Rosa roxburghii fermented product as well as preparation method and application thereof

Abstract

The invention provides a rosa roxburghii fermented product as well as a preparation method and application thereof, and belongs to the technical field of rosa roxburghii application. The preparation method of the rosa roxburghii tratt leavening provided by the invention comprises the following steps: mixing rosa roxburghii tratt, fructus momordicae and herba eupatorii, and pulping to obtain pulp; and mixing the slurry with a complex microbial inoculant composed of a rhizopus arrhizus CLF001 seed solution, yeast powder and lactobacillus plantarum powder, and fermenting to obtain the rosa roxburghii fermented product. The rosa roxburghii fermented product prepared by the preparation method not only has the effect of improving sleep, but also has the effect of preventing and / or treating osteosarcoma and urticaria. The preparation method of the rosa roxburghii fermentation product is a directional preparation scheme designed by aiming at the core effects of improving sleep and preventing and treating osteosarcoma and urticaria, and a key material basis and quality guarantee are laid for the subsequent effects of improving sleep and preventing and treating osteosarcoma and urticaria of the fermentation product through raw material proportioning, microbial inoculum synergy and process control.

Description

Technical Field

[0001] This invention belongs to the field of prickly pear application technology, specifically relating to a prickly pear ferment, its preparation method, and its application. Background Technology

[0002] With the fast pace of life and increasing social pressure, sleep disorders have become a global health problem. Sleep disorders and malignant tumors are two major public health issues threatening human health. They are related in their pathological mechanisms and often occur together, posing a dual challenge to patients' quality of life and clinical treatment. Osteosarcoma is the most common primary malignant bone tumor, occurring most frequently in adolescents and children, with 75% of patients developing the disease between the ages of 10 and 20, and the incidence rate is higher in males than in females. Typical symptoms include persistent pain in the tumor area, a rapidly growing mass, and frequent lung metastasis in advanced stages, seriously threatening the patient's life. Currently, the clinical treatment approach is a comprehensive treatment regimen combining surgical resection with chemotherapy and radiotherapy. Although this has increased the 5-year survival rate to 60%–80%, the long-term survival rate for patients with metastatic or recurrent osteosarcoma remains below 30%, and the toxic side effects of chemotherapy drugs and tumor drug resistance issues urgently need to be addressed.

[0003] Prickly pear, also known as ci pear or Wenxian fruit, is mainly produced in Guizhou and other regions. The fruit has a sweet, sour, and astringent taste and contains vitamin C, prickly pear polysaccharides, and other components, possessing stomach-strengthening, digestive, and heat-relieving properties. Existing prickly pear products in this field mostly target sleep improvement alone, failing to consider the comorbidity between sleep disorders and chronic diseases such as tumors, thus failing to meet the diverse health needs of patients. Furthermore, research on prickly pear in this field mainly focuses on common tumors such as lung cancer and gastric cancer, and there are currently no reports on its ability to prevent or treat osteosarcoma. In addition, no research in this field has focused on the synergistic application of prickly pear in both tumor prevention and sleep improvement, failing to meet the needs of osteosarcoma patients who often experience sleep disorders as a complication, and lacking targeted, multifunctional product development. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a prickly pear ferment that has the effects of improving sleep and preventing osteosarcoma and urticaria, and a method for preparing the same.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing prickly pear ferment, comprising the following steps: mixing and pulping prickly pear, monk fruit, and agastache rugosa to obtain a slurry; mixing the slurry with a compound microbial agent for fermentation to obtain prickly pear ferment; the compound microbial agent is composed of Rhizopus oryzae CLF001 seed liquid, yeast powder, and Lactobacillus plantarum powder, wherein the volume-to-weight ratio of each raw material in the compound microbial agent is 100mL of Rhizopus oryzae CLF001 seed liquid: 10-15g of yeast powder: 20-25g of Lactobacillus plantarum powder; the Rhizopus oryzae CLF001 seed liquid contains 1-3g of mycelial dry weight per 100mL; and the weight ratio of prickly pear, monk fruit, and agastache rugosa is 20:2-3:2-4.

[0006] Preferably, the weight ratio of slurry to compound microbial agent is 100:3~5; the fermentation conditions are 25~35℃, 100~150r / min, and fermentation for 20~30h.

[0007] The present invention also provides a prickly pear ferment obtained by the above preparation method.

[0008] The present invention also provides the application of the above-mentioned prickly pear ferment in the preparation of any of the following products, characterized in that (1) it helps to improve sleep; (2) it helps to prevent osteosarcoma; and (3) it helps to prevent urticaria.

[0009] The present invention also provides a composition having the effects of improving sleep, preventing osteosarcoma and / or preventing urticaria, comprising the above-mentioned prickly pear ferment.

[0010] Preferably, the composition further includes Astragalus membranaceus, Codonopsis pilosula, Prunus mume, corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita.

[0011] Preferably, the weight parts of each raw material in the composition are: 30-35 parts of prickly pear ferment, 10-15 parts of astragalus, 13-16 parts of codonopsis, 5-9 parts of dried plum, 6-10 parts of corn silk, 4-8 parts of chrysanthemum, 3-6 parts of licorice, 12-15 parts of pinellia, 16-18 parts of white peony root, and 7-10 parts of yam.

[0012] The present invention also provides a method for preparing the above composition, comprising the following steps: mixing Astragalus membranaceus, Codonopsis pilosula and Prunus mume to obtain mixture 1; mixing mixture 1 with water and decocting to obtain the decoction; mixing corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica and Dioscorea opposita to obtain mixture 2; mixing mixture 2 with an ethanol solution with a volume fraction of 70%~80% and refluxing to obtain an extract; mixing the decoction, the extract and the prickly pear ferment, and concentrating to a relative density of 1.25~1.29.

[0013] Preferably, the weight-to-volume ratio of the mixture 1 to water is 1g:20~25mL, and the decoction is to boil and then keep boiling for 30~40 minutes.

