CN100525799C - Pharmaceutical composition for preventing and/or treating oral disease caused by microbe - Google Patents
Pharmaceutical composition for preventing and/or treating oral disease caused by microbe Download PDFInfo
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- CN100525799C CN100525799C CNB2004100286791A CN200410028679A CN100525799C CN 100525799 C CN100525799 C CN 100525799C CN B2004100286791 A CNB2004100286791 A CN B2004100286791A CN 200410028679 A CN200410028679 A CN 200410028679A CN 100525799 C CN100525799 C CN 100525799C
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Abstract
The invention relates to a pharmaceutical composition for preventing and/or treating oral disease caused by microbe, which comprises Chinese angelica root, dried rehmannia root, goldthread root, bark of peony root and cimicifuga rhizome.
Description
Technical field
The present invention relates to prevent and/or treat the compositions and the method for the oral disease that causes because of microorganism.More particularly, the present invention relates to utilize traditional Chinese medical science prescription to prevent and/or treat the oral disease that causes because of microorganism.
Background technology
Modern medicine thinks that the morbidity main cause of periodontal disease is due to (1) oral cavity partial pessimal stimulation, and the bacterial plaque that forms as poor oral hygiene, tartar, tartar, regular food down plug, bad dummy stimulate.(2) endocrine regulation (as diabetes, hyperthyroidism), hematologic disease and immunological incompetence disease (leukemia, AIDS), malnutrition, chronic general wasting diseases, or the overtired resistance that causes descends, (see and cause infected by microbes such as streptococcus to draw the around teeth tissue and send out this disease, Cao Caifang, periodontal People's Health Publisher 2000; P.29-65,66-80).Common self awareness symptom has that gingiva is red, swollen, pain, hemorrhage and odontoseisis etc. (seeing Meitner, S.W.Identification of inflamedgingival surface.J.Clin.Periodontol.1979, (6), 93).S.S.Socransky proposed the standard of five periodontal disease infective pathogen bacterium in 1977: (1) this microorganism must be higher than non-disease location at the number of disease location.(2) eliminate this microorganism and can slow down this progression of disease.(3) this microorganism should take place with periodontal disease and the relevant property of toxin of progress.(4) cellular immunization or humoral immunization must have higher reaction to this microorganism, are enough to show the status of this bacterium uniqueness in disease.(5) pathology from animal produces the test, must be able to reason out the formation of human periodontal disease (sees, Socransky, S.S.Microbiology of periodontaldisease-Present status and future consideration.J.Periodontol.1977,48,497-504.).Dental plaque has been confirmed to be and has caused one of pathogenetic main cause of periodontal, the antibacterial of finding in the human now oral cavity is above 400 kinds, wherein the easiest initiation or cause the main plaque bacteria of periodontal inflammation to have porphyromonas gingivalis (Porphyromonas gingivalis), actinomyces viscosus (Actinomyces viscosus), Streptococcus sanguis (Streptococcus sanguis), Streptococcus mutans (Streptococcus mutans) etc. (to see, Ling Lizhen, the periodontal disease machine that causes a disease changes new concept, J.Dent Sci.2001; 21:7-14).
When periodontal tissue is invaded in the microorganism that causes oral disease, cause periodontal tissue to infect.The mankind are to infection, activate in the process of inflammatory response with the opposing bacterial invasion, and the participation of cytokine all need be arranged.Complementary T lymph corpuscle 1 (T
H1 cells) excretory interleukin-2 (IL-2), complementary T lymph corpuscle 2 (T
H2 cells) excretory interleukin-4 (IL-4), (see MosmannTR, Coffman RL.Two types of mouse T helper cell clones:implicationsfor immune regulation.Immunology Today 1987; 8:223-226.), the excretory tumor necrosis factor of macrophage (TNF) etc. is all the excretory cytokine of immunocyte.These a little cytokines all participate in the immunoreation of nature; Regulate lymph corpuscle growth, differentiation and cytoactive; Participate in immune inflammatory response; Stimulate leukocytic growth of immaturity and differentiation.So with human closely bound up to the anti-infective function.
The mode that is used for the treatment of periodontal disease now (is seen Niu Dongping, oral medicine, People's Health Publisher 1998 for removing dental calculus, root of the tooth face planification and oral drugs; P.121-136).The medicine that is usually used in treating periodontal disease is chemobiotic and anti-inflammatory and analgesic agents, though antibiotic and anti-inflammatory and analgesic agents can be alleviated the partial symptoms of periodontal disease acute inflammation phase, bacterial drug resistance that antibiotic and anti-inflammatory and analgesic agents are hidden and adverse side effect phenomenon are big secret worries that can not be ignored.People periodontal pathogenic bacterium (actinobacillus actinomycetem comitans (the Actinobacillus actinomycetemcomitanss different to 50 strains such as scholar Mandinier in 1999, the A.a bacterium)) do the antibiotic resistant test, find that all A.a bacterium are all to antibiotic amoxicillin sensitivity, 4% pair of erythromycin tool Drug resistance, 4% pair of tetracycline tool Drug resistance, 72% pair of antibiotic metronidazole tool Drug resistance (is seen, Mandinier IM, Fosse TB, Hitig C, Charbit Y, Hannoun LR.Resistance profile survey of 50 periodontal strains of Actinobacillusactinomyectomitans.J Periodontol.1999, Aug, 70 (8), 888-92.).So seeking the oral or external curing medicine of better periodontal disease is that its necessity and value are arranged.
