CN103705694B - A kind of greasing base Chinese medicine ointment formulation treating diabetic foot and its production and use - Google Patents

Abstract

The invention discloses a kind of greasing base Chinese medicine ointment formulation treating diabetic foot, be specifically related to the greasing base ointment being made up of Radix Arnebiae (Radix Lithospermi), Cinnabaris, Sanguis Draxonis, the Radix Astragali, Colla Corii Asini, Borneolum Syntheticum 6 taste Chinese medicine and specific substrate adjuvant.Additionally, the invention also discloses process of preparing and the purposes of said preparation.The external application Chinese medicine compound ointment agent of the present invention can control diabetic foot gangrene development, the granulation growth of promotion skin ulcer face, make gangrene healing.This ointment has good tack and coating, and each dispersion of medicine, steady quality, character are good, and denseness is suitable, is suitable for clinical practice, and clinic has no obvious adverse reaction, can be used for treating diabetic foot.

Description

A kind of greasing base Chinese medicine ointment formulation treating diabetic foot and preparation method thereof And purposes

Technical field

The invention belongs to the field of Chinese medicines, relate to the medicine treating diabetic foot.Being specifically related to one, to control diabetic foot bad Cellulitis development, promote the growth of skin ulcer face granulation, the external application Chinese medicine oil substrate ointment formulation making gangrene healing and preparation method thereof and Purposes.

Background technology

Diabetic foot (Diabetic Foot, DF) is also called diabetic acromelic gangrene, be diabetes main serious the most also Send out one of disease, be also the main cause that disables of diabetics.Diabetic foot difficulty is big, and the cycle is long, amputation rate, lethal Rate, relapse rate are high, also cause heavy financial burden to society while causing body and mind injury to patient.Although Chinese and western medicine medicine Medical treatment combines local debridement and Chinese medicine and western medicine dressing change in surgical department to part DF patient's satisfactory effect, reconstructive vascular operation and Endovascular Interventional technique is gradually carried out many hospitals, makes the amputation rate of DF decline, but the treatment of diabetic foot is still and faces at present A difficult problem for bed.Therefore active treatment diabetic foot ulcer is particularly important.

The pathogenesis of diabetic foot is multifactor participation, intricate.And focus on a certain single link or target spot The complex lesions of preventing and treating too many levels, relatively difficult, this is also that modern medicine does not seeks obtaining one of the major reason of ideal medicament so far. Chinese medicine has the curative effect advantage of uniqueness in terms of preventing and treating diabetic foot ulcer, and especially on medicine for external use, its advantage is brighter Aobvious.External treatment of Chinese medicine has simple to operate, the advantages such as easy, low cost of drawing materials, and has many abundant dosage forms available, Can select flexibly according to the situation in skin ulcer face.The traditional Chinese medical science morbidity machine of diabetic foot core how is extracted from sturdy clinical research accumulation System, and the preferable external therapy of Chinese herb preparation of excavation development is to fill up in current diabetic foot from Long-term clinical effective Couple herbs The blank of medicine external treatment, improves the key of diabetic foot external treatment integral level.Chinese medicine compound external preparation is by multipath, multilamellar Secondary effect realize, present comprehensive effect, with diabetic foot occur development complicated mechanism corresponding, this just Chinese medicine whole The advantage place of body idea.

Summary of the invention

It is an object of the invention to develop a kind of determined curative effect, easy to use, treatment sugar without obvious toxic-side effects The new medicine externally used compound preparation (greasing base ointment) of urine foot disease, sets up and stablizes its preparation method.It is specifically related to A kind of diabetic foot gangrene that can control develops, promotes the granulation growth of skin ulcer face, makes the gangrenous effective external prescription healed.

It is special that the external Chinese medicine ointment preparation for the treatment of diabetic foot of the present invention comes from China's famous traditional Chinese medical science diabetic foot The Empirical formula of nearly 50 years treatment diabetic foots of family professor Xi Jiuyi, verifies for a long time through clinic, determined curative effect.In early-stage Study On the basis of, optimize prescription further, define the warp being made up of Cinnabaris, Radix Arnebiae (Radix Lithospermi), the Radix Astragali, Sanguis Draxonis, Colla Corii Asini, Borneolum Syntheticum 6 taste Chinese medicine Proved recipe.There is heat-clearing and toxic substances removing, putrefaction removing blood stasis dispelling, effect of QI invigorating granulation promoting.Purple Zhu's ointment of clinical practice is in preparation processing technique There is preparation technology simple, preparation is coarse, in-convenience in use, the problems such as quality is unstable, and packaging storage is delayed.This Bright with traditional Chinese medicine theory for instructing, use modern pharmaceutical technology that purple Zhu's ointment is carried out process modification, develop a kind of peace The complete stable effective pharmaceutical preparation coordinating surgery of Chinese medicine debridement treatment diabetic foot ulcer.

The requirement of " Chinese medicine registration management way " Chinese medicine that the present invention promulgates according to SFDA, natural drug classification 6.By right The research of early stage effective prescription medical material, proper auxiliary materials research, preparation process research, ointment behavior study etc., develop one and have Heat-clearing and toxic substances removing, putrefaction-removing granulation-promoting, effect of invigorating qi and benefiting blood, without obvious toxic-side effects treatment diabetic foot Chinese medicine external oil Substrate ointment formulation.

In one aspect of the invention, it is provided that a kind of greasing base Chinese medicine ointment formulation treating diabetic foot, it be by Effective ingredient is made with substrate adjuvant, and described effective ingredient is made up of the crude drug of following weight portion: 7 parts of Cinnabaris, Radix Arnebiae (Radix Lithospermi) 3 parts, The Radix Astragali 3 parts, Sanguis Draxonis 6 parts, 5 parts of Colla Corii Asini, Borneolum Syntheticum 1 part；Described substrate adjuvant includes the component of following weight portion: lanoline 30 Part, 94.5 parts of vaseline, PLURONICS F87 24 parts, Macrogol 4000 1 part, tristerin 10.5 parts, propylene glycol 5 parts, 10 parts of water, ethylparaben 0.06 part.

