A plant polyphenol complex based on collagen microsphere wrapping and a preparation method and application thereof

Abstract

The present application relates to the technical field of oral care, in particular to a plant polyphenol compound based on collagen microspheres and a preparation method and application thereof.The plant polyphenol compound based on collagen microspheres comprises the following raw material components in percentage by weight: soluble collagen 0.5-6%, crosslinking agent 0.01-0.5%, notoginseng extract 0.1-3%, honeysuckle extract 0.1-3%, glycyrrhetinic acid 0.1-1%, hydroxypropyl-beta-cyclodextrin 0.1-3%, mannitol 1-8%, and the rest is an oral care acceptable auxiliary.The present application realizes the slow release and synergistic effect of plant polyphenols by forming a microsphere structure through collagen self-assembly, has good biocompatibility and significant anti-inflammatory, heat-clearing, cell adhesion and repair promoting functions, and can be stable during the storage period, mucosa resident after brushing, and responsive release in the oral environment.

Description

Technical Field

[0001] This invention relates to the field of oral care technology, specifically to a plant polyphenol complex based on collagen microsphere encapsulation, its preparation method, and its application. Background Technology

[0002] Traditional oral care products face multiple challenges, including the moist oral environment, high microbial load, and frequent mechanical stimulation (chewing, talking). Traditional liquid dressings also struggle to stay effectively on wounds and are easily washed away by saliva. Existing oral care products mainly suffer from the following deficiencies: 1) Difficulty in staying After brushing, the active ingredients are immediately lost with rinsing, failing to form a protective layer on the mucosal surface. The large volume of saliva in the mouth creates a strong rinsing effect, while traditional aqueous solutions or gel formulations lack a mucosal adhesion mechanism, resulting in insufficient retention time for the active ingredients and extremely low bioavailability.

[0003] 2) Uncontrollable release The active ingredients are released instantly, lacking a sustained-release effect. Most existing products use simple dissolution or physical mixing methods, lacking a sustained-release structure. The drug concentration rapidly reaches its peak in the oral cavity and then drops sharply, failing to maintain an effective therapeutic concentration and requiring frequent administration.

[0004] 3) Poor mechanical stability The shear force during brushing can damage the carrier structure, causing premature release of the contents. The high shear rate during brushing makes traditional microcapsules or liposomes prone to rupture under this mechanical action, resulting in leakage of contents, which not only reduces efficacy but also causes unpleasant taste.

[0005] 4) Insufficient biocompatibility Synthetic polymeric carriers (such as polyacrylates and polyvinyl alcohol) may trigger foreign body reactions and are difficult to biodegrade; long-term retention may cause mucosal irritation or fibrosis. Some chemical cross-linking agents (such as glutaraldehyde) residues are cytotoxic and detrimental to postoperative wound healing.

[0006] Furthermore, most plant extracts are currently used alone or in combination, which often faces problems such as poor stability, low solubility, high irritation, and weak synergistic effects. Existing encapsulation technologies mainly use cellulose gum, gelatin, and gum arabic for encapsulation, resulting in a single encapsulation mechanism, passive release mechanism, poor storage stability, and poor oral adaptability. It is difficult to achieve stable storage and responsive release to the oral microenvironment after use. Moreover, when used through rinsing, the active ingredients are immediately lost with water and cannot remain on the oral mucosa; even when using mouthwash, it is difficult to guarantee therapeutic efficacy, and the utilization rate of active ingredients is low. Summary of the Invention

[0007] This invention aims to solve at least one of the above-mentioned technical problems by providing a plant polyphenol complex based on collagen microsphere encapsulation, its preparation method, and its application. The microsphere structure is formed through collagen self-assembly, achieving sustained release and synergistic effects of plant polyphenols. This complex exhibits good biocompatibility and significant anti-inflammatory, heat-clearing, cell adhesion-promoting, and tissue repair-enhancing functions. It is stable during storage, mechanically tolerated during use, retains on the mucosa after brushing, and responds to release in the oral environment, making it suitable for oral care, wound repair, and other fields.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A first aspect of the present invention provides a plant polyphenol complex based on collagen microsphere encapsulation, comprising the following raw material components by weight percentage: Soluble collagen: 0.5–6% Crosslinking agent: 0.01–0.5% Panax notoginseng extract: 0.1-3% Honeysuckle extract: 0.1-3% Glycyrrhetinic acid: 0.1–1% Hydroxypropyl-β-cyclodextrin (HP-β-CD): 0.1–3% Mannitol: 1-8% Acceptable dressings in oral care: excess.

[0009] By adopting the above technical solution, this invention uses collagen as a carrier to self-assemble into microsphere structures and encapsulate plant polyphenols. In the oral environment, the positive charge on the surface of the collagen microspheres allows them to remain in the oral cavity through electrostatic adsorption. The use of a cross-linking agent to micro-crosslink the self-assembled collagen microspheres ensures their stability during storage, preventing premature release of active ingredients. After use, in the oral microenvironment, collagenases in saliva decompose the collagen, causing the self-assembled microspheres to swell and disintegrate, thus slowly releasing the encapsulated active ingredients. This achieves a stimulus-responsive, sustained-release mechanism in the oral microenvironment, extending the effective time for antibacterial and anti-inflammatory effects. The collagen also promotes cell adhesion and wound healing. Through the combination of multiple plant components, a comprehensive synergistic effect of antibacterial, anti-inflammatory, heat-clearing, and repairing effects can be achieved. The combination of plant active ingredients and the stimulus-responsive mechanism allows for synchronization of the release rate with collagen enzymatic cleavage, meaning that the antibacterial, anti-inflammatory, and heat-clearing effects of the drug are synergistically enhanced with the adhesion and repairing effects of collagen in a spatiotemporal manner.

