus plantarum in Examples 1-3. Therefore, those skilled in the art can reasonably infer that the above-mentioned technical feature, its subordinate concepts, its substantially equivalent technical means, and technical means that can be replaced within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention. For example, replacing 11-25 parts of Angelica sinensis with 14 parts or 12-24 parts of Angelica sinensis while keeping other technical features unchanged still falls within the protection scope of this invention.

[0064] Examples 1-3 of this invention at least support the protection scope of "the preparation method of the traditional Chinese medicine probiotic composition".

[0065] The technical feature “the preparation method of the Chinese herbal probiotic composition” is summarized by the foregoing explanation and / or the corresponding specific steps (1), (2), (3) and (4) in Examples 1-3. Therefore, those skilled in the art can reasonably infer that the above-mentioned technical features, their subordinate concepts, their basically equivalent technical means, and technical means that can be replaced by conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention. For example, if the alcohol reflux extraction in step (1) is replaced by 90%-95% ethanol reflux extraction, or the alcohol reflux extraction in step (2) is replaced by 10%-50% ethanol reflux extraction, while other technical features remain unchanged, they still fall within the protection scope of this invention.

[0066] Examples 1-3 and Detection Example 1 in this invention at least support the protection scope of "method to improve the storage stability of Lactobacillus plantarum".

[0067] The technical feature "method to improve the storage stability of Lactobacillus plantarum" is summarized by the foregoing explanation and / or the corresponding specific steps (1), (2), (3), and (4) in Examples 1-3. Furthermore, Example 1 demonstrates that the compound traditional Chinese medicine extract system of this invention can effectively improve the storage stability of Lactobacillus plantarum and has a good protective effect on the Lactobacillus plantarum cells. Therefore, those skilled in the art can reasonably infer that the above-mentioned technical features, their subordinate concepts, their essentially equivalent technical means, and technical means that can be replaced by conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0068] The beneficial effects of this invention are as follows: In this invention, *Lactobacillus plantarum* is used as the core of immune regulation to reshape the intestinal immune microecology; *Angelica sinensis* and *Rosa laevigata* strengthen the body's resistance and regulate the body's basic immunity; *Lonicera japonica* clears heat, detoxifies, and reduces inflammation; *Centella asiatica* and *Arctium lappa* clear turbidity, detoxify, and promote intestinal barrier repair; by combining herbal medicines with probiotics, multi-pathway synergy is achieved, thereby enhancing the body's immune capacity.

[0069] Experiments have shown that the combination of Lactobacillus plantarum and traditional Chinese medicine components in this invention produces a synergistic effect in enhancing immunity.

[0070] Meanwhile, experiments have shown that the compound Chinese herbal extract system of this invention can effectively improve the storage stability of Lactobacillus plantarum, has a good protective effect on Lactobacillus plantarum cells, and further enhances its intestinal protection effect.

[0071] Furthermore, based on the present invention: 1. Based on the comparison of Example 1 and Comparative Examples 1-3, it can be seen that the present invention adopts a differentiated extraction process combination of high-alcohol extraction of Angelica sinensis, medium-low-alcohol extraction of Rosa laevigata and Arctium lappa, and water extraction of Lonicera japonica and Centella asiatica, achieving new technical effects. Compared with the comparative examples that use a uniform solvent extraction method, the herbal extract obtained by the present invention can reduce the water activity of the system, reduce the damage of acidic components of the herbal medicine to Lactobacillus plantarum, and accelerate the survival rate of viable bacteria to 61.8%-67.2% after 6 months of storage. At the same time, it retains sufficient immune-active substances, effectively improves the phagocytosis and NO secretion levels of macrophages, and the combined technical effect is better than the sum of the effects of using each extraction method alone.

[0072] 2. Based on the comparison of Example 1 and Comparative Examples 4-5, this invention uses a limited weight proportion of five kinds of traditional Chinese medicine extracts combined with *Lactobacillus plantarum*. When the proportion of medicinal materials deviates or a component is missing, the storage stability of the *Lactobacillus plantarum* in the composition and its immune activation effect both decrease significantly. This demonstrates that there is a synergistic relationship between the various traditional Chinese medicine components and *Lactobacillus plantarum* in this invention. The traditional Chinese medicine extracts contribute to immunomodulatory activity and also protect *Lactobacillus plantarum*; *Lactobacillus plantarum* further amplifies the immunomodulatory effect, achieving a synergistic dual technical effect. Detailed Implementation

[0073] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0074] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0075] The *Lactobacillus plantarum* mentioned was purchased from Microcare Probiotics (Suzhou) Co., Ltd., strain number Lp05, with a live bacteria count of ≥6×10⁻⁶ in the powder. 11 CFU / g.

