CN104083466B - Preparation utilizing compatibility prescription of effective parts of rhizoma coptidis and rhizoma drynariae in treatment of periodontitis - Google Patents

Abstract

The invention relates to a preparation utilizing a compatibility prescription of the effective parts of rhizoma coptidis and rhizoma drynariae in treatment of periodontitis. The preparation comprises the following components by mass: 10-15 parts of a rhizoma coptidis effective part's water extract with a berberine content of more than 80%; and a rhizoma drynariae effective part's water extract with a naringin content of more than 30.71%. The preparation can be prepared into a ''rhizoma coptidis-rhizoma drynariae tooth strengthening capsule'' for systemic administration, or can be added with hydroxyethyl cellulose, concentrated glycerol, polyacrylic resin and other auxiliary materials to make a novel topical Chinese medicinal long-acting compound ointment ''rhizoma coptidis-rhizoma drynariae tooth strengthening ointment'', and a syringe administration mode is easy for operation at local parts of a periodontal pocket. With a good therapeutical effect on periodontitis, the preparation provided by the invention has the characteristics of small prescription, significant curative effect, no toxic or side effect. Especially in local application, after long-term use, the anti-inflammatory effect is similar to that of minocycline hydrochloride ointment (Periocline), but the osteogenic effect is obviously superior to that of Periocline. Thus, the preparation is a novel oral Chinese patent medicine with long-term efficacy and usability similar to western medicines.

Description

Utilize Rhizoma Coptidis and the preparation of Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis

Technical field

The present invention relates to application and the preparation thereof of a kind of Chinese patent medicine, utilize Rhizoma Coptidis and the effective portion of Rhizoma Drynariae particularly to one The preparation of position composition of prescription treatment periodontitis, uses periodontal pocket local injection formula to be administered or whole body Oral administration, is used for controlling Treat periodontitis.

Background technology

Periodontitis is the disease that field, oral cavity sickness rate is the highest, is also the difficult and complicated illness of oral medical field, it has also become become The Etiological of year people's loss of tooth.It is to many systemic diseases simultaneously, such as rheumatism, heart disease, hematopathy, diabetes etc. also There is the effect of induction it is considered to be threaten the third-largest killer of human body health after cancer, cardiovascular and cerebrovascular disease.Periodontal Inflammation is a kind of chronic bacterial infection disease, and periodontal tissue destruction is mainly by immunoprotection and the failure mechanism imbalance of host Caused, simultaneously as its open environment makes its inflammation healing and tissue regeneration relatively difficult, therefore the treatment of periodontitis needs Reach to control inflammation, regulate immunity, promotion soft or hard paradenlal tissue regeneration simultaneously.Drug therapy is the important auxiliary of periodontitis treatment Means, but simple western medicine is only capable of for partial symptoms such as inflammation etc., and it is difficult to long because being prone to produce drug resistance and toxic and side effects Phase medication.

The Chinese medicine of China always with low cost, side effect is little, evident in efficacy and is spoken approvingly of by world medicine, wherein Active substance have by Mutiple Targets, too many levels play multiple effect advantage.Motherland's medical science is thought, by determining the pathogenesis of ZANG-FU disease based on the differentiation of symptoms and signs tooth Belong to the category of kidney.The Etiological of periodontitis is gastrointestinal accumulated heat, weak constitution, debility, nephrons loss and the factor such as oral cavity is unclean, Main pathogenesis is stomach internal organs accumulated heat, and sun naked light is vehement, damp and hot fumigates in upper and form gingival atrophy, therefore will with the kidney invigorating void, clearing away stomach-heat, invigorate blood circulation Removing toxic substances opinion is controlled.A lot of Chinese medicines have proven to have anti-inflammation and sterilization, regulation immunity, the effect of balance bone metabolism simultaneously, may be suitable for In the treatment of periodontitis, mainly include Radix Et Rhizoma Rhei, Rhizoma Coptidis, Rhizoma Coptidis, Galla Chinensis, Rhizoma Drynariae, Herba Epimedii, Fructus Psoraleae, Radix Scrophulariae and Radix Angelicae Sinensis Deng.Accordingly, some Chinese patent medicine prescriptions can be used for the treatment of periodontitis, mainly includes that bushen guchi pills, FUKE QIANJIN PIAN, Calculus Bovis are clear Stomach ball, yunü decoction, Bolus as a Kidney-Yin-Tonic, Yougui Wan, the kidney-Yang-Reinforcing Bolus, the kidney tonifying side of relieving inflammation or internal heat etc., wherein Gu Chi Wan, teeth consolidating cream are also with ancient prescription LIUWEI DIHUANG WAN Based on preparation.

But the above prescription lacks the support of pharmaceutical research, and from the angle of the quality of the pharmaceutical preparations, this kind of medicine steady Qualitative, controllability also is difficult to realize.Effective ingredient in Chinese refers in the extract of a herb or herbal mixture, a class or several The content of class chemical composition reaches more than the 50% of total extract, is a class or the mixture of a few constituents.In recent years, in utilization Medicine effective site and components compatibility prescription, have become as Chinese patent medicine research recently and the feature of clinical development.Effective ingredient in Chinese Meet Chinese medicine application traditional theory: organic conception and dialectical treatmert, there is the feature of multicomponent, Mutiple Targets；Chinese medicine is effective Composition and effective site are to be obtained by improving of extracting method, can regard it as the Chinese medicine refined, and more traditional Chinese medicine is more For stable and controlled；Curative effect is more excellent compared with Chinese medicine, and toxic and side effects is relatively low；Also save medical material simultaneously.

Summary of the invention

In order to overcome above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of for treating in periodontitis Patent medicine preparation, from Rhizoma Coptidis, Rhizoma Drynariae single medicinal material, filters out its effective site and index components (effective ingredient) respectively, and then It is new medicament by effective site in optium concentration and ratio composition of prescription, can be used for clinical treatment periodontitis.

Utilizing Rhizoma Coptidis and the preparation of Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis, its raw material components wraps in mass ratio Include for:

Rhizoma Coptidis effective site water extract 10-15 part containing berberine more than 80%

Rhizoma Drynariae effective site water extract 5-10 part of naringin more than 30.71%

Utilizing Rhizoma Coptidis and the preparation of Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis, its raw material components wraps in mass ratio Include for:

Rhizoma Coptidis is cohosh, has heat clearing and damp drying, effect of eliminating fire and detoxication；Rhizoma Coptidis be mainly composed of multiple biology Alkali, including berberine also known as berberine (bcrberine), coptisine (coptisinc), 13-methyl-.psi.-coptisine. (worcnine) and Epiberberine (epibcrberine) etc..Rhizoma Drynariae is the dry rhizome of Plants of Polypodiaceae Mongolian oak Herba pteridii latiusculi, and effect is the kidney invigorating bone strengthening, continues Hinder pain relieving etc.；Rhizoma Drynariae water extract is water solublity effective site, and naringin is the most topmost a kind of flavone component.

Inventor first passes through Methyl thiazoly tetrazolium assay (MTT) colorimetry, enzyme linked immunosorbent assay (ELIISA) and semidefinite Amount RT-polymerase chain reaction (RT-PCR) the detection alkaline phosphatase activities of people's periodontal ligament cell, the table of NTx albumen Reaching and the expression of OPG mRNA confirms, the Rhizoma Drynariae Aqueous extracts (containing naringin 30.71%) of 1.0mg/L is to promoting periodontal ligament cell Breed and the strongest to skeletonization direction transformation；Also by hPDLCs/CMC-HPLC/MS on-line coupling method, filter out Rhizoma Coptidis Water position is effective site, and its active component is berberine hydrochloride (berberine), and mtt assay detection confirms that it can promote people's periodontal membrane The propagation of cell, ALP detection confirms that it has certain osteogenic activity.

Inventor and then establish SD Periodontal Tissues of Rat Periodontitis Model, effective with Rhizoma Coptidis effective site (33mg/kg/d), Rhizoma Drynariae The single medicinal material at position (18mg/kg/d) or prescription medicine gavage therapeutic intervention.The sulcular bleeding index of each group of rat, tooth is checked with crossing All bag degree of depth, tooth mobility and frontal resorption value, and carry out art district microscopic CT scanning and pathological study；EILISA The expression of method each group of rat blood serum CRP level of detection and level in gingival sulcus fluid IL-1 β, TNF α, the expression of IL-8, PGE2；Immune group The expression of RANKL and OPG in weave chemistry method detection rat periodontium.As a result, it was confirmed that compared with using single medicinal material, whole body Apply coptis root aqueous extract with Rhizoma Drynariae water extract effect more preferably, the most named " even mending Gu Chi Wan " (embodiment 1-6). In miniature pig Periodontitis Model, coptis root aqueous extract and Rhizoma Drynariae water extract are pressed 11:6 composition of prescription Formulations for systemic administration, logical Cross Periodontal Status (BI, PD), gingival crevicular fluid, level, x-ray and the CT image of level in gingival sulcus fluid intrinsic factor (IL-8, IL-1 β, PGE2) (bone relative volume, trabecular bone density and bone trabecula number), HE dyeing and immunohistochemical staining (TNF-α, IL-8 and IL-1 β) etc. The detection of index is it was confirmed " even mending Gu Chi Wan " (embodiment 1-6) has good systemic effect to periodontitis.

Inventor also have detected Rhizoma Coptidis water position with alkaline phosphoric acid enzyme method and Rhizoma Drynariae aqueous extract is independent and prescription local During application, the ossification to people's periodontal ligament cell, to point out compared with using single medicinal material, coptis root aqueous extract carries with osteomiosis moisturizing Take thing effect more preferably.Use for reference L further₁₆(4)⁴Orthogonal table carries out experiment packet, by the Rhizoma Coptidis of variable concentrations, Rhizoma Drynariae LPS is stimulated servant's periodontal ligament cell to produce the influence research of PGE2 it was confirmed there is the Rhizoma Coptidis of 3 concentration groups by compatibility of effective components The PGE2 that water section+osteomiosis moisturizing position compatibility produces is less；Select SD Periodontal Tissues of Rat Periodontitis Model further, apply 3 groups of medicines In carrying out periodontal pocket, local is treated, and carries out the examination of curative effect (method is ibid) of related drugs topical application, finally confirms The topical application curative effect of 0.1mg/L Rhizoma Coptidis water position and 1mg/L osteomiosis moisturizing position compatibility is best.

Inventor has also set up with hydroxyethyl cellulose, concentrated glycerin, and polyacrylic resin is Chinese medicine prepared by major auxiliary burden Compound sustained-released gel systems, it is first determined the prescription ratio of each composition in sustained-release gel, and then determine and even mend composition of prescription The optimum release concentration of (+1mg/L osteomiosis moisturizing position, 0.1mg/L Rhizoma Coptidis water position) sustained-release gel is 6%, and it is named " even Mend teeth consolidating cream " (embodiment 7-12).Preliminary toxicologic study confirms, slow release in-situ gel preparation, without obvious side effect, can be grown Phase is applied.In miniature pig Periodontitis Model, by a series of pharmacologys detection (method is ibid) it was confirmed embodiment 8 is at tooth In all bags during topical application, there is obvious antiinflammatory and ossification；And with positive control medicine pike change minocycline hydrochloride Ointment (minocycline hydrochloride) is compared, and the antiphlogistic effects of prolonged application is similar to pike change, but skeletonization effect is substantially better than Pike change, can be used for the local treatment of periodontitis.

