CN106619975B - A kind of Zusanli point injectable drug and its preparation method for being used to treat diabetes - Google Patents
A kind of Zusanli point injectable drug and its preparation method for being used to treat diabetes Download PDFInfo
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- CN106619975B CN106619975B CN201610904330.2A CN201610904330A CN106619975B CN 106619975 B CN106619975 B CN 106619975B CN 201610904330 A CN201610904330 A CN 201610904330A CN 106619975 B CN106619975 B CN 106619975B
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- 239000002674 ointment Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 238000000554 physical therapy Methods 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 235000019615 sensations Nutrition 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 230000004622 sleep time Effects 0.000 description 1
- -1 sorbierite Chemical compound 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 206010042772 syncope Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 239000003860 topical agent Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/80—Scrophulariaceae (Figwort family)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention belongs to the field of Chinese medicines, specifically related to a kind of LEMAHUI ZHUSHEYE and preparation method thereof, the LEMAHUI ZHUSHEYE of the present invention is made up of active medicine extract solution of reining in the horse back, the extract solution of reining in the horse back is sunk by alcohol extracting, water to be extracted, eluted and purified by resin column, it is eventually adding auxiliary material and LEMAHUI ZHUSHEYE is made, LEMAHUI ZHUSHEYE of the invention can significantly reduces the blood glucose value of mouse, and further experiment shows that LEMAHUI ZHUSHEYE acupoint injection therapy of the present invention can significantly treat diabetes.
Description
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 caused suffering to patient, and it is added huge financial burden.According to incompletely statistics, diabetic foot
Ulcer accounts for 20% or so.
Diabetic foot makes lower limb defencive function decline due to DPN, and big blood vessel and microangiopathies make artery
Hypoperfusion causes microcirculation disorder easily to occur diabetes.Wherein sensory nerve lesion 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 lost sensibility.From ground, shoes or other adjacent to the pressure or shearing force of toes cause ulcer shape
Into due to lacking normal Neuroprotective Mechanisms, ulcer is often aggravated because of the presence of apophysis.The lesion of autonomic nerves system is caused
Skin normally perspires regulatory function, skin temperature regulatory function and blood fortune regulating power and lost, and causes local organization pliability to drop
It is low, form thick callosity and more broken and cracking.In addition, the normal forfeiture for perspiring ability has blocked the water again of local organization
Change, cause tissue further to destroy so that deep tissue is easier to bacteria planting.Morbidity of the kinesitherapy nerve lesion 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 and the ulcerated risk increase in the sharp plantar side of toe, and vascular lesion causes the tissue of destruction to be difficult to heal.
The medicament for the treatment of diabetes is mainly outer wiping ointment in the prior art, and 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 speedwellPseudolysimachion linariifolium subsp. Dilatatum (Nakai ＆ Kitagawa) D.Y.HongGround herb.
The content of the invention
In view of the shortcomings of the prior art, the invention provides a kind of LEMAHUI ZHUSHEYE for treating diabetes and its preparation
Method, its rapid reliable decoction 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. influence, no first pass effect, and medicament contg is difficult loss, and curative effect is reliable.
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, the active medicine
For extract solution of reining in the horse back.
It is preferred that, the extract solution of reining in the horse back accounts for the 5% of parenteral solution cumulative volume.
It is preferred that, 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 for the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：
（1）Take it is dry rein in the horse back add 3~8 times of amount concentration in 65~85% ethanol water, to be warming up to 80~
100 DEG C, cold filtration after the h of heating and refluxing extraction 0.5~2 obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction,
Merge 3 gained decoctions, then vacuum distillation obtains alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 3~5 h, filtering takes supernatant, then with surpassing
Membrane filtration, filtrate concentration obtains relative density extract solution at the beginning of 1.05~1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, first with the pH of 2~10 times of amounts of column volume
1~5 aqueous solution is eluted, and collects eluent, then is eluted with 50% ethanol of 2~10 times of amounts of column volume, and collection is washed
De- liquid, eluent merging twice, concentration obtains concentrate, concentrate then is adjusted into pH to 10~11 with alkali, at 60~80 DEG C
1~2h of lower heating, then pH to 3~5 is adjusted, then it is handled through polyamide resin column, with 2~5 times of water elution, collection is washed
De- liquid, then distills at 70~80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6~7, obtains extract solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 6.5~8.0 to aequum, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes,
Produce LEMAHUI ZHUSHEYE.