[0076] Example 1: A Traditional Chinese Medicine Probiotic Composition and Its Preparation Method The herbal probiotic composition comprises, by weight: 18 parts Angelica sinensis, 14 parts Rosa laevigata, 12 parts Lonicera japonica, 6 parts Centella asiatica, 10 parts Arctium lappa, and 1.5 parts Lactobacillus plantarum.

[0077] The preparation method of the aforementioned traditional Chinese medicine probiotic composition includes the following specific steps: (1) Angelica sinensis was extracted twice by reflux with 92% ethanol (volume fraction), with a material-to-liquid ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted twice by reflux with 30% ethanol at a ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 2. (3) Honeysuckle and Centella asiatica were decocted twice with water at a ratio of 1:15, each time for 1 hour. The decoctions were then filtered, combined, concentrated, and dried to obtain dry extract 3. (4) After mixing dry extract 1, dry extract 2 and dry extract 3, cool to room temperature, add Lactobacillus plantarum and mix well.

[0078] Example 2: A Traditional Chinese Medicine Probiotic Composition and Its Preparation Method The herbal probiotic composition comprises, by weight: 11 parts Angelica sinensis, 7 parts Rosa laevigata, 6 parts Lonicera japonica, 2 parts Centella asiatica, 6 parts Arctium lappa, and 0.5 parts Lactobacillus plantarum.

[0079] The preparation method of the aforementioned traditional Chinese medicine probiotic composition includes the following specific steps: (1) Angelica sinensis was extracted three times by reflux with 90% ethanol (volume fraction), with a material-to-liquid ratio of 1:8 and an extraction time of 0.5 h each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted once by reflux with 50% ethanol at a ratio of 1:15, for 2 hours each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 2. (3) Honeysuckle and Centella asiatica were decocted three times with water at a ratio of 1:8, each time for 0.5 hours. The decoctions were then filtered, combined, concentrated, and dried to obtain dry extract 3. (4) After mixing dry extract 1, dry extract 2 and dry extract 3, cool to room temperature, add Lactobacillus plantarum and mix well.

[0080] Example 3: A Traditional Chinese Medicine Probiotic Composition and Its Preparation Method The herbal probiotic composition comprises, by weight: 25 parts Angelica sinensis, 21 parts Rosa laevigata, 16 parts Lonicera japonica, 10 parts Centella asiatica, 14 parts Arctium lappa, and 3 parts Lactobacillus plantarum.

[0081] The preparation method of the aforementioned traditional Chinese medicine probiotic composition includes the following specific steps: (1) Angelica sinensis was extracted once by reflux with 95% ethanol (volume fraction) at a material-to-liquid ratio of 1:15 for 2 hours each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted three times by reflux with 10% ethanol at a ratio of 1:8, for 0.5 h each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 2. (3) Honeysuckle and Centella asiatica were decocted once with water at a ratio of 1:20 for 3 hours each time. The decoction was filtered, and the decocted liquids were combined, concentrated and dried to obtain dry extract 3. (4) After mixing dry extract 1, dry extract 2 and dry extract 3, cool to room temperature, add Lactobacillus plantarum and mix well.

[0082] Comparative Example 1 The only difference between this comparative example and Example 1 is that the preparation method of the traditional Chinese medicine probiotic composition is different.

[0083] The specific steps are as follows: (1) Angelica sinensis, Rosa laevigata and Arctium lappa were extracted twice by reflux with 92% ethanol at a ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) Honeysuckle and Centella asiatica were extracted twice by reflux with 30% ethanol at a ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 2. (3) After mixing dry extract 1 and dry extract 2, cool to room temperature, add Lactobacillus plantarum and mix well.

[0084] Comparative Example 2 The only difference between this comparative example and Example 1 is that the preparation method of the traditional Chinese medicine probiotic composition is different.

[0085] The specific steps are as follows: (1) Angelica sinensis, Rosa laevigata, Arctium lappa, Lonicera japonica and Centella asiatica were decocted twice with water at a ratio of 1:15, each time for 1 hour. The decoctions were filtered, and the decocted liquids were combined, concentrated and dried to obtain a dry extract. (2) Cool the dry extract to room temperature, add Lactobacillus plantarum and mix well.

[0086] Comparative Example 3 The only difference between this comparative example and Example 1 is that the preparation method of the traditional Chinese medicine probiotic composition is different.