Finally, inventor have selected 87 example volunteers, on the basis of informed consent, has carried out preliminary clinical trial and has ground Study carefully.With Primary Care coordinate Chinese medicine compound sustained-release gel use treatment the moderate-severe periodontitis, and with positive drug pike change And periodental non-surgical treatment contrasts merely.As a result, it was confirmed that the present invention can make Effective Component of Chinese Medicine slowly discharge, and can the long period Ground keeps local higher concentration, plays its sterilization, antibacterial effect, can improve the effect of simple mechanical treatment, reduce or delay Recurrence, can be as effective supplementary means for the treatment of chronic periodontitis.

The present invention utilizes Chinese medicine to carry out periodontitis treatment, it then follows organic conception, the principle of determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, by Rhizoma Coptidis water Extract and Rhizoma Drynariae water extract compatibility, it is determined that index components (effective ingredient) and optimal prescription, by clinical research and Pharmacological experiment, it was demonstrated that it can produce multidimensional, multi-mode, many target position, efficient therapeutic effect to periodontitis.Result of study is demonstrate,proved Bright, this Chinese prescription is a kind of medicine that can be used for clinical treatment periodontitis.Before this research, effective to both the above Chinese medicine The aspects such as the composition of prescription at position, optium concentration and proportioning, pharmacological action and treatment periodontal disease clinical application there are no any Documents and materials are reported.

The present invention has the spy that prescription is little, preparation method is simple, processing cost is low, evident in efficacy, without any side effects Point, is a kind of novel form oral cavity Chinese patent medicine with the long-lasting and ease for use similar to Western medicine, has clinical practice widely Prospect.

Accompanying drawing explanation

Fig. 1 Rhizoma Coptidis and Rhizoma Drynariae compound preparation outward appearance, (A) embodiment 5 (even invigorating capsule)；(B) embodiment 8 (is even mended soft Cream).

The cultivation of Fig. 2 people's periodontal ligament cell (hPDLCs) and qualification: (A) original cuiture 1 week；(B) original cuiture 4 weeks；(C) HE dyes, light Microscopic observation；(D) Vimentin stained positive；(E) keratin stain is negative.

The impact that hPDLCs is bred by Fig. 3 mtt assay detection naringin: variable concentrations group naringin is in different time points pair The impact of hPDLCs propagation.(1.0mg/L group is the most notable (p < 0.01) to the proliferation function of cell, and in time in positive Close).

Fig. 4 naringin is on hPDLCs alkaline phosphatase activities and the impact of NTx protein expression: (A) variable concentrations group The naringin impact on hPDLCs alkaline phosphatase activities: 1.0mg/L group ALP activity is higher than other each group (p < 0.01)；(B) no With the impact on hPDLCs NTx protein expression of the concentration group naringin: the NTx expressing quantity of 1.0mg/L group wants height In other concentration groups (p < 0.01).

Fig. 5 naringin impact on hPDLCs OPG mrna expression: (A) 1.0mg/L naringin is to hPDLCsOPGmRNA Express the impact of relative quantity: before the relatively dosing of OPGmRNA relative quantity, have obvious difference, and prolongation over time and gradually increase By force；(B) naringin respectively organizes β-actin, OPG electrophoretogram after intervening periodontal ligament cell: the amplified production of OPG, β-actin is respectively 342bp、179bp.Note: each swimming lane is followed successively by from right to left: Bio Marker I；β-actin matched group (0,1,3,5,7,9 My god)；Naringin group (0,1,3,5,7,9 days).

Fig. 6 hPDLCs/CMC-HPLC/MS on-line coupling schematic diagram: the one-dimensional stream of Position A:hPDLCs/CMC: M1 (flowing phase 1)-P1 (chromatogram pump 1)-S1 (complex sample)-DUV (UV-detector)-C1 (hPDLCs/CMC post)-EC1 (enrichment Post 1)-W1 (waste liquid 1)；HPLC/MS two dimension stream: M2 (flowing phase 2)-P2 (chromatogram pump 2)-EC2 (enriching column 2)-C2 (C18 Post)-DMS (MS detector)-W2 (waste liquid 2).The one-dimensional stream of Position B:hPDLCs/CMC: M1 (flowing phase 1)-P1 (chromatograph Pump 1)-S1 (complex sample)-DUV (UV-detector)-C1 (hPDLCs/CMC post)-EC2 (enriching column 2)-W1 (waste liquid 1)； HPLC/MS two dimension stream: M2 (flowing phase 2)-P2 (chromatogram pump 2)-EC1 (enriching column 1)-C2 (C18 post)-DMS (MS detector)- W2 (waste liquid 2).

The effective ingredient of Fig. 7 hPDLCs/CMC-HPLC/MS on-line coupling method screening Chinese Drug Rhizomes of Coptis: A simvastatin exists Two-dimensional chromatogram (the A simvastatin chromatograph on hPDLCs/CMC analyzed in hPDLCs/CMC-online-HPLC/MS system Figure；Chromatogram on HPLC/MS after retained fraction R1 enrichment on B hPDLCs/CMC；S simvastatin directly divides through HPLC/MS The chromatogram of analysis；The R0 simvastatin not retained fraction on hPDLCs/CMC；R1 simvastatin guarantor on hPDLCs/CMC Stay chromatographic peak；The R1-1R1 main chromatographic peak after HPLC/MS analyzes)；Belit acid berberine is at hPDLCs/CMC-online- Two-dimensional chromatogram (the alite acid berberine chromatogram on hPDLCs/CMC analyzed in HPLC/MS system；B hPDLCs/CMC Chromatogram on HPLC/MS after upper retained fraction R1 enrichment；The chromatogram that S berberine hydrochloride is directly analyzed through HPLC/MS；R0 The berberine hydrochloride not retained fraction on hPDLCs/CMC；R1 berberine hydrochloride reservation chromatographic peak on hPDLCs/CMC； The R1-1R1 main chromatographic peak after HPLC/MS analyzes)；C Rhizoma Coptidis-water extract (HL-5) is at hPDLCs/CMC-online- Two-dimensional chromatogram (A Rhizoma Coptidis-the water extract chromatogram on hPDLCs/CMC analyzed in HPLC/MS system；B hPDLCs/ Chromatogram on HPLC/MS after CMC upper retained fraction R1 (between two lines) enrichment；S Rhizoma Coptidis-direct warp of water extract The chromatogram that HPLC/MS analyzes；R0 Rhizoma Coptidis-the water extract not retained fraction on hPDLCs/CMC；R1 Rhizoma Coptidis-water extract Reservation chromatographic peak on hPDLCs/CMC；The R1-1R1 main chromatographic peak after HPLC/MS analyzes).

Fig. 8 berberine and simvastatin are on hPDLCs propagation and the impact of alkaline phosphatase activities: (A-E) berberine and pungent Cut down the impact that hPDLCs is bred by statin；(F-I) berberine affects * P ＜ 0.05 and * * P to hPDLCs alkaline phosphatase activities ＜ 0.01 compares matched group；(J) berberine impact (ALP staining) on hPDLCs alkaline phosphatase activities.

Fig. 9 is each group rat mandible tissue pathological slice (10 ×) after treating 4 weeks.

Figure 10 treats 4 weeks, after 12 weeks, miniature pig respectively organizes Periodontal Status.A1: even benefit group 4 weeks, B1: even 4 weeks X-ray films of benefit group, C1: even 4 weeks CT sheets of benefit group；A2: periodontitis group 4 weeks, 4 weeks X-ray films of B2: periodontitis group, 4 weeks CT sheets of C2: periodontitis group；A3: even Benefit group 12 weeks, B3: even 12 weeks X-ray films of benefit group, C3: even 12 weeks CT sheets of benefit group；A4: periodontitis group 12 weeks, B4: periodontitis group 12 weeks X-ray film, 12 weeks CT sheets of C4: periodontitis group.

Figure 11 respectively organizes miniature pig and treats 4 weeks, tissue pathological slice (4 ×) after 12 weeks.A1: normal group 4 weeks, B1: periodontitis Organize 4 weeks, C1: even benefit group 4 weeks；A2: normal group 4 weeks, B2: periodontitis group 4 weeks, C2: even benefit group 4 weeks.

The preparation flow figure of Figure 12 effective ingredient in Chinese sustained-release gel in situ.

Figure 13 2%, 4%, 6% even mends sustained-release gel release in vitro situation.

Figure 14 rat Mucosa of lip HE dyeing Microscopic observation (x400).A connects benefit group；B Blank gel group；C blank group.

Figure 15 topical medications X-ray after 1 month and 3 months and CT image.

Figure 16 topical medications is each group HE dyeing Microscopic observation after 1 month and 3.A1 even mends 1 month group of sustained-release gel； 1 month group of B1 pike change；1 month group of C1 Blank gel；A2 even mends 3 months groups of sustained-release gel；3 months groups of B2 pike change；C2 is empty 3 months groups of white gel.

Detailed description of the invention

Embodiment 1

The raw material weight composition of the present embodiment is: 15 parts of coptis root aqueous extracts (berberine 80%) and 8 parts of osteomiosis moisturizings Extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

Embodiment 2

The raw material weight composition of the present embodiment is: 14 parts of coptis root aqueous extracts (berberine 80%) and 9 parts of osteomiosis moisturizings Extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

Embodiment 3

The raw material weight composition of the present embodiment is: 13 parts of coptis root aqueous extracts (berberine 80%) and 10 parts of Rhizoma Drynariae Water extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

Embodiment 4

The raw material weight composition of the present embodiment is: 12 parts of coptis root aqueous extracts (berberine 80%) and 5 parts of osteomiosis moisturizings Extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

Embodiment 5

The raw material weight composition of the present embodiment is: 11 parts of coptis root aqueous extracts (berberine 80%) and 6 parts of osteomiosis moisturizings Extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

Embodiment 6

The raw material weight composition of the present embodiment is: 10 parts of coptis root aqueous extracts (berberine 80%) and 7 parts of osteomiosis moisturizings Extract (naringin 30.71%)；1 part of gelatin, glycerol 2 parts.

The preparation method of above example, comprises the following steps:

1, by Rhizoma Drynariae raw material pulverizing, decocting three times, decocting liquid directly extracts with macroporous resin and separates, 70% Alcohol rinse, concentrates, upper spray dryer, and measuring naringin concentration is 30.71%；

2, Acidic distillation extracts coptis root aqueous extract, will invade bubble by Rhizoma Coptidis raw material acid solution, and regulation PH is to alkalescence And filter, filtrate regulation PH is to acid, with Sal dissolution filter, adjusts PH to filter to obtain crude product, and distilled water dissolves, filters, vacuum is done The dry finished product that obtains, measuring berberine concentration is 80%；

3, the fine powder that (1) prepares with (2) is mixed homogeneously, add 72% ethanol accounting for mixed powder weight 16%, make soft Material, pelletizes with 20 eye mesh screens in granulator, dries 2 hours with the temperature of 62 DEG C in the drying chamber, crosses 20 mesh sieve granulate, and mixing is all Even；

4, take 1 part of gelatin, glycerol 2 parts, pure water 2 parts, 50 DEG C of heating, be sufficiently stirred for, evacuation 1 hour, upper machine, warp Syringe chemical conversion rubber, adds molding by (3) raw material, shapes through rotating cage, dehumidifying, drying oral medicine.

Embodiment of the present invention 1-6 uses decocting method combination Amberlyst process extract and separate the effective site of Rhizoma Drynariae, its Effective ingredient (index components) is naringin；Extract and separate the effective site of Rhizoma Coptidis with Acidic distillation, its effective ingredient (refers to Mark composition) it is berberine.Rhizoma Coptidis and osteomiosis effective site can composition of prescription, make " even mending teeth consolidating capsule ", Formulations for systemic administration, i.e. mouth Take medicine.

