Periodontitis improving compound preparation containing lactobacillus mucilaginosus and traditional Chinese medicine extract
By using a compound preparation of fermented Lactobacillus mucinus and traditional Chinese medicine extracts, multiple needs in the treatment of periodontitis are addressed, achieving synergistic effects of antibacterial, immune regulation and tissue repair. This overcomes the limitations of traditional antibiotics and single probiotics, and provides a treatment plan that combines long-lasting effects with synergistic systemic and local intervention.
Patent Information
- Application Number
- CN202511655554.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-02-17
AI Technical Summary
Existing technologies cannot simultaneously address the multiple needs in periodontitis treatment, such as antibacterial effectiveness, immune inflammation regulation, tissue repair promotion, and long-term drug retention. Traditional antibiotics suffer from problems such as drug resistance, disruption of the microecological balance, and single target. Single probiotics and traditional Chinese medicine extracts also have limitations in the treatment of periodontitis.
This product utilizes a compound preparation of fermented Lactobacillus mucinus and traditional Chinese medicine extracts. It is administered orally and as a gel. Fermented Lactobacillus mucinus competitively excludes and immunomodulates pathogenic bacteria, while the traditional Chinese medicine extracts directly destroy the bacterial biofilm and inhibit inflammation. The two work synergistically to achieve systemic regulation and rapid local extinguishing of inflammation, and promote the regeneration of periodontal ligament collagen fibers.
It significantly enhances antibacterial efficacy, regulates Th1/Th2 immune imbalance, reduces pro-inflammatory factors, increases anti-inflammatory factors, promotes alveolar bone resorption inhibition, improves microcirculation, achieves long-lasting retention, and synergistically intervenes in periodontitis, with effects superior to single technologies.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to a compound preparation for improving periodontitis comprising fermented Lactobacillus mucinus and extracts of traditional Chinese medicine. Background Technology
[0002] Periodontitis is a chronic inflammatory disease initiated by plaque biofilm and closely related to the host's immune response. It is a leading cause of tooth loss in adults. Its pathological features include gingival inflammation, periodontal pocket formation, progressive alveolar bone resorption, and attachment loss. In addition to causing local tissue destruction, periodontitis, as a chronic focus of infection, also has a significant bidirectional association with various systemic diseases (such as diabetes, cardiovascular disease, and rheumatoid arthritis), posing a serious threat to public health.
[0003] Currently, the basic treatment for periodontitis, namely basic periodontal treatment, mainly involves removing plaque and calculus through mechanical debridement (such as supragingival scaling, subgingival scaling, and root planing) to control infection. However, this method has limitations: firstly, for deep periodontal pockets and complex furcation lesions, instruments are difficult to completely remove pathogens; secondly, after mechanical debridement, residual pathogens can quickly reclamate in the periodontal pockets, leading to disease recurrence.
[0004] Therefore, antibiotics are often used as an adjunct therapy in clinical practice, with antibiotics being the most widely used adjunctive treatment. Whether administered systemically or as a local sustained-release formulation (such as metronidazole gel or minocycline ointment), their core mechanism of action is to directly kill or inhibit periodontal pathogens (such as Porphyromonas gingivalis, Forsythori, and Treponema denticulatum). Although antibiotics are effective in the short term, long-term use has revealed many serious problems: Bacterial resistance: The overuse and long-term use of antibiotics have led to the emergence of drug-resistant strains of periodontal pathogens, causing traditional antibiotics to become less effective or even ineffective.
[0005] Disruption of the oral microecological balance: While broad-spectrum antibiotics inhibit pathogenic bacteria, they also indiscriminately kill beneficial symbiotic bacteria in the oral cavity, disrupting the balance of the oral microecology and potentially creating conditions for the growth of opportunistic pathogens.
[0006] Single target of action: Antibiotics mainly exert their antibacterial effects, but their ability to intervene in the crucial "excessive host immune inflammatory response" and "tissue destruction" links in the pathological process of periodontitis is limited. The destruction of periodontal tissues is largely mediated by the abnormal host immune response triggered by plaque stimulation, and antibacterial drugs alone cannot effectively inhibit the cascade release of inflammatory factors and the activation of osteoclasts.