[0014] Preferably, the weight-to-volume ratio of mixture 2 to ethanol solution is 1g:10~15mL; the reflux extraction temperature is 75~85℃, and the reflux extraction time is 1~1.5h.

[0015] The beneficial effects of this invention are: The prickly pear ferment obtained using the method of this invention not only improves sleep but also has preventive and / or therapeutic effects on osteosarcoma and urticaria. The method for preparing the prickly pear ferment of this invention is a targeted preparation scheme designed around the core efficacy goals of improving sleep and preventing osteosarcoma and urticaria. Through raw material ratios, synergistic effects of microbial agents, and process control, it lays a key material foundation and ensures quality for the subsequent efficacy of the ferment in improving sleep, preventing osteosarcoma, and preventing urticaria. Detailed Implementation

[0016] This invention provides a method for preparing prickly pear ferment, comprising the following steps: mixing and pulping prickly pear, monk fruit, and agastache rugosa to obtain a slurry; mixing the slurry with a compound microbial agent for fermentation to obtain prickly pear ferment; the compound microbial agent is composed of Rhizopus oryzae CLF001 seed liquid, yeast powder, and Lactobacillus plantarum powder, wherein the volume-to-weight ratio of each raw material in the compound microbial agent is 100mL of Rhizopus oryzae CLF001 seed liquid: 10-15g of yeast powder: 20-25g of Lactobacillus plantarum powder; the Rhizopus oryzae CLF001 seed liquid contains 1-3g of mycelial dry weight per 100mL; and the weight ratio of prickly pear, monk fruit, and agastache rugosa is 20:2-3:2-4.

[0017] This invention does not specifically limit the source of each raw material. Rhizopus oryzae CLF001 is a disclosed strain, detailed in invention patent CN 120442410 A, deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCCNo:66063, deposit date March 27, 2025. In this invention, the preferred weight ratio of prickly pear, monk fruit, and agastache rugosa is 20:2~3:2~4. In this invention, the weight ratio of the slurry to the compound microbial agent is preferably 100:3~5, more preferably 100:4; the fermentation conditions are preferably 25~35℃, 100~150r / min for 20~30h, more preferably 30℃, 120r / min for 25h. In this invention, the preferred method for preparing the Rhizopus oryzae CLF001 seed culture is as follows: Rhizopus oryzae CLF001 is inoculated in the center of a PDA plate and cultured until the mycelium is abundant and spores form. The culture conditions are preferably 28~30℃ for 24~48h. Then, mycelial blocks are picked and inoculated into PDB liquid culture medium, and cultured with shaking until each 100mL of liquid contains 1~3g of mycelial dry weight. The shaking culture conditions are preferably 28~30℃, 150~200rpm for 24h. In this invention, the preferred volume-to-weight ratio of the raw materials in the compound microbial agent is 100 mL of Rhizopus oryzae CLF001 seed liquid: 11-14 g of yeast powder: 21-24 g of Lactobacillus plantarum powder, more preferably 100 mL of Rhizopus oryzae CLF001 seed liquid: 12-13 g of yeast powder: 22-23 g of Lactobacillus plantarum powder. In some embodiments of this invention, the weight ratio of prickly pear, monk fruit, and agastache rugosa is 20:2:2, 20:3:2, 20:2:4, 20:2:3, or 20:3:3.

[0018] This invention utilizes prickly pear fermented product as a base ingredient. Prickly pear, rich in vitamin C, polyphenols, and flavonoids, is a key precursor for inhibiting tumor cell proliferation and clearing neurooxidative stress. Monk fruit glycosides not only neutralize the astringent taste of prickly pear but also possess anti-inflammatory and metabolic-regulating effects, further enhancing the inhibitory effect of prickly pear on tumor microenvironment inflammation. Simultaneously, they synergistically improve neurotransmitter balance with tryptophan in prickly pear. The aromatic components of agastache can soothe nervous tension, directly contributing to improved sleep. Furthermore, its medicinal components complement the active substances in prickly pear and monk fruit, further enriching the anti-tumor active ingredient library of the fermented product. This combination not only avoids the problems of limited efficacy and unpleasant flavor associated with single prickly pear ingredients but also constructs a complex system of anti-tumor and sleep-aiding components. In addition, the polyphenols and flavonoids in prickly pear can inhibit histamine release and block the generation of allergy mediators; monk fruit glycosides can regulate immune cell balance (reducing basophil activation); and agastache volatile oil can inhibit mast cell degranulation and alleviate allergic reactions.

[0019] The proportions of each ingredient in the prickly pear fermented product of this invention ensure that there is no antagonistic effect between the ingredients. Furthermore, the addition of Eupatorium fortunei can reduce the inhibition of subsequent bacterial activity by tannins in prickly pear. The high sweetness of Monk Fruit reduces the need for additional sugar during fermentation, avoiding the adverse effects of high sugar on the metabolism of cancer patients. This provides a safety guarantee for the fermented product to be suitable for target groups who want to improve sleep and prevent osteosarcoma.

