CN106728651A - The preparation method and its method of quality control of a kind of rhizoma cyperi SIWU KELI - Google Patents

The preparation method and its method of quality control of a kind of rhizoma cyperi SIWU KELI Download PDF

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CN106728651A
CN106728651A CN201710156960.0A CN201710156960A CN106728651A CN 106728651 A CN106728651 A CN 106728651A CN 201710156960 A CN201710156960 A CN 201710156960A CN 106728651 A CN106728651 A CN 106728651A
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methyl alcohol
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rhizoma cyperi
ether
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CN106728651B (en
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段金廒
钱大玮
刘培
宿树兰
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Nanjing University of Chinese Medicine
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Nanjing 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/89—Cyperaceae (Sedge family)
    • A61K36/8905—Cyperus (flatsedge)
    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232—Angelica
    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/236—Ligusticum (licorice-root)
    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/285—Aucklandia
    • 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)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • 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)
    • A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
    • 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)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • 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
    • 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
    • G01N30/90—Plate chromatography, e.g. thin layer or paper 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
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • 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
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • 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

Abstract

The invention discloses the preparation method and its method of quality control of a kind of rhizoma cyperi SIWU KELI, it by Radix Angelicae Sinensis, Ligusticum wallichii, rhizoma cyperi and banksia rose four traditional Chinese medicine material, using extraction by steam distillation volatile oil, volatile oil is ground inclusion using beta cyclodextrin, it is residue obtained after volatile oil extracting, plus cultivated land, the root of herbaceous peony and corydalis tuber, add water to cook extraction, extract solution carries out alcohol precipitation with 95% ethanol, stand, suction filtration, supernatant is concentrated under reduced pressure, dry, dry extract is obtained, Icing Sugar, dextrin is added, mixed, granulation, dry, whole grain, add inclusion compound, mix, be made.Its method of quality control includes that thin-layer qualitative differentiates and HPLC assays.Volatile oil part is individually extracted the present invention inclusion, with the double effects for reducing supplementary product consumption and performance therapeutic action, each flavour of a drug addition when taking of more current single granule, the advantage of drug matching can more be given full play to, and perfect quality standard is established, can effectively control the quality of compound granular.

Description

The preparation method and its method of quality control of a kind of rhizoma cyperi SIWU KELI
Technical field
The invention belongs to Chinese medicinal granule technical field, and in particular to a kind of preparation method of rhizoma cyperi SIWU KELI and its Method of quality control.
Background technology
Rhizoma cyperi Siwu Tang is made up of seven flavor medicines such as rhizoma cyperi, the banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, prepared rhizome of rehmannia, the root of herbaceous peonys, is promoting the circulation of qi One of stagnation resolvation side of representative, comes from the honest and clean husband of Qing Dynasty's beam《I do not know that doctor is necessary》Volume four, blood is adjusted with blood-nourishing, main the effect of promoting qi circulation and relieving pain Control the diseases such as caused by energy stagnation and blood stasis dysmenorrhoea, irregular menstruation.
The generation of dysmenorrhoea mainly synthesizes with menstrual period endometrium and release prostaglandin increase is relevant, uterus muscle reactivity mistake Height, secondary mesometrium ischemic causes pain.The traditional Chinese medical science thinks, the main pathogenesis of dysmenorrhoea be hindered by feelings will, daily life accidentally or six Smooth, mechanism of qi does not block the QI-blood circulation that the excessive cause of disease such as cause harm is caused, and general rule is not bitterly.QI-blood circulation is not freely topmost pathology base Plinth, it is clinically then common with the dysmenorrhoea of qi stagnation and blood stasis type.
Rhizoma cyperi Siwu Tang has functions that blood promoting qi circulation and relieving pain is adjusted in blood-nourishing by Siwu Tang perfuming is attached, the banksia rose, corydalis tuber are constituted, Cure mainly the diseases such as caused by energy stagnation and blood stasis dysmenorrhoea, irregular menstruation.It is one of regulating qi to disperse stagnation side of representative.Rhizoma cyperi taste is pungent, sweet, slight bitter, it is mild-natured Partial temperature, main return liver warp, pungent scattered temperature is led to, enter liver, and can pass through qi and blood, is apt to the strongly fragrant of conditioning irritability, and can adjust the stagnant of smooth menses, therefore is had Regulating liver and qi, the effect of menstruction regulating and pain relieving, Li Shizhen (1518-1593 A.D.) praises that it is " Directorate-General DG of gas disease, the chief commander of female section ", illustrates to qi depression to blood stasis Type gynaecological disease has special efficacy.Often shared with Radix Angelicae Sinensis, a main qi leel, a main blood system, qi and blood is simultaneously controlled, and the work(of vital energy regualting and blood circulation-promoting is played altogether. The banksia rose, taste is pungent, hardship are warm in nature, main returns spleen, stomach, large intestine, tri-jiao channel, and Xin Xiang rows dissipate, and hardship cooling is logical, and can rise to drop, and leads to reason three Jiao, there is a promoting qi circulation and relieving pain, effect of reinforcing spleen to promote digestion, is often shared with rhizoma cyperi, corydalis tuber etc., with dispersing liver and promoting blood circulation menstruction regulating and pain relieving.Corydalis tuber, Taste is pungent, bitterness temperature, the main thoughts of returning home, liver, spleen, lung channel, pungent scattered hardship let out warm logical, that is, enter conscience and enter spleen and lung through walking blood system, again through flat Point, can invigorate blood circulation, and the gas in energy promoting circulation of blood, and can relieve pain again, therefore be the key medicine of blood-activating and qi-promoting analgesic, all upper and lower Zhu Tong of the whole body Category and qi depression to blood stasis person, can use it.Radix Angelicae Sinensis etc. often is equipped with, with the stagnant pain of dispelling of dredging collateral row.
Biological effect research shows that rhizoma cyperi Siwu Tang has significant inhibitory action to overall dysmenorrhea model in mice, can be significantly Suppress the contraction frequency and contraction movement power of mouse isolated uterine, and inhibitory action to COX-2 enzymes is significantly increased；Can be obvious Improve qi depression to blood stasis rat model hemorheology index, the hydracetin-induced anemia mice model of intervention improves peripheral hemogram effect not substantially, In dysmenorrhoea effect is suppressed based on analgesia.Clinical research shows that the total effective of qi stagnation and blood stasis type primary dysmenorrhea treats in the party More than 80%, larger kind YUEYUESHU TONGJIBAO KELI, ibuprofen sustained release capsules can more improve patient's dysmenorrhoea symptom to rate.
Traditional Chinese herbal decoction as traditional tcm clinical practice medication main formulation, with card plus-minus, flexible prescription, be easy to suction The advantages of receiving, work very fast, it is deep to be trusted by many patients.But mould losing is susceptible to because allocating, carry, decoct, be long placed in temporarily The shortcomings of rotten, soup bitter, big amount, does not adapt to the life requirement of modern.Chinese medicinal granule maintains many of simple Efficacy, remains the active ingredient of medicine to greatest extent, but Chinese medicinal granule is all single medicinal material extract at present, is made Used time " single is extracted, and mixing is taken after mixing it with water ", there are the variability issues of " decocting altogether " and " divide and decoct ".Composite powdered extracts have passed on Chinese medicine The advantage of single granule, while it is contemplated that interaction of the medicine materical crude slice in decoction process, meets traditional Chinese medical science tradition pharmacology By with larger value.
The content of the invention
It is an object of the invention to provide the preparation method and its method of quality control of a kind of rhizoma cyperi SIWU KELI.
The present invention is achieved by the following technical solutions：
A kind of preparation method of rhizoma cyperi SIWU KELI, it is to compare 3 by weight:2:3:6:3:8:3 rhizoma cyperi, the banksia rose, prolong recklessly Rope, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony are made, and specifically include following steps：
A. Radix Angelicae Sinensis, Ligusticum wallichii, rhizoma cyperi and banksia rose medicine materical crude slice are weighed by above weight ratio, adds medicinal material 5-15 times of weight of gross weight Water, after soaking 10-15 hours, then steam distillation is extracted 8-12 hours, extracts volatile oil；
B. taking volatile oil adds beta-schardinger dextrin and water to be fully ground, and freeze-drying obtains volatile oil clathrate compound；
C. take it is residue obtained after volatile oil extracting, add above-mentioned weight than cultivated land, the root of herbaceous peony and corydalis tuber, add water to cook 2 ~3 times, each amount of water is 6-14 times of total medicinal material, is decocted 1-3 hours, merges liquid, filtration, drying under reduced pressure to relative density The concentrate of 1.00-1.15, concentration of alcohol is 80~85% in adding the ethanol of volumetric concentration 95% to concentrate, is stood, suction filtration, Filtrate is taken, is concentrated under reduced pressure, dried, get dry extract powder；
D. take dried cream powder and add dextrin, Icing Sugar by a certain percentage, mix, granulation is dried, and whole grain is eventually adding step b systems The volatile oil clathrate compound for obtaining, obtains final product.
Preferably, the preparation method of above-described rhizoma cyperi SIWU KELI, specifically includes following steps：
A. Radix Angelicae Sinensis, Ligusticum wallichii, rhizoma cyperi and banksia rose medicine materical crude slice are weighed by above weight ratio, add medicinal material 8 times of water of weight of gross weight, After immersion 12 hours, steam distillation is extracted 10 hours, extracts volatile oil；
B. taking volatile oil adds beta-schardinger dextrin and water to be fully ground, and freeze-drying obtains volatile oil clathrate compound；
C. it is residue obtained after volatile oil extracting, add above-mentioned weight than cultivated land, the root of herbaceous peony and corydalis tuber, add water to cook 2 times, Each amount of water is 10 times of total medicinal material, is decocted 3 hours, merges liquid, filtration, drying under reduced pressure to relative density 1.05-1.08 Concentrate, add volumetric concentration be 95% ethanol to concentrate in concentration of alcohol be 80%, stand 24 hours after, suction filtration, filter Liquid is concentrated under reduced pressure, and dries, and get dry extract powder；
D. take dried cream powder and add dextrin, Icing Sugar, mix, granulation is dried, whole grain, be eventually adding that step b prepares waves Hair oil inclusion compound, obtains final product.
Radix Angelicae Sinensis of the invention, Ligusticum wallichii, rhizoma cyperi and the banksia rose contain volatile oil, are effective component extracting, this hair to greatest extent Bright use volatile oil collection device, obtains volatile oil, and carry out inclusion treatment to it.It is preferred that step b in, the beta-schardinger dextrin Addition be to volatilize 5-20 times, particularly preferred 8-15 times of oil volume；The addition of water is 10-50 times of volatilization oil volume, Particularly preferably 20-40 times.
It is preferred that step d in, the addition of dextrin is 0-2 times of dried cream powder weight, particularly preferred 1~2 times, and Icing Sugar adds It is 0-2 times, particularly preferred 1~2 times of dried cream powder weight to enter amount.