Cinnabaris uses 200 mesh water Cinnabaris industrialization finished products in the present invention；Colla Corii Asini is 60 mesh with electric crusher pulverizing Powder；Radix Arnebiae (Radix Lithospermi), Borneolum Syntheticum are effective ingredient thermally labile medical material, use comminution by gas stream mode to process；Radix Astragali fibroid is relatively strong, step by step Use alcohol extraction and water extraction extractum；Sanguis Draxonis is prepared as solid dispersion to improve its dispersibility in adjuvant.

In another aspect of this invention, it is provided that the process of preparing of ointment formulation of the present invention, need to be by above-mentioned place The medical material managed and substrate, according to the technological process mixing optimized, make ointment formulation.Prepare especially by following step:

1. with 55～60 DEG C of water dissolution Radix Astragali extracts, add Macrogol 4000, liquid level is sprinkled into Colla Corii Asini uniformly Powder makes its molten in water, adds lanoline and absorbs water liquid, stirs；Rising solution temperature, to 80～85 DEG C, adds dragon Sanguis Draxonis solid dispersion so that it is melted, quickly stirring makes the mixture of formation mix, and continues stirring until mixture cooling；

2. white vaseline mixes with tristerin, is heated to melting, mixing, adds Cinnabaris and is sufficiently stirred for grinding, in About 40～45 DEG C add Arnebia Root, and dispersed with stirring is uniform；Borneolum Syntheticum is placed in third containing ethylparaben 12.5mg/ml In glycol solution, it is dissolved under 75～80 DEG C of water-baths without grains of sand sense, after cooling, adds above-mentioned white vaseline and glycerol stearate Ester admixture mixes；

3. the product of above-mentioned two step gained is mixed, stir, cool down and i.e. obtain ointment finished product.

Step 1. in, described Sanguis Draxonis solid dispersion is adopted and is prepared with the following method: Sanguis Draxonis is with appropriate 90～95% ethanol To being completely dissolved, add in 80～85 DEG C of melted PLURONICS F87s, stir, fling to 95% ethanol, obtain Sanguis Draxonis Solid dispersion.

Described Cinnabaris uses 200 mesh water Cinnabaris industrialization finished products；Described Colla Corii Asini is 60 mesh powder with electric crusher pulverizing End；Described Radix Arnebiae (Radix Lithospermi), Borneolum Syntheticum use comminution by gas stream mode to process；Described Radix Astragali extract substep uses alcohol extraction and water extraction leaching Cream, specifically adopts and prepares with the following method: first with 8 times amount 75% twice Radix Astragali of ethanol extraction, then by the medicinal residues after alcohol extraction with 6 times amount Water extraction three times, obtains alcohol extraction and water extraction extractum.

In another aspect of this invention, it is provided that described ointment formulation answering in the medicine of preparation treatment diabetic foot With.

The use of Chinese medicine external ointment formulation of the present invention, every day 1 time, each 1 ointment patch or in affected part uniform application Thin layer (average every cm²Ointment patch contains ointment 0.15 gram), it is a course for the treatment of with two weeks, 2 courses for the treatment of can be used continuously, Specifically used amount and cycle need skilled clinician to adjust according to dosage form, the body constitution of patient, the situation of wound surface.

External Chinese medicine ointment preparation of the present invention, mainly with diabetic foot as object of study, by Chinese medicine compound preparation Advantage and characteristic be combined with modern science and technology, applicating new process, new technique, new adjuvant, fill up at present without treatment diabetes The blank of foot disease that calls for specialized treatment medicine.On the basis of its pathogenesis clear and definite, develop treatment diabetic foot safely, effectively, stable, can The pure Chinese medicinal preparation of control.

Greasing base Chinese medicine ointment formulation prepared by the present invention is made up of, as ointment medicine and substrate adjuvant two parts Excipient and the carrier of medicine, substrate adjuvant has the biggest impact to the quality of ointment, the dispersion of medicine.Oleaginous Substrate is lubricious, nonirritant, the protected and emollescence to skin, and vaseline can play closing wound as substrate adjuvant With the effect maintaining the certain wettability of wound.This ointment has good tack and coating, each dispersion of medicine, quality Stable, character good, denseness is suitable, is suitable for clinical practice, and clinic has no obvious adverse reaction, has price low simultaneously Storage honest and clean, easy, advantage easy to use, be worth research further and clinical expansion to use.

The external application Chinese medicine compound ointment agent of the present invention can control diabetic foot gangrene development, promotion skin ulcer face granulation grows, Make gangrene healing.This ointment has good tack and coating, and each dispersion of medicine, steady quality, character are good, thick Degree is suitable, is suitable for clinical practice, and clinic has no obvious adverse reaction, can be used for treating diabetic foot.

Accompanying drawing explanation

Fig. 1 is the preparation technology flow chart of greasing base Chinese medicine ointment formulation of the present invention.

Fig. 2 is Liver and kidney HE result schematic diagram after moist diabetic foot rat model respectively organizes medication in test example 1 of the present invention, its In, A: normal ulcer group, B: diabetic groups, D: diabetic infection ulcer matched group, Z0: Matrix controls group group, little dose of Z1: Chinese medicine Amount group, Z2: dosage group in Chinese medicine, Z3: Chinese medicine high dose group, C: western medicine group.

Fig. 3 is Chinese drug-treated group different time sections blood Hg changes of contents schematic diagram in test example 1 of the present invention.

Fig. 4 is Elisa result schematic diagram in test example 1 of the present invention.

Fig. 5 A and Fig. 5 B is Western blot result schematic diagram in test example 1 of the present invention.

Fig. 6 A-Fig. 6 F and Fig. 7 is Real-time PCR result schematic diagram in test example 1 of the present invention.Wherein, Fig. 6 A is E- Ca amplification curve；Fig. 6 B is E-Ca solubility curve；Fig. 6 C is GAPDH amplification curve；Fig. 6 D is GAPDH solubility curve；Fig. 6 E is VEGF amplification curve；Fig. 6 F is VEGF solubility curve.

Fig. 8 and Fig. 9 is SABC (IHC) result schematic diagram in test example 1 of the present invention.

Detailed description of the invention

The invention will be further elaborated by the following examples:

Embodiment 1

Greasing base purple Zhu's ointment formulation prescription of the present invention and preparation method are as described below:

1 greasing base purple Zhu's ointment prescription medical material and substrate adjuvant

Medical material (with 40g ointment gauge): Cinnabaris 3.5kg, Radix Arnebiae (Radix Lithospermi) 1.5kg, Sanguis Draxonis 3kg, Radix Astragali 1.5kg, Colla Corii Asini 2.5kg, Borneolum Syntheticum 0.5kg.