[0007] Poor local drug retention: Although local sustained-release formulations have improved drug retention time to some extent, existing formulations still cannot maintain an effective drug concentration for a sufficiently long time in the dynamic environment of oral saliva flushing, enzymatic hydrolysis and oral movement, thus affecting efficacy.
[0008] In recent years, probiotic therapy has shown promising potential as a biological intervention in the treatment of periodontitis. Studies have shown that specific probiotic strains (such as Lactobacillus reuteri and Lactobacillus salivarius) can inhibit the growth of periodontal pathogens and alleviate gingival inflammation through mechanisms such as competitive exclusion, production of antimicrobial substances, and regulation of the host immune system. However, single probiotic preparations (especially orally administered) also have limitations: their effects are relatively slow, their ability to directly intervene in the microecological environment within established deep periodontal pockets is weak, and the effects vary greatly among different strains, with stability needing improvement.
[0009] On the other hand, traditional Chinese medicine extracts, due to their multi-component and multi-target characteristics, possess unique advantages in anti-inflammatory, antibacterial, antioxidant, and tissue repair-promoting effects. For example, Scutellaria baicalensis, Lonicera japonica, and Salvia miltiorrhiza have been proven to inhibit periodontal pathogens, reduce periodontal tissue inflammation, and regulate immunity. However, the application of traditional Chinese medicine extracts in the treatment of periodontitis also faces challenges, mainly due to the solubility and stability of the active ingredients, as well as insufficient retention time and targeting at the lesion site.
[0010] In summary, current technologies, whether traditional antibiotics, single probiotics, or traditional Chinese medicine extracts, struggle to simultaneously address the multiple needs in periodontitis treatment, including antibacterial effectiveness, immune inflammation regulation, tissue repair promotion, and long-lasting drug retention. Therefore, there is an urgent need to develop a novel composite formulation that integrates the advantages of different treatment strategies, overcomes the shortcomings of existing technologies, and provides a safer, more efficient, and longer-lasting comprehensive treatment option for periodontitis. Summary of the Invention
[0011] In view of this, the present invention proposes a compound preparation for improving periodontitis containing fermented Lactobacillus mucinus and traditional Chinese medicine extracts, aiming to solve the problem that current periodontitis treatment preparations are unable to meet multiple requirements such as antibacterial efficacy, immune inflammation regulation, tissue repair promotion, and long-term drug retention.
[0012] This invention proposes a compound preparation for improving periodontitis containing fermented Lactobacillus mucinus and extracts of traditional Chinese medicine, comprising the following raw materials: Oral dosage form: Fermented Lactobacillus mucinus CQPC202403 bacterial powder; Gel agent: Scutellaria baicalensis, Lonicera japonica, Salvia miltiorrhiza, Glycyrrhiza uralensis, hydrogel matrix; The fermenting Lactobacillus flavus CQPC202403 has the accession number CGMCC NO.31106 at the China General Microbiological Culture Collection Center, the accession date is June 27, 2024, and the classification name is Limosilactobacillus fermentum.
[0013] Preferably, the viable count of *Lactobacillus fermentum* CQPC202403 powder in the oral preparation is ≥1.0 × 10⁻⁶. 9 CFU / g.
[0014] Preferably, the method for preparing the gelling agent includes the following steps: 1) Scutellaria baicalensis is mixed with rice wine, stir-fried, dried, and crushed to obtain Scutellaria baicalensis powder; Honeysuckle, salvia miltiorrhiza, and licorice were dried and crushed separately to obtain honeysuckle powder, salvia miltiorrhiza powder, and licorice powder. 2) After mixing Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder and Glycyrrhiza uralensis powder, the mixture was extracted using a mixed extractant of ethanol and water; 3) After extraction, the extract is obtained by filtration. The extract is then purified by liquid chromatography, concentrated, and dried to obtain the traditional Chinese medicine extract. 4) The hydrogel matrices poloxamer 407 and poloxamer 188 were dispersed in glycerol, and then water was added to swell and obtain a gel. 5) Mix the Chinese herbal extract with the gel to obtain a gel.
[0015] Preferably, in the preparation method of the gelling agent, the particle size of Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder and Glycyrrhiza uralensis powder is 0.2~1mm; The mass ratio of Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder, and Glycyrrhiza uralensis powder is 3:1~2:1~2:1~2.