Summary of the invention
The traditional Chinese medical science has anxious, slow branch for the periodontal disease disease.The acute rising up of the stomach-fire that belongs to more, symptoms such as red swelling of gingiva burning pain, hemorrhage pyorrhea belong to heat symptom-complex, and treatment is earlier based on antiinflammatory, pain relieving, antibiotic.The chronic void that belongs to the spleen kidneys, tooth more dredge slit, the impractical atrophy of gingiva, dull pain or nothing, root of the tooth a surname reveals, and is the treatment basis with the invigorating spleen and kidney." spleen " of traditional Chinese medical science indication comprises spleen and pancreas, with stomach the exterior and the interior mutually.Major function is fortuneization, rise gas, system blood.Also comprise immune system, hormonal system and central nervous system except influencing digestive system.So function of systems such as spleen reinforcing can reinforced immunological, digestion, endocrine, hemopoietic, nerve.Pharmaceutical composition of the present invention can be in order to suppress the known microorganism that causes oral disease, and other can be used for improving the amount of the secreted cytokine of immune system, reaches the effect of prevention with the immune system that promotes self.
The present invention finds the new purposes of the oral disease that traditional traditional Chinese medical science prescription QINGWEI SAN causes in the treatment microorganism.On Chinese medicine, QINGWEI SAN is made up of Radix Angelicae Sinensis, the Radix Rehmanniae, Rhizoma Coptidis, Cortex Moutan and Rhizoma Cimicifugae.Inexpectancy of the present invention ground finds that the QINGWEI SAN based composition is in the new purposes that treats and/or prevents the oral disease that microorganism causes.
The pharmaceutical composition of the new purposes of tool of the present invention
The invention provides a kind of pharmaceutical composition that is used to prevent and/or treat the oral disease that microorganism causes, it comprises following traditional Chinese medicines: (a) Radix Angelicae Sinensis, (b) Radix Rehmanniae, (c) be selected from the Chinese medicine of Rhizoma Coptidis, Coptis Beltoidea C.Y.CHENG et HSIAO and Coptis Teeta Wall, (d) Cortex Moutan and (e) be selected from the Chinese medicine of Rhizoma Cimicifugae, big SANYE Rhizoma Cimicifugae and Xingan's Rhizoma Cimicifugae.
According to pharmaceutical composition of the present invention, Chinese medicine (a) refers to the dry root of umbelliferae angelica (Angelicasinesis DIELS).Chinese medicine (b) refers to the dry root and rhizome of XUANSHEN section plant Radix Rehmanniae (Rehanniaglutinosa LIBOSCH).Chinese medicine (c) refers to the dry rhizome of the removal fibrous root of ranunculaceae plant Rhizoma Coptidis (Coptis chinesis FRANCH), Coptis deltoidea C.Y.Cheng et Hsiao (Coptis beltoidea C.Y.Chenget Hsiao) or Coptis Teeta Wall (Coptis teeta Wall.).Chinese medicine (d) divests the dry root bark of woody part for ranunculaceae peony (Pooaeonia suffruticosa ANDR.) plant rip cutting.Chinese medicine (e) is the dry rhizome of ranunculaceae plant Rhizoma Cimicifugae (Cimicifuga foetidaLINN.), big SANYE Rhizoma Cimicifugae (Cimicifuga heraleifolia Kom.) or Xingan's Rhizoma Cimicifugae (rhizoma cimicifugae dahuricae) (Cimicifuga dahurica (Turcz.) Maxim.).Preferably, this Chinese medicine (c) is Rhizoma Coptidis, and this Chinese medicine (e) is Rhizoma Cimicifugae.
According to a preferred embodiment of the invention, the ratio of the Chinese medicine in the pharmaceutical composition of the present invention (a), Chinese medicine (b), Chinese medicine (c), Chinese medicine (d) and Chinese medicine (e) is 1.5-5.0:1.5-5.0:1.5-5.0:2.5-8.0:5.0-15.0, preferably, this ratio is 2.5-3.5:2.5-3.5:2.5-3.5:4.5-5.5:9.5-10.5, most preferably, this ratio is 3.6:3.6:3.6:6:12.