Substrate adjuvant includes (with 100kg ointment gauge): lanoline 15kg, vaseline 47.25kg, poloxamer 18812kg, Macrogol 4000 0.5kg, tristerin 5.25kg, propylene glycol 2.5kg, water 5kg, P-hydroxybenzoic acid second Ester 0.03kg.

Wherein, Cinnabaris uses 200 mesh water Cinnabaris industrialization finished products；Colla Corii Asini is 60 mesh powder with electric crusher pulverizing； Radix Arnebiae (Radix Lithospermi), Borneolum Syntheticum are effective ingredient thermally labile medical material, use comminution by gas stream mode to process；Radix Astragali fibroid is relatively strong, and substep uses Alcohol extraction and water extraction extractum, first with 8 times amount 75% ethanol extraction twice, then by the medicinal residues after alcohol extraction with 6 times amount water extraction three Secondary, obtain alcohol extraction and water extraction extractum；Sanguis Draxonis is prepared as solid dispersion to improve its dispersibility in adjuvant.

The Formulation of 2 ointment

Former Clinical practice prescription employing vaseline and lanoline are as prescription substrate, and add appropriate menthol as absorption Accelerator, its preparation method is: weigh Cinnabaris, Borneolum Syntheticum, Sanguis Draxonis, crosses 100 mesh sieves and become fine powder after pulverizing；Weigh Colla Corii Asini, the Radix Astragali, Radix Arnebiae (Radix Lithospermi), crosses 100 mesh sieves and becomes fine powder after pulverizing；By the traditional Chinese medicine fine powder mix homogeneously after above-mentioned sieving, standby；Weigh Mentholum to add Appropriate 95% ethanol, becomes menthol solutions after stirring and dissolving；Weigh Yellow Vaselin, heating in water bath, add hydroxyl after whole moltens Ethyl benzoate, stirring makes ethylparaben all dissolve；After the Yellow Vaselin of above-mentioned molten is the coldest, add with system Standby menthol solutions, stirs, and is placed on blender stirring, adds with the traditional Chinese medicine fine powder of mixing by several times while stirring, treat After medicated powder all adds, continue to stir to partly solidifying shape, to obtain final product.

The present invention considers infective use needs, still continues to use ointment type substrate, but to use the kind of adjuvant, proportioning with And preparation technology is redesigned.Simultaneously as crude drug has promoted through pre-treatment, dissolution and dispersive property, therefore Add absorption enhancer the most separately.

This prescription is applied white vaseline be conducive to moisturizing and the lubrication of wound surface, and have suitable toughness and painting exhibition Property, stable in properties nonirritant；Lanoline has stronger water absorption, the water of absorbable about 2 times of weight, can improve the suction of vaseline Aqueous, energy secure adhesion, with on skin, is difficult to wash away；Tristerin, as the stabilizer of substrate or thickening agent, can make base The sensitivity of confrontation variations in temperature reduces；PLURONICS F87 is the carrier of solid dispersion, due to large usage quantity, to ointment base The character of matter produces and significantly affects, and also should be considered major-minor.Containing a small amount of water in prescription, and add a small amount of PEG4000 and rise surely Determine and viscosifying action；Propylene glycol is in order to dissolve dispersion Borneolum Syntheticum.Rotten for preventing ointment microbial contamination from occurring, ointment needs add Enter suitable preservative, but owing to prescription containing the Cinnabaris of larger dose, itself i.e. have certain antisepsis, preservative Consumption can suitably reduce, ethylparaben as preservative, have low toxicity, odorless, tasteless, non-volatile, stable in properties without Irritating advantage, consumption is 0.03%.

3 ointment evaluation indexes

3.1 appearance character evaluation indexes

According to pharmacopoeial requirements, qualified ointment answers that denseness is suitable, exquisiteness uniformly, is easily coated with exhibition, easy cleaning, without denaturalization phenomenon.Root Requirement accordingly, needs in conjunction with Clinical practice, sets the ocular estimate standard of ointment, with total score 5 points (sophistication 1 point, viscosity 2 Point, coating 2 points) ointment outward appearance is given a mark.Standards of grading are shown in Table 1.

Table 1 ocular estimate index

3.2 dewatering ability indexs

Take ointment about 5g, be placed in 10ml centrifuge tube, respectively with centrifugal condition 2500r/min, 30min；3000r/min, 30min；4000r/min, 30min；5000r/min,30min；6000r/min, 30min are condition, after observation ointment is centrifugal Whether there are precipitation or composition separation situation to occur, prepare the dewatering ability of ointment with test.Standards of grading are shown in Table 2.

The experiment scoring of table 2 dewatering ability

3.3 resist cold heat-resistant experiment

Cold-resistant heat-resistant experiment: sealed by ointment, is respectively placed in 40 DEG C, in the baking oven of 60 DEG C, and in the refrigerator of-20 DEG C Take out after 24h, to be restored to room temperature, observe its form, Character change.Whether there are character, homogeneity to change according to ointment, are No have precipitation or lamination, evaluates its stability under high/low temperature condition.Evaluation criterion is shown in Table 3.

Table 3 resist cold heat-resistant experiment scoring

The single factor test prescription screening of 4 blank ointment bases

4.1 ointment water content are investigated

Due to the needs of dispersion medicine, containing a certain amount of water in ointment, the blank making a series of water content different is soft Cream base matter, during water content 5%-20%, the state of the blank ointment base of gained is as shown in table 4, when ointment water content is below 15%, The dewatering ability of ointment is preferable.

Table 4 ointment water content is investigated

4.2 lanoline additions are investigated

Ratio when lanoline addition makes agnolin mix with PLURONICS F87 in prescription produces change, Thus affect viscosity and the stability of ointment.Make the blank ointment base that a series of lanoline addition is different, table 5 see Go out when lanoline content is at 15%-22.5% the dewatering ability of ointment, cold-resistant heat-resistant stability preferable, and have conveniently viscous Denseness, is suitably coated with.