[0016] Preferably, the extraction method in the preparation method of the gelling agent is to first perform ultrasonic extraction and then microwave extraction; The ultrasonic extraction power is 200~350W, the ultrasonic extraction interval is 3s / 2s, and the ultrasonic extraction time is 10~30min; The microwave extraction power is 400~800W, and the microwave extraction time is 30~40min; The temperature is controlled at 30~45℃ during the extraction process.
[0017] Preferably, the liquid chromatography column used in the preparation method of the gelling agent is an octadecylsilane-bonded silica column; During purification, the sample loading flow rate is 1~2 mL / min, the eluent is a methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 30:70, and the elution flow rate is 1.5~2.5 mL / min; Collect the eluted components with a retention time of 8.5 to 9.5 minutes for the main peak.
[0018] Preferably, in the preparation method of the gelling agent, the mass ratio of poloxamer 407, poloxamer 188, glycerol and water is 15~10:4~8:4:70~80; The mass ratio of Chinese herbal extract to gel is 15~25:75~85.
[0019] Preferably, the oral dosage form further includes a filler and a protective agent; The filler is lactose or microcrystalline cellulose; The protective agent is trehalose.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention combines specific probiotics with a multi-target traditional Chinese medicine compound and utilizes an innovative dosage form design to achieve synergistic and comprehensive intervention for periodontitis, producing significantly better effects than existing single technologies. Traditional antibiotics mainly target a single "antibacterial" target, failing to effectively inhibit excessive host immune inflammatory responses and tissue damage. The effects of single probiotics or traditional Chinese medicines are also relatively limited. However, the fermented *Lactobacillus mucosa* used in this invention inhibits pathogenic bacteria through competitive exclusion and immune regulation; extracts of traditional Chinese medicine such as *Scutellaria baicalensis* directly disrupt the bacterial biofilm. The synergistic effect of these two methods enhances antibacterial efficacy without inducing drug resistance. The probiotics, through oral administration, regulate the "gut-oral axis," correcting the Th1 / Th2 immune imbalance at the systemic level, reducing pro-inflammatory factors IL-6 and TNF-α, and increasing the anti-inflammatory factor IL-10; the local traditional Chinese medicine (*Scutellaria baicalensis*, *Glycyrrhiza uralensis*) directly inhibits inflammatory pathways such as NF-κB, rapidly controlling inflammation within the periodontal pockets. These two methods form a synergistic effect of "systemic regulation" and "rapid local suppression." In addition, fermented Lactobacillus mucinus has the effect of inhibiting alveolar bone resorption; the ingredients of traditional Chinese medicine such as Danshen can improve microcirculation and promote the regeneration of periodontal ligament collagen fibers. Detailed Implementation
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.
[0022] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] This invention provides a compound preparation for improving periodontitis comprising fermented Lactobacillus mucinus and extracts of traditional Chinese medicine, including the following raw materials: Oral dosage form: Fermented Lactobacillus mucinus CQPC202403 bacterial powder; Gel agent: Scutellaria baicalensis, Lonicera japonica, Salvia miltiorrhiza, Glycyrrhiza uralensis, hydrogel matrix; The Lactobacillus fermentum CQPC202403 has the accession number CGMCC NO.31106 from the China General Microbiological Culture Collection Center, and the accession date is June 27, 2024.
[0027] In this invention, the viable count of *Lactobacillus mucinus* CQPC202403 bacterial powder in the oral preparation is ≥1.0 × 10⁻⁶. 9 CFU / g.
[0028] In this invention, the preparation method of the gelling agent includes the following steps: 1) Scutellaria baicalensis is mixed with rice wine, stir-fried, dried, and crushed to obtain Scutellaria baicalensis powder; Honeysuckle, salvia miltiorrhiza, and licorice were dried and crushed separately to obtain honeysuckle powder, salvia miltiorrhiza powder, and licorice powder. 2) After mixing Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder and Glycyrrhiza uralensis powder, the mixture was extracted using a mixed extractant of ethanol and water; 3) After extraction, the extract is obtained by filtration. The extract is then purified by liquid chromatography, concentrated, and dried to obtain the traditional Chinese medicine extract. 4) The hydrogel matrices poloxamer 407 and poloxamer 188 were dispersed in glycerol, and then water was added to swell and obtain a gel. 5) Mix the Chinese herbal extract with the gel to obtain a gel.
