CN116139172A - Effective fraction of gallnut, preparation method and application thereof - Google Patents
Effective fraction of gallnut, preparation method and application thereof Download PDFInfo
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- CN116139172A CN116139172A CN202211416725.XA CN202211416725A CN116139172A CN 116139172 A CN116139172 A CN 116139172A CN 202211416725 A CN202211416725 A CN 202211416725A CN 116139172 A CN116139172 A CN 116139172A
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Abstract
The invention belongs to the technical field of extraction of gallnuts, and particularly relates to a gallnut effective part, a preparation method and application thereof. The effective parts of the galls prepared by the method have good remineralization, can be attached to teeth to form a protective film, induce in-situ mineralization of hydroxyapatite on enamel, can block dentinal tubules, and effectively treat dentinal desensitization; in bacteriostasis, the growth of cariogenic bacteria streptococcus mutans and actinomyces viscosus can be inhibited, and the protective film formed on the teeth can reduce the adhesion of the biological film of the streptococcus mutans and actinomyces viscosus; meanwhile, the compound has good anti-inflammatory effect, has obvious treatment effect on gingivitis, and can obviously reduce the expression of inflammatory factors interleukin-6 and interleukin-1 beta and the enzyme activities of aspartic acid aminotransferase and alkaline phosphatase.
Description
Technical Field
The invention belongs to the technical field of extraction of gallnuts, and particularly relates to a gallnut effective part, a preparation method and application thereof.
Background
Dental caries (Dental caries) is a disease with extremely high incidence in humans, and has been classified by the world health organization and cancer and cardiovascular disease as a three major control of disease in humans. Caries formation is mainly caused by attachment of dental plaque to the tooth surface, in which cariogenic bacteria such as streptococcus mutans (s. Mutans) and actinomyces viscosus (a. Viscosus) metabolize sugar to produce acid, acid etch dental hard tissue and induce demineralization. In the absence of intervention, most of the initial unrecovered caries lesions continue to demineralize and progressively worsen, causing dentinal hypersensitivity and gingivitis. Traditional caries treatment methods require invasive treatment, including preparation of the oral tissue and its cavities, and restoration with traditional materials, such as amalgams and composite resins. However, due to the differences in properties of dental tissue and artificial materials, the long term performance and the prospect of the restored teeth are far below expected. Commercial products, such as toothpastes and mouthwashes, containing antibiotics, chlorhexidine, and fluoride chemicals are common agents for preventing biofilm formation by tooth decay pathogens. However, resistance and side effects limit the effectiveness of antibiotics for daily use. Therefore, a multifunctional drug which is effective and safe and has in-situ mineralization, bacteriostasis and anti-inflammatory effects is searched from natural products and becomes a new hot spot for preventing and treating dental caries.
The gallic acid is a gall produced by larvae of galls (Cynips galliae-tinctoria Oliv.) parasitized on young branches of galli tree (Quercus infectoria Oliv.) belonging to Fagaceae, and contains various physiologically active components such as 50-70wt% of tannins, 2-4wt% of gallic acid, a small amount of resin, etc. At present, the extracted effective parts of the galls only have an inhibitory effect on pathogenic bacteria of periodontal diseases, so that a need exists for finding effective parts of the galls which integrate mineralization, anti-biofilm and anti-inflammatory activities and can be used for treating and/or preventing dental caries.
Disclosure of Invention
The invention aims at solving the existing problems and provides a gallic effective part and a preparation method and application thereof. The effective part of the prepared gallnut can induce tooth remineralization, inhibit and kill cariogenic bacteria, and reduce the expression of inflammatory factors in gingival crevicular fluid.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a gallic effective part, which comprises the following steps:
soaking Galla Turcica powder in water, and reflux extracting to obtain extractive solution;
subjecting the extract to column chromatography to obtain eluent; the column chromatography comprises eluting and eluting sequentially, wherein the eluted solution is ethanol solution, and the volume fraction of ethanol in the ethanol solution is 70%;
concentrating and drying the eluent in sequence to obtain the effective part of the nutgall.
Preferably, the chromatographic column of the column chromatography is a polyamide column or a C18 column.
Preferably, the leaching is sequentially carried out by first leaching by using an ethanol solution with the volume fraction of 10 percent and second leaching by using an ethanol solution with the volume fraction of 30 percent.
Preferably, the mass ratio of the gallic powder to the water is 1:2-3.
Preferably, the particle size of the gallic powder is 40 to 60 mesh.
Preferably, the soaking time is 1-2 hours.
Preferably, the reflux extraction time is 2-3 hours.
Preferably, the number of times of the reflux extraction is 2 to 3.
The invention also provides the effective parts of the gallnut obtained by the preparation method of the technical proposal, which comprise tetra-O-galloyl glucose, penta-O-galloyl glucose, hexa-O-galloyl glucose and hepta-O-galloyl glucose.
The invention also provides application of the effective part of the gallnut obtained by the preparation method in preparing a medicine for treating and/or preventing dental caries.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a preparation method of effective parts of gallnut, which comprises the steps of soaking gallnut medicinal material powder in water, extracting under reflux, eluting an extracting solution by column chromatography in sequence to obtain an eluent, concentrating and drying the eluent to obtain the effective parts of the gallnut, wherein the eluent is ethanol solution with the volume fraction of 70 percent of ethanol. The gallic acid effective part prepared by the invention is adsorbed on teeth to form a protective film with in-situ mineralization, bacteriostasis and anti-inflammatory effects, can induce tooth remineralization, inhibit the biomembrane viscosity of cariogenic bacteria streptococcus mutans and actinomyces viscosus and effectively kill bacteria, and obviously reduces the expression of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) inflammatory factors and the enzyme activity of aspartic acid Aminotransferase (AST) and alkaline phosphatase (ALP) in gingival crevicular fluid, thereby being capable of being used for preventing and/or treating dental caries.
