CN106309671B - Longnan of Gansu Province Z-bungeanum Maxim extract components and preparation method thereof and detection method - Google Patents

Longnan of Gansu Province Z-bungeanum Maxim extract components and preparation method thereof and detection method Download PDF

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CN106309671B
CN106309671B CN201610847879.2A CN201610847879A CN106309671B CN 106309671 B CN106309671 B CN 106309671B CN 201610847879 A CN201610847879 A CN 201610847879A CN 106309671 B CN106309671 B CN 106309671B
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extract
solution
prickly ash
chinese prickly
water
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CN106309671A (en
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李越峰
严兴科
吴平安
杜丽东
刘安国
金辉
曹瑞
牛江涛
边甜甜
司昕蕾
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Gansu University of Chinese Medicine
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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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/758—Zanthoxylum, e.g. pricklyash
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
    • 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
    • A61K2236/30—Extraction of the material
    • 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
    • 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
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • 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
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

Abstract

The invention belongs to plant extracts field, it is related to a kind of anesthesiophore Longnan of Gansu Province Z-bungeanum Maxim extract components of tool and preparation method thereof and detection method.The extract is adopted to be prepared with the following method：(1) Chinese prickly ash is crushed, put after drying in oven, plus 50%~60% aqueous acetone solution, in water-bath refluxing extraction, extract solution reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution stirring and dissolving under water bath condition, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in a water bath；(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7~9, loading volume 6BV~8BV, wash 4BV~6BV removal of impurities, with ethyl acetate:Acetone=100:5 eluant, eluent 7BV~9BV elutions, flow velocity is 2BV/h~4BV/h, collects eluent, and eluent concentration is dried, produced.

Description

Longnan of Gansu Province Z-bungeanum Maxim extract components and preparation method thereof and detection method
Technical field：
The invention belongs to plant extracts field, and in particular to the anesthesiophore Longnan of Gansu Province pepper extract of one kind tool Component and preparation method thereof.
Technical background：
Chinese prickly ash (Pericarpium Zanthoxyli) is rutaceae green pepper (Zanthoxylum schinifolium Sieb.et Zucc.) or Chinese prickly ash (Zanthoxylum bungeanum Maxim.) dry mature skin, have long and thin hot pepper, pericarpium zanthoxyli, Tens kinds of kinds such as rock green pepper, to commonly use drugs for dispelling internal cold.《Pharmacopoeia of People's Republic of China》Middle regulation：Chinese prickly ash is traditional Chinese medicine, tool There is the drug effect of " warm in pain, desinsection is antipruritic ".
Modern study achievement shows that the chemical composition of domestic and international xanthoxylum mainly has alkaloid, acid amides, lignanoid, perfume (or spice) Legumin, volatile oil and aliphatic acid etc., to cardiovascular system, digestive system, immunity function, coagulation function, analgesia, calmness, anti-inflammatory, It is local anaesthesia, antibacterial and kill itch mite effect, antitumor etc. have stronger pharmacological activity.How effectively traditional literature to be recorded It is combined with current scientific achievement, that is, it is the current task of top priority to find both points of penetration of combination, it is therefore necessary to further examined Chinese prickly ash effect is demonstrate,proved, is that deep its medical value of excavation and raising corresponding product added value provide reference.
Longnan City is located in Southeast Gansu Province, the natural climate environment and long cultivation history for having suitable Chinese prickly ash growth. South of Gansu Province Chinese prickly ash is local famous-brand and high-quality Ways of Special Agricultural Products, is otherwise known as " Z-bungeanum Maxim ", wins outside country because best in quality within 1993 Import and export the prize of product high-quality in trade portion；Win within 1994 national forestry famous, special, excellent and new products fair gold medal；Win China within 2006 Yang Ling agricultural high-teches fair " god of agriculture's prize "." Chinese famous-particular-excellent in the first batch is authorized by the State Administration of Forestry in south of Gansu Province Wudu in 2000 The township of economic forest Chinese prickly ash ".2011 " Wudu Chinese prickly ash " obtains " Wudu Chinese prickly ash geographical sign is proved " that State Administration for Industry ＆ Commerce issues Certificate of registry.Under the overall background that featured agriculture industrialization is greatly developed in Gansu Province, south of Gansu Province Chinese prickly ash turns into local government's emphasis One of preponderant dinstinctive industry of development.
The section for the important unit Gansu university of TCM that the present inventor studies as Longnan of Gansu Province Z-bungeanum Maxim Personnel are ground, years of researches are had been carried out to Longnan of Gansu Province Chinese prickly ash, it is favorite in the lasting research to Longnan of Gansu Province Chinese prickly ash for many years Outer to find, the extract of Longnan of Gansu Province Chinese prickly ash has certain anesthetic effect, but scientific research personnel's research is found, is only carried specific The south of Gansu Province pepper extract take, prepared under purification process just has a preferable anesthetic effect, and conventional water extraction, the south of Gansu Province of alcohol extracting The anesthetic effect of pepper extract is unsatisfactory.Inventor has carried out years of researches to this problem, is ground by substantial amounts of experiment Study carefully, inventor is determined with the best south of Gansu Province pepper extract of anesthetic effect and its preparation technology.Verified through experimental study, should South of Gansu Province pepper extract has extraordinary anaesthetic effect.
In addition, inventor also found, the south of Gansu Province pepper extract that the present invention is provided is sweet in treatment rheumatic arthritis, preventing and treating Also there is excellent action effect in terms of injury of vein caused by revealing alcohol.
Chinese prickly ash used by the present invention is Longnan of Gansu Province Z-bungeanum Maxim medicinal material, is provided by south of Gansu Province Chinese prickly ash Co., Ltd.
The content of the invention：
It is an object of the invention to provide a kind of pepper extract.
It is a further object of the present invention to provide the preparation method of the extract.
It is a further object of the present invention to provide the quality determining method of the extract.
The invention provides application of the extract in anaesthetic is prepared.
Present invention also offers application of the extract in treatment rheumatoid arthritis agents are prepared.
Present invention also offers application of the extract in injury of vein medicine caused by preparing preventing and treating mannitol.
Present invention also offers application of the extract in preventing and treating tumour medicine is prepared.
The purpose of the present invention is achieved in the following ways：
The anesthesiophore Longnan of Gansu Province pepper extract of one kind tool, the pepper extract is to adopt to prepare with the following method 's：
(1) Chinese prickly ash is crushed, put in baking oven after 40 DEG C~50 DEG C drying 3h~5h, plus 15~25 times of amounts 50%~60% the third The ketone aqueous solution, in 60 DEG C~70 DEG C water-bath refluxing extractions 2 times~4 times, each 70min~80min merges extract solution, reclaims third Ketone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 40 DEG C~50 DEG C water Stirring and dissolving under the conditions of bath, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 40 DEG C~50 DEG C water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7~9, loading body Product 6BV~8BV, washes 4BV~6BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 7BV~9BV is eluted, and flow velocity is 2BV/h~4BV/h, collects eluent, and eluent concentration is dried, produced.
The pepper extract is preferably to adopt what is prepared with the following method：
(1) Chinese prickly ash is crushed, put in baking oven after 40 DEG C of drying 5h, plus 15 times of 55% aqueous acetone solutions of amount, in 60 DEG C of water-baths Refluxing extraction 4 times, each 70min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 50 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 50 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7, loading volume 8BV, washes 4BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 9BV elutions, flow velocity For 2BV/h, eluent is collected, eluent concentration is dried, produced.
The pepper extract can also be preferred to use following method and prepare：
(1) Chinese prickly ash is crushed, put in baking oven after 50 DEG C of drying 3h, plus 25 times of 50% aqueous acetone solutions of amount, in 70 DEG C of water-baths Refluxing extraction 2 times, each 80min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 40 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 40 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:9, loading volume 6BV, washes 6BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 7BV elutions, flow velocity For 4BV/h, eluent is collected, eluent concentration is dried, produced.
