CN106619975A - Medicine injected at Zusanli acupoint for treating diabetic foot and preparation method of medicine - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to dicranostigma leptopodum injection and preparation method thereof. The dicranostigma leptopodum injection is prepared from a dicranostigma leptopodum extracting solution which is an active drug, and the dicranostigma leptopodum extracting solution is obtained by extracting through alcohol extraction and water precipitation, eluting and purifying through a resin column, and finally adding auxiliary materials. By the dicranostigma leptopodum injection, the blood glucose level of mice can be reduced remarkably, and further experiments show that by acupoint injection of the dicranostigma leptopodum injection, diabetic foot can be treated remarkably.

Description

A kind of Zusanli point injectable drug and its preparation method for treating diabetes

Technical field

The present invention relates to LEMAHUI ZHUSHEYE and its preparation, and in particular to a kind of LEMAHUI ZHUSHEYE for treating diabetes And preparation method thereof.

Background technology

Diabetes are a kind of serious complication of diabetes, are that diabetic is disabled, or even lethal major reason One of, not only cause suffering to patient, and make it add huge financial burden.According to incompletely statistics, diabetic foot Ulcer accounts for 20% or so.

Diabetic foot makes lower limb defencive function go down due to DPN, and big blood vessel and microangiopathies make artery Hypoperfusion causes microcirculation disorder to be susceptible to diabetes.Wherein sensory nerve pathology merges too high mechanical stress, is to draw Play the main initiating agent of foot ulcers and infection.Inflammation and histologic lesion are that a certain degree of alternate stress acts on a spy The result in the fixed region for losing sensibility.From ground, shoes or other cause ulcer shape adjacent to the pressure or shearing force of toes Into due to lacking normal Neuroprotective Mechanisms, ulcer often increases because of the presence of apophysis.Autonomic pathology is caused Skin normally perspires regulatory function, skin temperature regulatory function and blood fortune regulating power and loses, and causes local organization pliability to drop It is low, form the callosity of thickness and more broken and cracking.Additionally, the forfeiture for normally perspiring ability has blocked the water again of local organization Change, cause to organize further destruction so that deep tissue is easier to bacteria planting.Morbidity of the kinesitherapy nerve pathology in diabetes In also play certain effect, the contracture of foot inherence flesh causes typical claw-like microdactyly.The hyperextension of articulationes metatarsophalangeae is also proved to Pressure under caput metatarsale can directly be increased so that the position is more likely formed ulcer.Nearly interphalangeal joint flexing is caused between the toe of projection Joint dorsal part increases with the ulcerated risk in toe point plantar side, and vascular lesion causes the tissue of destruction to be difficult to heal.

The predominantly outer wiping ointment of medicament of diabetes is treated in prior art, local treatment is reached by promoting blood circulation, sterilization The effect of diabetes, but the drug action of topical agent is slow, and cure the symptoms, not the disease, it is impossible to fundamentally cure diabetes Foot.

The inventor of this patent has been surprisingly found that in research work, will rein in the horse back to be prepared into parenteral solution and carry out acupoint injection therapy and controls Treat diabetes and taken unexpected effect, on this basis, inventor has been carried out after repeated multiple times research, is obtained Technical scheme.

It is of the present invention to rein in the horse back as Scrophulariaceae bastard speedwell Pseudolysimachion linariifolium subsp. The ground herb of dilatatum (Nakai ＆ Kitagawa) D.Y.Hong.

The content of the invention

For the deficiencies in the prior art, the invention provides a kind of LEMAHUI ZHUSHEYE for treating diabetes and its preparation Method, rapid reliable its liquid of its effect is directly injected into acupuncture point, and absorption process is very short, and without alimentary canal, not by pH, Enzyme, food etc. affect, and without first pass effect, medicament contg is difficult loss, curative effect reliability.

To realize object above, the present invention is achieved by the following technical programs：

A kind of LEMAHUI ZHUSHEYE for treating diabetes, is made up of active medicine and excipient substance, and the active medicine is Le Horse returns extract.

Preferably, the extract of reining in the horse back accounts for the 5% of parenteral solution cumulative volume.

Preferably, the excipient substance be sodium chloride, vitamin C, Sulfobutyl ether β _ cyclodextrin, glycerine, ethyl lactate, One or more in citric acid, mannitol, glycerine, glycine, sorbierite.

A kind of preparation method of the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：

（1）It is in 65～85% ethanol water, to be warming up to 80～100 to take dry 3～8 times of amount concentration of addition of reining in the horse back DEG C, cold filtration after the h of heating and refluxing extraction 0.5～2 obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then vacuum distillation obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 3～5 h, is filtered, take supernatant, then use milipore filter Filter, filtrate concentration, it is extract at the beginning of 1.05～1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the pH 1～5 of 2～10 times of amounts of column volume The aqueous solution eluted, collect eluent, then eluted with the 50% of 2～10 times of column volume amount ethanol, collect wash-out Liquid, eluent merging twice, concentration obtains concentrate, then concentrate is adjusted into pH to 10～11 with alkali, at 60～80 DEG C 1～2h of heating, then pH to 3～5 is adjusted, then its Jing polyamide resin column is processed, with 2～5 times of water elution, collect wash-out Liquid, then distills at 70～80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6～7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 6.5～8.0, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product LEMAHUI ZHUSHEYE.

Preferably, the preparation method of the LEMAHUI ZHUSHEYE of the treatment diabetes, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C to take dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, and then decompression is steamed Evaporate, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then use milipore filter mistake Filter, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

Preferably, step（1）Described reduced pressure distillation process condition be -0.05～-0.09MPa, 40～90 DEG C.