The effective part of the gallnut obtained from the natural product provides a multifunctional strategy integrating mineralization, anti-biofilm and anti-inflammatory activity for preventing and treating dental caries, and has clinical potential for effectively treating dental caries through simple gargling or smearing.
The invention also provides the effective parts of the gallnut obtained by the preparation method, which comprise tetra-O-galloyl glucose, penta-O-galloyl glucose, hexa-O-galloyl glucose and hepta-O-galloyl glucose, wherein the purity of 4 effective components is high, and the total purity is 92-98%.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a TIC diagram of the effective parts of galls;
FIG. 2 shows SEM analysis of a protective film formed on teeth by a gallic effective site;
FIG. 3 is a graph showing the static water contact angle of enamel before and after treatment of the galling effective area;
FIG. 4 shows XPS analysis of elemental and functional changes in tooth surface before and after treatment with galling sites;
FIG. 5 is an SEM image of the enamel and dentin surface morphology before and after treatment in an in vitro mineralization experiment;
fig. 6 is an XPS elemental analysis and raman spectrum of the enamel surface of the control and gallic active site-administered groups;
FIG. 7 is an experimental schematic diagram in an in vivo mineralization experiment and an SEM image of the surface morphology and cross section of tooth enamel after in vivo mineralization;
FIG. 8 is a mechanical test of enamel after mineralization in vivo;
FIG. 9 is a representation of the active ingredient-induced precipitate of gallnut;
FIG. 10 shows the bacteriostatic action of the effective fraction of gallnut on free Streptococcus mutans and Actinomyces viscosus;
FIG. 11 shows the biofilm inhibition of Streptococcus mutans and Actinomyces viscosus by the effective fraction of Galla Turcica;
FIG. 12 shows anti-inflammatory effect of the active fraction of Galla Turcica;
fig. 13 shows the biocompatibility of the gallic effective fraction.
Detailed Description
The invention provides a preparation method of a gallic effective part, which comprises the following steps:
soaking Galla Turcica powder in water, and reflux extracting to obtain extractive solution;
subjecting the extract to column chromatography to obtain eluent; the column chromatography comprises eluting and eluting sequentially, wherein the eluted solution is ethanol solution, and the volume fraction of ethanol in the ethanol solution is 70%;
concentrating and drying the eluent in sequence to obtain the effective part of the nutgall.
In the present invention, all materials used are commercial products in the art unless otherwise specified.
The invention pulverizes the gallnut to obtain the gallnut powder.
In the present invention, the particle size of the gallic powder is preferably 40 to 60 mesh. The specific mode of the pulverization is not particularly limited, and may be any mode known to those skilled in the art.
After the galling powder is obtained, the extraction liquid is obtained by soaking the galling powder in water and then carrying out reflux extraction.
In the present invention, the mass ratio of the gallic powder to the water is preferably 1:2 to 3.
In the invention, the soaking time is preferably 1-2 h, the soaking temperature is not particularly required in the invention, and in the specific embodiment of the invention, the normal temperature is adopted.
In the present invention, the time of the reflux extraction is preferably 2 to 3 hours, more preferably 2.5 hours, and the temperature of the reflux extraction is preferably 96 to 100 ℃, more preferably 98 ℃.
In the present invention, the number of extraction times of the reflux extraction is preferably 2 to 3.
The invention preferably combines the liquids obtained from each reflux extraction to obtain the extract.
After the extracting solution is obtained, the extracting solution is subjected to column chromatography to obtain the eluent; the column chromatography comprises eluting and eluting sequentially, wherein the eluted solution is ethanol solution, and the volume fraction of ethanol in the ethanol solution is 70%.
In the present invention, the column chromatography column is preferably a polyamide column or a C18 column.
In the present invention, the rinsing is preferably performed by sequentially performing a first rinsing with an ethanol solution having a volume fraction of 10% and a second rinsing with an ethanol solution having a volume fraction of 30%.
In the present invention, the volumes of the 10% by volume of the ethanol solution and the 30% by volume of the ethanol solution are independently preferably 2 to 3 times the column volume.
In the present invention, the amount of the eluted solution is preferably 2 to 3 times the column volume.
The invention has no special requirements on the elution rate and the elution rate, and the rate commonly used by the person skilled in the art is adopted.
After the eluent is obtained, the eluent is concentrated and dried in sequence to obtain the effective part of the nutgall.
In the present invention, the concentration is preferably reduced pressure concentration, the reduced pressure concentration is preferably rotary evaporation, and the parameters of the rotary evaporation preferably include: the pressure is 400-600 mmHg, the temperature is 70 ℃, and the rotating speed is 50-100 rpm.
In the present invention, the volume of the solution obtained after concentration is preferably 10 to 20% of the volume of the eluent.
In the present invention, the drying is preferably freeze-drying, and the temperature of the freeze-drying is preferably-20 to-10 ℃ and the time is preferably 8 to 10 hours.
The invention also provides the effective parts of the gallnut obtained by the preparation method of the technical proposal, which comprise tetra-O-galloyl glucose, penta-O-galloyl glucose, hexa-O-galloyl glucose and hepta-O-galloyl glucose.