According to the present invention, any method of using Chinese medicine all can be used for using of pharmaceutical composition of the present invention.Preferably, using of pharmaceutical composition of the present invention is after Chinese medicine (a), Chinese medicine (b), Chinese medicine (c), Chinese medicine (d) and Chinese medicine (e) are mixed, add an amount of solvent, leave standstill after 1 hour and be heated, treat that transferring little fire to after it seethes with excitement simmers, and filters and collects filtrate, so repeatedly for several times, merging filtrate promptly gets the crude extract of pharmaceutical composition of the present invention.Preferably, wherein this extractant is a water, and repeatable operation extracts composition number of times is four times.Perhaps, with Chinese medicine (a), Chinese medicine (b), Chinese medicine (c), Chinese medicine (d) and Chinese medicine (e), extract indivedual extracts respectively according to aforesaid way.Again the indivedual extracts of gained are mixed.
According to the present invention, pharmaceutical composition of the present invention can collutory or the capsule oral mode use.Preferably, can obtain preferable effect so that the collutory capsule-containing is oral in preventing and/or treating the oral disease that causes because of microorganism.Collutory is used, and gargles every day with the pharmaceutical composition of the present invention of concentration 50 mg/ml to 1500 mg/ml and can effectively suppress and eliminate the microorganism that causes oral disease for several times.Preferably, this collutory concentration is 250 mg/ml to 1000 mg/ml.Orally administered, give pharmaceutical composition of the present invention every day according to body weight 250 mg/ml to 750 mg/ml.Preferably, give pharmaceutical composition of the present invention every day according to body weight 500 mg/ml.
Pharmaceutical composition of the present invention carries out quantitative antibacterial experiment with dilution method, the minimum dose therapeutically effective (MIC) that inference is possible.Pharmaceutical composition of the present invention is established feasibility and curative effect with the treatment by Chinese herbs periodontal disease with periodontal disease morbid state zootype.Detect the variation of interleukin-2 (IL-2), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-a) in the female BALB/c mouse body that gives the present invention in 28 days continuously and form crude drug with ELISA, to inquire into the influence of the present invention to immunoregulation capability.
By the antibacterial QINGWEI SAN of discovering that branch is fried in shallow oil stronger bacteriostasis can be arranged, learn to close by the zoopery of periodontal morbid state and fry in shallow oil QINGWEI SAN and stronger antiinflammatory repair is arranged for Inflamed tissue, therefore on preparation uses in the future, our suggestion is fried in shallow oil QINGWEI SAN with branch and is controlled the oral environment of periodontal sufferer as the mode of external or rinsing the mouth, and the oral medication periodontal disease is excellent to close the mode of frying in shallow oil still.
Description of drawings
Fig. 1 for bring out morbid state the 5th day, the periodontal cosmetic variation, redness and dental plaque appear in the BALB/c mouse gingiva.
Fig. 2 is for bringing out morbid state the 7th, and periodontal cosmetic variation, BALB/c mouse occur and the similar symptom of human periodontal lesion tissue, as red swelling of gingiva, gingival atrophy, touching is hemorrhage, alveolar bone absorbs, tooth shakes etc.
Fig. 3 fries in shallow oil QINGWEI SAN 1000 mg/ml and treats periodontal cosmetic variation on the 5th, ill inflammation symptom that BALB/c mouse is previous such as red swelling of gingiva, gingival atrophy, the hemorrhage extinction tests that all occurred of touching for closing.
Fig. 4 treats periodontal cosmetic variation on the 5th for giving tetracycline 250 mg/ml, ill inflammation symptom that BALB/c mouse is previous such as red swelling of gingiva, gingival atrophy, the hemorrhage extinction tests that all occurred of touching.
Fig. 5 closes to fry in shallow oil the BALB/c mouse periodontal cosmetic variation that QINGWEI SAN 1000 mg/ml were treated the 7th, previous ill inflammation symptom such as red swelling of gingiva, gingival atrophy, the hemorrhage all complete obiterations of touching.
Fig. 6 gives the BALB/c mouse periodontal cosmetic variation that tetracycline 250 mg/ml were treated the 7th, previous ill inflammation symptom such as red swelling of gingiva, gingival atrophy, the hemorrhage all complete obiterations of touching.
Inflammation diseases such as Fig. 7 fries in shallow oil QINGWEI SAN 500 mg/ml and treats BALB/c mouse periodontal cosmetic variation on the 5th for closing, and red swelling of gingiva, touching property are hemorrhage all do not exist and the gingiva outward appearance is replied.
Inflammation diseases such as Fig. 8 fries in shallow oil QINGWEI SAN 500 mg/ml and treats BALB/c mouse periodontal cosmetic variation on the 5th for dividing, and red swelling of gingiva, touching property are hemorrhage all do not exist and the gingiva outward appearance is replied.
Fig. 9 closes histopathologic slide's microscopy of frying in shallow oil the BALB/c mouse of QINGWEI SAN 250 mg/ml after 7 days.
Figure 10 closes histopathologic slide's microscopy of frying in shallow oil the BALB/c mouse of QINGWEI SAN 500 mg/ml after 7 days.