Preferably, step（2）Milipore filter used by described ultrafiltration is 3000～6000 dalton, selected from diacetate fiber Plain film, three cellulose acetate membrane, cyanoethyl cellulose film, PS membrane, sulfonated polysulfone membrane, poly (ether sulfone) film, sulfonated polyether sulfone Film, Polysulfonamide, phenolphthalein side base polyarylsulfone (PAS) film, polyvinylidene fluoride film, polyacrylonitrile film, polyimide film, cellulose membrane, first Base methyl acrylate-acrylonitrile-based copolymers membrane, polyacrylonitrile-cellulose diacetate blend film.

A kind of application of LEMAHUI ZHUSHEYE in treatment diabetes medicine is prepared, the LEMAHUI ZHUSHEYE is by active drug Thing and excipient substance are constituted, and the active medicine is extract of reining in the horse back, accounts for the 5% of LEMAHUI ZHUSHEYE cumulative volume, the medicine Auxiliary material is sodium chloride, glycerine, glycerine, water, and the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C by dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then technique bar Part is -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

A kind of application of LEMAHUI ZHUSHEYE in medicament for treating insomnia is prepared, the LEMAHUI ZHUSHEYE by active medicine and Excipient substance is constituted, and the active medicine is extract of reining in the horse back, accounts for the 5% of LEMAHUI ZHUSHEYE cumulative volume, the excipient substance For sodium chloride, glycerine, glycerine, water, the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C by dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then technique bar Part is -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

Specific embodiment

To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention, Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one Divide embodiment, rather than the embodiment of whole.Based on the embodiment in the present invention, those of ordinary skill in the art are not making The every other embodiment obtained under the premise of creative work, belongs to the scope of protection of the invention.

Embodiment 1：

A kind of LEMAHUI ZHUSHEYE for treating diabetes, by account for parenteral solution cumulative volume 5% rein in the horse back extract and sodium chloride, Glycine, glycine, sorbierite, citric acid, water composition.

A kind of preparation method of the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：

（1）Take the dry 100g that reins in the horse back, add 3～8 times of amount concentration be in 65～85% ethanol water be warming up to 80～ 100 DEG C, cold filtration after the h of heating and refluxing extraction 0.5～2 obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, Merge 3 gained liquids, then vacuum distillation obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 3～5 h, is filtered, take supernatant, then use milipore filter Filter, filtrate concentration, it is extract at the beginning of 1.05～1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the pH 1～5 of 2～10 times of amounts of column volume The aqueous solution eluted, collect eluent, then eluted with the 50% of 2～10 times of column volume amount ethanol, collect wash-out Liquid, eluent merging twice, concentration obtains concentrate, then concentrate is adjusted into pH to 10～11 with alkali, at 60～80 DEG C 1～2h of heating, then pH to 3～5 is adjusted, then its Jing polyamide resin column is processed, with 2～5 times of water elution, collect wash-out Liquid, then distills at 70～80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6～7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 6.5～8.0, and then sintered glass funnel filtration rushes nitrogen embedding in 2mL ampoules, and sealing uses 100 DEG C flowing steam sterilization 30 minutes, obtain final product LEMAHUI ZHUSHEYE.

Embodiment 2：

A kind of LEMAHUI ZHUSHEYE for treating diabetes, by account for parenteral solution cumulative volume 5% rein in the horse back extract and sodium chloride, Sulfobutyl ether β _ cyclodextrin, sorbierite, ethyl lactate, water composition.

A kind of preparation method of the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：

（1）The dry 100g that reins in the horse back is taken, adds 6 times of amount concentration to be in 65% ethanol water, to be warming up to 80 DEG C, heated back Cold filtration after stream extraction 0.5, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, so Vacuum distillation afterwards, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 3h, is filtered, take supernatant, then with ultrafiltration membrance filter, Filtrate concentrates, and it is extract at the beginning of 1.05～1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first water-soluble with the pH 1 of 10 times of column volume amount Liquid is eluted, and collects eluent, then is eluted with the 50% of 2 times of column volume amount ethanol, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 11 with alkali, and 1h is heated at 80 DEG C, then adjusts pH to 5, Then its Jing polyamide resin column is processed, with 5 times of water elution, collects eluent, then distilled at 80 DEG C, remove ethanol Extremely without alcohol taste, then pH is adjusted to 6, obtain extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 6.5～8.0, and then sintered glass funnel filtration rushes nitrogen embedding in 2mL ampoules, and sealing uses 100 DEG C flowing steam sterilization 30 minutes, obtain final product LEMAHUI ZHUSHEYE.

Embodiment 3：

A kind of LEMAHUI ZHUSHEYE for treating diabetes, by 5% rein in the horse back extract and sodium chloride, glycerine, water are constituted.

A kind of preparation method of the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：

（1）The dry 100g that reins in the horse back is taken, adds 5 times of amount concentration to be in 75% ethanol water, to be warming up to 85 DEG C, heated back Cold filtration after 1.5 h is flowed, extract and the dregs of a decoction are obtained, aforesaid operations 2 times are repeated to the dregs of a decoction, merge 3 gained liquids, then Vacuum distillation, obtains alcohol extract, the reduced pressure distillation process be -0.05 MPa, 60 DEG C；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 3 h, is filtered, take supernatant, then use 3000 dalton The h of cyanoethyl cellulose membrane filtration 24, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing adsorption resin columns at the beginning of reining in the horse back are processed, and are first washed with the aqueous solution of the pH 3 of 5 times of amounts of column volume It is de-, then eluted with the ethanol of 5 times of amounts of column volume, eluent is collected, concentrate is concentrated to give, then by concentrate ammoniacal liquor PH to 10 is adjusted, 3 h are heated at 60 DEG C, then adjust pH to 5, then processed its Jing polyamide resin column, with 2 times of washing It is de-, then distill at 80 DEG C, ethanol is removed extremely without alcohol taste, then pH is adjusted to 7, obtain extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 7, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C of stream Logical steam sterilizing 30 minutes, obtains final product LEMAHUI ZHUSHEYE.