The invention also provides application of the effective part of the gallnut obtained by the preparation method in preparing a medicine for treating and/or preventing dental caries.
In the invention, the effective parts of the gallnut are applied to the preparation of the medicines for treating and preventing dental caries, and have the functions of tooth remineralization and oral antibacterial and anti-inflammatory.
In a specific embodiment of the present invention, for the purpose of studying the lack of foodThe tooth remineralization effect of the sub-effective part is achieved by constructing a tooth demineralization model by adopting a human third molar after acid etching by a phosphoric acid solution with the volume fraction of 37%, after the model is successfully formed, remineralization treatment is carried out by using the gallic effective part and artificial saliva, the appearance and the thickness of the sediment on the tooth surface are observed by using a scanning electron microscope, and the sediment is characterized by using a high-resolution transmission electron microscope. The in vitro test result shows that the effective part of the gallic acid can form a protective film on the surface of teeth to induce Ca in artificial saliva 2+ And PO (PO) 4 3- The compact hydroxyapatite sediment layer is formed on the surface of the teeth, the remineralization induced by the effective parts of the galls can improve the hardness of the teeth, and the newly formed hydroxyapatite sediment layer has better bonding strength with the teeth.
In the specific embodiment of the invention, in order to study the inhibition effect of the effective part of the galling on the mutans streptococcus and the actinomyces viscosus of the oral cavity, the inhibition effect is evaluated by using a inhibition zone, a flat plate count and a scanning electron microscope, and meanwhile, after the teeth and bacteria are co-cultured to form a biological film, the appearance of the bacteria is observed by using the scanning electron microscope, and the adhesion effect of the bacteria to the biological film is evaluated. The test result shows that the effective part of the gallnut has good inhibition effect on the mutans streptococcus and the stickness actinomycetes of the oral cavity cariogenic bacteria, the MIC is 5.00 mg/mL and 2.50mg/mL respectively, and the bacterial forms of the mutans streptococcus and the stickness actinomycetes are gradually destroyed along with the increase of the concentration of the effective part of the gallnut; in the anti-biomembrane adhesion test, the test result shows that the effective part of the gallic acid can effectively inhibit the adhesion of the streptococcus mutans and the actinomyces viscosus biomembrane on teeth and can effectively destroy the biomembrane.
In the specific embodiment of the invention, in order to study the anti-gingivitis effect of the edible effective parts, a rat gingivitis model is constructed by adopting a silk thread ligation and lipopolysaccharide method, and after successful modeling, the expression of interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta) inflammatory factors and the contents of aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) in the gingival crevicular fluid of the rat are detected, and the gingival tissues are subjected to H & E staining. The test results show that: the gingivitis of animals of the gallic effective part administration group is basically healed, compared with the animals of the model, the expression level of IL-6 in gingival crevicular fluid of the animals of the gallic effective part administration group is obviously reduced (P < 0.01), the expression level of IL-1 beta is obviously reduced (P < 0.001), and the contents of AST and ALP are obviously reduced (P < 0.001).
For further explanation of the present invention, the effective parts of the gallnut, the preparation method and application thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Experimental materials:
the galli medicinal material is purchased in Guangdong of China, identified as the galli by the professor Han Bo (university of Shihe medical college), and the evidence specimen (No. 20160305) is stored by the national medicine subject group (university of Shihe medical college);
the third molar is approved by the medical ethics committee of the first affiliated hospital of the university of Shihe medical college, and is collected in the stomatology of the first affiliated hospital of the university of Shihe medical college after written informed consent of the patient. Selecting third molar with normal enamel development and no caries, and removing soft tissue, dental calculus and pigment. The teeth were sterilized with 3% sodium hypochlorite solution, washed with PBS and cut perpendicular to the longitudinal axis of each tooth crown with a low-speed diamond saw to obtain enamel and dentin flat sections 1-2 mm thick and 3-5 mm in diameter. Mirror polishing the surface of the tooth slice by 400 mesh silicon carbide paper, then ultrasonically cleaning the tooth specimen by water, and drying at room temperature;
Sprague-Dawley (SD) rats (clean grade, male, 8 weeks old, 220-300 g) and Kunming mice (clean grade, male, 6 weeks old, 21-23 g) were supplied by Xinjiang laboratory animal research center (SCXK (xin) 2018-0002) and were subjected to adaptive feeding for one week prior to treatment after approval by the university of Shihe medical institute's first affiliated hospital animal protection committee;
streptococcus mutans (ATCC 25175) was aerobically cultured in brain-heart extract (BHI) at 37℃for 24 hours. Myxococcus (ATCC 27044) was anaerobic (85% N) at 37 DEG C 2 ,10％H2,5％CO 2 ) The culture was carried out in soybean casein medium (TSB) under the conditions for 24 hours. Centrifuging (5000 rpm,10 min) and collectingBacteria were collected, washed once with PBS, and suspended in BHI/TSB, respectively, with bacteria concentration adjusted to 1×10 7 CFU/mL。
Reagent:
acetonitrile (chromatographic grade, fisher Scientific); formic acid, ethanol, phosphoric acid (top grade, tianjin optical complex fine chemical institute); interleukin-6 (IL-6) kit, interleukin-1 beta (IL-1 beta) kit, aspartic acid Aminotransferase (AST) kit, and alkaline phosphatase (ALP) kit (shanghai zebra biotechnology limited); artificial saliva (Shanghai mu Biotech Co.); brain heart extract (BHI) and soy casein culture medium (TSB) (peninsula maribo biotechnology limited); CCK-8 kit, glycerol, glutaraldehyde, lipopolysaccharide (LPS) and Phosphate Buffer (PBS) (beijing soebao biotechnology limited); chlorhexidine (CHX) (jiangsu morning brand bond pharmaceutical company limited).