Figure 11 is histopathologic slide's microscopy that branch is fried in shallow oil the BALB/c mouse of QINGWEI SAN 500 mg/ml after 7 days.
Figure 12 is the histopathologic slide's microscopy that gives the BALB/c mouse of QINGWEI SAN 1000 mg/ml after 7 days.
Figure 13 is the histopathologic slide's microscopy that gives the BALB/c mouse of tetracycline 250 mg/ml after 7 days.
Figure 14 is the histopathologic slide's microscopy that gives the BALB/c mouse of deionized water after 7 days.
The specific embodiment
Embodiment 1 preparation of drug combination of the present invention
The composition medical material that this experiment is used, determine that through the pharmacognosy base is former as follows:
Radix Angelicae Sinensis (Angelicae Sinensis Radix): this product is the dry root of umbelliferae angelica (Angelica sinesis DIELS).
Radix Rehmanniae (Rehmanniae Radixet Rhizom): this product is the dry root and rhizome of XUANSHEN section plant Radix Rehmanniae (Rehannia glutinosa LIBOSCH).
Rhizoma Coptidis (Coptidis Rhizoma): this product is the dry rhizome that ranunculaceae plant Rhizoma Coptidis (Coptis chinesisFRANCH) is removed fibrous root.
Cortex Moutan (Moutan Radicis Cortex): this product is that ranunculaceae peony (Pooaeonia suffruticosa ANDR.) plant rip cutting divests the dry root bark of woody part.
Rhizoma Cimicifugae (Cimicifuga foetida): this product is ranunculaceae plant Rhizoma Cimicifugae (Cimicifugafoetida LINN.) and the dry rhizome that belongs to kindred plant together.
Above-mentioned experiment medical material is complied with
Chinese medicine committee of Department of Health of Executive YuanQINGWEI SAN benchmark prescription proportion of composing (Radix Angelicae Sinensis: the Radix Rehmanniae: Rhizoma Coptidis: Cortex Moutan: Rhizoma Cimicifugae=3.6: 3.6: 3.6: 6: 12), weigh the common 288g of above-mentioned crude drug in whole and add an amount of pure water, leave standstill after 1 hour and be heated, treat that transferring little fire to after its boiling simmers until volume and be less than or equal to 144 milliliters, filtered while hot is collected filtrate, so quadruplication, merging filtrate, reconcentration to 144 milliliter promptly gets QINGWEI SAN crude extract (being 2 grams per milliliter QINGWEI SAN extractums), and this fries in shallow oil QINGWEI SAN for closing.Its each single composition medicine is respectively got 100 grams and is made 50 milliliters of extractums according to the said method extraction, again according to Chinese angelica extraction liquid: Radix Rehmanniae extract: the Rhizoma Coptidis extract: the Cortex Moutan extract: the volume ratio of Rhizoma Cimicifugae extract 3.6: 3.6: 3.6: mix at 6: 12, this fries in shallow oil QINGWEI SAN for dividing.
The effect of 2 pairs of periodontal pathogen of embodiment
The main mode of this anti-periodontal disease pathogenic bacterium susceptibility experiment is dilution method and disk agar gel diffusion test (Disc agar diffusion test), the main purpose of dilution method is for the minimum inhibitory concentration that determines the anti-periodontal disease pathogenic bacterium of QINGWEI SAN (MIC), is convenient to the selection of therapeutic dose in the future.The disk agar gel diffusion test has operation simple and easy and interpretation advantage easily, and we can growth inhibited circle size be occurred and have or not judging the susceptibility of experimental strain to medicine by disk perimeter.This tests employed staphylococcus aureus (Staphylococcus aureus) and escherichia coli (Escherichia coli) bacterial strain is to be the ATCC reference culture that teacher Lin Shijie provides by middle mountain medical university medical technologies, and all the other bacterial strains are all preserved available from Foodstuff Industrial Development Inst. of Financial Group Legal Persons's living resources and the reference culture in research center: Streptococcus mutans ATCC 25175, CCRC10793, Streptococcus sanguis ATCC 49295, CCRC 15273, porphyromonas gingivalis (Porphyromonas gingivialis) ATCC 33277, CCRC 14417, staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922.
This tests employed culture medium all available from opening glad bio tech ltd, and kinds of culture medium is as follows:
The brain heart leaches culture fluid (BHI broth)
Thiosulfate culture fluid (Thioglycollate broth)
Bacterial alkaline phosphatase agar (BAP Agar)
Caseinhydrolysate agar (Mueller-Hinton Agar)
Actication of culture
The following step is all operated under gnotobasis: to speckle with the Cotton Gossypii of 70% ethanol, wiping outer tube, the heat insulation fiber front end of heating outer tube on flame.Drip in heating place, after the outer tube be full of cracks, crack with strong again.Take out heat insulation fiber and interior pipe, the tampon of pipe in taking out with tweezers.With aseptic straw, draw 0.3～0.5 milliliter of specified fluid medium, pipe washes thalline in splashing into; Suction is put for several times, makes the abundant homogenizing of bacterium liquid.Draw this thallus suspension liquid as early as possible, splash in the specified fluid medium, under specified temperature, cultivated 3～4 days.Culture fluid is muddiness and promptly represents strain growth.