It is preferred that, the preparation method of the LEMAHUI ZHUSHEYE of the treatment diabetes, step is as follows：
（1）Dry reining in the horse back is taken to add 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, heat back
Cold filtration after 1.5 h is flowed, extract solution and the dregs of a decoction is obtained, aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, then
Vacuum distillation, obtains alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 4 h, filtering takes supernatant, then use milipore filter
Filtering, filtrate concentration obtains relative density extract solution at the beginning of 1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to aequum, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, produces and reins in the horse
Return parenteral solution.
It is preferred that, step（1）Described reduced pressure distillation process condition be -0.05~-0.09MPa, 40~90 DEG C.
It is preferred that, step（2）Milipore filter used in 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 composition, the active medicine is extract solution 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）Dry reining in the horse back is added 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, heat back
Cold filtration after 1.5 h is flowed, extract solution and the dregs of a decoction is obtained, aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, 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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to aequum, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, produces and reins in the horse
Return parenteral solution.
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 solution 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）Dry reining in the horse back is added 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, heat back
Cold filtration after 1.5 h is flowed, extract solution and the dregs of a decoction is obtained, aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, 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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to aequum, and it is filling then to rush nitrogen, sealing, with 100 DEG C of flowing steam sterilization 30 minutes, produces and reins in the horse
Return parenteral solution.
Embodiment
To make the purpose, technical scheme and 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 whole embodiments.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 rein in the horse back extract solution and the chlorination that account for parenteral solution cumulative volume 5%
Sodium, glycine, glycine, sorbierite, citric acid, water composition.
A kind of preparation method for the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：
（1）The dry 100g that reins in the horse back is taken, adds 3~8 times of amount concentration in 65~85% ethanol water, to be warming up to
80~100 DEG C, cold filtration after the h of heating and refluxing extraction 0.5~2 obtains extract solution and the dregs of a decoction, and aforesaid operations 2 are repeated to the dregs of a decoction
It is secondary, merge 3 gained decoctions, then vacuum distillation obtains alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 3~5 h, filtering takes supernatant, then with surpassing
Membrane filtration, filtrate concentration obtains relative density extract solution at the beginning of 1.05~1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, first with the pH of 2~10 times of amounts of column volume
1~5 aqueous solution is eluted, and collects eluent, then is eluted with 50% ethanol of 2~10 times of amounts of column volume, and collection is washed
De- liquid, eluent merging twice, concentration obtains concentrate, concentrate then is adjusted into pH to 10~11 with alkali, at 60~80 DEG C
1~2h of lower heating, then pH to 3~5 is adjusted, then it is handled through polyamide resin column, with 2~5 times of water elution, collection is washed
De- liquid, then distills at 70~80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6~7, obtains extract solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 6.5~8.0 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed,
With 100 DEG C of flowing steam sterilization 30 minutes, LEMAHUI ZHUSHEYE is produced.
Embodiment 2：
A kind of LEMAHUI ZHUSHEYE for treating diabetes, by rein in the horse back extract solution and the chlorination that account for parenteral solution cumulative volume 5%
Sodium, Sulfobutyl ether β _ cyclodextrin, sorbierite, ethyl lactate, water composition.
A kind of preparation method for the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：
（1）Take the dry 100g that reins in the horse back, add 6 times of amount concentration in 65% ethanol water, to be warming up to 80 DEG C, plus
Cold filtration after circumfluence distillation 0.5, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained medicines
Liquid, then vacuum distillation, obtains alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 3h, filtering takes supernatant, then use milipore filter
Filtering, filtrate concentration obtains relative density extract solution at the beginning of 1.05~1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, first uses the pH's 1 of 10 times of amounts of column volume
The aqueous solution is eluted, and collects eluent, then is eluted with 50% ethanol of 2 times of amounts of column volume, collects eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 11 with alkali, 1h is heated at 80 DEG C, then adjust pH
To 5, then it is handled through polyamide resin column, with 5 times of water elution, eluent is collected, is then distilled at 80 DEG C, removed
Ethanol is extremely without alcohol taste, then adjusts pH to 6, obtains extract solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 6.5~8.0 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed,
With 100 DEG C of flowing steam sterilization 30 minutes, LEMAHUI ZHUSHEYE is produced.
Embodiment 3：
A kind of LEMAHUI ZHUSHEYE for treating diabetes, by 5% rein in the horse back extract solution and sodium chloride, glycerine, water group
Into.