[0010] According to an embodiment of the present invention, the degree of crosslinking of soluble collagen in the complex is 0.5 to 2%, calculated based on the percentage of crosslinked and uncrosslinked collagen amino groups.

[0011] According to an embodiment of the present invention, the soluble collagen includes one or more of bovine Achilles tendon collagen, porcine skin collagen, fish skin collagen, and recombinant humanized collagen.

[0012] Preferably, the soluble collagen is bovine Achilles tendon type I collagen.

[0013] According to an embodiment of the present invention, the number-average molecular weight of the soluble collagen is 30 to 300 kDa.

[0014] According to an embodiment of the present invention, the crosslinking agent includes one or more of transglutaminase (TGase), genipin, epigallocatechin gallate (EGCG), and natural tannins.

[0015] Preferably, the cross-linking agent is transglutaminase.

[0016] According to an embodiment of the present invention, the transglutaminase includes at least one of Streptomyces TGase, soybean TGase, and guinea pig liver TGase; preferably Streptomyces TGase.

[0017] According to an embodiment of the present invention, the Panax notoginseng extract includes at least one of Panax notoginseng root extract or Panax notoginseng leaf extract.

[0018] According to an embodiment of the present invention, the total saponin content in the Panax notoginseng extract is 3-80 wt%.

[0019] According to an embodiment of the present invention, the chlorogenic acid content of the honeysuckle extract is 3-10 wt%.

[0020] According to an embodiment of the present invention, the content of furanocoumarin in the honeysuckle extract is ≤0.001wt%.

[0021] According to an embodiment of the present invention, the molar ratio of glycyrrhetinic acid to hydroxypropyl-β-cyclodextrin is (0.1-5):1.

[0022] According to an embodiment of the present invention, the degree of hydroxypropyl substitution (DS) of the hydroxypropyl-β-cyclodextrin is 0.4 to 0.8.

[0023] According to an embodiment of the present invention, the weight-average molecular weight Mw of the hydroxypropyl-β-cyclodextrin is 1000-2000 Da.

[0024] According to an embodiment of the present invention, the hydroxypropyl-β-cyclodextrin is substituted at least one of 2-OH, 3-OH, and 6-OH.

[0025] According to an embodiment of the present invention, the oral care-acceptable excipients include one or more of the following: flavorings, preservatives, pH adjusters, rheology modifiers, moisturizing agents, abrasives, and deionized water.

[0026] A second aspect of the present invention provides a method for preparing the aforementioned plant polyphenol complex based on collagen microsphere encapsulation, comprising the following steps: Soluble collagen, moisturizing agent and water are mixed to obtain collagen dispersion; Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin were mixed and ground to obtain an inclusion complex; Add Panax notoginseng extract, Lonicera japonica extract and mannitol to the inclusion complex, stir, and obtain an active premix; The collagen dispersion was mixed with the active premix, the pH was adjusted, the mixture was sheared, and a crosslinking agent was added to carry out a crosslinking reaction to obtain a crosslinked microsphere suspension. Add the remaining oral care-acceptable excipients to the cross-linked microsphere suspension, homogenize, and obtain.

[0027] According to an embodiment of the present invention, the grinding temperature is 35–50°C.

[0028] According to an embodiment of the present invention, the grinding time is 1 to 4 hours.

[0029] According to an embodiment of the present invention, the grinding temperature is 35-50°C, and the grinding time is 1-4 hours.

[0030] According to an embodiment of the present invention, the endpoint of the pH adjustment is pH 6.0 to 8.0.

[0031] According to an embodiment of the present invention, the shearing is performed using high-speed shear emulsification, and the shearing rate is 5000 to 20000 rpm.

[0032] According to an embodiment of the present invention, the temperature of the crosslinking reaction is 35-55°C, and the time of the crosslinking reaction is 2-8 hours.

[0033] According to an embodiment of the present invention, the homogenization includes one or more of stirring, shearing, and grinding.

[0034] According to an embodiment of the present invention, the preparation method of the Panax notoginseng extract includes: The Panax notoginseng was crushed, extracted with an aqueous ethanol solution, filtered, concentrated, and dried to obtain the Panax notoginseng extract.

[0035] According to an embodiment of the present invention, the method for preparing the Panax notoginseng extract satisfies at least one of the following conditions: The ethanol concentration of the aqueous ethanol solution is 50-80%. The extraction temperature is 40–70°C; The extraction is performed 1 to 3 times; The drying process is either vacuum drying or spray drying. According to an embodiment of the present invention, the method for preparing the honeysuckle extract includes: Add deionized water to honeysuckle, extract, filter, concentrate, and dry to obtain honeysuckle extract.

[0036] According to an embodiment of the present invention, the method for preparing the honeysuckle extract satisfies at least one of the following conditions: The extraction temperature is 80–100℃.

[0037] The extraction is performed 1 to 3 times.

[0038] The drying process is either vacuum drying or spray drying.

[0039] A third aspect of the present invention provides an application of the aforementioned plant polyphenol complex based on collagen microsphere encapsulation in oral care.

[0040] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0041] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0045] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0046] Unless otherwise specified, the number of parts in this article refers to the number of mass parts.

