Drugs based on bilberry for improving oral microecology and their applications

Abstract

A drug based on red bilberry to improve oral microecology and its application, relates to a drug based on red bilberry to improve oral microecology and its application. The present invention is to solve the problem of harmful oral bacteria causing human systemic diseases. The drug is added to a juicer at a ratio of 1:5 between the mass of red bilberry and the volume of water, homogenized, and the filtrate is decompressed and concentrated before freeze-drying; the drug based on red bilberry to improve oral microecology is added to a juicer at a ratio of 1:5 between the mass of red bilberry and the volume concentration of 50% ethanol, homogenized, ultrasonically extracted for 30 minutes under an ultrasonic frequency of 40KHz, and the filtrate is decompressed and concentrated before freeze-drying. The drug based on red bilberry to improve oral microecology is used to increase oral mucosal immunoglobulin. The present invention increases the secretion of oral mucosal immunoglobulin and improves the oral microenvironment through red bilberry extract.

Description

Technical Field

[0001] The invention relates to a medicine for improving oral microecology based on red bean bilberry and application thereof. Background Art

[0002] Vaccinium vitis-idea, belonging to the Ericaceae family (Ericaceae) Vaccinium genus (Vaccinium.spp), commonly known as the northern red bean, has a high utilization value. It is rich in anthocyanins, flavonoids, terpenes and other active ingredients. It has antioxidant, antibacterial, anti-inflammatory, anti-cancer, weight loss, prevention of cardiovascular diseases, improving memory and improving vision and other physiological functions. It is a special forest berry resource with broad development prospects. The reserves of wild vaccinium vitis-idea resources in the Greater Khingan Range of my country are relatively large.

[0003] The oral cavity is a complex ecosystem second only to the intestine. The oral microbiome is crucial to the oral mucosal barrier and immune response, and plays a key role in resisting pathogen invasion and preventing and treating oral diseases. The oral microenvironment is regulated by the host and external environmental factors. However, a variety of factors such as unhealthy lifestyles, eating habits or diseases can cause dysbiosis of the oral microecology and cause oral-related diseases such as caries and oral ulcers. Oral immunoglobulin plays an important role in enhancing oral immune activity. As an important component of the oral immune system, oral immunoglobulin can specifically recognize and bind to pathogens in the oral cavity, such as bacteria and viruses, thereby preventing further infection. Oral immunoglobulin can also promote the proliferation and differentiation of oral mucosal cells and enhance the barrier function of the oral mucosa. In addition, oral immunoglobulin can also activate immune cells in the oral cavity, triggering a series of immune responses, such as phagocytosis and cell killing, thereby more effectively removing pathogens in the oral cavity. Therefore, oral immunoglobulin is of great significance for maintaining oral health and preventing oral diseases. At the same time, the antioxidant capacity in the mouth is essential to oral and dental health. Enhancing the antioxidant capacity can reduce the production of free radicals, prevent bacterial growth, and thus reduce the risk of oral and dental problems. 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 medicine and application based on cranberry for improving oral microecology.

[0005] A drug based on cranberry for improving oral microecology is prepared by adding cranberry to water in a ratio of 1:5 by volume into a juicer, homogenizing, and taking the filtrate to decompress and concentrate it, followed by freeze-drying.

[0006] A drug based on cranberry for improving oral microecology is prepared by adding cranberry to 50% ethanol in a volume ratio of 1:5 into a juicer, homogenizing, ultrasonically extracting for 30 minutes at an ultrasonic frequency of 40 KHz, and decompressing and concentrating the filtrate before freeze-drying.

[0007] Application of drugs based on cranberry for improving oral microecology, wherein the drugs based on cranberry for improving oral microecology are used to increase oral mucosal immunoglobulin.

[0008] Application of drugs based on cranberry for improving oral microecology, wherein the drugs based on cranberry for improving oral microecology are used to increase oral mucosal immunoglobulin.