Embodiment 7

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 9 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；22.5g glyceryl triacetate, 2.5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

Embodiment 8

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 10 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；22.5g glyceryl triacetate, 2.5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

Embodiment 9

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 11 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；22.5g glyceryl triacetate, 2.5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

Embodiment 10

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 9 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；20g glyceryl triacetate, 5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

Embodiment 11

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 10 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；20g glyceryl triacetate, 5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

Embodiment 12

The raw material weight composition of the present embodiment is: 1 part of coptis root aqueous extract (berberine 80%) and 11 parts of osteomiosis moisturizings Extract (naringin 30.71%), quantification of 3g after volatilizing；20g glyceryl triacetate, 5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g glycerol.

The preparation method of above example, comprises the following steps:

1, by Rhizoma Drynariae raw material pulverizing, decocting three times, decocting liquid directly extracts with macroporous resin and separates, 70% Alcohol rinse, concentrates, upper spray dryer, and distilled water dissolves, and measuring naringin concentration is 30.71%；

2, Acidic distillation extracts coptis root aqueous extract, will invade bubble by Rhizoma Coptidis raw material acid solution, and regulation PH is to alkalescence And filter, filtrate regulation PH is to acid, with Sal dissolution filter, adjusts PH to filter to obtain crude product, and distilled water dissolution filter obtains finished product, Measuring berberine concentration is 80%；

3, the glycerol taking recipe quantity is heated to 90-100 DEG C, constant temperature, the lower recipe quantity hydroxyethyl cellulose that adds of stirring, at a high speed Stirring to hydroxyethyl cellulose is completely dissolved；

4, the glycerol triacetate taking recipe quantity is heated to 45-50 DEG C, constant temperature, under agitation adds recipe quantity polyacrylic acid Resin Eudragit RS stirs to being completely dissolved；

5, the temperature of above-mentioned (3) solution is down to 45-50 DEG C, at the ratio addition in berberine naringin mass ratio 1:10 Side's amount Rhizoma Coptidis Rhizoma Drynariae water extract, stirring at low speed is to complete drug dissolution；

6, the solution obtained in (4th) step is joined under stirring at low speed in above-mentioned (5) drug solution, 45-50 DEG C of guarantor Stir under the conditions of temperature to homogenizing and i.e. obtain medicine for external use.

Embodiment of the present invention 7-12, by adding the adjuvants such as hydroxyethyl cellulose, concentrated glycerin, polyacrylic resin, makes one Plant Novel external Chinese medicine long-acting composite ointment and " even mend teeth consolidating ointment ", local application in periodontal pocket, i.e. medicine for external use.

Clinical trial

1, test medicine: Rhizoma Coptidis Rhizoma Drynariae sustained-release gel formulations (embodiment 8 is called for short and even mends ointment, with reference to Fig. 1)

2, test objective: evaluate embodiment 8 Chinese medicine compound periodontal sustained-release gel formulations auxiliary treatment periodontitis curative effect and Safety.

3, EXPERIMENTAL DESIGN

3.1 test indications: accept scaling for chronic periodontitis, scrape the treatment of the auxiliary after controlling root planing.

3.2 EXPERIMENTAL DESIGN principles: use single-blind randomized, the test method of parallel control, carry out clinical comparison checking；For protecting At the end of confirmatory test, test number of cases at least up to often organizes 20 examples, testing plan group 20+20 example, matched group 20+20 example, and altogether 80 Right；Test group is Rhizoma Coptidis Rhizoma Drynariae sustained-release gel, and matched group is Minocycline ointment and blank group；Because for the treatment of other diseases And respective record answered by the medicine used.

3.3 experimenters select

3.3.1 diagnostic criteria: chronic periodontitis (according to newly-built international periodontal disease Classification of Diseases in 1999)；Moderate And disease damage more than moderate, select the periodontal pocket observing tooth to visit and examine the degree of depth (PD) at least site >=5mm, but≤10mm.

3.3.2 inclusion criteria: moderate and the above chronic periodontitis of moderate；Reserved tooth >=10 in mouthful；PD:5-10m, BOP(+)；Age 25-65 year, male or female；This research of voluntary participation, obeys therapeutic scheme, comes institute and follow up a case by regular visits to；Signature informed consent Book；It is "No" such as any one answer above, then can not participate in test.

3.3.3 exclusion standard: used antibacterials in March before experiment；Periodontal treatment was accepted in June before experiment；Female Property gestation or suckling, or intend fertility person；There are severe cardiac, liver, kidney, endocrine and hematologic disease；There is drug allergy history person；Above If any one answer is "Yes", then can not participate in test.

3.3.4 standard is exited: test finds do not meet case standard person；The infull person of data；Do not follow the doctor's advice medication person； The person of automatically exiting from；Because of drug allergy or untoward reaction termination test person (do not list observation case statistics in, but should count bad instead Answer in case and evaluate)；Serious adverse events occurs and promptly takes off blind person；Person lost to follow-up；Other.

3.4 dosage regimens: carry out periodontal inspection when head examines, screen patient, after supragingival scaling, baseline inspection, determine Patient observes tooth position, carries out clinical examination and level in gingival sulcus fluid inspection, hematuria routine examination, hepatic and renal function and Electrocardioscopy.So After give Primary Care (i.e. scaling, scrape control, root planing), injection experiments medicine in periodontal pocket, instructs patient's mouth to defend subsequently Raw, test group and the same method of matched group.Further consultation arrange: before medication, medication 1 week, medication 2 weeks, medication 3 weeks, medication surrounding, medication 3 months, medication 6 months, medication 9 months, medication 12 months.Experimenter should periodically be followed up a case by regular visits to by doctor, supervises it correctly to hold Practise medicine and advise.Every experimenter can only participate in and once study.

3.5 observation index

3.5.1 chief complaint index: include gingival hemorrhage, red swelling of gingiva, odontoseisis etc..Clinical meaning judges: 1 nothing；2 have Slight abnormality；3 medium exceptions；4 severely subnormals.

3.5.2 clinical indices: take the observation tooth position of different quadrant, each 6 sites of observation tooth record；Periodontal pocket is visited and is examined deeply Degree (PD, mm): using standardization periodontal probe, the data obtained rounds up；Attachment level measures (CAL, mm): enamelo-cemental junction To the distance at the bottom of periodontal pocket；Bleeding index (BI): record site, three, cheek side and site, one, tongue side；Plaque index (PLI): bacterium Each bacterial plaque of tooth surface stained area is recorded after speckle dyeing；Root-bifurcating diseases, Movable degree, frontal resorption.

3.5.3 level in gingival sulcus fluid inspection: Waterman40# filter paper, is cut into 10mm × 2mm size, puts in 1.5mlEP pipe, High pressure steam sterilization, constant temperature roaster is dried, and weighs, and number record is standby.Aseptic filter paper bar is inserted gum gently along cheek side facing Ditch or periodontal pocket, the power that is hampered stagnation 30 seconds, after 30 seconds, taking-up is put immediately in aseptic EP pipe and (notices that filter paper bar is stained with blood and is then discarded not With), again weigh and record within the shortest time, calculate level in gingival sulcus fluid weight ,-70 DEG C are frozen standby.In level in gingival sulcus fluid The detection (ELISA method) of IL-8 content；The detection (ELISA method) of IL-1 β, PGE2 content in level in gingival sulcus fluid；The same IL-8 of measuring process The detecting step of content, after obtaining data, obtains the concentration of IL-1, PEG2 in sample by drawing standard curve.

3.5.4 lab testing: Electrocardioscopy；Blood routine examination, routine urianlysis；Hepatic and renal function inspection (ALT, AST、BUN、CR)；Detect when baseline (head examines) and at the end of medication respectively.

3.5.5 imaging examination: x line sheet；CBCT scans.

3.5.6 data statistic analysis: ELISA result uses the Curve Expert1.3 software kit that Reagent Company provides, and paints Standard curve processed, compares employing paired t-test two-by-two to data statistic analysis, the variance analysis LSD inspection that multiple samples compare. Represent that difference is statistically significant with P ＜ 0.05.Measurement data uses mean ± standard deviation to carry out statistics and describes, and uses t inspection Or variance analysis (ANOVA) or Wilcoxon rank test compare.Enumeration data uses frequency (constituting ratio) to add up Describe, use X²Inspection or non parametric tests.Ranked data rank test or Ridit analyze.

Group	M-F	Mean age	Include tooth number in
				Even benefit group	1:1	43.8 years old	166
Pike change group	3:2	43.9 years old	153
				Blank group	1:1	47.0 years old	159

4, case-data:

Case harmony is analyzed:

5, result of the test

5.1 safety analysiss: this experiment uses medicine for even mending two kinds of medicines such as sustained-release gel (embodiment 8), pike change. Actually used medicine number is 61 people, 18 people lost to follow-up, but all includes 61 people in safety analysis collection.Terminate to test, none The adverse events such as example generation allergy, idiosyncratic reaction, before treatment, treatment in and treatment after safety indexes inspection (routine blood test, Routine urinalysis, electrocardiogram, liver function, renal function) it is showed no obvious abnormalities, illustrate that drug safety is preferable, and zero difference between group.

5.2 therapeutic evaluation

Baseline period, treat latter 6 months, treat the change of rear 12 crescent moon weekly check indexs

Baseline period, treatment 6 months, the change of gingival crevicular fluid after 12 months

5.3 model case data

Case one: stone ××, man, 52 years old, Xi'an, Shaanxi Ba Qiao government functionaries, it is chief complaint medical with odontoseisis after upper right In Xi'an Communications University's stomatological hospital Periodontics & Oral Medicine, trial inspection finds that 16 tooth PD are 6mm-9mm, loosens III degree, and x-ray shows Alveolar bone horizontality absorbs and reaches root tip, and suggestion then is pulled out, and remaining sound of baby talk week, situation was according to newly-built international periodontal disease disease in 1999 Sick criteria for classification is diagnosed as chronic periodontitis, and the order of severity belongs to more than moderate；Meet inclusive criteria, patient's informed consent with After, give on gum scaling and carry out oral hygiene instyuctions, after one week, setting up baseline period, including according to random packet principle and even mend Group, numbering even mends No. 1.Baseline period starts to give full mouth subgingival debridement and root planing, and postoperative 3% hydrogen peroxide rinses, periodontal pocket Benefit ointment (embodiment 8), once in a week, continuous four times is connected on Nei.Medicine-feeding terminates check in rear January, finds that gingivitis substantially disappears Moving back, but oral hygiene is safeguarded not good enough, periodontal pocket is not detected by this time check.Thereafter March, the 6th month, the 9th Month, check in 12 months, spys of row dental pattern week examine inspection, if any periodontal pocket still >=4mm, give secondary and scrape and control, repeat first and treat Journey medicine-feeding process.After 1 year observation period, in addition to main suit's tooth is pulled out, remaining tooth PD, BI, CA are all decreased obviously, and CBCT checks and finds tooth Groove bone density increases, and height change is inconspicuous.