[0020] In this invention, Rhizopus oligosporus CLF001 can break through macromolecular barriers and release core active ingredients. For example, it can reduce the tannin content in prickly pear, thereby reducing the stimulation of nerve cells by tannins and eliminating interference with the subsequent neuroprotective effect of improving sleep. At the same time, its metabolites can regulate the pH value of the raw materials, providing a suitable environment for the growth of other microbial species. Yeast powder can catalyze the transformation of active ingredients to generate auxiliary functional substances, enhancing its apoptosis-inducing effect on tumor cells. In addition, the small amount of alcohols and esters generated by yeast metabolism can further optimize the flavor of fermented products and improve patient compliance, especially for the poor appetite problem often encountered by osteosarcoma patients. Lactobacillus plantarum powder enhances the stability of active ingredients and strengthens immunomodulatory effects. The lactic acid produced by Lactobacillus plantarum maintains the pH of the fermentation system at 3.8-4.5. This acidic environment inhibits contamination by other microorganisms and significantly improves the stability of prickly pear polysaccharides and flavonoids. The short-chain fatty acids produced by its metabolism not only regulate intestinal flora and improve intestinal dysfunction after chemotherapy in cancer patients, indirectly enhancing immunity, but also synergistically activate immune cells with the polysaccharides in prickly pear, enhancing the body's ability to recognize and attack osteosarcoma cells. Simultaneously, the neuroprotective effect of short-chain fatty acids can help improve sleep quality. In this invention, Rhizopus oligosporus CLF001 seed liquid, yeast powder, and Lactobacillus plantarum powder work synergistically. The content of key sleep-improving components in the fermented product, such as 5-hydroxytryptamine precursors and melatonin synthesis promoters, is significantly increased compared to unfermented raw materials. The bioavailability of active ingredients for preventing osteosarcoma is also significantly improved, and no harmful metabolites are generated, providing a high-activity and high-absorption material guarantee for subsequent efficacy. Furthermore, this invention utilizes a compound microbial agent for fermentation, transforming large molecules in the raw materials into smaller molecules, thereby improving bioavailability. Simultaneously, probiotic metabolites can regulate the balance of intestinal flora, improving the body's allergic constitution through the gut-immune axis and reducing the frequency and severity of urticaria attacks. The active ingredients from these three components, after fermentation, have a synergistic anti-inflammatory effect, which can alleviate inflammatory reactions such as skin redness, swelling, and itching during urticaria attacks.

[0021] The present invention also provides a prickly pear ferment obtained by the above preparation method.

[0022] This invention also provides the use of the above-mentioned prickly pear ferment in the preparation of products that help improve sleep and / or prevent osteosarcoma. In this invention, the products preferably include pharmaceuticals.

[0023] The present invention also provides a composition having the effects of improving sleep and preventing osteosarcoma, comprising the above-mentioned prickly pear ferment.

[0024] In this invention, the composition preferably further includes Astragalus membranaceus, Codonopsis pilosula, Prunus mume, corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita. The preferred weight proportions of each ingredient in the composition are: 30-35 parts of fermented prickly pear extract, 10-15 parts of Astragalus membranaceus, 13-16 parts of Codonopsis pilosula, 5-9 parts of Prunus mume, 6-10 parts of corn silk, 4-8 parts of chrysanthemum, 3-6 parts of licorice, 12-15 parts of Pinellia ternata, 16-18 parts of Ampelopsis japonica, and 7-10 parts of Dioscorea opposita; more preferably: 31-34 parts of fermented prickly pear extract, 11-14 parts of Astragalus membranaceus, Codonopsis pilosula, and Dioscorea opposita. The ingredients are: 14-15 parts ginseng, 6-8 parts dried plum, 7-9 parts corn silk, 5-7 parts chrysanthemum, 4-5 parts licorice, 13-14 parts pinellia, 17 parts white peony root, and 8-9 parts yam; further preferred ingredients are: 32-33 parts prickly pear ferment, 12-13 parts astragalus, 14 parts codonopsis, 7 parts dried plum, 8 parts corn silk, 6 parts chrysanthemum, 4 parts licorice, 13 parts pinellia, 17 parts white peony root, and 8 parts yam.

[0025] The composition of this invention uses the prickly pear fermented product prepared in this invention as the core, combined with Astragalus membranaceus, Codonopsis pilosula, Prunus mume, corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita. Each component exerts a synergistic effect in improving sleep and preventing osteosarcoma. The active ingredients in the prickly pear fermented product, after fermentation and transformation, can promote the production of serotonin and melatonin in the brain, regulate neurotransmitter balance, and improve nerve excitability, laying the foundation for improved sleep. Astragalus membranaceus and Codonopsis pilosula can invigorate qi and strengthen the spleen, enhance the body's immunity, and regulate organ function, providing a favorable internal environment for improved sleep. Prunus mume, with its sour taste, can astringe the lungs and intestines, promote body fluid production and quench thirst, and assist in regulating metabolism, alleviating sleep problems caused by physical discomfort. Corn silk and chrysanthemum have heat-clearing, diuretic, and neutralizing properties. This formula benefits the liver and eyesight, relieving dryness, anxiety, and other adverse states, soothing nerves, and promoting sleep. Licorice harmonizes the effects of other herbs, moderating their properties; its glycyrrhetinic acid and other components also have a calming effect, aiding in sleep improvement. Pinellia ternata dries dampness, resolves phlegm, and relieves nausea and vomiting, improving sleep disorders caused by internal phlegm and dampness. Ampelopsis japonica clears heat and detoxifies, reducing inflammatory responses and preventing inflammation from interfering with sleep. Dioscorea opposita tonifies the spleen and stomach, promotes fluid production, and benefits the lungs, regulating spleen and stomach function and improving insomnia caused by spleen and stomach disharmony. These multiple components, acting through different mechanisms, work synergistically to significantly enhance the sleep-improving effect of the composition. It is suitable for various types of sleep disorders, with high safety and no significant side effects.

[0026] In the prevention and treatment of osteosarcoma, the composition of this invention also exhibits excellent synergistic effects, achieving multi-target and multi-pathway action. The active ingredients in prickly pear ferment, such as polysaccharides and flavonoids, have enhanced bioactivity after fermentation, inhibiting the proliferation of osteosarcoma cells, inducing tumor cell apoptosis, and simultaneously regulating the body's immune function, enhancing the body's ability to recognize and attack tumor cells. Astragalus and Codonopsis not only enhance the body's immunity, but their components, such as astragalus polysaccharides and Codonopsis saponins, can also inhibit tumor cell growth, prevent tumor cell metastasis, and enhance the body's resistance to tumors. The umeboside and other components in dried plum have antioxidant and anti-inflammatory effects, reducing inflammatory responses in the tumor microenvironment and inhibiting the growth and spread of tumor cells. The flavonoids in corn silk... The ingredients in this product can inhibit tumor angiogenesis and reduce the nutrient supply to tumor cells, thereby inhibiting tumor growth. Chrysanthemum contains chrysanthemum lactones and other components with anti-tumor activity, which can help inhibit osteosarcoma cell proliferation. Licorice contains glycyrrhizic acid and other components that can enhance the body's immune function and also have a certain inhibitory effect on tumor cells. Pinellia contains pinellia protein and other components that can induce tumor cell apoptosis and inhibit tumor growth. Ampelopsis contains ampelopsis glycosides and other components with anti-tumor effects, which can help inhibit osteosarcoma cell proliferation and metastasis. Dioscorea contains yam polysaccharides that can enhance the body's immunity and regulate metabolism, providing a good physical foundation for tumor prevention and treatment. These multiple components work synergistically, targeting multiple points and pathways, including inhibiting tumor cell proliferation, inducing tumor cell apoptosis, regulating the body's immunity, inhibiting tumor angiogenesis, and reducing inflammatory responses, to jointly exert their efficacy in preventing and treating osteosarcoma. This can effectively delay the progression of osteosarcoma, improve patients' quality of life, and provide a new and effective solution for the prevention and treatment of osteosarcoma. Compared with traditional chemotherapy drugs, it has higher safety, fewer side effects, and better patient tolerance.