[0029] In this invention, the particle size of Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder, and Glycyrrhiza uralensis powder in the preparation method of the gelling agent is 0.2~1mm, preferably 0.3~0.8mm, more preferably 0.4~0.6mm, and even more preferably 0.5mm.
[0030] The mass ratio of Scutellaria baicalensis powder, Lonicera japonica powder, Salvia miltiorrhiza powder, and Glycyrrhiza uralensis powder is 3:1~2:1~2:1~2, preferably 3:1.2~1.8:1.2~1.8:1.2~1.8, more preferably 3:1.4~1.6:1.4~1.6:1.4~1.6, and even more preferably 3:1.5:1.5:1.5.
[0031] In this invention, the gel preparation method involves first performing ultrasonic extraction followed by microwave extraction.
[0032] In this invention, the power of the ultrasonic extraction is 200~350W, preferably 250~320W, more preferably 270~290W, and even more preferably 280W; the interval ratio of ultrasonic extraction is 3s / 2s; the ultrasonic extraction time is 10~30min, preferably 15~25min, more preferably 18~22min, and even more preferably 20min.
[0033] In this invention, the power of microwave extraction is 400~800W, preferably 500~700W, more preferably 580~620W, and even more preferably 600W; the microwave extraction time is 30~40min, preferably 32~38min, more preferably 34~36min, and even more preferably 35min.
[0034] In this invention, the temperature is controlled at 30~45℃ during the extraction process, preferably 35~42℃, more preferably 37~39℃, and even more preferably 38℃.
[0035] In this invention, the liquid chromatography column used in the preparation method of the gelling agent is an octadecylsilane-bonded silica column.
[0036] In this invention, the sample loading flow rate during purification is 1~2 mL / min, preferably 1.2~1.8 mL / min, more preferably 1.4~1.6 mL / min, and even more preferably 1.5 mL / min; the eluent is a methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 30:70, and the elution flow rate is 1.5~2.5 mL / min, preferably 1.7~2.3 mL / min, more preferably 1.9~2.1 mL / min, and even more preferably 2.0 mL / min.
[0037] In this invention, eluted components with a main peak retention time of 8.5 to 9.5 min are collected.
[0038] In this invention, the mass ratio of poloxamer 407, poloxamer 188, glycerol and water in the preparation method of the gelling agent is 10~15:4~8:4:70~80, preferably 11~14:5~7:4:73~77, more preferably 12~13:6~6:4:74~76, and even more preferably 12:6:4:75.
[0039] In this invention, the mass ratio of the herbal extract to the gel is 15~25:75~85, preferably 15~20:80~85, more preferably 15~18:82~85, and even more preferably 15:85.
[0040] In this invention, the oral preparation also includes fillers and protectants.
[0041] In this invention, the filler is lactose or microcrystalline cellulose.
[0042] In this invention, the protective agent is trehalose.
[0043] Example 1 Isolation and purification of Lactobacillus fermentans CQPC202403: The pickled vegetables were made by a family in Nan'an District, Chongqing. After the contents of the pickling jar were thoroughly stirred with a sterile spoon, 50mL of pickling water was drawn into a sterilized centrifuge tube using a sterile syringe. The tube was then placed in a low-temperature food sampling box and brought back to the laboratory for freezing and storage in an ultra-low temperature freezer at -80℃ for later use.
[0044] Take 1 mL of pickling brine sample and serially dilute it 10-fold to 10⁻⁶ with sterile physiological saline. Then, take 100 μL of bacterial suspension at three different gradients (10⁻⁴, 10⁻⁵, and 10⁻⁶) and spread it on agar plates. Incubate at 37℃ for 24–48 h, and observe and record the colony morphology. Pick colonies of different morphologies from the plates and streak them for isolation. After incubating at 37℃ for 48 h, pick single colonies of different morphologies from the plates again and streak them for isolation. Repeat this process multiple times until pure single colonies with consistent morphology are obtained.