Embodiment 4：Rein in the horse back the research of extracting factor

1st, concentration of alcohol

According to the experimental design of extraction process, the ethanol of many variable concentrations carries out preferably, testing respectively with 25%, 50%, 75% and 95% ethanol as Extraction solvent, the rate of transform with extract protocatechuic acid as inspection target, in combination with protocatechuic acid Content, different solvents are investigated.

Experimental technique：Weigh and rein in the horse back 5 parts, every part of 200g adds solvent to be heated to reflux 3 times, is heated to reflux temperature for 85 DEG C, the time is 1h, and the extract of three extractions is merged, and ethanol is reclaimed in vacuum distillation, and vacuum drying determines containing for protocatechuic acid Amount（Assay method reference：Chen Yong, Fang Cuifen, Xiang Zhimin, Tang steps on peak .RP-HPLC and determines containing for capsule protocatechuic acid of reining in the horse back Amount [J]. Chinese patent drug. 2007.29 (12):1867-1869. is similarly hereinafter）, as a result such as table 1.

Wherein experimental group solvent is respectively the ethanol of 5 times of amounts 25%, 50%, 75% and 95%, and control group solvent is that 5 times of amounts are steamed Distilled water.

Impact of the concentration of alcohol of table 1 to extraction process

As a result show, when being extracted with 75% ethanol, the content and the rate of transform of protocatechuic acid are above the ethanol of other concentration, because The ethanol of this selection 75% is used as Extraction solvent.

, heating-up temperature

After Extraction solvent is determined, also need it is further preferred to Extracting temperature, test using 75% ethanol as Extraction solvent, carry The time is taken for 1.5h, heating-up temperature be respectively 80 DEG C, 85 DEG C, 90 DEG C, 100 DEG C of rates of transform with extract protocatechuic acid be Inspection target, is carried out preferably to the heat time, as a result such as table 2.

Impact of the heating-up temperature of table 2 to extraction process

As a result show, the rate of transform of the protocatechuic acid of experimental group 2 selects heating-up temperature to be 85 DEG C apparently higher than other groups.

, the heat time

After Extraction solvent is determined, also need it is further preferred to extraction time, test using 75% ethanol as Extraction solvent, plus Hot reflux temperature is 85 DEG C, and extraction time is respectively the ethanol of 0.5h, 1h, 1.5h, 2h as Extraction solvent, with extract Central Plains The rate of transform of catechuic acid is inspection target, the heat time is carried out preferably, as a result such as table 3.

Impact of the heat time of table 3 to extraction process

As a result show, the rate of transform of the protocatechuic acid of experimental group 3 is substantially poor with the thing of experimental group 4 higher than enforcement group 1 and experimental group 2 It is different, therefore select to be heated to reflux the time for 1.5h.

Embodiment 5：LEMAHUI ZHUSHEYE treats the experimental study of diabetes

1 experiment material

1.1 experimental animals

Experiment is from SPF levels health male Wistar rat 50（There is provided by Heilongjiang University of Chinese Medicine's animal center）, body weight For（250±10）g.Raise in 22～25 DEG C of room temperature, humidity 50%, draughty environment, with cleaning grade feed（By black dragon River university of TCM animal center is provided）Single cage is fed, and is freely ingested, is drunk water.

1.2 reagents and medicine

Streptozotocin（Steptozotocin, STZ）：Sigma（Sigma）Aldrich Shanghai trade Co., Ltd；

LEMAHUI ZHUSHEYE：LEMAHUI ZHUSHEYE is made up of active medicine and excipient substance, and the active medicine is carried to rein in the horse back Liquid is taken, the 5% of LEMAHUI ZHUSHEYE cumulative volume is accounted for, the excipient substance is sodium chloride, glycerine, glycerine, water, the re-injection of reining in the horse The preparation method of liquid is penetrated, step is as follows：

（1）By the dry 100g that reins in the horse back, add 5 times of amount concentration to be in 75% ethanol water, to be warming up to 85 DEG C, heat back Cold filtration after 1.5 h is flowed, extract and the dregs of a decoction are obtained, aforesaid operations 2 times are repeated to the dregs of a decoction, merge 3 gained liquids, then Process conditions are -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 7, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C of stream Logical steam sterilizing 30 minutes, obtains final product LEMAHUI ZHUSHEYE.

1.3 prepare diabetes rat model

Randomly select 40 rats and diabetes rat model is set up using the method for STZ lumbar injections, it is former according to ischemia-reperfusion Reason manufacture diabetes rat model（Ulcer of foot is one of cardinal symptom of diabetes）.The right side to diabetic model group rat The same position of hind leg is ligatured, and using 10 extra section suture ligature rat ankle and foots, 2h is ligatured every time, decontrols 30min, daily Repeat ligation 3 times, 3d is ligatured altogether, operate 1 time every 1d.There is skin with ligated limbs to black, bubble, bleeding blister, ulcer are true Recognize the successful standard of modeling.During ligation, operated by same person, it is ensured that identical using strength during ligation.It is all ligated The equal Cheng Mo of rat.