The method of quality control of the rhizoma cyperi SIWU KELI prepared present invention also offers a kind of above-mentioned preparation method, using thin Layer chromatography Qualitive test and high performance liquid chromatography assay.
Specifically include one or more in following methods：
The method of quality control of described rhizoma cyperi SIWU KELI, it is characterised in that including one kind in following methods or several Kind：
(1) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus sodium bicarbonate solution, ultrasonically treated, centrifugation, take supernatant and adjusted with watery hydrochloric acid PH value is shaken with ether and extracted 2~3 times to 2~3, merges ether solution, is volatilized, and residue adds the methyl alcohol to make dissolving, used as Ligustilide Need testing solution；
Rhizoma cyperi SIWU KELI, plus EtOH Sonicate dissolving are taken, is put and concentrated in water-bath, shaken with ether and extracted 2~3 times, merged Ether extracted liquid, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, ether solution is discarded, extract solution is adjusted with watery hydrochloric acid Section pH value shakes extraction to 2~3, then with ether, merges ether extracted liquid, volatilizes, and residue adds methyl alcohol to dissolve, and is supplied as forulic acid Test sample solution；
Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material, plus EtOH Sonicate treatment are taken, are put and concentrated in water-bath, shaken with ether and extracted 2~3 times, Merge ether extracted liquid, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, discard ether solution, the dilute salt of extract solution Acid for adjusting pH value shakes extraction to 2~3, then with ether, merges ether extracted liquid, volatilizes, and residue adds methyl alcohol to dissolve, and is made control Medicinal material solution；
Rhizoma cyperi, the banksia rose, corydalis tuber, cultivated land and the root of herbaceous peony, plus EtOH Sonicate treatment are taken, is put and concentrated in water-bath, shaken with ether Extract 2~3 times, merge ether extracted liquid, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, discard ether solution, Extract solution watery hydrochloric acid adjusts pH value and shakes extraction to 2~3, then with ether, merges ether extracted liquid, volatilizes, and residue adds methyl alcohol Dissolving, is made double-negative contrast solution；
Forulic acid and ligustilide from rhizome are taken, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 5 kinds of solution is drawn, put on same silica gel g thin-layer plate respectively, be with volume ratio 30:1:3 benzene-glacial acetic acid-methyl alcohol is solvent, is launched, and is taken out, and airing is put under ultraviolet lamp, and 365nm is inspected, as a result, supplied On the relevant position of test product and reference substance, show the spot of same color, negative controls are noiseless；
(2) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add diethyl ether and be heated to reflux, filter, filtrate volatilizes, and residue adds ethyl acetate to dissolve, As need testing solution；
Ligusticum chuanxiong Hort powder is taken, is added diethyl ether and is heated to reflux, filtered, filtrate volatilizes, and residue adds ethyl acetate to dissolve, and it is right to be made According to medicinal material solution；
Rhizoma cyperi, the banksia rose, corydalis tuber, cultivated land, Radix Angelicae Sinensis and the root of herbaceous peony are taken, is added diethyl ether and is heated to reflux, filtered, filtrate volatilizes, residue Plus ethyl acetate dissolving, it is made negative control solution；
Levistilide A reference substance is taken, plus ethyl acetate is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica GF254 lamellae, with volume Than being 3:1 n-hexane-ethyl acetate is solvent, is launched, and is taken out, airing, and 254nm is inspected under putting ultraviolet lamp, as a result, supplied On the relevant position of test product and reference substance, show the spot of same color, negative controls are noiseless；
(3) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add diethyl ether, place 1~2 hour, constantly shake, filter, filtrate volatilizes, and residue adds Ethyl acetate makes dissolving, used as need testing solution；
Rhizoma cyperi control medicinal material powder is taken, is added diethyl ether, placed 1~2 hour, constantly shaken, filtered, filtrate volatilizes, and residue adds Ethyl acetate makes dissolving be made control medicinal material solution；
The banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and white Peony Root are taken, are added diethyl ether, placed 1~2 hour, constantly shaken, Filtration, filtrate volatilizes, and residue adds the ethyl acetate dissolving is made negative control solution；
α-cyperolone reference substance is taken, plus ethyl acetate is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica GF254 lamellae, use volume Than being 80:1:1 dichloromethane-ethyl acetate-glacial acetic acid is solvent, is launched, and is taken out, and airing is put under ultraviolet lamp 254nm Inspect, then spray with DNPH ethanol test solution, it is seen that inspected under light；As a result, the relevant position of test sample and reference substance On, showing the spot of same color, negative controls are noiseless；
(4) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus petroleum ether, cold soaking 30~60 minutes, constantly shake, filter, filtrate volatilizes, residual Slag adds the ethyl acetate to make dissolving, used as need testing solution；
The powder of banksia rose control medicinal material, plus petroleum ether are taken, cold soaking 30~60 minutes constantly shakes, filtered, filtrate volatilizes, Residue adds the ethyl acetate dissolving is made control medicinal material solution；
Rhizoma cyperi, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony, plus petroleum ether are taken, cold soaking 30~60 minutes constantly shakes, Filtration, filtrate volatilizes, and residue adds the ethyl acetate dissolving is made negative control solution；
Dehydro-α-curcumene reference substance and costunolide reference substance are taken, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put on same silica gel g thin-layer plate respectively, use volume ratio 15:5:1 with cyclohexane-ethyl acetate-formic acid as solvent, launches, and takes out, airing, sprays molten with 5% vanillin-sulfuric acid ethanol Liquid, heating, clear to spot development, daylight is inspected, as a result, on the relevant position of test sample and reference substance, shows same color Spot, negative controls are noiseless；
(5) thin-layer chromatography Qualitive test is used
Particle 15g is taken, finely ground, add water 100mL, warm makes it fully leach, and lets cool, filtered with absorbent cotton, filtrate second Ether shaking is extracted 2 times, each 25mL, discards ether solution, then shakes extraction 2 times with ethyl acetate, and each 25mL merges acetic acid second Ester liquid, is evaporated, and residue adds the methyl alcohol 1mL to make dissolving, used as need testing solution.The powder of 1g cultivated land medicinal materials is taken, control is made in the same way of Medicinal material solution；Scarce cultivated land sample 15g is taken, negative control solution is made in the same way of.5 hydroxymethyl furfural reference substance is taken, plus methyl alcohol is made The solution containing 0.290mg per 1mL, as reference substance solution.According to thin-layered chromatography (general rule 0502) experiment, above-mentioned 4 kinds of absorption is molten Liquid, puts on same silica gel g thin-layer plate, with dimethylbenzene-ethyl acetate (1 respectively:1) it is solvent, launches, takes out, airing, spray With DNPH ethanol solution, daylight is inspected.As a result, on the relevant position of test sample and reference substance, same color is shown Spot.Negative controls are noiseless.
(6) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add water, heating for dissolving is extracted 2~3 times with water-saturated n-butanol, merges n-butanol Liquid, with n-butanol saturation water washing, merges n-butanol liquid, and water-bath is volatilized, plus methyl alcohol dissolving, using silicagel column, first with volume ratio It is 10:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol wash-out, be preferably with volume ratio 8:1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol to dissolve, used as need testing solution；
Root of herbaceous peony control medicinal material powder is taken, is added water, it is ultrasonically treated, extracted 2~3 times with water-saturated n-butanol, merge n-butanol Liquid, with n-butanol saturation water washing, merges n-butanol liquid, and water-bath is volatilized, plus methyl alcohol dissolving, using silicagel column, first with volume ratio It is 10:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol wash-out, be preferably with volume ratio 8:1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol to dissolve, and is made control medicinal material molten Liquid；
Rhizoma cyperi, the banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii and cultivated land medicinal material are taken, is added water, it is ultrasonically treated, extracted with water-saturated n-butanol Take 2~3 times, merge n-butanol liquid, with n-butanol saturation water washing, merge n-butanol liquid, water-bath is volatilized, plus methyl alcohol dissolving, adopt It is first 10 with volume ratio with silicagel column:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol Wash-out, is 8 preferably with volume ratio:1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol Dissolving, is made negative control solution；
Precision weighs Paeoniflorin reference substance, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put on same silica gel g thin-layer plate respectively, be with volume ratio 40:5:10:0.2 chloroform-acetate-methanol-formic acid is solvent, is launched, and is taken out, airing, is sprayed with 5% vanillic aldehyde sulphur Sour ethanol solution, daylight is inspected, as a result, on the relevant position of test sample and reference substance, shows the spot of same color, negative control Product are noiseless.
(7) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus methyl alcohol, ultrasonically treated, filtration, filtrate is evaporated, and residue is dissolved in water, enriching ammonia Test solution is adjusted to alkalescence, is shaken with ether and extracted, and merges ether solution, is evaporated, and residue adds methyl alcohol to dissolve, used as need testing solution；
The powder of corydalis tuber control medicinal material, plus methyl alcohol are taken, ultrasonically treated, filtration, filtrate is evaporated, and residue is dissolved in water, plus Strong ammonia solution is adjusted to alkalescence, is shaken with ether and extracted, and merges ether solution, is evaporated, and it is molten that residue adds methyl alcohol dissolving to be made control medicinal material Liquid；
Rhizoma cyperi, the banksia rose, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony, plus methyl alcohol are taken, ultrasonically treated, filtration, filtrate is evaporated, and residue adds Water dissolves, enriching ammonia solution is adjusted to alkalescence, is shaken with ether and extracted, and merges ether solution, is evaporated, and residue adds methyl alcohol dissolving to be made the moon Property contrast solution；
Precision weighs tetrahydropalmatine reference substance, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put on same silica gel g thin-layer plate respectively, be with volume ratio 9:2 toluene-acetones are solvent, are launched, and are taken out, airing, are sprayed to improve bismuth potassium iodide solution, and daylight is inspected, as a result, test sample On the relevant position of reference substance, show the spot of same color, negative controls are noiseless.
The method of quality control of rhizoma cyperi SIWU KELI of the present invention, including one or more in following methods：
(1) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；Detection forulic acid when with Volume ratio 20:The glacial acetic acid solution of 80 acetonitrile -1% is mobile phase；With volume ratio 12 during detection vanillic acid:The 88 ice second of acetonitrile -1% Acid solution is mobile phase；Detection wavelength is respectively 323nm and 260nm；Theoretical cam curve presses forulic acid and vanillic acid is calculated not Less than 3000；
The preparation of reference substance solution takes forulic acid, vanillic acid reference substance in right amount, accurately weighed, adds methyl alcohol to be made often respectively Reference substance solutions of the 1mL containing 0.0316mg, 0.0295mg；
The preparation of need testing solution takes rhizoma cyperi SIWU KELI, accurately weighed, puts in conical flask with cover, and precision adds methyl alcohol, claims Weight, is stood overnight, ultrasound, is re-weighed, and the quality of loss is supplied with methyl alcohol, is shaken up, and is filtered, and precision measures subsequent filtrate, and water-bath is steamed It is dry；Residue sodium hydroxide solution dissolves, and is transferred in backflow flask, and water-bath backflow lets cool；Move into separatory funnel, use salt PH=2 is adjusted in acid, is extracted with ether, merges ether solution, reclaims ether, is dissolved with methyl alcohol, is shaken up, and is obtained final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, i.e., ；
This product contains Radix Angelicae Sinensis and Ligusticum wallichii per 15g particles in terms of forulic acid and vanillic acid, must not be less than 0.714mg and 0.439mg；
(2) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；With volume ratio 18:82 The acetic acid of acetonitrile -0.1% water is mobile phase；Detection wavelength is 230nm, and theoretical cam curve presses Paeoniflorin and albiflorin calculates equal It is not less than 2000；
The preparation of reference substance solution takes Paeoniflorin, albiflorin reference substance in right amount, accurately weighed, adds methyl alcohol to be made respectively The reference substance solution containing 0.300mg, 0.200mg per 1mL.