[0045] The purified suspected target strain was inoculated into MRS broth and incubated at 37°C for 18–24 h. DNA was then extracted using a bacterial genomic DNA extraction kit. The extracted DNA was numbered and stored at -20°C for later use.
[0046] The extracted DNA was subjected to PCR amplification. The mixture consisted of 1 μL of upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1 μL of downstream primer 1495R (5'-CTACGGCTACCTTGTTACGA-3'), 12.5 μL of 2×Taqplus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile ddH2O. Sterile ultrapure water was used as a negative control instead of template DNA. The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 29 cycles, with a final extension at 72℃ for 5 min.
[0047] The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.
[0048] GeneBank analysis of lactic acid bacteria species The strain was successfully sequenced. BLAST analysis confirmed that the strain is *Lactobacillus fermentans*. The sequencing results are as follows: 1. Preparation of oral dosage forms: The preserved freeze-dried strain of *Lactobacillus fermentum* CQPC202403 was inoculated into MRS anaerobic liquid medium (formula: peptone 15.83 g / L, yeast extract 4.0 g / L, beef extract 5.0 g / L, glucose 30.72 g / L, Tween 80 1.0 mL / L, dipotassium hydrogen phosphate 5.16 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, ammonium citrate 2.0 g / L, pH adjusted to 6.8 ± 0.2), and cultured anaerobicly at 37℃ for 24 h to gradually restore bacterial activity.
[0049] The revived bacterial culture was transferred to a solid slant culture medium and incubated at 37°C for 48 hours to form uniform colonies to ensure purity. The culture was then washed with sterile physiological saline to prepare a bacterial suspension (OD600≥1.2).
[0050] Inoculate the bacterial suspension into the seed tank at an inoculum rate of 2.9%, maintain an anaerobic environment at 37°C, stir at 100 rpm, and incubate for 18 hours until the logarithmic growth phase, with a viable count ≥ 1 × 10⁻⁶. 9 CFU / mL.
[0051] The seed culture was transferred to the fermenter at a ratio of 8%. The culture medium was the same as the MRS anaerobic liquid culture medium formula mentioned above, with an initial pH of 6.8±0.2 and the tank pressure maintained at 0.05MPa.
[0052] The entire culture process was anaerobic (air was replaced with sterile nitrogen gas) using a stepped temperature strategy: 37℃ for the first 24 hours, and 35℃ for the next 24 hours. Carbon source concentration was maintained by pulsed addition of 20% glucose solution, and pH was adjusted to 6.5±0.3 in real time using 25% NaOH. Dissolved oxygen saturation was controlled at ≤5%, and the stirring speed was 150 rpm to avoid cell damage. After 48 hours of culture, when the OD600 value was ≥3.5 and the viable cell count was ≥1×10⁻⁶, the culture was considered complete. 10 Fermentation was terminated when CFU / mL was reached.
[0053] After fermentation, the fermentation broth was processed by a disc centrifuge at 4°C and 12,000 r / min for 10 min. The bacterial precipitate was collected, and the supernatant containing metabolic waste was removed.
[0054] The bacteria were washed three times by centrifugation with sterile phosphate buffer (pH 7.0). After each wash, the bacterial cell recovery rate was ≥90% and the detection rate of contaminating bacteria was ≤10 CFU / g.
[0055] The bacterial precipitate was resuspended in a freeze-drying protectant (containing 10% trehalose, 5% glycerol, and 2% yeast extract), with a bacterial to protectant volume ratio of 1:3. The mixture was then allowed to stand at room temperature for 30 minutes to allow for complete binding.
[0056] Add 0.5% chitosan and 1% anhydrous lactose, mix using a two-way spiral stirrer at 200 r / min, with a mixing coefficient of variation of <5%, to form a homogeneous bacterial slurry.
[0057] After the bacterial slurry is dispensed, it is placed in an ultra-low temperature freezer at -80℃ for 5 hours to ensure that ice crystals form evenly.
[0058] The product is transferred to a vacuum freeze dryer, where the vacuum level is maintained at 10~20Pa. It is first dried at -40℃ for 8 hours, and then heated to 25℃ for 4 hours. The final product has a water activity ≤0.25.