A kind of preparation method for the LEMAHUI ZHUSHEYE for treating diabetes, step is as follows：
（1）Take the dry 100g that reins in the horse back, add 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, plus
Cold filtration after 1.5 h of heat backflow, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions,
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, is stood after 3 h, filtering takes supernatant, then use 3000 roads
You the h of cyanoethyl cellulose membrane filtration 24, obtain relative density extract solution at the beginning of 1.15 rein in the horse back；
（3）Extract solution is handled through adsorption resin column at the beginning of reining in the horse back, is first carried out with the pH 3 of 5 times of amounts of the column volume aqueous solution
Elution, then eluted with the ethanol of 5 times of amounts of column volume, eluent is collected, concentrate is concentrated to give, then by concentrate ammonia
Water adjusts pH to 10, and 3 h are heated at 60 DEG C, then adjusts pH to 5, then handles it through polyamide resin column, with 2 times of water
Elution, then distills at 80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 7, obtains extract solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C
Flowing steam sterilization 30 minutes, produce 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 various concentrations preferably, test respectively with 25%, 50%, 75%
Ethanol with 95% is as Extraction solvent, using the rate of transform of extract protocatechuic acid as inspection target, in combination with former catechu
Different solvents are investigated by the content of acid.
Experimental method：Weigh and rein in the horse back 5 parts, every part of 200g adds solvent and is heated to reflux 3 times, and it is 85 to be heated to reflux temperature
DEG C, the time is 1h, and the extract solution 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 step on peak .RP-HPLC and determine containing for capsule protocatechuic acid of reining in the horse back
Measure [J] Chinese patent drugs 2007.29 (12):1867-1869. 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.
Influence 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 second of other concentration
Alcohol, therefore the ethanol of selection 75% is used as Extraction solvent.
, heating-up temperature
After Extraction solvent is determined, also need it is further preferred to Extracting temperature, experiment using 75% ethanol as extract it is molten
Agent, extraction time is 1.5h, and heating-up temperature is respectively 80 DEG C, 85 DEG C, 90 DEG C, 100 DEG C of turning with extract protocatechuic acid
Shifting rate is inspection target, the heat time is carried out preferably, as a result such as table 2.
Influence 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 is apparently higher than other groups, therefore selection heating-up temperature is 85
℃。
, the heat time
After Extraction solvent is determined, also need it is further preferred to extraction time, experiment using 75% ethanol as extract it is molten
Agent, is heated to reflux temperature for 85 DEG C, extraction time be respectively 0.5h, 1h, 1.5h, 2h ethanol as Extraction solvent, to extract
The rate of transform of thing protocatechuic acid is inspection target, the heat time is carried out preferably, as a result such as table 3.
Influence 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 higher than implementation group 1 and experimental group 2, bright with the thing of experimental group 4
Significant difference is different, therefore selection is heated to reflux the time for 1.5h.
Embodiment 5：LEMAHUI ZHUSHEYE treats the experimental study of diabetes
1 experiment material
1.1 experimental animal
Experiment is from SPF grades of healthy male Wistar rats 50（By Heilongjiang University of Chinese Medicine, animal center is provided）,
Body weight is（250±10）g.Raise in 22~25 DEG C of room temperature, humidity 50%, draughty environment, with cleaning grade feed（By
Heilongjiang University of Chinese Medicine's 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 to rein in the horse
Extract solution 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 in 75% ethanol water, to be warming up to 85 DEG C, plus
Cold filtration after 1.5 h of heat backflow, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions,
Then process conditions be -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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C
Flowing steam sterilization 30 minutes, produce LEMAHUI ZHUSHEYE.
1.3 prepare diabetes rat model
Randomly select 40 rats and diabetes rat model is set up using the STZ methods being injected intraperitoneally, according to transient ischemia/reperfusion
Note principle manufacture diabetes rat model（Ulcer of foot is one of cardinal symptom of diabetes）.To diabetic model group rat
The same position of right hind ligatured, using 10 extra section suture ligature rat ankle and foots, ligature 2h every time, decontrol 30min,
Ligation 3 times is repeated daily, 3d is ligatured altogether, is operated 1 time every 1d.There is skin nigrescence with ligated limbs, bubble, bleeding blister, ulcer,
To confirm the successful standard of modeling.During ligation, operated by same person, it is ensured that identical using strength during ligation.All quilts
The equal Cheng Mo of Banded Rats.