[0047] A first aspect of the present invention provides a plant polyphenol complex based on collagen microsphere encapsulation, comprising the following raw material components by weight percentage: Soluble collagen: 0.5–6% Crosslinking agent: 0.01–0.5% Panax notoginseng extract: 0.1-3% Honeysuckle extract: 0.1-3% Glycyrrhetinic acid: 0.1–1% Hydroxypropyl-β-cyclodextrin (HP-β-CD): 0.1–3% Mannitol: 1-8% Acceptable dressings in oral care: excess.

[0048] By adopting the above technical solution, this invention uses collagen as a carrier to self-assemble into microsphere structures and encapsulate plant polyphenols. In the oral environment, the positive charge on the surface of the collagen microspheres allows them to remain in the oral cavity through electrostatic adsorption. The use of a cross-linking agent to micro-crosslink the self-assembled collagen microspheres ensures their stability during storage, preventing premature release of active ingredients. After use, in the oral microenvironment, collagenases in saliva decompose the collagen, causing the self-assembled microspheres to swell and disintegrate, thus slowly releasing the encapsulated active ingredients. This achieves a stimulus-responsive, sustained-release mechanism in the oral microenvironment, extending the effective time for antibacterial and anti-inflammatory effects. The collagen also promotes cell adhesion and wound healing. Through the combination of multiple plant components, a comprehensive synergistic effect of antibacterial, anti-inflammatory, heat-clearing, and repairing effects can be achieved. The combination of plant active ingredients and the stimulus-responsive mechanism allows for synchronization of the release rate with collagen enzymatic cleavage, meaning that the antibacterial, anti-inflammatory, and heat-clearing effects of the drug are synergistically enhanced with the adhesion and repairing effects of collagen in a spatiotemporal manner.

[0049] For example, the amount of soluble collagen used can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.5%, 3.0%, 3.2%, 3.5%, 3.8%, 4.0%, 4.2%, 4.5%, 5.0%, 5.5%, 6.0%, etc.

[0050] According to an embodiment of the present invention, the degree of crosslinking of soluble collagen in the complex is 0.5 to 2%, calculated based on the percentage of crosslinked and uncrosslinked collagen amino groups.

[0051] Therefore, the collagen microspheres maintain a solid spherical shape during storage, preventing disintegration. Salivary collagenases in the oral cavity preferentially cleave natural collagen sequences, and the cross-linking points become "stress concentration zones," accelerating network disintegration and achieving controlled release. In other words, the cross-linked microspheres exhibit minimal swelling in water, ensuring stability during storage. After use, they swell or disintegrate under the stimulation of collagenases in the oral cavity, releasing their encapsulated active ingredients. This maintains structural integrity without losing enzyme sensitivity due to excessive cross-linking.

[0052] It should be noted that the present invention does not impose any restrictions on the source and extraction process of the soluble collagen. For example, the source of the soluble collagen can be animal-derived or recombinant-derived; the extraction process can be enzymatic (pepsin / trypsin) extraction, acid extraction, etc.

[0053] According to an embodiment of the present invention, the soluble collagen includes one or more of bovine Achilles tendon collagen, porcine skin collagen, fish skin collagen, and recombinant humanized collagen.

[0054] Preferably, the soluble collagen is bovine Achilles tendon type I collagen. Therefore, the collagen has high triple helix integrity and high cross-linking efficiency, which is beneficial for improving the storage stability of the complex and its antibacterial, anti-inflammatory, heat-clearing, and repairing effects.

[0055] According to an embodiment of the present invention, the number-average molecular weight of the soluble collagen is 30–300 kDa, specifically 30 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, etc. This facilitates a balance between the processability and strength of the collagen, improving storage stability while retaining its rapid release capability.

[0056] For example, the amount of the crosslinking agent can be 0.01%, 0.02%, 0.03%, 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc.

[0057] According to an embodiment of the present invention, the crosslinking agent includes one or more of transglutaminase (TGase), genipin, epigallocatechin gallate (EGCG), and natural tannins.

[0058] By adopting the above technical solution, the cross-linking agent can catalyze or directly cross-link with collagen molecules to form a three-dimensional network. The three-dimensional network can maintain limited swelling in the aqueous toothpaste matrix, avoiding premature release of the active ingredients encapsulated in the collagen; after entering the oral cavity, salivary collagenase can recognize and cleave the natural collagen sequence, and the cross-linked network gradually disintegrates, realizing the intelligent behavior of "storage stability - enzyme response release".

[0059] Preferably, the cross-linking agent is transglutaminase.

[0060] By adopting the above technical solution, the transglutaminase can catalyze the formation of heteropeptide bonds (Gln-Lys crosslinking) between collagen molecules, and the soluble collagen can serve as a microsphere framework material, providing a triple helix structure and giving the self-assembled microspheres mechanical strength; it provides cell adhesion sites and promotes oral mucosal repair; its modified positively charged surface can also achieve electrostatic residence, which is beneficial to comprehensively improve the antibacterial, anti-inflammatory, heat-clearing and repairing effects of the complex.

[0061] It should be noted that the present invention does not limit the source of the transglutaminase, for example, it can be of microbial, plant or animal origin.

[0062] According to an embodiment of the present invention, the transglutaminase comprises at least one of Streptomyces TGase, soybean TGase, and guinea pig liver TGase; preferably Streptomyces TGase. Therefore, Ca... 2+ Non-dependent transglutaminase is beneficial in avoiding calcium deficiency. 2+ The effect on toothpaste stability.

[0063] For example, the dosage of the Panax notoginseng extract can be 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 2.8%, 3.0%, etc.