[0087] The specific steps are as follows: (1) Angelica sinensis, Rosa laevigata and Arctium lappa were extracted twice by reflux with 92% ethanol at a ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) Honeysuckle and Centella asiatica were decocted twice with water at a ratio of 1:15, each time for 1 hour. The decoctions were then filtered, combined, concentrated, and dried to obtain dry extract 2. (3) After mixing dry extract 1 and dry extract 2, cool to room temperature, add Lactobacillus plantarum and mix well.

[0088] Comparative Example 4 The only difference between this comparative example and Example 1 is the amount of raw materials used in the herbal probiotic composition.

[0089] Specifically, the herbal probiotic composition comprises, by weight: 32 parts Angelica sinensis, 12 parts Lonicera japonica, 6 parts Centella asiatica, 10 parts Arctium lappa, and 1.5 parts Lactobacillus plantarum.

[0090] The preparation method comprises the following specific steps: (1) Angelica sinensis was extracted twice by reflux with 92% ethanol (volume fraction), with a material-to-liquid ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 1. (2) Burdock seeds were extracted twice by reflux with 30% ethanol (volume fraction), with a material-to-liquid ratio of 1:12, for 1 hour each time. The extracts were filtered, combined, concentrated and dried to obtain dry extract 2. (3) Honeysuckle and Centella asiatica were decocted twice with water at a ratio of 1:15, each time for 1 hour. The decoctions were then filtered, combined, concentrated, and dried to obtain dry extract 3. (4) After mixing dry extract 1, dry extract 2 and dry extract 3, cool to room temperature, add Lactobacillus plantarum and mix well.

[0091] Comparative Example 5 The only difference between this comparative example and Example 1 is the amount of raw materials used in the herbal probiotic composition.

[0092] Specifically, the herbal probiotic composition comprises, by weight: 18 parts Angelica sinensis, 14 parts Rosa laevigata, 2 parts Lonicera japonica, 6 parts Centella asiatica, 20 parts Arctium lappa, and 1.5 parts Lactobacillus plantarum.

[0093] The preparation method is basically the same as in Example 1.

[0094] Detection Example 1 Evaluation of the storage stability of Lactobacillus plantarum The traditional Chinese medicine probiotic compositions prepared in the examples and comparative examples were stored for 6 months under sealed and light-proof conditions at a relative humidity of 50% and a temperature of 37°C. The number of viable bacteria was measured after the experiment.

[0095] Calculate the viable cell retention rate (%) = number of viable cells after storage / initial number of viable cells × 100%.

[0096] The results are shown in Table 1.

[0097] Table 1

[0098] As shown in Table 1, the probiotic compositions prepared in Examples 1-3 of this invention maintained a high survival rate of *Lactobacillus plantarum* after 6 months of accelerated storage at 37°C and 50% relative humidity, with minimal decline in viable count. In contrast, the probiotic compositions prepared in Comparative Examples 1-5 showed a significant decrease in *Lactobacillus plantarum* viable count with prolonged storage. This demonstrates that the compound herbal extract system of this invention can effectively improve the storage stability of *Lactobacillus plantarum* and provides good protection for the bacterial cells.

[0099] Detection Example 2 Evaluation of cellular immune efficacy This test used the Raw264.7 mouse macrophage cell line as an in vitro model to evaluate the immunomodulatory activity of the traditional Chinese medicine probiotic composition.

[0100] (1) Cell culture Raw264.7 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). The cells were passaged in a cell culture incubator at 37°C and 5% CO2 saturated humidity. Cells in the logarithmic growth phase were used for subsequent in vitro functional experiments.

[0101] (2) Assay of cell proliferation activity Cell seeding: Select cells in the logarithmic growth phase and seed them into 96-well plates, adding 100 μL of cell suspension to each well (seeding density 1×10⁻⁶). 5 Cells / mL were cultured overnight at 37℃ with 5% CO2 saturated humidity, and the supernatant was discarded. 100 μL of a traditional Chinese medicine probiotic combination solution with concentrations of 50, 100, 200, 400, 600, and 1000 μg / mL (prepared with culture medium) was added, along with a blank group (no cells inoculated) and a control group (cells inoculated with culture medium only). Three replicates were set up, and the cells were cultured overnight under the same conditions. 100 μL of 1 mg / mL MTT working solution was added, and the cells were incubated for another 4 hours. The supernatant was discarded, and 100 μL of LDMSO was added, gently shaken, and incubated at room temperature for 10 minutes. The absorbance (OD value) of each well at 570 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = [(OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0102] (3) Assay of cellular phagocytic activity Cell seeding: Raw264.7 cells in logarithmic growth phase were used, and the seeding density of the cell suspension was adjusted to 1×10⁻⁶. 5 Add 100 μL of cell suspension to each well of a 96-well plate, incubate overnight, and discard the culture medium; Sample preparation: A blank group (without cell inoculation), a control group, a positive control group, Example 1-Example 3 groups, and Comparative Examples 1-Comparative Examples 5 groups were set up, with 6 replicates in each group; 100 μL of culture medium (without sample) was added to the blank group and the control group, and an equal volume of culture medium containing 200 μg / mL of the corresponding Chinese herbal probiotic composition was added to each example and the comparative example group, and an equal volume of culture medium containing 1 μg / mL LPS was added to the positive control group.