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 mould
Type is divided into 2 groups, every group 20, is respectively：LEMAHUI ZHUSHEYE treatment group and diabetes blank control group.To reining in the horse, re-injection is penetrated
Liquid measure group is according to 300mg/（kg·d）（Equivalent to 5 times of clinical patients consumption per day）Dosage, to gastrocnemius position on the left of mouse
Put injection medicine, 1 time/d.Physiological saline gavage, 2 times/d are used to diabetes blank control group.Body weight is measured weekly, is changed
Dosage, 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
（Refer to including high shear rate, low shear rate, Plasma Viscosity, hematocrit, erythrocyte aggregation index, deformable index 6
Mark）.
1.5 statistical method
Database is set up with Excel, with SPSS13.0 statistical analysis software analyze datas.Measurement data with± s represents,
Compare between two groups and examined using t, multigroup is compared and use variance analysis；Enumeration data, which compares, uses χ2Examine, inspection level α=
0.05。
2 results
2.1 LEMAHUI ZHUSHEYE treatment groups, diabetes blank control group comparitive study
After being intervened by the LEMAHUI ZHUSHEYEs of 6 weeks, after the treatment of diabetes LEMAHUI ZHUSHEYE treatment group, foot symptom
It is clearly 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 experiment is treatment initial stage, FBG, body weight and the foot symptom score no significant difference of two groups of rats；Rein in the horse
Going back to parenteral solution treatment group, FBG is significantly reduced after the treatment, and FSI is significantly raised, high shear rate, low shear rate, blood plasma in lectin from hemolymph
Viscosity, hematocrit, erythrocyte aggregation index, deformable index work property are reduced.It is shown in Table 5.
The LEMAHUI ZHUSHEYE treatment group of table 5 and body weight, FBG, FSI and blood flow after the treatment of diabetes blank control group
The comparison of change（±s）
3 conclusions
This experiment is confirmed：（1）LEMAHUI ZHUSHEYE can improve the fasting blood-glucose and foot ulcers of diabetes B rat
Symptom, the effect with certain reduction blood glucose, treatment diabetes.（2）LEMAHUI ZHUSHEYE can significantly reduce diabetes
6 indexs, have improvement result to hemorheology in sufficient rat lectin from hemolymph.This experiment also demonstrates that LEMAHUI ZHUSHEYE has drop
The blood glucose of low diabetes rat, it is influential on diabetes to illustrate the medicine；In experiment the serum insulin level of rat with
There is obvious rise after medicine, 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 treatment is difficult, and treatment cycle is long, and medical expense is high,
White elephant is brought to patient and society.Inventor is with 0 grade of artificial research object of diabetic foot, using random packet pair
According to the research method of design, different treatment and nursing measures are given, the deterioration intervention to 0 grade of diabetes is studied, so that
A kind of more satisfactory treatment and nursing method of 0 grade of diabetes is explored, ulcer of foot is prevented.
1 object and method
1.1 object
In December, 2015 is to 0 grade of the diabetes B diabetes 24 of hospitalization in July, 2016, patient with diabetic feet
Meet the diagnostic criteria of the international clinical guidelines of diabetes.Wherein male 12, female 12 is 50 years old~65 years old age, average
（62.9±50.1）Year, diabetic duration（3.6±2.1）Year；Receive diabetes systematic treating, OHA or insulin
Treatment is effective.All patients feet's skins are complete, without open focus, are venation block through Chinese medical discrimination, show as foot skin
Skin is purplish red or purple dim, and limbs send out numbness cool, sharp ache, and fuyang pulse weakens.It is randomly divided into experimental group and control group, every group 12
Example, two groups of patient's ordinary circumstances（Sex, the age, the course of disease, symptom, sign, blood glucose, HbAle egg from, blood fat, blood pressure etc.）Nothing
Statistical significance（P>0.05）, with comparativity.
1.2 method
Related patient is pressed into the sequencing numbering of institute, is incorporated into two groups at random respectively.
Control group：Diabetic diet and exercise therapy add OHA and（Or）Insulin therapy controls blood glucose, makes sky
Abdomen blood glucose≤7.0mmoL/L, postprandial blood sugar≤10.0mmoL/L, using conventional foodcare and health education.