[0064] It should be noted that the Panax notoginseng extract of the present invention is rich in ginsenosides Rg1, Rb1, Rd and other components. Rg1 can quickly insert into the hydrophobic microdomains on the collagen surface, providing instant mucosal adhesion; Rb1 can penetrate deep into the triple helix of collagen to form a stable complex and delay release.

[0065] According to an embodiment of the present invention, the Panax notoginseng extract includes at least one of Panax notoginseng root extract or Panax notoginseng leaf extract.

[0066] According to an embodiment of the present invention, the total saponin content in the Panax notoginseng extract is 3-80 wt%, specifically such as 3 wt%, 5 wt%, 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, etc.

[0067] The total saponin content can be detected by high performance liquid chromatography (HPLC).

[0068] It should be noted that the present invention does not limit the extraction method of the Panax notoginseng extract, such as water extraction, ethanol extraction, supercritical CO2 extraction or macroporous resin purification, etc.

[0069] For example, the amount of honeysuckle extract can be 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 2.8%, 3.0%, etc.

[0070] It should be noted that the honeysuckle extract of the present invention is rich in chlorogenic acid (3-O-caffeoylquinic acid). The catechol group in its structure can form hydrogen bonds with collagen amino groups during storage to help stabilize microspheres. In the oral environment, it can be oxidized to generate o-benzoquinone, which forms a covalent anchor with mucosal epithelial cells, which is beneficial to prolonging the residence time. It can also form a pre-assembled complex with glycyrrhetinic acid through π-π stacking to regulate release kinetics.

[0071] By adopting the above technical solution, when in use, Rg1 in the Panax notoginseng extract can be rapidly released to promote local microcirculation, while Rb1 can be slowly released for continuous anti-inflammatory and repair effects; chlorogenic acid in the honeysuckle extract can be oxidized and cross-linked on the surface of collagen microspheres to form an outer anchoring layer; the Panax notoginseng extract focuses on promoting repair, while the honeysuckle extract focuses on antibacterial and anti-inflammatory effects, covering the entire healing process and having a synergistic effect.

[0072] According to an embodiment of the present invention, the chlorogenic acid content of the honeysuckle extract is 3-10 wt%, specifically 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, etc.

[0073] According to an embodiment of the present invention, the content of furanocoumarin in the honeysuckle extract is ≤0.001wt%.

[0074] It should be noted that the present invention does not limit the extraction method of the honeysuckle extract, such as water extraction, ethanol extraction, supercritical CO2 extraction or macroporous resin purification, etc.

[0075] For example, the amount of glycyrrhetinic acid can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc.

[0076] It should be noted that the glycyrrhetinic acid described in this invention has a pentacyclic triterpenoid structure and can achieve anti-inflammatory activity by inhibiting the 11β-HSD2 and NF-κB pathways; however, it is highly hydrophobic and has extremely low solubility in water, so it is necessary to increase its content in water-based toothpaste by encapsulation.

[0077] The chlorogenic acid in the honeysuckle extract can exert a broad-spectrum antibacterial effect by inhibiting bacterial protein synthesis and disrupting cell membranes; the glycyrrhetinic acid can have significant activity against Gram-positive bacteria by inhibiting cell wall synthesis; the Panax notoginseng extract can assist in antibacterial action by disrupting membrane structure and interfering with energy metabolism; the combination of the three achieves a broad-spectrum, mild, and comprehensive oral antibacterial effect, with a synergistic effect, while avoiding the drug resistance and irritation of traditional antibacterial agents.

[0078] For example, the amount of hydroxypropyl-β-cyclodextrin can be 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 2.8%, 3.0%, etc.

[0079] Therefore, the hydrophobic cavity of the hydroxypropyl-β-cyclodextrin of the present invention can accommodate the glycyrrhetinic acid steroid skeleton, thereby forming a 1:1 inclusion complex with glycyrrhetinic acid, which can significantly improve the apparent solubility of glycyrrhetinic acid.

[0080] By adopting the above technical solution, the HP-β-CD can disperse glycyrrhetinic acid in a hydrophilic collagen network, significantly increasing the apparent solubility of glycyrrhetinic acid in an aqueous system and preventing its aggregation and precipitation. The inclusion complex formed by the two has moderate stability and can slowly dissociate after the microspheres disintegrate. This slow dissociation may be due to the multiple effects of dynamic equilibrium characteristics, diffusion resistance, competitive substitution, and amphiphilic traps, which can synergize with the microsphere enzymatic hydrolysis process to achieve true "responsive sustained release," which is beneficial to prolonging the anti-inflammatory effect time. At the same time, the cyclodextrin cavity can also shield the hydrophobic surface of glycyrrhetinic acid, which is beneficial to reduce mucosal irritation.

[0081] According to an embodiment of the present invention, the molar ratio of glycyrrhetinic acid to hydroxypropyl-β-cyclodextrin is (0.1-5):1.

[0082] According to an embodiment of the present invention, the degree of hydroxypropyl substitution (DS) of the hydroxypropyl-β-cyclodextrin is 0.4 to 0.8, specifically 0.4, 0.5, 0.6, 0.7, 0.8, etc. This improves its water solubility and inclusion capacity.

[0083] According to an embodiment of the present invention, the weight-average molecular weight Mw of the hydroxypropyl-β-cyclodextrin is 1000-2000 Da, specifically 1000 Da, 1200 Da, 1500 Da, 1700 Da, 2000 Da, etc.

[0084] According to an embodiment of the present invention, the hydroxypropyl-β-cyclodextrin is substituted at least one of 2-OH, 3-OH, and 6-OH.