Case two: marquis's ××, female, 25 years old, Xi'an, Shaanxi engineering department high-ranking accountant, because tooth arrangement is uneven in west Stomatological hospital correction section of peace university of communications finds that Periodontal Status is poor when going to a doctor, and then changes the place of examination to Periodontics & Oral Medicine, and patient's readme was both Toward history the most once because periodontitis is in outer court's row dental pattern week flap operation.The situation inspection of dental pattern week finds that oral hygiene is not good enough, Debris index is 3, and universal depth of probe PD is 4mm-8mm, and x-ray display alveolar bone horizontality absorbs and reaches II degree, according to 1999 Newly-built international periodontal disease Classification of Diseases is diagnosed as chronic periodontitis, and the order of severity belongs to moderate；Meet inclusive criteria, suffer from After person's informed consent, give on gum scaling and carry out oral hygiene instyuctions, after one week, setting up baseline period, former according to random packet The then company's of including in benefit group, numbering even mends No. 2.Baseline period starts to give full mouth subgingival debridement and root planing, postoperative 3% hydrogen peroxide Rinse, in periodontal pocket on connect benefit ointment (embodiment 8), once in a week, continuous four times.Medicine-feeding terminates check in rear January, finds tooth Gingival inflammation substantially disappears, but oral hygiene is safeguarded not good enough, enjoins it to check after oral hygiene instyuctions is repeated on time, the most multiple Look into and periodontal pocket is not detected.Thereafter March, the 6th month, the 9th month, 12 months check, patient's oral hygiene is tieed up Protecting preferably, inspection is examined in the spy of row dental pattern week, and PD, BI, CA are all decreased obviously, and CBCT checks and finds that Alveolar bone density increases, highly It is slightly changed.

Case three: Lee's ××, female, 64 years old, people from Xi'an, Shaanxi, retired worker.Based on upper left posterior occlusion discomfort February Telling and go to a doctor in Xi'an Communications University's stomatological hospital Periodontics & Oral Medicine, trial inspection finds that 26 sound of baby talk gingival enlargements are obvious, gingival recession About 3mm, PD are 6mm-9mm, and palatine root bifurcated exposes, and can probe into and not lead to, and loosen II degree, and x-ray display alveolar bone horizontality absorbs Reaching the tip of a root 1/3, main suit's tooth is diagnosed as chronic periodontitis, II degree of Root-bifurcating diseases, the remaining sound of baby talk week situation newly-built according to 1999 International periodontal disease Classification of Diseases is diagnosed as chronic periodontitis, and the order of severity belongs to more than moderate；Meet inclusive criteria, patient After informed consent, give on gum scaling and carry out oral hygiene instyuctions, setting up baseline period after one week, according to random packet principle The company's of including in benefit group, numbering even mends No. 3.Baseline period starts to give full mouth subgingival debridement and root planing, postoperative 3% hydrogen peroxide punching Wash, in periodontal pocket on connect benefit ointment (embodiment 8), once in a week, continuous four times.Medicine-feeding terminates check in rear January, finds gingiva Inflammation substantially disappears, main suit's tooth transference cure, Movable degree I degree, but oral hygiene is safeguarded not good enough, and periodontal pocket is not entered by this time check Row is detected.Thereafter March, the 6th month, the 9th month, 12 months check, inspection, indivedual sounds of baby talk are examined in the spy of row dental pattern week Week bag still >=4mm, give secondary and scrape and control, repeated for first course for the treatment of and add medicine to process.After 1 year observation period, including main suit's tooth pulls out, PD, BI, CA are all decreased obviously, and CBCT measures and finds that Alveolar bone density increases, and height change is inconspicuous.

Case four: Lee's ××, man, 26 years old, Shaanxi Chang An University Weishui Campus junior, because labial teeth teeth space is the most medical When stomatological hospital correction section of Xi'an Communications University goes to a doctor, find that Periodontal Status is poor, changes the place of examination then to Periodontics & Oral Medicine.Dental pattern Week situation inspection finds that oral hygiene is not good enough, and dental pattern gum is red and swollen substantially, and gingival recession is about 2mm-3mm, universal depth of probe PD is 3mm-8mm, x-ray display alveolar bone horizontality absorbs and reaches II degree, according to newly-built international periodontal disease classification of diseases in 1999 Standard diagnostics is chronic periodontitis, and the order of severity belongs to moderate；Meet inclusive criteria, after patient's informed consent, give on gum Scaling also carries out oral hygiene instyuctions, sets up baseline period after one week, includes even benefit group according to random packet principle, numbering even benefit 4 Number.Baseline period starts to give full mouth subgingival debridement and root planing, and postoperative 3% hydrogen peroxide rinses, in periodontal pocket on connect benefit ointment (embodiment 8), once in a week, continuous four times.Medicine-feeding terminates check in rear January, finds that gingivitis substantially disappears, and patient's mouth Chamber Health Maintenance is good, and periodontal pocket is not detected by this time check.Thereafter March, the 6th month, the 9th month, 12 The moon is checked, and inspection is examined in the spy of row dental pattern week, and PD, BI, CA are all decreased obviously, and CBCT checks and finds that Alveolar bone density increases, highly Being slightly changed, within 1 year, observe and expire, Periodontal Status is stable, can carry out orthodontic treatment, and advises during patient's correction periodically Periodontal is checked.

Case five: Lee's ××, man, 37 years old, Xi'an high-tech zone, Shaanxi company software engineer, with the cold and hot thorn of upper right backteeth Swashing pain, Ou You spontaneous pain main suit goes to a doctor in Xi'an Communications University's stomatological hospital Periodontics & Oral Medicine, and trial inspection finds 16 sounds of baby talk Hat is complete, cold (+), kowtow (+), loosen II, gingival recession be 2mm-4mm, PD be 4mm-10mm, x-ray display alveolar bone horizontality Absorption reaches II degree, is then diagnosed as ascending pulpitis, chronic periodontitis.Remaining sound of baby talk week, situation was according to newly-built international tooth in 1999 Week, sick Classification of Diseases was diagnosed as chronic periodontitis, and the order of severity belongs to more than moderate；Meeting inclusive criteria, patient knows the inside story same After meaning, main suit's tooth gives combined periodonto-endodon-tic therapy, and remaining tooth gives on gum scaling and carries out oral hygiene instyuctions, a Zhou Houshe Vertical baseline period, includes even benefit group according to random packet principle, numbering even number picked up 5.Baseline period starts to give full mouth subgingival debridement And root planing, postoperative 3% hydrogen peroxide rinses, in periodontal pocket on connect benefit ointment (embodiment 8), once in a week, continuous four times. Medicine-feeding terminates check in rear January, finds that gingivitis substantially disappears, but oral hygiene is safeguarded not good enough, and this time check is not to periodontal pocket Detect.Thereafter March, the 6th month, the 9th month, 12 months check, the spy of row dental pattern week is examined inspection, is still had individual Other tooth periodontal pocket still >=4mm, give secondary and scrape and control, repeated for first course for the treatment of and add medicine to process.After 1 year observation period, main suit's tooth symptom disappears Losing, Movable degree reduces, and Periodontal Status is stable, and remaining tooth PD, BI, CA are all decreased obviously, and CBCT check measurement finds Alveolar bone density Increase, the most slightly raise.

6, conclusion (of pressure testing): test the use treatment the moderate-severe periodontitis coordinating Chinese medicine compound sustained-release gel with Primary Care, And doing not with positive drug pike change and simple Primary Care, ointment (embodiment 8) is even mended in result explanation can make Chinese medicine have Effect composition slowly discharges, and can keep local higher concentration for a long time, plays its sterilization, antibacterial effect, can improve single The effect of purely mechanic treatment, reduces or delays recurrence, can be as effective supplementary means for the treatment of chronic periodontitis.

Infrastest

1. the Rhizoma Drynariae active principle naringin experimentation to people's periodontal ligament cell (hPDLCs) function point analysis

1) original cuiture of hPDLCs and qualification

(1) choose 12～18 years old teenager need, because of orthodontic treatment, the premolars pulled out, scrape tooth with aseptic operation blade In root, the periodontal membrane tissue of 1/3, shreds, and adds containing 0.2% NTx enzymic digestion 30min, adds the DMEM containing 20%FBS and cultivates Liquid, puts in CO2 incubator (5%CO2,37 DEG C, saturated humidity) and cultivates.Cell is had to climb out of from piece of tissue seen from 1 week, and in Adherent growth；After 4 weeks, cell growth is intensive and has certain directivity.The cell attachment growth of original cuiture be paved with bottle at the bottom of about (Fig. 2 A-B) is passed on when 60%～80%.

(2) the 4th generation hPDLCs cultivated is inoculated in six well culture plates being equipped with coverslip, under 4% paraformaldehyde room temperature Fixing 10～15 minutes, HE dyes；ABC method dyes, and carries out anti-keratin and the dyeing of anti-Vimentin, and light Microscopic observation kytoplasm Pornographic condition.Visible Vimentin stained positive, cell cytosol is brownish discoloration, and keratin stain is negative (Fig. 2 C-E).Explanation The cell cultivated is derived from ectoblastic mesenchyme, rather than epithelium.Therefore according to position and the morphocytology sight of drawing materials Examine and may determine that this cell is hPDLCs.

2) naringin impact on hPDLCs function

(1) impact that hPDLCs is bred by mtt assay detection naringin: take the 4th generation hPDLCs, be inoculated in 96 orifice plates.According to Drug treating time difference is divided into 1,3,5,7,9 days groups after medicine effect.Add MTT solution 20 to every hole at corresponding time point μ l, every hole adds 200 μ lDMSO.Each hole absorbance value (Absorbency is measured with enzyme linked immunological tester under wavelength 490nm Value, A490).Result shows 100,10,1.0,0.1mg/L naringin all can promote cell proliferation, especially breed with 1.0mg/L Best results, and be proportionate with drug treating time；And under different time sections, the medicine pair of naringin each concentration group The cultivation effect of hPDLCs different (F=205.005, p < 0.01) (Fig. 3).

(2) naringin impact on hPDLCs alkaline phosphatase activities: take the 5th generation hPDLCs, is inoculated in 18 orifice plates, every hole It is separately added into 2ml variable concentrations medicine culture fluid.After hatching 5 days, collect cell culture supernatant, according to test kit operation instruction Operate, under wavelength 520nm, measure each pipe absorbance (A520) value with enzyme linked immunological tester, then converse each sample The activity of alkali phosphatase.Result shows, under the medicine effect of variable concentrations cell ALP activity value different (F=36.664, P ＜ 0.01), and ALP activity during 1.0mg/L is higher than other concentration group and matched group (Fig. 4 A).

(3) naringin impact on hPDLCs NTx protein expression: collect cell culture supernatant, according to people I type glue The operating procedure of former enzyme linked immunological assay kit is carried out, and measures each hole with enzyme linked immunological tester at wavelength 450nm Absorbance.Found that cell NTx expressing quantity under variable concentrations medicine effect different (F=392.384, P ＜ 0.01), the NTx expressing quantity of 1.0mg/L concentration group is higher than other concentration groups (Fig. 4 B).

(4) naringin impact on hPDLCs OPG mrna expression: cell is inoculated in 7 culture bottles cultivation 24h, HPDLCs is intervened in packet, and (matched group: 100 μ g/ml LPS, intervenes 1 bottle of cell；Naringin group: 1.0mg/L, intervenes 6 bottles of cells), 0,1,3,5,7,9 days harvestings are cultivated in experiment respectively, with the total serum IgE of BIOZOL traditional extraction hPDLCs, try according to reverse transcription Agent box operation instruction, it is thus achieved that cDNA.PCR reacted product electrophoresis in agarose gel, sees on automatic gel imaging instrument Examine result, carry out gradation of image analysis by image-pro plus6.0 image analysis software.Result is visible, OPG, β-actin's Amplified production is respectively 342bp, 179bp.RT-PCR result shows: have a small amount of expression before adding naringin seen from OPG mRNA, Addition 1.0mg/L naringin is after 1 day, and its OPG expresses and substantially raises, OPG genes of interest band gray scale and β-actin reference gene Have obvious difference before the relatively dosing of the ratio of band gray scale, and prolongation over time and gradually strengthen, reached at the 9th day High (Fig. 5).