Example 1
Preparation of effective parts of the gallnut:
1) Selecting galls and fruits of outer galling tree produced by larvae of Apis galli belonging to Apidae on young branches of Galla Turcica belonging to Fagaceae as galli medicinal materials;
2) Pulverizing gallnut, sieving with 60 mesh sieve, weighing 80g of gallnut powder, soaking in 3 times distilled water (240 mL) for 2 hr, reflux-extracting for 3 times, extracting for 3 hr each time, and mixing to obtain gallnut extractive solution;
3) Subjecting the extract obtained in the step 2) to column chromatography by using a polyamide column, eluting by using an ethanol solution with the volume fraction of ethanol of 10% and an ethanol solution with the volume fraction of ethanol of 30% which are 3 times of the column volume respectively, eluting by using an ethanol solution with the volume fraction of ethanol of 70% which is 3 times of the column volume, collecting the eluent with the volume fraction of ethanol of 70%, concentrating under reduced pressure to 20% of the volume, and freeze-drying the concentrated solution at-20 ℃ to obtain 19.8g of effective parts of the gallnut, wherein the mass of the effective parts is 24.75% of that of the powder of the gallnut.
Analyzing the effective parts of the gallnut by using a liquid chromatography-mass spectrometry technology:
the liquid chromatography conditions included: waters XBiridge C18 column (. Phi.19X150 mm,5 μm); the mobile phase A is formic acid aqueous solution with the formic acid volume fraction of 0.2%, and the mobile phase B is acetonitrile; gradient elution procedure: 0-8 min, the volume of the mobile phase A is kept at 93%; 8-35 min, the volume of the mobile phase A is reduced from 93% to 80%; 35-50 min, the volume of the mobile phase A is reduced from 80% to 70%; 50-60 min, the volume of the mobile phase A is reduced from 70% to 0%; flow rate: 1mL/min; sample injection amount: 20. Mu.L.
ESI-MS conditions: electrospray negative ion mode, mass range: 100-1250 amu, source temperature: desolventizing temperature at 150 ℃): 250 ℃, capillary voltage 2.64kV and cone voltage 50V.
The effective parts of the galls are tetra-O-galloyl glucose, penta-O-galloyl glucose, hexa-O-galloyl glucose and hepta-O-galloyl glucose, which are measured by liquid chromatography-mass spectrometry, and the total purity of 4 components is 98%.
FIG. 1 shows a TIC chart of the effective fraction of galls, and the specific components are shown in Table 1, and the content of the ingredients is detected, wherein the peak-to-charge ratio No. 1 is 787.07, the peak-to-charge ratio No. 2 is 936.36, the peak-to-charge ratio No. 3 is 1091.49, the peak-to-charge ratio is 1091.49, and the peak-to-charge ratio No. 4 is 1243.14, and the peak-to-charge ratio is seven-O-galloyl glucose.
TABLE 1 information on each component of the Galla Turcica extract
Example 2
1) Selecting galls and fruits of outer galling tree produced by larvae of Apis galli belonging to Apidae on young branches of Galla Turcica belonging to Fagaceae as galli medicinal materials;
2) Pulverizing gallnut, sieving with 40 mesh sieve, weighing 80g of gallnut powder, soaking in 2 times of distilled water (160 mL) for 1 hr, reflux-extracting for 2 hr each time, and mixing to obtain gallnut extractive solution;
3) And 2) carrying out column chromatography on the extract obtained in the step 2) by using a C18 column, eluting by using an ethanol solution with the volume fraction of 10% and an ethanol solution with the volume fraction of 30% which are 3 times of the volume of the column respectively, eluting by using an ethanol solution with the volume fraction of 70% which is 3 times of the volume of the column, collecting an eluent with the volume fraction of 70%, concentrating under reduced pressure to 20% of the volume, and freeze-drying the concentrated solution at-20 ℃ to obtain 13.2g of the effective part of the nutgall, wherein the mass of the effective part of the nutgall is 16.50% of that of the powder of the nutgall.
The effective parts of the gallnut are tetra-O-galloyl glucose, penta-O-galloyl glucose and hexa-O-galloyl glucose, and the total purity of the 4 components is 92 percent.
Application example 1
Remineralization experiments of the effective fraction of Galla Turcica obtained in example 1
1. Preparation and characterization of the gallic effective part coating:
the experimental method comprises the following steps: and (3) pickling enamel and dentin for 1min by adopting a phosphoric acid solution with the volume fraction of 37%, constructing a tooth demineralization model, and randomly dividing the model into a control group and a gallic effective part administration group after the model is successfully built. After the tooth enamel in the effective part administration group is soaked for 5min in the dark condition of 37 ℃ by 10mg/mL of the effective part of the gallnut, the appearance of the coating on the tooth enamel surface is observed by using a scanning electron microscope, the static water contact angle of the tooth enamel before and after the effective part of the gallnut is treated is measured, and the surface element and functional group analysis is carried out on the coating by using XPS and Raman.