[0085] For example, the amount of mannitol used can be 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, etc.

[0086] By employing the above technical solution, mannitol can form an amorphous glassy matrix, stabilizing the collagen microsphere structure. Upon contact with saliva, the mannitol dissolves rapidly, generating an osmotic pressure gradient that accelerates water entry into the microspheres, promoting swelling and release. Furthermore, the hydroxyl groups of mannitol can form multi-point hydrogen bonds with collagen side chains and saponin sugar chains, enhancing the mechanical strength of the microspheres. Therefore, the cross-linking of mannitol with transglutaminase forms a dual "physical-chemical" stable network, significantly improving storage stability and enabling rapid release within the oral microenvironment.

[0087] According to an embodiment of the present invention, the oral care-acceptable excipients include one or more of the following: flavorings, preservatives, pH adjusters, rheology modifiers, moisturizing agents, abrasives, and deionized water.

[0088] According to an embodiment of the present invention, the amount of the moisturizing agent is 15-35%.

[0089] According to an embodiment of the present invention, the moisturizing agent includes one or more of glycerin, sorbitol, mannitol, propylene glycol, and polyethylene glycol.

[0090] According to an embodiment of the present invention, the amount of the friction agent is 10-25%.

[0091] According to an embodiment of the present invention, the amount of the thickener is 1 to 1.5%.

[0092] According to an embodiment of the present invention, the amount of the preservative is 0.1% to 1%.

[0093] According to an embodiment of the present invention, the amount of water used is 10-35%.

[0094] A second aspect of the present invention provides a method for preparing the aforementioned plant polyphenol complex based on collagen microsphere encapsulation, comprising the following steps: Soluble collagen, moisturizing agent and water are mixed to obtain collagen dispersion; Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin were mixed and ground to obtain an inclusion complex; Add Panax notoginseng extract, Lonicera japonica extract and mannitol to the inclusion complex, stir, and obtain an active premix; The collagen dispersion was mixed with the active premix, the pH was adjusted, the mixture was sheared, and a crosslinking agent was added to carry out a crosslinking reaction to obtain a crosslinked microsphere suspension. Add the remaining oral care-acceptable excipients to the cross-linked microsphere suspension, homogenize, and obtain.

[0095] According to an embodiment of the present invention, the grinding temperature is 35–50°C.

[0096] According to an embodiment of the present invention, the grinding time is 1 to 4 hours.

[0097] According to an embodiment of the present invention, the grinding temperature is 35-50°C, and the grinding time is 1-4 hours.

[0098] According to an embodiment of the present invention, the endpoint of the pH adjustment is pH 6.0 to 8.0.

[0099] According to an embodiment of the present invention, the shearing is performed using high-speed shear emulsification, and the shearing rate is 5000 to 20000 rpm.

[0100] According to an embodiment of the present invention, the temperature of the crosslinking reaction is 35-55°C, and the time of the crosslinking reaction is 2-8 hours.

[0101] According to an embodiment of the present invention, the homogenization includes one or more of stirring, shearing, and grinding.

[0102] According to an embodiment of the present invention, the preparation method of the Panax notoginseng extract includes: The Panax notoginseng was crushed, extracted with an aqueous ethanol solution, filtered, concentrated, and dried to obtain the Panax notoginseng extract.

[0103] According to an embodiment of the present invention, the method for preparing the Panax notoginseng extract satisfies at least one of the following conditions: The ethanol concentration of the aqueous ethanol solution is 50-80%. The extraction temperature is 40–70°C; The extraction is performed 1 to 3 times; The drying process is either vacuum drying or spray drying. According to an embodiment of the present invention, the method for preparing the honeysuckle extract includes: Add deionized water to honeysuckle, extract, filter, concentrate, and dry to obtain honeysuckle extract.

[0104] According to an embodiment of the present invention, the method for preparing the honeysuckle extract satisfies at least one of the following conditions: The extraction temperature is 80–100℃.

[0105] The extraction is performed 1 to 3 times.

[0106] The drying process is either vacuum drying or spray drying.

[0107] A third aspect of the present invention provides an application of the aforementioned plant polyphenol complex based on collagen microsphere encapsulation in oral care.

[0108] According to an embodiment of the present invention, the oral care includes at least one of postoperative oral wound care, daily oral care, and mucosal repair.

[0109] According to an embodiment of the present invention, the postoperative oral wound care includes at least one of tooth extraction, dental implantation, and periodontal surgery wound protection.

[0110] According to an embodiment of the present invention, the daily oral care includes at least one of toothpaste, mouthwash, and oral gel.

[0111] According to an embodiment of the present invention, the mucosal repair includes at least one of the adjunctive treatments for oral ulcers, erosions, and burning mouth syndrome.

[0112] This invention does not restrict the use of the Panax notoginseng extract or honeysuckle extract. For example, the extract powder can be used directly, or the extract can be dissolved in a corresponding solvent to obtain an extract solution. The amount of the corresponding extract solution can then be calculated based on the solid content of the extract before use.