The preparation of need testing solution takes rhizoma cyperi SIWU KELI, accurately weighed, puts in conical flask with cover, and precision adds methyl alcohol, It is accurately weighed, it is ultrasonically treated, let cool, then it is weighed, less loss quality is supplied with methyl alcohol, shake up, filter, subsequent filtrate is taken as test sample Solution；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, i.e., .
This product contains the root of herbaceous peony per 15g in terms of Paeoniflorin and albiflorin, must not be less than 52.5mg and 17.9mg；
(3) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；It is 55 with volume ratio: The acetic acid water of 45 acetonitrile -0.1%, and pH=6.0 is adjusted as being mobile phase with triethylamine；Detection wavelength is 280nm；Theoretical cam curve Calculated by tetrahydropalmatine and Berberine hydrochloride and be not less than 3000；
The preparation of reference substance solution takes tetrahydropalmatine, Berberine hydrochloride reference substance in right amount, accurately weighed, and methyl alcohol is added respectively It is made reference substance solutions of every 1mL containing 0.246mg, 0.297mg；
The preparation of need testing solution takes rhizoma cyperi SIWU KELI 15g, accurately weighed, puts in boiling flask, and precision adds volume Than being 1:20 strong ammonia solution-methyl alcohol mixed solution, weighed weight, cold soaking is heated to reflux, lets cool, then weighed weight, uses volume Than being 1:20 strong ammonia solutions-methyl alcohol mixed solution supplies the weight of less loss, shakes up, filtration；
Precision measures subsequent filtrate 75mL, is evaporated, and residue adds methyl alcohol to dissolve, and is transferred in 10mL measuring bottles, and is diluted to scale, Shake up, filter, take subsequent filtrate, obtain final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, i.e., ；
This product per 15g particles containing corydalis tuber in terms of tetrahydropalmatine and Berberine hydrochloride, must not less than 2.67mg and 1.22mg。
The present invention compared with prior art, has the advantages that：
(1) classics recipe rhizoma cyperi Siwu Tang is conventionally closed and is fried into composite powdered extracts, more current single formula Each flavour of a drug are simply added when particle is taken, and can more give full play to the advantage of drug matching, embody the overall idea of traditional Chinese medicine, really Guarantor reaches the purpose of attenuation synergistic, for clinical application provides new selection.
(2) present invention collects volatile oil active component in extraction process is decocted, after volatile oil beta-cyclodextrin inclusion compound again Particle is made, volatility reduction, utilization rate increases, and can promote the absorption of involatile constituent, improves drug bioavailability.
(3) present invention uses chromatography, i.e. thin-layered chromatography and high performance liquid chromatography, active ingredient is carried out it is qualitative and Quantitative analysis, carries out quality control, can be very good to ensure product quality.
(4) present invention rhizoma cyperi SIWU KELI is established using forulic acid in hplc simultaneous determination particle, Vanillic acid, Paeoniflorin, albiflorin, the quality control of six kinds of leading indicator component contents of tetrahydropalmatine and Berberine hydrochloride Method processed, the quality standard is realized to the root of herbaceous peony in prescription, Radix Angelicae Sinensis, Ligusticum wallichii, the taste medicine materical crude slice content of corydalis tuber four while be controlled, With stronger specificity and good reappearance, real embodiment drug safety is effective, quality controllable.
Brief description of the drawings
Fig. 1 is the thin-layer chromatogram of embodiment 1, wherein 1 to lack Radix Angelicae Sinensis, the double-negative sample of Ligusticum wallichii, 2-4 is 3 batches for examination Product, 5 is Radix Angelicae Sinensis control medicinal material, and 6 is Ligusticum wallichii control medicinal material, and 7 is forulic acid reference substance；
Fig. 2 is the thin-layer chromatogram of embodiment 1, wherein 1 to lack Radix Angelicae Sinensis, the double-negative sample of Ligusticum wallichii, 2-4 is 3 batches for examination Product, 5 is Radix Angelicae Sinensis control medicinal material, and 6 is Ligusticum wallichii control medicinal material, and 7 is ligustilide from rhizome；
Fig. 3 is the thin-layer chromatogram of embodiment 2, wherein 1 to lack Ligusticum wallichii negative sample, 2-4 is 3 batch test samples, and 5 is river Rhizome of chuanxiong control medicinal material, 6 is Levistilide A reference substance；
Fig. 4 is the thin-layer chromatogram of embodiment 3, wherein 1 to lack rhizoma cyperi negative sample, 2-4 is 3 batch test samples, and 5 is perfume Attached control medicinal material, 6 is α-cyperolone reference substance；
Fig. 5 is the thin-layer chromatogram of embodiment 4, wherein 1 to lack banksia rose negative sample, 2-4 is 3 batch test samples, and 5 is wood Fragrant control medicinal material, 6 is dehydro-α-curcumene reference substance, and 7 is costunolide reference substance；
Fig. 6 is the thin-layer chromatogram of embodiment 5, wherein 1 to lack cultivated land negative sample, 2-4 is 3 batch test samples, and 5 is ripe Ground control medicinal material, 6 is 5 hydroxymethyl furfural reference substance；
Fig. 7 is the thin-layer chromatogram of embodiment 6, wherein 1 to lack root of herbaceous peony negative sample, 2-4 is 3 batch test samples, and 5 is white Chinese herbaceous peony control medicinal material, 6 is Paeoniflorin reference substance；
Fig. 8 is the thin-layer chromatogram of embodiment 7, wherein 1 to lack corydalis tuber negative sample, 2-4 is 3 batch test samples, and 5 are Corydalis tuber control medicinal material, 6 is tetrahydropalmatine reference substance；
Fig. 9 is the high-efficient liquid phase chromatogram of embodiment 8, and wherein A is forulic acid reference substance, and B is need testing solution, and C is scarce Radix Angelicae Sinensis and the double-negative sample of Ligusticum wallichii, 1 is forulic acid；
Figure 10 is the high-efficient liquid phase chromatogram of embodiment 8, and wherein A is vanillic acid reference substance, and B is need testing solution, and C is scarce Radix Angelicae Sinensis and the double-negative sample of Ligusticum wallichii, 1 is vanillic acid；
Figure 11 is the high-efficient liquid phase chromatogram of embodiment 8, and wherein A is Paeoniflorin and albiflorin mixing reference substance, and B is Need testing solution, C lacks root of herbaceous peony negative sample, and 1 is albiflorin, and 2 is Paeoniflorin；
Figure 12 is the high-efficient liquid phase chromatogram of embodiment 8, and wherein A is tetrahydropalmatine and Berberine hydrochloride reference substance, and B is Test sample, C is to lack corydalis tuber negative sample, and 1 is Berberine hydrochloride, and 2 is tetrahydropalmatine.
Specific embodiment
The present invention is further illustrated below by specific embodiment, following examples are the specific embodiment party of the present invention Formula, but embodiments of the present invention are not limited by following embodiments.
Embodiment 1：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 14kg is added, after soaking 12 hours, water Steam distillation is extracted 10 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 10 times of volatile oil volume) and water 30 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 30kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.05 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
Forulic acid and ligustilide from rhizome are taken, plus methyl alcohol is made solution of every 1mL containing 2.05mg and 2.00mg, as right According to product solution.
The preparation of 1.2 control medicinal material solution
Radix Angelicae Sinensis, each 3g of Ligusticum wallichii control medicinal material powder, plus 1% sodium bicarbonate solution 150mL, ultrasonically treated 10min are taken, from The heart, takes supernatant watery hydrochloric acid and adjusts pH value to 2~3, is shaken with ether and extracted 2 times, and each 40mL merges ether solution, volatilizes, Residue adds the methyl alcohol 1mL to make dissolving, used as control medicinal material solution.
The processing method of 1.3 need testing solutions
Need testing solution 1：Take above-mentioned rhizoma cyperi SIWU KELI 15g, plus 1% sodium bicarbonate solution 150mL, it is ultrasonically treated 10min, centrifugation takes supernatant watery hydrochloric acid and adjusts pH value to 2~3, is shaken with ether and extracted 2 times, and each 40mL merges ether Liquid, volatilizes, and residue adds the methyl alcohol 1mL to make dissolving, used as need testing solution 1；
Need testing solution 2：Above-mentioned rhizoma cyperi SIWU KELI 15g is taken in beaker, plus 80% ethanol 150mL ultrasonic dissolutions, put Ethanol to about 30mL is boiled off in water-bath, is shaken with ether and extracted 2 times, each 40mL merges ether extracted liquid, then to above-mentioned ether In solution plus the shaking of 1% sodium carbonate liquor is extracted 2 times, each 40mL discards ether solution, extract solution watery hydrochloric acid adjust pH value to 2~3, then extraction 2 times is shaken with ether, each 60mL merges ether extracted liquid, volatilizes, and residue adds methyl alcohol 1mL to dissolve, as Need testing solution 2.
The preparation of 1.4 negative control solutions
In above-mentioned prescription ratio and preparation method, the double-negative control sample of scarce Radix Angelicae Sinensis and Ligusticum chuanxiong Hort is made.It is molten by test sample The preparation method of liquid 1 and 2, is made double-negative contrast solution.
2. the condition optimizing of solvent
It is Optimal flattening agent, draws above-mentioned solution and put respectively on same silica gel g thin-layer plate, from two kinds of expansion systems, Launch, take out, dry, put and inspect under ultraviolet lamp (365nm), as a result show, forulic acid and Ligustilide are in development system A There is obvious spot, forulic acid spot is not obvious and separating effect is bad in development system B, therefore selection benzene-glacial acetic acid-methyl alcohol (30:1:3) it is forulic acid and the solvent of Ligustilide.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
The μ L of forulic acid reference substance solution each 1,2,4,6 are drawn respectively to put on same silica gel g thin-layer plate successively, are drawn respectively The μ L of ligustilide from rhizome solution 1,2,3,4 are put on same silica gel g thin-layer plate successively, and unrolling strips are analyzed with the TLC for determining Part is analyzed, and two kinds of reference substance difference point sample amounts have obvious spot, and forulic acid spot in 4 μ L is the most clear, in Jehol Ligusticum Rhizome Ester has relatively bright spot in 2 μ L.So selection 4 μ L and 2 μ L are respectively the point sample amount of forulic acid and Ligustilide.