The pepper extract can also be preferred to use following method and prepare：
(1) Chinese prickly ash is crushed, put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, in 65 DEG C of water-baths Refluxing extraction 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, produced.
Described pepper extract, can carry out assay using high performance liquid chromatography to single leaf rue product alkali：
(1) chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 20~40：60~80 acetonitrile-water； Detection wavelength：190~210nm；Column temperature：15~25 DEG C；Flow velocity：0.5~1.5mLmin-1；Sample size：5~20 μ L；
(2) prepared by reference substance solution：Precision weighs dry single leaf rue product alkali reference substance to constant weight in right amount, plus methanol is molten Reference substance solution is made in solution；
(3) preparation of need testing solution：Precision weighs pepper extract of the present invention, plus methanol, is heated to reflux, and extract solution is returned Stream solvent is simultaneously concentrated to dryness, and residue is dissolved in water, and is shaken and extracted with water saturated n-butanol, is merged n-butanol extracting liquid, is used ammonia Test solution is washed, and n-butanol extracting liquid recycling design is to doing, and residue adds methanol to dissolve, and shakes up, and filtration takes filtrate, produces test sample Solution；
(4) determine：Precision measures each 5~20 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high-efficient liquid phase color Spectrometer, is detected.
Wherein, it is preferred to use high-efficient liquid spectrum method carries out assay to single leaf rue product alkali：
(1) chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 30：70 acetonitrile-water；Detect ripple It is long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；
(2) prepared by reference substance solution：It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus first Reference substance solutions of every 1mL containing 0.2mg is made in alcohol dissolving；
(3) preparation of need testing solution：Precision weighs pepper extract 10mL of the present invention, plus methanol 40mL, is heated to reflux 4h, extract solution reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, every time 20mL, merges n-butanol extracting liquid, is washed with ammonia solution 3 times, each 15mL, n-butanol extracting liquid recycling design to dry, residue Plus methanol dissolves and is transferred in 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate, produces need testing solution；
(4) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected.
Described pepper extract, assay is carried out using gas chromatography to sabinene, Humuleno：
(1) chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 0.5~1.5mLmL-1；Hydrogen Gas, 30~50mLmL-1；Air, 300~500mLmin-1；Split ratio：5~15：1；Injector temperature：240~260 DEG C, Detector temperature：290~310 DEG C；Temperature programming：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
(2) preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute solution is made, shake up, as Inner mark solution；
(3) preparation of need testing solution：Precision measures this product, puts in volumetric flask, plus absolute ethyl alcohol is diluted to scale, then essence It is close to measure, put in volumetric flask, precision adds inner mark solution, plus absolute ethyl alcohol is diluted to scale, shakes up；
(4) preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus absolute ethyl alcohol Dissolve and dilute and the reference substance stock solution containing sabinene and Humuleno is made, it is standby；
(5) determine：Precision measures each 5~20 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas-chromatography Instrument, is detected.
Wherein, assay is carried out to sabinene, Humuleno using gas chromatography, preferred steps are as follows：
(1) chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 1.0mLmL-1；Hydrogen, 40mL·mL-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heating：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
(2) preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute every 1mL is made containing 12.5mg Solution, shake up, be used as inner mark solution；
(3) preparation of need testing solution：Precision measures pepper extract 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol Scale is diluted to, then precision measures 1.0mL, puts in 10mL volumetric flasks, precision addition inner mark solution 1.0mL, plus absolute ethyl alcohol are dilute Release to scale, shake up；
(4) preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus absolute ethyl alcohol Dissolve and dilute and 0.301mgmL containing sabinene is made-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
(5) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas chromatograph, enters Row detection.
Described pepper extract, can use pharmaceutical methods conventional in pharmacy to be prepared into external preparation or aerosol Agent.
The external preparation is preferably prepared as externally used paste, cataplasm, external use liquid.
Described pepper extract uses pharmaceutical methods conventional in pharmacy to be prepared into external medicine preparation
Described pepper extract can be used for preparing injury of vein medicine caused by preventing and treating mannitol.
Pepper extract of the present invention can be used for preparing treatment rheumatoid arthritis agents.
The technique effect of the present invention is verified by following experimental study：
Experiment one：Experimental study of the extract Neulized inhalation of the present invention to rat anesthesia effect
The anaesthetic effect of Isoflurane significantly, attempt by being compareed in existing anaesthetic by this experiment in existing anesthesia art On the basis of research, Animal Anesthesia experiment, observation are carried out with extract of the present invention contrast Western medicine preparation with reference to medicinal atomized standard Whether the curative effect of anaesthetic of the present invention can substitute Western medicine preparation.
1 material
1.1 experimental animal cleaning grade healthy SD rats 50, male and female half and half, weight (220 ± 20) g, by the Gansu traditional Chinese medical science Medicine university animal experimental center is provided.Raise in Experimental Animal Center, raising temperature (20 ± 2) DEG C, feed, water, bedding and padding are by special People adds in time to be changed.
1.2 experiment materials
1.2.1 extract of the present invention：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.4g of the present invention.
1.2.2 extract A is contrasted：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg, plus 10 times of amount 70% ethanol Soakage extraction 2 times at room temperature are taken, often Secondary 2 days, merge extract solution, filtering, decompression filtrate recycling ethanol concentrates, dries, must contrast extract A 21.5g.
1.2.3 extract B is contrasted：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration obtains Chinese prickly ash water extract, concentrated, dries, must contrast extract B 23.4.
1.2.4 extract C is contrasted：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration, filtrate concentration obtains the total medicinal extract of Chinese prickly ash；By above-mentioned total medicinal extract, column chromatography for separation, tree are carried out by polyamide post Fat post blade diameter length ratio is 1:7, loading volume 8BV, wash 4BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphates= 100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, collects eluent, and 5 times of 95% ethanol of amount are added into eluent, are admixed Diatomite, drying, puts in Soxhlet extractor, with acetone extract, and extract reclaims acetone in air-distillation, obtains concentrate, will be dense Contracting thing is in water-bath Back stroke acetone to the greatest extent, contrast extract C 8.3g.
1.2.5 Isoflurane：Produced by Hubei Xin Kang medication chemistries Co., Ltd.
1.3 equipment and equipment ultrasonic atomizer, toy suction-type gas anesthesia machine, intelligent non-invasive blood pressure measuring-mouse measurement Instrument BP-98A.
2 experimental methods
2.1 animal packet rats sub-cage rearing under the same conditions, it is considered to stocking density, per 5, cage, routine feeding standard Pellet, and 5 groups are randomly divided into, respectively：It is extract inhalation group of the present invention, contrast extract A inhalation group, right Than extract B inhalation group, contrast extract C inhalation group, Western medicine Isoflurane inhalation group.
The implementation experiment of 2.2 anesthesia starts preceding 30min and rat is placed on the experimental bench for be covered with woollen blanket into freely activity with suitable Answer experimental situation.Non-invasive blood pressure measuring, rat-tail observation, continuous monitoring arterial pressure and heart rate are applied simultaneously, and record basic value.Fiber crops Liquor-saturated operation is as follows.
Extract inhalation group of the present invention：By ultrasonic atomizer, ultrasonic frequency is adjusted, extraction materialization is not being destroyed In the case of learning property, Chinese herbal aerosol soakage in the control unit interval.Maintain Neulized inhalation 1h.
Contrast extract A inhalation group：By ultrasonic atomizer, ultrasonic frequency is adjusted, extraction materialization is not being destroyed In the case of learning property, Chinese herbal aerosol soakage in the control unit interval.Maintain Neulized inhalation 1h.