[0009] In the present invention, the red bean and bilberry 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

[0010] Figure 1 This is a comparative diagram of the effects of 50% ethanol extract of red bean bilberry 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;

[0011] Figure 2 This is a comparative chart of the effects of red bean lingonberry water extract on different 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

[0012] 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.

[0013] Specific implementation method 1: In this implementation method, the drug based on red bean bilberry to improve oral microecology is prepared by adding red bean bilberry to water in a ratio of 1:5 by volume into a juicer, homogenizing, taking the filtrate, decompressing and concentrating it, and then freeze-drying it.

[0014] The method for preparing a medicine for improving oral microecology based on red bean bilberry comprises: adding red bean bilberry to water in a ratio of 1:5 by volume into a juicer, homogenizing, taking the filtrate and concentrating it under reduced pressure, and then freeze-drying to obtain a red bean bilberry water extract.

[0015] Specific implementation method 2: This implementation method is based on the drug for improving oral microecology of red bean cranberry. The drug based on red bean cranberry for improving oral microecology is prepared by adding red bean cranberry to 50% ethanol in a volume ratio of 1:5 into a juicer, homogenizing, ultrasonically extracting for 30 minutes at an ultrasonic frequency of 40KHz, and taking the filtrate to reduce pressure, concentrate and then freeze-dry.

[0016] The method for preparing a drug for improving oral microecology based on red bean bilberry is as follows: red bean bilberry is added into a juicer at a volume ratio of 1:5 to 50% ethanol by volume, homogenized, ultrasonically extracted for 30 minutes under an ultrasonic frequency of 40 kHz, and the filtrate is concentrated under reduced pressure and freeze-dried to obtain a 50% ethanol extract of red bean bilberry.

[0017] Specific implementation method three: Specific implementation method one: the use of drugs based on red bean bilberry to improve oral microecology, wherein the drugs based on red bean bilberry to improve oral microecology are used to increase oral mucosal immunoglobulin.

[0018] Specific embodiment 4: Specific embodiment 2 The application of drugs based on cranberry to improve oral microecology, wherein the drugs based on cranberry to improve oral microecology are used to increase oral mucosal immunoglobulin.

[0019] The following experiments were used to verify the effect of the present invention:

[0020] Experiment 1

[0021] Effect of bilberry extract on oral immunoglobulin secretion:

[0022] Step 1. 70 SPF Kunming mice, half male and half female, with a body weight of 18-22g, were randomly divided into 7 groups, including a blank control group (normal saline), high-, medium- and low-dose groups of red bean bilberry water extract, and high-, medium- and low-dose groups of red bean bilberry 50% ethanol extract, with 10 mice in each group.

[0023] Step 2: Dilute the water extract of red bean bilberry and the 50% ethanol extract of red bean bilberry with distilled water, and inject the drug into the oral cavity of mice with a pipette, 4 times a day, 30 μL each time, for 10 consecutive days. The high, medium and low dose groups were administered 200 mg / kg·d, 100 mg / kg·d, and 50 mg / kg·d, respectively.

[0024] Step 3: Determine the content of slgA

[0025] The mice were killed 10 days after administration, and 0.2 mL of normal saline was placed in a centrifuge tube. The mouse mouth was repeatedly wiped 3 times with a small-headed cotton swab dipped in normal saline. The cotton swab head was collected and placed in a centrifuge tube containing 0.2 mL of normal saline and vortexed for 3-5 minutes. The supernatant was taken and the slgA content in the mouse mouth was detected by enzyme-linked immunosorbent assay.

[0026] Experiment 2

[0027] Study on the antioxidant capacity of bilberry extract in vitro:

[0028] 1. Determination of DPPH free radical scavenging ability:

[0029] Step 1: dilute the water extract of blueberry and the 50% ethanol extract of blueberry with anhydrous ethanol to the sample solutions with concentrations of 5.00 mg / mL, 2.50 mg / mL and 1.25 mg / mL.