Intervention of 1.4 LEMAHUI ZHUSHEYEs to diabetes rat model

40 diabetes rat models are numbered according to body weight, according to random digits table, by diabetes rat model point For 2 groups, 20 per group, respectively：LEMAHUI ZHUSHEYE treatment group and diabetes blank control group.To LEMAHUI ZHUSHEYE amount Group is according to 300mg/（kg·d）（Equivalent to 5 times of clinical patients consumption per day）Dosage, to mouse left side gastrocnemius position note Penetrate medicine, 1 time/d.To diabetes blank control group use physiological saline gavage, 2 times/d.Body weight is measured weekly, changes medication Amount, shares medicine 6 weeks.The fasting blood-glucose of each group rat is detected respectively（FBG）, Diagnostic Value of Fasting Serum insulin（FSI）, lectin from hemolymph（Including High shear rate, low shear rate, Plasma Viscosity, hematocrit, erythrocyte aggregation index, 6 indexs of deformable index）.

1.5 statistical methods

Database is set up with Excel, with SPSS13.0 statistical analysis software analyze datas.Measurement data with± s represents, two groups Between compare and checked using t, multigroup is compared and adopts variance analysis；Enumeration data compares and adopts χ²Inspection, inspection level α=0.05.

2 results

2.1 LEMAHUI ZHUSHEYE treatment groups, diabetes blank control group comparitive study

After the LEMAHUI ZHUSHEYEs of 6 weeks are intervened, after the treatment of diabetes LEMAHUI ZHUSHEYE treatment group, foot symptom is obvious Take a turn for the better；And the body weight of diabetes blank control group rat is decreased obviously before relatively treating, foot symptom aggravates.It is shown in Table 4.

The LEMAHUI ZHUSHEYE treatment group of table 4 and diabetes blank control group foot symptom score level condition（Only, %）

Note：Two groups are compared, χ²=10.489, P=0.015.Two groups have notable difference.

Each Testing index compares between 2.2 LEMAHUI ZHUSHEYE treatment groups and diabetes blank control group

Before the experiment initial stage treats, the FBG of two groups of rats, body weight and foot symptom score no significant difference；Rein in the horse re-injection After the treatment FBG is significantly reduced for She Ye treatment groups, and FSI is significantly raised, high shear rate in lectin from hemolymph, low shear rate, Plasma Viscosity, Hematocrit, erythrocyte aggregation index, deformable index work property is reduced.It is shown in Table 5.

Body weight, FBG, FSI and blood flow after the LEMAHUI ZHUSHEYE treatment group of table 5 and the treatment of diabetes blank control group The comparison of change（±s）

3 conclusions

This experiment confirms：（1）LEMAHUI ZHUSHEYE can improve the fasting blood-glucose of diabetes B rat and the disease of foot ulcers Shape, with certain effect for reducing blood sugar, treatment diabetes.（2）LEMAHUI ZHUSHEYE can significantly reduce diabetes 6 indexs, have improvement result to Hemorheology in rat lectin from hemolymph.This experiment also demonstrates that LEMAHUI ZHUSHEYE has and reduces The blood sugar of diabetes rat, illustrates that the medicine is influential on diabetes；The serum insulin level of rat is in medication in experiment After have significantly rising, illustrate that this medicine can improve the islet function of diabetes B rat of induction.

Embodiment 6：The clinical observation of 0 grade of diabetes of LEMAHUI ZHUSHEYE type Treated with Point-injection Therapy

Diabetes make the quality of life degradation of patient, and treat difficult, and treatment cycle is long, and medical expense is high, to trouble Person and society bring white elephant.Inventor is set with the artificial research object of 0 grade of diabetic foot using random packet control The research method of meter, gives different treatment and nursing measures, and the deterioration intervention to 0 grade of diabetes is studied, so as to explore A kind of more satisfactory treatment and nursing method of 0 grade of diabetes, prevents ulcer of foot from occurring.

1 object and method

1.1 objects

0 grade of the diabetes B diabetes 24 in December, 2015 to the hospitalization of in July, 2016, patient with diabetic feet is accorded with Close the diagnostic criteria of diabetes world clinical guidelines.It is wherein male 12, female 12,50 years old～65 years old age, averagely（62.9± 50.1）Year, diabetic duration（3.6±2.1）Year；Receive diabetes systematic treating, OHA or insulin therapy have Effect.All patients feet's skins are complete, and without open focus, Jing Chinese medical discriminations are venation block, show as pedal skin purplish red Or it is purple dim, limbs send out cool numbness, sharp ache, fuyang pulse decrease.It is randomly divided into experimental group and control group, per group 12, two groups Patient's ordinary circumstance（Sex, the age, the course of disease, symptom, sign, blood sugar, HbAle egg from, blood fat, blood pressure etc.）Without statistics Meaning（P>0.05）, with comparativity.

1.2 methods

Related patient is pressed into the sequencing numbering of institute, two groups are incorporated at random respectively.

Control group：Diabetic diet and exercise therapy add OHA and（Or）Insulin therapy controls blood sugar, makes sky Abdomen blood sugar≤7.0mmoL/L, postprandial blood sugar≤10.0mmoL/L, using conventional foodcare and health education.

Experimental group：LEMAHUI ZHUSHEYE acupoint injection therapy is adopted on the basis of former equal treatment and nursing.With reference to National Technical prison Superintend and direct People's Republic of China's standard of office's promulgation《Location of acupoints》, determine point st 36.Patient makes even clinostatism, and a parapodum three is taken every time In acupuncture point, left and right wheels are for using, after cave area skin routine disinfection, extracted with the 2mL sterilizing syringes for being furnished with No. 5 syringe needles and reined in the horse back Parenteral solution 2mL, straight thrust is subcutaneous 0.5 11 cun, and pumpback after bringing about the desired sensation is injected into re-injection of reining in the horse and penetrates without blood and impassivity Rediating Pain per cave Liquid 1mL, one time a day, and 15 times is a course for the treatment of, treats a course for the treatment of.

