Medicine for improving oral micro-ecology based on perilla leaves and application thereof
The medicine prepared from perilla leaf extract solves the problem of harmful oral bacteria causing systemic diseases. By increasing the secretion of mucosal immunoglobulins and regulating the oral microecology, it inhibits harmful bacteria and promotes beneficial bacteria, thereby improving oral health.
Patent Information
- Application Number
- CN202510915270.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-12-19
- Filing Date
- 2025-07-03
- Publication Date
- 2025-09-23
AI Technical Summary
Harmful oral bacteria cause systemic diseases in humans, and existing technologies are difficult to effectively improve the oral microecology.
The medicine is prepared from perilla leaf extract, which is extracted with water or ethanol to increase the secretion of immunoglobulin in the oral mucosa, inhibit the growth of harmful bacteria, and promote the growth of beneficial bacteria. The medicine can be made into sprays, liquids, lozenges, or mixed into oral products for use.
Increase the secretion of immunoglobulin in the oral mucosa, inhibit harmful bacteria, promote the growth of beneficial bacteria, improve the oral microenvironment, and reduce bad breath.
Smart Images
Figure CN120678827A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a medicine for improving oral microecology based on perilla leaves and application thereof. Background Art
[0002] Perilla leaves are the dried leaves (or young shoots) of Perilla frutescens (L.) Britt. Harvested in the summer when the foliage is lush, the leaves are often wrinkled, curled, or broken. Intact leaves, when flattened, are oval, with a long or acute apex, a rounded or broadly cuneate base, and serrated margins. They are purple on both sides or green on the upper surface and purple on the lower surface, sparsely covered with grayish-white hairs. The lower surface is covered with numerous pitted glandular scales. They have a delicate fragrance and a slightly pungent flavor. my country, one of the origins of the Perilla genus, boasts a cultivation history of over 2,000 years and a rich collection of germplasm resources. The Shennong Bencao Jing, my country's oldest extant pharmacological treatise, clearly describes the medicinal properties of perilla: "It has a pungent and slightly warming flavor, is used to dispel qi, ward off bad breath, and remove toxins and ward off evil spirits." Modern research indicates that perilla leaves contain a variety of chemical components, such as volatile oils, anthocyanins, and flavonoids, which exhibit immunomodulatory, antibacterial, antiviral, antipyretic, anti-inflammatory, and anti-allergic properties.
[0003] The oral cavity is a crucial window into the interaction between the human body and the environment. The oral microenvironment is regulated by both host and external environmental factors. A growing body of research supports the important association between the oral microbiome and human systemic diseases, such as rheumatoid arthritis, nephritis, enteritis, and cancer. Due to diverse oral colonization sites and variations in oxygen levels, nutrients, temperature, and the host immune system, oral microbes form a complex ecosystem. Streptococcus mutans, a recognized major cariogenic bacterium, produces acid through anaerobic fermentation in dental plaque, thereby initiating caries. Fusobacterium nucleatum subsp. nucleatum, a resident oral microbiome, typically coexists with its host but can also cause a variety of opportunistic infections. Furthermore, the oral cavity is colonized by systemic pathogens, such as Staphylococcus aureus and Pseudomonas aeruginosa. Summary of the Invention
[0004] The purpose of the present invention is to solve the technical problem that harmful oral bacteria cause systemic diseases in humans, and to provide a method for extracting a perilla leaf extract and an application of the perilla leaf extract.
[0005] The preparation method of the medicine for improving oral microecology based on perilla leaves is as follows:
[0006] The perilla leaves were crushed and added with distilled water in a mass ratio of 1:20. After soaking in a sealed container for 12 hours, they were decocted in water for 3 hours at a decoction temperature of 90±5℃. The filtrate was taken and the remaining drug was extracted once. The filtrates were combined, concentrated under reduced pressure and freeze-dried. A solution with a drug content of 1 mg / mL was prepared with distilled water and sterilized by filtering through a 0.22 μm filter membrane to obtain a perilla leaf-based drug for improving oral microecology (perilla leaf water extract).