Contrast extract B inhalation group：By ultrasonic atomizer, ultrasonic frequency is adjusted, extraction materialization is not being destroyed In the case of learning property, Chinese herbal aerosol soakage in the control unit interval.Maintain Neulized inhalation 1h.
Contrast extract C inhalation group：By ultrasonic atomizer, ultrasonic frequency is adjusted, extraction materialization is not being destroyed In the case of learning property, Chinese herbal aerosol soakage in the control unit interval.Maintain Neulized inhalation 1h.
Western medicine Isoflurane inhalation group：By suction-type gas anesthesia machine, Isoflurane flow is set to be set to 6L/min.Tested Isoflurane inhaled concentration is adjusted according to breathing pattern and heart rate in journey, exit connects gas monitor, dense with Monitored anesthesia gas Degree, oxygen concentration and gas concentration lwevel.Oxygen flow is adjusted, fraction of inspired oxygen 30% is maintained.Respiratory time is 1h.
Anesthesia case is opened wide ingress of air by anesthesia after terminating, and is put back to after rat regains consciousness naturally in cage.
2.3 observation index
2.3.1 ordinary circumstance
Rat keeps autonomous respiration, respiratory rate, acra and the lip color of continuous observation mouse in anaesthesia process.
2.3.2 blood pressure, heart rate measurement
Non-invasive blood pressure measuring, continuous monitoring arterial pressure (mm/Hg) and heart rate (beat/min).Measurement anesthesia before 30min blood pressures and 30min blood pressures and heart rate after mean arterial pressure and heart rate, revival in heart rate, anaesthesia process.
2.3.3 onset time, duration
Onset time：Record is since the medicine suction to rat anesthesia time (min) (the root of the tail portion of clamp rat, with anti- Should be done nothing standard).Duration：Remove the time (min) revived after nebulization equipment to rat.
2.4 statistical procedures
Data after statistical procedures, statistics are carried out with statistics software SPSS15.0 to useRepresent, carry out normal distribution Examine, measurement data compare two-by-two between the unilateral variance analyses of ANOVA, multigroup mean, and heterogeneity of variance person then uses sum of ranks Examine.Represent that difference is statistically significant with P ＜ 0.05, P ＞ 0.05 represent no significant difference.
3 results
3.1 ordinary circumstances
Rat inhalation anesthesia model is successfully established, whole experiment is smoothly completed, nothing is dropped by the wayside and dead.In experimentation Rat lip acra position is without purple group, and breathing, palmic rate are without substantially slowing down.Monitoring result is shown in Table 1-1.Frequency is breathed between two groups Rate and body temperature are without significant difference (P ＞ 005).
Table 1-1 two groups of respiratory rates, body temperature
3.2 monitoring of blood pressure
Mean arterial pressure (MAP) change is compared in 30min before two groups of anesthesia, anaesthesia process after mean arterial pressure and revival 30min blood pressure, monitoring result is shown in Table 1-2.Comparative evaluation angiosthenia is without significant difference (P ＞ 0.05) between group, in group.
Two groups of angiosthenia mm/Hg of table 1-2
The measure of 3.3 hearts rate
Compare each group heart rate, monitoring result is shown in Table 1-3.Compare heart rate without significant difference (P ＞ 0.05) between group, in group.
Two groups of hearts rate of table 1-3 compare beat/min
3.4 onset times, duration
Compared with Western medicine Isoflurane inhalation group, it is contrast extract A inhalation group, contrast extract B inhalation group, right Onset time, duration than extract C inhalation group, there were significant differences (P ＜ 0.05).With Western medicine Isoflurane inhalation group Compare, onset time of extract inhalation group of the present invention, duration are without significant difference (P>0.05), illustrate that the present invention is carried Take the anaesthetic effect of thing suitable with Western medicine Isoflurane, Western medicine Isoflurane can be substituted.Monitoring result is shown in Table 1-4.
Two groups of anesthesia onset times of table 1-4, duration min (n=10)
Note：Compared with Western medicine Isoflurane inhalation group, * P<0.05
4 discuss and conclusion
By the blood pressure of preliminary Monitored anesthesia rat, heart rate, Sucked medicine anaesthetic effect is assessed, by experimental study, As a result show, the anaesthetic effect of south of Gansu Province pepper extract of the present invention is suitable with Western medicine comparison medicine Isoflurane, can substitute Western medicine different Fluorine ether, is used as anaesthetic.This research provides for the application of south of Gansu Province pepper extract Neulized inhalation anesthesia of the present invention from now on Theory and practice foundation.
Extract formulation of the present invention is pure natural traditional Chinese medicine, and anaesthetic effect is good and not yet finds that its bad poison is secondary anti- Should.Extract of the present invention has when treating the patient that some merge other diseases and is better than in addition to having preferable anaesthetic effect The effect of Isoflurane, such as injury of vein caused by treating rheumatic arthritis, preventing and treating mannitol etc..
Experiment two：The experimental study of pepper extract treatment rheumatic arthritis effect of the present invention
Rheumatic arthritis belongs to the illness Chinese traditional treatment of impediment syndrome category with dispelling wind and removing obstruction in the meridians, based on promoting blood circulation and stopping pain, takes orally There is good therapeutic effect with outer usage.Further to evaluate the Pharmacodynamics of extract emplastrum of the present invention, as Clinical practice is carried For foundation, carry out result of the test and be reported as follows.
1 data and method
1.1 Experimental agents and reagent
1.1.1 extract emplastrum of the present invention：
Prescription：Chinese prickly ash 20Kg
Preparation method：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.3g of the present invention；
(4) pepper extract of the present invention is taken, matrix (rubber 16g, rosin 16g, lanolin 4g, vaseline 1.5g, liquid is added Body paraffin 1g, zinc oxide 20g, gasoline 45g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
1.1.2 extract A (Chinese prickly ash ethanol extract emplastrum) is contrasted：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg, plus 10 times of amount 70% ethanol Soakage extraction 2 times at room temperature are taken, often Secondary 2 days, merge extract solution, filtering, decompression filtrate recycling ethanol concentrates, dries, must contrast extract A 21.3g.
(2) Chinese prickly ash ethanol extract, adds matrix (rubber 30g, rosin 30g, lanolin 8g, vaseline 3g, atoleine 2g, zinc oxide 30g, gasoline 80g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
1.1.3 extract B (Chinese prickly ash water extract emplastrum) is contrasted
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration obtains Chinese prickly ash water extract, concentrated, dries, must contrast extract B 23.5.
(2) Chinese prickly ash water extract, adds matrix (rubber 30g, rosin 30g, lanolin 8g, vaseline 3g, atoleine 2g, zinc oxide 30g, gasoline 80g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
1.1.4 extract C (Chinese prickly ash acetone extract emplastrum) is contrasted
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration, filtrate concentration obtains the total medicinal extract of Chinese prickly ash；By above-mentioned total medicinal extract, column chromatography for separation, tree are carried out by polyamide post Fat post blade diameter length ratio is 1:7, loading volume 8BV, wash 4BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphates= 100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, collects eluent, and 5 times of 95% ethanol of amount are added into eluent, are admixed Diatomite, drying, puts in Soxhlet extractor, with acetone extract, and extract reclaims acetone in air-distillation, obtains concentrate, will be dense Contracting thing is in water-bath Back stroke acetone to the greatest extent, contrast extract C 8.5g.
(2) Chinese prickly ash acetone extract, adds matrix (rubber 16g, rosin 16g, lanolin 4g, vaseline 1.5g, liquid stone Wax 1g, zinc oxide 20g, gasoline 45g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
1.2 animals
SD rats, male and female half and half, 160~180g of body weight is provided by Gansu university of TCM Experimental Animal Center.