The determination of 3.2 need testing solution point sample amounts
Need testing solution 1 each 4,6,8,10 μ L is drawn respectively, need testing solution 2 each 2,4,6,8 μ L is drawn respectively and is divided successively Other point is analyzed in same silica gel g thin-layer plate with the TLC conditions for determining, the spot in 4~10 μ L of need testing solution 1 is all clear Clear, point sample amount can be 4 μ L；The spot in 8 μ L of need testing solution 2 is most obvious, the optional 8 μ L of point sample amount.
4. sample survey
According to the TLC discrimination conditions for determining, 3 batch rhizoma cyperi SIWU KELIs are carried out with the TLC mirror of forulic acid and Ligustilide Not, as a result as illustrated in fig. 1 and 2, on the relevant position of test sample and reference substance, the spot of same color is shown.Negative controls without Interference.
Embodiment 2：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 16.8kg is added, after soaking 15 hours, Steam distillation is extracted 8 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 8 times of volatile oil volume) and water 20 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 28kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.13 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
Weigh Levistilide A reference substance in right amount, plus ethyl acetate is made solution of every 1mL containing 0.116mg and (puts brown amount In bottle), as reference substance solution.
The preparation of 1.2 control medicinal material solution
Ligusticum chuanxiong Hort powder 1g is taken, add diethyl ether 100mL, be heated to reflux l h, filtered, filtrate volatilizes, and residue adds ethyl acetate 2mL makes dissolving, used as control medicinal material solution.
The processing method of 1.3 need testing solutions
Above-mentioned rhizoma cyperi SIWU KELI 15g is taken, add diethyl ether 100mL, be heated to reflux l h, filtered, filtrate volatilizes, and residue adds second Acetoacetic ester 2mL makes dissolving, used as need testing solution.
The preparation of 1.4 negative control solutions
According to the ratio and preparation method of above-mentioned prescription, preparation lacks the negative control sample of Ligusticum wallichii, by the test sample under 1.3 The preparation method of solution, is made negative control solution.
2. the condition optimizing of solvent
Compare n-hexane-ethyl acetate (3:1), n-hexane-ethyl acetate-formic acid (3:1:0.05), n-hexane-acetic acid second Ester-formic acid (7:1:0.05) three kinds of expansion systems, select n-hexane：Ethyl acetate (3:1) it is the solvent of Levistilide A.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
The μ L of Levistilide A reference substance solution each 1,2,4,5 are drawn respectively to put successively in same gel GF 254 plate.Control Product difference point sample amount has obvious spot, is that spot has been apparent from 4 μ L, and 4 μ L of selection are the point sample amount of Levistilide A.
The determination of 3.2 need testing solution point sample amounts
The μ L of need testing solution each 2,4,8,10 being drawn respectively, being analyzed with the TLC conditions for determining, need testing solution is in 8 μ Spot is more clear regular during L, so the selection of point sample amount is 8 μ L.
4. sample survey
According to the TLC conditions for determining, the thin layer that 3 batch rhizoma cyperi SIWU KELIs are carried out with Levistilide A differentiates, as a result such as Shown in Fig. 3, on the relevant position of test sample and reference substance, show the spot of same color.Negative controls are noiseless.
Embodiment 3：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 16.8kg is added, after soaking 15 hours, Steam distillation is extracted 8 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 8 times of volatile oil volume) and water 20 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 28kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.13 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
α-cyperolone reference substance is taken, plus ethyl acetate is made solution of every 1mL containing 2.2mg, as reference substance solution.
The preparation of 1.2 control medicinal material solution
Rhizoma cyperi control medicinal material powder 1g is taken, add diethyl ether 75mL, place 1h, constantly shaken, filtered, filtrate volatilizes, and residue adds Ethyl acetate 1mL makes dissolving, used as control medicinal material solution.
The processing method of 1.3 need testing solutions
Above-mentioned rhizoma cyperi SIWU KELI 15g is taken, add diethyl ether 75mL, place 1h, constantly shaken, filtered, filtrate volatilizes, and residue adds Ethyl acetate 1mL makes dissolving, used as need testing solution.
The preparation of 1.4 negative control solutions
According to the ratio and preparation method of above-mentioned prescription, preparation lacks the negative control sample of rhizoma cyperi, by the system of need testing solution Preparation Method, is made negative control solution.
2. the optimization of solvent and color condition
Dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) it is the most suitable solvent of α-cyperolone.Gel GF 254 plate and Silica G plate gained spot effect is accurate refinement quite, and selection silica GF254 launches, and uviol lamp (254nm) is inspected, then is sprayed with 2, 4- dinitrophenylhydrazine ethanol test solution watches spot development.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
The μ L of α-cyperolone reference substance solution each 1,2,4,5 are drawn respectively to put successively in same silica GF254 lamellae, with true The TLC conditions set are analyzed, and spot is most regular during 2 μ L, and 2 μ L of selection are point sample amount.
The determination of 3.2 need testing solution point sample amounts
The μ L of need testing solution each 4,6,8,10 being drawn respectively, being analyzed with the TLC conditions for determining, need testing solution is 10 Spot is more clear regular during μ L, so the selection of point sample amount is 10 μ L.
4. sample survey
According to the TLC conditions for determining, the thin layer that 3 batch rhizoma cyperi SIWU KELIs are carried out with α-cyperolone differentiates, as a result such as Fig. 4 It is shown, on the relevant position of test sample and reference substance, show the spot of same color.Negative controls are noiseless.
Embodiment 4：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 14kg is added, after soaking 12 hours, water Steam distillation is extracted 10 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 10 times of volatile oil volume) and water 30 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 30kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.05 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
Take dehydro-α-curcumene reference substance, costunolide reference substance appropriate, plus methyl alcohol is respectively prepared every 1mL and contains The solution of 2.28mg, 1.85mg, as reference substance solution.
The preparation of 1.2 control medicinal material solution
The powder of 0.5g banksia rose control medicinal materials, plus petroleum ether (30~60 DEG C) 75mL, cold soaking 30min are taken, is constantly shaken, filtered Cross, filtrate volatilizes, residue adds the ethyl acetate 1mL to make dissolving, takes filtrate and compares medicinal material solution.
The processing method of 1.3 need testing solutions
Above-mentioned rhizoma cyperi SIWU KELI 15g, plus petroleum ether (30~60 DEG C) 75mL, cold soaking 30min are taken, are constantly shaken, filtered, Filtrate volatilizes, and residue adds the ethyl acetate 1mL to make dissolving, used as need testing solution.
The preparation of 1.4 negative control solutions
In above-mentioned prescription ratio and preparation method, the negative control sample of scarce banksia rose medicinal material is made.By the preparation of need testing solution Method, is made double-negative contrast solution.
2. the condition optimizing of solvent
It is Optimal flattening agent, draws above-mentioned solution and put respectively on same silica gel g thin-layer plate, from three kinds of expansion systems, Launch, take out, dry, solvent B and C gained spot are relatively obscured, exhibition is away from close to forward position, and solvent A gained clear spots are regular, Exhibition is away from more suitable, therefore selection cyclohexane-ethyl acetate-formic acid (15:5:1) it is the exhibition of costunolide and dehydro-α-curcumene Open agent.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
Respectively draw the μ L of dehydro-α-curcumene reference substance solution each 1,2,3,4, costunolide reference substance solution each 2,4, 6th, 8 μ L, put in same silica gel g thin-layer plate respectively successively, are analyzed with the TLC conditions for determining, dehydro-α-curcumene control Product difference point sample amount has obvious spot, and in 2 μ L, spot is most regular, so 2 μ L of selection are its point sample amount.Costunolide pair In 8 μ L it is most obvious spot according to product, therefore 8 μ L of selection are its point sample amount.
The determination of 3.2 need testing solution point sample amounts
The μ L of need testing solution each 4,6,8,10 are drawn, is put respectively in same silica gel g thin-layer plate successively, with the TLC bars for determining Part is analyzed, as a result need testing solution all display dot in 4~10 μ L, and its point sample amount can be 10 μ L.
4. sample survey
According to the TLC discrimination conditions for determining, dehydro-α-curcumene and costunolide are carried out to 3 batch rhizoma cyperi SIWU KELIs Thin layer differentiate, as a result as shown in figure 5, on the relevant position of test sample and reference substance, showing the spot of same color.Negative control Product are noiseless.
Embodiment 5：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 14kg is added, after soaking 12 hours, water Steam distillation is extracted 10 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 10 times of volatile oil volume) and water 30 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 30kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.05 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
5 hydroxymethyl furfural reference substance is taken, plus methyl alcohol is made solution of every 1mL containing 0.29mg, as reference substance solution.
The preparation of 1.2 control medicinal material solution
The powder of 1g cultivated land medicinal materials is taken, add water 100mL, warm makes it fully leach, and lets cool, filtered with absorbent cotton, filtrate Shaken with ether and extracted 2 times, each 25mL discards ether solution, then shakes extraction 2 times with ethyl acetate, and each 25mL merges second Acetoacetic ester liquid, is evaporated, and residue adds the methyl alcohol 1mL to make dissolving, used as control medicinal material solution.
The processing method of 1.3 need testing solutions
Particle 15g is taken, add water 100mL, warm makes it fully leach, and lets cool, filtered with absorbent cotton, filtrate is shaken with ether Extract 2 times, each 25mL, discard ether solution, then extracted 2 times with ethyl acetate shaking, each 25mL, combined ethyl acetate liquid, It is evaporated, residue adds the methyl alcohol 1mL to make dissolving, used as need testing solution.
The preparation of 1.4 negative control solutions
In prescription ratio and preparation method, the negative control sample of scarce prepared rhizome of rehmannia medicinal material is made.By the preparation side of need testing solution Method, is made double-negative contrast solution.
2. the condition optimizing of solvent
Different Optimization condition：GF254 plates and solvent A, ultraviolet (254nm) is inspected；G plates and solvent B, spray developer, Daylight is observed；G plates and solvent C, spray developer, and daylight is inspected.Developer：2,4 dinitrophenyl hydrazine ethanol solution.By this three Plant and launch to be compared with color development system.Result shows that the Rf of solvent A is smaller, the exhibition of solvent B away from close to forward position, so Selection solvent C：Dimethylbenzene-ethyl acetate (1:1) it is the solvent of 5 hydroxymethyl furfural, DNPH ethanol solution It is developer.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
The μ L of 5 hydroxymethyl furfural reference substance solution each 6,8,10,12 are drawn respectively, are put successively in same silica gel g thin-layer plate, With the TLC condition analysis for determining, reference substance difference point sample amount has obvious spot, and spot is more regular during 8 μ L, so selection 8 μ L are point sample amount.
The determination of 3.2 need testing solution point sample amounts
Need testing solution 2 each 1,2,3,4 μ L is drawn respectively, is put successively in same silica gel g thin-layer plate, with the TLC bars for determining Part is analyzed, and need testing solution all shows obvious spot, the optional 2 μ L of point sample amount in 1~4 μ L.