Table 5 lanoline addition is investigated

The mixing temperature of 4.3 agnolins and PLURONICS F87 is investigated

PLURONICS F87 can be the most melted more than 60 DEG C, uses higher mixing temperature, is persistently sufficiently stirred for contributing to PLURONICS F87 forms less colloidal particle in the mixture.Make the mixed of a series of agnolin and PLURONICS F87 Close the bare substrate that temperature is different, table 6 find out along with the viscosity raising prepared ointment of mixing temperature has increased, but from Heart stability is more preferable, and mixing temperature is substantially similar 80 DEG C-85 DEG C prepared ointment character.

The mixing temperature of table 6 agnolin and PLURONICS F87 is investigated

The ratio of 4.4 glyceryl monostearates and white vaseline is investigated

The addition of glyceryl monostearate can make ointment more stable, but ointment also can be made more sticky, affects ointment Appearance index.Make the bare substrate that a series of glyceryl monostearate is different from the ratio of white vaseline, table 7 find out if not The dewatering ability adding glyceryl monostearate ointment is poor, i.e. can be observed there is oil under the centrifugal condition of 3500r/min The phenomenon of layer emersion, has preferable dewatering ability when the mass ratio of glyceryl monostearate Yu white vaseline is 2:8, and thick Can spend still, be suitably coated with.

The ratio of table 7 glyceryl monostearate and white vaseline is investigated

5 orthogonals

According to early stage experiment of single factor examination result, ointment character is had considerable influence because have: in ointment Water content (A)；Addition (B) agnolin of lanoline and the mixing temperature (C) of PLURONICS F87；Stearic acid in prescription Glyceride and the ratio (D) of white vaseline, each factor takes 3 levels, by L₉3⁴Orthogonal table experiment arrangement, factor level table is shown in Table 8.With the mode of appearance (S1) of ointment, dewatering ability (S2), cold-resistant heat-resistant stability (S3) is inspection target, respectively with power Weight coefficient 4:3:3 carries out comprehensive grading S=S1+S2+S3.The face shaping of ointment is the best, energy under the highest centrifugal rotational speed Keeping stable, after high/low temperature condition, still molding is good, then the scoring of S will be the highest, otherwise, the lowest.

Table 8 factor level table

Table 9L₉3⁴Orthogonal table

Table 10 variance analysis

Note: F(0.05)=19

To the scoring of indices, comprehensive grading intuitively analyzes and variance analysis (be shown in Table 9, table 10).Analysis directly perceived Result shows that mode of appearance, the dewatering ability of ointment are all had aobvious with the ratio of white vaseline (factor D) by tristerin Writing impact, the scoring with the former is negative correlation；The cold-resistant heat-resistant stability of ointment is affected relatively by the water content (factor A) in ointment Greatly；Indices is all had an impact, according to the results of analysis of variance by the temperature (factor C) when lanoline mixes with PLURONICS F87 The comprehensive grading of ointment is had a significant impact by it；The impact that indices is marked by the addition (factor B) of lanoline is the most not Greatly.According to orthogonal experiment comprehensive grading result A₁B₃C₁D₃As optimum optimizing condition.Stearic acid is found in actual mechanical process The higher meeting of additional proportion of glyceride makes the viscosity of ointment excessive, and the production affecting finished product is canned, therefore adjusts tristerin Interpolation percentage ratio in white vaseline mixture is 10%.I.e. water content is the 5% of ointment total amount, and lanoline is ointment total amount 5%, lanolin mixture and Sanguis Draxonis solid dispersion mixing temperature are 80 DEG C, and tristerin is at white vaseline mixture In interpolation percentage ratio be 10%.

The preparation technology of 6 greasing base purple Zhu's ointment

6.1 with 60 DEG C of water dissolution Radix Astragali extracts (can use 70 DEG C of alcohol reflux of Radix Astragali decoction pieces), adds PEG4000, is sprinkled into Colla Corii Asini 60 mesh fine powder on liquid level uniformly and makes its molten in water, add lanoline and absorb water liquid, stirring Uniformly.Rising solution temperature is to 80 DEG C, and (Sanguis Draxonis solid dispersion is adopted and made with the following method to add Sanguis Draxonis solid dispersion Standby: Sanguis Draxonis to being completely dissolved with appropriate 95% ethanol, adds in 80 DEG C of melted PLURONICS F87s, stirs, fling to 95% Ethanol, obtains the solid dispersion of Sanguis Draxonis) so that it is melted, quickly stirring makes the mixture of formation mix, and continues stirring until Mixture cools down.

6.2 white vaseline mix with tristerin, are heated to melting, mixing, add water Cinnabaris 200 mesh powder and fill Dividing agitation grinding, add the Radix Arnebiae (Radix Lithospermi) 1000 mesh powder through comminution by gas stream in about 40 DEG C, dispersed with stirring is uniform；Through comminution by gas stream Borneolum Syntheticum 1000 mesh powder is placed in the propylene glycol solution containing ethylparaben 12.5mg/ml, dissolves under 80 DEG C of water-baths To without grains of sand sense, add above-mentioned white vaseline after being cooled to 55 DEG C and mix in glyceryl stearate mixture.

The product of above-mentioned two step gained is mixed under the conditions of 55 DEG C by 6.3, stirs, cools down and i.e. obtain ointment finished product. Fig. 1 is shown in the technological process of greasing base purple Zhu's ointment of the present invention.

7 conclusions

Greasing base purple Zhu's ointment prepared by the present invention is made up of, as the tax of ointment medicine and substrate adjuvant two parts Shape agent and the carrier of medicine, substrate adjuvant has the biggest impact to the quality of ointment, the dispersion of medicine.Oleaginous substrate Lubricious, nonirritant, the protected and emollescence to skin, and vaseline can play closing wound and dimension as substrate adjuvant Hold the effect of the certain wettability of wound.This ointment has good tack and coating, and each dispersion of medicine, quality are steady Fixed, character good, denseness is suitable, is suitable for clinical practice, and clinic has no obvious adverse reaction, have simultaneously cheap, Easily storage, advantage easy to use, be worth research further and clinical expansion utilization.

Below by way of the test of pesticide effectiveness, the purposes of invention formulation is further elaborated:

Test example 1 invention formulation is used for treating diabetic foot rat experiment model

(1) materials and methods

1. material

1.1 animals: SD male rat, SPF level, body weight (180 ± 10) g, by Shanghai Univ. of Traditional Chinese Medicine's Experimental Animal Center There is provided.