[0030] Step 2: Weigh 9.86 mg DPPH and dilute to 250 mL with anhydrous ethanol to obtain a 0.1 mmol / L DPPH solution.

[0031] Step 3: Determination of DPPH free radical scavenging ability: Add 2 mL of DPPH solution and 2 mL of the sample solution to be tested into the test tubes respectively, shake well, react in the dark at room temperature for 30 minutes, and measure the absorbance at 517 nm. Repeat 3 times for each group.

[0032] Step 4: DPPH free radical scavenging rate (%) = [1 – (A 1 –A 2 ) / A 0 ]×100%, where A 0 is the absorbance value of anhydrous ethanol solution, A 1 is the absorbance value of the red bean bilberry sample solution, A 2 It is the absorbance value without adding DPPH solution (replaced by anhydrous ethanol).

[0033] 2. Determination of ABTS free radical scavenging ability:

[0034] Step 1: dilute the water extract of blueberry and the 50% ethanol extract of blueberry with distilled water to the concentrations of 5.00 mg / mL, 2.50 mg / mL and 1.25 mg / mL sample solutions.

[0035] Step 2: Weigh 38.41 mg of ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and dilute to 10 mL with distilled water to obtain an ABTS solution with a concentration of 7 mmol / L.

[0036] Step 3: Weigh 6.62 mg of potassium persulfate and dilute to 10 mL with distilled water to obtain a potassium persulfate solution with a concentration of 2.45 mmol / L.

[0037] Step 4: Take 2 mL of freshly prepared ABTS solution and potassium persulfate solution, mix well, and react at room temperature in the dark for 16 hours. Dilute the mixed solution of ABTS and potassium persulfate with distilled water before use so that its absorbance at a wavelength of 734 nm is 0.70±0.02 to obtain ABTS working solution.

[0038] Step 5. Determination of ABTS free radical scavenging ability: Add 50 μL of the test solution and 200 μL of ABTS working solution to the test tubes respectively, react at room temperature in the dark for 10 minutes, and measure the absorbance at 734 nm. Repeat 3 times for each group.

[0039] Step 6: ABTS free radical scavenging rate (%) = (A 0 –A 1 ) / A 0 ×100%; where A 0 is the absorbance value of the blank control (distilled water), A 1 is the absorbance value of the blueberry sample solution.

[0040] Experiment 3

[0041] Effects of Red Bean Bilberry Extract on Different Bacteria:

[0042] 1. Resuscitation of strains

[0043] 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).

[0044] Step 2: Culture medium and culture conditions:

[0045] 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 to culture Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, cultured at 37 ° C for 18 h;

[0046] Brain heart infusion broth: peptone 10.0g, dehydrated calf brain infusion powder 12.5g, dehydrated ox heart infusion powder 5.0g, sodium chloride 5.0g, glucose 2.0g, disodium hydrogen phosphate 2.5g, distilled water 1000mL, pH value 7.4±0.2, used to culture Streptococcus mutans and Fusobacterium nucleatum subsp. nucleatum. Streptococcus mutans was cultured at 37℃ for 18h, and Fusobacterium nucleatum subsp. nucleatum was cultured at 37℃ under anaerobic conditions for 3d;

[0047] MRS broth medium: peptone 10.0g, beef powder 8.0g, yeast powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1.0g, distilled water 1000mL, pH 5.7±0.2, used to cultivate Lactobacillus plantarum and Lactobacillus acidophilus, Lactobacillus plantarum was cultured at 37°C for 18h, and Lactobacillus acidophilus was cultured at 42°C under anaerobic environment for 24-48h.

[0048] 2. Effects of Red Bean and Bilberry Extract on Different Bacteria

[0049] Step 1: Prepare a 1 g / mL red bean and bilberry solution with distilled water, and sterilize it by filtering it through a 0.22 μm filter membrane.

[0050] Step 2: Prepare culture medium with red bean and bilberry extract. The content of red bean and bilberry extract is 25%, 12.5% ​​and 6.25% respectively. The culture medium with an equal amount of physiological saline is used as a control. The total volume of the culture medium is 10 mL.