LEMAHUI ZHUSHEYE：LEMAHUI ZHUSHEYE is made up of active medicine and excipient substance, and the active medicine is to rein in the horse Extract is returned, the 5% of LEMAHUI ZHUSHEYE cumulative volume is accounted for, the excipient substance is sodium chloride, glycerine, glycerine, water, and this is reined in the horse The preparation method of parenteral solution is returned, step is as follows：

（1）By the dry 100g that reins in the horse back, add 5 times of amount concentration to be in 75% ethanol water, to be warming up to 85 DEG C, heat back Cold filtration after 1.5 h is flowed, extract and the dregs of a decoction are obtained, aforesaid operations 2 times are repeated to the dregs of a decoction, merge 3 gained liquids, then Process conditions are -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 7, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C of stream Logical steam sterilizing 30 minutes, obtains final product LEMAHUI ZHUSHEYE.

1.3 clinical observation on the therapeutic effect

1.3.1 judgment criteria

With reference to 2002《New Chinese medicine guideline of clinical investigations》In《New Chinese medicine physiotherapy diabete（Diabetes）Clinic Will be with regard to guideline》.

Recovery from illness：Pain, numbness, the symptom treatment such as send out cool after all disappear, skin color recovers normal；

It is effective：Pain, numbness, the symptom treatment such as send out cool after be obviously improved, skin color is obviously improved；

Effectively：Pain, numbness, the symptom treatment such as send out cool after take a turn for the better, skin color improves；

It is invalid：Pain, numbness, the symptom treatment such as send out cool after change not substantially, skin color does not improve.

All objects are responsible for observing related symptoms and sign and recording by special messenger（Observer is not involved in treatment, does not also know point Group situation）, observe and be detected on treatment the previous day for the first time and last time is treated and carried out one day after.

1.3.2 school work rheology Hygienic monitoring on hands of childhood

The arteria dorsalis pedis hemorheology ginseng before and after Case treatment is determined using Acuson28XP/10 type color Doppler echocardiographies instrument Number, is carried out one day after in treatment the previous day first time and last time treatment.

1.4 side reactions are observed

After injection 0.5h, 2h, 6h, 12h, 24h, three days, seven days, observation whether there is the local reactions such as itching, redness, pain And the general reaction such as have a fainting spell during acupuncture treatment, have a headache, suffering a shock.

1.5 statistical procedures

All market demand SPSS11.0 statistical softwares are processed, and using inspection, measurement data is with for the inspection of enumeration data and rate Number ± standard deviation（）Represent, checked using t.

2 results

2.1 two groups of patient's biped therapeutic effects compare

6 two groups of patient's biped therapeutic effects of table compare

The result of table 6 shows：The treatment total effective rate of experimental group is 100.00%, and the treatment total effective rate of control group is 58.33%, two Compare that there were significant differences between group（=12.596, * P<0.01）.

2.2 two groups of patient blood rheological parameters compare

7 two groups of patient's arteria dorsalis pedis blood rheology parameters of table compare（）

The result of table 7 shows：The front instep Diameter of two groups of Case treatments, VPV and CBF numeric ratio are compared with without statistics Learn difference（P>0.05）.2. itself compare before and after two groups of Case treatments, instep Diameter expands after treatment group's Case treatment, blood Flow velocity degree is accelerated, and CBF increases, compares with statistical significance with before treatment（P<0.01）, and become after control group Case treatment Change not statistically significant（P>0.05）.3. compare after two groups of Case treatments, each Experiment Parameter group is superior to control group, with notable Statistical significance (P<0.01）.

3 conclusions

After intervening two groups of diabetes Bs, 0 grade of diabetes difference treatment and nursing, clinical efficacy and arteria dorsalis pedis blood stream Become correlation study to show：Experimental group is substantially better than control group, is more beneficial for improving pedal blood circulation, alleviates diabetes Foot symptom and sign, desirably prevent the generation of ulcer of foot.

Diabetes are one of diabetes important complications, and according to external investigation, about 15% diabetic will occur foot Ulcer, more than 80% diabetic cuts toe or amputation due to ulcer of foot, so if can rationally control 0 phase diabetes Treat and carry out nursing and interfering in time, prevent the generation of ulcer of foot, patient suffering will be reduced, mitigate burden on society, this exactly traditional Chinese medical science The preferably embodiment of " not treating the disease affected, preventive treatment of disease " theory.The pathogenesis of diabetes is related to DPN, vascular lesion and sense Dye, and blood circulation disorder is the basis of diabetes morbidity.Thus on Primary Care, using the treatment side of tcm characteristic Method, catches blood circulatory disorder this pathologic process, and being reached by promoting blood circulationization addiction medicine acupoint injection therapy improves blood circulation, eliminates induction Factor, so as to intervene the deterioration of 0 grade of diabetes, prevents ulcer of foot from occurring.

This result of study shows, to 0 grade of diabetes of diabetes B using LEMAHUI ZHUSHEYE in Zusanli, Sanyinjiao Acupoint injection therapy, can promote 0 grade of diabetes remission, prevent the generation of ulcer of foot, and patient has no adverse reaction, and is a kind of peace Entirely, 0 grade of effective, simple diabetes intervening measure.

Embodiment 7：Therapeutic action of the LEMAHUI ZHUSHEYE to Diabetic Nephropathy is studied

1 materials and methods

1.1 animals, medicine, reagent and instrument

Cleaning grade Wistar rats 45, purchased from Heilongjiang University of Chinese Medicine's Experimental Animal Center.