1.3 methods
Extract emplastrum of the present invention is inhibited to rat adjuvant polyarthirtis, from 60 points of SD rats For model control group, cortisone group, extract emplastrum group of the present invention, contrast extract A emplastrum group, contrast extract B emplastrum group, Contrast extract C emplastrum group, every group 10.Inflammation is caused with Freund Freund's complete adjuvants, from causing be administered within scorching first 2 days, method is：Each group is pasted Each group emplastrum is refetched 0.1ml by every 100 grams of animal bodies by 4.0g/Kg dosage and drops in the coated scorching foot of cause on absorbent cotton by cream, keeps 3h, 1 time/d；Using excipient as model control group, positive control 1 time/2d, is caused scorching from hydrocortisone 25mg/Kg is subcutaneously injected The 21st day each group animal is discontinued afterwards, later observation 3d.
1.4 observation index volumetric methods measurement primary causes scorching foot and Secondary cases to cause volume before and after inflammation to parapodum, calculates swelling Degree.
1.5 statistical methods use SPSS19.0 statistics software processing datas.Measurement data is with mean ± standard deviationShow, examined using t.P<0.05 represents that difference is statistically significant.
2 results
Extract emplastrum group of the present invention can significantly reduce different time (3h, 7d, 14d, 21d) after cause inflammation and cause scorching foot swelling Degree, and can significantly reduce cause it is scorching after different time (7,12d, 18d, 21d) cause scorching paw swelling, compared respectively with model control group Statistically significant (the P of difference<0.05).It is shown in Table 2-1, table 2-2.
The influence of table 2-1 rat assist agent arthritis novo lesions is compared
Note：Compared with model control group,aP<0.05
The influence of table 2-2 rat assist agent arthritis secondary arthritis is compared
Note：Compared with model control group,aP<0.05
3 discuss
Rheumatic arthritis belongs to allergic disease, is the main performance of rheumatic fever, more with febris acuta and joint Pain is disease symptom, and clinical manifestation is light or moderate heating, wandering arthritis, and affected joints are the high pointes such as knee, ankle, shoulder, elbow Section, common different joint transfers, lesion locally has red, swollen, hot, pain to show.Rheumatic arthritis is common chronic immunity disease Disease, most course of disease is longer, and is difficult to cure, and various clinical symptoms to have a strong impact on control outside quality of life, traditional Chinese medicine and rheumatism is closed The scorching aspect of section has preferable advantage.
Extract emplastrum of the present invention is traditional Chinese medicine for external application, and experimental result is shown：Extract emplastrum confirmability of the present invention Suppressing rat assist agent arthritis causes local Earlier period of inflammation to react and uses rear different time points swelling, can also suppress to parapodum Because of the local swelling that delayed type hypersensitivity, DTH induces, dispelling wind and eliminating dampness, antalgic and the work of extract emplastrum of the present invention are embodied Blood dredging collateral effect, the drug effect foundation of science is provided for Clinical practice external preparation treatment rheumatic arthritis.Meanwhile, experiment knot Fruit also illustrates that pepper extract action effect of the present invention is substantially better than the component of other pepper extracts.
Experiment three：Extract emplastrum external application of the present invention prevents the experimental study of mannitol caused rabbit ear Damage of superficial veins
Mannitol is widely used in clinical treatment treatment, due to its hypertonicity, when quick, high concentration is stayed through peripheral vein To put cause when pin is inputted and puncture local injury of vein, the rubescent, swelling of local organization appearance, scorching hot, pain, streak red line, Sometimes with constitutional symptoms such as chilly, heatings.The generation of phlebitis not only increases patient suffering and medical expense, and delay is suffered from The diagnosis and treatment of person.To find effective prevention and controls of phlebitis, inventor's early-stage Study is tried out south of Gansu Province Chinese prickly ash of the present invention and carried Take thing Local out dressing prevent hyperosmotic drugs caused by Damage of superficial veins animal experiment study, from substantially performance and histopathology South of Gansu Province pepper extract of the present invention is have rated to reducing the generation of phlebitis with good prevention effect Deng microcosmic angle.
1 materials and methods
1.1 experimental animals
Healthy New Zealand's adult White Rabbit 20, male and female half and half, body are provided by Gansu university of TCM animal experimental center 2.5~3.0Kg is weighed, rabbit ear has lopsided person to exclude, and adaptability is fed 1 week before experiment.Body weight sorts, uses random digit from light to heavy Experimental rabbit is divided into experimental group and control group, every group of 10 rabbit (20 ears) by table method.Two groups of rabbit ordinary circumstances compare no statistics Difference (P ＞ 0.05), with comparativity.
1.2 experiment materials and instrument and equipment
H-2 type paraffin slicing machines, light microscope, microscopic image analysis software.20% mannitol, vulcanized sodium, penta bar Than appropriate sodium.
Extract emplastrum of the present invention：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.2g of the present invention；
(4) pepper extract of the present invention is taken, matrix (rubber 16g, rosin 16g, lanolin 4g, vaseline 1.5g, liquid is added Body paraffin 1g, zinc oxide 20g, gasoline 45g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
1.3 methods
Experimental group 30min before Transfusing Mannitol, will be covered when being transfused mannitol at venipuncture with sterile cotton balls After lid is fixed, centered on venipuncture point, around point of puncture and along vein traveling, extract of the present invention is pasted with spatula Patch is at rabbit ear, and diameter is more than 2~3cm, and 2 times a day, each 1h wipes external application before each topical application of drug with physiological saline cotton balls Medicine, observation local organization and vascular manifestations, then using same medicine, method external application.
Control group normal venous is transfused mannitol, any medicine of not external application.
Daily observation experiment rabbit diet, active situation, detaining time of intravenous indwelling needle 5d is boundary, two groups of rabbit ears of observed and recorded Whether there is phlebitis and the order of severity.
The foundation of 1.4 venoclysis models
Operations meet experimental animal Ethical Principles in experiment.Modeling is carried out using general mannitol infusion pattern, Yellow Jackets are injected by per kilogram of body weight 30mg dosage abdominal cavity, it is quiet in two rabbit ear edge respectively after anesthesia comes into force rabbit peace and quiet Arteries and veins at have sharp ears 2cm with gentian violet solution away from being marked, and row vein retained needle puncture art to puncture and use Kang Erhui sterile transparents after once succeeding Patch is fixed, and is modeled successfully after animal regains consciousness naturally, at room temperature the mannitol solution of venoclysis 20%, by 2.5ml/Kg dosage Venoclysis, injection speed 8ml/min, injection is finished with 50U/ml heparin saline 3~5ml positive pressure tube sealings, daily defeated 2 times, and two Minor tick 8h, continuous infusion 5d.Close observation whether there is drug extravasation during venoclysis, it is found that drug leakage is thrown-away model.
The making of 1.5 Pathologic specimens
The 5th day after last venoclysis intervention, every group of 12 rabbits are handled respectively, with hemostasis clamp toward auricular vein Distal end, rapidly centered on the centripetal end 2cm in front of venipuncture point, cuts needle body position auricular vein and auricle tissue 1cm2Sample, is put into 4% formalin and fixes after 24h, send Gansu pharmaceutical college of university of TCM pharmacological evaluation room to do routine pathology Section.
1.6 evaluation criterions
1.6.1 phlebitis evaluation criterion
It is daily to visually observe whether part occurs phlebitis, phlebitis time of origin and its order of severity, phlebitis standard According to American Nursing Society's version in 2006《Infusion treatment nursing practice standard》Judged, remove and ache with reference to zoopery feature Pain index, is divided into 5 grades by phlebitis：0 grade, without symptom；It is I grade, local rubescent；II grade, local erythema or oedema；Ⅲ Level, erythema, oedema, streak vein is formed；IV grade：Significantly, streak vein forms ＞ 2.5cm, or has fester for erythema, oedema Outflow.