[0051] Step 3: 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 2. 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; Fusobacterium nucleatum subspecies nucleatum is cultured at 37°C in an anaerobic environment for 3 days.

[0052] Step 4: Take the culture obtained in step 3, 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.

[0053] Step 5: The absorbance value of the culture without red bean bilberry extract is A 1 The absorbance value of the drug-containing culture is A 2 , with A 1 / A 2Calculate the promoting or inhibiting effect of the drug-containing culture medium on the strain. If the result is greater than 1 and P is less than 0.05, it means that the red bean bilberry extract has a significant promoting effect on the strain; if the result is less than 1 and P is less than 0.05, it means that the red bean bilberry extract has a significant inhibitory effect on the strain; otherwise, it means that the red bean bilberry extract has no significant promoting or inhibiting effect on the strain.

[0054] Experimental results:

[0055] Experiment 1

[0056] Red bean and bilberry extracts have a certain promoting effect on the secretion of slgA in mice. The slgA content of each group of mice after administration is shown in Table 1.

[0057] Table 1 Mouse slgA secretion ( n=10)

[0058]

[0059] Note: Compared with the blank control group, * P<0.05

[0060] Experiment 2

[0061] The antioxidant capacity of bilberry extract was investigated by DPPH and ABTS free radical scavenging rates. Vaccinium vitis-idaea had significant antioxidant activity, and the antioxidant capacity of 50% ethanol extract of Vaccinium vitis-idaea was superior to that of water extract. The results are shown in Table 2.

[0062] Table 2 DPPH and ABTS free radical scavenging rates of blueberry

[0063]

[0064] Experiment 3

[0065] Red bean bilberry extract has different degrees of inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Fusobacterium nucleatum subspecies nucleatum, and Streptococcus mutans, and the antibacterial effect of 50% ethanol extract is stronger than that of water extract; Red bean bilberry extract has different degrees of promoting effect on Lactobacillus plantarum and Lactobacillus acidophilus, and the promoting effect on Lactobacillus acidophilus is more significant. The results of the effect of 50% ethanol extract of red bean bilberry on different strains are shown in Figure 1 The results of the effects of the water extract of red bean bilberry on different strains are shown in Figure 2 .

[0066] Experiments have shown that bilberry extract can promote the secretion of slgA in mice through oral mucosal administration. The secretion of slgA increases with the increase of administration dosage, and the effect of 50% ethanol extract of bilberry is better than that of water extract of bilberry. bilberry has significant antioxidant properties, and the antioxidant capacity increases with the increase of concentration, and the antioxidant effect of 50% ethanol extract of bilberry is better than that of water extract of bilberry. bilberry extract has a significant inhibitory effect on common oral harmful bacteria, and has a certain promoting effect on common oral beneficial bacteria Lactobacillus.

Claims

1. A drug for improving oral microecology based on red bean bilberry, characterized in that The bilberry-based drug for improving oral microecology is prepared by adding bilberry to water in a ratio of 1:5 by volume into a juicer, homogenizing, and taking the filtrate to decompress and concentrate it, followed by freeze-drying.

2. A drug for improving oral microecology based on red bean bilberry, characterized in that The drug for improving oral microecology based on red bean bilberry is prepared by adding red bean bilberry to 50% ethanol in a volume ratio of 1:5 into a juicer, homogenizing, ultrasonically extracting for 30 minutes under an ultrasonic frequency of 40KHz, and decompressing and concentrating the filtrate before freeze-drying.

3. The use of the drug based on bilberry to improve oral microecology according to claim 1, characterized in that The medicine for improving oral microecology based on red bean bilberry is used for increasing oral mucosal immunoglobulin.

4. The use of the drug based on bilberry to improve oral microecology according to claim 2, characterized in that The medicine for improving oral microecology based on red bean bilberry is used for increasing oral mucosal immunoglobulin.

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