[0007] The preparation method of the medicine for improving oral microecology based on perilla leaves is as follows:
[0008] Perilla leaves were crushed and added with 50% ethanol by volume at a mass ratio of 1:20. The leaves were soaked in a sealed container for 3 hours and then extracted under an ultrasonic frequency of 40 kHz for 30 minutes. The filtrate was taken and the remaining drug was extracted once. The filtrates were combined, concentrated under reduced pressure and freeze-dried. A solution with a drug content of 1 mg / mL was prepared with distilled water and sterilized by filtering through a 0.22 μm filter membrane to obtain a drug based on perilla leaves for improving oral microecology (50% ethanol extract of perilla leaves).
[0009] The medicine for improving oral microecology based on perilla leaves is used for increasing oral mucosal immunoglobulin.
[0010] The present invention increases the secretion of oral mucosal immunoglobulin and improves the oral microenvironment through the perilla leaf extract, which can be prepared into a spray, an aqueous solution, a lozenge or other dosage forms, and can also be mixed into mouthwash, toothpaste, chewing gum, chewing candy or oral spray for use. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 This is a comparative chart of the effects of 50% ethanol extract of Perilla leaves on different bacterial strains. In the figure, A represents Escherichia coli, B represents Staphylococcus aureus, C represents Pseudomonas aeruginosa, D represents Fusobacterium nucleatum subsp. nucleatum, E represents Streptococcus mutans, F represents Lactobacillus plantarum, and G represents Lactobacillus acidophilus.
[0012] Figure 2 This is a comparative chart of the effects of perilla leaf water extract on different bacterial strains. In the figure, A represents Escherichia coli, B represents Staphylococcus aureus, C represents Pseudomonas aeruginosa, D represents Fusobacterium nucleatum subsp. nucleatum, E represents Streptococcus mutans, F represents Lactobacillus plantarum, and G represents Lactobacillus acidophilus. DETAILED DESCRIPTION
[0013] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.
[0014] Specific embodiment 1: The preparation method of the drug for improving oral microecology based on Perilla leaves in this embodiment is as follows:
[0015] The perilla leaves were crushed and added with distilled water in a mass ratio of 1:20. After soaking in a sealed container for 12 hours, they were decocted in water for 3 hours. The filtrate was taken and the remaining drug was extracted repeatedly. The filtrates were combined, concentrated under reduced pressure and freeze-dried. A solution with a drug content of 1 mg / mL was prepared with distilled water and sterilized by passing through a 0.22 μm filter membrane to obtain a perilla leaf-based drug for improving oral microecology (perilla leaf water extract).
[0016] Specific embodiment 2: The preparation method of the drug for improving oral microecology based on Perilla leaves in this embodiment is as follows:
[0017] Perilla leaves were crushed and added with 50% ethanol by volume at a mass ratio of 1:20. The leaves were soaked in a sealed container for 3 hours and then extracted under an ultrasonic frequency of 40 kHz for 30 minutes. The filtrate was taken and the remaining drug was extracted once. The filtrates were combined, concentrated under reduced pressure and freeze-dried. A solution with a drug content of 1 mg / mL was prepared with distilled water and sterilized by filtering through a 0.22 μm filter membrane to obtain a drug based on perilla leaves for improving oral microecology (50% ethanol extract of perilla leaves).
[0018] Specific embodiment three: Specific embodiment one: The drug for improving oral microecology based on perilla leaves is used to increase oral mucosal immunoglobulin.
[0019] Specific embodiment 4: The drug for improving oral microecology based on perilla leaves described in specific embodiment 2 is used to increase oral mucosal immunoglobulin.
[0020] The following experiments were used to verify the effects of the present invention:
[0021] Experiment 1
[0022] Effects of Perilla Leaf Extract on Local Immunoglobulin Secretion in the Oral Cavity:
[0023] Step 1. 70 SPF-grade Kunming mice, half male and half female, with a body weight of 20-22 g, were randomly divided into 7 groups, namely, a blank control group (normal saline), high-, medium-, and low-dose groups of perilla leaf water extract, and high-, medium-, and low-dose groups of perilla leaf alcohol extract, with 10 mice in each group.
[0024] Step 2: Dilute the drug solution and inject 30 μL of the drug into the mice's mouths four times daily for 10 consecutive days. The high-, medium-, and low-dose groups received 976 mg / kg / d, 488 mg / kg / d, and 244 mg / kg / d of the drug, respectively.