4. sample survey
According to the TLC discrimination conditions for determining, the thin layer that 3 batch rhizoma cyperi SIWU KELIs are carried out with 5 hydroxymethyl furfural differentiates, Result is as shown in fig. 6, on the relevant position of test sample and reference substance, show the spot of same color.Negative controls are noiseless.
Embodiment 6：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 16.8kg is added, after soaking 15 hours, Steam distillation is extracted 8 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 8 times of volatile oil volume) and water 20 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 28kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.13 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
Weigh Paeoniflorin reference substance in right amount, plus methyl alcohol is made solution of every 1mL containing 1.03mg, as reference substance solution.
The preparation of 1.2 control medicinal material solution
0.5g root of herbaceous peony control medicinal material powder is taken, is dissolved with 1mL methyl alcohol, using silicagel column (300-400 mesh, about 20g, internal diameter About 2-2.5cm), with chloroform：Methyl alcohol (10:Isosorbide-5-Nitrae 4mL；9:1,50mL；8:Isosorbide-5-Nitrae 5mL) wash-out, preceding 15mL eluents are discarded, Collect follow-up 8:1 eluent.It is evaporated, plus 1mL methyl alcohol dissolved residues, it is control medicinal material solution.
The processing method of 1.3 need testing solutions
Rhizoma cyperi SIWU KELI 15g, plus 50mL water are taken, heating for dissolving, water-saturated n-butanol is extracted 2 times, each 25mL merges N-butanol liquid, with n-butanol saturation water washing 2 times, each 25mL merges n-butanol liquid, and water-bath is volatilized, and obtains residue.Residue is used 1mL methyl alcohol dissolves, using silicagel column (300-400 mesh, about 20g, internal diameter about 2-2.5cm), with chloroform：Methyl alcohol (10:1, 44mL；9:1,50mL；8:Isosorbide-5-Nitrae 5mL) wash-out, preceding 15mL eluents are discarded, collect follow-up 8:1 eluent, is evaporated, and residue adds 1mL Methyl alcohol makes dissolving, used as need testing solution.
The preparation of 1.4 negative control solutions
According to the ratio and preparation method of prescription, preparation lacks the negative control sample of the root of herbaceous peony, by the preparation side of need testing solution Method, is made negative control solution.
2. the determination of point sample amount
The determination of 2.1 reference substance solution point sample amounts
The μ L of Paeoniflorin reference substance solution each 4,6,8,10 are drawn respectively, is put successively in same silica G plate, with the TLC for determining Condition is analyzed, and Paeoniflorin reference substance difference point sample amount has obvious spot, and in 10 μ L, spot is more regular, and color is more Clearly, so 10 μ L of selection are the point sample amount of Paeoniflorin.
The determination of 2.2 need testing solution point sample amounts
The μ L of need testing solution each 2,4,8,10 being drawn respectively, being analyzed with the TLC conditions for determining, need testing solution is in 8 μ Spot is more clear regular during L, so the selection of point sample amount is 8 μ L.
3. sample survey
According to the TLC conditions for determining, the thin layer that 3 batch rhizoma cyperi SIWU KELIs are carried out with Paeoniflorin differentiates, as a result such as Fig. 7 institutes Show, on the relevant position of test sample and reference substance, show the spot of same color.Negative controls are noiseless.
Embodiment 7：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 16.8kg is added, after soaking 15 hours, Steam distillation is extracted 8 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 8 times of volatile oil volume) and water 20 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 28kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.13 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Thin-layer chromatography Qualitive test is used to rhizoma cyperi SIWU KELI, specific method is as follows：
1. the preparation of sample
The preparation of 1.1 reference substance solutions
Weigh tetrahydropalmatine reference substance in right amount, plus methyl alcohol is made solution of every 1mL containing 1.25mg, it is molten as reference substance Liquid.
The preparation of 1.2 control medicinal material solution
The powder of 1g corydalis tuber control medicinal materials, plus methyl alcohol 100mL, ultrasonically treated 30min are taken, is filtered, filtrate is evaporated, residue The 20mL that adds water makes dissolving, and enriching ammonia solution is adjusted to alkalescence, is shaken with ether and extracted 3 times, and each 20mL merges ether solution, is evaporated, Residue adds the methyl alcohol 1mL to make dissolving, used as control medicinal material solution.
The processing method of 1.3 need testing solutions
Particle 15g, plus methyl alcohol 100mL, ultrasonically treated 30min are taken, are filtered, filtrate is evaporated, the residue 20mL that adds water makes dissolving, Enriching ammonia solution is adjusted to alkalescence, is shaken with ether and extracted 3 times, and each 20mL merges ether solution, is evaporated, and residue adds the methyl alcohol 1mL to make Dissolving, as need testing solution.
The preparation of 1.4 negative control solutions
According to prescription ratio and preparation method, the negative control sample for lacking corydalis tuber medicinal material is made.Then need testing solution is pressed Preparation method, obtain negative control solution.
2. the optimization of coloration method
It is optimization coloration method, draws above-mentioned each 10 μ L of solution, puts respectively on same silica gel g thin-layer plate, method one：With Toluene-acetone (9:2) it is solvent, sprays to improve bismuth potassium iodide solution, daylight is inspected；Method two：With toluene-acetone (9:2) It is solvent, takes out, volatilize, put in iodine cylinder and taken out after about 1min, after waving the iodine adsorbed on most plate, puts ultraviolet lamp (365nm) Under inspect, spray can obtain obvious spot to improve bismuth potassium iodide solution, and uviol lamp is inspected down, corydalis tuber in need testing solution The spot of B prime is difficult to differentiate, and is covered by other spot, so the method for selection chromogenic reagent is analyzed.
3. the determination of point sample amount
The determination of 3.1 reference substance solution point sample amounts
The μ L of tetrahydropalmatine reference substance solution each 4,6,8,10 are drawn respectively, are put successively in same silica G plate, to determine Condition be analyzed, tetrahydropalmatine reference substance difference point sample amount all spottiness, in 8 μ L, spot is more regular, institute It is the point sample amount of tetrahydropalmatine to select 8 μ L.
The determination of 3.2 need testing solution point sample amounts
The μ L of need testing solution each 4,6,8,10 are drawn respectively, is put successively in same silica G plate, entered with the bar TLC parts for determining Row analysis.Need testing solution all spottiness, the optional 6 μ L of point sample amount in 4~10 μ L.
4. sample survey
According to the TLC discrimination conditions for determining, the thin layer that 3 batch rhizoma cyperi SIWU KELIs are carried out with tetrahydropalmatine differentiates, knot Fruit is as shown in figure 8, on the relevant position of test sample and reference substance, show the spot of same color.Negative controls are noiseless.
Embodiment 8：
A kind of preparation method of rhizoma cyperi SIWU KELI, comprises the following steps：
A. rhizoma cyperi 300g, banksia rose 200g, Radix Angelicae Sinensis 600g, Ligusticum wallichii 300g are taken, the water of 16.8kg is added, after soaking 15 hours, Steam distillation is extracted 8 hours, extracts volatile oil.
B. (addition is volatilization oil body for volatile oil addition beta-schardinger dextrin (addition is 8 times of volatile oil volume) and water 20 times of accumulated amount) it is fully ground 2 hours, freeze-drying obtains volatile oil clathrate compound.
C. it is residue obtained after volatile oil extracting, plus corresponding proportion cultivated land 800g, root of herbaceous peony 300g and corydalis tuber 300g, add water Decoct 2 times, each amount of water 28kg, decoct 2 hours, extract liquid out, merge liquid, filtration, drying under reduced pressure to relative density 1.13 concentrate, concentration of alcohol is 80% (v/v) in adding 95% ethanol to system, and after standing 24 hours, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder.
D. dried cream powder adds dextrin (with dried cream powder equivalent) and Icing Sugar (with dried cream powder equivalent), mixes, and granulation is dried, whole Grain, is eventually adding volatile oil clathrate compound, obtains final product rhizoma cyperi SIWU KELI.
Assay is carried out to rhizoma cyperi SIWU KELI, using high performance liquid chromatography, specific method is as follows：
1. the assay of forulic acid and vanillic acid
1.1 instruments and reagent
The highly effective liquid phase chromatographic systems of WatersAlliance 2695 (quaternary gradient pump, automatic sampler, column temperature system, 2998 photodiode array detectors and the chromatographic work stations of Empower 2, Waters, US)；
KH-500 types supersonic wave cleaning machine (Kunshan He Chuan ultrasonic instruments Co., Ltd)；
HH-S type digital displays thermostat water bath (Medical Instruments factory of Jintan City)；
MS105 assay balances (Mettler-Toledo Instrument Ltd.)；
EPED ultrapure water systems (Nanjing Yi Puyida developments in science and technology Co., Ltd).
Rhizoma cyperi SIWU KELI sample (presses the present embodiment above method self-control sample, lot number：20151101、20151102、 20151103)；
Reference substance forulic acid (lot number：110773-201313), vanillic acid reference substance (lot number：110776-200402), Purchased from National Institute for Food and Drugs Control.It is pure that remaining reagent is analysis.
1.2 methods and result
1.2.1 chromatographic condition
AlltimaTM C18Chromatographic column (250mm × 4.6mm, 5 μm, GRACE)；
Mobile phase：, (glacial acetic acid solution of acetonitrile -1% volume ratio is 20 to forulic acid:80), the vanillic acid (glacial acetic acid of acetonitrile -1% Liquor capacity ratio is 12:88)；
Volume flow 1.0mL/min；Column temperature is 30 DEG C；The μ L of sample size 10；
Detection wavelength：Forulic acid is 323nm, and vanillic acid is 260nm；
Appearance time：Forulic acid is 16min, and vanillic acid is 18min.
1.2.2 the preparation of solution
The preparation of reference substance solution takes forulic acid, vanillic acid reference substance in right amount, accurately weighed, adds methyl alcohol to be made often respectively Reference substance solutions of the 1mL containing 0.0316mg, 0.0295mg, stepwise dilution is configured to 6 reference substance solutions of various concentrations.
The preparation of need testing solution takes rhizoma cyperi SIWU KELI 15g, accurately weighed, in putting conical flask with cover, precision plus methyl alcohol 150mL, is weighed, and stands overnight, ultrasonic 1h, is re-weighed, and the quality of loss is supplied with methyl alcohol, is shaken up, and filtration, precision measures continuous filter Liquid 50mL, water bath method.Residue is transferred in backflow flask, water-bath with 0.5mol/L sodium hydroxide solutions 50mL points of 3 dissolving Backflow 2h, lets cool.Move into separatory funnel, pH=2 is adjusted with hydrochloric acid, extracted 4 times with ether, each 25mL, merge ether solution, return Ether is received, 10mL is dissolved to methyl alcohol, shaken up, obtained final product.
The preparation of negative controls solution is prepared and is lacked Radix Angelicae Sinensis and Ligusticum wallichii the moon by the prescription and preparation technology of rhizoma cyperi SIWU KELI Property control sample.Double-negative solution is obtained by the processing method of need testing solution again.