[0027] In the prevention and treatment of urticaria, the composition of this invention uses prickly pear ferment as its core ingredient. Through the small-molecule flavonoids and polyphenols produced during fermentation, it directly inhibits mast cell degranulation and histamine release, blocking the source of urticaria attacks. Simultaneously, its probiotic metabolites can regulate the intestinal flora, improving allergic constitution through the gut-immune axis, laying the foundation for the composition's anti-allergic efficacy. Combined with nine traditional Chinese medicines, which assist in treatment from four dimensions—anti-inflammatory, immune-regulating, wind-dispelling and itch-relieving, and spleen-strengthening—it synergizes with the prickly pear ferment, ultimately achieving a precise and enhanced effect in the prevention and treatment of urticaria.

[0028] The present invention also provides a method for preparing the above composition, comprising the following steps: mixing Astragalus membranaceus, Codonopsis pilosula and Prunus mume to obtain mixture 1; mixing mixture 1 with water and decocting to obtain the decoction; mixing corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica and Dioscorea opposita to obtain mixture 2; mixing mixture 2 with an ethanol solution with a volume fraction of 70%~80% and refluxing to obtain an extract; mixing the decoction, the extract and the prickly pear ferment, and concentrating to a relative density of 1.25~1.29.

[0029] In this invention, preferably, Astragalus membranaceus, Codonopsis pilosula, and Prunus mume are first pulverized before being mixed with water and decocted. This invention does not have specific limitations on the specific pulverization method or particle size. In this invention, the weight-to-volume ratio of the mixture 1 to water is preferably 1g:20-25mL, more preferably 1g:22-24mL; the decocting is preferably maintained at boiling for 30-40 minutes, more preferably 34-38 minutes. In this invention, preferably, corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita are first pulverized before being mixed with an ethanol solution and refluxed for extraction. This invention does not have specific limitations on the specific pulverization method or particle size. In this invention, the weight-to-volume ratio of the mixture 2 to the ethanol solution is preferably 1g:10-15mL, more preferably 1g:11-14mL, and even more preferably 1g:12-13mL; the reflux extraction temperature is preferably 75-85℃, more preferably 77-82℃, and the reflux extraction time is preferably 1-1.5h, more preferably 1.1-1.4h. In this invention, the volume fraction of the ethanol solution is preferably 72%-78%, more preferably 74%-76%.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the following embodiments are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] The Lactobacillus plantarum powder used in the following examples was purchased from Shandong Pingao Pharmaceutical Co., Ltd., with product number PG-LPZ131; the yeast powder was Angel Yeast High-Activity Dry Yeast.

[0034] The values ​​in the following examples are expressed as averages.

[0035] Example 1 A prickly pear ferment, the preparation method consists of the following steps: The prickly pear, monk fruit and agastache rugosa were mixed and pulped in a weight ratio of 20:2:2 to obtain a pulp. The pulp was then mixed with a compound microbial agent in a weight ratio of 100:3 and fermented under the conditions of 25°C, 100r / min for 30h to obtain prickly pear fermented product. The compound microbial agent consists of Rhizopus oryzae CLF001 seed culture, yeast powder, and Lactobacillus plantarum powder. The volume-to-weight ratio of each raw material in the compound microbial agent is 100mL of Rhizopus oryzae CLF001 seed culture: 10g of yeast powder: 20g of Lactobacillus plantarum powder. The Rhizopus oryzae CLF001 seed culture contains 1g of mycelial dry weight per 100mL. The preparation method of the Rhizopus oryzae CLF001 seed culture is as follows: Take the slant culture from a 4℃ refrigerator, cut a piece (3mm×3mm) in the center of a PDA plate, and incubate at 28℃ for 48h until the mycelium is abundant and spores are formed. Directly pick a piece of mycelium (5mm in diameter) and inoculate it into PDA liquid culture medium, and incubate at 28℃ and 150rpm for 24h with shaking until each 100mL of seed culture contains 1g of mycelial dry weight, forming a uniform mycelial suspension, also known as Rhizopus oryzae CLF001 seed culture.

[0036] Example 2 A prickly pear ferment, the preparation method consists of the following steps: The prickly pear, monk fruit and agastache were mixed and pulped in a weight ratio of 20:3:4 to obtain a pulp. The pulp was then mixed with a compound microbial agent in a weight ratio of 100:5 and fermented under the conditions of 35℃, 150r / min for 20h to obtain prickly pear fermented product. The compound microbial agent consists of Rhizopus oryzae CLF001 seed culture, yeast powder, and Lactobacillus plantarum powder. The volume-to-weight ratio of each raw material in the compound microbial agent is 100mL of Rhizopus oryzae CLF001 seed culture: 15g of yeast powder: 25g of Lactobacillus plantarum powder. The Rhizopus oryzae CLF001 seed culture contains 3g of mycelial dry weight per 100mL. The preparation method of the Rhizopus oryzae CLF001 seed culture is as follows: Take the slant culture from a 4℃ refrigerator, cut a piece (3mm×3mm) in the center of a PDA plate, and incubate at 30℃ for 24h until the mycelium is abundant and spores are formed. Directly pick a piece of mycelium (5mm in diameter) and inoculate it into PDA liquid culture medium, and incubate at 30℃ and 200rpm for 24h with shaking until each 100mL of seed culture contains 3g of mycelial dry weight, forming a uniform mycelial suspension, also known as Rhizopus oryzae CLF001 seed culture.