[0025] Step 3: Determine the content of SIgA
[0026] Ten days after administration, the mice were killed and 0.2 mL of normal saline was placed in a centrifuge tube. The mouse oral cavity was repeatedly wiped three times with a small-headed cotton swab dipped in normal saline. The cotton swab tip was collected and placed in a centrifuge tube containing 0.2 mL of normal saline and vortexed for 3-5 min. The supernatant was centrifuged at 3000 rpm / min for 10 min, and the SIgA content in the mouse oral cavity was detected by enzyme-linked immunosorbent assay.
[0027] Experiment 2
[0028] Resuscitation of strains:
[0029] Step 1. Bacteria strains: Escherichia coli (CMCC(B)44102), Staphylococcus aureus (CMCC(B)26003), Pseudomonas aeruginosa (CMCC(B)10104), Streptococcus mutans (ATCC25175), Fusobacterium nucleatum subsp. nucleatum (ATCC25586), Lactobacillus plantarum (CICC22696), Lactobacillus acidophilus (CICC20248).
[0030] Step 2. Culture medium and culture conditions: Nutrient broth medium: peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, distilled water 1000 mL, pH 7.2±0.2, used for culturing Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, cultured at 37°C for 18 h; Brain heart infusion broth: peptone 10.0 g, dehydrated calf brain extract powder 12.5 g, dehydrated beef heart extract powder 5.0 g, sodium chloride 5.0 g, glucose 2.0 g, sodium hydrogen phosphate 2.5 g, distilled water 1000 mL, pH 7.4±0.2, used for culturing Streptococcus mutans and Fusobacterium nucleatum subsp. nucleatum, Streptococcus mutans cultured at 37°C for 18 h, Fusobacterium nucleatum subsp. nucleatum cultured at 37°C under anaerobic conditions for 3 d; MRS broth medium: peptone 10.0 g, beef extract powder 8.0 g, sodium chloride 5.0 g, distilled water 1000 mL, pH 7.4±0.2, used for culturing Streptococcus mutans and Fusobacterium nucleatum subsp. nucleatum, Streptococcus mutans cultured at 37°C for 18 h, Fusobacterium nucleatum subsp. nucleatum cultured at 37°C under anaerobic conditions for 3 d. g, yeast powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH 5.7±0.2, used to culture Lactobacillus plantarum and Lactobacillus acidophilus, Lactobacillus plantarum was cultured at 37°C for 18 h, and Lactobacillus acidophilus was cultured at 42°C under anaerobic conditions for 24-48 h;.
[0031] Effects of Perilla Leaf Extract on Different Bacteria:
[0032] Step 1: Prepare culture medium with perilla extract. The content of perilla leaf extract is 25%, 12.5%, and 6.25%, respectively. A culture medium with an equal amount of physiological saline is used as a control. The total volume of the culture medium is 10 mL.
[0033] Step 2: Take 100 μL each of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, Fusobacterium nucleatum subsp. nucleatum, Lactobacillus plantarum, and Lactobacillus acidophilus and add them to the culture medium from Step 1. Incubate E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, and Lactobacillus plantarum at 37°C for 18 hours. Incubate Lactobacillus acidophilus at 42°C under anaerobics for 24-48 hours. Incubate Fusobacterium nucleatum subsp. nucleatum at 37°C under anaerobics for 3 days.
[0034] Step 3: Take the culture from step 2, add it to a centrifuge tube, centrifuge at 12000 rpm / min for 10 min, remove the supernatant and resuspend, repeat 4 times to remove the culture medium.
[0035] Step 4: Take the liquid from step 3, add it to a 96-well plate, and measure the absorbance at 600 nm.
[0036] Step 5: The absorbance value of the culture without Perilla extract is A1, and the absorbance value of the culture containing Perilla extract is A2. The promoting or inhibiting effect of the drug-containing culture on the strain is calculated as A1 / A2. A result greater than 1 and P < 0.05 indicates that the Perilla extract has a significant promoting effect on the strain; a result less than 1 and P < 0.05 indicates that the Perilla extract has a significant inhibitory effect on the strain; otherwise, it indicates that the Perilla extract has no significant promoting or inhibiting effect on the strain.