1.3 determination methods
Reference substance solution, need testing solution and each 10 μ L of negative controls solution are drawn respectively, by the chromatogram under 1.2.1 Condition is analyzed, and as a result sees Fig. 9 and Figure 10.From figure knowable to, in sample chromatogram figure, with forulic acid reference substance, vanillic acid pair According on the corresponding position of product chromatogram, there is the chromatographic peak of an identical retention time, and negative controls solution is at this retention time It is noiseless.
1.4 methodological studies
1.4.1 standard curve and the range of linearity
The accurate each 10 μ L of reference substance for drawing 6 various concentrations, inject liquid chromatograph respectively, by 1.2.1 lower chromatogram Condition is determined.With reference substance peak area value Y (μ Vs) as ordinate, with mass concentration X (μ g/mL) as abscissa, carry out linear Return, draw regression equation.
Forulic acid：Y=5.04 × 107X-5185.4, r=0.9999, in the range of 6.32~47.4 μ g/mL mass concentrations It is good with peak area linear relationship；
Vanillic acid：Y=3.83 × 104X+12993, r=0.9999, in the range of 5.90~29.5 μ g/mL mass concentrations It is good with peak area linear relationship.
1.4.2 precision test
Same reference substance solution is taken, by continuous sample introduction under 1.2.1 lower chromatographic condition 6 times, 10 μ L, observe its repetition every time Property.Result shows that the RSD of forulic acid and vanillic acid reference substance peak area is respectively 0.620% and 0.560%, display instrument essence Density is good.The results are shown in Table 1.
The Precision test result of the forulic acid of table 1 and vanillic acid
1.4.3 reappearance test
6 parts of rhizoma cyperi SIWU KELI (lot number 20151101) is taken, it is accurately weighed respectively, by the preparation method system of need testing solution Into need testing solution, precision draws the μ L of need testing solution 10 injection liquid chromatographs, is determined by 1.2.1 lower chromatographic condition, meter The RSD for calculating forulic acid and vanillic acid in need testing solution is respectively 0.363% and 0.844%, and method reappearance is good.Result is shown in Table 2.
The reproducible test results of the forulic acid of table 2 and vanillic acid
1.4.4 stability test
Liquid is injected respectively at 0,2,4,6,8,10,12, the accurate each 10 μ L of forulic acid, vanillic acid reference substance solution that draw of 24h Chromatography is determined, and is determined by the condition under 1.2.1.Result shows that the RSD of forulic acid reference substance solution peak area is 1.27%, the RSD of vanillic acid reference substance solution peak area is 2.66%, shows two reference substance solutions stabilization within 24h.Knot Fruit is shown in Table 3.
The stability test result of the forulic acid of table 3 and vanillic acid
1.4.5 average recovery experiment
The accurately weighed rhizoma cyperi SIWU KELI sample (lot number for having determined：20151101) 6 parts, forulic acid, perfume (or spice) are separately added into Oxalic acid reference substance is appropriate, according to the processing method of need testing solution, need testing solution is obtained, by 1.2.1 lower described condition Determine, calculate average recovery.Result shows that the average recovery rate of forulic acid and vanillic acid is respectively 99.6% and 101%, RSD is respectively 0.822% and 1.07%, and the two has the good rate of recovery.The results are shown in Table 4 and table 5.
The average recovery result of the test of the forulic acid of table 4
The average recovery result of the test of the vanillic acid of table 5
1.5 sample determinations
Precision weighs 3 batches of rhizoma cyperi SIWU KELI sample (lot numbers：20151101st, 20151102,20151103), by test sample The preparation method of solution is made need testing solution, is determined by 1.2.1 lower chromatographic condition, and calculates content, the results are shown in Table 6.
The assay result of the forulic acid of table 6 and vanillic acid
2. the assay of Paeoniflorin and albiflorin
2.1 instruments and reagent
2.1.1 instrument
With 1.1.1 Suo Shu.
2.1.2 reagent
Rhizoma cyperi SIWU KELI sample (presses the present embodiment above method self-control sample, lot number：20151101、20151102、 20151103)；
Reference substance Paeoniflorin (lot number：110736-201438), purchased from National Institute for Food and Drugs Control；
Reference substance albiflorin (lot number：39011-90-0), purchased from Nanjing Spring ＆ Autumn Biological Engineering Co., Ltd..Remaining examination It is pure that agent is analysis.
2.2 methods and result
2.2.1 chromatographic condition
AlltimaTM C18Chromatographic column (250mm × 4.6mm, 5 μm, GRACE)；
Mobile phase：The acetic acid of acetonitrile -0.1% water (18:82)；
Volume flow 1.0mL/min；
Column temperature is 30 DEG C；
Detection wavelength is 230nm；
The μ L of sample size 10；
The appearance time of Paeoniflorin and albiflorin is about 12min and 10min.
2.2.2 the preparation of solution
The preparation of reference substance solution takes Paeoniflorin, albiflorin reference substance in right amount, accurately weighed, adds methyl alcohol to be made respectively The reference substance solution containing 0.300mg, 0.200mg per 1mL, stepwise dilution is configured to 6 reference substance solutions of various concentrations.
The preparation of need testing solution takes rhizoma cyperi SIWU KELI 15g, accurately weighed, puts in conical flask with cover, and precision adds first Alcohol 75mL, it is accurately weighed.Ultrasonically treated 25min, is let cool, then weighed, and less loss quality is supplied with methyl alcohol, is shaken up, filtration, takes continuous filter Liquid is used as need testing solution.
The preparation of negative sample solution prepares the negative system for lacking the root of herbaceous peony by the prescription and preparation technology of rhizoma cyperi SIWU KELI Agent.The preparation method for pressing need testing solution again is obtained feminine gender solution.
2.3 determination methods
Reference substance solution, need testing solution and each 10 μ L of negative controls solution are drawn respectively, by the chromatogram under 2.2.1 Condition is analyzed, and as a result sees Figure 11.In sample chromatogram figure, in position corresponding with Paeoniflorin, albiflorin reference substance chromatogram Put, there is the chromatographic peak of an identical retention time.And negative controls solution at this retention time without peak.
2.4 methodological studies
2.4.1 standard curve and the range of linearity
The accurate each 10 μ L of reference substance for drawing 6 various concentrations, inject liquid chromatograph respectively, by 2.2.1 lower chromatogram Condition is determined.With reference substance peak area value Y (μ Vs) as ordinate, with mass concentration X (μ g/mL) as abscissa, carry out linear Return, draw regression equation.
Paeoniflorin：Y=1.39 × 104X+98826, r=0.9992, in the range of 150~750 μ g/mL mass concentrations with Peak area linear relationship is good.
Albiflorin：Y=1.24 × 104X+48408, r=0.9996, in 100~500 μ g/mL mass concentration scopes It is interior good with peak area linear relationship.
2.4.2 precision test
Same reference substance solution is taken, by continuous sample introduction under 2.2.1 lower chromatographic condition 6 times, 10 μ L, observe its repetition every time Property.Result shows that the RSD of Paeoniflorin reference substance and albiflorin reference substance peak area is respectively 1.19% and 0.820%, table Bright instrument precision is good.The results are shown in Table 7.
The Precision test result of the Paeoniflorin of table 7 and albiflorin
2.4.3 reappearance test
Take rhizoma cyperi SIWU KELI (lot number：20151101) it is 6 parts, accurately weighed respectively, according to the preparation side of need testing solution Method is made need testing solution.Precision draws 10 μ L need testing solutions injection liquid chromatograph, is determined by the condition under 2.2.1, The RSD for obtaining Paeoniflorin and albiflorin in need testing solution is respectively 1.43% and 1.24%, shows, method reappearance is good It is good.The results are shown in Table 8.
The reproducible test results of the Paeoniflorin of table 8 and albiflorin
2.4.4 stability test
Noted respectively at 0,2,4,6,8,10,12, the accurate each 10 μ L of Paeoniflorin, albiflorin reference substance solution that draw of 24h Enter hplc determination, determined by the condition under 2.2.1.Result shows, Paeoniflorin and albiflorin reference substance solution The RSD of peak area points is 1.05% and 1.48%, is shown, two kinds of reference substance solution stabilizations within 24h.The results are shown in Table 9.
The stability test result of the Paeoniflorin of table 9 and albiflorin
2.4.5 average recovery experiment
Take the rhizoma cyperi SIWU KELI sample (lot number for having determined：20151101) it is 6 parts, accurately weighed, it is separately added into Paeoniflorin It is appropriate with albiflorin reference substance, according to the preparation method of need testing solution, need testing solution is obtained.By under 2.2.1 Condition is measured, and calculates the rate of recovery.Result shows that the rate of recovery of Paeoniflorin and albiflorin is 101% and 102%, RSD It is 1.74% and 1.52%, the two has the good rate of recovery.The results are shown in Table 10 and table 11.
The average recovery experimental result of the Paeoniflorin of table 10
The average recovery experimental result of the albiflorin of table 11
2.5 sample determinations
Precision weighs 3 batches of rhizoma cyperi SIWU KELI sample (lot numbers：20151101st, 20151102,20151103), by test sample The preparation method of solution is made need testing solution, is determined by the chromatographic condition under 2.2.1, and calculates content, the results are shown in Table 12.
The assay result of the Paeoniflorin of table 12 and albiflorin
3. the assay of tetrahydropalmatine and Berberine hydrochloride
3.1 instruments and reagent
3.1.1 instrument
With 1.1.1 Suo Shu.
3.1.2 reagent
Rhizoma cyperi SIWU KELI sample (presses the present embodiment above method self-control sample, lot number：20151101、20151102、 20151103)；
Reference substance tetrahydropalmatine (lot number：110726-201414) with reference substance Berberine hydrochloride (lot number：110713- 201212) it is purchased from National Institute for Food and Drugs Control.It is pure that remaining reagent is analysis.
3.2 methods and result
3.2.1 chromatographic condition
AlltimaTM C18Chromatographic column (250mm × 4.6mm, 5 μm, GRACE)；
Mobile phase is that volume ratio is 55:The acetic acid water of 45 acetonitrile -0.1%, triethylamine adjusts pH=6.0；
Volume flow 1.0mL/min；
Column temperature is 30 DEG C；Detection wavelength is 280nm；The μ L of sample size 10；
The appearance time of tetrahydropalmatine and Berberine hydrochloride is about 11min and 7min.
3.2.2 the preparation of solution
The preparation of reference substance solution：Take tetrahydropalmatine, Berberine hydrochloride reference substance appropriate, it is accurately weighed, first is added respectively Alcohol is made reference substance solutions of every 1mL containing 0.246mg, 0.297mg, and stepwise dilution, the reference substance for being configured to 6 various concentrations is molten Liquid.
The preparation of need testing solution takes rhizoma cyperi SIWU KELI 15g, accurately weighed, puts in boiling flask, and precision adds dense ammonia Test solution-methyl alcohol (1:20) mixed solution 150mL, weighed weight is heated to reflux 1h after cold soaking 1h, lets cool, then weighed weight, with dense Ammonia solution-methyl alcohol (1:20) mixed solution supplies the weight of less loss, shakes up, filtration.Precision measures subsequent filtrate 75mL, is evaporated, residual Slag adds methyl alcohol to dissolve, and is transferred in 10mL measuring bottles, and is diluted to scale, shakes up, filtration, takes subsequent filtrate, obtains final product.