[0113] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0114] Example 1 A plant polyphenol complex based on collagen microsphere encapsulation, comprising the following raw material components by weight percentage: Enzymatic extraction of type I collagen (Mn=100kDa) from bovine Achilles tendon: 4.0% Transglutaminase (from Streptomyces mobara) 0.2% Panax notoginseng extract (total saponins approximately 30 wt%) 2.0% | Honeysuckle extract (approximately 6 wt% chlorogenic acid) 2.0% Glycyrrhetinic acid (purity ≥98%) 0.4% Hydroxypropyl-β-cyclodextrin (DS=0.6) 1.5% Mannitol 5.0% Glycerin 30.0% Sorbitol 25.0% 18.0% silicon dioxide Sodium carboxymethyl cellulose 1.2% Phenoxyethanol / p-hydroxyacetophenone 0.6% Fragrance 1.0% Deionized water balance; The preparation method of the plant polyphenol complex based on collagen microsphere encapsulation includes the following steps: S1. Disperse soluble collagen in a portion of deionized water to obtain a collagen solution; S2. Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin are mixed and ground at 40°C for 2 hours to form an inclusion complex. Panax notoginseng extract, honeysuckle extract and mannitol are added and stirred evenly to obtain the active premix. S3. Mix the collagen solution obtained in S1 with the active premix obtained in S2, adjust the pH to 7.0, shear to form microspheres, add transglutaminase, and cross-link at 45℃ for 3 hours to obtain a cross-linked microsphere suspension. S4. Add glycerol, sorbitol, silica, sodium carboxymethyl cellulose, phenoxyethanol / p-hydroxyacetophenone, fragrance and the balance deionized water to the cross-linked microsphere suspension obtained in S3, homogenize, and obtain the plant polyphenol complex based on collagen microsphere encapsulation. The preparation method of the Panax notoginseng extract includes: The Panax notoginseng was pulverized, and extracted three times at 50°C with 70% ethanol aqueous solution. The mixture was then filtered, concentrated, and dried under reduced pressure to obtain the Panax notoginseng extract. The preparation method of the honeysuckle extract includes: Add deionized water to honeysuckle, extract three times at 80℃, filter, concentrate, and dry under reduced pressure to obtain honeysuckle extract.

[0115] Example 2 A plant polyphenol complex based on collagen microsphere encapsulation, comprising the following raw material components by weight percentage: Enzymatic extraction of fish skin collagen (Mn=150kDa) 5.0% Transglutaminase (from Streptomyces mobara) 0.4% Panax notoginseng extract (total saponins approximately 30 wt%) 1.5% | Honeysuckle extract (approximately 6 wt% chlorogenic acid) 3.0% Glycyrrhetinic acid (purity ≥98%) 0.6% Hydroxypropyl-β-cyclodextrin (DS=0.5) 3.0% Mannitol 6.0% Glycerin 25.0% Sorbitol 20.0% 15.0% silicon dioxide Sodium carboxymethyl cellulose 1.0% Phenoxyethanol / p-hydroxyacetophenone 0.5% Fragrance 0.8% Deionized water balance; The preparation method of the plant polyphenol complex based on collagen microsphere encapsulation includes the following steps: S1. Disperse soluble collagen in a portion of deionized water to obtain a collagen solution; S2. Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin are mixed and ground at 35°C for 2 hours to form an inclusion complex. Panax notoginseng extract, honeysuckle extract and mannitol are added and stirred evenly to obtain the active premix. S3. Mix the collagen solution obtained in S1 with the active premix obtained in S2, adjust the pH to 7.0, shear to form microspheres, add transglutaminase, and cross-link at 45℃ for 2.5h to obtain a cross-linked microsphere suspension. S4. Add glycerol, sorbitol, silica, sodium carboxymethyl cellulose, phenoxyethanol / p-hydroxyacetophenone, fragrance and the balance deionized water to the cross-linked microsphere suspension obtained in S3, homogenize, and obtain a plant polyphenol complex based on collagen microsphere encapsulation. The preparation methods of Panax notoginseng extract and honeysuckle extract in this embodiment are the same as in Embodiment 1.

[0116] Example 3 A plant polyphenol complex based on collagen microsphere encapsulation, comprising the following raw material components by weight percentage: Recombinant humanized type I collagen (RHCP-I) (Mn=50kDa) 3.5% Transglutaminase (from Streptomyces mobara) 0.4% Panax notoginseng extract (total saponins approximately 30wt%) 2.5% | Honeysuckle extract (approximately 6 wt% chlorogenic acid) 2.5% Glycyrrhetinic acid (purity ≥98%) 0.8% Hydroxypropyl-β-cyclodextrin (DS=0.4) 2.0% Mannitol 4.0% Glycerin 20.0% Sorbitol 25.0% 20.0% silicon dioxide Sodium carboxymethyl cellulose 1.0% Phenoxyethanol / p-hydroxyacetophenone 0.5% Fragrance 0.8% Deionized water balance; The preparation method of the plant polyphenol complex based on collagen microsphere encapsulation includes the following steps: S1. Disperse soluble collagen in a portion of deionized water to obtain a collagen solution; S2. Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin are mixed and ground at 45°C for 2 hours to form an inclusion complex. Panax notoginseng extract, honeysuckle extract and mannitol are added and stirred evenly to obtain the active premix. S3. Mix the collagen solution obtained in S1 with the active premix obtained in S2, adjust the pH to 7.0, shear to form microspheres, add transglutaminase, and cross-link at 45℃ for 3 hours to obtain a cross-linked microsphere suspension. S4. Add glycerol, sorbitol, silica, sodium carboxymethyl cellulose, phenoxyethanol / p-hydroxyacetophenone, fragrance and the balance deionized water to the cross-linked microsphere suspension obtained in S3, homogenize, and obtain a plant polyphenol complex based on collagen microsphere encapsulation. The preparation methods of Panax notoginseng extract and honeysuckle extract in this embodiment are the same as in Embodiment 1.

[0117] Example 4 This embodiment is based on Embodiment 1, and the only difference from Embodiment 1 is that: in this embodiment, an equal amount of EGCG is used instead of transglutaminase in Embodiment 1.