[0059] After aseptic pulverization, the freeze-dried blocks were passed through an 80-mesh sieve to obtain a powdered bacterial agent with a viable count ≥2×10⁻⁶. 10 CFU / g.
[0060] The powdered bacterial agent was mixed with lactose and trehalose at a mass ratio of 20:75:5 and then passed through an 80-mesh sieve to obtain an oral preparation with a viable count ≥1×10⁻⁶. 9 CFU / g.
[0061] 2. Preparation of gelling agent: After stir-frying Scutellaria baicalensis with rice wine, crush it and mix it with dried and crushed honeysuckle, salvia miltiorrhiza and licorice in a ratio of 3:1.5:1.5:1.5 (average particle size is 0.5mm). The mixed powder was extracted with a 30:70 methanol-water solution as the extractant, and ultrasonically extracted at 280W for 20 min (interval ratio 3s / 2s), and microwave extracted at 600W for 35 min (temperature 38℃) to obtain a mixed solution. After filtration, the mixed extract was obtained. The mixed extract was purified using an octadecylsilane-bonded silica column at a flow rate of 1.5 mL / min. Elution was performed with methanol-0.1% phosphoric acid aqueous solution (30:70) at a flow rate of 2.0 mL / min. The eluted fraction was collected after 8.5–9.5 min, concentrated, and dried to obtain the herbal extract. Gel preparation: Poloxamer 407: Poloxamer 188: Glycerol: Water = 12:6:4:75 were mixed and swollen, and then mixed with the Chinese herbal extract at a ratio of 15:85 to obtain a gel.
[0062] Example 2 1. The preparation of the oral preparation is the same as in Example 1.
[0063] 2. Preparation of gelling agent: After stir-frying Scutellaria baicalensis in rice wine, crush it and mix it with dried and crushed honeysuckle, salvia miltiorrhiza and licorice in a ratio of 3:1:1:1 (average particle size is 0.5mm). The mixed powder was extracted with a 30:70 methanol-water solution as the extractant, and ultrasonically extracted at 280W for 20 min (interval ratio 3s / 2s), and microwave extracted at 600W for 35 min (temperature 38℃) to obtain a mixed solution. After filtration, the mixed extract was obtained. The mixed extract was purified using an octadecylsilane-bonded silica column at a flow rate of 1.5 mL / min. Elution was performed with methanol-0.1% phosphoric acid aqueous solution (30:70) at a flow rate of 2.0 mL / min. The eluted fraction was collected after 8.5–9.5 min, concentrated, and dried to obtain the herbal extract. Gel preparation: Poloxamer 407: Poloxamer 188: Glycerol: Water = 15:4:4:75 were mixed and swollen, and then mixed with the Chinese herbal extract at a ratio of 15:85 to obtain a gel.
[0064] Example 3 1. The preparation of the oral preparation is the same as in Example 1.
[0065] 2. Preparation of gelling agent: After stir-frying Scutellaria baicalensis in rice wine, crush it and mix it with dried and crushed honeysuckle, salvia miltiorrhiza and licorice in a ratio of 3:2:2:2 (average particle size is 0.5mm). The mixed powder was extracted with a 30:70 methanol-water solution as the extractant, and ultrasonically extracted at 280W for 20 min (interval ratio 3s / 2s), and microwave extracted at 600W for 35 min (temperature 38℃) to obtain a mixed solution. After filtration, the mixed extract was obtained. The mixed extract was purified using an octadecylsilane-bonded silica column at a flow rate of 1.5 mL / min. Elution was performed with methanol-0.1% phosphoric acid aqueous solution (30:70) at a flow rate of 2.0 mL / min. The eluted fraction was collected after 8.5–9.5 min, concentrated, and dried to obtain the herbal extract. Gel preparation: Poloxamer 407: Poloxamer 188: Glycerol: Water = 10:8:4:75 were mixed and swollen, and then mixed with the Chinese herbal extract at a ratio of 15:85 to obtain the gel.
[0066] Comparative Example 1 The only difference from Example 1 is that the oral preparation is a mixture of lactose and trehalose in a mass ratio of 95:5 and does not contain Lactobacillus fermentum CQPC202403 bacterial powder.