Experimental group：LEMAHUI ZHUSHEYE acupoint injection therapy is used 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 is made 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 and reined in the horse back with the 2mL sterilizing syringes equipped with No. 5 syringe needles
Parenteral solution 2mL, thrusts straightly subcutaneous 0.5 11 cun, and pumpback is injected into re-injection of reining in the horse per cave and penetrated without blood and impassivity Rediating Pain after bringing about the desired sensation
Liquid 1mL, one time a day, and 15 times are a course for the treatment of, treat 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 solution 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 in 75% ethanol water, to be warming up to 85 DEG C, plus
Cold filtration after 1.5 h of heat backflow, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions,
Then process conditions be -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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 7 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed, with 100 DEG C
Flowing steam sterilization 30 minutes, produce 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）'s
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 observation related symptoms and sign by special messenger and recorded（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 treatment is carried out one day after.
1.3.2 school work rheology Hygienic monitoring on hands of childhood
The arteria dorsalis pedis hemorheology before and after Case treatment is determined using Acuson28XP/10 type color Doppler echocardiographies instrument
Parameter, is carried out one day after in treatment the previous day first time and last time treatment.
1.4 side reactions are observed
Respectively at 0.5h after injection, 2h, 6h, 12h, 24h, three days, seven days, it is local that observation whether there is itching, redness, pain etc.
The general reaction such as react and have a fainting spell during acupuncture treatment, have a headache, suffering a shock.
1.5 statistical procedures
All data application SPSS11.0 statistical softwares processing, the inspection of enumeration data and rate is using inspection, measurement data
With mean ± standard deviation（）Represent, examined 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%, compare that there were significant differences between two groups（=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 preceding instep Diameter of two groups of Case treatments, VPV and CBF numeric ratio are compared with nothing
Significant difference（P>0.05）.2. itself compare before and after two groups of Case treatments, instep Diameter expands after treatment group's Case treatment
Greatly, VPV is accelerated, and CBF increases, with being compared before treatment with statistical significance（P<0.01）, and control group patient controls
Change after treatment not statistically significant（P>0.05）.3. compare after two groups of Case treatments, each Experiment Parameter group is superior to control group, have
There is significant statistical significance (P<0.01）.
3 conclusions
After intervening the different treatment and nursings of two groups of diabetes Bs, 0 grade of diabetes, clinical efficacy and arteria dorsalis pedis blood
Fluid rheology correlation study shows：Experimental group is substantially better than control group, is more beneficial for improving pedal blood circulation, alleviates diabetes
The foot symptom and sign of foot, are conducive to preventing the generation of ulcer of foot.
Diabetes are one of diabetes important complications, according to external investigation, and foot will occur for about 15% diabetic
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, will reduce patient suffering, 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 this pathologic process of blood circulatory disorder, and improvement blood circulation is reached by promoting blood circulationization addiction medicine acupoint injection therapy, eliminates and induces
Factor, so as to intervene the deterioration of 0 grade of diabetes, prevents ulcer of foot.
This result of study is shown, 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, patient has no adverse reaction, and is a kind of peace
Entirely, 0 grade of diabetes intervening measure effective, simple and easy to apply.
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 carries 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 in 75% ethanol water, to be warming up to 85 DEG C, plus
Cold filtration after 1.5 h of heat backflow, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions,
Then process conditions be -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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 6.5~8.0 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed,
With 100 DEG C of flowing steam sterilization 30 minutes, LEMAHUI ZHUSHEYE is produced.
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 reality
Test group and give Streptozotocin 55mg/kg intraperitoneal injections and set up Diabetic nephropathy animal model, normal group gives Isodose
Physiological saline intraperitoneal injection.After modeling successfully, experimental group rat gives LEMAHUI ZHUSHEYE 300mg/kg intraperitoneal injections, 1
Secondary/d；Normal group and control group give the physiological saline intraperitoneal injection of Isodose, 1 time/d.Three groups of rat free waters are entered
Food, any hypoglycemic processing is not given.
1.3 blood glucose, twenty-four-hour urine albumen, serum creatinine and urea nitrogen detection
Before three groups of rat injections（Before treatment）And when injecting 12 weeks（After treatment）Its blood glucose, twenty-four-hour urine egg are detected respectively
In vain, serum creatinine and urea nitrogen.
1.4 statistical method
Using the statistical softwares of Stata 10.0.Measurement data is used± s represented, three groups are compared and use variance analysis, between group
Compare two-by-two and examined using t, correlation analysis Pearson relevant function methods.P ＜ 0.05 are that difference is statistically significant.