[0118] Comparative Example 1 This comparative example is based on Example 1, except that an equal amount of Panax notoginseng extract is added to replace the honeysuckle extract in Example 1.

[0119] Comparative Example 2 This comparative example is based on Example 1, except that an equal amount of honeysuckle extract is added to replace the Panax notoginseng extract in Example 1.

[0120] Comparative Example 3 This comparative example is based on Example 1, except that an equal amount of hydroxypropyl-β-cyclodextrin is added to replace glycyrrhetinic acid in Example 1.

[0121] Comparative Example 4 This comparative example is based on Example 1, and differs from Example 1 only in that: an equal amount of glycyrrhetinic acid is added to replace the hydroxypropyl-β-cyclodextrin in Example 1.

[0122] Effect test Test Example 1: Storage Stability Test The composites of the examples and comparative examples were placed in a sealed, light-proof environment at 40°C and 75% RH for 90 days for accelerated aging. Subsequently, the composition was observed by the naked eye for oil separation and sedimentation. The pH was measured with a pH meter to characterize whether the pH shift occurred, etc., and the results are recorded in Table 1.

[0123] Table 1 Stability test results ; As can be seen from the results in Table 1, Examples 1-4 of the present invention showed good storage stability in terms of visual observation and pH drift. Comparative Example 4 showed only slight sedimentation visible to the naked eye, indicating that hydroxypropyl-β-cyclodextrin and glycyrrhetinic acid can form an inclusion structure, which effectively improves the stability of the complex.

[0124] Test Example 2: Stimulus-Response Release Test Take 1g of the complexes from Examples 1-4 and Comparative Examples 1-4 respectively, preheat at 37°C for 1 min, add 10ml of artificial saliva with pH 7.0, and maintain the temperature in a water bath shaker at 37°C for 12 h; the artificial saliva contains 6.4mM NaCl, 3.6mM KCl, 1.5mM CaCl2·2H2O, 0.5mM NaHCO3, and 100 μL of... -1 An aqueous solution of α-amylase and 50 μg / mL collagenase was prepared. Samples were then taken and the contents of Panax notoginseng extract, Lonicera japonica extract, and glycyrrhetinic acid before and after release were measured using high-performance liquid chromatography-ultraviolet (HPLC-UV) technology. The release rates were calculated, and the results are recorded in Table 2. Table 2 Results of stimulus-responsive release test ; As shown in Table 2, the complexes of Examples 1-4 of the present invention can all undergo stimulus-responsive release in the oral microenvironment containing collagenase. At 12 hours, the release rates of Panax notoginseng extract, Lonicera japonica extract, and glycyrrhetinic acid were all higher than 65%. This indicates that the complexes of the present invention have the ability to be controlled and released sustainably in the oral microenvironment containing collagenase. Among them, Example 4... -The release rate was significantly lower than that in Example 1, indicating that the complex using transglutaminase had a faster release rate than the complex using EGCG.

[0125] Test Example 3: Cell Proliferation Test Human gingival fibroblasts (HGF-1) were used at a concentration of 5 × 10⁻⁶. 3 / wells were seeded into 96-well plates and cultured for 24 h; culture medium containing the complexes from Examples 1-4 and Comparative Examples 1-4 (100 μL / well) was added, with 5 replicates; after 24 h of culture, 20 μL of LTT solution (5 mg / mL) was added to each well, and cultured for another 4 h; the supernatant was discarded, and 150 μL of DMSO was added, and the mixture was shaken for 10 min to dissolve the crystals; the absorbance at 570 nm was measured using a microplate reader (the cell-free sample was used as the blank group, and the sample containing cells cultured only with culture medium without any complex was used as the control group), and the cell proliferation rate was calculated according to the following formula and recorded in Table 3: The cell proliferation rate (%) = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance A570) × 100% Test Example 4: Scratch Test HGF-1 at 2×10 5 / wells were seeded into 6-well plates and cultured until monolayer confluence; vertical scratches were made using a 200 μL pipette tip, detached cells were washed away with PBS, and the scratches at 0 h were photographed and recorded; culture medium containing the aforementioned complex was added, and after 24 h of culture, the scratches were photographed again, and the scratch area before and after culture was measured using ImageJ software. The scratch healing rate was calculated according to the following formula and recorded in Table 3: The scratch healing rate (%) = (A0 - A) 24 ) / A0 × 100% Where A0 is the scratch area at time 0h; A 24 The area of ​​the scratch is 24 hours later.

[0126] Test Example 5: Anti-inflammatory Performance Test RAW264.7 cells were grown at a rate of 1×10⁻⁶. 5 Cells were seeded in 24-well plates and cultured for 24 h. An inflammation model was established by stimulation with 1 μg / mL lipopolysaccharide (LPS) for 4 h. Culture medium containing the complex was added and the cells were treated for 24 h. The supernatant was collected and processed according to the ELISA kit instructions. Cells were measured using a microplate reader, and the concentrations of inflammatory factors TNF-α and IL-6 were quantified using a standard curve, and the inhibition rate was calculated. The results are recorded in Table 3. Samples stimulated with LPS and then added to normal culture medium, without the complex, served as the model group. Cells cultured in normal culture medium, without LPS or the complex, served as the blank group.

[0127] The formula for calculating the inhibition rate is: Inhibition rate (%) = (Model group concentration - Experimental group concentration) / (Model group concentration - Blank group concentration) × 100% 1) Use SEM cross-sectional counting to test and calculate the dentinal tubule occlusion rate; 2) Optical coherence tomography (OCT) was used to test the demineralized area and calculate the demineralized area reduction rate; 3) Test the hardness of the model using a microhardness tester and calculate the hardness recovery rate.