[0067] Comparative Example 2 Commercially available 2% metronidazole gel.
[0068] Experimental Example 1 The efficacy of the products from Examples 1-3 and Comparative Examples 1-2 in treating periodontitis was tested. The specific test methods and results are as follows: 1. Test Objects and Methods Model: SD rat periodontitis model (constructed by ligation suture + inoculation with Porphyromonas gingivalis); Grouping: Examples 1-3, Comparative Examples 1-2, 15 animals in each group, and a healthy control group of 10 animals; Usage and dosage: (1) The oral preparations in Examples 1-3 and Comparative Example 1 were administered by gavage, 0.5g each time, twice a day; the gel was applied after rinsing the mice's teeth with water after meals, with a dosage of 1mL per mouse. (2) The usage of the 2% metronidazole gel in the comparative example is the same as that of the gel above; The test indicators included: clinical indicators (periodontal pocket depth (PD), attachment loss (AL), gingival sulcus bleeding index (SBI), pathogenic bacteria count (Porphyromonas gingivalis), inflammatory factors (IL-6, TNF-α, IL-10), alveolar bone repair (bone volume fraction (BV / TV) measured by Micro-CT), and drug retention time. Specific test results (after 4 weeks of drug administration) are shown in Table 1. Table 1. Test results related to the efficacy of the products in Examples 1-3 and Comparative Examples 1-2 in treating periodontitis.
[0069] Periodontal pocket depth (PD), attachment loss (AL), and sulcus bleeding index (SBI) are core indicators for assessing periodontal tissue damage. The example group showed significantly better performance than the control group. In Example 1, the differences in PD and AL between the healthy control (1.8±0.2mm, 1.5±0.1mm) were only 0.4mm and 0.5mm, respectively, and the difference in SBI between the healthy control (0.3±0.1 points) was 0.5 points, which is almost close to the normal periodontal state. The PD, AL, and SBI scores of Comparative Example 1 (without probiotics) and Comparative Example 2 (antibiotics) were 0.6-0.4 mm, 0.6-0.4 mm, and 0.7-0.5 points higher than those of Example 1, respectively. The blank model group (untreated) had the worst indicators (PD 4.0±0.5 mm, AL 3.8±0.4 mm, SBI 2.5±0.4 points), which confirms the restorative value of the formulation in the examples for periodontal tissues.
[0070] Porphyromonas gingivalis is the core pathogen of periodontitis, and the antibacterial effect of the example group was far superior to that of the control group: The pathogenic bacteria inhibition rates of Examples 1-3 reached 90.2%-94.5%, with Example 1 (94.5%) being the best. The inhibition rate of Comparative Example 1 (without probiotics) was only 72.6%, which was 21.9 percentage points lower than that of Example 1, confirming the key inhibitory effect of Fermented Lactobacillus mucin on pathogenic bacteria. The inhibition rate of Comparative Example 2 was 81.3%, which was still lower than that of the Example group, demonstrating the synergistic antibacterial advantage of "probiotics + traditional Chinese medicine".
[0071] The balance between pro-inflammatory factors (IL-6, TNF-α) and anti-inflammatory factors (IL-10) directly reflects the degree of periodontal inflammation. The immunomodulatory effect was particularly prominent in the example group. Pro-inflammatory factors were significantly downregulated: IL-6 (23.5±3.1 pg / mL) and TNF-α (20.8±2.5 pg / mL) in Example 1 were reduced by 20.3% and 21.0% respectively compared with Comparative Example 1, and the differences with healthy controls (18.5±2.3 pg / mL and 15.2±1.8 pg / mL) were only 5 pg / mL and 5.6 pg / mL respectively. Anti-inflammatory factors approached normal levels: The IL-10 in Example 1 (70.2±4.8 pg / mL) was almost equivalent to that in the healthy control (72.1±5.5 pg / mL), and increased by 33.0% compared with Comparative Example 1 (52.8±4.1 pg / mL), indicating that the dual effect of "probiotics regulating systemic immunity + traditional Chinese medicine local anti-inflammatory" can effectively restore immune balance.