Experimental results: fig. 2 shows a protective film formed on teeth by SEM analysis of galling effective parts, wherein a is a normal Enamel surface morphology, the surface is relatively smooth (enamal), B is an Acid etched Enamel surface morphology, the surface is relatively rough (Acid-etched), C is an Enamel surface morphology after galling effective part treatment, and a dense protective film (TGE coating) is formed on the surface.
Fig. 3 shows the static water contact angle of enamel before and after treatment of the galling active site, the contact angle before treatment was 73.91 ° and the contact angle after treatment was 59.32 °.
Fig. 4 is an XPS spectrum of a tooth surface analyzed for changes in elements and functional groups on the surface before and after treatment with a galling effective site, wherein a is an XPS spectrum of a galling effective site (TGE), enamel (enamal) and Enamel covered with a galling effective site coating (TGE & enamal), peak areas of C and O elements in the Enamel after treatment with the galling effective site are significantly increased, and B and E are XPS peak-splitting treatment result graphs of C and O elements, respectively, which indicate that c=o groups in the galling effective site are successfully introduced into the Enamel, demonstrating successful attachment of the galling effective site to the Enamel, C and D are XPS peak-splitting treatment result graphs of Ca and P elements, respectively, and electric potentials of Ca and P elements are increased, indicating that electron densities around calcium ions and phosphate ions are changed, possibly participating in an adsorption process.
2. Gallic effective part coating in vitro and in vivo remineralization experiments:
the experimental method comprises the following steps: in an in vitro remineralization test, enamel and dentin of a gallic effective part administration group are treated with the gallic part for 5min, a control group is treated with pure water for 5min, and after mineralization in artificial saliva for 3 days and 7 days, a scanning electron microscope is used for observing the change of the tooth surface morphology, and XPS is used for element content change analysis; in the in vivo remineralization test, tooth hardness, deposit thickness on the surface and bond strength of the deposit to the tooth were observed after fixing the demineralized enamel in the oral cavity of a rat for 3 days and 7 days.
Experimental results: fig. 5 is an SEM image of enamel and dentin surface morphology before and after treatment in an in vitro mineralization experiment. Wherein a1 and a2 are the surface morphology of the tooth enamel after demineralization, and a2 is an enlarged view of a 1; a3 to a5 are the dentin surface morphology after demineralization, a4 is an enlarged view of a3, and a5 is a sectional view of dentin tubules; b1-b 5 and c 1-c 5 are SEM scanning images of the surface morphology of the enamel and dentin of the control group after being respectively mineralized in artificial saliva for 3 days and 7 days; d 1-d 5, e 1-e 5 are surface morphology SEM scanning pictures of enamel and dentin of the effective part administration group of the gallic acid after being respectively mineralized in artificial saliva for 3 days and 7 days; the results show that the coating formed on the enamel surface by the gallic effective parts can induce the remineralization of the enamel, and the sediment on the enamel surface is more and more increased along with the prolongation of mineralization time; in dentin remineralization experiments, the effective parts of the galls induce dentin remineralization, and the precipitate can effectively block dentin tubules.
Fig. 6 is an XPS elemental analysis and raman spectrum of the enamel surface of the control group and the gallic effective site administration group. A is XPS result graph, the result shows that after artificial saliva remineralization treatment, the peak areas of Ca, O and P elements are increased to different degrees, but compared with the control group, the peak areas of Ca, O and P elements in the administration group of the effective parts of the galls are increased more rapidly, the remineralization effect of the effective parts of the galls is obvious, B is Raman spectrum, 4 typical phosphate vibration peaks are generated, and the peak areas are respectively 960cm -1 、426cm -1 、1045cm -1 And 584cm -1 In the Enamel, control 3d,Control 7d,TGE 3d and TGE 7d spectra, the average peak area of phosphate was 4.83×10, respectively 5 、5.68×10 5 、6.12×10 5 、6.35×10 5 、2.55×10 5 . As mineralization time increases, the average peak area of phosphate increases, indicating that gallic effectiveness can accelerate phosphate deposition on tooth surfaces. .
Fig. 7 is a schematic diagram of experiments in an in vivo mineralization experiment and SEM images of the surface morphology and cross section of enamel after in vivo mineralization. Wherein A is a schematic preparation diagram of enamel sheets; b is a schematic diagram of fixing enamel sheets in the oral cavity of a rat; c and D are surface morphology SEM scanning images of the enamel of the control group and the effective gallic part administration group after mineralizing for 3 days and 7 days in the oral cavity of the rat respectively, and compared with the control group, the sediment in the effective gallic part administration group is obviously increased; e is a cross-sectional SEM scan of enamel of control and gallic active site-administered groups after mineralization in the oral cavity of rats for 3 and 7 days, respectively, no significant precipitate layer was observed after mineralization in the control for 3 days, and the precipitate thickness after mineralization for 7 days was about 12.51 μm, and the precipitate thickness after mineralization in the gallic active site-administered groups was about 16.72 μm and 25.64 μm, respectively.
Fig. 8 is a mechanical test of enamel after mineralization in vivo, wherein a is an indentation mark of the vickers hardness test of the surface after mineralization of the control group and the administration group of the gallic effective part for 3 days and 7 days, B is a corresponding hardness statistical chart, and the hardness after acid etching is 82.31HV compared with the hardness 219.77HV of the natural enamel. In addition, after the treatment of the gallic effective part coating, the tooth enamel hardness is obviously enhanced in 3 days and 7 days, and reaches 230.64HV and 327.34HV, which are respectively improved by 4.12 percent and 48.42 percent compared with natural tooth enamel; c is nanometer scratch analysis after mineralization of the control group and the effective part administration group for 7 days, and the result shows that the bonding strength of the sediment in the control group and the tooth enamel is 32.24mN, and the bonding strength of the sediment in the effective part administration group and the tooth enamel is 43.77mN, thus showing that the sediment induced by the effective part of the gallnut is easier to be attached on the tooth enamel.