LEMAHUI ZHUSHEYE：LEMAHUI ZHUSHEYE is made up of active medicine and excipient substance, and active medicine is carried to rein in the horse back Liquid is taken, the 5% of LEMAHUI ZHUSHEYE cumulative volume is accounted for, excipient substance is sodium chloride, glycerine, glycerine, water, the LEMAHUI ZHUSHEYE Preparation method, step is as follows：

（1）By the dry 100g that reins in the horse back, add 5 times of amount concentration to be in 75% ethanol water, to be warming up to 85 DEG C, heat back Cold filtration after 1.5 h is flowed, extract and the dregs of a decoction are obtained, aforesaid operations 2 times are repeated to the dregs of a decoction, merge 3 gained liquids, then Process conditions are -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 6.5～8.0, and then sintered glass funnel filtration rushes nitrogen embedding in 2mL ampoules, and sealing uses 100 DEG C flowing steam sterilization 30 minutes, obtain final product LEMAHUI ZHUSHEYE.

The mono- grand antibody of sheep anti mouse CTGF, purchased from CellSignaling companies of the U.S.；Sheep anti mouse β-actin antibody, is purchased from Sigma companies；Streptozotocin, purchased from Sigma companies.Small table centrifuge（Mierofuge18）, purchased from Bechman Coulter companies；Turbula shaker（Vortex-GenieZ）, purchased from Scilnd companies of the U.S.；Automatic clinical chemistry analyzer, purchase From Beckman Coulter Inc. of the U.S..

The packet of 1.2 rats, modelling and administration

45 cleaning grade Wistar rats are randomly divided into normal group, control group and experimental group, each 15.Control group and experimental group Give Streptozotocin 55mg/kg intraperitoneal injections and set up Diabetic nephropathy animal model, normal group gives the life of Isodose Reason salt solution lumbar injection.After modeling success, experimental group rat gives LEMAHUI ZHUSHEYE 300mg/kg intraperitoneal injections, 1 time/d； Normal group and control group give the physiological saline intraperitoneal injection of Isodose, 1 time/d.Three groups of rat free water feeds, no Give any hypoglycemic to process.

The detection of 1.3 blood sugar, twenty-four-hour urine albumen, serum creatinine and urea nitrogen

Before three groups of rat injections（Before treatment）And when injecting 12 weeks（After treatment）Its blood sugar, twenty-four-hour urine albumen, blood are detected respectively Clear creatinine and urea nitrogen.

1.4 statistical methods

Using the statistical softwares of Stata 10.0.Measurement data is used± s represents, three groups are compared and adopt variance analysis, between group two-by-two Relatively checked using t, correlation analysis Pearson relevant function methods.P ＜ 0.05 are that difference is statistically significant.

2 results

Three groups of rat blood sugars, twenty-four-hour urine albumen, serum creatinine and urea nitrogen levels compare before and after 2.1 treatments

Experimental group and control rats twenty-four-hour urine albumen, serum creatinine and urea nitrogen levels are higher than normal group before treatment（The equal ＜ of P 0.05）；Without substantially reduction before experimental group Rat 24 h Urine proteins, serum creatinine and urea nitrogen are relatively treated after treatment（The equal ＞ of P 0.05）.Refer to table 8.

Blood sugar, twenty-four-hour urine albumen, serum creatinine and urea nitrogen levels compare before and after 8 three groups of rat treatments of table（±s）

3 discuss and conclusion

After LEMAHUI ZHUSHEYE 12 weeks, its 24 hours Urine proteins, serum creatinine and urea nitrogen levels are not apparent from reducing, and After being not below treatment of control group, illustrate that LEMAHUI ZHUSHEYE can not reduce diabetes rat albuminuria, Renal Function in Rats is not had Protective effect, it is impossible to treat diabetic nephropathy.

Embodiment 8：The experimental study of LEMAHUI ZHUSHEYE treatment insomnia

1 experiment material

Medicine：

LEMAHUI ZHUSHEYE：LEMAHUI ZHUSHEYE：LEMAHUI ZHUSHEYE is made up of active medicine and excipient substance, and active medicine is Rein in the horse back extract, account for the 5% of LEMAHUI ZHUSHEYE cumulative volume, excipient substance is sodium chloride, glycerine, glycerine, water, and this is reined in the horse The preparation method of parenteral solution is returned, step is as follows：

（1）By the dry 100g that reins in the horse back, add 5 times of amount concentration to be in 75% ethanol water, to be warming up to 85 DEG C, heat back Cold filtration after 1.5 h is flowed, extract and the dregs of a decoction are obtained, aforesaid operations 2 times are repeated to the dregs of a decoction, merge 3 gained liquids, then Process conditions are -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely 1000mL, adjusts pH value 6.5～8.0, and then sintered glass funnel filtration rushes nitrogen embedding in 2mL ampoules, and sealing uses 100 DEG C flowing steam sterilization 30 minutes, obtain final product LEMAHUI ZHUSHEYE.

Yellow Jackets：Beijing land for growing field crops Feng Tuo chemical technologies Co., Ltd produces；

Strychnine nitrate parenteral solution：Beijing Double-Crane Pharmaceutical Co., Ltd produces；

Diazepam：Tianjin KingYork Amino Acid Co., Ltd. produces；

Animal：Kunming mouse, body weight 18g～22g, female dual-purpose is provided by Heilongjiang University of Chinese Medicine experimental center.

2 experimental techniques and result

The research of 2.1 pairs of spontaneous activity in mice

Mouse 30, male and female are regardless of, and are randomly divided into 3 groups, i.e. saline control group, LEMAHUI ZHUSHEYE group, and it is positive right to stabilize According to group, 10 per group.

After adapting to 5 minutes in mouse input animal activity case before administration, animal walking time and the forelimb of 2 minutes are recorded The numerical value of number of times is praised upwards as normal value.

