Medicine for improving oral micro-ecology based on perilla leaves and application thereof

Abstract

The invention relates to a medicine for improving oral micro-ecology based on perilla leaves and application thereof, in particular to a medicine for improving oral micro-ecology based on perilla leaves and application thereof. The invention aims to solve the problem of human systemic diseases caused by oral harmful bacteria. The method comprises the following steps: crushing perilla leaves, adding distilled water or ethanol with a volume concentration of 50% according to a mass ratio of the perilla leaves to the distilled water or the ethanol with a volume concentration of 50% of 1: 20, soaking the distilled water in a closed container for 12 h, decocting with water for 3 h, and taking a filtrate; extracting with ethanol with the volume concentration of 50%, soaking in a closed container for 3 hours, performing ultrasonic extraction for 30 minutes, and taking filtrate; and repeatedly extracting the remaining medicines once, merging the filtrate, carrying out vacuum concentration on the filtrate, freeze-drying, preparing a solution with the medicine content of 1g / mL by using distilled water, and sterilizing by using a 0.22 mu m filter membrane to obtain the traditional Chinese medicine composition. The medicine for improving the oral micro-ecology based on the perilla leaves is used for improving oral mucosa immune globulin. Through the perilla leaf extract, the secretion amount of immune globulin in oral mucosa is increased, and the oral microenvironment is improved.

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 branches) of Perilla frutescens (L.) Britt. They are harvested in summer when the branches and leaves are lush. The leaves are mostly wrinkled, curled, and broken. The intact leaves are oval when flattened, with long or sharp tips, round or wide wedge-shaped bases, and rounded edges. Both sides are purple or the upper surface is green, the lower surface is purple, sparsely covered with grayish-white hairs, and the lower surface has many concave glandular scales. The smell is fresh and the taste is slightly spicy. my country is one of the origins of Perilla plants, with a cultivation history of more than 2,000 years and rich germplasm resources. The earliest existing pharmaceutical monograph in my country, "Shennong's Herbal Classic", has a clear record of the medicinal effects of Perilla: "It tastes spicy and slightly warm, mainly for lowering qi, eliminating bad breath, removing toxins and eliminating evil." Modern research shows that Perilla leaves contain a variety of chemical components, such as volatile oils, anthocyanins, flavonoids, etc., which have pharmacological activities such as immunomodulation, antibacterial, antiviral, antipyretic, anti-inflammatory, and anti-allergic.

[0003] The oral cavity is one of the important windows for the interaction between the human body and the environment. The oral microenvironment is regulated by the host and external environmental factors. More and more studies support the important association between the oral microbiome and human systemic diseases. Such as rheumatoid arthritis, nephritis, enteritis, cancer, etc. Due to the different colonization sites in the oral cavity and the differences in oxygen content, nutrients, temperature and host immune system, oral microorganisms form a complex ecosystem. Streptococcus mutans can undergo anaerobic metabolism in dental plaque, ferment and produce acid, thereby causing caries, and is recognized as the main oral caries bacteria; Fusobacterium nucleatum is a resident member of the oral microbiome, and can form a symbiotic relationship with its host, which can cause a variety of opportunistic infections. Systemic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are also colonized in the oral cavity. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that harmful oral bacteria lead to human systemic diseases, 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 based on perilla leaf for improving oral microecology 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, the leaves were decocted in water for 3 hours, and the filtrate was taken. The remaining drug was extracted repeatedly, and the filtrates were combined. The filtrates were concentrated under reduced pressure and freeze-dried. A solution containing 1 g / mL of the drug was prepared with distilled water, and the solution was 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 based on perilla leaf for improving oral microecology is as follows:

[0008] The perilla leaves were crushed, and 50% ethanol by volume was added, the mass ratio of the perilla leaves to the 50% ethanol by volume was 1:20, and the leaves were immersed in a sealed container for 3 hours, and then extracted for 30 minutes under an ultrasonic frequency of 40 kHz, and the filtrate was taken, and the remaining drugs were extracted repeatedly once, and the filtrates were combined, and the filtrates were concentrated under reduced pressure and freeze-dried, and then a solution with a drug content of 1 g / mL was prepared with distilled water, and the solution was 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] In the present invention, the perilla leaf extract is used to increase the secretion of oral mucosal immunoglobulin and improve the oral microenvironment. The extract can be prepared into a spray, an aqueous solution, a lozenge or other dosage forms, and can also be mixed into a mouthwash, a toothpaste, a chewing gum, a chewing candy or an oral spray for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a comparative diagram of the effects of 50% ethanol extract of perilla leaves on different strains, in which A represents Escherichia coli, B represents Staphylococcus aureus, C represents Pseudomonas aeruginosa, D represents Fusobacterium nucleatum subspecies 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 implementation modes listed below, but also includes any combination of the specific implementation modes.