[0037] Example 3 A prickly pear ferment, the preparation method consists of the following steps: The prickly pear, monk fruit and agastache rugosa were mixed and pulped in a weight ratio of 20:3:3 to obtain a pulp. The pulp was then mixed with a compound microbial agent in a weight ratio of 100:4 and fermented under the conditions of 30℃, 120r / min for 25h to obtain prickly pear fermented product. The compound microbial agent consists of Rhizopus oryzae CLF001 seed culture, yeast powder, and Lactobacillus plantarum powder. The volume-to-weight ratio of each raw material in the compound microbial agent is 100mL of Rhizopus oryzae CLF001 seed culture: 12g of yeast powder: 22g of Lactobacillus plantarum powder. The Rhizopus oryzae CLF001 seed culture contains 2g of mycelial dry weight per 100mL. The preparation method of the Rhizopus oryzae CLF001 seed culture is as follows: Take the slant culture from a 4℃ refrigerator, cut a piece (3mm×3mm) in the center of a PDA plate, and incubate at 29℃ for 30h until the mycelium is abundant and spores are formed. Directly pick a piece of mycelium (5mm in diameter) and inoculate it into PDB liquid culture medium, and incubate at 29℃ and 180rpm for 24h with shaking until each 100mL of seed culture contains 2g of mycelial dry weight, forming a uniform mycelial suspension, also known as Rhizopus oryzae CLF001 seed culture.

[0038] Example 4 A composition having the effects of improving sleep, preventing osteosarcoma and preventing urticaria is made from the following raw materials in parts by weight: 30 parts of prickly pear ferment obtained in Example 1, 10 parts of astragalus, 13 parts of codonopsis, 5 parts of dried plum, 6 parts of corn silk, 4 parts of chrysanthemum, 3 parts of licorice, 12 parts of pinellia, 16 parts of white peony root and 7 parts of yam.

[0039] The preparation method is as follows: After pulverizing Astragalus membranaceus, Codonopsis pilosula and Prunus mume, they are passed through a 100-mesh sieve and mixed to obtain mixture 1. Mixture 1 is then decocted with water at a weight-to-volume ratio of 1g:20mL. After boiling, the mixture is kept boiling for 30 minutes. The decoction is then collected for later use. After pulverizing corn silk, chrysanthemum, licorice, pinellia, white peony root and yam, they were passed through a 100-mesh sieve and mixed to obtain mixture 2. Mixture 2 was then mixed with a 70% ethanol solution, with the weight-to-volume ratio of mixture 2 to ethanol solution being 1g:10mL. The mixture was then refluxed at 75℃ for 1.5h to obtain the extract. The decoction, extract and prickly pear ferment obtained in Example 1 were mixed and concentrated to a relative density of 1.25 (60°C).

[0040] Example 5 A composition having the effects of improving sleep, preventing osteosarcoma and preventing urticaria is made from the following raw materials in parts by weight: 35 parts of prickly pear ferment obtained in Example 2, 15 parts of astragalus, 16 parts of codonopsis, 9 parts of dried plum, 10 parts of corn silk, 8 parts of chrysanthemum, 6 parts of licorice, 15 parts of pinellia, 18 parts of white peony root and 10 parts of yam.

[0041] The preparation method is as follows: After pulverizing Astragalus membranaceus, Codonopsis pilosula and Prunus mume, they are passed through a 100-mesh sieve and mixed to obtain mixture 1. Mixture 1 is then decocted with water at a weight-to-volume ratio of 1g:25mL. After boiling, the mixture is kept boiling for 40 minutes. The decoction is then collected for later use. After pulverizing corn silk, chrysanthemum, licorice, pinellia, white peony root and yam, they were passed through a 100-mesh sieve and mixed to obtain mixture 2. Mixture 2 was then mixed with an 80% ethanol solution, with the weight-to-volume ratio of mixture 2 to ethanol solution being 1g:15mL. The mixture was then refluxed at 85℃ for 1h to obtain the extract. The decoction, extract and prickly pear ferment obtained in Example 2 were mixed and concentrated to a relative density of 1.29 (60°C).

[0042] Example 6 A composition having the effects of improving sleep, preventing osteosarcoma and preventing urticaria is made from the following raw materials in parts by weight: 32 parts of prickly pear ferment obtained in Example 3, 13 parts of astragalus, 14 parts of codonopsis, 7 parts of dried plum, 8 parts of corn silk, 6 parts of chrysanthemum, 5 parts of licorice, 13 parts of pinellia, 17 parts of white peony root, and 8 parts of yam.

[0043] The preparation method is as follows: After pulverizing Astragalus membranaceus, Codonopsis pilosula and Prunus mume, they are passed through a 100-mesh sieve and mixed to obtain mixture 1. Mixture 1 is then mixed with water and decocted. The weight-to-volume ratio of mixture 1 to water is 1g:22mL. After boiling, the mixture is kept boiling for 35 minutes. The decoction is then collected for later use. Corn silk, chrysanthemum, licorice, pinellia, white peony root and yam are pulverized and passed through a 100-mesh sieve to obtain mixture 2. Mixture 2 is then mixed with a 75% ethanol solution at a weight-to-volume ratio of 1g:12mL. The mixture is then refluxed at 80℃ for 1.3h to obtain the extract. The decoction, extract and prickly pear ferment obtained in Example 3 were mixed and concentrated to a relative density of 1.27 (60°C).