3. Characterization of remineralization:
the experimental method comprises the following steps: the sediment is subjected to ultrasonic dispersion and remineralization by absolute ethyl alcohol, the morphology, the crystal lattice particle size, the diffraction ring and the element content of the sediment are characterized by a high-resolution transmission electron microscope, and the particle size distribution of the sediment is measured by a Markov laser particle sizer.
Experimental results: FIG. 9 is a representation of the active ingredient-induced precipitate of gallnut. Wherein A is a high-resolution transmission image of sediment, the sediment of the administration group of the effective parts of the galls is rod-shaped crystal, and the sediment of the control group is irregular round particles; b is a lattice image of the precipitate, the rod-shaped mineral crystals induced by the administration group of the gallic effective part have uniform lattice fringes, the lattice fringe spacing is about 0.391nm, and no obvious lattice fringes are observed in the control group; c is a selected area electron diffraction pattern of the precipitate, the specific dispersion points and the crystal patterns of the rings of the gallic effective part administration group correspond to the (002), (211) and (300) crystal face reflections, and only the crystal face (002) is observed in the control group; d is a particle size distribution diagram, the structural length of the rod-shaped mineral of the gallnut administration group is 139+/-58 nm, and the particle size of the control group is 532+/-176 nm; e is EDX diagram, the calcium-phosphorus atomic ratio (Ca: P) of the rod-like mineral is 1.67, which is the same as the theoretical value of Hydroxyapatite (HAP), and the calcium-phosphorus atomic ratio of the irregular round particles is 0.97, which is similar to the theoretical value of Amorphous Calcium Phosphate (ACP); f is XRD result diagram, and the result shows that the rod-shaped mineral crystal has (213) crystal face and (004) crystal face, which are similar to natural enamel, besides (002), (211) and (300), and the (002) crystal face and (004) crystal face are observed in the control group, and cannot be confirmed as HAP, so that the coating of the effective part of the gallnut is likely to induce the conversion of ACP into HAP.
Application example 2
Experimental investigation of the antibacterial action of Streptococcus mutans and Actinomyces viscosus on the Galla Turcica effective fraction obtained in example 1
Mic and MBC assay:
the experimental method comprises the following steps: the effective parts of the gallnut are prepared into solutions with different concentrations (0.31-40.00 mg/mL) and are dripped into 96-well cell culture plates (100 mu L/well). Each well was inoculated with 100. Mu.L of the bacterial suspension (1X 10) 7 CFU/mL). The positive control group is the effective fraction of the gallnut, the negative control group is BHI culture medium, and the blank control group is TSB culture medium. Bacteria growth was assessed after 24 hours of incubation. mu.L of the bacterial suspension was applied to BHI or TSB agar plates, respectively, and incubated at 37℃for 48h. The Minimal Inhibitory Concentration (MIC) was defined for macroscopic no bacterial growth in the drug sensitive assay, and the Minimal Bactericidal Concentration (MBC) was defined as the minimal concentration at which no colonies formed on the agar plates after incubation.
TABLE 2 MIC and MBC of the Galla Turcica effective fraction against Streptococcus mutans and Actinomyces viscosus
2. Test of zone of inhibition
The experimental method comprises the steps of continuously diluting the effective parts of the gallnut by pure water by a double-ratio dilution method, wherein the final concentration is 0.31-40.00 mg/mL. 3 or 4 oxford cups were placed in each dish and marked on the bottom in advance. After sterilization, the BHI or TSB agar culture medium is cooled to about 50 ℃, and two bacterial liquids (1×10) are added into the culture medium according to the volume ratio of 1:100 7 CFU/mL), and poured into sterile petri dishes. After the solution had completely solidified, the oxford cup was removed and 100 μl of the solution to be tested was added to each well. The petri dishes were transferred to a 37 ℃ incubator for 24 hours, and the diameter of each zone was measured 3 times with a vernier caliper.
3. Bacterial morphology observations
The experimental method comprises the following steps: co-culturing solutions (0.31-40.00 mg/mL) of different concentrations of effective parts of the gallnut for 5 hours, centrifuging the bacterial solution, washing the bacterial precipitate for 1 time by using PBS buffer solution, centrifuging the bacterial precipitate again, adding glutaraldehyde, standing in a refrigerator at 4 ℃ for 8 hours, taking out the solution, centrifuging, and washing the bacterial precipitate once by using the PBS buffer solution. Bacterial pellet was centrifuged again and, after gradient dehydration with 10%, 30%, 50%, 70%, 90% and 100% ethanol, the bacterial morphology was observed with a scanning electron microscope.