[0014] Specific implementation method 1: The method for preparing a drug for improving oral microecology based on perilla leaves in this implementation method 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, the leaves were decocted in water for 3 hours, and the filtrate was taken. The remaining drug was extracted repeatedly, and the filtrates were combined. The filtrates were concentrated under reduced pressure and freeze-dried. A solution containing 1 g / mL of the drug was prepared with distilled water, and the solution was 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).

[0016] Specific implementation method 2: The method for preparing a drug for improving oral microecology based on perilla leaves in this implementation method is as follows:

[0017] The perilla leaves were crushed, and 50% ethanol by volume was added, the mass ratio of the perilla leaves to the 50% ethanol by volume was 1:20, and the leaves were immersed in a sealed container for 3 hours, and then extracted for 30 minutes under an ultrasonic frequency of 40 kHz, and the filtrate was taken, and the remaining drugs were extracted repeatedly once, and the filtrates were combined, and the filtrates were concentrated under reduced pressure and freeze-dried, and then a solution with a drug content of 1 g / mL was prepared with distilled water, and the solution was 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 implementation method three: Specific implementation method one The drug based on perilla leaves for improving oral microecology 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 effect 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 Kunming mice, half male and half female, with a body weight of 20-22g, were randomly divided into 7 groups, namely, a blank control group (normal saline), high-, medium- and low-dose groups of perilla water extract, and high-, medium- and low-dose groups of perilla alcohol extract, with 10 mice in each group.

[0024] Step 2: Dilute the drug solution and inject the drug into the mouse's mouth with a pipette, 4 times a day, 30 μL each time for 10 consecutive days. The high, medium and low dose groups were given 975 mg / kg·d, 488 mg / kg·d and 244 mg / kg·d, respectively.

[0025] Step 3: Determine the content of slgA

[0026] The mice were killed 10 days after administration, and 0.2 mL of saline was placed in a centrifuge tube. The mouse mouth was repeatedly wiped 3 times with a small-headed cotton swab dipped in saline. The cotton swab head was collected and placed in a centrifuge tube containing 0.2 mL of saline and vortexed for 3-5 minutes. The supernatant was centrifuged at 3000 rpm / min for 10 minutes, and the slgA content in the mouse mouth 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 10.0g, beef extract powder 3.0g, sodium chloride 5.0g, distilled water 1000mL, pH 7.2±0.2, used to culture Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, cultured at 37℃ for 18h; brain heart infusion broth: peptone 10.0g, dehydrated calf brain extract powder 12.5g, dehydrated beef heart extract powder 5.0g, sodium chloride 5.0g, glucose 2.0g, disodium hydrogen phosphate 2.5g, distilled 1000mL of water, pH 7.4±0.2, used for culturing Streptococcus mutans and Fusobacterium nucleatum subsp. nucleatum. Streptococcus mutans was cultured at 37°C for 18h, and Fusobacterium nucleatum subsp. nucleatum was cultured at 37°C under anaerobic conditions for 3d; MRS broth medium: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 1.0g of Tween 80, 1000mL of distilled water, pH 5.7±0.2, used for culturing Lactobacillus plantarum and Lactobacillus acidophilus. Lactobacillus plantarum was cultured at 37°C for 18h, and Lactobacillus acidophilus was cultured at 42°C under anaerobic conditions for 24-48h;.

[0031] Effects of Perilla Leaf Extract on Different Bacteria:

[0032] Step 1: Prepare culture medium with perilla extract. The contents of perilla leaf extract are 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 subspecies nucleatum, Lactobacillus plantarum, and Lactobacillus acidophilus, and add them to the culture medium in step 1. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, and Lactobacillus plantarum are cultured at 37°C for 18 hours, Lactobacillus acidophilus is cultured at 42°C in an anaerobic environment for 24-48 hours, and Fusobacterium nucleatum subspecies nucleatum is cultured at 37°C in an anaerobic environment for 3 days.