2 results
The front and rear three groups of rat blood sugars of 2.1 treatments, twenty-four-hour urine albumen, serum creatinine and urea nitrogen levels compare
Experimental group and control rats twenty-four-hour urine albumen, serum creatinine and urea nitrogen levels are higher than normal group before treatment（P
Equal ＜ 0.05）；Experimental group Rat 24 h Urine proteins, serum creatinine and urea nitrogen are not substantially reduced before relatively treating after treatment（P
Equal ＞ 0.05）.Refer to table 8.
Blood glucose, 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 reduction,
And be not below after control group treatment, illustrate that LEMAHUI ZHUSHEYE can not reduce diabetes rat albuminuria, to Renal Function in Rats
There is no 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, active drug
Thing is extract solution of reining in the horse back, accounts for the 5% of LEMAHUI ZHUSHEYE cumulative volume, and excipient substance is sodium chloride, glycerine, glycerine, water, is somebody's turn to do
The preparation method of LEMAHUI ZHUSHEYE, step is as follows：
（1）By the dry 100g that reins in the horse back, add 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, plus
Cold filtration after 1.5 h of heat backflow, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions,
Then process conditions be -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, is stood after 4 h, filtering takes supernatant, then with 3000~
The cellulose diacetate ultrafiltration membrance filter of 6000 dalton, filtrate concentration obtains relative density and extracted at the beginning of 1.15 rein in the horse back
Liquid；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, is first 3 with the pH of 5 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 5 times of column volume amount, collection eluent, twice
Eluent merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then
With salt acid for adjusting pH to 5, then it is handled through polyamide resin column, with 3 times of water elution, eluent is collected, at 80 DEG C
Eluent is distilled, ethanol is removed extremely without alcohol taste, then adjusts pH to 7, extract solution of reining in the horse back is obtained；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented injection
Water adjusts pH value 6.5~8.0 to 1000mL, and then sintered glass funnel filtration is rushed nitrogen embedding in 2mL ampoules, sealed,
With 100 DEG C of flowing steam sterilization 30 minutes, LEMAHUI ZHUSHEYE is produced.
Yellow Jackets：Beijing crop field Feng Tuo chemical technologies Co., Ltd produces；
Strychnine nitrate parenteral solution：Beijing Double-Crane Pharmaceutical Co., Ltd is produced；
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 methods 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, stabilize sun
Property control group, every group 10.
After being adapted to 5 minutes in mouse input animal activity case before administration, the animal walking time and forelimb of 2 minutes is recorded
The numerical value of number of times is praised as normal value upwards.
Then give respectively：
LEMAHUI ZHUSHEYE group：LEMAHUI ZHUSHEYE 1mL is injected, is injected intraperitoneally.
Stable positive controls：Diazepam 1mL is injected, is injected intraperitoneally；
Saline control group：0.9% sodium chloride injection of same volume is injected, is injected intraperitoneally.
Each group mouse determined mouse walking time by the same method and double forelimbs praises number of times upwards in 60 minutes after administration.Knot
Fruit with± S is represented, the results are shown in Table 9.
Influence of the table 9 to spontaneous activity in mice number of times（± S, n=10）
Note：Compared with saline control group, * P< 0.01；Compared with stable group：# P > 0.05；
Result is shown in table：LEMAHUI ZHUSHEYE has obvious sedation to mouse, with saline control group ratio
Compared with there were significant differences（P < 0.01）.LEMAHUI ZHUSHEYE group sedation substantially, is compared indifference with stable group（P>
0.05）.As a result show：LEMAHUI ZHUSHEYE has obvious sedation.
2.2 researchs influenceed on mice sleep
2.2.1 to the influence 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, stabilize sun
Property control group, every group 10.
Then give respectively：
LEMAHUI ZHUSHEYE group：LEMAHUI ZHUSHEYE 1mL is injected, is injected intraperitoneally；
Stable positive controls：Diazepam 1mL is injected, is injected intraperitoneally；
Saline control group：0.9% sodium chloride injection of same volume is injected, is injected intraperitoneally.
After administration 60 minutes, every group of mouse gives yellow Jackets 40mg/kg respectively, intraperitoneal injection.Record sleeping for mouse
The dormancy time（It is sleep time from righting reflex loss to recovery time using righting reflex loss as time for falling asleep）.As a result
It is shown in Table 10.
Influence of the table 10 to the mouse yellow Jackets length of one's sleep（± S, n=10）
2.2.2 to the influence 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, stabilize sun
Property control group, every group 10.