4. Antimicrobial adhesion capability assay
The experimental method comprises the following steps: streptococcus mutans and actinomyces viscosus biofilms were cultured on enamel working surfaces in 96-well plates. The enamel sections were autoclaved and divided into 4 groups of 3 samples each, wherein both groups were immersed in 10mg/mL galling effective part solution to prepare enamel with galling effective part coating. The bacterial solutions (1×10) of Streptococcus mutans and Actinomyces viscosus are added into each hole 7 CFU/mL) 200 μl, after 24 hours incubation at 37 ℃, the enamel sections were removed, rinsed once with PBS, the biofilm-attached enamel sections were placed in a new 96-well plate, 10mg/mL of gallic active site solution or BHI/TSB (control) was added and incubated for 5 hours at 37 ℃. Each group was randomly sectioned for enamel and the details of the bacteria were observed with a scanning electron microscope.
Experimental results: FIG. 10 shows the bacteriostatic action of the effective fraction of gallnut on free Streptococcus mutans and Actinomyces viscosus. Wherein A is the diameter of the inhibition zone of the treatment of the streptococcus mutans and the actinomyces viscosus by the effective parts of the galls with different concentrations, and when the concentrations are the MIC and the MBC of the streptococcus mutans, the diameters of the inhibition zone are 13.16+/-1.05 mm and 24.80+/-1.21 mm respectively. When the concentration is MIC and MBC of actinomyces viscosus, the diameter of the inhibition zone is 13.58+/-1.54 mm and 22.80+/-1.83 mm respectively; b is the antibacterial rate, and the result shows that the inhibition effect of the effective part of the gallnut on the streptococcus mutans and the myxobacteria is concentration-dependent, and the inhibition rate on the streptococcus mutans and the myxoactinomyces viscosus is increased along with the increase of the concentration; c is a streptococcus mutans plate count test result, D is a actinomycete viscosus plate count test result, and the result shows that the colony number of the two bacteria is reduced along with the increase of the concentration of the effective parts of the galls, the streptococcus mutans grows at 40mg/mL, and the actinomycete viscosus grows at 20 mg/mL;
FIG. 11 shows the biofilm inhibition of S.mutans and Actinomyces viscosus by the active area of the gallic acid, wherein A is a typical morphological image of S.mutans SEM treated with different concentrations of the active ingredients of gallic acid, and B is a typical morphological image of Actinomyces viscosus SEM, resulting in the surface that as the concentration of the active area of gallic acid increases, the morphology of S.mutans and Actinomyces viscosus is destroyed, the integrity and permeability of cell membrane is destroyed, and the cytoplasm flows out, resulting in bacterial death; c is an SEM representative picture of a mutans streptococcus biological film growing on the enamel surface, D is an SEM representative picture of an actinomyces viscosus biological film, a dense and thick bacterial biological film is visible on the enamel surface of a control group, and bacteria growing on the enamel surface of the gallic effective part coating layer are fewer than those of the control group, so that the result shows that the gallic effective part coating layer can effectively inhibit adhesion of the mutans streptococcus and the actinomyces viscosus biological film.
Application example 3
Experimental investigation of anti-inflammatory Properties of the effective fraction of the gallnut obtained in example 1
The experimental method comprises the following steps: SD rats were randomly divided into 4 groups, namely, a negative control group, a positive control group, a model group and a dosing group, 6 each. Except for the negative control group, the remaining 3 groups of rats were anesthetized with 10% chloral hydrate, silk-sutured to the rat's double lower incisor neck, and gum injected with lipopolysaccharide 10 μl. Rats in the negative control group are only anesthetized with 10% chloral hydrate, the dental necks are not sutured, 10% sucrose solution and normal rat grains are fed after the operation is completed, 7 days are continued, normal feeding is performed after the modeling is successful, physiological saline is coated on the negative control group and the model group in the morning and evening, chlorhexidine (CHX) is coated on the positive control group in the morning and evening, and a gallic effective part solution (10 mg/mL) is coated on the administration group in the morning and evening. After 7 days of treatment, all mice were euthanized. A2 mm by 4mm filter paper was placed on the gingival mucosal surface between the lower incisors of the rat for 60s, placed in a freezer tube containing 0.5mL of physiological saline, shaken for 60s, and stored at-80 ℃. IL-6, IL-1β, ALP, AST were detected within 1 week using the corresponding kit. Simultaneously, the gingival tissue at the front gingival margin of 4mm multiplied by 3mm multiplied by 1mm is taken out, washed by normal saline and immediately fixed in 10 percent formaldehyde solution for more than 48 hours. Decalcification with mixed acid, gradient dehydration with ethanol, paraffin embedding, slice thickness of 5 μm, conventional H & E staining, and optical observation.
Experimental results: fig. 12 shows the anti-inflammatory effect of the active fraction of gallnut. Wherein A is a schematic diagram of gingivitis modeling and treatment flow; b is a change image of gums during molding and after different groups of treatments, and the results show that obvious inflammation occurs to the gums and food residues appear to gingival sulcus and teeth during 7 days of molding. After 7d of CHX rinse, food residues were significantly reduced and swelling was improved compared to the model group. After the effective part of the gallnut is treated for 7 days, the gingiva of the rat is basically the same as that of a normal group, and the effective part of the gallnut can be proved to be effective in treating gingivitis; c is a pathological section result, the normal group gingival tissue structure is basically normal, the gingival sulcus wall structure is normal, the natural layer has no inflammatory cell distribution, collagen fibers are rich and orderly arranged, the model group rat tissue structure is moderately abnormal, the epithelial layer structure is normal, a large number of inflammatory cells can be seen around the natural layer capillary, neutrophils and a small number of new blood vessels can be seen, the CHX group gingival tissue structure is normal, the gingival sulcus wall has no inflammatory cell infiltration, a large number of new collagen fibers are consistent with the normal gingival tissue structure, the effective part group rat gingival sulcus wall of the gallnut has no inflammatory cell infiltration, and the collagen fibers travel consistent; d is the weight change trend of the rat in the molding and treatment process of gingivitis; e and F are the expression levels of IL-6 and IL-1 beta after each group treatment, and the CHX and the effective parts of the galls obviously reduce the content of IL-6 and IL-1 beta, but the difference between groups is not obvious; g and H are AST and ALP enzyme activities after treatment of each group, and the effective parts of the gallnut and CHX can obviously reduce the expression level of ALP and AST, so that the results show that the effective parts of the gallnut have good anti-inflammatory activity.