1.6.2 liver histopathological analysis
Routine pathology section HE dyeing, om observation and description.Histopathology carries out pathology damage degree in terms of 4 Judgement, blood vessel endothelium swelling, blood vessel peripheral edema, cell infiltration and thrombosis, each project point 4 grades, nothing (-), slight (+), moderate (++), severe (+++).Judged by professional pathological experiment person's blind.
1.7 statistical procedures
Using SPSS17.0 statistics softwares.Two groups of phlebitis incidences, which compare, uses χ2Examine, pathological tissue damage journey The Wilcoxon rank tests that degree is compared using two independent sample ranked data, inspection level α=0.05.
2 results
2.1 two groups of rabbit ear veins are scorching, and a situation arises compares (table 3-1)
Two groups of rabbit auricular veins of table 3-1 are scorching, and a situation arises compares (only)
2.2 two groups of rabbit ear group pathology damage degree compare (table 3-2)
Two groups of rabbit ear veins of table 3-2 and surrounding tissue pathology damage degree compare (only)
3 discuss and conclusion
20% mannitol is to be clinically used for treating encephaledema, reduce the choice drug of intracranial pressure., can because its osmotic pressure is high Cause the rise of blood plasma and tissue infiltration pressure, cause vascular endothelial cell to be dehydrated, promote local platelet aggregation, and release is respectively Plant inflammation-causing substance；And mannitol needs fast drip to can be only achieved the effect of dehydration and diuresis when in use, and then aggravate to blood The damage of pipe and tissue, can make local vein occur pain, tube wall be hardened, the phenomenon such as elasticity loss, obliteration, even result in Sterile phlebitis.
Research shows that extract emplastrum principle active component list leaf rue product alkali of the present invention is a kind of natural, With antiphlogistic antibacterial, astringing to arrest bleeding, clearing heat and detoxicating, promotion organization regeneration and reparation, and blood circulation speed can be accelerated, dropped Low blood viscosity, suppress platelet aggregation, prevent thrombosis and the extensive and important life of protection vascular endothelial cell etc. Thing activity, inventor's early-stage Study also demonstrates extract emplastrum of the present invention to be had well to the phlebitis caused by mannitol Prevention effect.But how to select optimal external application opportunity to have no empirical theory foundation, this result of study is found, extract of the present invention During emplastrum external application the 3rd day, experimental group phlebitis incidence is less than control group (P ＜ 0.05), points out 30min before infusion mannitol to make Inflammation is can control with extract emplastrum external application of the present invention to develop, effectively the incidence of reduction phlebitis.The result of table 2 shows Show, the swelling of experimental group blood vessel endothelium, blood vessel peripheral edema, cell infiltration pathology are damaged after extract emplastrum external application 5d of the present invention Hinder degree less than control group (P ＜ 0.05), point out before infusion mannitol that 30min external applications extract emplastrum of the present invention more can be effective Its anti-inflammatory, the effect of detumescence are played, the generation of inflammatory pathologies reaction is reduced.Analysis reason is probably to shift to an earlier date when being transfused mannitol Using extract emplastrum external application of the present invention, the effective active composition of extract emplastrum of the present invention can sufficiently penetrate into it is subcutaneous and In tissues surrounding vascular, finite concentration is reached in the blood vessel, when Transfusing Mannitol, so that it may timely and effectively play it and disappear Scorching analgesic, expansion of blood vessels, the fragility of reduction vascular wall are to protect vein endothelial cell from the effect such as stimulation, so as to reach Prevent the purpose of injury of vein.Therefore infer that 30min is probably that prevention is sweet with extract emplastrum external application of the present invention before infusion mannitol Reveal alcohol phlebitis caused optimum opportunity.
Experiment four：Pepper extract high performance liquid chromatography quality determining method research of the present invention
1 instrument and reagent
1.1 instruments
(Agilent110 high performance liquid chromatographs and work station, G1311Aquat pumps, G1314 are purple for high performance liquid chromatograph External detector).
1.2 reagents
Single leaf rue product alkali reference substance (Chinese pharmaceutical biological product examines and determine research institute)；Pepper extract of the present invention：Reference It is prepared by the embodiment 1 in description of the invention embodiment；South of Gansu Province Chinese prickly ash medicinal material (offer of south of Gansu Province Chinese prickly ash Co., Ltd)；First Alcohol (chromatogram alcohol, Shanghai biochemistry work auxiliary reagent factory)；Other reagents are pure to analyze.
2 methods and result
2.1 prescriptions：Chinese prickly ash 20Kg
2.2 preparation methods：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.4g of the present invention.
The assay of 2.3 single leaf rue product alkali
2.3.1HPLC chromatographic condition
Using HypersilDs (4.0mm × 125mm, 5 μm) chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Inspection Survey wavelength：200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；Under the chromatographic condition, reference substance and sample Product chromatographic peak is good, and no Chinese prickly ash negative control is noiseless to determining.
2.3.2 the preparation of reference substance solution
It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus every 1mL is made containing 0.2mg in methanol Solution.
2.3.3 the preparation of need testing solution and negative controls
Precision weighs pepper extract 10g of the present invention, plus methanol 40mL, is heated to reflux 4h, and extract solution reflux solvent is simultaneously dense Be reduced to it is dry, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, each 20mL, merge n-butanol extract Liquid, is washed 3 times, each 15mL with ammonia solution, and n-butanol extracting liquid recycling design is to doing, and residue adds methanol to dissolve and be transferred to In 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate to produce；Separately prepared as stated above with ratio and be free of Chinese prickly ash Negative controls, be made in the same way of negative controls.
2.3.4 the drafting of standard curve
It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, and 10.4,20.8 are made of methanol, 41.6,83.2,166.4 μ gmL-1The solution of series concentration, respectively precision measure above-mentioned each 10 μ L of 5 kinds of strength solutions, injection is efficient Liquid chromatograph is measured.
Linear regression is carried out with peak area ratio and concentration, obtaining regression equation is：A=21.2764C-0.1394, r= 0.9998.Show single leaf rue product alkali in 10.1~166.9 μ gmL-1It is in good linear relationship in concentration range.
2.3.5 stability test
Precision draws the μ L of need testing solution 10, respectively at 0,1,2,4,8h sample introduction, and calculates single leaf rue product alkali content.Knot RSD is 0.34% (n=5) in fruit 8h.Show that sample solution is stable in 8h.
2.3.6 replica test
By 5 parts of need testing solution preparation method parallel processing sample, single leaf rue product alkali content is determined in accordance with the law and is calculated.Knot Fruit measures single leaf rue product alkali average content for 0.11mgg-1, RSD is 1.1%.
2.3.7 Precision Experiment
Precision draws single leaf rue product alkali reference substance solution, repeats sample introduction 5 times, and peak area is determined in accordance with the law.As a result RSD is 0.23% (n=5).Show that precision is preferable.
2.3.8 the rate of recovery is tested
Precision weighs 6 parts of the sample of the same lot number of known single leaf rue product alkali content, smart respectively by high, medium and low concentration It is close to add appropriate single leaf rue product alkali reference substance solution, determine and operated under item by sample, determine in accordance with the law, calculate the rate of recovery.Knot Fruit average recovery rate is that 100.2%, RSD is 0.44% (n=5).
2.3.9 sample size is determined
Reference substance solution and appropriate need testing solution are measured respectively, are filtered with miillpore filter, each μ L of sample introduction 10, by above-mentioned color Spectral condition determines 3 batches of samples, parallel determination 5 times.By external standard method containing with calculated by peak area need testing solution list leaf rue product alkali Amount.This product should be the 95%~105% of sign content containing single leaf rue product alkali, in terms of every 1g this product rue product alkali of the leaf containing list, no 0.11mg must be less than.3 batches of sample sizes are respectively 100.2% (RSD=1.1%), 101.6% (RSD=1.2%), 99.8% (RSD=1.2%).