Then give respectively：
LEMAHUI ZHUSHEYE group：LEMAHUI ZHUSHEYE 1mL is injected, is injected intraperitoneally；
Stable positive controls：Diazepam 1mL is injected, is injected intraperitoneally；
Saline control group：0.9% sodium chloride injection of same volume is injected, is injected intraperitoneally.
After administration 30 minutes, each group mouse gives yellow Jackets 30mg/kg respectively, intraperitoneal injection, observes and is recorded into
Sleep number of animals（Mouse righting reflex loss is used as sleep number of animals up to the mouse number on 1 minute in 15 minutes）, calculate sleep rate.Knot
Fruit is shown in Table 11.
Influence of the table 11 to mouse pentobarbital sodium sub-threshold lull dosage
The above two, which is tested, illustrates that LEMAHUI ZHUSHEYE has obvious syngignoscism to mouse, with saline control
Group compares that 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, stabilize sun
Property control group, every group 10.
Then give respectively：
LEMAHUI ZHUSHEYE group：LEMAHUI ZHUSHEYE 1mL is injected, is injected intraperitoneally.
Stable positive controls：Diazepam 1mL is injected, is injected intraperitoneally；
Saline control group：0.9% sodium chloride injection of same volume is injected, is injected intraperitoneally.
Each group mouse gives strychnine nitrate parenteral solution 1.5mg/kg respectively in after administration 60 minutes, is subcutaneously injected, note
Record mice convulsion number（There is generalized tonic with mouse to faint from fear for index）.It the results are shown in Table 12.
The effect of the anti-strychnine nitrate convulsive attack of table 12（n= 10）
Note：Compared with saline control group：*P < 0.01；Compared with stable group：※P > 0.05
As a result show, stable group plays the role of to resist strychnine nitrate convulsive attack, each group with LEMAHUI ZHUSHEYE group
Compared with saline control group, be statistically analyzed the equal significance of difference.And find the anticonvulsion of LEMAHUI ZHUSHEYE
Effect is most strong, and curative effect preferably, illustrates that LEMAHUI ZHUSHEYE has anticonvulsant effect.
3 conclusions
The study find that, LEMAHUI ZHUSHEYE can suppress spontaneous activity in mice；Influence and amobarbital to mice sleep
Sodium has synergy；Play the role of to resist strychnine nitrate convulsive attack.Illustrate that LEMAHUI ZHUSHEYE has obvious calmness, urged
Dormancy, anticonvulsant action, 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 deposited between operating
In any this actual relation or order.Moreover, term " comprising ", "comprising" or its any other variant are intended to
Nonexcludability is included, so that process, method, article or equipment including a series of key elements not only will including those
Element, but also other key elements including being not expressly set out, or also include being this process, method, article or equipment
Intrinsic key element.In the absence of more restrictions, the key element 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.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those within the art that：It still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these modification or
Replace, the essence of appropriate technical solution is departed from the present invention.
Claims (6)
1. a kind of LEMAHUI ZHUSHEYE for treating diabetes, is made up of active medicine and excipient substance, the active medicine is
Rein in the horse back extract solution, extract solution of reining in the horse back accounts for the 5% of parenteral solution cumulative volume, excipient substance be sodium chloride, vitamin C, sulphur butyl-
One or more in beta-schardinger dextrin, glycerine, ethyl lactate, citric acid, mannitol, glycerine, glycine, sorbierite, it is special
Levy and be, it is as follows the step of preparation method：
（1）Dry reining in the horse back is taken to add 3~8 times of amount concentration in 65~85% ethanol water, to be warming up to 80~100
DEG C, cold filtration after the h of heating and refluxing extraction 0.5~2 obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges
3 gained decoctions, then vacuum distillation obtains alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 3~5 h, filtering takes supernatant, then use milipore filter
Filtering, filtrate concentration obtains relative density extract solution at the beginning of 1.05~1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, first with the pH 1~5 of 2~10 times of amounts of column volume
The aqueous solution eluted, collect eluent, then eluted with 50% ethanol of 2~10 times of column volume amount, collect and elute
Liquid, eluent merging twice, concentration obtains concentrate, concentrate then is adjusted into pH to 10~11 with alkali, at 60~80 DEG C
1~2h is heated, then adjusts pH to 3~5, then handles it through polyamide resin column, with 2~5 times of water elution, elution is collected
Liquid, then distills at 70~80 DEG C, removes ethanol extremely without alcohol taste, then adjusts pH to 6~7, obtains extract solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented 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, produces
LEMAHUI ZHUSHEYE.