Application example 4
Experimental investigation of the effective fraction of the Galla Turcica obtained in example 1 on biocompatibility
Hemolysis experiment:
the experimental method comprises the following steps: fresh mouse erythrocytes were resuspended in PBS (20%, v/v). 100. Mu.L of the red blood cell suspension and 100. Mu.L of the gallic acid effective part are mixed in PBS solutions with different concentrations (0.31-40.00 mg/mL), incubated at 37 ℃ for 30min and centrifuged at 2000rpm for 10min. OD of the supernatant at 540nm was measured by a microplate reader. Meanwhile, a control group co-cultured with pure water was set. The rate of hemolysis is expressed as the OD540 ratio of the supernatant of the experimental group to the supernatant of the pure water control group.
Cytotoxicity assay:
the experimental method comprises the following steps: mouse fibroblasts (L929) and mouse mononuclear macrophages (RAW 264.7) were inoculated into 96-well plates and incubated with different concentrations of gallic acid active sites (0.31-40.00 mg/mL) for 6h. After removal of the medium, 180. Mu.L of LPBS and 20. Mu.L of CCK-8 reagent were added to each well, incubated for 2h, and OD490 was measured.
Toxicity in vivo experiment:
the experimental method comprises the following steps: 6 Kunming mice were orally administered 10mg/mL of the gallic active site for 7 consecutive days. Water was used as a negative control test. Body weight of each rat was measured daily during the experiment. All mice were sacrificed on day 7 and their organs such as heart, liver, spleen, lung, kidney were histologically stained.
Experimental results: fig. 13 shows the biocompatibility of the gallic effective fraction. Wherein A is the hemolysis rate of the solution of the effective parts of the galls with different concentrations, and compared with pure water (hemolysis 100 percent), the solution of the effective parts of the galls with different concentrations has no obvious hemolysis phenomenon; b is the cytotoxicity of different concentrations of the effective part solution of the gallnut to the L929 and the RAW264.7, the concentration of the effective part solution of the gallnut is 3.12-400.00 mug/mL, the effective part solution of the gallnut has no obvious cytotoxicity to the L929 cells, and the effective part solution of the gallnut has cytotoxicity to the RAW264.7 cells; c and D are weight distribution and histological evaluation (scale bar 200 μm) of mice after oral administration of pure water and 10mg/mL of the effective fraction of the galls, the weight of the mice is not significantly different during the administration, in terms of in vivo toxicity, the effective fraction of the galls is sacrificed 7 days after administration, and histological examination of the mice shows that other major organs have no obvious inflammatory reaction and cell damage. The biocompatibility experiment result shows that the effective parts of the gallnut have good blood compatibility and biocompatibility.
The effective part of the gallnut is effective for preventing and treating dental caries: a protective film can be formed on the teeth to induce tooth remineralization, inhibit the biomembrane viscosity of cariogenic bacteria streptococcus mutans and actinomyces viscosus and effectively kill bacteria; can remarkably reduce the expression of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) inflammatory factors in gingival crevicular fluid, and remarkably reduce the contents of aspartic acid Aminotransferase (AST) and alkaline phosphatase (ALP).
While the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments of the invention can be made and still fall within the scope of the invention without undue effort.
Claims (10)
1. A preparation method of a gallic effective part is characterized by comprising the following steps:
soaking Galla Turcica powder in water, and reflux extracting to obtain extractive solution;
subjecting the extract to column chromatography to obtain eluent; the column chromatography comprises eluting and eluting sequentially, wherein the eluted solution is ethanol solution, and the volume fraction of ethanol in the ethanol solution is 70%;
concentrating and drying the eluent in sequence to obtain the effective part of the nutgall.
2. The method according to claim 1, wherein the column chromatography column is a polyamide column or a C18 column.
3. The preparation method according to claim 1 or 2, wherein the leaching is performed sequentially by a first leaching with an ethanol solution with a volume fraction of 10% and a second leaching with an ethanol solution with a volume fraction of 30%.
4. The preparation method according to claim 1, wherein the mass ratio of the gallic powder to the water is 1:2-3.
5. The method according to claim 1 or 4, wherein the particle size of the gallic powder is 40 to 60 mesh.
6. The method according to claim 1 or 4, wherein the soaking time is 1 to 2 hours.
7. The method according to claim 1, wherein the time of the reflux extraction is 2 to 3 hours.
8. The method according to claim 1 or 7, wherein the number of extraction times of the reflux extraction is 2 to 3.
9. The gallic effective fraction obtained by the production method according to any one of claims 1 to 8, which comprises tetra-O-galloyl glucose, penta-O-galloyl glucose, hexa-O-galloyl glucose and hepta-O-galloyl glucose.
10. Use of the gallic acid-effective fraction of claim 9 in the preparation of a medicament for the treatment and/or prophylaxis of dental caries.
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