[0034] Step 3: Take the culture obtained in 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 with perilla extract is A2. The promoting or inhibiting effect of the drug-containing culture medium on the strain is calculated by A1 / A2. If the result is greater than 1 and P<0.05, it means that the perilla extract has a significant promoting effect on the strain; if the result is less than 1 and P<0.05, it means that the perilla extract has a significant inhibitory effect on the strain; otherwise, it means 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 8 groups, namely blank control group, model control group, high dose of water extract of perilla leaf group, medium dose of water extract of perilla leaf group, low dose of water extract of perilla leaf group, high dose of alcohol extract of perilla leaf group, medium dose of alcohol extract of perilla leaf group, and low dose of alcohol extract of perilla leaf group. After 1 week of adaptive feeding, the whole microbial community of saliva of patients with oral halitosis was used to colonize the rat halitosis model. The teeth and mucosa of the rats were cleaned with normal saline for 3 times, and the cleaning was repeated 3 times after 1 week of conventional feeding. The oral mucosa and teeth were rinsed with 0.1% NaOCl for 5 minutes, and then rinsed with 23μmoL / L sodium ascorbate for 10 minutes. The whole microbial community of saliva of patients with oral halitosis was applied to the teeth and oral mucosa of the rats once a day. After application, the rats were fasted for 1 hour and given 10% high sugar water every day. The blank control group was given normal saline, and the high, medium, and low doses of the drug-treated groups were given 675 mg / kg·d, 338 mg / kg·d, and 169 mg / kg·d, respectively. After 6 weeks, the breath detector was used to detect the oral gas values ​​of the rats. During the experiment, the breath test was performed once every 2 weeks and repeated 3 times.

[0039] Experimental results:

[0040] Experiment 1

[0041] Perilla leaf extract has a certain promoting effect on the secretion of slgA in mice. The slgA content in each group of mice after administration is shown in Table 1.

[0042] Table 1 Mouse slgA secretion ( n=10)

[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, and the promoting effect on Lactobacillus acidophilus is more significant. The effect of 50% ethanol extract of perilla leaves on different strains is shown in Figure 1 The results of the effects of water extract of Perilla leaves on different strains are shown in Figure 2 .

[0047] Experiment 3

[0048] After the mice were colonized with the whole microbiome of the saliva of patients with oral halitosis to construct a rat halitosis model, the breath values ​​increased. After drug administration, the breath values ​​of the mice decreased, as shown in Table 2.

[0049] Table 2 Mouse breath values ​​( n=10)

[0050]

[0051] Note: Compared with the model control group, * P<0.05

[0052] Experiments have shown that perilla leaf extract can promote the secretion of slgA in mice through oral mucosal administration, and the secretion of slgA increases with the increase of the dosage, and the effect of 50% ethanol extract of perilla leaf is better than that of water extract of perilla leaf. Perilla leaf extract has a significant inhibitory effect on common oral harmful bacteria and has a certain promoting effect on common oral beneficial bacteria Lactobacillus. Perilla leaf extract has an improvement effect on the breath of mice, and the effect of 50% ethanol extract of perilla leaf on improving the breath of mice is better than that of water extract of perilla leaf.

Claims

1. A drug for improving oral microecology based on perilla leaves, characterized in that The method for preparing the medicine for improving oral microecology based on Perilla leaf is as follows: 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, and the filtrate was taken. The remaining drugs were extracted repeatedly, and the filtrates were combined. The filtrates were concentrated under reduced pressure and freeze-dried. A solution with a drug content of 1 g / mL was prepared with distilled water, and the solution was sterilized by filtering 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 method for preparing the medicine for improving oral microecology based on Perilla leaf is as follows: The perilla leaves were crushed, and 50% ethanol by volume was added, with the mass ratio of the perilla leaves to the 50% ethanol by volume being 1:

20. The leaves were soaked in a sealed container for 3 hours, and then extracted for 30 minutes under an ultrasonic frequency of 40 kHz. The filtrate was taken, and the remaining drug was extracted repeatedly once. The filtrates were combined, concentrated under reduced pressure, and then freeze-dried. A solution with a drug content of 1 g / mL was prepared with distilled water, and the solution was sterilized by filtering through a 0.22 μm filter membrane to obtain a perilla leaf-based drug for improving oral microecology.

3. The use of the drug for improving oral microecology based on Perilla leaf according to claim 1, characterized in that The medicine for improving oral microecology based on perilla leaves is used for increasing oral mucosal immunoglobulin.

4. The use of the drug for improving oral microecology based on Perilla leaf according to claim 2, characterized in that The medicine for improving oral microecology based on perilla leaves is used for increasing oral mucosal immunoglobulin.

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