Then give respectively：

LEMAHUI ZHUSHEYE group：Injected LEMAHUI ZHUSHEYE 1mL, lumbar injection.

Stable positive controls：Injected diazepam 1mL, lumbar injection；

Saline control group：Injected 0.9% sodium chloride injection of same volume, lumbar injection.

Each group mouse determines mouse walking time and double forelimbs praises upwards number of times by the same method 60 minutes after administration.Knot Fruit with± S represents, the results are shown in Table 9.

Impact of the table 9 to spontaneous activity in mice number of times（± S, n=10）

Note：Compare with saline control group, * P< 0.01；Compare with stable group：# P > 0.05；

Result shows in table：LEMAHUI ZHUSHEYE has obvious sedation to mouse, and comparing with saline control group has Significant difference（P < 0.01）.LEMAHUI ZHUSHEYE group sedation substantially, with stable group indifference is compared（P> 0.05）.Knot Fruit shows：LEMAHUI ZHUSHEYE has obvious sedation.

2.2 researchs that mice sleep is affected

2.2.1 to the impact of the mouse yellow Jackets length of one's sleep

Mouse 30, male and female are regardless of, and are randomly divided into 3 groups, i.e. saline control group, LEMAHUI ZHUSHEYE group, and it is positive right to stabilize According to group, 10 per group.

Then give respectively：

LEMAHUI ZHUSHEYE group：Injected LEMAHUI ZHUSHEYE 1mL, lumbar injection；

Stable positive controls：Injected diazepam 1mL, lumbar injection；

Saline control group：Injected 0.9% sodium chloride injection of same volume, lumbar injection.

After administration 60 minutes, every group of mouse gives respectively yellow Jackets 40mg/kg, lumbar injection.Record sleeping for mouse The dormancy time（It is sleep time from righting reflex loss to recovery time with righting reflex loss as time for falling asleep）.As a result It is shown in Table 10.

Impact of the table 10 to the mouse yellow Jackets length of one's sleep（± S, n=10）

2.2.2 to the impact of mouse yellow Jackets sub-threshold dose

Mouse 30, male and female are regardless of, and are randomly divided into 3 groups, i.e. saline control group, LEMAHUI ZHUSHEYE group, and it is positive right to stabilize According to group, 10 per group.

Then give respectively：

LEMAHUI ZHUSHEYE group：Injected LEMAHUI ZHUSHEYE 1mL, lumbar injection；

Stable positive controls：Injected diazepam 1mL, lumbar injection；

Saline control group：Injected 0.9% sodium chloride injection of same volume, lumbar injection.

After administration 30 minutes, each group mouse gives respectively yellow Jackets 30mg/kg, and lumbar injection is observed and is recorded into Sleep number of animals（In 15 minutes mouse righting reflex loss up to the mouse number on 1 minute as sleep number of animals）, calculate sleep rate.Knot Fruit is shown in Table 11.

Impact of the table 11 to mouse pentobarbital sodium sub-threshold lull dosage

The above two is tested and illustrate LEMAHUI ZHUSHEYE and have obvious syngignoscism to mouse, with saline control group ratio Compared with there were significant differences（P < 0.01）.LEMAHUI ZHUSHEYE group has obvious syngignoscism.

The research of 2.3 anti-strychnine nitrate convulsive attack effects

Mouse 30, male and female are regardless of, and are randomly divided into 3 groups, i.e. saline control group, LEMAHUI ZHUSHEYE group, and it is positive right to stabilize According to group, 10 per group.

Then give respectively：

LEMAHUI ZHUSHEYE group：Injected LEMAHUI ZHUSHEYE 1mL, lumbar injection.

Stable positive controls：Injected diazepam 1mL, lumbar injection；

Saline control group：Injected 0.9% sodium chloride injection of same volume, lumbar injection.

Each group mouse gives respectively strychnine nitrate parenteral solution 1.5mg/kg, hypodermic injection, note after administration 60 minutes Record mice convulsion number（There is generalized tonic with mouse to faint from fear as index）.The results are shown in Table 12.

The effect of the anti-strychnine nitrate convulsive attack of table 12（n= 10）

Note：Compare with saline control group：*P < 0.01；Compare with stable group：※P > 0.05

As a result show, stable group plays the role of to resist strychnine nitrate convulsive attack, each group and life with LEMAHUI ZHUSHEYE group Reason saline control group compares, and is statistically analyzed the equal significance of difference.And the anticonvulsant action of discovery LEMAHUI ZHUSHEYE Most strong, curative effect preferably, illustrates that LEMAHUI ZHUSHEYE has anticonvulsant effect.

3 conclusions

The study find that, LEMAHUI ZHUSHEYE can suppress spontaneous activity in mice；Impact to mice sleep has with yellow Jackets Synergy；Play the role of to resist strychnine nitrate convulsive attack.Illustrate that LEMAHUI ZHUSHEYE has obvious calmness, hypnosis, resists Convulsion effect, the prospect with exploitation medicament for treating insomnia.

It should be noted that herein, such as first and second or the like relational terms are used merely to a reality Body or operation make a distinction with another entity or operation, and not necessarily require or imply these entities or deposit between operating In any this actual relation or order.And, term " including ", "comprising" or its any other variant are intended to Nonexcludability is included, so that a series of process, method, article or equipment including key elements not only will including those Element, but also including other key elements being not expressly set out, or also include for this process, method, article or equipment Intrinsic key element.In the absence of more restrictions, the key element for being limited by sentence "including a ...", it is not excluded that Also there is other identical element in process, method, article or equipment including the key element.

Above example only to illustrate technical scheme, rather than a limitation；Although with reference to the foregoing embodiments The present invention has been described in detail, it will be understood by those within the art that：It still can be to aforementioned each enforcement Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic；And these modification or Replace, the essence for not making appropriate technical solution departs from the present invention.