The preparation of negative sample solution is made the moon of scarce corydalis tuber medicinal material according to rhizoma cyperi SIWU KELI prescription and preparation technology Property preparation.The preparation method for pressing need testing solution again is obtained feminine gender solution.
3.3 determination methods
Reference substance solution, need testing solution and each 10 μ L of negative controls solution are drawn respectively, by the condition under 3.2.1 It is analyzed, as a result sees Figure 12.In sample chromatogram figure, position corresponding with tetrahydropalmatine and Berberine hydrochloride reference substance chromatogram Put, there is the chromatographic peak of an identical retention time.And negative controls solution at this retention time without peak.
3.4 methodological studies
3.4.1 standard curve and the range of linearity
It is accurate respectively to draw 6 μ L of the reference substance of various concentrations 10, liquid chromatograph is injected, by the condition under 3.2.1 Determine.With reference substance peak area value Y (μ Vs) as ordinate, as abscissa, linearly returned with mass concentration X (μ g/mL) Return, draw regression equation.
Tetrahydropalmatine：Y=8.42 × 103X-12481, r=0.9999, in 98.4~492 μ g/mL mass concentration scopes It is interior good with peak area linear relationship.
Berberine hydrochloride：Y=3.08 × 104X-134654, r=0.9999, in 59.4~446 μ g/mL mass concentration models It is good with peak area linear relationship in enclosing.
3.4.2 precision test
Same reference substance solution is taken, by continuous sample introduction under 3.2.1 lower chromatographic condition 6 times, 10 μ L, observe its repetition every time Property.Result shows that the RSD of tetrahydropalmatine reference substance and Berberine hydrochloride reference substance peak area is respectively 1.52% He 1.00%, show that instrument precision is good.The results are shown in Table 13.
The Precision test result of the tetrahydropalmatine of table 13 and Berberine hydrochloride
3.4.3 reappearance test
6 parts of rhizoma cyperi SIWU KELI (lot number 20151101) is taken, it is accurately weighed respectively, according to the preparation side of need testing solution Method, is made need testing solution.Precision draws 10 μ L, is determined by the condition under 3.2.1, calculates corydalis tuber in need testing solution The RSD of B prime and Berberine hydrochloride is respectively 0.943% and 1.14%, shows, method reappearance is good.The results are shown in Table 14.
The reproducible test results of the tetrahydropalmatine of table 14 and Berberine hydrochloride
3.4.4 stability test
Tetrahydropalmatine, each 10 μ of Berberine hydrochloride reference substance solution are drawn respectively at 0,2,4,6,8,10,12,24h precisions L presses 3.2.1 lower condition and determines.Result shows that the RSD of tetrahydropalmatine and Berberine hydrochloride reference substance solution peak area distinguishes It is 0.830% and 1.09%, shows, two kinds of reference substance solution stabilizations in 24h.The results are shown in Table 15.
The stability test result of the tetrahydropalmatine of table 15 and Berberine hydrochloride
3.4.5 average recovery experiment
Take the rhizoma cyperi SIWU KELI sample (lot number for having determined：20151101) it is 6 parts, accurately weighed, it is separately added into corydalis tuber B prime and Berberine hydrochloride reference substance are appropriate, by the preparation method of need testing solution, need testing solution are made, by under 3.2.1 Condition determine, calculate the rate of recovery.Result shows that the average recovery rate of tetrahydropalmatine and Berberine hydrochloride is 99.8% He 100%, RSD are 0.454% and 0.835%, and the two has the good rate of recovery.The results are shown in Table 16 and table 17.
The average recovery result of the test of the tetrahydropalmatine of table 16
The average recovery result of the test of the Berberine hydrochloride of table 17
3.5 sample determinations
Precision weighs 3 batches of rhizoma cyperi SIWU KELI sample (lot numbers：20151101st, 20151102,20151103), by test sample The preparation method of solution, is made need testing solution, is determined by the chromatographic condition under 3.2.1, and calculates content, the results are shown in Table 18.
The assay result of the tetrahydropalmatine of table 18 and Berberine hydrochloride
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (6)

1. a kind of preparation method of rhizoma cyperi SIWU KELI, it is characterised in that it is to compare 3 by weight:2:3:6:3:8:3 rhizoma cyperi, The banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony are made, and specifically include following steps：
A. Radix Angelicae Sinensis, Ligusticum wallichii, rhizoma cyperi and banksia rose medicine materical crude slice are weighed by above weight ratio, add the water of medicinal material 5-15 times of weight of gross weight, After immersion 10-15 hours, then steam distillation is extracted 8-12 hours, extracts volatile oil；
B. taking volatile oil adds beta-schardinger dextrin and water to be fully ground, and freeze-drying obtains volatile oil clathrate compound；
C. take it is residue obtained after volatile oil extracting, add above-mentioned weight than cultivated land, the root of herbaceous peony and corydalis tuber, add water to cook 2~3 Secondary, each amount of water is 6-14 times of total medicinal material, is decocted 1-3 hours, merges liquid, filtration, drying under reduced pressure to relative density The concentrate of 1.00-1.15, concentration of alcohol is 80~85% in adding the ethanol of volumetric concentration 95% to concentrate, is stood, suction filtration, Filtrate is taken, is concentrated under reduced pressure, dried, get dry extract powder；
D. take dried cream powder and add dextrin, Icing Sugar by a certain percentage, mix, granulation is dried, and whole grain is eventually adding obtained in step b Volatile oil clathrate compound, obtains final product.
2. the preparation method of rhizoma cyperi SIWU KELI according to claim 1, it is characterised in that specifically include following steps：
A. Radix Angelicae Sinensis, Ligusticum wallichii, rhizoma cyperi and banksia rose medicine materical crude slice are weighed by above weight ratio, adds medicinal material 8 times of water of weight of gross weight, immersion After 12 hours, steam distillation is extracted 10 hours, extracts volatile oil；
B. taking volatile oil adds beta-schardinger dextrin and water to be fully ground, and freeze-drying obtains volatile oil clathrate compound；
C. it is residue obtained after volatile oil extracting, add above-mentioned weight than cultivated land, the root of herbaceous peony and corydalis tuber, add water to cook 2 times, every time Amount of water is 10 times of total medicinal material, is decocted 3 hours, merges liquid, and filtration, drying under reduced pressure is dense to relative density 1.05-1.08's Contracting liquid, add volumetric concentration be 95% ethanol to concentrate in concentration of alcohol be 80%, stand 24 hours after, suction filtration, filtrate subtracts Pressure concentration, dries, and get dry extract powder；
D. take dried cream powder and add dextrin, Icing Sugar, mix, granulation is dried, and whole grain is eventually adding the volatile oil that step b is prepared Inclusion compound, obtains final product.
3. the preparation method of rhizoma cyperi SIWU KELI according to claim 1, it is characterised in that in step b, beta-schardinger dextrin Addition is 5-20 times of volatilization oil volume；The addition of water is 10-50 times of volatilization oil volume.
4. the preparation method of rhizoma cyperi SIWU KELI according to claim 1, it is characterised in that in step d, the addition of dextrin It is 0-2 times of dried cream powder weight to measure, and the addition of Icing Sugar is 0-2 times of dried cream powder weight.
5. the method for quality control of the rhizoma cyperi SIWU KELI described in any one of claim 1-4, it is characterised in that including with lower section One or more in method：
(1) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus sodium bicarbonate solution, ultrasonically treated, centrifugation, take supernatant watery hydrochloric acid regulation pH value To 2~3, shaken with ether and extracted 2~3 times, merge ether solution, volatilized, residue adds the methyl alcohol to make dissolving, as Ligustilide for trying Product solution；
Rhizoma cyperi SIWU KELI, plus EtOH Sonicate dissolving are taken, is put and concentrated in water-bath, shaken with ether and extracted 2~3 times, merge ether Extract solution, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, ether solution is discarded, extract solution adjusts pH with watery hydrochloric acid Value shakes extraction to 2~3, then with ether, merges ether extracted liquid, volatilizes, and residue adds methyl alcohol to dissolve, used as forulic acid test sample Solution；
Radix Angelicae Sinensis, Ligusticum wallichii control medicinal material, plus EtOH Sonicate dissolving are taken, is put and concentrated in water-bath, shaken with ether and extracted 2~3 times, merged Ether extracted liquid, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, ether solution is discarded, extract solution is adjusted with watery hydrochloric acid Section pH value shakes extraction to 2~3, then with ether, merges ether extracted liquid, volatilizes, and residue adds methyl alcohol to dissolve, and is made control medicinal material Solution；
Rhizoma cyperi, the banksia rose, corydalis tuber, cultivated land and the root of herbaceous peony, plus EtOH Sonicate dissolving are taken, is put and concentrated in water-bath, shaken with ether and extract 2 ~3 times, merge ether extracted liquid, then to adding sodium carbonate liquor shaking to extract in above-mentioned diethyl ether solution, discard ether solution, extract solution PH value being adjusted with watery hydrochloric acid and shaking extraction to 2~3, then with ether, merge ether extracted liquid, volatilized, residue adds methyl alcohol to dissolve, system Negative control solution in pairs；
Forulic acid and ligustilide from rhizome are taken, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 5 kinds of solution is drawn, put respectively on same silica gel g thin-layer plate, be 30 with volume ratio: 1:3 benzene-glacial acetic acid-methyl alcohol is solvent, is launched, and is taken out, and airing is put under ultraviolet lamp, and 365nm is inspected, as a result, test sample On the relevant position of reference substance, show the spot of same color, negative controls are noiseless；
(2) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add diethyl ether and be heated to reflux, filter, filtrate volatilizes, and residue adds ethyl acetate to dissolve, as Need testing solution；
Ligusticum chuanxiong Hort powder is taken, is added diethyl ether and is heated to reflux, filtered, filtrate volatilizes, and residue adds ethyl acetate to dissolve, and is made comparison medicine Material solution；
Rhizoma cyperi, the banksia rose, corydalis tuber, cultivated land, Radix Angelicae Sinensis and the root of herbaceous peony are taken, is added diethyl ether and is heated to reflux, filtered, filtrate volatilizes, and residue adds second Acetoacetic ester dissolves, and is made negative control solution；
Levistilide A reference substance is taken, plus ethyl acetate is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica GF254 lamellae, be with volume ratio 3:1 n-hexane-ethyl acetate is solvent, is launched, and is taken out, airing, and 254nm is inspected under putting ultraviolet lamp, as a result, test sample On the relevant position of reference substance, show the spot of same color, negative controls are noiseless；
(3) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add diethyl ether, place 1~2 hour, constantly shake, filter, filtrate volatilizes, and residue adds acetic acid Ethyl ester makes dissolving, used as need testing solution；
Rhizoma cyperi control medicinal material powder is taken, is added diethyl ether, placed 1~2 hour, constantly shaken, filtered, filtrate volatilizes, and residue adds acetic acid Ethyl ester makes dissolving be made control medicinal material solution；
The banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and white Peony Root are taken, are added diethyl ether, placed 1~2 hour, constantly shaken, filtered, Filtrate volatilizes, and residue adds the ethyl acetate dissolving is made negative control solution；
α-cyperolone reference substance is taken, plus ethyl acetate is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica GF254 lamellae, be with volume ratio 80:1:1 dichloromethane-ethyl acetate-glacial acetic acid is solvent, is launched, and is taken out, airing, puts inspection under ultraviolet lamp 254nm Depending on, then spray with DNPH ethanol test solution, it is seen that inspected under light；As a result, on the relevant position of test sample and reference substance, The spot of aobvious same color, negative controls are noiseless；
(4) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus petroleum ether, cold soaking 30~60 minutes, constantly shake, filter, filtrate volatilizes, and residue adds Ethyl acetate makes dissolving, used as need testing solution；
The powder of banksia rose control medicinal material, plus petroleum ether are taken, cold soaking 30~60 minutes constantly shakes, filtered, filtrate volatilizes, residue Plus ethyl acetate makes dissolving be made control medicinal material solution；
Rhizoma cyperi, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony, plus petroleum ether are taken, cold soaking 30~60 minutes constantly shakes, filtered, Filtrate volatilizes, and residue adds the ethyl acetate dissolving is made negative control solution；
Dehydro-α-curcumene reference substance and costunolide reference substance are taken, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica gel g thin-layer plate, with volume ratio 15:5:1 With cyclohexane-ethyl acetate-formic acid as solvent, launch, take out, airing, spray is heated with 5% vanillin-sulfuric acid ethanol solution, Clear to spot development, daylight is inspected, as a result, on the relevant position of test sample and reference substance, shows the spot of same color, negative Reference substance is noiseless；
(5) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI 15g is taken, finely ground, add water 100mL, warm makes it fully leach, and lets cool, filtered with absorbent cotton, filtrate Shaken with ether and extracted 2 times, each 25mL discards ether solution, then shakes extraction 2 times with ethyl acetate, and each 25mL merges second Acetoacetic ester liquid, is evaporated, and residue adds the methyl alcohol 1mL to make dissolving, used as need testing solution.The powder of 1g cultivated land medicinal materials is taken, is made in the same way of Control medicinal material solution；Scarce cultivated land sample 15g is taken, negative control solution is made in the same way of.Take 5 hydroxymethyl furfural reference substance, plus methyl alcohol Solution of every 1mL containing 0.290mg is made, as reference substance solution.According to thin-layered chromatography (general rule 0502) experiment, above-mentioned 4 are drawn Solution is planted, is put respectively on same silica gel g thin-layer plate, with dimethylbenzene-ethyl acetate (1:1) it is solvent, launches, takes out, it is cool It is dry, spray with DNPH ethanol solution, daylight is inspected.As a result, on the relevant position of test sample and reference substance, show identical The spot of color.Negative controls are noiseless.