2.hPDLCs/CMC-HPLC/MS on-line coupling method screening Chinese Drug Rhizomes of Coptis treats the effective site of periodontitis and has Effect composition

1) the membrane flexibility post of people's periodontal ligament cell and fluorimetric HPLC system (hPDLCs/CMC-online- HPLC/MS) foundation (Fig. 6)

(1) the membrane flexibility column system (hPDLCs/CMC) of people's periodontal ligament cell: the cultivation of periodontal ligament cell and qualification Ditto.4-6 is selected to be not less than 1 × 10 for hPDLCs cell total amount⁷Individual, under the conditions of 4 DEG C, ultrasonic rupture of membranes 30min, 12000rpm are high The centrifugal 20min of speed, obtains cell membrane suspension.Learnt from else's experience pretreated macro porous silica gel 0.05g, is dynamically added cell membrane suspension, 4 DEG C Stand overnight, obtain cell membrane and fix phase suspension.Take gc column tube (10mm × 2mm I.D.), note after fixing phase suspension is mixed Enter packing column machine to run, with ultra-pure water for flowing phase, obtain membrane flexibility post.

(2) foundation of hPDLCs/CMC-online-HPLC/MS combined system: hPDLCs/CMC model (the first dimension): stream Dynamic phase (M1) is ultra-pure water, flow velocity 0.2mL/min, column temperature 37 DEG C, detection wavelength: 238nm (simvastatin)；345nm (Rhizoma Coptidis). HPLC/MS system (the second dimension): flowing phase (M2): simvastatin is 0.2% glacial acetic acid: acetonitrile (20:80)；Rhizoma Coptidis is acetonitrile: 0.5% glacial acetic acid-1.2% triethylamine aqueous solution (40:60)；Electric spray ion source (ESI), N2 (purity 99.99%) as carrier gas, Flow velocity 1.5L/min, detector voltage 1.5KV, mass-to-charge ratio sweep limits: 50-1000m/z, scan mode: positive ion mode, add Thermal modules temperature 200 DEG C, bent row Desolventizing apparatus temperature 250 DEG C.

VP-ODS enriching column: first enriching column (EC1) is connected in parallel to 10 with second enriching column (EC2) and leads on valve, EC1 and EC2 can the most alternately be in the first dimension or two-dimentional system, and 2 enriching columns in parallel can alternately be enriched with, wash De-, to realize the one-dimensional on-line preconcentration being eluted thing.Ten direction changeover valves are used hPDLCs/CMC and HPLC/MS system to be entered Row connects, and establishes hPDLCs/CMC-online-HPLC/MS on-line coupled system.

2) application hPDLCs/CMC-online-HPLC/MS screening system Rhizoma Coptidis effective site and effective ingredient (Fig. 7)

(1) prepared by the sample of each extractive part of medicine: Chinese crude drug Rhizoma Coptidis, from the big pharmacy of easy shrine, is divided with simvastatin The extracting solution of discrete piece is as Positive mode medicine；The Chinese crude drug being dried pulverizer is pulverized, with 200ml methanol-water (70:30, V/ V) solution supersound extraction, redissolves with chromatograph alcohol methanol.Take methanol total extract, add ultra-pure water after volatilizing and make it dissolve, successively Add the organic solvent (petroleum ether, ether, chloroform, ethyl acetate) that polarity is different, supersound extraction, collect the upper and lower respectively Extracting solution, and final remaining water-soluble position extracting solution, i.e. can get the extracting solution at 5 kinds of different positions of polarity, after volatilizing Redissolve with Chromatographic Pure Methanol.

(2) application hPDLCs/CMC screening system Rhizoma Coptidis effective site: by equal for the flowing of hPDLCs/CMC chromatographic system Weighing apparatus 2-3h, after baseline is steady, draws each need testing solution 5uL injection chromatographic system and is analyzed measuring, and record chromatogram. With selected model drug simvastatin for comparison, investigate the reservation in hPDLCs/CMC system of each need testing solution special Property, found that Rhizoma Coptidis total extract has the obvious chromatograph similar with simvastatin after by hPDLCs/CMC system Peak；And the petroleum ether of Rhizoma Coptidis, ether, water position all have the obvious chromatographic peak similar with simvastatin to occur.

(3) application hPDLCs/CMC-online-HPLC/MS screening system Rhizoma Coptidis effective ingredient: chromatographic system flowing phase After balancing about 2h, after baseline is steady, by test product sample introduction 1uL, while one-dimensional membrane flexibility " identifies ", by retained fraction Be enriched on enriching column (EC1 or EC2), and alternately switch into two dimension reversed phase chromatography carry out " separate and identify ".Rhizoma Coptidis water carries It is berberine hydrochloride through Mass Spectrometric Identification to take the active component of thing.

3) the biological function detection of Rhizoma Coptidis effective ingredient berberine hydrochloride

(1) mtt assay detects the impact that hPDLCs is bred by each Rhizoma Coptidis active ingredient hydrochloric acid berberine: by berberine hydrochloride Storing solution is diluted to: 1mg/L, 0.1mg/L, 0.01mg/L totally 3 Concentraton gradient groups；Absorbance at 1,3,5,7,9 days cells (A490) value.Result shows, compares with the negative control group not adding medicine, hPDLCs drug level be 0.1mg/L, The propagation of people's periodontal ligament cell can be promoted during 0.01mg/L, and be proportionate with action time, but 0.1mg/L concentration Under berberine hydrochloride effect, cultivation effect is optimal (Fig. 8 A-E).

(2) the Rhizoma Coptidis active ingredient hydrochloric acid berberine impact on hPDLCs alkaline phosphatase activities: method is the same, by hydrochloric acid The storing solution of berberine is diluted to: 1mg/L, 0.1mg/L, 0.01mg/L totally 3 Concentraton gradient groups；Live at 5,9 days detection ALP Property.Result shows, under 0.1mg/L berberine hydrochloride effect, when 5 and 9 days, ALP activity was the strongest (Fig. 8 F-J).

3. whole body application Rhizoma Coptidis and the preliminary study of Rhizoma Drynariae active regions treatment periodontitis

1) foundation of stable SD Periodontal Tissues of Rat Periodontitis Model

(1) using cleaning grade male SD rat, chloral hydrate intraperitoneal injection of anesthesia, with the 4-0 silk thread ligation bilateral upper jaw the Two grind one's teeth in sleep cervical region, raise soft diet, high sucrose solution (glucose 100g/L) from ligation same day, continuous 8 weeks.

(2) modeling the 4th week and check as follows for the 8th weekend: gross examination of skeletal muscle, i.e. observe each treated animal active state, Diet, drinking-water and hair color change；Sulcular bleeding index (SBI), i.e. check whether hemorrhage with periodontal probe；Depth of pocket (PD), i.e. visit with periodontal probe and examine the closely middle cheek of experiment tooth, cheek, remote middle 3 some depth of pocket of cheek, take average；Tooth mobility (TM), tooth mobility classification are i.e. checked；Frontal resorption value (FR), i.e. takes upper jaw bone, and 1% aqueous solution of methylene blue dyes, really Determine the different tooth position enamelo-cemental junction length to alveolar ridge crest, take average；Microscopic CT scanning, Index for examination includes bone volume (BV/ TV), i.e. Trabecula Bone Volume and sample volume ratio；Bone surface amass volume ratio (BS/BV), i.e. bone trabecula surface area and bone volume it Ratio；Bone trabecula thickness (Tb Th), unit μm；Bone trabecula gap (Tb Sp), unit μm；Bone trabecula number (Tb N), unit mm- 1；Periodontal tissue's pathology are observed.

(3) result shows, within 8 weeks, can set up stable SD Periodontal Tissues of Rat Periodontitis Model.When modeling 4 weeks, each modeling rat with just Often group is compared, and sulcular bleeding index, depth of pocket and tooth mobility all dramatically increase (P < 0.05), but situation shakiness Fixed, between group, differ greatly in group.During 8 weekend, no difference of science of statistics (P > 0.05) between every statistical indicator；Micro-CT scanning shows Root of the tooth crotch region frontal resorption is modeled in root 1/2 after showing modeling 8 weeks.

2) whole body application Rhizoma Coptidis and Rhizoma Drynariae effective site single medicinal material, the general treatment of compound medicines treatment SD Periodontal Tissues of Rat Periodontitis Effect:

(1) animal packet: by 60 rat numberings, random packet, often group 12.Normal group: normally feed, does not do and locates Reason, uses distilled water gavage, gavage within 4 weeks, to put to death and observe after 8 weeks；Periodontitis group: stopping periodontal after modeling 8 weeks stimulates and use distilled water Gavage, gavage is put to death and is observed for 4 weeks；Rhizoma Coptidis group: stopping periodontal after modeling 8 weeks stimulates and gavage coptis root aqueous extract solution (33mg/ Kg/d), gavage is put to death and is observed for 4 weeks；Rhizoma Drynariae group: stopping periodontal after modeling 8 weeks stimulates and gavage Rhizoma Drynariae water extract solution (18mg/kg/d), gavage is put to death and is observed for 4 weeks；Even benefit group, stopping periodontal after modeling 8 weeks stimulates, and gavage coptis root aqueous extract is molten Liquid (33mg/kg/d) and Rhizoma Drynariae water extract solution (18mg/kg/d), gavage puts to death observation after 4 weeks.

(2) gross examination of skeletal muscle, clinical indices inspection, microscopic CT scanning and pathological study: method is the same.Result shows Show, connect benefit group gingiva color form quality seen from after treating 4 weeks and substantially returned to normal level, depth of pocket again smaller than 0.5mm, with Compare difference for other each group and there is significance (P < 0.05)；In periodontitis group, petechial hemorrhage, PD also can be visited in most tooth sites , there is the odontoseisis of more than 1 degree in >=1.5mm.The comparison therapy each group frontal resorption value after 4 weeks is visible: periodontitis group The nearly 3mm of bone resorption value, even benefit group average bone resorption value is respectively 0.88 ± 0.34mm, and difference has significantly compared with other each group Property (P < 0.05).

Table 3-1 respectively organizes rat gingival hemorrhage index (SBI)

Table 3-2 respectively organizes the spy of rat periodontal ligament bag and examines the degree of depth

Table 3-3 respectively organizes rats during tooth Movable degree (TM)

Table 3-4 treats 4 weeks respectively group bone resorption measured values

Note: * is statistically significant with periodontitis group comparing difference after representing treatment 4 weeks, P ＜ 0.05

# represents that difference is statistically significant compared with other each group, P ＜ 0.05

Treat the Micro-CT scanning reconstruction figure after 4 weeks to confirm that the bone resorption level of each group of rat has when the most relatively modeling 8 weeks and substantially change Kind.Benefit group Micro-CT scanning middle-distant direction profile Reconstruction figure shows: though these two groups of rat second molar buccal root crotch regions also can be shown in Bone absorption to a certain extent, but degree the most relatively remaining each group wants light, close to normal group level；Rhizoma Drynariae group, Rhizoma Coptidis There is certain bone resorption level in furcation area district seen from the middle-distant direction profile of group, but degree relatively periodontitis group substantially alleviates, and The area of new bone ridge of each group can be clearly seen that；Micro-CT scanning bone trabecula relevant parameter scanning analysis (table 3-5) of rat upper jaw bone shows Show: after modeling 8 weeks, each group rat compared with normal group is compared, bone trabecula number, thickness, bone surface amass volume ratio, bone volume notable under Fall (P < 0.05), bone trabecula gap dramatically increases (P < 0.05).After treating 8 weeks, each group bone trabecula number, thickness have increased Adding, wherein benefit group every bone trabecula parameter level is all substantially better than other each group (P < 0.05)；Rhizoma Coptidis group, Rhizoma Drynariae group and tooth Week, scorching group compared also have clear improvement (P < 0.05).