[0044] Comparative Example 1 The difference from Example 3 is that the compound microbial agent does not contain Rhizopus spp. CLF001 seed liquid, but all other aspects are the same as in Example 3.

[0045] Comparative Example 2 The difference from Example 3 is that it does not contain prickly pear; otherwise, it is the same as Example 3.

[0046] Comparative Example 3 The difference from Example 3 is that the weight ratio of prickly pear, monk fruit and agastache is 20:4:5, while the rest are the same as in Example 3.

[0047] Comparative Example 4 The difference from Example 3 is that the volume weight ratio of each raw material in the compound microbial agent is: 100mL of Rhizopus spp. CLF001 seed liquid: 9g of yeast powder: 28g of Lactobacillus plantarum powder, and the rest are the same as in Example 3.

[0048] Comparative Example 5 The difference from Example 6 is that it does not contain the prickly pear ferment obtained in Example 3, but otherwise it is the same as Example 6.

[0049] Comparative Example 6 The difference from Example 6 is that the weight parts of each raw material in the composition are as follows: 40 parts of prickly pear ferment, 8 parts of astragalus, 17 parts of codonopsis, 4 parts of dried plum, 12 parts of corn silk, 2 parts of chrysanthemum, 8 parts of licorice, 10 parts of pinellia, 20 parts of white peony root, and 5 parts of yam. The rest are the same as in Example 6.

[0050] Comparative Example 7 The difference from Example 6 is that all raw materials are prepared by boiling in water, while the other parameters, such as the weight-volume ratio of the mixture to water and the specific boiling method, are the same as in Example 6.

[0051] Comparative Example 8 The difference from Example 6 is that the prickly pear ferment obtained in Example 3 is replaced with the prickly pear ferment obtained in Comparative Example 3, and the rest are the same as in Example 6.

[0052] Comparative Example 9 The difference from Example 6 is that the volume fraction of the ethanol solution is 90%, while the rest are the same as in Example 6.

[0053] Experimental Example 1 130 Balb / c nude mice, aged 6-8 weeks and weighing 18-22g, were selected, with half males and half females. The mice were housed in an SPF (specific pathogen-free) animal room at a temperature controlled at 22-25℃ and a relative humidity of 40%-60%, with a 12-hour light / dark cycle. Sterile feed and drinking water were provided, and experiments began one week after acclimatization.

[0054] Human osteosarcoma cell line U-2OS was purchased from Shanghai Jingkang Biotechnology Co., Ltd., catalog number JK-R2550. After resuscitation, the cell line was cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and incubated at 37°C in a 5% CO2 cell culture incubator. When the cell confluence reached 80%–90%, the cells were passaged using 0.25% trypsin-EDTA digestion solution.

[0055] Experimental Design: 1. Grouping Planning 130 Balb / c nude mice were randomly divided into 13 groups of 10 mice each using a random number table: Model group: A mouse model of osteosarcoma was established by inoculating the human osteosarcoma cell line U-2OS, but no drug intervention was given, to observe the growth and development of osteosarcoma under natural conditions. Positive control group: After establishing the osteosarcoma cell line U-2OS, the patients were treated with the positive control drug cisplatin. Example 3: After establishing a human osteosarcoma cell line U-2OS, the prickly pear fermentation solution obtained in Example 3 was administered by gavage to explore the preventive and therapeutic effects of prickly pear fermentation on osteosarcoma.

[0056] The composition obtained in Example 6: After establishing a model by inoculating human osteosarcoma cell line U-2OS, the composition solution obtained in Example 6 was administered by gavage to explore the preventive and therapeutic effects of the composition on osteosarcoma.

[0057] Comparative Example 1: After establishing the model by inoculating human osteosarcoma cell line U-2OS, the prickly pear fermentation solution obtained in Comparative Example 1 was administered by gavage.

[0058] Comparative Examples 1 to 4: After establishing the model by inoculating human osteosarcoma cell line U-2OS, the prickly pear fermentation solutions obtained in Comparative Examples 1 to 4 were administered by gavage.

[0059] The compositions obtained in Comparative Examples 5 to 9: After establishing the model by inoculating human osteosarcoma cell line U-2OS, the compositions obtained in Comparative Examples 5 to 9 were administered by gavage.

[0060] 2. Dosing regimen Model group: Mice in the model group were given an equal volume of sterile saline by gavage every day, with a gavage volume of 0.2 mL per mouse, for 21 consecutive days to maintain normal physiological intake in mice and ensure consistency of experimental procedures. Positive control group: Mice in the positive control group were given intraperitoneal injections of cisplatin at a dose of 2 mg / kg, twice a week, starting on day 7 after inoculation with osteosarcoma cells, for a total of 3 weeks.

[0061] Example 3 prickly pear fermentation group: Starting on the 7th day after inoculation with osteosarcoma cells, the prickly pear fermentation solution obtained in Example 3 was administered by gavage at a dose of 2 mg / kg, with a gavage volume of 0.2 mL / animal, once a day for 21 consecutive days.

[0062] The composition group obtained in Example 6: Starting on the 7th day after inoculation with osteosarcoma cells, the composition solution obtained in Example 6 was administered by gavage at a dose of 2 mg / kg, with a gavage volume of 0.2 mL / animal, once a day for 21 consecutive days.

[0063] Comparative Examples 1 to 4: Starting on the 7th day after inoculation of human osteosarcoma cell line U-2OS, each animal was given a dose of 2 mg / kg of the prickly pear fermentation solution obtained in Comparative Examples 1 to 4 by gavage. The gavage volume was 0.2 mL / animal, and the administration was once a day for 21 consecutive days.

[0064] The compositions obtained in Comparative Examples 5 to 9: Starting on the 7th day after inoculation with human osteosarcoma cell line U-2OS, the patients were given a gavage of 2 mg / kg of the composition solutions obtained in Comparative Examples 5 to 9, with a gavage volume of 0.2 mL / animal, once a day for 21 consecutive days.