Minimum inhibitory concentration (MIC): broth dilution method
With QINGWEI SAN extract 2 grams per milliliters, do the twice serial dilution, making its ultimate density respectively is 2 * (1000 mg/ml), 4 * (500 mg/ml), 8 * (250 mg/ml).Aseptic standard medium BHI broth is done following configuration: A pipe 5cc culture fluid+5cc sterilized water, B pipe 5cc culture fluid+5cc QINGWEI SAN extract 2gm/ milliliter, C pipe 5cc culture fluid+5cc QINGWEI SAN extract (1000 mg/ml), D pipe 5cc culture fluid+5cc QINGWEI SAN extract (500 mg/ml), E pipe 5cc culture fluid+5cc QINGWEI SAN extract (250 mg/ml), F pipe 5cc culture fluid+5cc is equivalent to the tetracycline of 500 mg/ml.Add 50 μ L bacterium liquid in A～F pipe with being cultured to the bacterium liquid that turbidity is equivalent to 0.5 reduced turbidity, place 37 ℃ of incubators to cultivate.When A pipe turbidity was equivalent to 0.5 reduced turbidity, the bacterium amount so that inoculating loop is got 1 inoculating loop in A～F pipe was inoculated on the BAP culture medium, place 37 ℃ of incubators to cultivate after, calculate clump count, calculate the QINGWEI SAN Mlc.
We adjust blank group (deionized water) growth bacterium colony number in being lower than 200 via trial test.As shown in table 1 below, QINGWEI SAN can be observed in 8X dilution (being equivalent to 125 mg/ml QINGWEI SAN) has growth inhibiting phenomenon to bacterium such as Streptococcus mutans, Streptococcus sanguis, staphylococcus aureus, escherichia coli, then is to have bactericidal effect for porphyromonas gingivalis.Staphylococcus aureus, escherichia coli can be suppressed fully in QINGWEI SAN 8X dilution (being equivalent to 250 mg/ml QINGWEI SAN) growth; Can be observed the complete repressed phenomenon of growth of Streptococcus mutans and Streptococcus sanguis in QINGWEI SAN 4X dilution (being equivalent to 500 mg/ml QINGWEI SAN) and 2X dilution (being equivalent to 1000 mg/ml QINGWEI SAN).Matched group tetracycline 250 mg/ml all have Streptococcus sanguis, porphyromonas gingivalis, staphylococcus aureus, escherichia coli and suppress the growth effect fully, but then can't suppress fully Streptococcus mutans.Tetracycline 250 mg/ml are equally matched with QINGWEI SAN 8X dilution (being equivalent to 250 mg/ml QINGWEI SAN) to the bacteriostasis of Streptococcus mutans, but QINGWEI SAN 4X dilution (being equivalent to 500 mg/ml QINGWEI SAN) then obviously is better than tetracycline 250 mg/ml to the bacteriostasis of Streptococcus mutans.
Table 1
Porphyromonas gingivalis cultivation temperature and time are 37 ℃, 72 hours; Streptococcus sanguis, Streptococcus mutans cultivation temperature and time are 37 ℃, 24 hours; Staphylococcus aureus, escherichia coli cultivation temperature and time are 37 ℃, 12 hours.
Bacteriostasis rate %=(blank group growth clump count-experimental group growth clump count) ÷ blank group growth clump count * 100%
The disk agar gel diffusion test
To close and fry in shallow oil QINGWEI SAN (500 mg/ml), divide and to fry in shallow oil QINGWEI SAN (500 mg/ml) and each 10 μ L of deionized water and be incorporated in sterilization after the 6mm diameter disk.
Because the disk agar gel diffusion test is not suitable for speed of growth porphyromonas gingivalis more slowly, therefore we only select Streptococcus mutans, the research of Streptococcus sanguis two strain periodontal disease pathogenic bacterium in the disk agar gel diffusion test.
To be cultured to Streptococcus mutans, the Streptococcus sanguis bacterium liquid that turbidity is equivalent to 0.5 reduced turbidity, add 50 μ L bacterium liquid on culture medium, on average be applied in agar surface in three directions, make the former average mark cloth of inoculation with aseptic cotton rod.Cover the lid of agar, in regular turn that above-listed pastille disk is smooth in agar surface after about 3～5 minutes, place 37 ℃ of incubators to cultivate after, measure it and suppress the circle size.Aseptic disk diameter is 6mm, suppresses loop diameter and represents that greater than 6mm this medicine has fungistatic effect (+); Suppress loop diameter and represent that less than 6mm this medicine do not have a fungistatic effect ().Cultivation temperature and time are 37 ℃, and 24 hours, the control group was the disk that contains sterilized water, suppress the numerical value of circle and represent (n=3) with Mean ± SD.Branch as shown in table 2 below is fried in shallow oil the QINGWEI SAN bacteriostasis and obviously is better than closing and fries in shallow oil QINGWEI SAN.