Table 3-5 Rat Experimental periodontitis jawbone microscopic CT scanning bone trabecula parameter

After note: * represents treatment 4 weeks, P < 0.05 compared with periodontitis group

# represents P < 0.05 compared with other each group

After treating 4 weeks, still having a large amount of inflammatory cell infiltration in periodontitis group periodontal tissue, periodontal membrane fiber bundle is disorderly, ditch Interior epithelial reticular becomes, and alveolar ridge absorbs to the tip of a root 1/3, and alveolar ridge crest is uneven, it is seen that multiple cores in Bone resoiption pit Osteoclast.Each medication group rat periodontium inflammatory cell quantity significantly reduces, even benefit group phatnoma bone height with normal group No significant difference, area of new bone visible a large amount of osteoblast along the line, the bone of formation is close to ripe (Fig. 9).

(3) whole body application single medicinal material, the compound medicines effect to SD Periodontal Tissues of Rat Periodontitis bone resorption correlation factor:

A. method: packet is the same.Immunoassay: after each group is treated 4 weeks, intraperitoneal anesthesia, abdominal aortic blood.Adopt With the expression of CRP in ELISA kit test experience animal serum.Level in gingival sulcus fluid immunologic test: rat models 4 weeks, builds Within after mould 8 weeks and treatment 4 weeks, gather SD rat level in gingival sulcus fluid.Use IL-1 β in ELISA kit test experience animal level in gingival sulcus fluid, TNF α, the expression of IL-8, PGE2.Immunohistochemical detection: measure each group according to the explanation of immunohistochemical staining test kit The expression of OPG, RANKL albumen in laboratory animal periodontal tissue, basic step such as test kit explanation.Statistical analysis: use SPSS13.0 is to carrying out One-Way ANOVA inspection or the Kruskal after experimental result row homogeneity test of variance Wallistest checks.

B. result: respectively organize serum CRP level, normal rats blood after being treated 4 weeks by the detection of EILISA euzymelinked immunosorbent assay (ELISA) Clear CRP mean concentration is 342.65 ± 100.54ng/ml, periodontitis group serum CA125 mean concentration is 1000.54 ± 124.56ng/ml, there were significant differences between the two (P < 0.05).Even benefit group serum CRP level relatively periodontitis group be remarkably decreased (P < 0.01), Rhizoma Coptidis group serum CRP level difference compared with periodontitis group also has significance (P < 0.05).

Table 3-6 is each group rat blood serum CRP average level (ng/ml) after treating 4 weeks

Note: * represents that difference is statistically significant compared with periodontitis group, P ＜ 0.05

After treating 4 weeks, the inflammatory factor level of each treated animal all decreases, respectively group rat level in gingival sulcus fluid IL-1 β table The level that reaches is visible, and have significant difference with periodontitis group is Rhizoma Coptidis group and even benefit group (P < 0.05)；Level in gingival sulcus fluid TNF-α level with Even benefit group (P < 0.05) that what periodontitis group compared that there were significant differences is；Each medication group level in gingival sulcus fluid IL-8 level is compared with periodontitis group What there were significant differences is Rhizoma Coptidis group and connects benefit group (P < 0.05)；Each group level in gingival sulcus fluid PGE2 level compares display to be had aobvious with periodontitis group Write difference is to connect benefit group (P < 0.05).The comprehensive each cytokine levels of level in gingival sulcus fluid is visible: even benefit group various inflammatory factor level The most relatively periodontitis group is remarkably decreased (P < 0.05), has obvious antiinflammatory advantage.

Table 3-7 is each group rat level in gingival sulcus fluid IL-1 β, TNF-α, IL-8, PGE after treating 4 weeks₂Average level (pg/ml)

Note: * represents that difference is statistically significant compared with periodontitis group, P < 0.05

Under optical microscope visible: RANKL and the OPG positive is brownish discoloration, is positioned in endochylema.Normal rats RANKL and OPG in periodontal tissue is all in weak positive expression, and the osteoblast and the periodontal membrane that are uniformly distributed in alveolar bone surface are thin In the middle of born of the same parents.In periodontitis group periodontal tissue, OPG is weak positive expression, no significant difference compared with the gray value of normal group (P > 0.05), RANKL is that strong positive is expressed.In Lian Buzu periodontal tissue, OPG is that strong positive is expressed, its gray value and other each group Compare and there is significant difference (P<0.05), this group RANKL expression compared with normal group no significant difference (P> 0.05).In Rhizoma Coptidis group, Rhizoma Drynariae, RANKL expression is less than periodontitis group (P < 0.05) but higher than even benefit group (P < 0.05), yellow Even group and Rhizoma Drynariae group OPG expression are higher than periodontitis group (P < 0.05) but less than even benefit group (P < 0.05)

Table 3-8 respectively organizes the image analysis result (average gray difference value) of positive strength

Note: * represents significant difference compared with normal group, P ＜ 0.05

# represents significant difference, P ＜ 0.05 compared with other each group

3) foundation of stable miniature pig Periodontitis Model

(1) miniature pig intragluteal injection ketamine, power moon west add Su Mian Xin induced anesthesia, and 3.5% pentobarbital sodium maintains Anesthesia.

On every miniature pig bilateral, lower jaw the 3rd premolars, the 4th premolars, first molar are for modeling experiment tooth.In often Otch in ditch is done in P3, P4, M1 cheek side of miniature pig, and periosteotome fully opens mucoperiosteum lobe, clear exposes art district tooth cheek The alveolar bone in furcation area district, side.Vulgar split brill remove enamelo-cemental junction to root side about 6mm, middle-distant direction 5mm, the alveolus of deep 5mm Bone bone amount, persistently uses physiological saline solution flush operation district during boning, cool down district of boning.Thoroughly strike off and be exposed on root face Periodontal membrane, with normal saline flushing wound to oozing out without obvious blood.Then thermoplastic gutta-percha is inserted Cranial defect district, 4# silk thread adds tinsel ligation neck portion, makes ligature put into gingival sulcus, mucoperiosteum lobe reset suspension suture as far as possible.

(2) observation index: gross examination of skeletal muscle, observes the active state of each treated animal, diet, drinking-water and hair color change；Oral cavity Interior inspection, including sulcular bleeding index (SBI), depth of pocket (PD), attachment loss (AL)；Imaging examination, shooting modeling Animal art district's Dental X-ray film and CBCT sheet, observe art district frontal resorption and reconstruction situation；Periodontal tissue's pathology are observed, embedding group Knit block, use flat push type microtome to make cheek-tongue direction 4-5 μm slab.

(3) result shows, use local bone+tinsel ligatures+raises the method for soft diet and can be successfully established miniature pig in 8 weeks Periodontitis Model.Before modeling, miniature pig gingiva toner is red, matter is tough, examines hemorrhage without gingival recession or spy, and Jin Yinyuan district has on a small quantity Bacterial plaque tartar, visible art district red swelling of gingiva after modeling 8 weeks, matter is soft, has obvious gingival hyperplasia；Facing piles up a large amount of tartar, tooth A large amount of soft dirts seen from cervical region, visit and examine hemorrhage even hematostaxis, and depth of pocket meansigma methods reaches (6.59 ± 0.32) mm.These Clinical manifestation can be as determining one of mark that miniature pig Periodontitis Model is successfully established.

4) whole body application Rhizoma Coptidis and the general observation of curative effect of Rhizoma Drynariae effective part compound medicine treatment miniature pig periodontitis:

(1) animal packet: 6 miniature pigs are randomly divided into 3 groups, and every two is a group；Normal group: normally feed, does not does Process, put to death respectively at 12 weeks and 30 weekends；Periodontitis group: model 8 weeks, started to remove local excitation thing from the 9th week, enter weekly Row periodental non-surgical treatment, often organizes each execution 1 after treating 4 weeks and 12 weeks；Even benefit group: periodontal ligatured 8 weeks, from the 9th week Start to remove local excitation thing, raise every day with coptis root aqueous extract (33mg/kg/d), Rhizoma Drynariae water extract (18mg/kg/d)； Carry out weekly periodental non-surgical treatment, after treating 4 weeks and 12 weeks, often organize each execution 1.

(2) gross examination of skeletal muscle, clinical indices inspection, microscopic CT scanning and pathological study: method is the same.Result shows Showing, after treating 4 weeks, even benefit group gradually recovers normal, and periodontitis group compare with other each group have obvious fur loosen have some setbacks, Lacklustre performance.After treating 12 weeks, even benefit group with normal group no significant difference, periodontitis group still shows compared with other each group The lacklustre performance of obvious fur is shown.

Treat 4 weeks backteeth weekly checks visible: each treated animal gingivitis situation has taken a turn for the better (Figure 10 A1-A4), periodontal The spy of scorching group is examined rear hemorrhage spilling gum edge or extends along gum edge, and periodontal pocket mean depth is 5.78 ± 0.32mm, and even benefit group periodontal is visited Examining depth-averaged value is 4.13 ± 0.34mm, visits and shows petechial hemorrhage after examining or extend along gum edge, difference compared with periodontitis group There is significance (P < 0.05).After treating 12 weeks, even the depth of pocket of benefit group and sulcular bleeding index with normal group without bright Significant difference different (P>0.05), compared with the depth of pocket of periodontitis group 3.67 ± 0.32mm, difference statistically significant (P< 0.05)。

Table 3-9 respectively organizes miniature pig gingival hemorrhage index (SBI)

Table 3-10 respectively organizes miniature pig spy and examines the degree of depth

Table 3-11 respectively organizes miniature pig attachment loss level

Note: & represents that difference has significance, P ＜ 0.05 compared with before modeling；

* represent that difference has significance, P ＜ 0.05 compared with periodontitis group

X-ray image display normal group alveolar bone is fine and close, and alveolar ridge crest is without absorbing；After modeling 8 weeks, experiment root of the tooth crotch region can See the high density shadow that gutta-percha is formed.After treating 4 weeks, the stimulus object in furcation area district is eliminated, and periodontitis group furcation area district can See obvious wedge-like shade；The bone density in Lian Buzu furcation area district and height increase, it is seen that fuzzy area of new bone girder impact.Control After treating 12 weeks, the bone density in Ge Zu furcation area district and the most all increased, even the furcation area of benefit group can see normal bone Trabecularism, bone density is close to normal level (Figure 10 B1-C4).