[0103] After 24 hours of cell culture, the supernatant was discarded. Each group was then incubated with 20 μL of 0.1% neutral red solvent for 30 min, followed by discarding the supernatant. Cells were washed three times with sterile PBS, and then 100 μL of cell lysis buffer (ethanol:acetic acid = 1:1, v / v) was added. The cells were shaken and incubated at room temperature in the dark for 120 min. The OD values ​​of each well at 540 nm were measured and calculated using the following formula: Phagocytic activity (%) = [(OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0104] (4) Measurement of nitric oxide (NO) secretion levels Cell seeding: Raw264.7 cells in logarithmic growth phase were used, and the seeding density of the cell suspension was adjusted to 1×10⁻⁶. 5 Add 100 μL of cell suspension to each well of a 96-well plate, incubate overnight, and discard the culture medium; Sample processing: A control group, a positive control group, Example 1-Example 3 groups, and Comparative Examples 1-Comparative Examples 5 groups were set up, with 4 replicates in each group; 100 μL of culture medium was added to the control group, and an equal volume of culture medium containing 200 μg / mL of the corresponding Chinese herbal probiotic composition was added to each example and comparative example group, and an equal volume of culture medium containing 1 μg / mL LPS was added to the positive control group.

[0105] After culturing cells for 24 hours, the supernatant was collected, and the NO concentration in each well was determined and calculated using a nitric oxide kit (Shanghai Beyotime).

[0106] Table 2 shows the results of the cell proliferation activity assay.

[0107] Table 2

[0108] The results showed that the traditional Chinese medicine probiotic composition prepared in this invention has good biocompatibility with macrophages in the range of 50-1000 μg / mL, and can promote macrophage proliferation, which is beneficial to exerting an immunomodulatory effect. The traditional Chinese medicine probiotic compositions prepared in other examples and comparative examples are basically the same as those in Table 2, and will not be repeated here.

[0109] The results of cell phagocytic activity assay and NO secretion level assay are shown in Table 3.

[0110] Table 3

[0111] Note: In Table 3, different letters in the same column represent significant differences in data between groups, P < 0.05.

[0112] The results showed that the phagocytic activity of cells in the control group was 100.2±1.2%, and the NO release concentration was 8.1±0.4 μmol / L, indicating that the cells were in a normal physiological state. After LPS stimulation, the phagocytic activity and NO secretion level of macrophages in the positive control group were significantly increased, and the macrophages were significantly activated, thus establishing an effective cell activation model.

[0113] The macrophage phagocytic activity of each example group reached 132.1%-136.5%, and the NO concentration was 17.0-17.9 μmol / L, which was comparable to the positive control group (P>0.05). This indicates that the traditional Chinese medicine probiotic composition prepared in Examples 1-3 of the present invention can significantly enhance the macrophage phagocytic capacity, promote NO secretion, and has excellent immune activation effect, reaching the level of positive drug.

[0114] The phagocytic activity of each comparative example ranged from 102.4% to 114.0%, and the NO concentration ranged from 9.8 to 13.1 μmol / L, which were significantly lower than those of the positive control group (P < 0.05). This indicates that the immunoactivating effect of the traditional Chinese medicine probiotic compositions prepared in comparative examples 1-5 of this invention is significantly weaker than that of the positive control group.

[0115] In summary, compared with the other examples, the traditional Chinese medicine probiotic composition prepared in the embodiments of the present invention can significantly enhance the phagocytic activity of macrophages, increase NO secretion levels, and has a significantly better immune activation effect on macrophages, which confirms that there is a synergistic effect among the components of the formulation of the present invention, and that it can effectively activate the immune function of macrophages.