2. the LEMAHUI ZHUSHEYE of diabetes is treated as claimed in claim 1, it is characterised in that the treatment diabetes
LEMAHUI ZHUSHEYE preparation method, step is as follows：
（1）Dry reining in the horse back is taken to add 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, be heated to reflux 1.5
Cold filtration after h, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, and then decompression is steamed
Evaporate, obtain alcohol extract；
（2）Distilled water is added in alcohol extract, after being well mixed, is stood after 4 h, filtering takes supernatant, then use milipore filter mistake
Filter, filtrate concentration obtains relative density extract solution at the beginning of 1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, the water for being first 3 with the pH of 5 times of amounts of column volume
Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, is collected eluent, is eluted twice
Liquid merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then use salt
Then acid for adjusting pH is handled it through 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 solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented 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, produces re-injection of reining in the horse
Penetrate liquid.
3. the LEMAHUI ZHUSHEYE for the treatment of diabetes as claimed in claim 2, it is characterised in that the step of preparation method
（1）Described reduced pressure distillation process condition be -0.05~-0.09MPa, 40~90 DEG C.
4. the LEMAHUI ZHUSHEYE for the treatment of diabetes as claimed in claim 2, it is characterised in that the step of preparation method
（2）Milipore filter used in 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.
5. 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 solution 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）Dry reining in the horse back is added 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, be heated to reflux 1.5
Cold filtration after h, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, 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, is stood after 4 h, filtering takes supernatant, then with 3000~6000
The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration obtains relative density extract solution at the beginning of 1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, the water for being first 3 with the pH of 5 times of amounts of column volume
Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, is collected eluent, is eluted twice
Liquid merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then use salt
Then acid for adjusting pH is handled it through 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 solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented 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, produces re-injection of reining in the horse
Penetrate liquid.
6. a kind of application of LEMAHUI ZHUSHEYE in medicament for treating insomnia is prepared, it is characterised in that the LEMAHUI ZHUSHEYE by
Active medicine and excipient substance composition, the active medicine is extract solution of reining in the horse back, accounts for the 5% of LEMAHUI ZHUSHEYE cumulative volume, institute
Excipient substance is stated for sodium chloride, glycerine, glycerine, water, the preparation method of the LEMAHUI ZHUSHEYE, step is as follows：
（1）Dry reining in the horse back is added 5 times of amount concentration in 75% ethanol water, to be warming up to 85 DEG C, be heated to reflux 1.5
Cold filtration after h, obtains extract solution and the dregs of a decoction, and aforesaid operations are repeated 2 times to the dregs of a decoction, merges 3 gained decoctions, 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, is stood after 4 h, filtering takes supernatant, then with 3000~6000
The cellulose diacetate ultrafiltration membrance filter of dalton, filtrate concentration obtains relative density extract solution at the beginning of 1.15 rein in the horse back；
（3）Extract solution at the beginning of reining in the horse back is handled through D101B types macroporous absorbent resin, the water for being first 3 with the pH of 5 times of amounts of column volume
Solution is eluted, and collects eluent, then is eluted with 50% ethanol of 5 times of amounts of column volume, is collected eluent, is eluted twice
Liquid merges, concentration, obtains concentrate, and concentrate then is adjusted into pH to 10 with ammoniacal liquor, 1.5 h are heated at 60 DEG C, then use salt
Then acid for adjusting pH is handled it through 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 solution of reining in the horse back；
（4）Sodium chloride is added in extract solution of reining in the horse back and other drugs auxiliary material is uniformly mixed, and is supplemented 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, produces re-injection of reining in the horse
Penetrate liquid.
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| CN201610904330.2A CN106619975B (en) | 2016-10-18 | 2016-10-18 | A kind of Zusanli point injectable drug and its preparation method for being used to treat diabetes |
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|---|---|---|---|
| CN201610904330.2A CN106619975B (en) | 2016-10-18 | 2016-10-18 | A kind of Zusanli point injectable drug and its preparation method for being used to treat diabetes |
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Effective date of registration: 20180207 Address after: 150040 Heping Road, Xiangfang District, Heilongjiang, Harbin, China, 24 Patentee after: Heilongjiang University of Chinese Medicine Address before: Oblitera.ns 150009 in Heilongjiang province Harbin city Nangang District guogeli Street No. 411 the Second Affiliated Hospital of Heilongjiang University Of Chinese Medicine Patentee before: Wang Jing |