Table 2
The assessment of embodiment 3 periodontal disease therapeutic effects
Select 60 of the female BALB/c mouse in eight ages in week for use, 10 every group, be divided into 6 groups.This tests employed animal all available from National Science Council's Animal Experimental Study and breeding center SPF level experimental mouse, male eight age in week ICR be mice, female eight age in week ICR be mice and female 6 age in week BALB/c mouse.Raise in 25 ± 1 ℃ of room temperatures relative humidity 55 ± 5%, irradiation and dark each middle mountain medical university animal center of 12 hours.
Dentition correcting steel wire on its lower jaw labial teeth is pricked on the 1st, and in periodontal tissue inoculation periodontal disease pathogenic bacterium.The 5th, as shown in Figure 1, redness and dental plaque appearred in the BALB/c mouse gingiva.The 7th, as shown in Figure 2, BALB/c mouse occurred and the similar symptom of human periodontal lesion tissue, as red swelling of gingiva, gingival atrophy, touching is hemorrhage, alveolar bone absorbs, tooth shakes etc.Unmuzzle this moment, and beginning gives following medicine with oral way according to body weight: close and fry in shallow oil QINGWEI SAN 250 mg/ml, close and fry in shallow oil QINGWEI SAN 500 mg/ml, close and fry in shallow oil QINGWEI SAN 1000 mg/ml, divide and to fry in shallow oil 2 milliliters/milliliter of QINGWEI SAN 500 mg/ml, tetracycline 250 mg/ml and deionized waters.As shown in table 3, after administration the 3rd day, can obviously find to close and fry in shallow oil QINGWEI SAN (250 mg/ml, 500 mg/ml, 1000 mg/ml), divide the BALB/c mouse of frying in shallow oil QINGWEI SAN (500 mg/ml) and tetracycline (250 mg/ml) all can find the appearance of re-epithelialize in inflammation place.After administration, observed in 5th, close previous ill inflammation symptom of the BALB/c mouse of frying in shallow oil QINGWEI SAN 1000 mg/ml and giving tetracycline 250 mg/ml as shown in Figure 4 such as red swelling of gingiva, gingival atrophy, the hemorrhage extinction tests that all occurred of touching as shown in Figure 3.After administration, observed in 7th, as shown in Figure 5, close previous ill inflammation symptom of the BALB/c mouse of frying in shallow oil QINGWEI SAN 1000 mg/ml such as red swelling of gingiva, gingival atrophy, the hemorrhage all complete obiterations of touching; As shown in Figure 6, give previous ill inflammation symptom of the BALB/c mouse of tetracycline 250 mg/ml such as red swelling of gingiva, gingival atrophy, the hemorrhage all complete obiterations of touching; Close as shown in Figure 7 and fry in shallow oil that inflammation diseases such as QINGWEI SAN 500 mg/ml and the red swelling of gingiva that divides the BALB/c mouse fry in shallow oil QINGWEI SAN 500 mg/ml as shown in Figure 8, touching property be hemorrhage all do not exist and the gingiva outward appearance is replied.By BALB/c mouse that gives QINGWEI SAN 250 mg/ml and the BALB/c mouse that gives deionized water compare the inflammation diseases such as red swelling of gingiva that can find BALB/c mouse all give deionized water BALB/c mouse gentle many.Behind drug treatment the 7th day, can see by histopathologic slide's microscopy, as shown in Figure 9, close histopathologic slide's microscopy of the BALB/c mouse of frying in shallow oil QINGWEI SAN 250 mg/ml; As shown in figure 10, close histopathologic slide's microscopy of the BALB/c mouse of frying in shallow oil QINGWEI SAN 500 mg/ml; As shown in figure 11, the histopathologic slide's microscopy that divides the BALB/c mouse of frying in shallow oil QINGWEI SAN 500 mg/ml; The histopathologic slide's microscopy that gives the BALB/c mouse of QINGWEI SAN 1000 mg/ml as shown in figure 12 also or as shown in figure 13 gives all visible inflamed sites of histopathologic slide's microscopy of the BALB/c mouse of tetracycline 250 mg/ml and dwindles and tissue repair, with as shown in figure 14, the tissue inflammation's situation that gives the BALB/c mouse of deionized water has notable difference.
Table 3
(red swelling of gingiva, touching property are hemorrhage, gingival atrophy, gingiva suppurate, dental plaque) is defined as the appearance of periodontal disease table disease when following three kinds of symptoms appear in mice.(red swelling of gingiva, touching property are hemorrhage, gingival atrophy, gingiva suppurate, dental plaque) is defined as the disappearance of periodontal inflammation table disease when following transference cure.Periodontal inflammation table disease disappearance degree is divided into 10 grades, represents with n, when n=10, the complete obiteration of expression periodontal inflammation table disease.