[0116] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A traditional Chinese medicine probiotic composition, characterized in that, By weight, it includes 11-25 parts of Angelica sinensis, 7-21 parts of Rosa laevigata, 6-16 parts of Lonicera japonica, 2-10 parts of Centella asiatica, 6-14 parts of Arctium lappa, and 0.5-3 parts of Lactobacillus plantarum.

2. The traditional Chinese medicine probiotic composition according to claim 1, characterized in that, The total viable count of *Lactobacillus plantarum* in the herbal probiotic composition is (1-10)×10⁻¹⁰. 10 CFU / g.

3. The traditional Chinese medicine probiotic composition according to claim 1, characterized in that, By weight, it includes 15-21 parts of Angelica sinensis, 10-18 parts of Rosa laevigata, 8-14 parts of Lonicera japonica, 4-8 parts of Centella asiatica, 8-12 parts of Arctium lappa, and 1-2 parts of Lactobacillus plantarum.

4. The traditional Chinese medicine probiotic composition according to claim 1, characterized in that, By weight, it includes 18 parts Angelica sinensis, 14 parts Rosa laevigata, 12 parts Lonicera japonica, 6 parts Centella asiatica, 10 parts Arctium lappa and 1.5 parts Lactobacillus plantarum.

5. A method for preparing the traditional Chinese medicine probiotic composition according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Angelica alcohol was refluxed for extraction, solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 1; (2) The extracts of Rosa laevigata and Arctium lappa were extracted by reflux extraction with alcohol, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 2; (3) Honeysuckle and Centella asiatica were decocted in water, the solid and liquid were separated, the decoctions were combined and concentrated and dried to obtain dry extract 3; (4) After mixing dry extract 1, dry extract 2 and dry extract 3, add Lactobacillus plantarum and mix well.

6. The preparation method according to claim 5, characterized in that, The alcohol mentioned in step (1) is ethanol with a volume fraction of 90%-95%; And / or the reflux extraction described in step (1) is: a material-to-liquid mass ratio of 1:8-15, and extraction is performed at least once.

7. The preparation method according to claim 5, characterized in that, The alcohol mentioned in step (2) is ethanol with a volume fraction of 10%-50%; And / or the reflux extraction described in step (2) is: a material-to-liquid mass ratio of 1:8-15, and extraction is performed at least once.

8. The preparation method according to claim 5, characterized in that, The decoction process described in step (3) is as follows: the ratio of material to liquid mass is 1:8-20, and the decoction is performed at least once.

9. The use of the traditional Chinese medicine probiotic composition according to any one of claims 1-4 or the traditional Chinese medicine probiotic composition prepared by the preparation method according to any one of claims 5-8 in the preparation of products that help enhance immunity.

10. The application according to claim 9, characterized in that, The products include health foods, special dietary foods, or dietary supplements.

11. A product that helps boost immunity, characterized in that, This includes the traditional Chinese medicine probiotic composition according to any one of claims 1-4 or the traditional Chinese medicine probiotic composition prepared by the preparation method according to any one of claims 5-8.

12. The product according to claim 11, characterized in that, It also includes acceptable excipients.

13. A method for improving the storage stability of Lactobacillus plantarum, characterized in that, The process includes the following steps: Mixing *Lactobacillus plantarum* with the herbal extract until homogeneous to obtain a herbal probiotic composition, then sealing and storing it away from light; wherein, The herbal probiotic composition comprises, by weight, 11-25 parts of Angelica sinensis, 7-21 parts of Rosa laevigata, 6-16 parts of Lonicera japonica, 2-10 parts of Centella asiatica, 6-14 parts of Arctium lappa, and 0.5-3 parts of Lactobacillus plantarum.

14. The method according to claim 13, characterized in that, The preparation method of the traditional Chinese medicine extract includes the following steps: (1) Angelica sinensis was extracted by reflux with 90%-95% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 1; (2) The fruit of Rosa laevigata and the fruit of Arctium lappa were extracted by reflux with 10%-50% ethanol by volume, the solid and liquid were separated, the extracts were combined, concentrated and dried to obtain dry extract 2; (3) Honeysuckle and Centella asiatica were boiled in water, the solid and liquid were separated, the boiled liquid was combined, concentrated and dried to obtain dry extract 3; (4) Mix dry extract 1, dry extract 2 and dry extract 3 evenly.

15. The method according to claim 14, characterized in that, The reflux extraction described in steps (1) and (2) is as follows: the material-liquid mass ratio is 1:8-15, and the extraction is performed at least once.

16. The method according to claim 14, characterized in that, The decoction process described in step (3) is as follows: the ratio of material to liquid mass is 1:8-20, and the decoction is performed at least once.

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