1.2 reagent: PMSF-Amresco company, RIPA lysate-green skies biological engineering company limited, BCA albumen is dense Spend and measure test kit-green skies biological engineering company limited, PAGE gel-Shanghai Wen Yuange company, Tris-BBI company, Glycine Glycine-Amresco company, methanol-Shanghai traditional Chinese medicines group, sodium chloride-Shanghai traditional Chinese medicines group, Tween-20- Sigma company, SDS-PAGE sample-loading buffer-green skies biological engineering company limited, Ponceaux-Shanghai traditional Chinese medicines group, GAPDH Antibody-Santa Cruze company, E-Cadherin antibody, p-P65 antibody, P65 antibody, p-PI3K antibody, PI3K antibody, p- JNK antibody, JNK antibody, HRP labelling, goat antirabbit Ig, GHRP labelling, goat anti-mouse IgG-Santa Cruze company, CST Santa Cruze company of CST company of CST company of CST company of CST company of CST company of company Santa Cruze company Marker- Fermentas company, NC film-Millipore company, 3M filter paper (20*20cm)-Whatman company, ECL chemical illuminating reagent Box-Millipore company, reverse transcription M-MLV1 test kit (RT-PCR)-TAKARA company, Universal SYBR Green- TAKARA company, DEPC (burnt ethylene carbonate)-Sigma company, TRIzol Reagent-ambion company, chloroform-traditional Chinese medicines collection Group, isopropanol-traditional Chinese medicines group, ethanol-traditional Chinese medicines group.30% stannous chloride, absorbing liquid, hydrargyrum titer, hydrargyrum Standard Reserving Solution, hydrargyrum Standard solution-traditional Chinese medicines group, dimethylbenzene-traditional Chinese medicines group (Shanghai examination), hematoxylin dye liquor-traditional Chinese medicines group (Shanghai examination), neutral gum- Traditional Chinese medicines group (Shanghai examination), two anti-in-China fir Golden Bridge, ethanol at different levels-traditional Chinese medicines group (Shanghai examination).

1.3 experimental apparatus: electrophresis apparatus-BIO-RAD company, transferring film instrument-BIO-RAD company, microplate reader-Thermo company, Shaking table (TS-8)-Shanghai precision instrument manufacturing company, chemiluminescence imaging system-upper sea-gull Xiang scientific instrument company limited, glimmering Fluorescent Quantitative PCR instrument-Applied Biosystems, 7900HT low-temperature and high-speed centrifuge-big dragon emerging wound limited public affairs of experimental apparatus Department ,-80 DEG C of refrigerator-SANYO GS companies, mercury vapourmeter (F 1 type), electric tube furnace, paraffin slicing machine-Leica.

2. experimental technique

2.1 rat skins, hepatic and renal tissue (hematoxylin-eosin staining, HE) tissue slice

Hematoxylin eosin stain method 1, draw materials with fixing: take off piece of tissue (Liver and kidney is 0.5cm × 0.5cm × 0.5cm, Skin histology is 0.5cm × 0.5cm holostrome), 10% formalin is fixed.2, dehydration.3, waxdip embedding.4, section and paster (thickness: 8 microns).5, dewaxing dyeing: dewaxing 2min × 3 time；Dehydration of alcohol, 100% ethanol 2min × 2 time, 95% ethanol 2min × 1 time；Water rinses；Dyeing hematoxylin 1-2min, water rinses to without blue Yihong 20s, and water rinses；Dehydration, 95% ethanol 2min × 1 time, 100% ethanol 2min × 2 time；Transparent dimethylbenzene, 2min × 3；Mounting, neutral gum 80uL, coverslip 24X50mm.

Om observation: take 100 times and 200 times of visuals field.

2.2ELISA detection

Principle: it uses antigen to be connected with enzyme by determinand with the specific reaction of antibody, is then produced by enzyme-to-substrate Color reaction, is used for quantitative determining.Measure object can be antibody can also be antigen.During measurement, antigen (antibody) is first tied It is combined on solid phase carrier, but still retains its immunocompetence, then add the conjugate (labelling that a kind of antibody (antigen) is combined into enzyme Thing), this conjugate still retains its former immunocompetence and enzymatic activity, when conjugate reacts knot with the antigen (antibody) on solid phase carrier After conjunction, add the corresponding substrate of enzyme, i.e. play catalyzing hydrolysis or redox reaction and in color.Its shade generated It is directly proportional to antigen (antibody) content to be surveyed.This color products can with the naked eye, optical microscope, electron microscope observation, Can also be measured with spectrophotometer (microplate reader).Its method is simple, convenient news speed, high specificity.

Operational approach: take after rat blood 3000 revs/min, centrifugal 20min, draws upper serum ,-20 DEG C of preservations.IL- 6, TNF-a, IL-1 β, AGES, bFGF, hCRP, NF, NO, ELISA detection kit measures (corresponding instructions is shown in concrete operations), Substantially flow process is as follows:

1), before experiment, test kit is taken out equilibrium at room temperature 30min.

2) in blank well, gauge orifice and sample well are separately added into 50 μ l Sample dilution, then in blank well, gauge orifice and Sample well is separately added into 50 μ l Sample dilution, standard substance and sample.

3) every hole adds 50 μ l Biotin-Conjugate.

4) room temperature 200rpm hatches 2h.

5) washing liquid is used 4 times.

6) every hole adds 100 μ lStreptavidin-HRP.

7) room temperature 200rpm hatches 1h.

8) washing liquid is used 4 times.

9) every hole adds 100 μ lTMB Substrate Solution.

10) lucifuge room temperature 200rpm hatches 10min.

11) every hole adds 100 μ l stop buffers.

12) at 450nm, light absorption value (OD value) is surveyed.

13) calculate: calculate standard curve linear regression equation with OD value, by the OD value generation of sample according to the concentration of reference material Enter equation, calculate sample concentration, then be multiplied by extension rate and be the reagent concentration of sample.

2.4 real-time polymerase chain reactions (Real-time PCR)

Principle: Real-Time round pcr, also known as real-time quantitative fluorescence PCR, refers to add in PCR reaction system glimmering Light group, utilizes fluorescence signal accumulation to monitor whole PCR process in real time, finally by standard curve, unknown template is carried out total amount The method analyzed or by Ct value, template is carried out relative quantitative assay.