Experiment five：Pepper extract gas chromatography Simultaneous Determination sabinene of the present invention, the content of Humuleno
1 instrument and reagent
Agilent7890N gas chromatographs：Fid detector, A.01.12.1 chromatographic work station；SGH-300 high-purity hydrogens Generator (Beijing Orient elite science and technology garden Science and Technology Ltd.)；Chromatographic column fused-silica capillary column (30m × 0.25mm, 0.25μm)；The a ten thousandth electronic analytical balance of plum Teller-support benefit ten；Sabinene reference substance (content 99.9%, Chinese food Drug assay research institute)；Humuleno reference substance (content 99.9%, National Institute for Food and Drugs Control)；Chinese prickly ash of the present invention is carried Take thing (being prepared with reference to the embodiment 1 in description of the invention embodiment), reagent：Cyclohexanone, absolute ethyl alcohol is color Spectrum is pure.
2 chromatographic conditions
Chromatographic column：ZB-WAX fused-silica capillary columns (30m × 0.25mm, 0.25 μm)；Carrier gas：N2, 1.0mLmL-1； Hydrogen, 40mLmL-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature； Temperature programming：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method.
3 test methods and result
The preparation of 3.1 inner mark solutions
Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute solution of every 1g containing 12.5mg is made, shake up, be used as internal standard Solution.
The preparation of 3.2 need testing solutions
Precision measures this product 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol is diluted to scale, then precision measures 1.0mL, Put in 10mL volumetric flasks, precision adds inner mark solution 1.0mL, plus absolute ethyl alcohol is diluted to scale, shakes up.
The preparation of 3.3 reference substance stock solutions
It is appropriate that precision weighs sabinene reference substance, Humuleno reference substance, plus anhydrous alcohol solution and diluting is made containing fragrant Chinese juniper Alkene 0.301mgmL-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
The preparation of 3.4 negative control solutions
The negative controls without Chinese prickly ash separately are prepared in preparation method under above-mentioned " 3.2 " item and ratio, feminine gender is made in the same way of Comparison liquid.
The investigation of 3.5 linear relationships
Respectively precision pipette 0.2,0.5,1.0,1.5,3.5mL reference substances storing solution in 10mL volumetric flasks, plus internal standard is molten Liquid 1.0mL, plus absolute ethyl alcohol are diluted to scale, shake up, as reference substance solution, and 1 μ L sample introductions are taken respectively, record chromatogram, with Sabinene, Humuleno are ordinate (Y) with interior target peak area ratio, and concentration (C) is abscissa (X), standard is drawn respectively bent Line, obtaining regression equation is respectively：
Y (sabinene)=1.1148X-0.0016, R2=0.9999, sabinene concentration is in 0.144~2.552mgmL-1 In the range of, linear relationship is good；
Y (Humuleno)=1.1345X+0.0034, R2=0.9999, Humuleno concentration is in 0.194~3.465mgmL-1 In the range of, linear relationship is good.
3.6 precision tests
It is 0.230mgmL to take sabinene concentration-1It is 0.290mgmL with Humuleno concentration-1Reference substance solution, weight Sample introduction 6 times, records peak area again, and 2 kinds of compositions and interior target peak area ratio (A/A internal standards), sabinene, Humuleno are calculated respectively RSD be respectively 0.22% and 1.3% (n=6).
3.7 replica tests
Take same batch of sample, the method replication 6 times under item determined by sample, as a result sabinene, RSD points of Humuleno Wei not 0.34%, 1.4% (n=6).
3.8 stability tests
Same batch of sample solution is taken, is determined at room temperature after placement 0,2,4,6,8,12h respectively, as a result by sabinene, humulus The RSD of oxalene is respectively 0.36%, 1.8%, illustrates that sample solution is determined in 12h, as a result stablizes.
3.9 average recoveries are tested
9 parts of the sample solution of known content is taken, and adds appropriate basic, normal, high reference substance solution, is surveyed by sample determination method Fixation sabinene, Humuleno content, calculate the rate of recovery, the results are shown in Table 4-1 respectively.
Table 4-1 determination of recovery rates result (n=9, %)
As a result show, preferably, the rate of recovery of sabinene is respectively in 99.5%~100.1%, humulus grass for the rate of recovery of this method The rate of recovery of alkene is between 98.5%~100.6%, and relative standard deviation is respectively 0.29% and 0.87%, this assay method energy Meet the assay of sabinene and Humuleno in pepper extract of the present invention.
3.10 quantitative limits and test limit
The quantitative limit and test limit of this research are determined using " signal to noise ratio method ", line taking standard liquid is appropriate, using adding Progressively dilution method is diluted absolute ethyl alcohol, when sample introduction concentration is 6.27,9.90 μ gmL-1When, take 1 μ L sample introductions, continuous sample introduction 3 It is secondary, sabinene, internal standard, Humuleno signal to noise ratio average value are obtained respectively close to 10.0, can be using this concentration as quantitative limit；Continue dilute Sample introduction is released, when sample introduction concentration is 1.044,1.65 μ gmL-1, continuous sample introduction 3 times obtains sabinene, internal standard, Humuleno signal to noise ratio Average value, can be using this concentration as test limit close to 3.0.
3.11 serviceability tests
Investigated through different chromatographic columns and stability of solution is investigated, and column temperature, injector temperature and detector temperature investigation, Show this method good tolerance, it is adaptable to the assay of two components in pepper extract of the present invention.
3.11.1 the influence of chromatographic column
From the chromatographic column of 3 different commercial specifications, the content of same batch of sample is determined, RSD% points of content value are calculated Wei 1.3,1.7,1.6.As a result show, sample determines content with different PEG chromatographic columns, and sabinene, Humuleno and internal standard are equal It can efficiently separate, illustration method good tolerance.
3.11.2 the influence of column temperature
Influence of the column temperature to separation predominantly influences the appearance time of main peak, and temperature is higher, and main peak appearance time is shorter, When first stage is 80 DEG C, sabinene main peak sabinene and impurity peaks can guarantee that baseline separation, Humuleno during 120 DEG C of second stage Main peak can guarantee that baseline separation with impurity peaks, and the RSD of content is less than 2.0% at each temperature.
3.11.3 the influence of injector temperature
When injector temperature is higher than column temperature, sabinene ensure that baseline separation with impurity peaks, and Humuleno is separated with impurity Well, and at each temperature the RSD of content is less than 1.8%.
3.11.4 the influence of detector temperature
When detector temperature is higher than injector temperature, sabinene and impurity peaks ensure that baseline separation, Humuleno with it is miscellaneous Matter separation is good, and the RSD of content is less than 1.9% at each temperature.
3.12 sample size measurement results
By Method validation, this content assaying method is easy to operate, the degree of accuracy is high, favorable reproducibility, can more effectively control Product quality processed.Therefore application this method carries out assay using internal standard method according to preceding method, as a result seen to 10 batches of samples Table 4-2.
Table 4-2 sample size measurement results
4 discuss
4.1 system suitability tests
Under this experiment gas chromatography system, respectively pipette samples determine with mixed reference substance solution, need testing solution with Each 1 μ L of negative control solution, record chromatogram.2 kinds of components can be separated preferably with internal standard compound, negative noiseless.This is System adaptability the results are shown in Table 4-3.
Table 4-3 system suitability tests
The selection of 4.2 internal standard compounds
Once cyclohexanone, naphthalene, biphenyl, gaultherolin etc. were tried out, because sample volatile ingredient is more, as a result with cyclohexanone Retention time and separating effect are most suitable.
The selection of 4.3 column temperatures
The boiling point difference of sabinene, cyclohexanone and Humuleno is than larger, and when column temperature is low, the retention time of Humuleno is long, When column temperature is high, sabinene can not be efficiently separated with impurity, while through that can meet two kinds of compositions using two sections of temperature-programmed modes Analysis.