The influence of 4 pairs of BALB/c mouse serum cytokines of embodiment
Select 36 of the female BALB/c mouse in six ages in week for use, 12 every group, be divided into 3 groups.Mice source is as described in the embodiment 2.Give following medicine with oral way according to body weight every day: close and fry in shallow oil QINGWEI SAN 500 mg/ml, divide and to fry in shallow oil 2 milliliters/milliliter of QINGWEI SAN 500 mg/ml and deionized waters.Respectively at carotid artery blood sampling in 7,14,21,28 after the administration,, after under 4 ℃ the condition centrifugal 10 minutes, get upper serum detecting IL-2, IL-4, TNF-α concentration change at 3000r.p.m..
Preparation of ELISA culture dish and flow process
100 μ l have been diluted good anti-Mus cytokine capture antibody (anti-mouse cytokinecapture antibody) to add a trace dish (microwell) and leave standstill an evening under 4 ℃ of environment.The next day, (0.005% Tween 20, in 1X PBS, good trace drips dish flushing 3 times pH7.4) will to apply (coating), so that unnecessary or loose capture antibody (capture antibod) is removed with dcq buffer liquid (wash buffer).Then with 200 μ l experiment diluents (assay diluent) adding trace dish and after leaving standstill 1 hour under the room temperature, with dcq buffer liquid flushing 3 times.Again 100 μ l standards or sample are added a trace dish, after leaving standstill 2 hours under the room temperature, with dcq buffer liquid flushing 5 times.Then, add 100 μ l operation detection liquid (working detector) (detecting antibody+Avidin-horseradish peroxidase (avidin HRP)) and drip dish, after leaving standstill 1 hour under the room temperature, with dcq buffer liquid flushing 7 times in trace.Drip in the dish in trace and to add 100 μ l substrate solutions (3,3 ', 5,5 '-tetramethyl benzidine (TMB)+hydrogen peroxide) as colour former, shading leave standstill through 30 minutes samples by transparent transfer to blue.At last with 50 μ l stop buffer (2N H
2SO
4) add, sample is surveyed light absorption value with little dish analyser (microplate reader) at the 450nm wavelength by the blue yellow that transfers to.Each experiment is all compared through triple redoubling and standard inspection amount line, calculates to record sample concentration.This research, empirical value is represented with meansigma methods ± standard deviation.
As shown in table 4, close and fry in shallow oil QINGWEI SAN 500 mg/ml and divide BALB/c mouse serum il-2 rapid rising on the 7th after administration of frying in shallow oil QINGWEI SAN 500 mg/ml, be tending towards subsequently relaxing.IL-2 is the multiplicaiton factor of T cell and the activation of TCR also can be brought out production of cytokines, most CD4
+Th cell and CD8
+The T cell is can be in 1-2 days of short duration produces IL-2, during the period the reciprocal action of IL-2 and high affinity IL-2 receiver and cause the activation and the growth of T cell.This high affinity IL-2 receiver approximately only occurred about 1 week, promptly stimulated the propagation of TCR restricted T cells afterwards.So we see QINGWEI SAN 500 mg/ml and the situation of the IL-2 value of dividing the BALB/c mouse of frying in shallow oil QINGWEI SAN 500 mg/ml in the appearance decline of the 2nd week of frying in shallow oil of closing.But close and fry in shallow oil QINGWEI SAN 500 mg/ml and fry in shallow oil QINGWEI SAN 500 mg/ml and regulate the BALB/c mouse that the ability that increases all gives deionized water in IL-2 and obviously increase with dividing.
Table 4
| The 1st week | The 2nd week | The 3rd week | The 4th week | |
| Close and fry in shallow oil QINGWEI SAN | 49.75±22.52 * | 27.9±4.35 | 29.2±4.74 | 35.39±6.71 |
| Divide and fry in shallow oil QINGWEI SAN | 54.19±26.94 * | 32.58±3.06 | 30.85±8.6 | 26.85±0.54 |
| Deionized water | 7.99±0.81 | 14.18±11.53 | 29.97±0.38 | 29.43±1.38 |
The numerical value of IL-2 is represented with Mean ± SD, unit: the pg/ milliliter
As shown in table 5, take to close when BALB/c mouse and fry in shallow oil in the QINGWEI SAN 500 mg/ml serum on the 7th IL-4 numerical value and divide and fry in shallow oil 7 days BALB/c mouse height of QINGWEI SAN 500 mg/ml, even exceed two times more than, the 2nd week played both and promptly reaches unanimity, but with the BALB/c mouse that gives deionized water remarkable increase was arranged more then.