Claims (10)

1. a kind of LEMAHUI ZHUSHEYE for treating diabetes, it is characterised in that be made up of active medicine and excipient substance, it is described Active medicine is extract of reining in the horse back.

2. the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 1, it is characterised in that the extract of reining in the horse back Account for the 5% of parenteral solution cumulative volume.

3. the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 1, it is characterised in that the excipient substance is chlorine Change sodium, vitamin C, Sulfobutyl ether β _ cyclodextrin, glycerine, ethyl lactate, citric acid, mannitol, glycerine, glycine, sorbierite In one or more.

4. it is a kind of as described in claims 1 to 3 is arbitrary treatment diabetes LEMAHUI ZHUSHEYE preparation method, its feature It is that step is as follows：

（1）It is in 65～85% ethanol water, to be warming up to 80～100 to take dry 3～8 times of amount concentration of addition of reining in the horse back DEG C, cold filtration after the h of heating and refluxing extraction 0.5～2 obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then vacuum distillation obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 3～5 h, is filtered, take supernatant, then use milipore filter Filter, filtrate concentration, it is extract at the beginning of 1.05～1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the pH 1～5 of 2～10 times of amounts of column volume The aqueous solution eluted, collect eluent, then eluted with the 50% of 2～10 times of column volume amount ethanol, collect wash-out Liquid, eluent merging twice, concentration obtains concentrate, then concentrate is adjusted into pH to 10～11 with alkali, at 60～80 DEG C 1～2h of heating, then pH to 3～5 is adjusted, then its Jing polyamide resin column is processed, with 2～5 times of water elution, collect wash-out Liquid, then distills at 70～80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6～7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 6.5～8.0, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product LEMAHUI ZHUSHEYE.

5. the preparation method of the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 4, it is characterised in that described to control The preparation method of the LEMAHUI ZHUSHEYE of diabetes is treated, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C to take dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, and then decompression is steamed Evaporate, obtain alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then use milipore filter mistake Filter, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

6. the preparation method of the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 5, it is characterised in that step （1）Described reduced pressure distillation process condition be -0.05～-0.09MPa, 40～90 DEG C.

7. the preparation method of the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 6, it is characterised in that step （2）Milipore filter used by described ultrafiltration is 3000～6000 dalton, selected from cellulose diacetate film, Triafol T Film, cyanoethyl cellulose film, PS membrane, sulfonated polysulfone membrane, poly (ether sulfone) film, sulfonated polyether sulfone film, Polysulfonamide, phenolphthalein Side base polyarylsulfone (PAS) film, polyvinylidene fluoride film, polyacrylonitrile film, polyimide film, cellulose membrane, methyl methacrylate-propylene Lonitrile copolymer film, polyacrylonitrile-cellulose diacetate blend film.

8. application of a kind of LEMAHUI ZHUSHEYE in treatment diabetes medicine is prepared, it is characterised in that the re-injection of reining in the horse is penetrated Liquid is made up of active medicine and excipient substance, and the active medicine is extract of reining in the horse back, accounts for LEMAHUI ZHUSHEYE cumulative volume 5%, the excipient substance is sodium chloride, glycerine, glycerine, water, and the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C by dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then technique bar Part is -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

9. application of a kind of LEMAHUI ZHUSHEYE in medicament for treating insomnia is prepared, it is characterised in that the LEMAHUI ZHUSHEYE by Active medicine and excipient substance are constituted, and the active medicine is extract of reining in the horse back, accounts for the 5% of LEMAHUI ZHUSHEYE cumulative volume, institute It is sodium chloride, glycerine, glycerine, water to state excipient substance, and the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C by dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then technique bar Part is -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.

10. a kind of application of LEMAHUI ZHUSHEYE in treatment medicine for treating diabetic nephropathy is prepared, it is characterised in that the re-injection of reining in the horse Penetrate liquid to be made up of active medicine and excipient substance, the active medicine is extract of reining in the horse back, accounts for LEMAHUI ZHUSHEYE cumulative volume 5%, the excipient substance is sodium chloride, glycerine, glycerine, water, and the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：

（1）It is in 75% ethanol water, to be warming up to 85 DEG C by dry 5 times of amount concentration of addition of reining in the horse back, is heated to reflux 1.5 Cold filtration after h, obtains extract and the dregs of a decoction, and to the dregs of a decoction aforesaid operations 2 times are repeated, and merges 3 gained liquids, then technique bar Part is -0.05～-0.09MPa, vacuum distillation under conditions of 40～90 DEG C, obtains alcohol extract；

（2）Distilled water is added in alcohol extract, after being well mixed, after standing 4 h, is filtered, take supernatant, then with 3000～6000 The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration, it is extract at the beginning of 1.15 rein in the horse back to obtain relative density；

（3）Extract Jing D101B types macroporous absorbent resin at the beginning of reining in the horse back is processed, first with the water that the pH of 5 times of amounts of column volume is 3 Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, collects eluent, is eluted twice Liquid merges, concentration, obtains concentrate, then concentrate is adjusted into pH to 10 with ammoniacal liquor, and 1.5 h are heated at 60 DEG C, then uses salt Then acid for adjusting pH is processed its Jing polyamide resin column to 5, with 3 times of water elution, eluent is collected, to washing at 80 DEG C De- liquid is distilled, and removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract of reining in the horse back；

（4）Add sodium chloride and other drugs auxiliary material to be uniformly mixed in extract of reining in the horse back, be supplemented with water for injection extremely Aequum, adjusts pH value 7, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, obtains final product re-injection of reining in the horse Penetrate liquid.