Internal reference method: add the most quantitative internal standard and primer, internal standard gene engineering method in different PCR reaction tubes Synthesis.Forward primer fluorescent labeling, downstream primer not labelling.While template amplification, internal standard is also amplified.Produce at PCR In thing, owing to internal standard is different from the length of target template, the amplified production of the two can be separated with electrophoresis or high-efficient liquid and come, respectively Measure its fluorescence intensity, within be designated as compareing quantitative template to be detected.

Operational approach: take skin histology after rat healing, measure the content of itself VEGF and Ecadherin.

2.3.1Total the extraction of RNA and qualification

1) in sample cell, 1ml Trizol is added.

2) tissue refiner 60Hz grinds 90s, until lysate obvious little granule at noon.

3) room temperature stands 5min.

4) adding 1/5 volume of chloroform (200 μ l), acutely vibrate 15s, treats solution fully emulsified no phase separation phenomenon, and room temperature is quiet Put 5min.

5) 12,000g4 DEG C of centrifugal 15min.

6) carefully taking out centrifuge tube, Aspirate supernatant is transferred to another without in RNase centrifuge tube.

7) adding isopyknic isopropanol (500 μ l), turn upside down after fully mixing, room temperature stands 10min.

8) 12,000g4 DEG C of centrifugal 10min, supernatant discarded, low-speed centrifugal 5s, discard residual isopropanol.

9) 75% ethanol l ml washing, 12,000g4 DEG C of centrifugal 5min, carefully discard ethanol.

10) drying at room temperature 2-3min, adds 20 μ L sterilizing DEPC water dissolution precipitations, flicks bottom EP pipe to complete with finger tip CL.

2.3.2cDNA synthesis

I.PCR centrifuge tube is prepared mixed liquor, full dose 12 μ L, operates on ice.

Table 11 system

Cooled on ice 2min after ii.65 DEG C of insulation 5min.

Iii., after being centrifuged the several seconds, PCR pipe is prepared inverse transcription reaction liquid.

Table 12 reverse transcription reaction system

Iv.42 DEG C of insulation 1h.

DEG C v.70 cooled on ice after insulation 5min, the cDNA obtained can be directly used for 2nd-Strand cDNA synthesis or PCR expands ,-20 DEG C of preservations.During PCR amplification, the recommendation maximum usage amount of cDNA is 1 μ L.

2.3.3PCR reaction

1) primer sequence designed and analysis condition such as table 13 and table 14:

2) PCR pipe is prepared PCR reaction mixture, operate on ice.

Table 13real-time PCR reaction system

3) being divided into 3 parts after mixing, each sample does 3 and repeats comparison.

4) upper machine augmentation detection.

Primer sequence:

Table 14

2.4 Western blottings (Western-Blot)

Principle: Western Blot i.e. western blotting.It is in molecular biology, biochemistry and immunogenetics Conventional a kind of experimental technique.Its ultimate principle is the cell or biological tissue processed gel electrophoresis by specific antibody Sample colours.The position coloured by analysis and color depth obtain specified protein in the cell or tissue analyzed The information of expression.

Western Blot method uses polyacrylamide gel electrophoresis, and detected material is protein, and " probe " is anti- Body, " colour developing " resists with the two of labelling.Through the protein example that PAGE (polyacrylamide gel electrophoresis) separates, transfer to solid phase On carrier, solid phase carrier is with non-covalent bond form adsorbed proteins, and can keep polypeptide forms and the biology thereof of electrophoretic separation Activity is constant.Using the protein on solid phase carrier or polypeptide as antigen, play immunoreation with corresponding antibody, then with enzyme or with The second antibody of position element labelling reacts, through the specificity purpose base that substrate colour developing or autoradiography are separated by electrophoresis with detection Because of the protein ingredient expressed.This technology is also widely used in detecting the expression of protein level.

Operational approach: take rat skin tissue after healing, measure its E-Cadherin, p-P65, P65, p-PI3K, PI3K, P-JNK, JNK content.Step is as follows:

2.4.1. protein extracting and concentration measure

2.4.1.1 protein extracting

(1) tissue ice PBS is washed 3 times.

(2) taking the lysate of appropriate amount, add PMSF before use in several minutes, the ultimate density making PMSF is 1mM.

(3) every 10mg tissue adds 100 μ l lysates, shakes 2 minutes cracking tissues in 4 DEG C of homogenizers.

(4), after fully cracking, 10000g is centrifuged 5 minutes, takes supernatant.

2.4.1.2 quantification of protein

(1) according to sample size, add 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepare appropriate BCA work Liquid, fully mixes.

(2) add proper volume sample in 1.5ml centrifuge tube, complement to 100 μ l with 0.9%NaCl solution.

(3) each hole adds BCA working solution 1ml, places 30 minutes for 37 DEG C.

(4) measure A562, calculate protein concentration according to standard curve.

2.4.2.SDS-PAGE electrophoresis

1) prepare sample liquid: sample and 5 × sample-loading buffer 4:1 mixing, boil 5 minutes.

2) by ready sample and protein marker, loading respectively.

3) electrophoresis: use BioRad electrophoretic apparatus, sample first under 120V constant voltage electrophoresis to dyestuff close to separate Glue top.Then 160V constant voltage electrophoresis is to bottom the firm plastic emitting of bromophenol blue.

2.4.3. Protein transfer

(1) take out gel, remove all concentration glue.Gel is immersed in transfering buffering liquid 5 minutes.

(2) film is prepared: in advance NC film is submerged initially in transfering buffering liquid balance 5 minutes.

(3) transfer interlayer is assembled according to the order of foam, filter paper, gel, transfer film, filter paper, foam.

(4) transfer is folded up in transfer groove, it is ensured that transfer folder gel-side to negative electrode (-) film side to anode (+), in transfer groove, add appropriate amount of buffer solution, it is ensured that be totally submerged by transfer folder.

(5), during membrane-transferring device is placed in frozen water, turns on the power 400mA and transfer 2 hours.

(6), after having shifted, from groove, take out transfer folder, carefully open laminate layer transfer with tweezers, rinse in distilled water Film.

2.4.4. protein imaging

(1) film is placed in reaction box (the one of trace albumen faces up)

(2) add 0.5ml Ponceaux to dye 30 seconds, observe transferring effect.

(3) removing dye liquor, distilled water washes film three times, each 5 minutes.