The content limit of 4.4 this product
By 10 batch products measurement results, the content limit of tentative this product is：This product must not be less than per 1g containing sabinene 0.200mg, 0.200mg must not be less than containing Humuleno.
This method is to 2 kinds of compositions while being separated and being detected, method is quick, sensitive, and separating degree is good, specificity is good, energy Efficiently control drug quality.
Experiment six：Single leaf rue product alkali, sabinene, the assay of Humuleno in different pepper extracts
1 testing sample
1.1.1 pepper extract of the present invention
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.6g of the present invention.
1.2 contrast extract As：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg, plus 10 times of amount 70% ethanol Soakage extraction 2 times at room temperature are taken, often Secondary 2 days, merge extract solution, filtering, decompression filtrate recycling ethanol concentrates, dries, must contrast extract A 21.6g.
1.3 contrast extract Bs：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration obtains Chinese prickly ash water extract, concentrated, dries, must contrast extract B 23.6.
1.4 contrast extract Cs：
Prescription：Chinese prickly ash 20Kg
Preparation method：Chinese prickly ash meal (40 mesh) 20Kg is taken, is added water to cook three times, (pan-fried add water 6 times is measured, and decocts 90min； Two pan-fried add water 4 times are measured, and decoct 60min；Three pan-fried add water 4 times are measured, and decoct 30min)；It is dense with decoction that decoction merging is concentrated into medicinal material amount The ratio of contracting liquid measure is 1:1, plus ethanol is to alcohol content up to 60%, stands 24h, filtration is reclaimed ethanol to without alcohol taste, is dissolved in water, 36h is stood, filtration, filtrate concentration obtains the total medicinal extract of Chinese prickly ash；By above-mentioned total medicinal extract, column chromatography for separation, tree are carried out by polyamide post Fat post blade diameter length ratio is 1:7, loading volume 8BV, wash 4BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphates= 100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, collects eluent, and 5 times of 95% ethanol of amount are added into eluent, are admixed Diatomite, drying, puts in Soxhlet extractor, with acetone extract, and extract reclaims acetone in air-distillation, obtains concentrate, will be dense Contracting thing is in water-bath Back stroke acetone to the greatest extent, contrast extract C 8.3g.
2 assay methods
The method of the 2.1 high effective liquid chromatography for measuring list leaf rue product alkali contents provided using present invention experiment three is carried out Determine.
The 2.2 gas chromatography Simultaneous Determination sabinenes provided using present invention experiment four, the method for the content of Humuleno It is measured.
3 measurement results
Measurement result is shown in Table 5-1.
Table 5-1 sample size measurement results
4 discuss and conclusion
From table 5-1 as can be seen that invention pepper extract in single leaf rue product alkali, sabinene, humulus grass The content of alkene is apparently higher than other pepper extracts, and this may be also that pepper extract of the present invention has anesthetic effect, Yi Ji The reason for effect in terms for the treatment of rheumatic arthritis is better than other extracts.
Embodiment：
Embodiment 1：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.6g of the present invention.
Assay：
Assay is carried out to single leaf rue product alkali using high-efficient liquid spectrum method：
(1) chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 30：70 acetonitrile-water；Detect ripple It is long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；
(2) prepared by reference substance solution：It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus first Reference substance solutions of every 1mL containing 0.2mg is made in alcohol dissolving；
(3) preparation of need testing solution：Precision weighs pepper extract 10mL of the present invention, plus methanol 40mL, is heated to reflux 4h, extract solution reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, every time 20mL, merges n-butanol extracting liquid, is washed with ammonia solution 3 times, each 15mL, n-butanol extracting liquid recycling design to dry, residue Plus methanol dissolves and is transferred in 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate, produces need testing solution；
(4) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；
(5) measurement result：It is 0.15mg in terms of every 1g this product rue product alkali of the leaf containing list.
Assay is carried out to sabinene, Humuleno using gas chromatography：
(1) chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 1.0mLmL-1；Hydrogen, 40mL·mL-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heating：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
(2) preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute every 1mL is made containing 12.5mg Solution, shake up, be used as inner mark solution；
(3) preparation of need testing solution：Precision measures pepper extract 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol Scale is diluted to, then precision measures 1.0mL, puts in 10mL volumetric flasks, precision addition inner mark solution 1.0mL, plus absolute ethyl alcohol are dilute Release to scale, shake up；
(4) preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus absolute ethyl alcohol Dissolve and dilute and 0.301mgmL containing sabinene is made-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
(5) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas chromatograph, enters Row detection；
(6) measurement result：This product is 0.210mg containing sabinene per 1g, is 0.212mg containing Humuleno.
Embodiment 2：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.2g of the present invention.
Assay：
Assay is carried out to single leaf rue product alkali using high-efficient liquid spectrum method：
(1) chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 30：70 acetonitrile-water；Detect ripple It is long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；
(2) prepared by reference substance solution：It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus first Reference substance solutions of every 1mL containing 0.2mg is made in alcohol dissolving；
(3) preparation of need testing solution：Precision weighs pepper extract 10mL of the present invention, plus methanol 40mL, is heated to reflux 4h, extract solution reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, every time 20mL, merges n-butanol extracting liquid, is washed with ammonia solution 3 times, each 15mL, n-butanol extracting liquid recycling design to dry, residue Plus methanol dissolves and is transferred in 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate, produces need testing solution；
(4) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；
(5) measurement result：It is 0.14mg in terms of every 1g this product rue product alkali of the leaf containing list.
Assay is carried out to sabinene, Humuleno using gas chromatography：
(1) chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 1.0mLmL-1；Hydrogen, 40mL·mL-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heating：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
(2) preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute every 1mL is made containing 12.5mg Solution, shake up, be used as inner mark solution；
(3) preparation of need testing solution：Precision measures pepper extract 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol Scale is diluted to, then precision measures 1.0mL, puts in 10mL volumetric flasks, precision addition inner mark solution 1.0mL, plus absolute ethyl alcohol are dilute Release to scale, shake up；
(4) preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus absolute ethyl alcohol Dissolve and dilute and 0.301mgmL containing sabinene is made-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
(5) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas chromatograph, enters Row detection；
(6) measurement result：This product is 0.211mg containing sabinene per 1g, is 0.213mg containing Humuleno.
Embodiment 3：
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 9.8g of the present invention.
Assay：
Assay is carried out to single leaf rue product alkali using high-efficient liquid spectrum method：
(1) chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 30：70 acetonitrile-water；Detect ripple It is long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；
(2) prepared by reference substance solution：It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus first Reference substance solutions of every 1mL containing 0.2mg is made in alcohol dissolving；
(3) preparation of need testing solution：Precision weighs pepper extract 10mL of the present invention, plus methanol 40mL, is heated to reflux 4h, extract solution reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, often Secondary 20mL, merges n-butanol extracting liquid, is washed with ammonia solution 3 times, each 15mL, and n-butanol extracting liquid recycling design is residual to dry Slag adds methanol to dissolve and be transferred in 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate, produces test sample molten Liquid；
(4) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；
(5) measurement result：It is 0.14mg in terms of every 1g this product rue product alkali of the leaf containing list.
Assay is carried out to sabinene, Humuleno using gas chromatography：
(1) chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 1.0mLmL-1；Hydrogen, 40mL·mL-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heating：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
(2) preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute every 1mL is made containing 12.5mg Solution, shake up, be used as inner mark solution；
(3) preparation of need testing solution：Precision measures pepper extract 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol Scale is diluted to, then precision measures 1.0mL, puts in 10mL volumetric flasks, precision addition inner mark solution 1.0mL, plus absolute ethyl alcohol are dilute Release to scale, shake up；
(4) preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus absolute ethyl alcohol Dissolve and dilute and 0.301mgmL containing sabinene is made-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
(5) determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas chromatograph, enters Row detection；
(6) measurement result：This product is 0.223mg containing sabinene per 1g, is 0.218mg containing Humuleno.