Table 5
| The 1st week | The 2nd week | The 3rd week | The 4th week | |
| Close and fry in shallow oil QINGWEI SAN | 2.28±1.36 | 1.24±0.44 | 0.72±0.25 | 1.55±0.64 |
| Divide and fry in shallow oil QINGWEI SAN | 1.33±0.67 | 1.24±0.36 | 0.72±0.24 | 1.55±1.11 |
| Deionized water | 0.75±0.48 | 0.62±0.25 | 1.28±0.63 | 0.94±0.07 |
The numerical value of IL-4 is represented with Mean ± SD, unit: the pg/ milliliter
As shown in table 6, as if observe BALB/c mouse serum TNF-α concentration in the TNF-α concentration change of BALB/c mouse has situation about raising along with increasing age in week to occur, no matter and be to close to fry in shallow oil QINGWEI SAN 500 mg/ml or branch is fried in shallow oil QINGWEI SAN 500 mg/ml, all can stimulate BALB/c mouse serum TNF-α concentration to raise and the BALB/c mouse that gives deionized water significantly increases.
Table 6
| First week | Second week | The 3rd week | Around | |
| Close and fry in shallow oil QINGWEI SAN | 25.15±17.21 | 220±129.9 | 319.05±118.34 | 691.89±109.71 * |
| Divide and fry in shallow oil QINGWEI SAN | 28.63±11.42 | 300.43±169.45 | 309.54±67.83 | 679.72±54.86 * |
| Deionized water | 14.02±3.89 | 152.28±20.15 | 243.37±29.75 | 387.03±24.13 |
The numerical value of TNF-α is represented with Mean ± SD, unit: the pg/ milliliter
Claims (6)
1. a compositions is used for promoting the purposes of the medicine of IL-2, IL-4 or TNF-α in preparation, wherein said composition is made up of following traditional Chinese medicines: (a) Radix Angelicae Sinensis, (b) Radix Rehmanniae, (c) be selected from the Chinese medicine of Rhizoma Coptidis, Coptis deltoidea C.Y.Cheng et Hsiao and Coptis Teeta Wall, (d) Cortex Moutan reaches the Chinese medicine that (e) is selected from Rhizoma Cimicifugae, big SANYE Rhizoma Cimicifugae and Xingan's Rhizoma Cimicifugae, and wherein the ratio of this Chinese medicine (a), Chinese medicine (b), Chinese medicine (c), Chinese medicine (d) and Chinese medicine (e) is 1.5-5.0:1.5-5.0:1.5-5.0:2.5-8.0:5.0-15.0.
2. purposes as claimed in claim 1, wherein this Chinese medicine (c) is Rhizoma Coptidis, and this Chinese medicine (e) is Rhizoma Cimicifugae.
3. purposes as claimed in claim 1, wherein the ratio of this Chinese medicine (a), Chinese medicine (b), Chinese medicine (c), Chinese medicine (d) and Chinese medicine (e) is 3.6:3.6:3.6:6:12.
4. purposes as claimed in claim 1, wherein said composition is an extract.
5. purposes as claimed in claim 4, wherein this extract is made by following method: Radix Angelicae Sinensis, the Radix Rehmanniae, Rhizoma Coptidis, Cortex Moutan and Rhizoma Cimicifugae are mixed the back with this mixture of solvent extraction, or respectively with solvent extraction, indivedual extracts are mixed.
6. purposes as claimed in claim 5, wherein this solvent comprises water.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100286791A CN100525799C (en) | 2004-03-12 | 2004-03-12 | Pharmaceutical composition for preventing and/or treating oral disease caused by microbe |
| HK06103257.6A HK1083199A1 (en) | 2004-03-12 | 2006-03-14 | A pharmaceutical composition for use in the prevention and/or treatment of the oral cavity diseases caused by microorganisms |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100286791A CN100525799C (en) | 2004-03-12 | 2004-03-12 | Pharmaceutical composition for preventing and/or treating oral disease caused by microbe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1666773A CN1666773A (en) | 2005-09-14 |
| CN100525799C true CN100525799C (en) | 2009-08-12 |
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| CNB2004100286791A Expired - Fee Related CN100525799C (en) | 2004-03-12 | 2004-03-12 | Pharmaceutical composition for preventing and/or treating oral disease caused by microbe |
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| HK (1) | HK1083199A1 (en) |
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| CN104257864A (en) * | 2014-10-27 | 2015-01-07 | 济南舜景医药科技有限公司 | Medicine for treating toothache, and preparation method |
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2006
- 2006-03-14 HK HK06103257.6A patent/HK1083199A1/en not_active IP Right Cessation
Non-Patent Citations (2)
| Title |
|---|
| 清胃散治疗牙周炎49例. 尹沂平.辽宁中医杂志，第20卷第12期. 1993 |
| 清胃散治疗牙周炎49例. 尹沂平.辽宁中医杂志，第20卷第12期. 1993 * |
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| Publication number | Publication date |
|---|---|
| HK1083199A1 (en) | 2006-06-30 |
| CN1666773A (en) | 2005-09-14 |
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