(6) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, add water, heating for dissolving is extracted 2~3 times with water-saturated n-butanol, merges n-butanol liquid, With n-butanol saturation water washing, merge n-butanol liquid, water-bath is volatilized, plus methyl alcohol dissolving, using silicagel column, it is with volume ratio first 10:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol wash-out, is finally 8 with volume ratio: 1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol to dissolve, used as need testing solution；
Take root of herbaceous peony control medicinal material powder, add water, it is ultrasonically treated after, with water-saturated n-butanol extract 2~3 times, merge n-butanol liquid, With n-butanol saturation water washing, merge n-butanol liquid, water-bath is volatilized, plus methyl alcohol dissolving, using silicagel column, it is with volume ratio first 10:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol wash-out, is finally 8 with volume ratio: 1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol to dissolve, and is made control medicinal material solution；
Take rhizoma cyperi, the banksia rose, corydalis tuber, Radix Angelicae Sinensis, Ligusticum wallichii and cultivated land medicinal material, add water, it is ultrasonically treated after, extracted with water-saturated n-butanol 2~3 times, merge n-butanol liquid, with n-butanol saturation water washing, merge n-butanol liquid, water-bath is volatilized, plus methyl alcohol dissolving, use Silicagel column, is first 10 with volume ratio:1 chloroform and methyl alcohol wash-out, then with volume ratio be 9:1 chloroform and methyl alcohol is washed It is de-, it is finally 8 with volume ratio:1 chloroform and methyl alcohol wash-out, collects follow-up 8:1 eluent, is evaporated, and residue adds methyl alcohol molten Solution, is made negative control solution；
Precision weighs Paeoniflorin reference substance, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica gel g thin-layer plate, be 40 with volume ratio: 5:10:0.2 chloroform-acetate-methanol-formic acid is solvent, is launched, and is taken out, airing, is sprayed with 5% vanillin-sulfuric acid Ethanol solution, daylight is inspected, as a result, on the relevant position of test sample and reference substance, shows the spot of same color, negative controls It is noiseless.
(7) thin-layer chromatography Qualitive test is used
Rhizoma cyperi SIWU KELI is taken, it is finely ground, plus methyl alcohol, ultrasonically treated, filtration, filtrate is evaporated, and residue is dissolved in water, enriching ammonia solution Alkalescence is adjusted to, is shaken with ether and extracted, merge ether solution, be evaporated, residue adds methyl alcohol to dissolve, used as need testing solution；
The powder of corydalis tuber control medicinal material, plus methyl alcohol are taken, ultrasonically treated, filtration, filtrate is evaporated, and residue is dissolved in water, enriching ammonia Test solution is adjusted to alkalescence, is shaken with ether and extracted, and merges ether solution, is evaporated, and residue adds methyl alcohol dissolving to be made control medicinal material solution；
Rhizoma cyperi, the banksia rose, Radix Angelicae Sinensis, Ligusticum wallichii, cultivated land and the root of herbaceous peony, plus methyl alcohol are taken, ultrasonically treated, filtration, filtrate is evaporated, and residue adds water molten Solution, enriching ammonia solution is adjusted to alkalescence, is shaken with ether and extracted, and merges ether solution, is evaporated, and it is negative right that residue adds methyl alcohol dissolving to be made According to solution；
Precision weighs tetrahydropalmatine reference substance, plus methyl alcohol is made reference substance solution；
According to thin-layered chromatography experiment, above-mentioned 4 kinds of solution is drawn, put respectively on same silica gel g thin-layer plate, be 9 with volume ratio:2 Toluene-acetone is solvent, launch, take out, airing, spray to improve bismuth potassium iodide solution, daylight is inspected, as a result, test sample with On the relevant position of reference substance, show the spot of same color, negative controls are noiseless.
6. the method for quality control of the rhizoma cyperi SIWU KELI described in any one of claim 1-4, it is characterised in that including with lower section One or more in method：
(1) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；With volume during detection forulic acid Than 20:The glacial acetic acid solution of 80 acetonitrile -1% is mobile phase；With volume ratio 12 during detection vanillic acid:88 glacial acetic acid of acetonitrile -1% is molten Liquid is mobile phase；Detection wavelength is respectively 323nm and 260nm；Theoretical cam curve is calculated by forulic acid and vanillic acid and is not less than 3000；
The preparation of reference substance solution takes forulic acid, vanillic acid reference substance in right amount, accurately weighed, adds methyl alcohol to be made every 1mL respectively and contains The reference substance solution of 0.0316mg, 0.0295mg；
The preparation of need testing solution takes rhizoma cyperi SIWU KELI, accurately weighed, puts in conical flask with cover, and precision adds methyl alcohol, weighs, Stand overnight, ultrasound is re-weighed, and the quality of loss is supplied with methyl alcohol, is shaken up, and is filtered, and precision measures subsequent filtrate, water bath method； Residue sodium hydroxide solution dissolves, and is transferred in backflow flask, and water-bath backflow lets cool；Move into separatory funnel, adjusted with hydrochloric acid PH=2, is extracted with ether, merges ether solution, reclaims ether, is dissolved with methyl alcohol, is shaken up, and is obtained final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, and obtains final product；
This product per 15g rhizoma cyperis SIWU KELIs containing Radix Angelicae Sinensis and Ligusticum wallichii in terms of forulic acid and vanillic acid, must not less than 0.714mg and 0.439mg；
(2) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；With volume ratio 18:82 second The acetic acid of nitrile -0.1% water is mobile phase；Detection wavelength is 230nm, and theoretical cam curve presses Paeoniflorin and albiflorin is calculated not Less than 2000；
The preparation of reference substance solution takes Paeoniflorin, albiflorin reference substance in right amount, accurately weighed, adds methyl alcohol to be made often respectively Reference substance solutions of the 1mL containing 0.300mg, 0.200mg.
The preparation of need testing solution takes rhizoma cyperi SIWU KELI, accurately weighed, puts in conical flask with cover, and precision adds methyl alcohol, accurate It is weighed, it is ultrasonically treated, let cool, then it is weighed, less loss quality is supplied with methyl alcohol, shake up, filter, take subsequent filtrate molten as test sample Liquid；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, and obtains final product.
Rhizoma cyperi SIWU KELI contains the root of herbaceous peony per 15g in terms of Paeoniflorin and albiflorin, must not be less than 52.5mg and 17.9mg；
(3) assay of rhizoma cyperi SIWU KELI, using high performance liquid chromatography, comprises the following steps that：
Chromatographic condition is with system suitability test with octadecylsilane chemically bonded silica as filler；It is 55 with volume ratio:45 second The acetic acid water of nitrile -0.1%, and pH=6.0 is adjusted as being mobile phase with triethylamine；Detection wavelength is 280nm；Theoretical cam curve is by prolonging Hu Suo Yisu and Berberine hydrochloride are calculated and are not less than 3000；
The preparation of reference substance solution takes tetrahydropalmatine, Berberine hydrochloride reference substance in right amount, accurately weighed, and methyl alcohol system is added respectively Into reference substance solutions of every 1mL containing 0.246mg, 0.297mg；
The preparation of need testing solution takes rhizoma cyperi SIWU KELI 15g, accurately weighed, and in putting boiling flask, precision adds the volume ratio to be 1:20 strong ammonia solution-methyl alcohol mixed solution, weighed weight, cold soaking is heated to reflux, lets cool, then weighed weight, is with volume ratio 1:20 strong ammonia solutions-methyl alcohol mixed solution supplies the weight of less loss, shakes up, filtration；
Precision measures subsequent filtrate 75mL, is evaporated, and residue adds methyl alcohol to dissolve, and is transferred in 10mL measuring bottles, and is diluted to scale, shakes Even, filtration takes subsequent filtrate, obtains final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, and obtains final product；
Rhizoma cyperi SIWU KELI per 15g particles containing corydalis tuber in terms of tetrahydropalmatine and Berberine hydrochloride, must not less than 2.67mg and 1.22mg。
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