Embodiment 4：Emplastrum
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.1g of the present invention；
(4) pepper extract of the present invention is taken, matrix (rubber 16g, rosin 16g, lanolin 4g, vaseline 1.5g, liquid is added Body paraffin 1g, zinc oxide 20g, gasoline 45g), coating is made, painting cream is carried out, cut-out, lid lining is cut into small pieces, produced.
Embodiment 5：Aerosol
Prescription：Chinese prickly ash 20Kg
Preparation method：
(1) Chinese prickly ash meal (40 mesh) 20Kg is taken, is put in baking oven after 45 DEG C of drying 4h, plus 20 times of 60% aqueous acetone solutions of amount, In 65 DEG C of water-bath refluxing extractions 3 times, each 75min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
(2) by obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 45 DEG C of water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 45 DEG C of water-baths；
(3) column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:8, loading volume 7BV, washes 5BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 8BV elutions, flow velocity For 3BV/h, eluent is collected, eluent concentration is dried, obtains pepper extract 10.1g of the present invention；
(4) pepper extract of the present invention is taken, water 900mL is added, propane diols 100mL, appropriate essence is added, mixed, filtration, Obtain decoction；Above-mentioned decoction is sub-packed in aerosol container in desinfection chamber quantification, valve member is fixed on container, Ran Hou Under 780Pa pressure, the propellant F filtered through miillpore filter is pressed into bottle122g, produces solution aerosol.100 are made altogether Bottle.

Claims (5)

1. the anesthesiophore Longnan of Gansu Province pepper extract of one kind tool, it is characterised in that the pepper extract is using as follows Prepared by method：
（1）Chinese prickly ash is crushed, put in baking oven after 40 DEG C~50 DEG C drying 3h~5h, plus 15~25 times of 50%~60% acetone of amount are water-soluble Liquid, in 60 DEG C~70 DEG C water-bath refluxing extractions 2 times~4 times, each 70min~80min merges extract solution, reclaims acetone, obtains Chinese prickly ash total extract medicinal extract；
（2）By obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is in 40 DEG C~50 DEG C water bath conditions Lower stirring and dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 40 DEG C~50 DEG C water-baths；
（3）Column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7~9, loading volume 6BV ~8BV, washes 4BV~6BV removal of impurities, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 7BV~ 9BV is eluted, and flow velocity is 2BV/h~4BV/h, collects eluent, and eluent concentration is dried, produced；
Assay is carried out to single leaf rue product alkali in the pepper extract using high performance liquid chromatography, assay method is：
（1）Chromatographic condition：Using HypersilDs chromatographic columns；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection wavelength： 200nm；Column temperature：20℃；Flow velocity：1.0mL·min-1；Sample size：10μL；
（2）It is prepared by reference substance solution：It is appropriate to single leaf rue product alkali reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol is molten Reference substance solutions of every 1mL containing 0.2mg is made in solution；
（3）The preparation of need testing solution：Precision weighs pepper extract 10mL, plus methanol 40mL, is heated to reflux 4h, and extract solution is returned Stream solvent simultaneously be concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butanol shaking extract 5 times, each 20mL, merge just Butanol extract solution, is washed 3 times, each 15mL with ammonia solution, and n-butanol extracting liquid recycling design is to doing, and residue adds methanol dissolving simultaneously It is transferred in 10mL volumetric flasks, plus methanol is to scale, shakes up, filtration, takes filtrate, produces need testing solution；
（4）Determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects high performance liquid chromatograph, enters Row detection；
Assay is carried out to the sabinene in the pepper extract, Humuleno using gas chromatography, assay method is：
（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary columns；Carrier gas：N2, 1.0mLmin-1；Hydrogen, 40mL min-1；Air, 400mLmin-1；Split ratio：8：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Temperature programming： Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；
（2）The preparation of inner mark solution：Take cyclohexanone appropriate, plus anhydrous alcohol solution and dilute every 1mL is made containing the molten of 12.5mg Liquid, shakes up, and is used as inner mark solution；
（3）The preparation of need testing solution：Precision measures pepper extract 1.0g, puts in 10mL volumetric flasks, plus absolute ethyl alcohol dilution 1.0mL is measured to scale, then precision, is put in 10mL volumetric flasks, precision adds inner mark solution 1.0mL, plus absolute ethyl alcohol is diluted to Scale, shakes up；
（4）The preparation of reference substance solution：Precision weighs sabinene reference substance, Humuleno reference substance in right amount, plus anhydrous alcohol solution And 0.301mgmL containing sabinene is made in dilution-1And Humuleno 0.901mgmL-1Reference substance stock solution, it is standby；
（5）Determine：Precision measures each 10 μ L of above-mentioned need testing solution, reference substance solution respectively, injects gas chromatograph, is examined Survey.
2. pepper extract as claimed in claim 1, it is characterised in that the pepper extract is to adopt to prepare with the following method 's：
（1）Chinese prickly ash is crushed, put in baking oven after 40 DEG C of drying 5h, plus 15 times of 55% aqueous acetone solutions of amount, carried in 60 DEG C of water-bath backflows Take 4 times, each 70min, merge extract solution, reclaim acetone, obtain Chinese prickly ash total extract medicinal extract；
（2）By obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is stirred under 50 DEG C of water bath conditions Dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 50 DEG C of water-baths；
（3）Column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7, loading volume 8BV, water 4BV removal of impurities is washed, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, Eluent is collected, eluent concentration is dried, produced；
3. the pepper extract as described in claim 1~2 any one, it is characterised in that the pepper extract uses pharmacy Middle conventional pharmaceutical methods are prepared into external medicine preparation or aerosol.
4. application of a kind of pepper extract in treatment rheumatoid arthritis agents are prepared, it is characterised in that the Chinese prickly ash is extracted Thing is adopted to be prepared with the following method：
（1）Chinese prickly ash is crushed, put in baking oven after 40 DEG C of drying 5h, plus 15 times of 55% aqueous acetone solutions of amount, carried in 60 DEG C of water-bath backflows Take 4 times, each 70min, merge extract solution, reclaim acetone, obtain Chinese prickly ash total extract medicinal extract；
（2）By obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is stirred under 50 DEG C of water bath conditions Dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 50 DEG C of water-baths；
（3）Column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7, loading volume 8BV, water 4BV removal of impurities is washed, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, Eluent is collected, eluent concentration is dried, produced.
5. a kind of application of pepper extract in injury of vein medicine caused by preparing preventing and treating mannitol, it is characterised in that the flower Green pepper extract is adopted to be prepared with the following method：
（1）Chinese prickly ash is crushed, put in baking oven after 40 DEG C of drying 5h, plus 15 times of 55% aqueous acetone solutions of amount, carried in 60 DEG C of water-bath backflows Take 4 times, each 70min, merge extract solution, reclaim acetone, obtain Chinese prickly ash total extract medicinal extract；
（2）By obtained Chinese prickly ash total extract medicinal extract with chloroform:Methanol=50:5 mixed solution is stirred under 50 DEG C of water bath conditions Dissolving, filtering, filtrate carries out column chromatography for separation after being concentrated under reduced pressure in 50 DEG C of water-baths；
（3）Column chromatography for separation is carried out from D101B types macroporous absorbent resin, resin column blade diameter length ratio is 1:7, loading volume 8BV, water 4BV removal of impurities is washed, with ethyl acetate:0.2mol/L sodium dihydrogen phosphate=100:5 eluant, eluent 9BV elutions, flow velocity is 2BV/h, Eluent is collected, eluent concentration is dried, produced.
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