[0037] Experiment 3
[0038] Healthy 6-8-week-old male Wistar rats were randomly divided into eight groups: blank control, model control, high-dose perilla leaf aqueous extract, medium-dose perilla leaf aqueous extract, low-dose perilla leaf aqueous extract, high-dose perilla leaf ethanol extract, medium-dose perilla leaf ethanol extract, and low-dose perilla leaf ethanol extract. After one week of adaptive feeding, a rat halitosis model was established using the whole saliva microbiota from patients with oral halitosis. The rats' teeth and mucosa were washed three times with normal saline. After one week of conventional feeding, the washing was repeated three times. The oral mucosa and teeth were rinsed with 0.1% sodium octyl chloride for 5 minutes, followed by a 10-minute rinse with 23 μmol / L sodium ascorbate. The rats' teeth and oral mucosa were smeared with the whole saliva microbiota from patients with oral halitosis once daily. After smearing, the rats were deprived of food or water for 1 hour and given 10% high-sugar water daily. The blank control group received normal saline, while the high, medium, and low dose groups received 675 mg / kg / day, 338 mg / kg / day, and 169 mg / kg / day, respectively. Six weeks later, oral breath measurements were performed using a breath detector. During the experiment, breath measurements were performed every two weeks for three replicates.
[0039] Experimental results:
[0040] Experiment 1
[0041] Perilla leaf extract has a certain promoting effect on the secretion of SIgA in mice. The SIgA content of each group of mice after administration is shown in Table 1.
[0042] Table 1 SIgA secretion in mice ( )
[0043]
[0044] Note: Compared with the blank control group, * P<0.05
[0045] Experiment 2
[0046] Perilla leaf extract has different degrees of inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Fusobacterium nucleatum subspecies nucleatum, and Streptococcus mutans, and has different degrees of promoting effect on Lactobacillus plantarum and Lactobacillus acidophilus. The results of the effect of 50% ethanol extract of perilla leaves on different strains are shown in Figure 1 The results of the effects of water extract of Perilla leaves on different bacterial strains are shown in Figure 2 .
[0047] Experiment 3
[0048] After rats were colonized with the whole saliva microbiome of patients with oral halitosis to establish a rat halitosis model, the breath values increased. After drug administration, the breath values of rats decreased, as shown in Table 2.
[0049] Table 2 Rat breath values ( )
[0050]
[0051] Note: Compared with the model control group, * P<0.05
[0052] Experiments have shown that oral administration of perilla leaf extract can promote SIgA secretion in mice. SIgA secretion increases with increasing dosage, and a 50% ethanol extract of perilla leaves is more effective than an aqueous extract. Perilla leaf extract has a significant inhibitory effect on common oral harmful bacteria and a moderately stimulating effect on common beneficial oral bacteria. Perilla leaf extract improves bad breath in rats, with a 50% ethanol extract being more effective than an aqueous extract.
Claims
1. A drug for improving oral microecology based on perilla leaves, characterized in that The preparation method of the medicine for improving oral microecology based on Perilla leaves is as follows: Perilla leaves were crushed and added with distilled water at a mass ratio of 1:
20. The leaves were soaked in a sealed container for 12 hours and then decocted in water for 3 hours at a decoction temperature of 90±5°C. The filtrate was taken and the remaining drug was extracted once again. The filtrates were combined, concentrated under reduced pressure, and freeze-dried. A solution containing 1 mg / mL of the drug was then prepared with distilled water and sterilized through a 0.22 μm filter membrane to obtain a drug based on perilla leaves for improving oral microecology.
2. A drug for improving oral microecology based on perilla leaves, characterized in that The preparation method of the medicine for improving oral microecology based on Perilla leaves is as follows: Perilla leaves were crushed and added with 50% ethanol by volume at a mass ratio of 1:
20. The leaves were soaked in a sealed container for 3 hours and then extracted under an ultrasonic frequency of 40 kHz for 30 minutes. The filtrate was taken and the remaining drug was extracted once. The filtrates were combined, concentrated under reduced pressure, and freeze-dried. A solution with a drug content of 1 mg / mL was prepared with distilled water and sterilized by filtering through a 0.22 μm filter membrane to obtain a drug based on perilla leaves for improving oral microecology.
3. The use of the medicament for improving oral microecology based on Perilla leaves according to claim 1, characterized in that The medicine for improving oral microecology based on perilla leaves is used for increasing oral mucosal secretory immunoglobulin A.
4. The use of the medicament for improving oral microecology based on Perilla leaves according to claim 2, characterized in that The medicine for improving oral microecology based on perilla leaves is used for increasing oral mucosal secretory immunoglobulin A.