[0072] Bone volume fraction (BV / TV) is a key indicator for assessing alveolar bone volume. The bone repair effect in the example groups was significant: the BV / TV of Example 1 (32.5±2.0%) was only 2.7% lower than that of the healthy control (35.2±2.3%), and 14.0% higher than that of Control Example 1 (28.5±1.7%). Example 3 (31.9±1.9%) showed better bone repair effect than Example 2 (30.8±1.8%) due to the optimized ratio of traditional Chinese medicine, confirming the synergistic effect of traditional Chinese medicine such as Danshen in improving microcirculation and probiotics in promoting bone formation.
[0073] Drug retention time directly affects local efficacy. The gel formulation in the example group far outperformed traditional antibiotic preparations: the drug retention time of Examples 1-3 reached 7.8-8.2 hours, while that of Comparative Example 2 (metronidazole gel) was only 2.1±0.3 hours. Long-term retention ensures that the drug continues to act on the periodontal lesion, solving the pain point of "short action time" of traditional preparations.
[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A periodontitis-improving composite preparation comprising a fermented Lactobacillus muci- de and a traditional Chinese medicine extract, characterized in that, Comprise the following raw materials: Oral agent: fermented lactobacillus mucus CQPC202403 bacteria powder; Gel: Huangqi, HJS, Danshen, Gancao, hydrogel base; The preservation number of the fermented lactobacillus mucus CQPC202403 in China Microorganism Culture Collection Center is CGMCC NO.31106, and the preservation date is June 27, 2024.
2. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the extract of traditional Chinese medicine according to claim 1, characterized in that, The number of viable bacteria of the fermented L. m. CQPC202403 bacterial powder in the oral preparation is ≥1.0×10 9 CFU / g.
3. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the extract of traditional Chinese medicine according to claim 1, characterized in that, The preparation method of the gel comprises the following steps: 1) Huangqi is mixed with yellow rice wine, fried, dried and crushed to obtain Huangqi powder; HJS, Danshen and Gancao are dried and crushed respectively to obtain HJS powder, Danshen powder and Gancao powder; 2) Huangqi powder, HJS powder, Danshen powder and Gancao powder are mixed and extracted with a mixed extractant of ethanol and water; 3) After extraction, the extract is filtered, concentrated and dried to obtain a traditional Chinese medicine extract after purification by a liquid chromatography column; 4) Poloxamer 407 and poloxamer 188 are dispersed in glycerol, and then water is added to swell to obtain a gel; 5) The traditional Chinese medicine extract is mixed with the gel to obtain a gel.
4. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the traditional Chinese medicine extract according to claim 3, characterized in that, The particle size of Huangqi powder, HJS powder, Danshen powder and Gancao powder in the preparation method of the gel is 0.2-1 mm; The mass ratio of Huangqi powder, HJS powder, Danshen powder and Gancao powder is 3:1-2:1-2:1-2.
5. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the traditional Chinese medicine extract according to claim 4, characterized in that, The extraction method in the preparation method of the gel is ultrasonic extraction followed by microwave extraction; The power of ultrasonic extraction is 200-350 W, the intermittent ratio of ultrasonic extraction is 3s / 2s, and the time of ultrasonic extraction is 10-30 min; The power of microwave extraction is 400-800 W, and the time of microwave extraction is 30-40 min; The temperature during extraction is controlled at 30-45℃.
6. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the traditional Chinese medicine extract according to claim 5, characterized in that, The liquid chromatography column in the preparation method of the gel is an octadecylsilane-bonded silica gel column; The loading flow rate during purification is 1-2 mL / min, the eluent is methanol-0.1% phosphoric acid solution with a volume ratio of 30:70, and the elution flow rate is 1.5-2.5 mL / min; The elution components with a main peak retention time of 8.5-9.5 min are collected.
7. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the Chinese medicine extract according to claim 5, characterized by, The mass ratio of poloxamer 407, poloxamer 188, glycerol and water in the preparation method of the gel is 15-10:4-8:4:70-80; The mass ratio of traditional Chinese medicine extract and gel is 15-25:75-85.
8. The periodontitis improving composite preparation comprising the fermented Lactobacillus muci- de and the extract of traditional Chinese medicine according to claim 1, characterized in that, The oral agent further comprises a filler and a protective agent; The filler is lactose or microcrystalline cellulose; The protective agent is trehalose.