[0065] 3. Specific methods Human osteosarcoma cell line U-2OS in logarithmic growth phase was digested with 0.25% trypsin-EDTA digestion solution. After the cells became rounded and detached, the digestion was terminated by adding DMEM high-glucose medium containing 10% fetal bovine serum, and the cells were pipetted to prepare a single-cell suspension. The cell suspension was transferred to centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with sterile PBS. After centrifugation again, the cells were resuspended in sterile physiological saline and the cell density was adjusted to 5 × 10⁶ cells / mL. 6 per mL. Inoculation procedure: Balb / c nude mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (100 mg / kg) and fixed in a supine position on the operating table. After disinfecting the skin under the right axilla of the nude mouse, 0.2 ml of the prepared cell suspension was slowly injected subcutaneously into the axilla using a 1 ml syringe, containing 1 × 10⁻⁶ cells. 6 After injection, disinfect the injection site with povidone-iodine and gently press for a moment to prevent the cell suspension from spilling out. Tumor identification: Closely observe the general condition of mice and changes at the inoculation site after inoculation. Approximately 7-10 days later, a noticeable mass can be felt at the inoculation site, indicating successful tumor inoculation. Wait until the tumor grows to approximately 100 mm². 3 At that time, 1-2 mice were randomly selected, euthanized by cervical dislocation, and tumor tissue was extracted for pathological examination. The tumor tissue was fixed in 4% paraformaldehyde for 24 hours, then routinely dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (HE). The morphology and structure of tumor cells were observed under a microscope to further confirm the successful establishment of the osteosarcoma model. After confirming the successful establishment of the osteosarcoma model, the following drug administration procedures were performed: Model group: Mice in the model group were administered an equal volume (0.2 ml / mouse) of sterile saline via gavage at a fixed time each day. During gavage, the mice were gently restrained so that their heads and bodies were in a straight line. The gavage needle was slowly inserted along the side wall of the mouse's mouth until slight resistance was felt, indicating that the needle had reached the stomach. Then, the saline was slowly injected. Care should be taken to be gentle during the gavage process to avoid damaging the mouse's mouth and esophagus. Positive control group: Mice in the positive control group received intraperitoneal injections of cisplatin starting on day 1 after successful establishment of the osteosarcoma model. Cisplatin was prepared into a solution of the required concentration using physiological saline. An appropriate amount of solution was drawn using a 1ml syringe, and air was expelled. After fixing the mice, the cisplatin solution was slowly injected at a 45° angle into the lower third of the mouse's abdomen, avoiding the bladder and intestines. The dose was 2 mg / kg, administered twice weekly for 3 weeks. The mice's response was closely monitored during injection; if any abnormalities occurred, the injection was immediately stopped and appropriate measures were taken. The prickly pear fermented products obtained in Example 3 and Comparative Examples 1-4 were administered via gavage starting on the first day after successful establishment of the osteosarcoma model. According to the experimental design, the prickly pear fermented product powder was prepared into a 10 mg / ml solution using sterile physiological saline. The corresponding dose of prickly pear fermented product solution was administered via gavage at a fixed time each day, with a gavage volume of 0.2 ml / kg. The administration procedure was the same as that for the control group, and care was taken to avoid injury to the mice.

[0066] The compositions obtained in Example 6 and Comparative Examples 5-9 were administered via gavage starting on day 1 after successful establishment of the osteosarcoma model. According to the experimental design, the dried prickly pear ferment powder was prepared into a 10 mg / ml solution using sterile physiological saline. The corresponding dose of prickly pear ferment solution was administered via gavage at a fixed time each day, with a gavage volume of 0.2 ml / kg. The administration procedure was the same as for the control group, and care was taken to avoid injury to the mice.

[0067] Starting from the first day after the successful establishment of the osteosarcoma model, the long axis (a) and short axis (b) of the mouse tumor were measured every 3 days using vernier calipers. During measurement, the mouse was gently fixed to fully expose the tumor, and the longest and shortest axes were accurately measured with vernier calipers to an accuracy of 0.1 mm. The values ​​were calculated using the formula V = 1 / 2 × a × b. 2 Tumor volume was calculated and data recorded. The results are expressed as averages, and are shown in Table 1. Table 1 shows that both the prickly pear ferment and the composition containing prickly pear ferment have a significant inhibitory effect on osteosarcoma growth.

[0068] Table 1. Statistical results of tumor volume in different groups

[0069] Experimental Example 2 1. Test materials Experimental animals: 130 male SPF-grade SD rats, weighing 220-250g, were acclimatized for 7 days (temperature 22±2℃, humidity 50±5%, 12h light-dark cycle). Insomnia model construction: The rat insomnia model was constructed by intraperitoneal injection of p-chlorophenylalanine (PCPA) at a dose of 300 mg / kg for two consecutive days to inhibit the synthesis of 5-hydroxytryptamine (5-HT) in the rat brain (successful modeling criteria: the number of nocturnal activities of rats increased by ≥50% and the sleep latency was prolonged by ≥40% compared with the normal group). Test substances: prickly pear fermentation solutions obtained in Example 3 and Comparative Examples 1-4, with a concentration of 1 g / mL and physiological saline as the solvent; and composition solutions obtained in Example 6 and Comparative Examples 5-9, with a concentration of 1 g / mL and physiological saline as the solvent. A blank control group (physiological saline) and a positive control group (melatonin, dose 5 mg / kg) were also set up.

[0070] 2. Experimental grouping and drug administration After the insomnia model was successfully constructed, it was divided into 13 groups with 10 animals in each group.

[0071] Blank control group: administered physiological saline by gavage at a rate of 10 mL / kg for 14 days; Positive control group: Melatonin solution was administered orally at a dose of 5 mg / kg for 14 days; Example 3 and Comparative Examples 1-4: prickly pear fermentation group: administered physiological saline by gavage at a dose of 5 mg / kg for 14 days; Compositions of Example 6 and Comparative Examples 5 to 9: were administered physiological saline by gavage at a dose of 5 mg / kg for 14 days.