2.4.5. immune detection

(1) close: Fresh confining liquid: in TBST, add defatted milk powder extremely final concentration of 5%(w/v), during closing, Being immersed in confining liquid by film, vibrate under room temperature 1h.

(2) adding the anti antibody diluted, 4 DEG C overnight.

Table 15

Antibody Designation	Species origin	Concentration	Corresponding two resist
				E-Cadherin antibody	Rabbit	1:500	Goat antirabbit
P-P65 antibody	Rabbit	1:1000	Goat antirabbit

P65 antibody	Rabbit	1:1000	Goat antirabbit
				P-PI3K antibody	Rabbit	1:1000	Goat antirabbit
PI3K antibody	Rabbit	1:1000	Goat antirabbit
				P-JNK antibody	Rabbit	1:1000	Goat antirabbit
JNK antibody	Rabbit	1:1000	Goat antirabbit
				GAPDH antibody	Mice	1:1000	Goat anti-mouse

(3) TBST washes film 3 times (each 5 minutes).

(4) two anti antibodys (1:5000) diluted, 1 hour oscillation incubation of room temperature are added.

(5) TBST washes film 3 times (each 5 minutes).

2.4.6. chemiluminescence detection

(1) after washed film, take out ECL luminescence reagent, take A liquid and B liquid each 1ml mixing.

(2) film is put in exposure apparatus, drip luminescent solution, expose three times, each 5min, select the overlap exposed three times Value.

2.5 hydrargyrum (Hg) cold-vapour atomic absorption method

Principle: mercury vapour has strong Absorption to the ultraviolet light of wavelength 253.7nm, after sample is appropriately processed, The hydrargyrum of various forms is transformed into mercury ion, with stannous chloride, mercury ion is reduced into element mercury, then is measured with mercury vapourmeter, Hydrargyrum concentration is directly proportional to absorption value.Instrument and reagent: mercury vapourmeter (F 1 type), electric tube furnace, 30% stannous chloride, absorb Liquid, hydrargyrum titer, hydrargyrum Standard Reserving Solution, hydrargyrum standard solution.

Operational approach:

2.5.1. sample treatment: accurately weigh and handle sample well and be placed in quartz boat (upper cover one fritter filter paper), by quartz Boat is put in the quartz ampoule of electric tube furnace, and one end of quartz ampoule is connected with oxygen cylinder, the other end and the suction filling 20ml absorbing liquid Closed tube connects.By Control for Kiln Temperature in 750 ± 50 DEG C of logical oxygen (flow velocity 2L/min), sectional combustion 3min, the hydrargyrum enrichment in sample In absorbing liquid, for measuring.

2.5.2. measure: accurately absorption certain volume absorbing liquid (after burning) is in hydrargyrum reaction bulb, add 2m130% chlorine Stopper bottle stopper immediately after changing stannous solution, start circulating pump, read obtained the maximum absorption.

2.6 immunohistochemistry technologies (immunohistochemistry, IHC)

Principle: SABC i.e. immunohistochemistry technology (immunohistochemistry) or immunocytochemistry skill Art (immunocytochemistry), is applied immunology ultimate principle antigen antibody reaction, i.e. antigen is special with antibody Property combine principle, by chemical reaction make traget antibody developer (fluorescein, enzyme, metal ion, isotope) colour developing come Determine histiocyte endoantigen (peptide and protein), it is positioned, qualitative and determine quantifier elimination.Between antibody and antigen Combination have height specificity, immunohistochemistry make use of this principle just.First by certain in tissue or cell Chemical substance extracts, and in this, as antigen or hapten, by obtaining specific antibody after immune animal, then resists with this Body removes the similar antigenic substance in detection tissue or cell.By means of histochemical method, antigen-antibody is combined simultaneously Position shows, reaches to carry out qualitative to the unknown antigen in tissue or cell with it, positions or determine quantifier elimination.

Operational approach:

(1) dewaxing

30min in 60 DEG C of baking boxs.

Put into dimethylbenzene I---10min while hot.

(2) aquation

100% ethanol I---5min, II---5min；

95% ethanol I---5min, II---5min；

85% ethanol---3min；

75% ethanol---2min；

Washing---1min.

(3) endogenic catalase is removed

Put into 3%H immediately₂O₂Methanol (30%H₂O₂(4 degree of lucifuges) 1ml+ methanol 9ml, now with the current) soak 10min.Washing 3min × 2 time.

(4) antigen retrieval

Pressure cooker is repaired: put into citrate buffer solution immediately, waits until boiling, and float rises, and timing 2min naturally cools to Room temperature.

(5) serum is closed

PBS--5min/ time, washing 2 times, dry periphery, SABC pen hooks circle, air-dries 15s.

Dropping 5%BSA confining liquid, is then placed in half an hour in 37 DEG C of incubators.Get rid of surplus liquid, do not wash.

(6) add one anti-

Dry the serum around microscope slide reverse side and face weave with absorbent paper, add 1:100 dilution one resist, in wet box in 4 DEG C of refrigerators preserve overnight.

(7) add two anti-

After returning to room temperature, PBS washes 3 times, each 5min, and it is anti-to add two after drying the PBS around tissue, is placed in 37 in wet box 0.5h in DEG C incubator.

(8) DAB developer is added

Dripping developer, incubated at room 5min after drying tissue PBS around immediately, water is slightly washed termination reaction, is steeped in water In.

(9) haematoxylin is redyed

Dropping Mayer ' s haematoxylin redyes 3～about 10min, immerses saturated Na2HPO4 molten after distilled water immersion several minutes 2min in liquid, steeps in water after specimen color becomes basket.

(10) be dehydrated transparent

Successively microscope slide is put into 75% ethanol-85% ethanol-95% ethanol-95% ethanol-100% ethanol-100% ethanol-two Toluene-xylene.Each reagent is placed 2min, is finally immersed in dimethylbenzene, moves in ventilated chamber.

(11) mounting

Drop in tissue side with neutral gum, then with on coverslip lid, will first set level side, put down another the most gently Side, in order to avoid producing bubble, sealing slice, thin piece and being placed in ventilated chamber and dry.

(12) statistical analysis

Utilize OPTPro software to take pictures, analyze positive rate.