[0128] Test Example 6: Antibacterial Effect Test The complexes of Examples 1-4 and Comparative Examples 1-4 were contacted with a bacterial suspension of Streptococcus mutans ATCC 25175 for 24 hours. The antibacterial rate of the samples was calculated by measuring metabolic inhibition using XTT. Five parallel experiments were conducted for each sample, and the average value was recorded in Table 3.

[0129] Table 3 Cell test results ; As shown in Table 3, the cell proliferation rate, scratch healing rate, TNF-α inhibition rate, IL-6 inhibition rate, and antibacterial rate of Examples 1-4 of the present invention are significantly higher than those of the comparative example, indicating that the complex described in the present invention can significantly improve migration, repair, anti-inflammatory and antibacterial effects. Among them, the cell proliferation rate, scratch healing rate, TNF-α inhibition rate, IL-6 inhibition rate, and antibacterial rate of Example 4 are significantly lower than those of Example 1, indicating that the complex using transglutaminase has better migration, repair, anti-inflammatory and antibacterial effects than the complex using EGCG, producing unexpected technical effects. Compared with Example 1, the cell proliferation rate, scratch healing rate, TNF-α inhibition rate, IL-6 inhibition rate, and antibacterial rate of Comparative Examples 1-4 were significantly reduced. This indicates that Panax notoginseng extract and honeysuckle extract have synergistic repair and anti-inflammatory effects; cyclodextrin can improve the solubility of glycyrrhetinic acid, thereby enhancing the repair and anti-inflammatory effects of the complex; Panax notoginseng extract, honeysuckle extract, and glycyrrhetinic acid have synergistic antibacterial effects, and none of them can be omitted.

[0130] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A plant polyphenol complex based on collagen microsphere encapsulation, characterized in that, By weight percentage, it includes the following raw material components: Soluble collagen: 0.5–6% Crosslinking agent: 0.01–0.5% Panax notoginseng extract: 0.1-3% Honeysuckle extract: 0.1-3% Glycyrrhetinic acid: 0.1–1% Hydroxypropyl-β-cyclodextrin: 0.1–3% Mannitol: 1-8% Acceptable dressings in oral care: excess.

2. The plant polyphenol complex according to claim 1, characterized in that, At least one of the following conditions must be met: The degree of cross-linking of the soluble collagen in the complex is 0.5%–2%; The soluble collagen includes one or more of bovine Achilles tendon collagen, porcine skin collagen, fish skin collagen, and recombinant humanized collagen; The number-average molecular weight of the soluble collagen is 30–300 kDa.

3. The plant polyphenol complex according to claim 2, characterized in that, The cross-linking agent includes one or more of transglutaminase, genipin, epigallocatechin gallate, and natural tannins.

4. The plant polyphenol complex according to claim 3, characterized in that, The Panax notoginseng extract must meet at least one of the following conditions: the Panax notoginseng extract includes at least one of Panax notoginseng root extract or Panax notoginseng leaf extract; The total saponin content in the Panax notoginseng extract is 3-80 wt%.

5. The plant polyphenol complex according to claim 3, characterized in that, At least one of the following conditions must be met: The chlorogenic acid content of the honeysuckle extract is 3-10 wt%. The content of furanocoumarin in the honeysuckle extract is ≤0.001wt%.

6. The plant polyphenol complex according to claim 3, characterized in that, At least one of the following conditions must be met: The degree of hydroxypropyl substitution of the hydroxypropyl-β-cyclodextrin is 0.4 to 0.8; The weight-average molecular weight (Mw) of the hydroxypropyl-β-cyclodextrin is 1000–2000 Da; The hydroxypropyl-β-cyclodextrin is substituted at least one of 2-OH, 3-OH, and 6-OH. The molar ratio of glycyrrhetinic acid to hydroxypropyl-β-cyclodextrin is (0.1-5):

1.

7. The plant polyphenol complex according to any one of claims 1 to 6, characterized in that, The oral care-acceptable excipients include one or more of the following: flavorings, preservatives, pH adjusters, rheology modifiers, moisturizing agents, abrasives, and deionized water.

8. A method for preparing a plant polyphenol complex as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Soluble collagen, moisturizing agent and water are mixed to obtain collagen dispersion; Glycyrrhetinic acid and hydroxypropyl-β-cyclodextrin were mixed and ground to obtain an inclusion complex; Add Panax notoginseng extract, Lonicera japonica extract and mannitol to the inclusion complex, stir, and obtain an active premix; The collagen dispersion was mixed with the active premix, the pH was adjusted, the mixture was sheared, and a crosslinking agent was added to carry out a crosslinking reaction to obtain a crosslinked microsphere suspension. Add the remaining oral care-acceptable excipients to the cross-linked microsphere suspension, homogenize, and obtain.

9. The preparation method according to claim 8, characterized in that, At least one of the following conditions must be met: The grinding temperature is 35–50°C, and the grinding time is 1–4 hours. The endpoint for adjusting the pH is pH 6.0–8.0; The shearing process employs high-speed shear emulsification, with a shearing rate of 5000–20000 rpm. The cross-linking reaction is carried out at a temperature of 35–55°C for 2–8 hours.

10. The application of a plant polyphenol complex as described in any one of claims 1 to 7 or a plant polyphenol complex prepared by the preparation method described in claim 8 or 9 in oral care.