Miniature pig periodontal ligament cell marshalling is shown in normal group section, and direction is parallel with periodontal membrane fiber Shu Fangxiang, periodontal Intermembrane space, without broadening, has no that there is bone resorption phenomenon in furcation area district.After treating 4 weeks, substantial amounts of inflammation seen from periodontitis group furcation area district Disease cellular infiltration, periodontal membrane fiber arrangement disorder, alveolar ridge surface absorbs in nibbling shape, and bone resorption edge is visible the most multiple The osteoclast of core；Lian Buzu periodontal tissue inflammatory cell is limited only to epithelial peg and dashes forward or epithelium, and blood capillary proliferation expands , it is seen that newborn epithelial tissue or the epithelial tissue hypertrophy in periodontal membrane space that drops, and visible a small amount of new bone formation With new bone edge osteoblast.After treating 12 weeks, the most visible a large amount of inflammatory cell infiltrations in periodontitis group furcation area district, blood vessel expands Open hyperemia；Even the visible a large amount of area of new bone of benefit group are formed, and area of new bone has covered with furcation area district the most, alveolar ridge crest substantially recover to Normal level level, trabecular bone structure convergence is ripe；Even benefit group epithelium can see a small amount of inflammatory cell infiltration, mineralising journey Degree is significantly stronger than periodontitis group (Figure 11).

(3) the whole body application compound medicines effect to miniature pig periodontitis bone resorption correlation factor:

Detection method is the same.Detected by EILISA euzymelinked immunosorbent assay (ELISA) before treating, model 8 weeks, treat 4 weeks and treat 12 weeks Respectively organizing serum CRP level, normal group miniature pig serum CA125 mean concentration is 100.54 ± 30.68ng/ml, periodontitis group blood Clear CRP mean concentration is 3635.76 ± 50.97ng/ml, and there were significant differences between the two (P < 0.01).After treating 4 weeks, even mend Group serum CRP level relatively periodontitis group is remarkably decreased (P < 0.05).After treating 12 weeks, even benefit group serum CRP level and periodontitis Group is compared difference and is had significance (P < 0.05).

Table 3-12 modeling is front and models 8 weeks miniature pigs serum CA125 average level (ng/ml)

Table 3-13 treats 4 weeks and treatment respectively organizes miniature pig serum CA125 average level (ng/ml) in 8 weeks

Note: * represents that difference has significance, P ＜ 0.05 compared with periodontitis group

By EILISA euzymelinked immunosorbent assay (ELISA) detect each group of miniature pig model before, modeling 8 weeks, treatment 4 weeks and after treating 12 weeks Level in gingival sulcus fluid IL-1 β, TNF-α, the expression of IL-8, PGE2.Each treated animal inflammatory factor level the most relatively models after modeling 8 weeks Before significantly increase (P < 0.05).After treating 4 weeks, benefit group level in gingival sulcus fluid TNF-α, IL-8, PGE2 level are all notable compared with periodontitis group Decline.After treating 12 weeks, even benefit group level in gingival sulcus fluid IL-1 β, TNF-α, the expression relatively periodontitis group of IL-8, PGE2 are remarkably decreased (P<0.05).Experimental result is visible: periodental non-surgical treatment also can substantially lower inflammatory Cytokines Expression level, and even benefit group has certain anti- Scorching therapeutical effect.

Table 3-13 modeling is front and models 8 weeks miniature pig level in gingival sulcus fluid IL-1 β, TNF-α, IL-8, PGE₂Average level (pg/ml)

Table 3-14 is each group miniature pig level in gingival sulcus fluid IL-1 β, TNF-α, IL-8, PGE after treating 4 weeks₂Average level (pg/ml)

Table 3-15 is each group miniature pig level in gingival sulcus fluid IL-1 β, TNF-α, IL-8, PGE after treating 12 weeks₂Average level (pg/ml)

Note: * represents that treatment group difference compared with periodontitis group is statistically significant, P ＜ 0.05,

During in miniature pig periodontal tissue, RANKL and OPG is expressed in alveolar bone and periodontal ligament cell, in yellow, brown color Color.In normal group periodontal tissue, RANKL, OPG are all in weak positive expression, after treating 4 weeks, and the weak positive expression of periodontitis group OPG, with Normal group gray value no difference of science of statistics (P>0.05), RANKL is that strong positive expresses (P<0.05)；Even benefit group OPG expression Higher than periodontitis group (P < 0.05), RANKL expression is less than periodontitis group (P < 0.05).After treating 12 weeks, each group OPG expresses Level is relatively treated 4 weeks and is significantly increased (P < 0.05), and RANKL expression is relatively treated 4 weeks and is remarkably decreased (P < 0.05)；Compare two-by-two Result shows: periodontitis group OPG expression minimum (P < 0.05), RANKL expression the highest (P < 0.05).

Table 3-16 respectively organizes the image analysis result (average gray difference value of positive strength

Note: * represents that compared with normal group difference has a significance, P ＜ 0.05,

# represents that same time difference compared with periodontitis group has a significance, P ＜ 0.05,

4. topical application Rhizoma Coptidis and the preliminary study of Rhizoma Drynariae effective part compound sustained-release gel formulations treatment periodontitis

1) the topical application optimal prescription of Chinese medicine compound based on Rhizoma Coptidis and Rhizoma Drynariae effective site is determined

(1) alkaline phosphoric acid enzyme method detection topical application single and the Chinese medicine compound impact on hPDLCs ossification: people's tooth Original cuiture and the qualification of all theca cells are the same.Packet: coptis root aqueous extract group: berberine (100mg/L, 10mg/L, 1.0mg/ L、0.lmg/L、0.01mg/L)；Rhizoma Drynariae water extract group: naringin (10mg/L, 1mg/L, 0.1mg/L)；Rhizoma Coptidis Rhizoma Drynariae Group, i.e. 1.0mg/L coptis root aqueous extract+Rhizoma Drynariae water extract (10mg/L, 1mg/L, 0.1mg/L), 0.1mg/L Rhizoma Coptidis water carries Take thing+Rhizoma Drynariae water extract (10mg/L, 1mg/L, 0.1mg/L), 0.01mg/L coptis root aqueous extract+Rhizoma Drynariae water extract (10mg/L、1mg/L、0.1mg/L)；Separately set blank group.

After each group is continued to hatch 5 days under standard environment, collect cell culture supernatant with aseptic EP pipe；It is separately added into phenol Standard Applying Solution, buffer and matrix liquid；Add developer, with UV detector, under 520nm wavelength, measure each Pipe absorbance (A520) value.Result shows, Chinese medicine compound variable concentrations prescription is different to the alkaline phosphatase activities of cell, but general All over higher than the single medicine alkaline phosphatase activities to cell, the prescription that wherein ALP activity value is the highest is Rhizoma Coptidis-berberine 0.1mg/l and Rhizoma Drynariae-naringin 1mg/l, compares it with matched group and single medicinal material group, has significant difference.

Table 4-1 variable concentrations single and compound medicine prescription affect result to people periodontal ligament cell ALP activity

* compared with matched group, difference is statistically significant, p < 0.05；**p<0.01

(2) LPS is stimulated servant's periodontal ligament cell to produce the shadow of PGE2 by topical application Rhizoma Coptidis, Rhizoma Drynariae compatibility of effective components Ring: people

Original cuiture and the qualification of periodontal ligament cell are the same.L is used for reference in packet₁₆(4)⁴Orthogonal table carries out experiment packet.

Table 4-2 Rhizoma Drynariae and Rhizoma Coptidis effective site concentration table

Take the logarithm the 6th generation people's periodontal ligament cell of trophophase, be inoculated in 96 orifice plates, suck supernatant, be separately added into each group Medicine+1 μ g/mlLPS200 μ l, sets blank group simultaneously, carries out ELISA detection according to the operating procedure of test kit.Result shows Showing 3 compatibility groups, the respectively the 3rd, 13,14 3 groups of effects are more apparent, can be as the foundation of follow-up animal-use drug.

LPS is stimulated servant's periodontal ligament cell to produce PGE by table 4-3 Rhizoma Drynariae and Rhizoma Coptidis compatibility of effective components group₂Result is converged Summary table

(3) the preferable compatibility of effective components group of topical application 3 groups observation of curative effect in Experimental Periodontitis SD rat: SD rat Periodontitis Model method for building up is the same.Each experimental group often organizes each 10 of 12 SD rats, model control group and blank group.In Inject each group of medicine 50 μ l respectively by group at the bottom of the gingival sulcus of bilateral maxillary second molar palate side, every injection in 1 day once, totally 4 times. After medication 1 week, take rat level in gingival sulcus fluid, saliva；Take blood sample simultaneously.Row Micro-CT three-dimensional measurement is analyzed and histological stain Observing, method is the same.

Rat level in gingival sulcus fluid, saliva, the ELISA experimental result of PGE2, IL-1 β, TNF-α, CRP in blood sample Wherein 1-3 group refers to that the 3rd, 13,14 assemble 5 groups respectively, and 4 groups refer to model control group, and 5 groups refer to Normal group.Result shows Showing, the ELISA testing result of PGE2 in level in gingival sulcus fluid and blood, the 3rd group minimum；The ELISA testing result of sample IL-1 β, is shown in the 3rd Group IL-1 β amount is minimum；The ELISA testing result of sample TNF-α, is that the 3rd group of TNF-α amount is minimum；The ELISA detection of sample CRP As a result, also it is that the 3rd group of CRP amount is minimum.

PGE in table 4-4 level in gingival sulcus fluid, saliva and blood₂Statistic analysis result

IL-1 β statistic analysis result in table 4-5 level in gingival sulcus fluid, saliva

TNF-α statistic analysis result in table 4-6 level in gingival sulcus fluid, saliva

CRP statistic analysis result in table 4-7 blood

* compared with model control group, p < 0.05.

Select bilateral maxillary second molar furcation area root side 0.7mm, size be the region of 1.1 × 1.7 × 1.1mm3 be me Analyzed area, and use VIVID software, analyze the parameter of bone, including BV/TV, BS/BV, Tb.Th, Tb.N, Tb.Sp And Tb.Pf, and One-Way ANOVA is used to analyze difference between the parameter between seven groups.Analysis result is shown in that the 3rd group has the most obvious Effect.

The analysis of table 4-8 bone parameter

Compared with model control group (the 5th group), * refers to that p < 0.05, * * refers to p < 0.01

In HE dyeing and PGE2 and MCP staining analysis, it was observed that new bone formation and arranging along alveolar bone surface linear The osteoblast of row.Compare below furcation area 2mm simultaneously²The amount of interior PGE2 and MCP-1, result show the 3rd group effective.

Table 4-9PGE₂With MCP-1 SABC comparision contents

* compared with model control group, P < 0.05.

2) optimum concentration of topical application Chinese medicine compound sustained-release gel is screened

(1) preparation and the prescription strength of slow release situ-gel screens: selection acrylic resin (Eudragit RS PO) is Main slow-release material, prepares solvent sensitivity situ-gel (Figure 12).Finally determine the prescription of each composition in sustained-release gel Ratio, show that hydroxyethyl cellulose accounts for the 0.3% of total gel component, and Eudragit RSPO accounts for the 5-10% of total gel component, wherein Medicine glycerol adding adds hydroxyethyl cellulose and glyceryl triacetate and adds Eudragit RS PO mass ratio when being 1:1, at optimum Side.And then it is prepared for the Rhizoma Coptidis Rhizoma Drynariae sustained-release gel of 2%, 4%, 6% concentration respectively.

Table 4-10 drug extract sustained-release gel prescription composition table

(2) slow release in-situ gel preparation is evaluated: coptis root aqueous extract and Rhizoma Drynariae water extract press berberine and Pericarpium Citri grandis Sustained-release gel prepared by glycosides mass ratio 1:10, outward appearance is dark brown emulsifiable paste shape, and the color of inclined Rhizoma Drynariae has certain denseness, Become the fluid state of non-free flowing.PH value is detected as 7.2～7.4, meets the gel preparation PH that 2010 editions Chinese Pharmacopoeias specify The value requirement less than 8.0.By variable concentrations medicament slow release gel centrifugal stability, mobility, freezing Heating Experiment are examined Looking into, result see table.