(2) result

1.HE result

Moist diabetic foot rat model respectively organizes Liver and kidney HE result after medication as in figure 2 it is shown, A: normal ulcer group, B: glycosuria Sick group, D: diabetic infection ulcer matched group, Z0: Matrix controls group group, Z1: Chinese medicine small dose group, Z2: dosage group in Chinese medicine, Z3: Chinese medicine high dose group, C: western medicine group.Can be illustrated by Fig. 2: each group glomerule structural integrity, renal tubules form is normal, liver Door is normal, and hepatocyte form is normal, is showed no abnormal change.Do not find the sign of mercurialism.

2. Chinese drug-treated group different time sections blood Hg changes of contents

As it is shown on figure 3, explanation: Chinese medicine is respectively organized Hg assay result in blood and is shown, high, medium and low dosage group respectively organizes blood Hg content in liquid, in rising trend at the 3rd day to the 10th day, the 10th day front on a declining curve to putting to death, each group blood Hg content It is proportionate with dosage height.After 10th day may adapt to Organism of Rats to the front each group Hg changes of contents of execution, drug metabolism Accelerate relevant.

3.Elisa result

As shown in Figure 4, illustrate: Chinese medicine is respectively organized to compare with model control group and all can be reduced serum NO, and HCRP, NF contain Amount, wherein middle dosage group reduction is the most obvious.

4.Western blot result

As fig. 5 a and fig. 5b, illustrate: each group non-phosphorylating PI3K, JNK, P65, Ecadherin zero difference (P > 0.05), illustrate that each group of baseline is identical, there is comparability.Each Chinese drug-treated group compares with model group, except high dose group P-JNK is without substantially Outside difference, P-PI3K, P-JNK, P-P65 are respectively provided with difference (P < 0.05).Chinese drug-treated group PI3K, JNK, P65 protein phosphorylation are described Level is higher.

5.Real-time PCR result

As shown in Fig. 6 A-Fig. 6 F, illustrate: same template carries out the amplification curve diagram of 96 amplifications in same PCR instrument Destination county detection product amount is non-constant；The most great repeatability of Ct value.

As it is shown in fig. 7, explanation: Chinese drug-treated group Z1, Z2, Z3 group compares VEGF mRNA and Ecadherin with model control group Mrna expression has diversity (P < 0.05), and wherein in Z2 Chinese medicine, dosage group vegf expression is the most obvious；Z1 Chinese medicine low dose group Ecadherin expresses the most obvious.

5. SABC (IHC) result

As shown in Figure 8 and Figure 9, illustrating: result above shows, brown is positive expression, VEGF labelling, Z0, Z1 and Z3 group Expressing the lowest, express hardly, Z2 group is slightly expressed.CD34 antibody labeling, Z0 group is expressed the lowest, almost without the positive, with Z0 Relatively, Z1 expresses rising, and Z2 group is expressed the highest, and Z3 group is decreased slightly as low.

(3) conclusion

Summary index, verifies by the pharmacodynamics of non-infection diabetic foot rat model, Chinese drug-treated group of the present invention can Reduce the inflammatory reaction of local, improve the expression of the local anti-inflammatory factor, thus promote the healing of wound surface, it is seen that invention formulation (purple Zhu's ointment) treatment diabetic foot determined curative effect, draws purple Zhu's ointment medicament curative effect of middle dosage group relatively by Integrated comparative Good, this is consistent with the Medicines screening and gradingup result of early stage.

Claims (4)

1. treating a greasing base Chinese medicine ointment formulation for diabetic foot, it is made up with substrate adjuvant of effective ingredient, It is characterized in that, described effective ingredient is made up of the crude drug of following weight portion: 7 parts of Cinnabaris, Radix Arnebiae (Radix Lithospermi) 3 parts, the Radix Astragali 3 parts, sanguis draconis Exhaust 6 parts, 5 parts of Colla Corii Asini, Borneolum Syntheticum 1 part；Described substrate adjuvant includes the component of following weight portion: lanoline 30 parts, vaseline 94.5 Part, PLURONICS F87 24 parts, Macrogol 4000 1 part, tristerin 10.5 parts, propylene glycol 5 parts, 10 parts of water, to hydroxyl Yl benzoic acid ethyl ester 0.06 part.

2. the process of preparing of the greasing base Chinese medicine ointment formulation as described in claim 1, it is characterised in that under including State step:

1. with 55～60 DEG C of water dissolution Radix Astragali extracts, add Macrogol 4000, liquid level is sprinkled into Colla Corii Asini powder uniformly Make its molten in water, add lanoline and absorb water liquid, stir；Rising solution temperature, to 80～85 DEG C, adds Sanguis Draxonis Solid dispersion so that it is melted, quickly stirring makes the mixture of formation mix, and continues stirring until mixture cooling；Described dragon Sanguis Draxonis solid dispersion is adopted and is prepared with the following method: Sanguis Draxonis to being completely dissolved with appropriate 95% ethanol, adds 80～85 DEG C and melts In the PLURONICS F87 melted, stir, fling to 95% ethanol, obtain the solid dispersion of Sanguis Draxonis；Described Radix Astragali extract Substep uses alcohol extraction and water extraction extractum, specifically adopts and prepares with the following method: first with twice Huang of 8 times amount 75% ethanol extraction Stilbene, then by the medicinal residues after alcohol extraction with 6 times amount water extraction three times, obtain alcohol extraction and water extraction extractum；

2. vaseline mixes with tristerin, is heated to melting, mixing, adds Cinnabaris and is sufficiently stirred for grinding, in 40～45 DEG C add Arnebia Root, dispersed with stirring is uniform；Borneolum Syntheticum is placed in the propylene glycol solution containing ethylparaben 12.5mg/ml In, it is dissolved under 75～80 DEG C of water-baths without grains of sand sense, adds after cooling in above-mentioned vaseline and glyceryl stearate mixture Mixing；

3. the product of above-mentioned two step gained is mixed, stir, cool down and i.e. obtain ointment finished product.

3. the method as described in claim 2, it is characterised in that described Cinnabaris uses 200 mesh water Cinnabaris industrialization finished products；Institute Stating Colla Corii Asini with electric crusher pulverizing is 60 mesh powder；Described Radix Arnebiae (Radix Lithospermi), Borneolum Syntheticum use comminution by gas stream mode to process.

4. the application in the medicine of preparation treatment diabetic foot of the preparation as described in claim 1.