[0072] 3. Detection Indicators and Methods Behavioral indicators (nighttime light-dark cycle, 20:00-8:00 the next day): Sleep latency: The time it takes for rats to transition from an active state to a resting sleep state (curled up, breathing steadily, and unresponsive to mild stimuli) is recorded using a video tracking system (EthoVision XT 16). Total sleep duration: The cumulative sleep time of rats within 12 hours was recorded; Number of awakenings: The number of times the rat transitioned from sleep to activity during the sleep process was recorded.

[0073] The results, as shown in Table 2, indicate that the prickly pear ferment and composition provided by this invention have a significant effect on improving sleep.

[0074] Table 2 Statistical results of behavioral indicators for different groups

[0075] Experimental Example 3 SPF-grade guinea pigs (250-300g, half male and half female) were used and kept in an environment with a temperature of 22-26℃ and a humidity of 50%-60%, with free access to food and water. The experiment began after the guinea pigs had been acclimatized to the environment for 3 days.

[0076] Preparation of sensitized serum: Ten guinea pigs were injected intraperitoneally with ovalbumin (OVA) (1 mg / pig) + Freund's complete adjuvant. A booster immunization was given every 7 days. Blood was collected from the orbital cavity 10 days after the last immunization, and the serum was separated by centrifugation (stored at -20℃ for later use).

[0077] Grouping for drug administration: The experimental guinea pigs were randomly divided into 13 groups (n=10 per group): Model control group: physiological saline; Positive control group: Cetirizine hydrochloride administered by gavage (10 mg / kg); Example 3 and Comparative Examples 1-4: prickly pear fermentation group: administered by gavage at a dose of 10 mg / kg; Compositions of Example 6 and Comparative Examples 5 to 9 were administered by gavage at a dose of 5 mg / kg.

[0078] Administration: Administer once daily for 7 consecutive days.

[0079] Skin sensitization and irritation: On the 7th day after administration, the hair on the back of the guinea pig was removed (area 2cm×2cm), and the sensitized serum was injected intradermally (0.1mL / point, 1 point on each side of the back).

[0080] Forty-eight hours after serum injection, OVA (1 mg / animal) + Evans blue staining solution (1%, 0.1 mL / 100 g body weight) was injected via the tail vein to stimulate the animal.

[0081] Thirty minutes after stimulation, the guinea pigs were euthanized, and the skin on their backs was peeled off to observe the formation of blue spots. The diameter of the blue spots was measured (accurate to 0.1 cm), and the area of ​​the blue spots was calculated. The formation of blue spots is a direct manifestation of the pathological mechanism of urticaria. Evans blue staining solution will seep into the skin tissue as vascular permeability increases, forming visible blue spots. The larger the area of ​​the blue spots, the more severe the local allergic reaction of the skin, and the higher the vascular permeability, which means that the core pathological symptoms of urticaria are more obvious.

[0082] The results are shown in Table 3, indicating that the prickly pear ferment and composition provided by the present invention have the effect of preventing and treating urticaria.

[0083] Table 3. Statistical results of urticaria prevention and treatment in different groups

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a prickly pear ferment, characterized in that, The process includes the following steps: mixing and pulping prickly pear, monk fruit, and agastache rugosa to obtain a pulp; mixing the pulp with a compound microbial agent for fermentation to obtain prickly pear fermented product; the compound microbial agent consists of Rhizopus oryzae CLF001 seed liquid, yeast powder, and Lactobacillus plantarum powder, with the volume-to-weight ratio of each raw material in the compound microbial agent being 100mL of Rhizopus oryzae CLF001 seed liquid: 10-15g of yeast powder: 20-25g of Lactobacillus plantarum powder; the Rhizopus oryzae CLF001 seed liquid contains 1-3g of mycelial dry weight per 100mL; the weight ratio of prickly pear, monk fruit, and agastache rugosa is 20:2-3:2-4.

2. The preparation method according to claim 1, characterized in that, The weight ratio of slurry to compound microbial agent is 100:3~5; the fermentation conditions are 25~35℃, 100~150r / min, and fermentation for 20~30h.

3. The prickly pear ferment obtained by the preparation method according to claim 1 or 2.

4. The use of the prickly pear ferment as described in claim 3 in the preparation of any one of the following products, characterized in that, (1) Helps improve sleep; (2) Prevents osteosarcoma; (3) Prevents urticaria.

5. A composition having the effects of improving sleep, preventing osteosarcoma and / or preventing urticaria, characterized in that, Includes the prickly pear ferment as described in claim 3.

6. The composition according to claim 5, characterized in that, The composition also includes Astragalus membranaceus, Codonopsis pilosula, Prunus mume, corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita.

7. The composition according to claim 6, characterized in that, The weight parts of each raw material in the composition are as follows: 30-35 parts of prickly pear ferment, 10-15 parts of astragalus, 13-16 parts of codonopsis, 5-9 parts of dried plum, 6-10 parts of corn silk, 4-8 parts of chrysanthemum, 3-6 parts of licorice, 12-15 parts of pinellia, 16-18 parts of white peony root, and 7-10 parts of yam.

8. A method for preparing the composition according to claim 6 or 7, characterized in that, The process includes the following steps: mixing Astragalus membranaceus, Codonopsis pilosula, and Prunus mume to obtain mixture 1; decocting mixture 1 with water to obtain the decoction; mixing corn silk, chrysanthemum, licorice, Pinellia ternata, Ampelopsis japonica, and Dioscorea opposita to obtain mixture 2; mixing mixture 2 with a 70%~80% ethanol solution and refluxing to obtain the extract; mixing the decoction, the extract, and the prickly pear ferment, and concentrating to a relative density of 1.25~1.

29.

9. The preparation method according to claim 8, characterized in that, The weight-to-volume ratio of the mixture 1 to water is 1g:20~25mL, and the decoction is to boil and then keep boiling for 30~40 minutes.

10. The preparation method according to claim 8, characterized in that, The weight-to-volume ratio of mixture 2 to ethanol solution is 1g:10~15mL; the reflux extraction temperature is 75~85℃, and the reflux extraction time is 1~1.5h.

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