Table 4-112%, 4%, the Chinese medicine compound sustained-release gel study on the stability result of 6% 3 kind of variable concentrations

Take different prescription and carry out release in vitro, parallel 3 groups of every kind of prescription, calculate the meansigma methods of each time point.With the time pair Accumulative release rate is mapped, and during seen from result, sustained-release gel is mended by the company of variable concentrations, the preparation of Testing index berberine is not With, the company of 4%, 6% concentration mends 7 days preparations of berberine in sustained-release gel and reaches more than 80%, and concentration is the highest, and accumulation is released Put rate the highest.First day berberine of company's benefit sustained-release gel of 4% concentration is slightly dashed forward and is released, and the 7 of 2% concentration days preparation deficiencies 80%.Therefore finally connect benefit sustained-release gel and finally select conduct optimum prescription (Figure 13) of 6% concentration.

(3) toxicologic study of slow release in-situ gel preparation:

(1) acute toxicological experiment: 30 kunming mices are divided into 3 groups, and (6% even mends sustained-release gel group, Blank gel group, sky White matched group), often group 10.Fasting gave mice Cmax (100%) maximum volume by 0.4ml/20g after 12 to 24 hours Gavage, gives in one day twice, Continuous Observation 7 days.Result shows, in addition to two are lethal because of gavage operation (all become celestial confirmation), Remaining equal activity normally has no adverse reaction, and the mental status is good, and hair color is glossy, feces no abnormality seen.Body weight increases basically identical, Each experimental group compares no difference of science of statistics with matched group.

The impact that Mouse Weight (g) is increased by table 4-12 Chinese medicine compound sustained-release gel

After 7 days, execution mice is to each organ gross anatomy procuratorial work, changes without obvious profile, has no congested, hemorrhage, edema etc. Change.Mice is 25g/kg to the maximum tolerated dose of Chinese medicine compound slow releasing agent, calculates by kg body weight, is equivalent to 50kg adult one 312 times of secondary consumption (assuming that the Chinese medicine compound sustained-release gel of 0.1ml is placed in 32 equal medications of tooth, every tooth).In explanation The sustained-release gel safety of recurrence due to taking drug side is bigger.

(2) local mucous membrane stimulation test: select 30 SD rats to be randomly divided into four groups and be respectively 6% concentration even benefit sustained-release gel Group, Blank gel group, matched group, often organize each 10, tests front chloral hydrate by rat anesthesia, fixing, its mucous membrane of lower lip It is stitched into blind bag with esthetic line, then sustained-release gel is slowly injected in blind bag, and smear its lower gum mucosa, make medicine with viscous Film at least contacts 4 hours (if medicine exposes, medicine-feeding by several times in 4 hours), observes animal and is administered latter 24 hours overall health of patients and office Portion's mucosa change, Continuous Observation 7 days, put to death animal, take oral cavity partial mucosa, observe mucosa and change with or without edema hyperemia etc., and Make histopathological examination.

Result shows, each group erythema and edema did not all occur in 72 hours, and in 7 days, mucosa is complete, without ulcer and erosion, sick Reason sections observation basal cell marshalling, form is normal, upper subcutaneous without obvious edema.Body weight increases basically identical, respectively tests Group compares no difference of science of statistics (Figure 14) with matched group.

Table 4-13 herbal mixture sustained-release gel local mucous membrane stimulates the impact on SD rat body weight (g)

(3) chronic toxicity: select 60 SD rats (120g ± 10g) be classified as Rhizoma Coptidis Rhizoma Drynariae water extract high, In, low dose group；Blank gel group；Matched group often organizes each 10.Adult presses 50kg and calculates, and full mouth clinical medicine dose is about 3.2ml/50kg.High dose group is 150 times of crude drug contained by clinical medicine dose, and middle dosage group is raw contained by clinical medicine dose 100 times of medicine, low dose group is 5 times of crude drug contained by clinical medicine dose.By 1ml/100g dosage gavage.Blank gel group is pressed 100 times of clinical dosage give i.e. every 100g mouse and give 0.6ml Blank gel.Timing every day gavage, continuous gavage 3 months, Weigh weekly once, in order to adjust given low according to body weight change.Carry out outward appearance sign, behavioral activity, meal situation, feces And the overview such as body weight change.Putting to death animal after experiment terminates, abdominal aortic blood carries out routine blood test and biochemical indicator Detection, every rat, to its heart, liver, spleen, lung, 5 substantial viscera elder generation gross examination of skeletal muscle of kidney, then takes same position routine and fixes, Embedding, section, HE dyes, Microscopic observation.

Result shows, the activity in 90 days experiment periods of medicine group, Blank gel group, matched group is all normal, and behavior has no different Often, hair luster, have no that feces is abnormal, none is the most dead；It is basically identical that body weight increases change, and each group male and female Rat Development is good Good；Routine blood test, biochemical indicator check unknown significance difference, and part small group is in biochemical indicator numerically slightly above comparison Group, but there was no significant difference, and in range of normal value, it is impossible to illustrate that the change of these indexs and test medicine toxicity have directly Relation.

90 days rat body weight (g) change tables of table 4-14 gavage

The impact on each group of rat serum routine examination after 90 days of table 4-15 gavage

T inspection is compared with matched group, P ＞ 0.05

The impact on each group of rats'liver renal function after 90 days of table 4-16 gavage

T inspection is compared with matched group, P ＞ 0.05

3) the topical application Chinese medicine compound sustained-release gel therapeutical effect to miniature pig Experimental Periodontitis

(1) method for building up of miniature pig Experimental Periodontitis model is the same.Choose on 4 miniature pig bilaterals before lower jaw the 3rd Grind one's teeth in sleep, the 4th premolars, first molar are experiment tooth, totally 48 teeth, are randomly divided into even benefit group, pike change by district according to statistics Group and Blank gel group, often 16 teeth of group.Inject corresponding medicine in being empirically grouped in periodontal pocket, even mend slow release including a kind of reed mentioned in ancient books Gel, pike change, Blank gel, it is advisable filling periodontal pocket, about 0.1ml, add medicine to weekly once, continuously medicine-feeding 1 month, 3 Month.The acquisition of level in gingival sulcus fluid and level in gingival sulcus fluid intrinsic factor detection method are the same, and iconography and histological observation method are the same.

(2) before and after miniature pig modeling topical application Drug therapy, periodontal checks that change is learned in result, level in gingival sulcus fluid and impact: treatment After 1 month, experimental group gingivitis slightly takes a turn for the better, gingival hemorrhage index, and gingival crevicular fluid change compares reduction with matched group to be had significantly Sex differernce (P ＜ 0.05), but there is no notable difference between each experimental group, minimum with pike change group bleeding index, pike change is described There is certain anti-inflammatory effects.But attachment loss is even to mend, and sustained-release gel group is minimum has significant difference (P ＜ with Blank gel group 0.05), x Xian Shi furcation area district low-density shadow alleviates.

After local treatment 3 months, even benefit group gingivitis, gingival crevicular fluid substantially alleviate, and depth of pocket meansigma methods is 3.4 ± 0.88mm, relatively substantially shoals after modeling, and the rarely seen a small amount of bacterial plaque in neck portion is piled up；Pike change group gingivitis substantially alleviates, but It is high that depth of pocket relatively connects benefit group.Matched group red swelling of gingiva, visit examine hemorrhage substantially, depth of pocket and attachment loss are without bright Showing change, seen from neck portion, bacterial plaque is piled up.X-ray and Micro-CT display treatment connected root after benefit group is relatively treated 1 month after 3 months The density shadow of crotch region increases, and phatnoma bone height raises.Carry out Micro-CT scanning discovery after treating 3 months and connect benefit group bone photo pair Volume, bone trabecula thickness, bone trabecula number apparently higher than modeling after, have significant difference (P ＜ 0.05).The company of comparing between group Benefit group ossification is preferably and pike change group has significant difference (P ＜ 0.05), and pike change group compares with matched group does not has difference Property, the company's of explanation benefit group has a clear superiority in (Figure 15) in terms of ossification.

Before table 4-17 modeling, after modeling, treat 1 month, the change of 3 months backteeth weekly check indexs for the treatment of

Before table 4-18 modeling, after modeling, treat 1 month, the change of gingival crevicular fluid after 3 months

Before table 4-19 modeling, after modeling, treat 1 month, the level change of inflammatory factor in level in gingival sulcus fluid after 3 months

(3) histology and Immunohistochemical detection result: Microscopic observation after treating 1 month, even mending sustained-release gel group can See that bone matrix is formed, bone Mineral nodules, but also show a small amount of osteoclast and be present in bone lacuna；Pike change group, Lan Ranqu At inflammatory cell place, alleviate than inflammation with after matched group and modeling, but have no the formation of bone matrix；Blank gel group is visible Substantial amounts of inflammatory cell infiltration, periodontal membrane fiber arrangement disorder, alveolar bone has destruction in various degree, and osteoclast is positioned at bone and inhales Receive in lacuna (Figure 16).

Microscopic observation after local treatment 3 months, even mends sustained-release gel group thickness of periodontal membrane uniform, have more bone matrix and Bone Mineral nodules is formed, and has no that obvious osteoclast exists；Pike change group inflammation alleviates after relatively treating one month, rarely seen epithelium Slightly hypertrophy, inflammatory cell significantly reduces, but has not yet to see obvious new bone formation；Seen from Blank gel group furcation area significantly Cranial defect, periodontal membrane forfeiture, epithelial proliferation, elongation, substantial amounts of inflammatory cell infiltration.

After local is treated 1 month, IL-8, IL-1 β and PGE2 level all reduces with comparing after modeling, has significant difference (P ＜ 0.05), there is significant difference with matched group than pike change group, even benefit sustained-release gel group, minimum with pike change level, group is described Beautiful anti-inflammatory effects difficult to understand is best, secondly for even mending sustained-release gel group.After local treatment 3 months, even mend sustained-release gel group and pike change Between group, difference is inconspicuous, not statistically significant, and the same pike change of long-term anti-inflammatory effects of sustained-release gel group is mended by the company of explanation.TNF-α、 IL-1 β, IL-8 immunohistochemical staining result becomes strong positive to express after showing modeling, after local is treated 1 month, respectively uses after 3 months Medicine group compares expression and reduces with matched group, the most weak even to mend the expression of sustained-release gel group.

Table 4-20IL-1 β, IL-8 and the absorbance of TNF-α positive cell

Claims (4)

1. utilize Rhizoma Coptidis and the preparation of Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis, it is characterised in that it is the most medicinal Component is in mass ratio:

Rhizoma Coptidis effective site water extract 10-15 part containing berberine more than 80%

Rhizoma Drynariae effective site water extract 5-10 part of naringin more than 30.71%.

The preparation utilizing Rhizoma Coptidis and Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis the most according to claim 1, its Being characterised by, its component is in mass ratio:

3. utilize Rhizoma Coptidis and the preparation of Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis, it is characterised in that its component presses matter Amount ratio is:

The preparation utilizing Rhizoma Coptidis and Rhizoma Drynariae compatibility of effective components prescription therapeutic periodontitis the most according to claim 3, its It is characterized in that, its component is in mass ratio:

The Rhizoma Drynariae water extract of the coptis root aqueous extract of 1 part of berberine 80% and 10 parts of naringins 30.71%, after volatilizing quantitatively For 3g；22.5g glyceryl triacetate, 2.5g polyacrylic resin Eudragit RS, 0.15g hydroxyethyl cellulose, 21.85g is sweet Oil.